319
American Society of Cytopathology
53rd Annual Scientific Meeting
Platform and Poster Presentations
Supplement to Cancer Cytopathology
ASC Foundation Cytopathology
Research Seed Award
Platforms
1 November 7, 2005
10:00 am – 10:20 am
Cytologic Preparations can be used to
Monitor Tumor Cells for Response to
Targeted Drug Therapy
Brian S. Chang, M.D., Ph.D., Tao Yang, M.D., Ph.D.,
Jonathan A. Fletcher, M.D., Edmund S. Cibas, M.D.
INTRODUCTION: Targeted drug therapy is the Holy Grail of on-
cology. Through the understanding of key molecular path-
ways involved in tumor growth, specific inhibitors can be
designed to inactivate these pathways, thereby maximizing
therapeutic response. One of the most compelling examples
of successful targeted therapy for a metastatic solid tumor is
imatinib (Gleevec), which inhibits the KIT tyrosine kinase in
gastrointestinal stromal tumors (GIST). Dramatic clinical re-
sponses are seen routinely after initiation of Gleevec therapy,
but most patients eventually relapse as the tumors develop
resistance to the drug. Additional specific inhibitors of the KIT
signaling pathway are currently undergoing clinical trials. The
aim of this study was to develop a robust, cytology-based
assay to measure tumor susceptibility to target-specific small
molecule inhibitors.
MATERIALS AND METHODS: We created an immortal GIST cell
line (GIST882), which was treated, in vitro, with the Gleevec
inhibitor of KIT, and the RAD001 inhibitor of the KIT down-
stream signaling protein, mTOR. (The signaling pathway pro-
teins activated by the GIST KIT mutations are sequentially:
KIT!!AKT!!mTOR!!S6.) Treatment response was assessed in
cytologic preparations (ThinPrep and cytospin) by immunocy-
tochemical staining with antibodies to KIT, phospho-KIT, phos-
pho-AKT, and phospho-S6. Optimization was performed to max-
© 2005 American Cancer Society
DOI 10.1002/cncr.21457
Published online in Wiley InterScience (www.interscience.wiley.com).
imize staining in the absence of inhibitor, and minimize staining
in the presence of inhibitor.
RESULTS: GIST882 cells demonstrated strong, robust phos-
pho-S6 expression in the absence of inhibitor treatment. This
expression was completely inhibited by treatment with up-
stream signaling pathway inhibitors (Gleevec and RAD001.)
Other phospho-specific antibodies (phospho-AKT and Phospho-
KIT) had weaker baseline reactivity in the absence of inhibitor.
The accuracy of the immunocytochemical results on the cyto-
logic preparations was validated by immunoblotting studies.
CONCLUSION: Cytologic phospho-S6 staining can be used to
gauge GIST response to KIT or mTOR inhibitors. This approach
provides a straightforward strategy to measure biochemical re-
sponsiveness (or lack thereof) in patients receiving targeted che-
motherapy and will be a useful tool for evaluating the efficacy of
targeted therapies. Our study demonstrates the feasibility of
cytologic methods to monitor labile biochemical responses in
tumor cells during drug therapy. Such approaches will be en-
hanced by the development of additional activation state-spe-
cific antibodies, particularly those optimized for use in cytologic
preparations.
Brigham and Women’s Hospital, Boston, Massachusetts
2 November 7, 2005
10:20 am to 10:40 am
Improved FISH Assay for RET
Translocations in Papillary Thyroid
Carcinoma
Frank Chen, M.D., Ph.D., Anita Hawkins, Erik Kincaid, CT,
MP(ASCP), Constance Griffin, Douglas P. Clark, M.D.
INTRODUCTION/PURPOSE: Fifteen different RET rearrangements
(10q11.2) have been reported in papillary thyroid carcinoma
(PTC). RET/PTC1, resulting from a paracentric inversion, has-
been shown to be the most common type in the United States.
We have reported the development of break-apart FISH
320 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
probes as a tool to detect translocations/inversions involving
the RET gene.
MATERIALS AND METHODS: Multiple FISH probes located on
either side of the RET gene were prepared from mapped BACs
by DOP PCR (Telenius, Genomics 1992;13:718) and labeled
with Spectrum Red-dUTP and biotin-dUTP (AMCA"blue de-
tection). (1) BAC probes were characterized and optimized
individually by FISH to normal lymphocytes; the 2 best probes
from each side of RET were combined to maximize signal
intensity. (2) DOP-PCR labeled probes were DNase treated to
obtain segments less than 400 bp. A “permanent template”
was prepared by re-PCR after DNase to eliminate repeated
DNase steps with each PCR. (3) To enhance the assay speci-
ficity, a probe from the 5# end of the H4 gene (involved
by RET/PTC1 inversion) was used to add a fusion element to
the break-apart probe. TPC-1 cells (RET/PTC1 positive, con-
firmed by RT-PCR for RET/H4) were used to test the new
probe. TSH was added to TPC-1 culture medium to obtain
metaphases for FISH to chromosomes and G-banded karyo-
typing.
RESULTS: Combined probes gave stronger FISH signals on
interphase nuclei from both normal lymphocytes and TPC-1
cells than any individual probe. Probes from “permanent tem-
plates” showed similar results to probes from a previous
DNase method, while significantly enhancing the reproduc-
ibility of the assay. After the above modification, 97% of
normal interphase nuclei showed 2 paired red-blue signals
together with a low false positive rate (2%). Ninety-three
percent of TPC-1 cells showed the expected signal pattern
(one set of red-blue signals separated and one set paired) with
a low false negative rate (4%); however, wider separation of
signals was seen than expected for the inversion. Examination
of TPC-1 chromosomes showed the inv(10) was further rear-
ranged in a 3-way translocation (1;inv10;21) (consistent with
reported partial karyotype, Jossart, Surgery 1995;118:1018),
resulting in the red (centromeric to RET) signal moving to a
derivative chromosome, while the blue (telomeric to RET)
remained. When the green (H4) probe was added (all 3 probes
together on 10q in normals), blue and green signals colocal-
ized in TPC-1, indicating the inv(10) breakpoint in t(1;inv10;
21) is telomeric to the red probe and centromeric to (or at) the
inversion breakpoint in RET.
CONCLUSION: The break-apart FISH assay for RET gene trans-
locations was greatly enhanced by combining BAC probes.
Adding the third probe (H4) gave additional information on
metaphase chromosomes, but did not improve interphase
results. This assay can potentially detect all of the clinically
relevant translocations involving the RET gene. After further
development, this assay could be used to study PTC biology or
as an adjunct to thyroid FNA.
The Johns Hopkins University, Baltimore, Maryland
Platform Presentations
1 November 5, 2005
10:00 am – 10:15 am
ASC-US in the Community: Do HPV Results
and ASCCP Consensus Guidelines Actually
Direct Clinical Management?
Dawn K. Riedy, M.D., Elizabeth Parker, B.S., CT(ASCP),
Sharyn Rezsnyak, CT(ASCP)IAC, Lisa Livoti Struble, B.S.,
CT(ASCP)
INTRODUCTION/PURPOSE: Reflex HPV testing and ASCCP Man-
agement Guidelines (JAMA 2002) are intended to provide cost-
effective, evidence-based triage of ASC-US patients, avoiding
unnecessary repeat cytologies and colposcopies for women neg-
ative for high risk HPV and prompting colposcopy to exclude
underlying CIN2/3 in HPV positive women. The purpose of this
study is to determine whether or not in community practice HPV
test results are effectively utilized and ASCCP Guidelines are
routinely followed.
MATERIALS AND METHODS: With a volume of 111,300 gyn cytol-
ogies in 2004 (99% SurePath™), our laboratory has an ASC-US
rate of 2.3% (ASC-US: SIL ratio 1.1:1.0) and offers reflex testing
for high risk HPV (Digene HC2®) with a positive rate of 52%.
Using our computer data base and telephone follow-up, we
undertook a retrospective analysis of all cases of ASC-US diag-
nosed from January-March 2004 (658 cases total). Patients were
divided into three groups by HPV status (209$, 242- and 207
unknown) and results of cytologies and colposcopies over the
subsequent 12-15 months were recorded and analyzed.
RESULTS: The cumulative incidence of CIN2/3 in the HPV$
group was 20%, similar to the ALTS results, yet only 50% of these
patients had colposcopy within 3 months (55% by 12-15 months)
and half of the patients not colposcoped were high-risk by virtue
of prior or subsequent abnormal cytologies and/or biopsies. Of
note, the 20% incidence of CIN2/3 in this group was higher than
the 15% cumulative incidence of CIN2/3 in patients with LGSIL
cytologies over the same time period, yet more LGSIL patients
were colposcoped. No patients in the HPV negative group had
documented CIN2/3 during the follow-up period. While these
patients were rarely colposcoped, many had repeat cytologies
(1-4 repeat cytologies over 12-15 months) beginning as early as
1 month after the negative HPV result. 74% of HPV negative
patients with follow-up had early repeat cytologies (%10
months), on average 5.1 months after the index Pap, sooner even
than the recommendation for patients whose HPV status is not
known.
CONCLUSIONS: Clinicians do not consistently follow the colpos-
copy recommendation for ASC-US HPV$ patients despite the
significant incidence of CIN2/3 in this population. Conversely,
clinicians do not demonstrate confidence in negative HPV re-
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
sults, frequently performing repeat cytologies sooner than the
recommended 12 months. Whether or not clinical experience
and education will over time improve the efficacy of reflex HPV
testing and adherence to ASCCP Guidelines remains to be seen.
Via Health Rochester General Hospital, Rochester, New York
2 November 5, 2005
10:15 am – 10:30 am
The Monoprep® Pap Test for the Detection
of Cervical Cancer and its Precursors:
Results of a Multi-Center Clinical Trial
Carol Eisenhut, M.D.1, R. Marshall Austin, M.D., Ph.D.2,
David Bolick, M.D., FCAP, MIAC3, Michael Glant, M.D.1,
Michael Henry, M.D.4, E. Blair Holladay, Ph.D., CT(ASCP)2,
Ann Moriarty, M.D.5, Sally Slowman, M.D.1, Roosevelt
Torno, M.D.6, Norman Pressman, Ph.D.7, Edmund Cibas,
M.D.8
INTRODUCTION: The MonoPrep® Pap Test (MPPT) is a specimen
collection and processing kit specifically designed for preparing
gynecological specimens for Pap test and other testing. The
MPPT is processed on the MonoPrep® Processor (MonoGen,
Inc), an automated, multi-sample instrument for processing liq-
uid-based cytology specimens. MPPT’s utility in the detection of
cervical cancer and its precursors was evaluated in a multi-
center, masked, adjudicated, split-sample study.
MATERIALS AND METHODS: After preparation of a conventional
smear, the residuum on the collection device was rinsed into a
collection vial from which an MPPT slide was prepared at the
laboratory. The study was conducted at 4 U.S. laboratories, and
patients were recruited from the U.S. (72%) and abroad (28%)
under IRB supervision with subject consent. All slides from any
subject found to have an abnormality by smear and/or MPPT,
and at least 5% of all negative slides, were submitted for masked
adjudication (by one of five pathologists with added qualifica-
tion in cytopathology) and HPV testing with the Digene hc2 HPV
DNA test (high risk probe only). For data analysis, ASC-US cases
were stratified into low- and high-risk atypia. High-risk atypia
(HR-AT) included ASC-H, AGC, and ASC-US with a positive HPV
test.
RESULTS: Evaluable samples were obtained from 10,752 women
at 88 collection sites. Compared with the conventional smear,
MPPT showed increased adjudicator-diagnosed abnormalities at
all clinically significant diagnostic thresholds: HR-AT$ (29% in-
crease; p%0.0001), LSIL$ (59% increase, p%0.0001), HSIL$ (59
increase%, p%0.0001), and cancer (48% increase, p%0.059). Ad-
judicator diagnoses of glandular disease (AGC, AIS and adeno-
carcinoma) were more frequent than with the matched smear
(total 27 vs.17). The specificities of smear and MPPT diagnoses
were essentially identical. There was a 50% decrease in unsatis-
factory cases with MPPT as compared with the conventional
321
smear (p%0.0001), and MPPT slides demonstrated no difference
in the adequacy of the endocervical/transformation zone com-
ponent (p"0.223). The inter-observer agreement between labo-
ratories and adjudicators was evaluated with the kappa statistic
and level of exact agreement. MPPT showed improved diagnos-
tic agreement between observers for HR-AT$ (7%), LSIL (19%),
HSIL (33%) and cancer (44%).
CONCLUSIONS: In this study, detection of cervical cancer precur-
sors was significantly improved with MPPT-based screening
compared with the conventional smear. Based on these data, the
MPPT is a promising alternative liquid-based technology for
cervical cancer screening.
1
DCL Medical Laboratories, Indianapolis, Indiana, 2Medical Uni-
versity of South Carolina, Charleston, South Carolina, 3Amer-
iPath Utah, Sandy, Utah, 4Cleveland Clinic Florida Hospital,
Naples, Florida, 5AmeriPath, Indianapolis, Indiana, 6Universal
Diagnostic Laboratories, New York, New York, 7MonoGen, Inc.,
Vernon Hills, Illinois, 8Brigham and Women’s Hospital and Har-
vard Medical School, Boston, Massachusetts
3 November 5, 2005
10:30 am – 10:45 am
Evaluation of the CINtec™ Assay for
Detection of p16INK4a in ThinPrep®
Specimens and Correlation to Biopsy
Follow-up Results
Jane Meyer, CT(ASCP)1, Kirsten Bisgaard2, Birthe
Andersen2, Ole F. Rasmussen2, David Hanlon, Ph.D.1,
James Linder, M.D.1,3, Edmund Cibas, M.D.4
INTRODUCTION: Overexpression of p16INK4a in dysplastic epithe-
lial cells of the uterine cervix is a consequence of the oncogenic
activity of high-risk HPV E7 protein rather than viral presence
only and therefore remains a biomarker of interest. In published
studies that examined p16INK4a expression in cervical cytology
specimens, sample numbers were modest. The aim of this study
was to examine p16INK4a protein expression in ThinPrep® (Cytyc
Corporation) specimens utilizing the CINtec™ p16INK4a Cytol-
ogy Kit (DakoCytomation, Denmark). p16INK4a was evaluated as
a potential marker to complement cytological diagnosis. The
ability of this assay to accurately identify underlying lesions of
CIN II or greater was also assessed using available biopsy fol-
low-up data.
MATERIALS AND METHODS: A total of 398 residual ThinPrep
samples were collected from two clinical sites; 175 WNL, 60 BCC,
49 ASC-US, 6 ASC-H, 57 LSIL, and 51 HSIL samples. Two slides
were prepared from each sample; one slide was stained with the
ThinPrep Stain and a second slide immunostained using the
CINtec™ p16INK4a Cytology Kit. Both slides were prepared
within six weeks of the sample collection date and immunostain-
ing was completed within three weeks from the date the slide
was prepared. p16INK4a immunoreactivity was assigned to mor-
322 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
phologically defined categories. Staining intensity and the num-
ber of positive cells were recorded. High-risk HPV testing was
performed on all specimens using the Hybrid Capture® 2 High-
Risk HPV DNA Test™ (hc2) (Digene, Gaithersburg, MD). Avail-
able histological follow-up data was retrieved for abnormal cy-
tology specimens.
RESULTS: Of the 163 cytologically abnormal samples within this
study, six-month biopsy follow-up data was available for 73
specimens (45%). Specifically, follow-up data was obtained for
16/55 (29%) cases defined cytologically as ASC-US or ASC-H,
28/57 (49%) LSIL, and 29/51 (57%) HSIL. At initial evaluation 21
of the 26 cases with CIN II/III follow-up were positive for
p16INK4a, yielding an overall sensitivity of 81%, while 29 of the 47
cases diagnosed as CIN I or less were p16INK4a negative, yielding
a specificity of 62%. In contrast, the hc2 test results indicated a
sensitivity of 100% with a specificity of 15%. Following a review
of selected cases with CIN II/III follow-up, 25 of 26 slides exhib-
ited positivity for p16INK4a.
CONCLUSIONS: The CINtec™ p16INK4a Cytology Kit, in combina-
tion with ThinPrep cervical samples, allowed for clear evaluation
of p16INK4a protein overexpression with virtually no background
staining. Specificity of the p16INK4a CINtec™ assay was signifi-
cantly improved relative to hc2. However, because weak p16INK4a
immunostaining was missed in some abnormal cells, a modified
kit version with improved staining performance is currently be-
ing evaluated.
1
Cytyc Corporation, Marlborough, Massachusetts, 2DakoCytoma-
tion, Glostrup, Denmark, 3University of Nebraska Medical Center,
Omaha, Nebraska, 4Brigham & Women’s Hospital and Harvard
Medical School, Boston, Massachusetts
4 November 5, 2005
10:45 am – 11:00 am
Evaluation of Molecular Biomarkers for the
Detection of High Grade Cervical Disease
(CIN2!) in Routinely Collected Cervical
Cytology Samples
Margaret R. Parker, B.A., SCT(ASCP), Susan J. Sampson,
B.S., CT(ASCP), Janice J. Hessling, M.D., Ph.D., Arun
Chatterjee, M.D., Dominica K. Prpic, HT(ASCP), Laura B.
Hall, B.A., HTL(ASCP), Shay Wingate, B.S., Adriann J.
Taylor, B.A., Timothy J. Fischer, B.S.
INTRODUCTION: Recently it has been found that aberrant induc-
tion into S-phase represents the molecular consequence of per-
sistent HPV infection and the neoplastic transformation of cer-
vical cells. A hallmark of this process is the over-expression of
those genes responsible for DNA synthesis and replication in-
cluding the genes for minichromosome maintenance protein
(MCM2) and topoisomerase IIa (TOP2A). Additionally, advance-
ments in immunocytochemistry methodologies have facilitated
the ability to study biomarkers in the routine cytology laboratory
setting.
MATERIALS AND METHODS: 1609 residual specimens
(SurePathTM, TriPath Imaging, Inc., Burlington, NC) were col-
lected from geographically diverse locations within the United
States. All samples were collected under IRB and all patient
samples were delinked and deidentified. Pap results were pro-
vided for all specimens and biopsy results were provided for all
abnormal specimens. Two slides were prepared from each sam-
ple; one stained with the traditional Pap stain and one stained
with ProExTM C (TriPath Imaging, Inc.), a reagent that contains
antibodies directed to MCM2 and TOP2A. The ProEx C slides
were stained on a DakoCytomation Autostainer (DakoCytoma-
tion Inc, Carpinteria, CA) using SureDetect general use reagents
(TriPath Imaging, Inc.). Immunocytochemistry slides were re-
viewed by a cytotechnologist and a pathologist and given a score
of Positive, Negative, or Unsatisfactory for biomarker immuno-
reactivity. Sample distribution was: NILM: 671 (41.7%); ASC-US:
349 (21.68%); ASC-H: 38 (2.36%); AGC: 6 (.37%); LSIL: 395
(24.53%); HSIL: 150 (9.32%); and Sq Cell Ca: 1 (.06%).
RESULTS: Overall the sensitivity and PPV for detecting high grade
cervical disease (CIN2$) was significantly greater for ProEx C
assay results compared to the Pap results. The sensitivity for
ProEx C assay in the ASCUS$ population was 96.2% with a PPV
of 46.9% for identifying women with CIN2$ on biopsy. The Pap
Test had a sensitivity of 83.3% and PPV of 40.7% when the Pap
result was LSIL or greater.
CONCLUSIONS: Biomarkers (ProEx C) were incorporated into an
ICC assay designed to detect aberrant S-Phase induction that
can be performed in the routine cytology setting. The ICC assay
was superior (higher sensitivity and PPV) for the detection of
high grade cervical disease (CIN2$) when compared to the PAP
test in the ASCUS$ population.
TriPath Imaging, Inc., Burlington, North Carolina
5 November 5, 2005
11:00 am – 11:15 am
The Evolution of Pap Testing at a
Community Hospital – A Ten Year
Experience
Keith V. Nance, M.D., Pamella B. Godwin, CT(ASCP)
INTRODUCTION: During the last ten years the Pap program at our
community hospital laboratory progressed from the use of 100%
conventional smears (CS) through a period of both CS and
ThinPrep®(TP) use to a period of CS, TP and SurePath™ (SP)
use and now to nearly 100% SP combined with FocalPoint™
automated prescreening. During this time period the annual Pap
volume grew from less than 20,000 to nearly 50,000. This retro-
spective review focuses on improvements in high grade squa-
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting 323

mous intraepithelial lesion (HSIL) detection rates and specimen All reactive lesions and diagnoses of ASCUS $ were referred to
adequacy rates during the ten-year period. cytopathologists. At an interval of 4-12 weeks later, the same slides
MATERIALS AND METHODS: Statistical data from January 1, 1995 were imaged by the TIS and reviewed by cytotechnologists. This
through December 31, 2004 is reviewed and sorted according to review included initial review of 22 FOV, followed by full automated
type of Pap specimen, rate of HSIL detection and rate of unsat- review for reactive/abnormal and referral to a single cytopatholo-
isfactory specimens. gist. Manual and TIS diagnoses were compared. All 1-2 step dis-
RESULTS: crepancies between TIS-R and MR were adjudicated.
RESULTS: MR and TIS-R was performed on a cohort of 5,596
Total Total% CS% TP% SP% CS% TP% SP% cases with following results: NIL (84.33% MR; 87.76% TIS-R);
Paps HSIL HSIL HSIL HSIL Unsat Unsat Unsat ASC/AGC (7.49% MR, 5.15% TIS-R); LSIL (6.20% MR, 5.16%
TIS-R); HSIL (1.98% MR, 1.93% TIS-R). 664 discrepant cases were
1995 17356 0.3 0.3 NA NA 0.11 NA NA
1996 19526 0.26 0.26 NA NA 0.10 NA NA adjudicated and a “true diagnosis” established for each Bethesda
1997 14278 0.23 0.23 NA NA 0.11 NA NA category. (NIL 84.77%, ASC 6.95%, LSIL 6.00%, and HSIL/CA
1998* 16576 0.45 0.45 0.41 NA 0.10 0.50 NA 2.27%) Based on this adjudicated review the sensitivity for MR
1999 23502 0.26 0.18 0.35 NA 0.12 0.54 NA vs. TIS-R for ASC$ was (84.15% vs. 78.40%); Specificity (96.63%
2000 33515 0.43 0.38 0.47 NA 0.24 0.33 NA
vs. 99.66%); PPV (81.76 vs. 97.66%); NPV (97.14% vs. 96.25%).
2001* 47734 0.37 0.34 0.37 0.42 0.26 0.33 0.05
2002* 49034 0.32 0.12 0.30 0.42 0.89 0.55 0.14 Based on this adjudicated review data, CT Misses on the TIS Arm
2003* 40689 0.57 0.34 0.44 0.65 2.18 0.85 0.20 showing abnormal cell in the 22 FOV were factored out to
2004 47416 0.43 0.05 NA 0.44 2.37 NA 0.17 display sensitivity of the imager based on “true Imager misses”,
those cases missed by the TIS and not present in the 22 FOV. The
* In June of 1998 use of TP began, in May of 2001 use of SP began, in May of 2002 Bethesda 2001
adjusted measures were as follows for TIS-R for ASC$ as follows:
adequacy criteria were adopted, and in August of 2003 use of FocalPoint™ began. Beginning January
of 2003 all unsatisfactory TP specimens were reprocessed using the SP method, which reduced the TP sensitivity 88.7%, specificity 99.6%, PPV 97.9%, and NPV 98%.
unsatisfactory rate by nearly 60%. The MR missed 6.6 % HSIL vs. 1.05% by TIS-R. Of the 4 HSIL
Annual HSIL biopsy confirmation rates ranged from 90% in 1995 to 97% in 2004. cases missed on TIS-R only 1 was TIS miss and 3 were CT misses.
CONCLUSION: Based on the above data the TIS assisted primary
CONCLUSIONS: Annual HSIL detection rates vary regardless of Pap screening for ThinPrep pap test performs as well as MR. There is
methodology used, however, liquid-based Pap tests provide a preliminary evidence that the sensitivity and PPV for HSIL is
higher HSIL rate when compared to conventional smears. Sure- better on TIS-R than MR. Appropriate training and adjustment
Path™ consistently yields the highest HSIL detection rate and the to a new method of screening is essential in adaptation of this
highest specimen adequacy rate of the three Pap Test methods. new technology.
1
University of Texas Southwestern Medical Center, Dallas, Texas,
Rex Hospital, Raleigh, North Carolina 2
Parkland Health & Hospital System, Dallas, Texas

6 November 5, 2005 7 November 5, 2005

11:15 am – 11:30 am 11:30 am – 11:45 am

Validation of the ThinPrep® Imaging
System (TIS) for Primary Screening in
Routine Clinical Practice
Ann Marie West, BS, CT (ASCP)2, M.H. Saboorian, M.D.1,
Kusu Vettimattam, CT (ASCP)2, Beatrice Solares, SCT
(ASCP)2, Huiping Chen, CT (ASCP)2, Cindi Vela, CT
(ASCP)2, S.T. Gokaslan, M.D.1, Raheela Ashfaq, M.D.1
INTRODUCTION: The Thin Prep Imaging System (TIS) is an auto-
mated system that assists the cytotechnologist with the primary
screening of ThinPrep® Pap Test (TPPT) slides. The premise of TIS
assisted screening is based on low prevalence of disease in the US
population, reduced fatigue, and improved focus on abnormalities
identified on TIS. The purpose of this study is to report clinical
validation of TIS-assisted primary screening in our institution.
MATERIALS AND METHODS: TPPT Slides stained with ThinPrep®
Pap stain were first manually reviewed (MR) by 4 cytotechnologists.
Will CLIA Gyn Cytology Proficiency Test
Scoring Negatively Impact Test Takers Pass
Rates?
Roger W. Wall, M.S., CT(ASCP)1,2, Rhonda L. Metzler,
SCT(ASCP)1, Gary W. Gill, CT(ASCP)CFIAC1,2, Michael D.
Glant, M.D.1,2, Carol C. Eisenhut, M.D.2
INTRODUCTION/PURPOSE: An 11-year provider of glass-slide
based gynecologic cytology continuing education and quality
assessment programs maps results obtained on its slides by 3
scoring systems: 1) its own ADR (Acceptable Diagnostic Range),
CAP UNP (Unsatisfactory, Negative, Positive), and CLIA PT (Pro-
ficiency Testing) scoring grids for cytotechnologists and pathol-
ogists. The results of such a comparison provide insight into
whether test participants who pass non-regulatory continuing
education programs might also be expected to pass CLIA gyn
cytology proficiency testing using similar slides.
324 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5

MATERIALS AND METHODS: Nearly 1000 Pap test slides (conven-

Conventional Thin Prep™ SurePath®
tional and LBP) that were categorized as routine in difficulty
were distributed among nearly 7,000 participants who provided Criteria CT (n) Path (n) pa CT (n) Path (n) pa CT (n) Path (n) pa
more than 322,000 responses. Routine-difficulty slides are field-
CMS 3.9 (2220) 6.3 (3351) %.001 3.1 (5490) 4.9 (5478) %.001 3.4 (1239) 4.6 (1286) 0.15
validated with more than 90% agreement among participants on CAP
(FV) 1 (4381) 1.5 (4479) 0.07 1.7 (1541) 2.6 (1699) 0.08 * *
an ongoing basis. The total number of correct responses (i.e.,
pb %.001 %.001 0.002 %.001
answers falling within the ADR) divided by the total number of
slides graded equals the mean correct responses. Mapping the a
Chi-square test on difference between CT and Path
b
same responses against the CAP UNP scoring system and CLIA Chi-square test between CMS and CAP standard
* Too few for analysis
PT scoring grids for cytotechnologists and pathologists allowed
similar calculations for comparison of results.
CONCLUSION: Failure rates were above 3% in all CMS standard
RESULTS: ADR scoring, CAP UNP scoring, and CLIA PT scoring
validation categories, and below 3% for CAP standard slides.
grids produced virtually identical results when mapped to
Participants overall (CT and Path combined) performed better
322,000 responses. All three mean scores exceeded 90%.
on liquid based than conventional preparations with CMS stan-
CONCLUSIONS: CLIA gynecologic cytology PT scoring grid should
dard validation (4.0% vs. 5.3%, p%.001). However, participants
not significantly adversely affect pass rates of cytotechnologists
performed better on conventional than liquid based prepara-
and pathologists. However, evaluating performance using a Be-
tions with CAP field validation standard (1.2% vs. 2.2%, p%.001).
thesda-based categorization system with broader ADR scoring
Poor performance of pathologists relative to cytotechnologists
may help identify more specific diagnostic problems areas to
may reflect lack of prescreening by cytotechnologists prior to
guide focused educational efforts to achieve diagnostic improve-
pathologist review, in some laboratories. As in actual PT, this
ment at national, laboratory, and individual practitioner levels
data, which is based upon retrospective performance in a quality
1
Midwest Institute for Medical Education, Indianapolis Indiana, assurance program, may not reflect true performance of pathol-
2
DCL Medical Laboratories, Indianapolis, Indiana ogists and cytotechnologists in clinical practice.

8
November 5, 2005
11:45 am – 12:00 pm
Potential Failures in Gynecologic
Proficiency Testing (PT): Results from the
College of American Pathologists (CAP)
Interlaboratory Comparison in Gynecologic
Cytology (PAP) Program
Nancy A. Young, M.D.1,2, Ann T. Moriarty, M.D.1,3,
Edward Wang, Ph.D.1, David C. Wilbur, M.D.1,4
INTRODUCTION: The current PT scoring system results in an
automatic failure for identifying high grade squamous intraepi-
thelial lesion (HSIL) as negative for intraepithelial lesion or ma-
lignancy (NILM). CAP PAP data from January 2004 to April 2005
was analyzed to estimate the percentage of failure based upon
this single parameter.
MATERIALS AND METHODS: Over 15,000 participants received
field validated (FV) and educational slide sets (CMS standard) for
conventional (CP), ThinPrep™ (TP) and SurePath® (SP) mod-
ules. The CMS standard slides fulfilled the minimum criteria of
CMS (review by three pathologists reaching a consensus diag-
nosis on biopsy HSIL). The field validated (CAP standard) slides
required at least 20 responses, with 90% matched to HSIL$
(standard error %0.05). Minimum PT failure estimates were
based upon incorrect NILM responses for the reference category
of HSIL.
RESULTS: Percentage of HSIL identified as NILM
1
College of American Pathologists, Northfield, Illinois, 2Fox Chase
Cancer Center, Philadelphia, Pennsylvania, 3AmeriPath Indiana,
Indianapolis, Indiana, 4Massachusetts General Hospital, Boston,
Massachusetts
9 November 6, 2005
8:00 am – 8:15 am
Epigenetic Alterations in Bladder Cancer: A
Role of Methylation Profiling in Detection of
Urothelial Carcinoma in Urine
Dana Weber, B.S., CT(ASCP), Lin Wang, CT(ASCP), David
Deeds, B.S., CT(ASCP), Charles Biscotti, M.D., Bin Yang,
M.D., Ph.D.
BACKGROUND: Urine cytology is the most common clinical screen-
ing method for bladder cancer. Although high grade non-papillary
urothelial carcinoma (HGTCC) can be readily detected in urine
cytology, detection of low grade papillary urothelial carcinoma
(LGTCC) in urine is challenging due to the overlapping morpho-
logic features also seen in benign reactive processes. Recent genetic
studies have revealed that LGTCC and HGTCC are two distinct
biologic processes involving different cellular signaling pathways.
However, epigenetic alterations between these two subtypes of
TCC have not yet been well studied. We have studied the promoter
methylation profiles of 14 tumor suppressor genes (TSG) of both
LGTCC and HGTCC in 30 urine specimens.
DESIGN: Urine specimens include 13 voided and 17 instru-
mented urines. Based on initial cytologic diagnoses, 30 cases
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
included 8 negative/benign, 10 TCC and 12 atypical changes.
Genomic DNA was extracted using Gentra system’s Puregene kit.
Promoter methylation of 14 candidate TSGs (APC, RAR-beta,
p14, p15, p16, p73, RASSF1a, hMLH1, DAPK, MGMT, BRCA1,
SOCS1, GSTP and FHIT) was analyzed by methylation-specific
PCR (MSP). Methylation profile was correlated with initial cyto-
pathologic and final surgical diagnoses.
RESULTS: Based on methylation frequencies between benign
and malignancies, fourteen TSGs can be classified into three
groups. First group showed insignificant difference in methyl-
ation frequencies between TCC and benign (p!0.05), such as
p15 (100% vs. 100%), BRCA1 (71% vs. 43%), p16 and GSTP (14%
vs. 12%), and hMLH1 and RAR-beta (5% vs. 0%). Second group
had significantly higher methylation frequencies in TCC than
benign (p%0.05). These include methylation of p14 (50% vs. 0%),
p73 and MGMT (46% vs. 0%), FHIT (68% vs. 12%), and DAPK
(55% vs. 12%). Third group was those methylated more fre-
quently in HGTCC than LGTCC (p%0.05), which includes
SOCS-1 (50% vs. 0%), APC (36% vs. 0%), and RASSF1a (43% vs.
12%). Twenty-eight cases had subsequent surgical follow-up.
Final diagnoses include 14 HGTCC, 7 LGTCC and 7 negative/
benign. Among 14 HGTCC cases, 6 were cytologically diagnosed
as either atypical or negative. All 6 cases (100%) showed meth-
ylation of three or more TSG promoters, with both second and
third group TSGs. Among 7 LGTCC cases, all except one was
diagnosed as atypical cytologically. Four cases (57%) showed
methylation of three or more TSG promoters, with mainly sec-
ond group TSGs. Among 7 negative/benign cases, 4 were cyto-
logically diagnosed as atypical. Methylation of three and more TSG
promoters was seen in only one case. The remaining negative/
benign cases had methylation of either p15 or BRCA1 or both.
CONCLUSIONS: Our study suggests that analysis of methylation
profiling of a panel of TSGs can assist in differentiation between
benign and malignant urothelial cells. Although sharing meth-
ylation of some TSGs, there are differential methylation profil-
ings between LGTCC and HGTCC. Methylation of SOCS-1, APC
and RASSF1a was seen more frequently in HGTCC than in
LGTCC. Our study indicates that detection of methylation pro-
files of TSGs in urine may facilitate early and accurate detection
of urothelial carcinoma.
Cleveland Clinic Foundation, Cleveland, Ohio
10 November 6, 2005
8:15 am – 8:30 am
The Clinical Utility of Telomerase Detection
of Bladder Cancer Using Cell Block
Preparation from Urinary Cytology
Specimens
Walid Khalbuss, M.D., Ph.D., Steve Goodison
BACKGROUND: Urinary cytology is the most commonly used
non-invasive test for the detection of urothelial carcinoma (UC).
325
However, the relatively low diagnostic sensitivity of urinary cy-
tology warrants the development of improved non-invasive di-
agnostic techniques. Telomeres are the noncoding termini of
eukaryotic chromosomes and function to stabilize and maintain
chromosomal structure. Telomeric DNA is lost at each cell divi-
sion until they reach a critical point thought to signal cell cycle
arrest and entry into cellular senescence. The enzyme telomer-
ase can repair and extend telomeres, thus the presence of func-
tional telomerase is necessary for cells to be capable of extended
proliferation. Accordingly, telomerase activity has been detected
in the majority of malignant tumor specimens tested. The aim of this
study was to test the clinical utility of telomerase detection in the
diagnosis of bladder cancer using cellblock preparation of urinary
cytology specimens.
METHODS: We evaluated retrospectively 65 urine samples avail-
able as formalin-fixed, paraffin embedded cellblocks. Specimens
included 21 cases of urothelial carcinoma, 3 cases of squamous
cell carcinoma, 1 case of small cell carcinomas, 15 cases con-
taining atypical cells, and 25 negative/benign cases. The cata-
lytic subunit of the telomerase holoenzyme (hTERT) was de-
tected immunohistochemically and the presence/absence of
hTERT expression (nucleolar stain) was correlated with cytolog-
ical diagnosis, bladder biopsy histology diagnosis (if available),
and clinical follow-up information.
RESULTS: The hTERT protein was detected in 14 cases (66%) of
samples obtained from patients with primary bladder cancer.
Conversely, hTERT was immunohistochemically detectable in
only 5 cases (20%) of benign/negative samples. Eight of 15 spec-
imens categorized as atypical were found to display urothelial
hTERT, however, 4 of these were subsequently found to have
come from patients with recurrent bladder cancer. The presence
of hTERT protein was indicative of the transformation of urothe-
lia to a malignant phenotype.
CONCLUSION: In this study, the immunohistochemical detection
of hTERT, the key component of the telomerase complex, cor-
related with urothelial malignancy. Thus, the detection of hTERT
protein coupled with cytology has the potential to aid the diag-
nosis of benign and malignant bladder lesions in urine cytology
specimens.
University of Florida, Jacksonville, Florida
11 November 6, 2005
8:30 am – 8:45 am
Superior Sensitivity for Bladder Cancer by
Combined NMP-22 and Cytology: Review of
954 cases.
Ibrahim Mansoor, M.D.1, Roger Calam1, Basim Al-Khafaji,
M.D.1, Edward Scherwish2
OBJECTIVES: To determine and compare the diagnostic utility of
nuclear matrix protein-22 (NMP22) marker versus urine cytology
to detect Urothelial Neoplasms and follow-up.
326 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
METHODS: Nine-hundred and fifty-four patients were included
in this study (over a 28 month period 1/2002 to 4/2004), all
patients had a urine cytological examination, plus a subsequent
NMP-22 marker testing plus a follow-up Cystoscopy with surgi-
cal biopsy. The NMP-22 test was considered positive when the
marker signal was higher than 10 U/ml. Cystoscopy with surgical
biopsy was considered the reference test.
RESULTS: Mean age of the patients was 68 years (range from 19
to 96), 83% were males and 17% females. Review of the 954
surgical biopsies revealed high-grade invasive Urothelial cell
carcinoma (HGIUC) in 17 cases, HG non-invasive UC in 31
(HGNOIUC), flat carcinoma-in situ in 18 (UCinsitu), low-
grade UC in 63 (LGUC), papilloma in 4, reactive in 118 and no
significant pathological diagnosis in 703 cases. Meanwhile,
results for NMP-22 testing confirmed the presence of an
Urothelial Neoplasm with an overall sensitivity of 64% (LGUC
50%, HGNOU 71%, HGIUC 100%, UCinsitu 67%), and a 94%
specificity (Reactive 88%, negative 95%), a positive predictive
value (PPV) of 63% and a negative predictive value (NPV) of
94%. When excluding all 139 inconclusive urine cytology stud-
ies {For which the corresponding histological diagnosis in-
cluded 7 atypia of unknown significance, 23 LGUC, 7 HG-
NOUC, 2 HGIUC, 8 UCinsitu, 25 reactive and 74 negative} and
considering atypical cells favor reactive as negative (55 cases)
plus atypical cells favor malignant as positive (60 cases), urine
cytology showed a sensitivity of 74% (LGUC 47%, HGNOUC
83%, HGIUC 80%, UCinsitu 90%), a specificity of 96% (reactive
90%, negative 98%), PPV 65% and NPV 97%. However, the
combined evaluation of both tests improved the sensitivity to
78% (LGUC 67%, HGNOUC 85%, HGIUC 100%, UCinsitu
83%), the specificity to 99.8% (reactive 99.8%, negative 99.2%),
with a PPV of 98% and a NPV of 97%.
CONCLUSION: Urinary Cytology is plagued with a high number of
inconclusive diagnoses that included 254 (26%) in our study.
Meanwhile, the utility of NMP 22, as a sole screening marker
shows a lower sensitivity and specificity. However, the combined
evaluation of urine cytology and NMP-22 resolved all the cyto-
logical inconclusive cases and showed a significant increase in
the overall sensitivity and positive predicted value. The combi-
nation of both these tests is highly recommended for a more
effective surveillance and diagnostic test for Urothelial Neo-
plasms.
Tests Sensitivity Specificity PPV NPV Inconclusive
NMP22 64 94 63 94 0
Cytology 74 96 65 97 139
NMP22$Cytology 78 99.8 98 96.6 0 values between 11000 and 15000 were identified to differentiate
1
St. John Hospital and Medical Center, Detroit, Michigan, 2Mich-
igan Institute of Urology, Detroit, Michigan
12 November 6, 2005
8:45 am – 9:00 am
Detection of Metastatic Mammary
Carcinoma by SELDI-TOF Mass
Spectrometry in Lymph Node Cytological
Smears
Erik Kincaid, CT, MP (ASCP)1, Heather Mezzadra1,
Rosemary Tambouret, M.D.2, Soner Altiok, M.D., Ph.D.1
INTRODUCTION/PURPOSE: This study explores the feasibility of
using highly sensitive surface-enhanced laser desorption/ioniza-
tion time-of-flight (SELDI-TOF) mass spectrometry (MS) to
identify protein expression profiles of metastatic breast cancer
cells in lymph node smears as an ancillary approach to increase
the sensitivity of conventional morphologic evaluation.
MATERIALS AND METHODS: Thirty two samples of direct and
archival cytological smears including primary ductal carcinoma
of breast (n"10), benign lymph node (n"11), sentinel lymph
node with atypical (n"4) or metastatic ductal carcinoma (n"4),
and peripheral blood (n"3) were used. All breast and lymph
node samples had follow-up histological diagnosis. Cellular pro-
teins, isolated from air dried/Diff-Quick (AD/DQ)-stained cyto-
logical smears after morphological evaluation, were applied on
Proteinchips with weak cationic surface properties (WCX) to
obtain proteomic spectra generated by SELDI-TOF MS to iden-
tify a collection of m/z peaks.
RESULTS: AD/DQ-stained samples of direct and archival cyto-
logical samples provide adequate cellular materials to analyze
protein profiles by SELDI-TOF MS. Using this proteomics
technology, two significant peaks were identified at m/z val-
ues between 11000 and 15000 that are found in all breast
cancer samples and in the extracts of T47D and MDA-MB-231
mammary carcinoma cell lines, but not in benign lymph node
smear samples. To validate these findings a retrospective
study of archival sentinel node smear samples were per-
formed. Three cases were examined in which the same patient
had two sentinel lymph node smears, one with a benign and
the other with a metastatic mammary carcinoma diagnosis. In
each case, based on the proteomic profiles, it was possible to
accurately differentiate the benign from the malignant sam-
ple. Additionally, with the aid of this approach the presence of
carcinoma cells were demonstrated in three cases with the
cytology diagnosis of “atypia” and follow-up histological di-
agnosis of “metastatic breast carcinoma” in archival cytolog-
ical slides.
CONCLUSIONS: In this study two major protein peaks at m/z
benign lymph nodes from those with metastatic mammary car-
cinoma. Thus, proteomic profiling of lymph node samples may
provide a new ancillary diagnostic tool to increase the sensitivity
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
of morphologic evaluation of metastatic breast carcinoma in
cytological smears.
1
Johns Hopkins Hospital, Baltimore, Maryland, 2Massachusetts
General Hospital, Boston, Massachusetts
13 November 6, 2005
10:30 am – 10:45 am
FISH Analysis Evaluating the
Bronchioloalveolar Pattern in Pulmonary
Adenocarcinoma
Narasimhan Agaram, Kathleen Cieply, N. Paul Ohori, M.D.
BACKGROUND: Bronchioloalveolar pattern (BA) in pulmonary
adenocarcinoma has been well recognized in cytologic prepara-
tions. This pattern may be present in pure bronchioloalveolar
carcinoma (p-BAC), adenocarcinoma with bronchioloalveolar
pattern (AD-BAC) and papillary adenocarcinoma (AD-PAP). In
this study we analyzed the molecular abnormalities in these
neoplasms by Fluorescence in situ hybridization using probes
for chromosomes 5, 6, 7 and 8 and compared them to that of
conventional adenocarcinoma without BA pattern (AD-NOS).
METHODS: Thirty-four cases of pulmonary cytology specimens
(bronchial brushings, bronchial washings, bronchioloalveolar la-
vage, sputum and fine needle aspirates) with corresponding
surgical pathology specimens comprised of adenocarcinoma
with a BA pattern [p-BAC (6), AD-BAC (16), AD-PAP (6)] and
without a BA pattern [AD-NOS (6)] were identified. FISH using
LAVysion (Vysis, Inc.) multicolor probe set (5p15, 7p12 – EGFR,
8q24 – C-MYC, and centromere 6) was performed on one rep-
resentative slide from each cytology case after destaining. Twen-
ty-five tumor cells were counted and aneusomy of any one of the
4 markers was considered abnormal. The percentage of abnor-
mal cells in each case and the percentage of abnormality of
individual genes or centromere copy number per case were
recorded. Ten benign bronchial brushing samples in patients
with no evidence of malignancy were used as controls.
RESULTS: Of the 34 cases, 21 were informative – P-BAC (3),
AD-BAC (9), AD-PAP (3) and AD-NOS (6). Chromosomal abnor-
malities were identified in all 21 informative cases. The abnor-
malities involved between 44% and 100% of cells in any given
case. The ranges of aneusomy for each probe in tumors with a
BA pattern were - 5p15 (12% - 100%, mean-68%), 7p12 (12% -
100%, mean-71.2%), 8q24 (0 – 100%, mean-65%) and 6p11 (4% -
100%, mean-68%). In comparison, the ranges of aneusomy in
tumors without a BA pattern were 5p15 (76% - 100%, mean-
90.6%), 7p12 (84% - 100%, mean-94%), 8q24 (64 – 100%, mean-
89%) and 6p11 (4% - 100%, mean-62%). The difference in the
pattern of chromosomal abnormality between the 2 groups was
not significant. None of the samples in the control group showed
abnormalities in any of the chromosomes.
CONCLUSIONS: These results indicate that chromosomal abnor-
327
malities by FISH using the LAVysion panel are useful in detecting
the malignant BA component of adenocarcinoma in cytologic
samples. However, FISH does not discriminate adenocarcino-
mas with and without a BA component.
University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania
14 November 6, 2005
10:45 am – 11:00 am
Quantitative Nuclear Grade Scores by Image
Analysis of Cells Collected from Biliary Tree
Strictures Without Mass Assist Routine
Cytology in the Diagnosis of Clinically
Occult Carcinoma
Emily Barr Fritcher, CT(ASCP), Benjamin Kipp, M.S., CT,
MP(ASCP), Johnita Ihrke, CT(ASCP), Haugen De Anna, B.S.,
Kevin Halling, M.D., Ph.D., Thomas Sebo, M.D., Ph.D.
INTRODUCTION: Routine cytology is relatively insensitive in de-
tecting carcinomas arising from biliary strictures not producing
a mass. This study assessed the role quantitative nuclear grading
by image analysis (IA) has in increasing the detection rate of
carcinoma in biliary strictures.
METHOD: Conventional cytology and IA were performed in tan-
dem on 141 biliary tract specimens (81 males, 60 females) with a
mean age of 57.3 years (range, 9-87). Cytology was classified as
negative, atypical/suspicious, or positive. For IA, up to 50 of the
most atypical appearing nuclei from each sample were quanti-
fied using the CAS 200 image analyzer. All patients had 6 months
to 2 years of clinical follow-up to assess for outcomes. Univariate
analysis identified 13 (of 32) nuclear features that differentiated
malignant from non-malignant specimens (significance based
on Bonferroni correction with p%0.05/32 " 0.0016). A nuclear
grade score (NGS) with the CAS 200 Nuclear Grade software was
calculated for each specimen using the 13 nuclear morphomet-
ric features which were significant to a level of p%0.0016 in
univariate analysis. These scores were expressed as the number
of standard deviations from the reference group (score 0.0)
which represents the cells collected from patients who, to date,
have no clinicopathologic evidence of carcinoma. Comparison
of the NGS between the patients with and without cancer on
follow-up was done by two-tailed t-test, and p-values %0.05
were considered statistically significant.
RESULTS: Fifty-nine of the 141 (41.8%) patients had pathologic
or clinical evidence of cancer on follow-up. There were signifi-
cant differences (p%0.001) between the NGS of specimens from
patients with and without carcinoma (carcinoma mean"4.43,
median"2.98; benign mean, 0.00, median, 0.03). All patients in
this study with a NGS !4.82 (18/59 patients, 30.5%) had cancer
on follow-up. Of these patients, the cytology results were nega-
tive in 5 (27.8%), atypical/suspicious in 6 (33.3%) and positive in
7 (38.9%). Thus, 11 (18.6%) more patients were captured as
328 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
positive for carcinoma using this method without compromise
to specificity. Significant differences (p%0.001) were also found
between the NGS of specimens called negative (mean, 0.58;
median, 0.53), atypical/suspicious (mean, 3.94; median, 3.54)
and positive (mean, 8.49; median, 9.40) by cytology.
CONCLUSION: A nuclear grade program can quantify the nuclear
features of cells from biliary tract specimens captured by IA.
Data from this study suggest nuclear grade information may be
clinically useful in distinguishing benign from malignant biliary
tract strictures when the cytologic evaluation is negative or in-
conclusive.
Mayo Clinic, Rochester, Minnesota
15 November 6, 2005
11:00 am – 11:15 am
Profiling of DNA Promoter Methylation
Facilitates Detection of Cholangiocarcinoma
in Biliary Brushing Specimens
Inga Kink, CT(ASCP), Lin Wang, M.S., Xiaotong Zhang,
M.S., Charles Biscotti, M.D., Jennifer Brainard, M.D., Bin
Yang, M.D., Ph.D.
BACKGROUND: Cholangiocarcinoma is an aggressive malignancy
with dismal 5-year survival rate. Early and accurate detection of
cholangiocarcinoma in biliary brushing samples is often chal-
lenging cytomorphologically. Our previous study on DNA pro-
moter methylation profiling in surgically resected cholangiocar-
cinoma showed that there is a distinct methylation profiling in
cholangiocarcinoma compared to benign biliary epithelium. To
test whether such a DNA methylation profiling can be applied
and useful in cytologic samples, we have studied promoter
methylation profiles of 14 tumor suppressor genes (TSG) in 31
biliary brushing samples.
DESIGN: Thirty-one biliary brushings, consisting of 15 negative/
benign, 5 adenocarcinoma and 11 atypical change, were retro-
spectively collected with IRB approval. Genomic DNA was ex-
tracted using Puregene kit from Gentra System. DNA was
chemically converted by bisulfite. Promoter methylation of 14
candidate TSGs (APC, CDH1, p14, p15, p16, p73, RASSF1a,
hMLH1, TIMP3, MGMT, BRCA1, SOCS1, RUNX3 and FANCF1)
was analyzed by methylation-specific PCR (MSP). Methylation
profile was correlated with cytopathologic diagnoses and clini-
copathologic follow-up data.
RESULTS: Although methylation of all TSGs, except FANCF1, was
seen in cholangiocarcinoma, methylation of five core TSGs dif-
ferentiated cholangiocarcinoma from benign biliary epithelium.
Methylation of APC, CDH1, p14, SOCS1 and RUNX3 in cholan-
giocarcinoma was significantly more frequent (60-100%) than in
benign biliary epithelium (0-13%) (P%0.01). Concerted methyl-
ation of six and more of 14 TSGs tested are seen in all cholan-
giocarcinoma, but rarely seen in benign group. Three adenocar-
cinomas were diagnosed in subsequent surgical biopsy/
resection among those cytologically diagnosed as atypical (27%).
All three malignant cases showed methylation of at least 3 of five
core TSGs (APC, EDH1, p14, SOCS1 and RUNX3). Three brush-
ings from primary sclerosing cholangitis (PSC) patients were also
tested. Although cytomorphologically benign appearance, 2/3
PSC brushings showed methylation of one of five core TSGs.
CONCLUSIONS: Our study demonstrates that MSP is applicable in
biliary brushing samples. Methylation of a panel of TSG promot-
ers distinguishes malignant from benign biliary epithelium. Our
study indicates that promoter methylation profiling of selected
TSGs may be useful in facilitating detection of cholangiocarci-
noma in cytologic specimens.
Cleveland Clinic Foundation, Cleveland, Ohio
16 November 6, 2005
11:15 am – 11:30 am
Cumulative Sum (CUSUM) Procedure in
Evaluation of EUS-guided FNA Cytology: The
Learning Curve and Diagnostic Performance
Beyond Sensitivity and Specificity
Isam Eltoum, M.D.1, David Chhieng, M.D.1, Darshana
Jhala, M.D.1, Nirag Jhala, M.D., MIAC1, Ralph Crowe1,
Shyam Varadarajulu2, Mohamad Eloubeid2
INTRODUCTION/PURPOSE: EUS-FNA is increasingly used to sam-
ple a variety of deep-seated organs. The objective of this study is
to evaluate EUS-FNA performance using Cumulative Sum
(CUSUM) chart. We assessed two trends: (1) how EUS-FNA
service, over time, would maintain an acceptable rate of incon-
clusive results defined as technical failures, unsatisfactory spec-
imens and atypical and suspicious diagnoses; (2) how EUS-FNA
service, over time, would maintain an acceptable rate of diag-
nostic errors.
MATERIALS AND METHODS: The study included all consecutive
patients who underwent EUS-FNA from July 2000 to April 2004
and followed up till December 2004. Using a simple spread
sheet, we constructed CUSUM to assess performance at a preset
acceptable rate of 10% and a preset non-acceptable rate of 15%
for inconclusive results and for diagnostic errors. We then strat-
ified results by lesion site, lesion size and cytopathologist.
RESULTS: Of the total 876 patients, the EUS-FNA was inconclu-
sive in 83 (9.5%) patients. The majority of inconclusive cases
were attributed to atypical diagnoses 43 (51%); 27 (33%) cases
were suspicious for malignancy, eight (10%) had insufficient
material for diagnosis and in five (6%) EUS-FNA technically
failed. Of the 585 cases with definitive diagnosis {benign (58%) or
malignant (42%)} and had adequate follow up, there were 26
(6.3%) diagnostic errors: 3 (0.5%) were false positive and 23 (3.1)
were false negative. The CUSUM charts for both inconclusive
results and diagnostic errors started with a small period of learn-
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting 329

ing then competency at preset levels was achieved at case num-

ber 121 and 97, respectively. Performance was best in lymph Patient Biopsy FISH % FISH Cytology
# Specimen Location Result Result Abn. Cells Result
nodes, followed by the pancreas. Performance was not signifi-
cantly different for lesions %25 mm compared to lesion !25 mm 3 L. Renal Pelvis $ $ 16 $
in diameter. For individual cytopathologist, 50-153 and 50-113 4 L. Ureter $ $ 92 Atypical
cases were needed to achieve performance at the pre-set levels 5 L. Renal Pelvis $ $ 9 Atypical
(see above) for inconclusive results and error rate respectively. 6 L. Renal Pelvis $ $ 15 Atypical
7 R. Ureter $ $ 4 Atypical
CONCLUSIONS: In this time of proficiency testing, error tracking
8 L. Ureter $ $ 41 -
and competence evaluation, a great potential exists for the use of 9 L. Ureter $ $ 32 NA
CUSUM chart to assess procedure failure and error tracking in 10 L. Ureter $ - - $
quality control programs particularly when a new procedure, 11 R. Renal Pelvis - - - -
such as EUS-FNA is introduced in the laboratory. Additionally, it 12 R. Ureter - - - -
13 L. Ureter - - - -
can be used as method to assess trainee competency and track
If Atypical " ($) (-)
proficiency of practicing cytologists. FISH: Sensitivity 8/9 " 89% Cytology: Sensitivity 7/8"87%; 4/8"50%
1 Specificity 3/4 " 75% Specificity 3/4"75%; 4/4"100%
Division of, Anatomic Pathology, Department of Pathology, Uni-
versity of Alabama at Birmingham, Birmingham, Alabama, 2Di- CONCLUSION: The results of this study show that the sensitivity
vision of Gastroentrology, Department of Medicine, Birmingham,
and specificity of FISH and cytology are similar if atypical cytol-
Alabama
ogy diagnoses are considered positive. However, if atypical cy-
tology results are considered negative, FISH has a higher sensi-

Poster Presentations tivity. FISH may be able to offer a more definitive diagnosis in
patients diagnosed with an atypical UUT specimen by cytology.
Although this limited data looks promising, additional studies
are required to better evaluate the usefulness of FISH for assess-
1 ing UC in UUT samples.

Fluorescence in situ Hybridization (FISH) Mayo Clinic, Rochester, Minnesota

with the UroVysion™ Probe Set for the
Detection of Urothelial Carcinoma (UC) of 2
Upper Urinary Tract Specimens
Jesse Voss, CT, MP(ASCP), Benjamin Kipp, M.S., CT,
A Potential Utility of P504S in Diagnosis of
MP(ASCP), Michael Blute, M.D., Zincke Horst, M.D., Ph.D., Adenocarcinoma of the Prostate in
Kevin Halling, M.D., Ph.D. Cytologic Specimens
INTRODUCTION/PURPOSE: Upper urinary tract (UUT) carcinomas Fan Lin, M.D., Ph.D.1, John Danella, M.D.1, Ting Shen,
represent approximately 10% of all urothelial cancers. Studies M.D., Ph.D.2
show that UroVysion™ has a higher sensitivity than cytology for
detecting UC, while maintaining the high specificity of cytology INTRODUCTION: P504S/!-methylacyl CoA racemase is a recently
in urine specimens. The goal of this project was to assess the identified tumor-associated protein, and its expression has been
utility of FISH for detecting UC in UUT specimens. reported in over 90% of adenocarcinoma of the prostate, and
MATERIALS AND METHODS: Institutional records were searched to usually negative or only focally weakly positive in benign pros-
identify UUT specimens with matching (same day) FISH and cytology tatic tissues. The diagnostic value of P504S in cytologic speci-
results, as well as surgical pathology results within six months follow- mens of the prostate has not been explored.
ing the cytology/FISH specimen. FISH specimens were considered MATERIALS AND METHODS: Two hundred forty slides (from 20
positive if five or more cells demonstrated polysomy (i.e. gain of 2 or prostatectomy specimens, positive for adenocarcinoma) were
more chromosomes in a cell). In FISH positive specimens, one hun- included. Six passes of fine needle aspiration biopsies from the
dred consecutive non-inflammatory/non-squamous cells were apex, middle gland and base of both sides of each prostate were
counted to determine the percentage of abnormal cells. performed. Two methanol-fixed slides were obtained from each
RESULTS: needle pass. Pap stain was performed on 120 slides prepared for
the cytologic evaluation. Immunocytochemical staining with the
Patient Biopsy FISH % FISH Cytology monoclonal antibody to P504S was done in the other 120 slides.
# Specimen Location Result Result Abn. Cells Result Positive and negative controls were included. The immunostain-
ing results are rendered into 1) positive for carcinoma - strong
1 R. Ureter $ $ 71 $
cytoplasmic staining in more than one group of epithelial cells,
2 R. Renal Pelvis $ $ 46 $
and 2) negative for carcinoma - no staining or very weak equiv-
330 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
ocal staining. Three categories were assigned to the cytologic
diagnoses: negative, positive, and atypical/suspicious. Twenty
prostatectomy specimens and 18 cases with available surgical
core biopsies were reviewed.
RESULTS: A diagnosis of adenocarcinoma was made in only 55%
(n"11) of cases when based on the cytologic features alone. In
contrast, adenocarcinoma was identified in 100% of cases
(n"20) when stained with P504S. On the basis of positive slides
screened, the sensitivity was also enhanced using the P504S
staining (i.e. 85 versus 25 out of 120 slides in each group respec-
tively). The positive diagnoses on the P504S stained slides were
confirmed by reviewing the corresponding core biopsies and
prostatectomy specimens.
CONCLUSIONS: Our preliminary data suggest that P504S is a
useful marker in assisting in the diagnosis of adenocarcinoma of
the prostate in cytologic specimens. A fine needle aspiration
biopsy of the prostate in conjunction with applying P504S im-
munostain may be considered as an alternative for a patient who
is a non-surgical candidate (high stage cancer or other medical
illness) or cannot well tolerate multiple core biopsies. A larger
series of cases on cytologic specimens will be necessary to con-
firm our observations.
1
Geisinger Medical Center, Danville, Pennsylvania, 2Quest Diag-
nostics, Teterboro, New Jersey
3 Urothelial Carcinoma
Urinary Cytopathologic Findings in Renal
Allograft Recipients: An Institutional
Experience
Aziza Nassar, M.D., Imrana Qureshi, M.D., Melinda Lewis,
M.D., Sanjay Logani, M.D., Momin Siddiqui, M.D.
INTRODUCTION/PURPOSE: Urinary cytology for renal transplant
patients is usually performed to assess graft rejection, infections
or malignancy. Due to immunosuppression, infections may be
more life threatening than graft rejection. Commonest infections
encountered in this setting include cytomegalovirus, herpes and
polyoma BK-virus. We undertook a cross-sectional study of uri-
nary cytology in renal transplant patients over a six-year period
(1999-2004) in our institution, to determine the spectrum of
cytopathologic changes detected in this unique patient popula-
tion.
MATERIALS AND METHODS: The patients’ records were searched
for renal transplant patients who had undergone urinary cyto-
logic examination, over a six year period. Patient demographics
and follow-up information was retrieved from medical records
and tabulated for cytopathologic findings. Slides which had been
prepared by cytospin and centrifugation methods and stained
using Papanicolaou stain, were reviewed.
RESULTS: A total of 28 renal transplant patients were found in
our patient records, 11 (39.2%) had urinary cytologic evaluation.
The average age was 52.5 years (range 35 – 69). Nine patients
were male and two were female. The duration of kidney trans-
plant ranged from 1 – 14 years (average 6.3 yrs). Specimen types
examined included: seven voided urines and four catheterized
urine. Of these, one (9.1%) had papillary type renal cell carci-
noma, three (27.3 %) had cytopathic changes consistent with BK
virus, two (18.2%) had marked acute inflammation, and the
remaining six (54.5%) did not show any significant pathology.
The two patients with acute inflammatory cells in the urine grew
bacterial micro-organisms on culture (Escherichia coli and Ser-
ratia marcescens). No patient with evidence of graft rejection
was identified.
CONCLUSIONS: Urinary cytology is a useful screening tool for
detection of infections in kidney transplant patients, specifically
viral infections. In addition, it can be utilized to investigate other
conditions, specifically malignancy in this unique patient pop-
ulation.
Emory University Hospital, Atlanta, Georgia
4
DNA Ploidy Evaluation in 76 Ileal Conduit
Urine Specimens is Highly Accurate in
Diagnosing Subclinical Recurrence of
Abha Khanna, M.A., CT(ASCP), Linda Payne, CT(ASCP),
Hua-Zhong Zhang, M.D., Nancy Caraway, M.D., Ruth
Katz, M.D.
INTRODUCTION: Ileal conduit urines (ICU) are difficult to evalu-
ate cytologically due to the presence of columnar epithelial cells,
neutrophils, lymphocytes, histiocytes, bacteria, degenerated
cells and debris. Recurrence of urothelial carcinoma (UC) is a
major concern as it is a multifocal disease that may affect the
remaining ureters, urethra and renal pelvis. Recurrence in these
sites is associated with high morbidity and mortality due to
ureteric or renal pelvic obstruction, impaired renal function and
ultimately death. A sensitive and specific method to diagnose
early urinary tract recurrence (UTR) is needed.
MATERIALS AND METHODS: From 2002-2005, 76 ileal conduit
specimens from 58 post radical cystectomy patients who clini-
cally had no evidence of disease, were evaluated for cytology
(CY) and DNA ploidy. Patients’ ages ranged from 51-93 years
(mean 70 years), M: F ratio was 44:14. Three cytospin slides, 2
Pap stained for CY and 1 Feulgen stained for DNA ploidy, were
prepared. 100 of the most atypical cells were interactively digi-
tized (SAMBA Image Analyzer) and evaluated for ploidy, per-
centage of proliferating cells (PI) and cells with DNA content !
5c.
RESULTS: On follow-up 51/58 patients with normal ICU and
DNA ploidy had no evidence of UTR. 7/58 (12%) patients devel-
oped UTR of whom 6/7 had cells with DNA content !5c or
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting 331

abnormal ploidy pattern. Cytologic evaluation was suboptimal p16 gene at 9p21) were used. The number of probe signals of
compared to DNA analysis. each color were then recorded to aneusomy of chromosomes 3,
7, or 17, or deletion of the 9p21 locus.
CY Diagnosis Ploidy PI Cells>5c F/U* Outcome RESULTS: We reviewed 122 cases referred for analysis. Of those
cases, the FISH assay results on 11 (9%) were not informative
1 UC Tetraploid 4 24 4 DOD
due to specimen quality/artifact. The reported factors hindering
2 Degenerated cells Abnormal Diploid 19 8 10 AWD
3 No malignant cells Aneuploid 1 0 6 DOD UroVysion® analysis includes inflammation, crystals, degener-
4 Negative x2 Diploid x2 0 x2 0 x2 18 AWD ation, blood and low cellularity. In addition, we have observed
UC Aneuploid 2 30 12 that lidocaine jelly obstructs signal overlay.
5 No malignant cells Aneuploid 10 0 9 DOD CONCLUSIONS: When specimen quality interferes with the
6 Negative, Atypical, Abnormal Diploid 2 7 8 AWD
UroVysion Assay it is important to identify the inhibiting factor
Suspicious x3 9 17
18 19 to determine the appropriate corrective action for optimal re-
7 Suspicious Abnormal Diploid 12 25 23 AWD sults. Some of these problems are correctible, while others re-
quire obtaining a new specimen.
* Follow-up time from CY Dx to clinical recurrence of UC (months); DOD: died of disease; AWD: alive
with disease Department of Pathology, University of Pittsburgh Medical Cen-
ter, Pittsburgh, Pennsylvania
CONCLUSIONS: Detection of aneuploid cells in ICU is highly
specific for malignancy and can detect early recurrence up to 23
months before clinical presentation. Thus, regular monitoring of 6
ICU with CY and DNA ploidy is a reliable way to follow these
high-risk patients for recurrent disease.
An Assessment of Cytyc ThinPrep®
M.D. Anderson Cancer Center, Houston, Texas UroCyte™ Filters for the Preparation of
Slides for FISH Analysis
5 Benjamin Kipp, M.S., CT, MP(ASCP)1, Michael Campion,
B.A.1, Aaron Harwood, B.A., CLSp(CG)1, John Tomisek,
B.S.2, Jeffrey Panella, M.S.2, Gerry Browne, B.S.2, Rutuja
Specimen Artifacts Inhibiting UroVysion®
Desai, B.S.2, Erin Coffman, M.S.3, Arthur Smith, M.B.A.3,
Assay Results
Kevin Halling, M.D., Ph.D.1
Peggy Whigham, CT(ASCP), Kathleen Cieply, M.D., Rajiv
INTRODUCTION/PURPOSE: ThinPrep® UroCyte™ filters are newly
Dhir, M.D., N. Paul Ohori, M.D., Sheldon Bastacky, M.D.
designed one cm diameter filters that utilize the T2000 ThinPrep
INTRODUCTION/PURPOSE: UroVysion® (Vysis) represents the first instrument to create a monolayer of cells from urine specimens.
noninvasive gene-based test available for both diagnosis and It is hoped that these filters will simplify the preparation of slides
monitoring of bladder cancer recurrence. The test is designed to for fluorescence in situ hybridization (FISH) analysis. The pur-
detect genetic changes in urothelial cells in urine specimens pose of this study was to assess the performance characteristics
using fluorescence in situ hybridization (FISH) analysis. While of ThinPrep UroCyte filters for FISH testing on urine specimens
the test is highly sensitive, the pitfalls affecting the assay have in comparison to a manual preparation technique.
not been widely reported. During the first four months of testing, MATERIALS AND METHODS: Forty-three urine specimens with a
we have identified specimen artifacts that inhibit FISH assay minimum of 70 mL urine were evenly split and one portion was
results. utilized to create a slide with the UroCyte filter method (UFM)
MATERIALS AND METHODS: Concurrently along with routine and the other portion was used to create a slide with a manual
cytologic preparations, additional cytospin slides fixed in 100% dropping method (MDM). FISH analysis was then concurrently
ethanol were made from urine samples sent for routine Cytology performed on the slides using the UroVysion probe set and the
on patients under surveillance for recurrent bladder cancer. results were categorized using the following criteria: overall FISH
FISH analysis was performed according to Vysis (Abbott Labo- diagnosis (positive, negative, inadequate for analysis), cellularity
ratories) protocol. Morphologically abnormal cells were ana- (!200 cells, 25-200 cells, %25 cells,), DAPI staining quality (good,
lyzed using the following filter with an epifluorescence micro- fair, poor), and probe signal quality (good, fair, poor). In addi-
scope equipped with a 100-watt mercury lamp: DAPI single tion, timings for the processing of batch runs of 10 slides using
bandpass, Aqua single bandpass (chromosome 17), Yellow (gold) each method were calculated.
single bandpass (9p21), and red/green dual-bandpass (chromo- RESULTS: Forty-two of the 43 specimens had concordant FISH
some 3 and 7). CEP 3, CEP 7 and CEP 17 (hybridize to the diagnoses with the UFM and MDM. The one discordant case was
centromere regions of chromosomes 3, 7 and 17, respectively) interpreted as negative with the MDM and not analyzable with
and a unique sequence probe, LSI p16 (9p21) (hybridizes to the the UFM due to inadequate cellularity. Thirty of the 43 speci-
332 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
mens had similar cellularity with the two methods. Of the 13
cases with dissimilar cellularity results, 7 had more cells with the
UFM and 6 had more cells with the MDM. The FISH probe signal
quality was similar for both methods for 39 of 43 specimens. Two
specimens had better probe signal quality with the UFM and 1
specimen had better probe signal quality with the MDM. The
quality of the DAPI staining was similar for 37 of the 43 speci-
mens. Four cases had better DAPI staining quality with the UFM
and 2 had better DAPI staining quality with the MDM. An overall
assessment of which slide was preferable using all criteria dem-
onstrated that 23 specimens were similar, the MDM was pre-
ferred in 7 specimens due to increased cellularity, and the UFM
was preferred in the remaining 13 cases due to cleaner back-
grounds and crisper signals (8), increased cellularity (4), and less
cell clumping (1). The average time taken to create slides figuring
for a batch of 10 using the UFM and MDM was 136 min (13.6 min
per case) and 181 min (18.1 min per case), respectively.
CONCLUSIONS: Data from this study suggest that the UroCyte
filter method of preparing slides for FISH analysis may signifi-
cantly reduce the time required to prepare these slides and
produce similar if not better overall results than the currently
utilized manual dropping method.
1
Mayo Clinic, Rochester, Minnesota, 2Vysis, Inc., Downers Grove,
Illinois, 3Cytyc Corporation, Marlborough, Massachusetts
7 Instrumented Versus Voided Urines
ThinPrep UroCyte™ Slide Preparation for
Urine Cytology
Joel S. Bentz, M.D.1, Leslie R. Rowe, M.S., CT(ASCP)2,
Evelyn V. Gopez, M.D.1, C. Jay Marshall, M.D.1
INTRODUCTION: We report a prospective study comparing results
of urine cytology using standard non-gynecologic slide prepara-
tions versus ThinPrep UroCyte (Cytyc) urine cytology process-
ing.
MATERIALS AND METHODS: The ThinPrep UroCyte Slide Prepa-
ration System, for use with urine cytology processing or slide-
based molecular testing, is a modification of the ThinPrep Tran-
sCyt filter (20mm) used with ThinPrep 2000 non-gynecologic
cytology sample processing. Consecutive urine samples were
received fresh and unfixed immediately from the outpatient
clinics at the study institution. Upon receipt, each urine sample
received in the study laboratory was split evenly. A minimum
sample volume of 20 mL was required for the study. One half of
the split-sample was processed as a non-gynecologic slide prep-
aration (cytospin, membrane filter, or ThinPrep) as used rou-
tinely in our laboratory. The other half of the split sample was
processed as a UroCyte slide preparation. A UroCyte filter
(10mm) was run on the ThinPrep 2000 Processor using Sequence
5 and fixed in 95% ethanol prior to staining. All slides were
stained using modified Papanicolaou staining. Each slide pair
was evaluated for overall slide quality, cellularity, and diagnosis.
RESULTS: A total of 25 split-sample urine cytology samples were
evaluated. Final interpretation included 1/25 (4%) unsatisfac-
tory, 19/25 (76%) negative, 3/25 (12%) atypical/dysplastic, and
2/25 (8%) positive for malignant cells. All cases showed exact
concordance for diagnosis. 17/25 (68%) of cases demonstrated
equivalent cellularity on UroCyte, 5/25 (20%) improved, and
3/25 (12%) showed decreased cellularity. There were two cases
with abundant fresh blood that produced a paucicellular deposit
of cells in the center region of the UroCyte slide.
CONCLUSIONS: The ThinPrep UroCyte Slide Preparation System
is an acceptable alternative for cytopathologic evaluation of
urine cytology samples. The UroCyte slide preparation appears
to be easier and less time consuming to screen and interpret,
because the cells are limited to smaller area (10 mm), the Thin-
Prep method provides a cleaner background, and the case ma-
terial is limited to a single slide preparation. Cellular presenta-
tion is highly dependent upon careful adjustment of staining
parameters and prompt attention to sample fixation.
1
Department of Pathology, University of Utah, Salt Lake City,
Utah, 2Institute for Clinical and Experimental Pathology, ARUP
Laboratories, Salt Lake City, Utah
8
Diagnostic Significance of “Atypia” in
Umesh Kapur, M.D., Christine Booth, M.D., Alia Salhadar,
M.D., Razan Massarani-Wafai, M.D., Eva Wojcik, M.D.
INTRODUCTION/PURPOSE: Atypical cells are a well-defined entity
in cervical cytology (ASC-US) with established interpretive cri-
teria and follow-up strategies. Atypical cells in urine cytology,
although often seen in a daily practice, have not been investi-
gated to the same extent. The aim of our study was to obtain
follow-up on cases that received a diagnosis of atypia and de-
termine if the type of submitted specimen could be associated
with different outcomes.
MATERIALS AND METHODS: All urine cytology cases during a
four-year time period with a diagnosis of atypia were obtained
from the cytopathology computer database. Only cases with
surgical follow-up were included for the purposes of this study.
The following data points were recorded and compared: 1) type
of urine specimen, 2) follow-up histologic diagnosis.
RESULTS: A total of 88 urine cytology cases (instrumented urines
– 70, voided urines –18) were diagnosed as atypical during a
four-year period and had follow-up histology. Of 70-instru-
mented urines, follow-up biopsies revealed malignancy in 17
(24%) cases (seven low-grade urothelial carcinomas, five high-
grade urothelial carcinomas, two poorly differentiated carcino-
mas and four carcinomas in situ). Mild to moderate dyplasia was
diagnosed in 6/70 cases. Of the 18 voided urines, follow-up
biopsy revealed malignancy in eight (44%) cases (four low-grade
urothelial carcinoma, two high-grade urothelial carcinomas, one
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
poorly differentiated carcinoma and one carcinoma in situ).
Mild dysplasia was present in one case. Two cases showed
treatment effect and one case revealed a nephrogenic adenoma.
The remainder of the cases showed inflammation on follow-up
histology.
CONCLUSIONS: Atypical cells have different implications in
voided versus instrumented urine as clearly demonstrated in this
study. On follow-up histology, 44% of voided urine cases were
malignant as compared to 24% of instrumented urine. The pres-
ence of atypical cells in voided urine specimens requires much
closer follow-up since the likelihood of malignancy is much
more significant.
Loyola University Medical Center, Maywood, Illinois
9
Biomarker Analysis on Breast Ductal
Lavage Cells in Chinese Women with and
without Breast Cancer.
JianYu Rao, M.D.1, Wei Zhang2, BaoNing Zhang2, QinJing
Pan2, YuChin Yung1, Steven Hart1, Karen Duvall1
BACKGROUND: Currently there are no effective means to deter-
mine a woman’s risk of developing breast cancer. Ductal lavage
(DL) method provides a potential tool for individual risk assess-
ment of breast cancer. However, cytomorphological analysis
alone on ductal lavage sample is a rather difficult task. Biomar-
ker based analysis may be used as an adjunct test for cytological
evaluation of DL samples. Our previous study showed that cel-
lular biomarkers including DNA 5c exceeding rate (DNA 5cER)
and G-actin measured on achieved Fine Needle Aspiration ma-
terials by Quantitative Fluorescence Image Analysis might be
markers for breast cancer risk. The objective of the current study
was to determine whether these markers could be measured on
the same DL slides after cytomorphological analysis was per-
formed.
METHODS: A total of 51 women were recruited from Cancer
Hospital, Chinese Academy of Medical Sciences, Beijing, China
for the study. Among them, 23 had a clinical diagnosis of breast
cancer, 7 had intraductal papilloma, and 21 had other benign
breast diseases. DL sample was processed by ThinPrep (CYTYC,
Inc). Cytomorphological analysis was carried out independently
by two cytopathologists. Biomarker analysis was performed on a
total of 34 specimens, first on a learning set of 24 specimens, 6
from women with benign breast diseases, 7 from women with
intraductal papilloma, and 11 from women with cancer. Subse-
quent analysis was performed on a test of samples including 6
benign and 6 cancers.
RESULTS: Cytological analysis showed that there were 4 unsat-
isfactory (%10 ductal cells), 14 benign, 5 mild atypia and 2
marked atypia in benign and papilloma control group and 8
unsatisfactory, 1 benign, 2 mild atypia, 8 marked atypia, and 5
333
malignant in cancer group. Biomarker analysis on the learning
set showed a cut off threshold for DNA5cER at 10.5 yielded 92%
specificity and 90% sensitivity and a cut-off threshold for G-actin
at 200 yielded 84% specificity and 100% sensitivity for cancer.
When the two markers were combined, the sensitivity and spec-
ificity for cancer were both at 100%. The finding was confirmed
in the test set of samples.
CONCLUSION: Cellular biomarker analysis of G-actin and
DNA5cER is feasible on achieved ductal lavage slides. Further
studies are warranted to determine the value of these biomark-
ers in prospective trials.
1
UCLA, Los Angeles, California, 2Cancer Institute/Hospital, CAMS,
Beijing, China
10
Cytopathologic Analysis of Breast Masses
in the Pediatric Population
Jun Zhang, M.D., Ph.D.1, Laurentia Nodit2, Anil Parwani,
M.D., Ph.D.2, Syed Ali, M.D.1
BACKGROUND: Breast masses in patients aged 21 and under are
relatively uncommon. As a result, fine needle aspiration (FNA) of
the breast is seldom performed in this age group and the clinical
utility of the procedure has not been adequately addressed.
MATERIALS AND METHODS: A 16-year (1989 –2005) retrospective
review of the cytopathology archives of two large teaching insti-
tutions revealed 128 cases of breast FNA performed in pediatric
patients. A total of 4,166 breast FNAs were performed in patients
older than 21 years. The subsequent surgical pathology material
(where available), as well as the medical records of all cases were
additionally reviewed. FNA was performed with or without im-
age-guidance and slides were stained with Diff Quik and/or
Papanicolaou stains. Cell block sections were prepared in se-
lected cases.
RESULTS: There were 123 females and 5 males (F/M ratio 26:1)
with an age range of 1 to 21 years (mean17). Most common
clinical indication of FNA included palpable, painful or tender
breast mass and nipple discharge. Of the 128 FNAs, 13% (16/128)
were considered non-diagnostic due to scant cellularity, 46%
(59/128) were categorized as non-neoplastic (inflammation
NOS, fibrocystic changes [FCC], gynecomastia and galactocele),
41% (53/128) were diagnosed as benign neoplasms (fibroadeno-
ma-36%, 46/128, lactating adenoma-3%, 4/128, papilloma-1%,
2/128 and lipoma -%1%, 1/128). No cancers were encountered
in the entire series. In 39/128 patients, a histologic follow-up was
available and in all cases displayed concordant results with the
FNA findings (fibroadenoma-33/39, lactating adenoma-2/39,
FCC 3/39 and galactocele-1/39). The sensitivity and specificity of
FNA as a method of assessing neoplasia of pediatric breast
masses is 100%. Two (2/39) FNAs with subsequent biopsy
showed cytologic atypia and turned out to be benign (both
fibroadenomas, one with ductal hyperplasia).
334 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
CONCLUSION: 1. Breast FNA is rarely performed in the pediatric
patients (3%, 128/4166). 2. Breast masses in pediatric patients
generally represent non-neoplastic lesions (46%). 3. Of the neo-
plastic cases (41%), benign tumors account for 100% of the
lesions. 4. Cytopathologic analysis on FNA is highly accurate
with a sensitivity/specificity of 100% for a neoplastic diagnosis.
5. Rate of unsatisfactory FNA due to scant cellularity is low
(13%). 6. Cytologic atypia in the ductal/lobular epithelium is rare
and may accompany fibroadenoma. 7. Approximately 30% of
FNAs in this age group are followed by tissue biopsy/resection. 8.
Male breast lesions are distinctly uncommon and mostly repre-
sent gynecomastia (4/5, 80%).
1
The Johns Hopkins Hospital, Baltimore, Maryland, 2University of
Pittsburgh Medical Center, Pittsburgh, Pennsylvania
11
The Value of Fine Needle Aspiration Biopsy
in Assessment of Palpable Breast Lesions:
A Review of 724 Cases
Asif Loya, M.D., Marilyn Bui, M.D., Ph.D., Amir
Mohammadi, Walid Khalbuss, M.D., Ph.D., Shahla
Masood, M.D.
BACKGROUND: The value of Fine Needle Aspiration Biopsy
(FNAB) of the breast as the initial diagnostic procedure for the
evaluation of patients with palpable breast lesions is already well
established. However, recently a renewed interest in the use of
ultrasound guided core needle biopsy has resulted in a decline in
the performance of FNAB. This study was conducted to assess
our experience in the department of Pathology at the University
of Florida/HSC, Jacksonville in utilization of FNAB in providing
diagnostic and prognostic/predictive information.
METHODS: A 5-year data of 724 cases of fine-needle aspiration
(FNAB) of breast lesions at the University of Florida Health
Science Center were reviewed and analyzed to determine the
diagnostic accuracy and effectiveness of this procedure. The
followup surgical biopsy specimens were available in 218 cases
for correlation.
RESULTS: The results of FNAB showed that 426 cases (59%) were
negative for malignancy, 171 cases (24%) of the cases were
positive for malignancy, 63 cases (8%) were suspicious for ma-
lignancy, and 64 cases (9%) were insufficient for diagnosis. There
were 218 surgical specimens (30% of the cases) available for
review. There were 12 false negative cases. The sensitivity of
breast FNAB was 90% and the specificity was 100%. The positive
predictive value and negative predictive value were 100% and
79% respectively. Out of the 171 malignant cases diagnosed by
FNAB, sufficient material was available to perform a panel of
prognostic/predictive testing, such as tumor ploidy, hormone
receptors, oncogenes, tumor suppressor genes and proliferation
markers in 159 cases.
CONCLUSIONS: This study supports the findings of other inves-
tigators that FNAB is the most cost effective and minimally
invasive procedure that provides a rapid diagnosis, and an op-
portunity for pretreatment planning. In addition, the ability to
provide prognostic/predictive factors utilizing the FNAB sample
offers the opportunity for administration of presurgical neoad-
juvant chemotherapy. This approach is critically important for
patients with large tumors who desire to undergo conservative
therapy.
University of Florida, Jacksonville, Florida
12
Cytologic Atypia Within Radiologically
Resolved Breast Cysts
Walter Blumenfeld, M.D., Quintus Chess, M.D.
INTRODUCTION: The need for cytologic examination of breast
cyst fluids is generally accepted, but has also been questioned.
One still finds reports in the current medical literature that
advocate discarding non-hemorrhagic breast cyst fluids without
benefit of cytologic examination. The vast majority of cytologic
exams are negative. However, on occasion, important findings
are present which are of vital clinical significance. Our labora-
tory receives a large number of breast FNAs (3990 in calendar
year 2004). Of these, the majority are from radiographically and
grossly unremarkable breast cyst aspirates. If clinical/radiologic
resolution occurs following aspiration of the cyst, a feature gen-
erally favoring a benign process, many of the submitting radiol-
ogists and surgeons are in the habit of reporting the resolution
on the requisition slip.
MATERIALS AND METHODS: Over a fifteen-month period, nine
breast fluids from cysts which were reported to have fully re-
solved were diagnosed with cytologic atypia. Only one of these
fluids was grossly hemorrhagic. Five fluids were grossly turbid or
slightly turbid. Volume ranged from 7.5 cc to 14 cc. (Three
specimens were received as prepared smears, precluding gross
description and measurement.) The nine fluids were from seven
patients. Two of the patients had repeat aspiration following the
report of cytologic atypia, after intervals of six weeks and eight
months. In each case fluid re-accumulated at the same site
following initial complete sonographic resolution of the cyst.
RESULTS: Surgical pathology follow up is available for five of the
seven patients, (including the two patients who each contributed
two specimens). Two patients had invasive carcinoma, one had
DCIS, one had an intraductal papilloma with associated atypical
ductal hyperplasia, and one had a radial scar. In each of the two
patients who contributed more than one specimen, the cyst
recurred after resolution, with similar atypical cytologic findings.
These two patients had DCIS and invasive carcinoma, respec-
tively.
CONCLUSION: Breast cysts which resolve following needle aspi-
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
ration may yield cytologically significant findings, including car-
cinoma. Localization of resolved cysts for biopsy is problematic,
but if the patient is followed the fluid will recur. Although the
frequency of such cases is low, their occurrence justifies cyto-
logic examination of breast cysts, including simple, non-hemor-
rhagic and fully resolved breast cysts.
CBL Path, Mamaroneck, New York
13
Utility of Quantitative Methylation-Specific
PCR for Categorization of Epithelial Cells in
Nipple Aspirate Fluid: A Preliminary
Feasibility Study
Savitri Krishnamurthy, M.D.1, Mary Jo Fackler2, Kelly K
Hunt1, Henry M Kuerer1, Saraswati Sukumar2
INTRODUCTION: There is renewed interest currently in using the
cytologic changes in intraductal fluid specimens for risk strati-
fication of women at increased risk for developing breast cancer.
To overcome the subjectivity in the interpretation, there is a
need for ancillary tests that can be used in conjunction with
conventional cytology for a more objective categorization of
epithelial cells in these specimens. We investigated the feasibility
and utility of quantitative methylation-specific PCR (QM-MSP)
as an ancillary test to conventional cytology for categorization of
epithelial cells in NAF.
MATERIALS AND METHODS: We selected 19 cases of NAF that
were classified as benign (2), mild atypia (10), marked atypia (2)
and malignant (3) based on conventional cytologic criteria. Ep-
ithelial cells were scrapped after removing the coverslip from
Papanicolaou stained cytospin smears and used for QM-MSP.
QM-MSP was performed in a blinded fashion without any
knowledge of the cytologic diagnosis of each of the cases. Perti-
nent primers were used for QM-MSP to detect methylated alleles
of genes known to be most commonly affected in breast cancer
including : RASSF1A, Twist, Hin1, CyclinD2, p16, RAR, ER alpha,
APC1, BRCA1,BRCA2.
RESULTS: QM-MSP could be performed from DNA extracted
from scrapped epithelial cell clusters in 18 of the 19 cases of NAF
( 95%). We found methylated alleles of one of more genes in-
cluding RASSF1A, APC1 and RAR in all the 3 malignant cases; 1
of the 2 markedly atypical cases and in 1 of the 11 mildly atypical
cases. Both the cases NAF classified as benign and 10 of the 11
miLdly atypical cases were negative for methylated alleles by
QM-MSP.
CONCLUSION: 1 QM-MSP has the potential to be utilized as an
ancillary molecular test in conjunction with conventional cytol-
ogy for categorization of epithelial cells in NAF. 2. QM-MSP can
be used for confirmation of a benign and malignant diagnosis
and for further categorization of the atypical cases. 3. QM-MSP
335
can be utilized for the selection of the truly atypical cases for
appropriate therapeutic intervention.
1
M.D. Anderson Cancer Center, Houston, Texas, 2Sidney Kimmel
Comprehensive Cancer Center, The Johns Hopkins Hospital, Bal-
timore, Maryland
14
The Cytotechnologist as an Interactive
Member of the Team in a Modern Day
Breast Center
Lacee Iselo, Lisa LaVigne, B.A., CT(ASC), Lonnie
DeCavallas, CT(ASCP), Jean Taylor, MSEd., SCT(ASCP)IAC
INTRODUCTION: “The literature on the effectiveness of immediate
adequacy assessment of FNA biopsy specimens in general is
controversial” (Eedes 2004). When the pathologist is present for
rapid assessment it has been reported that specimen adequacy
rates and sensitivity are generally higher than when they are
absent (Schwartz 2004). Small community hospital Breast Cen-
ters are finding that with the replacement of the pathologist with
a cytotechnologist, they are saving time and money while giving
the same patient satisfaction, diagnostic accuracy and yield. A
consecutive review of 4 years of data from St. Peter’s Hospital
Breast Center in Albany, NY was obtained to determine if the
breast center can offer an adequate biopsy service in the absence
of a pathologist for on-site specimen adequacy assessment.
METHODS: Data of non-diagnostic and diagnostic rates were
analyzed.
RESULTS: It was found that non-diagnostic rates were very low,
2.5% and the diagnostic rates were very high, 97.5%.
CONCLUSIONS: In conclusion, all breast center procedures done
at St. Peter’s Hospital Breast Center in Albany, NY utilize the
breast center team approach. This team approach yields an
adequate specimen in the absence of an on-site pathologist
effectively. This practice maximizes the detection and diagnosis
of disease, in addition to decreasing the likelihood of a repeat
procedure or unnecessary surgeries, all while minimizing the
cost.
Albany College of Pharmacy, Albany, New York
15
Intra-operative Touch Imprints of Axillary
Sentinel Lymph Nodes in Breast Cancer:
Analysis of 222 Cases.
Latha Pisharodi, M.D.1, 2, 3, Michele Lomme, M.D.2, 3, Li
Juan Wang, M.D.1, 2, Dilip Giri, M.D.1, 3
INTRODUCTION: Touch imprints (TI) are frequently used in the
intraoperative evaluation of axillary sentinel lymph nodes (SLN)
336 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
in patients with breast cancer. The purpose of our study was to
examine if accurate TI interpretation was related to the experi-
ence of the pathologist in cytopathology.
MATERIALS AND METHODS: TI of 222 SLN from 74 patients,
previously examined by staff surgical pathologists were re-
viewed. Imprints were stained using either H&E or Diff-Quik
stain. Three pathologists (2 cytopathologists and 1 surgical pa-
thologist with interest in breast pathology) independently re-
viewed all smears in a blinded fashion. Results were compared
with the original interpretation and with the final histologic
diagnoses.
RESULTS: Of a total of 222 cases, 26 (12%) SLNs were positive for
metastases on final histologic evaluation. Intra-operatively 15
(58%) TI were correctly identified as positive (true positive, TP),
11 (42%) were diagnosed negative (false negative, FN). One case
was diagnosed as positive on TI but negative on final histology
(false positive, FP). The overall agreement between TI and final
histologic diagnoses was 94.6% (210/222) with a discrepancy rate
5.4% (12/222). All the three reviewers interpreted the FP TI as
negative and all 15 TP TI as positive. One ore more reviewer
interpreted 5 of the 11 FN TI (46%) as positive and, 2 (18%) as
suspicious. The remaining 4 were interpreted as negative by all
three reviewers. The FN rate of the reviewers was 23% (6/26).
Interestingly, the number of malignant cells on the TI did not
accurately reflect the volume of carcinoma in the lymph node on
permanent sections.
CONCLUSIONS: False positive diagnosis on TI is extremely rare. In
the intra-operative setting, the false negative rate is significantly
higher (42%) for general surgical pathologists compared to cy-
topathologists. The number of malignant cells on TI do not
provide a reliable index of the volume of metastases in the
lymph node.
1
Rhode Island Hospital, Providence, Rhode Island, 2Women and
Infants Hospital, Providence, Rhode Island, 3Brown Medical
School, Providence, Rhode Island
16
A Prospective Cytology-Histology
Correlation Study of Breast Ductal Lavage
Specimens with Mild Atypia
1 2 1
Jing Zhai, M.D., Ph.D. , Jill R. Dietz , Horacio Maluf ,
Antonella Rastelli2, Rosa M. Davila, M.D.1
INTRODUCTION: Breast ductal lavage (BDL) is considered a prom-
ising tool for further stratification of women at high risk of
developing breast cancer. Patients with marked atypia or malig-
nancy in BDL are recommended to consider hormonal therapy
and undergo further testing using ductoscopy or ductography,
and in some instances magnetic resonance imaging. Patients
with mild atypia in BDL are recommended to repeat BDL, and
are counseled for risk reduction intervention. Our prospective
study’s goal is to assess the histologic correlate of ducts with
mild atypia by BDL.
MATERIALS AND METHODS: Women at high risk for breast cancer
were identified by the breast surgery clinic and BDL was offered
as a potential screening test. All BDL were performed by a
physician, following the manufacturer’s recommendations. Thin
prep slides were made from each sample, following the manu-
facturer’s recommendations. All samples were reviewed by a
cytopathologist trained to examine BDL samples. The samples
were diagnosed as benign, mild atypia, marked atypia, malig-
nant, or inadequate for diagnosis. Patients with BDL samples
diagnosed as atypical or malignant underwent ductoscopy di-
rected duct excision. The tissue was submitted to surgical pa-
thology for routine processing and histologic evaluation.
RESULTS: We examined 226 BDL samples from 95 women with a
mean age of 54 (range, 35 to 70). The cytologic diagnoses in-
cluded: inadequate (69 samples); benign (110); mild atypia (45);
marked atypia (2). Cyto-histologic correlation was available in
35/45 samples with mild atypia and 2/2 samples with marked
atypia. The histologic diagnoses of cases with mild atypia in BDL
included: fibrocystic change (16); papilloma (5); normal (4); duct
ectasia (2); infarcted papilloma & sclerosis (1); papilloma, duct
ectasia & fibrocystic change (1); papilloma, sclerosis & fibrocys-
tic change (1); papilloma, duct ectasia & sclerosis (1); duct ecta-
sia & atypical lobular hyperplasia (2); papilloma & atypical duc-
tal hyperplasia (1); invasive ductal carcinoma (1).
CONCLUSIONS: Most tissue biopsies (88.6%) from ducts with mild
atypia in BDL showed benign breast lesions or normal histology.
However, in 11.4% of ducts with mild atypia by BDL, tissue
biopsy revealed atypical hyperplasia or invasive ductal carci-
noma. Ductoscopic evaluation and intraductal biopsy may be
helpful in distinguishing benign proliferative from premalignant
causes of epithelial atypia.
1
Deparment of Pathology & Immunology, Washington University
Medical Center, St. Louis, Missouri, 2Department of Surgery,
Washington University Medical Center, St. Louis, Missouri
17
Cryoablation Induced Changes in a
Fibroadenoma, Diagnosed by FNA Biopsy
Ronald Balassanian, M.D.1, Madeline Vazquez, M.D.2
BACKGROUND: Cryoablation is a new therapeutic technique cur-
rently under investigation for the treatment of benign and ma-
lignant breast tumors. According to published patient education
material from the manufacturer (SanarusTM), the VisicaTM sys-
tem for cryoablation of fibroadenomas allows treatment of be-
nign tumors with a simple quick and virtually pain-free proce-
dure. The cyto-morphologic effects of this procedure on benign
breast tissue have not been well described.
DESIGN: We present a case report of a patient who initially
presented with a mass in the right breast along the 1:00 o clock
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
axis. Ultrasound examination demonstrated a 3.2 cm lobulated
mass. Core biopsy revealed a fibroadenoma with cellular myxoid
stroma. With the patient‘s consent, cyroablation of the lesion
with the VisicaTM system was subsequently performed. The first
cycle did not cover the entire area and a second cycle was
required. Four months later, the patient reported a new adjacent
nodule. By clinical exam there was only a slight decrease in the
size of the original lesion. Ultrasound examination demon-
strated a 2.2 cm anterior nodule representing an area of fat
necrosis and changes possibly due to prior therapy. A second
adjacent posterior 2.2 cm hypoechoic nodule was also noted.
Ultrasound guided FNA biopsy of both nodules was performed.
RESULTS: Aspirates from both nodules demonstrated a back-
ground of degenerating fibroadipose tissue and epitheliod his-
tiocytes with chronic inflammation typical of fat necrosis of the
breast. Aspirates from the anterior nodule also demonstrated
scattered cohesive clusters of reactive ductal cells admixed with
numerous large lobulated fragments of acellular, apparently ne-
crotic, stroma. The epithelial cells demonstrated striking cellular
features which may be confused with a sarcoma but actually
reflected reactive changes similar to those typically associated
with chemotherapy or radiation therapy of the breast. There was
marked cytomegally with abundant glassy vacuolated cyto-
plasm. The nuclei were enlarged with moderate contour irregu-
larities, hyperchromasia and prominent central macronucleoli.
The nucleus to cytoplasm ratio was low. Myoepithelial cells were
not readily identified.
CONCLUSION: Our case report shows that FNA biopsy of an area
of prior cryoablation of the breast may result in necrosis and
epithelial changes that mirror the effects of chemotherapy or
radiation therapy; special care must be taken in this setting, to
take into account the clinical history and not interpret the
changes as atypia or malignancy.
1
University of Pittsburgh, School of Medicine, Pittsburgh, Penn-
sylvania, 2Weil Medical College of Cornell University, New York,
New York
18
A Gene Expression Signature Associated
with Epidermal Growth Factor Receptor
(EGFR) Expression in Breast
Adenocarcinomas
Mark Watson, M.D., Ph.D.1, Rebecca Aft, M.D., Ph.D.2,
Lourdes Ylagan, M.D., FIAC1
INTRODUCTION: EGFR (ErbB1) and Her2Neu (ErbB2) are two
interacting members of the receptor tyrosine kinase (RTK) gene
family whose over-expression is associated with both overall
survival and response to new targeted therapeutics in human
cancers, including breast carcinomas. We analyzed breast ade-
nocarcinomas from a cohort of patients in a clinical trial to test
337
the hypothesis that EGFR protein expression could be correlated
with the tumor cell molecular phenotype (i.e. ‘luminal cell’ vs.
‘basal cell’) and a unique gene expression signature, by concur-
rent gene expression microarray analysis.
MATERIALS AND METHODS: Breast tumor core biopsies were
selected from 33 patients undergoing treatment for clinical Stage
II-III disease. Sixteen patients have ER/PR and Her2Neu negative
tumors (triple negative- TN, or ‘basal cell’ type), while 17 have
ER/PR positive, Her2NEu negative tumors (ER$/PR$, or ‘lumi-
nal cell’ type). Frozen samples were histologically reviewed, se-
rial sectioned for RNA isolation, target preparation and hybrid-
ization to Affymetrix U133Plus2 GeneChip™ microarrays.
Corresponding formalin-fixed paraffin embedded tissues were
immunostained (IHC) with an anti-EGFR and scored from (0 to
3$) for intensity of staining.
RESULTS: By IHC, 7 of 16 (44%) TN tumors, while 0 of 17
ER$/PR$ tumors were EGFR immunopositive (IP$). Eleven
different probe pairs for EGFR represented on the GeneChip™
Microarray showed 14 of 16 (88%) TN tumors were EGFR tran-
script profile “positive” (TP$) while 5 of 17 (29%) ER$/PR$
tumors were similarly classified. Not all tumors showed concor-
dance by IP and TP. One tumor that was IP$ was called TP- for
EGFR, while ten tumors (7 TN and 3 ER$/PR$) that were IP-
were called TP$ for EGFR. More significantly, two patients who
had macroscopic residual disease (!1 cm) after neoadjuvant
therapy had a robust EGFR IP staining and a unique EGFR TP.
Supervised analysis of all 33 tumors, based upon EGFR TP,
shows signature genes that could distinguish EGFR $ vs. EGFR -
tumors (p % 0.05, corrected).
CONCLUSIONS: A subset of TN breast carcinomas express EGFR at
both the transcript and protein level. Discordance between IP and
TP as measured by 11 different microarray probe sets suggests
different alternatively spliced transcripts may provide additional
molecular information not available in routine IHC. A unique gene
expression signature of these TN tumors, which are usually unre-
sponsive to conventional therapy, implicates the EGFR signaling
pathway, which could be a helpful diagnostic tool in stratifying
patients to protocols involving targeted therapies to EGFR.
1
Department of Pathology and Immunology, Washington Univer-
sity, St. Louis, Missouri, 2Department of Surgery, Washington
University, St. Louis, Missouri
19
Ductal Lavage Cytology in Chinese Women
with Abnormal Nipple Discharge –
Comparison to Malignant Ductal Lavage
Samples
Steven Hart1, Qinjing Pan2, Wei Zhang2, Baoning Zhang2,
Karen Duvall1, Sharon Hirschowitz, M.D.1, JianYu Rao, M.D.1
INTRODUCTION: Spontaneous nipple discharge is second only to
the breast mass as the chief complaint of patients undergoing
338 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
breast surgery. Cytology from nipple discharge fluid usually con-
tains few epithelial cells and therefore may not be accurate for
determining the potential causes. Breast ductal lavage (DL) cy-
tology may increase the yield of epithelial cells and provides a
minimally invasive diagnostic tool to evaluate women with ab-
normal nipple discharge.
METHODS: A total of 23 women with spontaneous nipple dis-
charge were recruited for DL examination. For the purpose of
comparison, 14 women with mass lesions clinically suspicious
for malignancies were also lavaged. DL was performed on the
diseased breast (breast with nipple discharge or mass lesion),
and the specimen was processed by Thinprep. Cytological fea-
tures including epithelial cell uniformity, cohesiveness, pres-
ence/absence of myoepithelial cells, presence/absence of pap-
illary fragments, apocrine changes, background, nuclear
overlapping, nuclear enlargement, N/C-ratio, hyperchromasia,
nucleoli, and presence of mitotic figures, were scored from 1 to
3 or 4 according to the degree of alteration. The scoring data was
analyzed by the multi-variant Spiegelhalter-Knill-Jones (SKJ)
method, which is based on the Bayesian theorem. Patient was
followed up to two years with mammogram and clinical exam-
ination for benign conditions, and surgery for malignant lesions.
RESULTS: In 23 women with spontaneous nipple discharge, DL
produced 2 unsatisfactory samples (%10 epithelial cells), 12 benign,
8 mild atypia, and 1 marked atypia. Among them, histological
examination was available for 12 women with 8 benign intraductal
papilloma, 1 intraductal papilloma with focal carcinoma in situ, 1
fibroadenoma, and 2 fibrocystic diseases. Notably, the specimen
with marked atypia was derived from the patient with carcinoma in
situ. The remaining 11 women had negative mammogram and
clinical examination 2 years after DL. In 14 women with suspicious
mass lesions, DL showed 4 unsat, 4 marked atypia, and 6 malig-
nancy, all had histological confirmation of malignancy. ROC plot
based on the SKJ score showed 92% sensitivity and 91% specificity
to discriminate benign from malignant DL samples, and 63% sen-
sitivity and 93% specificity to discriminate DL samples from intra-
ductal papilloma from other benign DL samples.
CONCLUSION: DL may be a useful tool to evaluate women with
spontaneous nipple discharge.
1
University of California, Los Angeles, Los Angeles, California,
2
Cancer Institute/Hospital, Chinese Academy of Medical Sciences,
Beijing, China
20
Do Mammographic Findings Allow Invasive
Breast Carcinoma to be Distinguished from
Non-Invasive Carcinoma?
Ritu Bhalla, Raman Danrad, Stephen Lau, M.D., George
Birdsong, M.D.
INTRODUCTION/PURPOSE: In-situ carcinoma (ISC) may be de-
tected either as a palpable lesion or as mammographic abnor-
mality. Many of these lesions demonstrate characteristic find-
ings on mammograms. Fine needle aspiration (FNA) has been
used in the diagnosis and triage of palpable breast lumps, how-
ever one of the drawbacks of FNA is that ISC and invasive
carcinoma (IC) cannot be distinguished with certainty. Given the
less aggressive management options for ISC and better progno-
sis it is important to differentiate ISC from IC. This study inves-
tigated the extent to which mammographic findings could help
to distinguish these two entities.
MATERIALS AND METHODS: This retrospective study examined a
consecutive series of patients with histologically proven ISC or
IC for whom a suspicious or malignant FNA, concurrent mam-
mogram report, and subsequent histologic specimen were avail-
able. There were in total 67 patients. All patients underwent
mammogram and FNA followed by either surgical resection or
biopsy. The cytology and mammogram reports were reviewed.
Surgical pathological diagnosis was considered as gold standard.
Simple chi square test was used for data analysis.
RESULTS: The average patient age was 60.1, and ranged from 23
to 92 years. There were 21 cases that showed spiculated mass on
mammogram; 20 were IC and 1was IS. There were 47 cases that
showed nonspiculated mass on mammogram; 32 were IDC and
15 were DCIS. Simple chi-square test showed statistically signif-
icant result with P value %0.02.
CONCLUSION: Findings of carcinoma on FNA of the breast from
a spiculated mass on mammogram strongly predicts invasive
carcinoma, even in this small study. A larger study would be
beneficial to bring greater statistical power to these findings. For
patients in whom chemotherapy or radiation therapy prior to
definitive surgical therapy would be appropriate, a obtaining
histologic may be considered.
Emory University School of Medicine, Atlanta, Georgia
21
Cytologic Features of Meningiomas on
Crush Preparations
Brett Mahon, M.D., David Cimbaluk, M.D., Deepa
Kasuganti, M.D., Vijaya Reddy, M.D., Elizabeth Cochran,
M.D., Paolo Gattuso, M.D.
PURPOSE: Meningiomas are rarely subjected to aspiration. How-
ever, since they may occur in extradural locations, it is important
to recognize them as such to avoid misdiagnosis. The goal of this
study was to examine the cytological features of meningiomas in
crush preparations and cytologic imprints prepared at the time
of frozen section.
MATERIALS AND METHODS: From 1999 to 2004, a total of 97 cases
of meningioma were submitted for frozen section evaluation at
our institution. The concomitant crush preparation and cyto-
logic imprint prepared in each case were reviewed retrospec-
tively. The cytologic material had been stained with H&E and
Diff-Quik methods.
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
RESULTS: The patient population included 65 females with a
mean age of 56 years and 32 males with a mean age of 57 years.
Thirty cases (30.9%) were located in the frontal region, 16
(16.5%) in the temporal region, 14 (14.4%) in the parietal region,
and 11 (11.3%) in the posterior fossa. Twenty-six cases (26.8%)
occurred in diverse sites (5 skull base, 4 cervical spine, 3 olfac-
tory groove, 3 ventricular, 3 clival, 3 sphenoid, 2 suprasellar, 2
ethmoid sinus, and 1 olfactory carotid triangle). Histological
subtypes included 78 classical, 7 atypical, 5 psammomatous, 5
microcystic, and 2 clear cell. The most common cytologic fea-
tures among these subtypes included large cohesive clusters of
densely packed cells showing peripheral feathering and polygo-
nal spindle cells with abundant indistinct cytoplasm. Intranu-
clear cytoplasmic inclusions, uniform nuclei, inconspicuous nu-
cleoli, single cells, and naked nuclei in the background were also
frequently seen. Additional features noted, though not consis-
tently, were focal whorling, psammoma bodies, marked capillary
proliferation, and rare clear cell and microcystic changes. Due to
overlapping of cytologic features between subtypes, a clear dis-
tinction could often not be made. However, the psammomatous
variant could be separated with certainty due to the presence of
numerous psammoma bodies and a prominent whorling pat-
tern. Also, the cytologic smears of the atypical meningiomas
often displayed mitotic activity, nuclear anaplasia, prominent
nucleoli, and sheet-like growth pattern, features not appreciated
in other histologic subtypes.
CONCLUSIONS: (1) The cytologic features of meningiomas may
not reflect their histologic subtype. (2) The psammomatous vari-
ant can be easily recognized in touch preps/imprints. (3) The
presence of nuclear anaplasia, macronucleoli, mitotic activity,
and sheet-like growth may suggest an atypical meningioma at
the time of frozen section. (4) It is important to be familiar with
the cytologic features of meningiomas since they may occur in
extradural locations and be mistaken for other neoplasms.
Department of Pathology, Rush University Medical Center, Chi-
cago, Illinois
22
Cytologic Features of Cerebrospinal Fluid
(CSF) in Patients with Multiple Myeloma
(MM)
Murat Gokden, M.D., Charles Surber, Robert Glenn,
CT(ASCP)
INTRODUCTION/PURPOSE: Involvement of the central nervous
system (CNS) by MM is rare and indicates aggressive behavior
and poor outcome. In addition to direct involvement, MM can
cause complications by involving the adjacent structures, met-
abolic and hematological alterations, and treatment-related
complications, including infections. Here, we evaluated the cy-
tologic findings of CSF from patients with MM.
339
MATERIALS AND METHODS: Of the 2101 CSFs identified in our
files in the period from1997 to 2005, 182 were obtained from 119
patients with MM. The patient population consisted of 67 men
(age range: 37-79; mean: 59) and 52 women (age range: 27-77;
mean: 57), who have been previously diagnosed with and un-
dergoing treatment for MM. Two air-dried, Diff-Quik-stained,
and two alcohol-fixed, Papanicolaou-stained cytospin slides
were prepared and reviewed from each specimen. Specimens
were evaluated for the presence of myeloma cells, inflammation,
infection, and any other cytologic feature.
RESULTS: A total of 18 specimens from 11 patients (9%) were
positive for malignant plasma cells. One specimen from each of
6 other patients (5%) showed atypical plasma cells, which were
suspicious, but not diagnostic for involvement by MM. Seven
specimens from 6 patients (6%) had increased lymphocytes and
4 others from 2 patients (1.6 %) showed cocci and accompanying
acute inflammation. One specimen had a large number of ap-
parently non-viable neoplastic cells consistent with chemother-
apy effect. No opportunistic infections or evidence of subarach-
noid hemorrhage were seen.
CONCLUSIONS: Although leptomeningeal myelomatosis has been
reported in 1.1% of patients with MM, our results indicate that,
of those patients, whose clinical and radiologic findings warrant
cytologic examination of CSF, 9% show definitive cytologic evi-
dence of such involvement. A subpopulation of specimens
shows atypical cells, increased lymphocytes, and bacterial infec-
tion.
Department of Pathology, University of Arkansas for Medical
Sciences, Little Rock, Arkansas
23
Evaluation of Papanicolaou (PAP), Diff-Quik
(DQ) and Hematoxylin-Eosin (H&E) Staining
Methods in Neurocytopathology
Neslihan Cetin, M.D., Murat Gokden, M.D.
INTRODUCTION/PURPOSE: H&E-stained squash preparation is
routine in neuropathology intraoperative consultation (IOC).
Cytologic features of the central nervous system (CNS) tumors
with this technique are well known by neuropathologists. These
features have not been well characterized by other techniques
more familiar to cytopathologists and cytotechnologists. Here,
we analyzed the cytologic features of CNS tumors by techniques
routinely used in cytopathology.
MATERIALS AND METHODS: Fifty intracranial tumors, consisting
of major histologic types (glioma, meningioma, pituitary ade-
noma, craniopharyngioma, neuronal/neurocytic, hemangio-
blastoma, schwannoma, chordoma, metastatic carcinoma) and
various grades, have been evaluated. Alcohol-fixed, H&E-
stained, alcohol-fixed, PAP-stained and air-dried, DQ-stained
squash preparations were made during IOC. They were evalu-
340 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
ated for nuclear, cytoplasmic, background, and architectural
features. Histologic diagnoses were used for correlation. The
cytologic features identified by these stains were analyzed.
RESULTS: PAP provided the best nuclear detail. Glial processes,
cytoplasmic membranes, squamous cells and keratin were better
highlighted compared to DQ. Staining quality was suboptimal in
air-dried and thick areas. DQ better highlighted the extracellular
matrix in glioma, mucin in metastatic adenocarcinoma, micro-
vacuolar background and cytoplasm in metastatic renal cell
carcinoma, myxoid matrix in chordoma, microvacuolar cyto-
plasm of stromal cells in hemangioblastomas, and tumor dia-
thesis. It was not satisfactory for nuclear detail and glial pro-
cesses. H&E was superior for cytoplasmic, including
gemistocytic, features and glial processes, and was satisfactory,
but not as good as DQ for extracellular material. The nuclear
features were acceptable, although not as good as in PAP. Arti-
facts due to air-drying and thickness were less prominent.
CONCLUSIONS: PAP and DQ provided the well-known advan-
tages in cytology, i.e., nuclear and cytoplasmic/extracellular de-
tail, respectively. H&E appears to combine their advantages sat-
isfactorily, in a single method, although with some degree of
compromise, saving time and limited tissue at the time of neu-
ropathology IOC. Since squash preparations are made from tis-
sue fragments, and neuroglial tissue is resistant to smearing, the
resultant microfragments provide the ability to evaluate cellu-
larity and architectural features. This, together with the simul-
taneous frozen sections, compensates for the compromise in the
nuclear detail in this particular setting.
Department of Pathology, University of Arkansas for Medical
Sciences, Little Rock, Arkansas
24
Comparison Between Cytomorphology
(CYTO) and Flow Cytometry Analysis (FC) in
the Evaluation of Cerebrospinal Fluid (CSF),
in Patients with Known or Suspected
Hematological Malignancies
Thaira Oweity, M.D., Cynthia Liu, M.D., Sherif Ibrahim,
M.D., Ph.D.
INTRODUCTION: CYTO evaluation of CSF in patients with neurological
symptoms is often performed to exclude CNS involvement by malig-
nancy. FC evaluation of CSF has been increasingly utilized in patients
with known or suspected hematological malignancies. Although the
value of FC in the diagnosis and classification of hematopoietic neo-
plasms has been well established in tissue and fine needle aspiration
material, this has not been well studied in CSF samples.
MATERIALS AND METHODS: We identified 206 consecutive CSF sam-
ples with concurrent CYTO and FC evaluation in the Cytology and
Hematopathology laboratories, respectively. Two cytospins (1 Diff-
Quik and 1 Papanicolaou- stained) were examined in each cytology
case. Occasionally, immunostains on additional cytospins were at-
tempted. A panel of monoclonal antibodies was tested in each FC
sample. This was selected based on the number of cells recovered and
the type of neoplastic cells (if known).
RESULTS: Comparison between the two methods is shown in
(Table 1). The results were concordant in about 94%. In 3.4% of
the cases (7) FC was diagnostic for hematopoietic neoplasm
while CYTO was negative. Four of these cases were Acute Lym-
phoblastic Leukaemia (ALL), 2 were non-Hodgkin lymphoma
(NHL) and 1 was Multiple Myeloma. In 2.9% of the cases (6)
CYTO evaluation detected malignant cells while FC was negative
in 2 and it did not have sufficient cells for analysis in 4. In 3 of
these 6 cases the malignant cells were proven to be non-hema-
topoietic (1 adenocarcinoma, 2 Merkel cell carcinoma). In 2, the
malignant cells were hematopoietic (1 ALL, 1 NHL). In 1 case, the
nature of the neoplastic cells was undetermined cytologically
due to the limited sample.
CYTO Negative Atypical Suspicious Positive Total
FC
Insufficient 58 8 1 4 (1.9 %) 71
Negative 62 24 2 2 (1%) 90
Suspicious 9 1 1 2 13
Positive 7 (3.4%) 3 2 20 32
Total 136 36 6 28 206
CONCLUSION: Flow cytometry and cytology evaluations of CSF are
complementary and should be performed concurrently for defini-
tive diagnosis of malignancy. In 3.4% of our cases, only FC detected
a hematopoietic neoplasm and in 1% only CYTO detected malig-
nant hematopoietic cells. In addition, CYTO had the advantage of
detecting non-hematopoietic malignancies not identified on FC.
New York University Medical Center, New York, New York
25
Imaging, Morphologic, and
Immunohistochemical Correlation in
Gastrointestinal Stromal Tumors (GIST)
Punam Bhanot, M.D.1, Li Cao, M.D.1, Charles Chaya,
M.D.2, Manoop S. Bhutani, M.D.2, Robert Logroño, M.D.1
INTRODUCTION/PURPOSE: Gastrointestinal stromal tumor (GIST)
has been recently distinguished morphologically, immunohisto-
chemically, and genetically from other gastrointestinal-tract
spindle-cell neoplasms. Purpose of study is to correlate clinical
and imaging findings with morphology and immunohistochem-
istry to diagnose GIST and differentiate it from other spindle-cell
lesions in the gastrointestinal tract.
MATERIALS AND METHODS: We reviewed 9 cases diagnosed as
GIST by image-guided and endosonographic-guided FNA with/
without core biopsy (7 stomach, 2 intraabdominal), M:F ratio
3:6, age range 33-80 years. On-site evaluation, preliminary cyto-
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
logic evaluation, and immunohistochemistry were provided for
5 cases. Immunostains were performed, depending on sample
size, on aspirates and/or core biopsy.
RESULTS: Upon imaging tumors were smooth and homogenous
consistent with GIST. Tumor size ranged from 1.8 to 22 cm. The
largest neoplasm showed solid/cystic and necrotic components.
Aspirates consisted of spindle-cell neoplastic proliferation ar-
ranged in fascicles exhibiting focal nuclear palisading, indistinct
cytoplasmic borders, and no significant atypia/mitosis. Focal
epithelioid changes or cytologic atypia and mitoses were ob-
served in 2 cases. Immunostains revealed tumor expression of
CD117 and CD34 in 4/5, actin in 2/5, and desmin in 1/5 cases. All
cases were diagnosed as GIST (or consistent with GIST for cases
lacking immunochemical analysis). Surgical excision was per-
formed in 4 cases; GIST diagnosis was confirmed in 3 while
another case proved to be neurofibroma. No surgical follow-up
was available for the remaining 5 cases, which had imaging and
morphologic findings consistent with GIST.
CONCLUSION: In the setting of consistent clinical and radiological
findings, the combined use of cytomorphology and immunohis-
tochemistry on FNA and/or core biopsy in most instances pro-
vides a reliable pathological diagnosis of GIST. The need of
sufficient material for performing ancillary studies and the usual
impossibility of excluding malignancy in GIST aspirates consti-
tute limitations to this approach.
1
Department of Pathology, 2Department of Medicine, University
of Texas Medical Branch, Galveston, Texas
26
Clinicopathologic Significance of “Signet-
Ring Cells” in Routine Cytology
Julie Wu, M.D., Syed Ali, M.D.
INTRODUCTION: “Signet ring cell” (SRC) is a phenotypic designa-
tion for a cell with a large clear cytoplasmic vacuole displacing
the nucleus to the periphery. SRCs are most commonly found in
gastric, ovarian, and breast carcinomas, and have associated
prognostic significance. Our study focuses on the cytopathologic
occurrence of SRCs in the context of diagnostic range, ancillary
studies, and clinical prognosis.
MATERIALS AND METHODS: Retrospective review of our cytopa-
thology files revealed 83 cases of SRCs diagnosed in a 16-year
period (1989-2004). Cytologic material was obtained by FNA, se-
rous cavity fluid (SCF) aspiration and miscellaneous other means.
Clinical data and ancillary studies were additionally reviewed.
RESULTS: Patients ranged in age from 13 to 87 yrs (mean 59.3) with
a M:F ratio of 1:1.24. Most common specimen types included SCFs
(45, 54%) [abdominal 30 and pleural 15], gastric brushings (8, 10%),
and breast FNA (5, 6%). Of the 83 cases, 13 (16%) were benign, 65
(78%) malignant, and 5 (6%) indeterminate. Benign lesions mostly
comprised of reactive mesothelial cells (9 cases, 69%). Of the ma-
341
lignant lesions, 47 (72%) were metastases, 14 (22%) were primary
cancers and 4 (6%) were local cancer recurrences. At the time of
diagnosis, 23 (49%) of metastatic cases had known primaries, the
most common of which included gastric (7, 30%), esophageal (6,
23%), and breast (5, 22%). Adenocarcinoma was the most prevalent
malignant diagnosis (53, 82%), followed by carcinoma, NOS (8%). All
FNAs with SRCs had a malignant diagnosis. Twenty-eight (34%) cases
received special stains at the time of primary diagnosis– 7 (25%) had
mucicarmine, PAS with/without diastase, or alcian blue only, 9 (32%)
had immunostains, and 12 (43%) had a combination of histochemical
and immunostains. For benign lesions, there was one case of mortality
due to end stage liver disease. For indeterminate lesions, there were
two cases of mortality due to later manifestations of cancers. For
malignant lesions, there were eight cases of mortality, all with associ-
ated survival times of less than seven months.
CONCLUSIONS: The most common site for occurrence of SRCs is
abdominal fluid and their presence usually indicates malignancy
(78%). Most cancers with SRCs are metastatic in origin (72%)
with a significant proportion from unknown primaries (51%).
The most common cancer with SRCs is gastric adenocarcinoma.
In 16% of cases, SRCs are associated with benign disease, most
often with reactive mesothelial cells. SRCs in an FNA sample
signify cancer in 100% of cases. Malignant lesions are associated
with significant mortality rate (18%) and short survival time (%7
mos). Cytologic diagnoses of SRCs for cancer have 97% sensitiv-
ity and 100% specificity.
The Johns Hopkins Hospital, Baltimore, Maryland
27
Probabilistic Reporting of EUS-FNA Cytology:
Towards an Improved Communication and
Better Clinical Decision Making.
Isam Eltoum, M.D.1, Victor Chen1, David Chhieng, M.D.1,
Darshana Jhala, M.D.1, Nirag Jhala, M.D., MIAC1, Ralph
Crowe, M.D.1, Shyam Varadarajulu, M.D.2, Mohamad
Eloubiedi, M.D., M.P.H.2
INTRODUCTION/PURPOSE: The estimates of probability of cancer
determine to a large extent the decision to perform EUS-FNA and
then to treat the patient. Using the threshold approach to clinical
decision making, we explicitly determined the threshold probabil-
ities needed to perform EUS-FNA and those needed to treat patient
suspected to have malignancy. We then compared these thresholds
to the pre- and post-test probability of malignancy associated with
benign, atypical, suspicious and malignant diagnoses. Our goal was
to help endoscopists to make appropriate clinical decision based
on quantitative and qualitative approach.
MATERIALS AND METHODS: The study included all consecutive
patients who underwent EUS-FNA from July 2000 to October
2003 and followed up till December 2004. Based on surgical
findings or close clinical follow as a reference standard, we
342 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
determined the positive likelihood ratios for each cytologic di-
agnostic category for all EUS-FNA or as stratified by organ site,
lesion size, or cytopathologist. A decision tree was constructed to
estimate the “treatment” threshold using pancreatic cancer as
the worse case scenario. The “no-test-test” and “test-treatment”
thresholds were determined and compared to the pre-EUS FNA
probability to decide whether EUS-FNA should be performed.
For treatment decision after EUS-FNA, we compared the post-
EUS FNA probability of malignancy for each diagnostic category
with the “treatment” threshold.
RESULTS: EUS-FNA has a wide range of pre-test probability
where it could be performed (0.014 to 0.93); below 0.014, the
decision would be watchful waiting; above 0.93 it should be
appropriate treatment. The positive likelihood ratios (95% CI) for
a positive/negative EUS-FNA (71, 24-207)/ (0.06, 0.04-.09) were
conclusive for the presence/absence of malignancy. The likeli-
hood ratios for suspicious (11, 1.9-64.0) and atypical (0.96 (0.45-
2.07) diagnoses were inconclusive of malignancy. The post-test
probabilities (95% CI) for malignancy, 0.99 (0.967-.996)/ 0.09
(0.057-0.126) after a positive/negative test were significantly dif-
ferent than the treatment threshold, 0.90; but not those of sus-
picious, 0.92 (0.767-0.994), and atypical, 0.60 (0.407-0.772), diag-
noses. These results did not vary by lesion size, organ site or
cytopathologist who examined the specimen.
CONCLUSIONS: This is the first study of its kind that used the
threshold approach to clinical decision in cytodiagnosis, a field
that often thought of by clinicians to give absolute and final
results. We demonstrated the uncertainty associated with EUS-
FNA diagnostic categories and used threshold model to qualify
quantitatively the decision to perform EUS-FNA and treat pa-
tients suspected of having malignancy.
1
Division of Anatomic Pathology, Department of Pathology, Uni-
versity of Alabama at Birmingham, Birmingham, Alabama, 2Di-
vision of Gastroentrology and Hepatology, Department of Medi-
cine, Birmingham, Alabama
28
Endoscopic Ultrasound-Guided Fine Needle
Aspiration of Intrathoracic and
Intraabdominal Spindle Cell and
Mesenchymal Lesions
Sarah Bean, M.D., Allyson Baker, Mohamad Eloudeidi, M.D.,
M.P.H., Isam Eltoum, M.D., Nirag Jhala, M.D., MIAC, Ralph
Crowe, M.D., Darshana Jhala, M.D., David Chhieng, M.D.
BACKGROUND: The clinical utility of endocoscopic ultrasound-
guided fine needle aspiration (EUS-FNA) as a minimally invasive
method for diagnosis of intrathoracic and intraabdominal masses
has been well-accepted. Its role in the evaluation of spindle cell and
mesenchymal lesions is less clear. This study reviewed our experi-
ence in diagnosing spindle and mesenchymal cell lesions encoun-
tered in EUS-FNA at an academic institute.
METHODS: All EUS-FNA specimens from Aug 2000 to Dec 2004 and
demonstrating spindle or mesenchymal cells as a significant com-
ponent were retrieved. Follow-up consisted of clinical correlation,
chart review, or evaluation of subsequent tissue specimens, includ-
ing FNAs, biopsies, and/or surgical resections. We classified the
lesions into two categories: inflammatory/reactive and neoplastic.
RESULTS: Forty-eight EUS-FNA cases that contained a significant
spindle cell/mesenchymal component were retrieved, accounting
for 2% of all EUS-FNA examined during the studied period. They
were from 42 patients: 23 males and 19 females with a median age
of 61 years (range: 20 to 88). The distribution of the lesions was as
follows: 22 lymph nodes, 15 gastrointestinal tract, 7 pancreas, 4
others. Four cases had no follow up and were excluded from the
study. Twenty-two cases were categorized as inflammatory/reac-
tive conditions including: 17 granulomatous lesions and 7 cases of
chronic pancreatitis with significant fibrosis. Twenty-two cases
were categorized as neoplasias including: 13 gastrointestinal stro-
mal tumors (GIST), 2 smooth muscle tumors, 2 sarcomatoid carci-
nomas, 2 melanomas, 2 sarcomas, and 1 solitary fibrous tumor. A
specific cytologic diagnosis was rendered in 30 cases (81%). Immu-
nocytochemistry was performed on 21 neoplastic cases and con-
tributed to the differential diagnosis in 18 cases. Follow-up was
unavailable for 5 spindle cell neoplasms. No false positive cases were
encountered, but three false negative cases were identified. Two pa-
tients with EUS-FNAs that were initially diagnosed as chronic pancre-
atitis with marked fibrosis were found to have pancreatic adenocarci-
noma on subsequent tissue sampling. No evidence of malignancy was
noted on retrospective review; we attributed the false negatives to
sampling error. In the third case, the EUS-FNA of a mediastinal mass
showed rare atypical cells and dense fibrocollagenous which was
interpreted as non-specific finding. Surgical resection of the medias-
tinal mass revealed a solitary fibrous tumor.
CONCLUSIONS: Spindle cell neoplasms are rarely encounted on
EUS-FNA. The differential diagnosis encompasses a wide variety
of benign and neoplastic entities. Correlation of cytomorphology
and ancillary studies yields a high diagnostic accuracy of spindle
cell and mesencyhmal lesions on EUS-FNA.
University of Alabama at Birmingham, Birmingham, Alabama
29
Analysis of 496 EUS-FNA Samples with
Special Emphasis on Atypical and
Suspicious Diagnoses
Nirag Jhala, M.D., MIAC1, Isam Eltoum, M.D., M.B.A.1,
Mohamad Eloubeidi, M.D., M.P.H.2, Regina Meara, M.D.1,
David Chhieng, M.D., M.B.A.1, D. Ralph Crowe, M.D.1,
Selwyn Vickers, M.D.3, Shyam Varadarajulu, M.D.2,
Darshana Jhala, M.D., B.Mus.1
INTRODUCTION: Endosonography (EUS) guided fine needle as-
piration (FNA) is now one of the commonly used modality to
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
obtain preoperative diagnosis from a clinically suspected ma-
lignancy for a deep seated lesion. An accurate and unequiv-
ocal cytologic diagnosis is essential for successful manage-
ment of patients with suspected malignancy. An equivocal
diagnosis (atypical or suspicious) may require additional
studies to provide a disease specific patient management.
Evaluation of EUS-FNA samples poses unique challenges for
cytopathologist in that regards. This study was undertaken
with to determine the frequency of indeterminate diagnosis
and their association with sample type and lesion size as well
as the underlying cytologic cause.
MATERIALS AND METHODS: Four hundred ninety six EUS-FNA
from adult patients (age range: 21-94 years; mean age: 63
years) were evaluated. Diff quik and Papanicolaou stained
smears were evaluated and cytologic diagnosis were catego-
rized into non diagnostic, benign/ reactive, atypical/ suspi-
cious for malignancy and malignant. All cases with diagnosis
of either atypical or suspicious for malignancy were further
evaluated for their frequency, association with the sample
type, lesion size and cytologic features. The standard for com-
parison for all diagnoses was clinical and/ or imaging follow
up and/ or surgical resection
RESULTS: Aspirations were obtained from pancreas (solid: 268;
cystic: 38), lymph nodes/ spleen (n"91), hepato-biliary tree
(n"58), gastrointestinal tract (n"15) and adrenal glands
(n"26). A 13% (63/496) frequency of equivocal diagnoses
(atypical " 42; suspicious for malignancy " 21) was noted in
our study. Diagnosis of atypical cells was significantly more
frequently (p " 0.02) rendered for pancreas and biliary tree in
comparison to other sites. A diagnosis of atypical cells for
pancreas was rendered more frequently for large (" 25 mms)
solid lesions (27/33, 82%) than for small (%25 mms) and cystic
pancreatic lesions (6/33, 18%). In 7/42 (17%) cases with diag-
nosis of atypical cells, a subsequent re aspiration confirmed
clinical suspicion of carcinoma. Tissue confirmation was
available in 28/42 cases and included pancreatic adenocarci-
noma (n"15), chronic pancreatitis (n"3), lymphoma (n"3)
and 1 each were islet cell tumor, endometriosis, GIST and
metastatic carcinoma. Lack of atypical cells was the most
frequent cause for indeterminate diagnosis for cases which
were diagnosed as carcinoma on follow up. Gastric epithelial
contamination led to a diagnosis of possible mucinous cystic
lesion in a patient with pseudo cyst in the tail of the pancreas.
All cases diagnosed as suspicious for malignancy were con-
firmed malignant on subsequent follow up.
CONCLUSION: Equivocal diagnoses are infrequently encoun-
tered in EUS-FNA specimens. EUS- FNA of pancreas and
biliary tree are the most frequent sites associated with equiv-
ocal diagnosis.
Departments of 1Pathology, 2Gastroenterology and Hepatology,
and 3Gastrointestinal Tract Surgery, University of Alabama at
Birmingham, Birmingham, Alabama
343
30
Is There a Need for Immediate Adequacy
Evaluation when Using Endoscopic
Ultrasound-Guided Fine Needle Aspiration
Biopsy?
Reina Tarabey, M.D., Eva Wojcik, M.D., MIAC, Michael
Laudermith, Alia Salhadar, M.D., Razan Massarani-Wafai,
M.D., Rasheed Hammadeh, M.D., Christine Booth, M.D.
INTRODUCTION: It has been shown that the time required for
pathologists in the immediate adequacy evaluation of endo-
scopic ultrasound-guided fine needle aspiration biopsies
(EUS-FNAB) is greater than the time for frozen section eval-
uation, clinician performed FNAB with pathologist immediate
adequacy evaluation and pathologist-performed FNAB. The
aim of this study was to determine whether or not immediate
evaluation of EUS-FNAB material made a difference in the
number of patient samples deemed “unsatisfactory for inter-
pretation”.
MATERIALS AND METHODS: All EUS-FNAB performed during
the 22-month period from 11/2002 – 9/2004 were retrieved
from the cytopathology computer database. The diagnostic
team included two gastroenterologists and four board-
certified cytopathologists. The following data points were re-
corded in each case: 1) clinical history, 2) site of FNA, 3)
gender, 4) patient age, 5) immediate evaluation, 6) cyto-
logic diagnosis, 7) follow-up procedure, and 8) histologic di-
agnosis.
RESULTS: A total of 132 EUS-FNAB from numerous sites were
performed during the 22-month time period. Of these, 69
(52.3%) had an immediate adequacy evaluation performed by
the pathologist. The majority of the EUS-FNAB were from the
pancreas (101/132; 76.5%) with the remainder from the lymph
nodes, stomach, esophagus, duodenum, bile duct and gall-
bladder. With immediate evaluation of adequacy in the GI
endoscopy suite, the unsatisfactory rate was 16.67%. Without
an immediate adequacy evaluation, the unsatisfactory rate
was 18.03%, which showed no statistically significant differ-
ence (p" 0.446).
CONCLUSIONS: There was no significant difference in the un-
satisfactory rate of EUS-FNAB whether or not an immediate
evaluation for adequacy was performed. Given the significant
amount of pathologist time required for the immediate eval-
uation of EUS-FNAB, and the lack of obvious benefits in
regard to specimen adequacy, the data suggests that there is
no need for a pathologist to be routinely present at the time of
the EUS-FNAB procedure.
Loyola University Medical Center, Maywood, Illinois
344 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
31
Anal Cytology: Cytologic, Virologic, and
Histologic Correlation in a Population of HIV
Positive Patients
Sarah M. Bean, M.D., David C. Chhieng, M.D., M.B.A.,
Janie Roberson, CT(ASCP), James Raper, D.S.N., Gregg
Shore, M.D., Ge Jin, B.S., Thomas Broker, Ph.D., Craig
Hoesley, M.D., Isam A. Eltoum, M.D., M.B.A.
INTRODUCTION/PURPOSE: The incidence of anal-rectal squamous
cell carcinoma in HIV positive men who have anal-receptive
intercourse with men rivals that of cervical carcinoma in women
prior to implementation of routine cervical screening. Anal rec-
tal cytology is now the recommended method for screening this
at-risk patient population. The purpose of this study is to de-
scribe the cytologic distribution of disease and to correlate cy-
tologic diagnoses with HPV DNA status as well as with surgical
biopsy.
MATERIALS AND METHODS: A retrospective search of the com-
puterized database between January 01, 2000 and November 30,
2004 identified patients who had undergone anal-rectal cytol-
ogy. Follow-up data including repeat anal Pap tests and surgical
biopsy diagnoses were obtained when available. HPV DNA PCR-
based genotyping data performed on a subset of the study pop-
ulation were also included.
RESULTS: At least one anal-rectal Pap test was performed on 118
HIV positive patients (116 men and 2 women; mean age 42;
range 23-68). Cytologic diagnoses included: 3% unsatisfactory
for diagnosis, 41% NILM, 23% ASCUS, 31% LSIL, and 2% HSIL
(ASCUS:SIL 0.7:1). Overall, 13 high grade lesions (2 AIN II, 10 AIN
III, and 1 invasive squamous cell carcinoma) were detected in
11% of the population. The percentage of high grade lesions in
each cytologic category is as follows: NILM (6%), ASCUS (7%),
LSIL (19%), and HSIL (50%). The majority of AIN II$ was pre-
ceded by a cytologic diagnosis of LSIL 54%, ASCUS 15%, and
HSIL 8%. The false negative fraction (high grade lesions detected
in NILM) was 23% (3/13). Seven of nine (78%) of the ASCUS
patients tested for HPV were positive. All 13 patients with LSIL
were HPV positive. Forty-eight percent (12/25) of NILM patients
were HPV positive. The CD4 counts (mean, SD) tend to be lower
in patients with high grade lesions (243, 65) than low grade
lesions (400, 261) than normal (428, 232).
CONCLUSIONS: Anal rectal cytology is a useful screening test for
detecting high grade squamous intraepithelial neoplasia. A high
percentage of AIN II$ lesions were detected in this at-risk pop-
ulation, and the majority were detected following cytologic ab-
normality. The trend of a lower CD4 count seen with high grade
lesions may explain some of these findings. The false negative
fraction is high.
University of Alabama at Birmingham, Birmingham, Alabama
32
Anal Cytology and Reflex HPV in Screening
for High Grade Anal Lesions in Patients
with Anal Condylomas
Raheela Ashfaq, M.D.1, Ann Marie West, BS, CT (ASCP)2,
S.T. Gokaslan, M.D.1, M.H. Saboorian, M.D.1, A. Lee,
M.D.1, H. Papaconstantinou1
INTRODUCTION: Anal Condylomas are caused by anal HPV in-
fection and is a primary risk factor for development of anal
carcinoma (AC) and its putative precursors, anal LSIL and
anal HSIL. The reported incidence of Anal Squamous Carci-
noma (SCC) in patients with anal condylomas is 3-4%. Early
detection of Anal HSIL may decrease incidence, morbidity,
and mortality from Anal SCC. The purpose of this study is to
determine whether anal cytology can detect HSIL in anal
condylomas.
MATERIALS AND METHODS: Over a 6-month study period, all
patients with anal condylomas were periodically evaluated for
anal cytology by TPPT, HPV typing, and tissue biopsy. Anal
cytology findings were reported using Bethesda 2001. Histo-
logic results were grouped as low grade, high grade, or carci-
noma. Histologic results served as truth. HPV testing was done
on residual cytologic material in Preservcyt® using the Digene
Hybrid Capture (HC) II test.
RESULTS: Fifty-three patients between the ages of 19-62
(mean age 38) undergoing operative treatment for anal con-
dylomas comprised the patient population. On surgical biop-
sies 64% (34/53) were low grade lesions and 19 (32%) were
high-grade lesions/carcinoma. The anal Paps had the follow-
ing diagnosis NIL 12 (35.2%), ASC-US 10 (20.5%), LSIL 19
(35.8%) and HSIL/CA 12 (31.5%) The overall sensitivity for
cytology was 47.37%, specificity 91%, PPV 78% and NPV
75.6%. A cytologic HSIL result on anal cytology is 60% sensi-
tive, and 86% specific with a PPV of 58% and a NPV of
90%. However, when an HPV test is added to cytology the
sensitivity for identifying Anal HSIL increases to 89.5% with an
NPV of 86.6%. Overall, 17 of the 19 high grade anal lesions (90
%) were positive for HPV compared to 20 of 33 (60%) for CIN
I lesions.
CONCLUSION: Anal cytology alone is not accurate for detecting
high-grade lesions in patients with anal condylomas. However
combining oncogenic HPV with cytology is more sensitive in
detecting high grade lesions and may be a better screening
strategy. Our recommendation would be to do reflex HPV test
in all patients with anal condylomas.
1
University of Texas Southwestern Medical Center, Dallas, Texas,
2
Parkland Health & Hospital System, Dallas, Texas
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
33
Endoscopy Ultrasound Guided FNA for the
Detection of Distant Metastasis to the
Celiac Axis Lymph Nodes in Lung Cancer
Eric Rachut, M.D.1, Renu Khode, M.D.1, Shanthi Gopal,
M.D.1, Edwin Johnson, M.D.1, Douglas Toler, Ph.D.,
CT(ASCP)1, Yashodeep Jadhav, M.D.1, Phalguni
Mukhopadhyay, M.D.1, Brian Camazine, M.D.1, Richard
Erickson, M.D.2, Shyam Narayana, M.D.1, Pankaj Singh,
M.D.1
BACKGROUND: Celiac axis lymph nodes are not a common site
for distant metastases in lung carcinoma and are therefore not
included in current staging. Staging is based on CT scan and
thoracotomy findings, which are inadequate for examination of
the celiac axis region.
AIM: To determine the utility of EUS guided fine needle aspira-
tion (EUS – FNA) in the detection of metastases to the celiac
lymph node in patients with lung cancer.
METHODS: In a prospective study, consecutive patients with
newly detected lung mass, suspicious for malignancy, were in-
cluded. EUS - FNA of lymph node at celiac axis was performed,
if lymph node could be identified. Primary outcome was to
determine the ability of the EUS-FNA and CT scan for the de-
tection of the metastases to lymph nodes at celiac axis. Second-
ary outcome was to determine the proportion of cases in which
EUS-FNA had an impact on management by changing the stage
of the tumor. On site rapid interpretation of the adequacy was
provided by a cytopathologist in every case. Air-dried, Diff Quik
stained smears were used for on site assessment of adequacy of
sample and preliminary diagnosis. Papanicolaou stained smears
along with the cell block made from pooled material were
viewed for the final diagnosis. Immunostains were done as in-
dicated.
RESULTS: 91 consecutive patients of lung carcinoma (mean age
65 years) underwent EUS examination of the celiac axis. EUS-
FNA was performed in 20 cases. 13/91 cases (14%) were cyto-
logically confirmed to have metastases to the lymph nodes at the
celiac axis. Lymph nodes in five of the 13 cases were less than 1
cm and were not detected by the CT scan. The median number
of needle passes was 3 (range 1-4). On site diagnosis was pro-
vided by the cytopathologist in 13/20 (65%) cases. The cellularity
was adequate in 17/20 samples, scant in 3/20 samples and
insufficient for diagnosis in 0 samples. Immunohistochemistry
was used in 7/20 (35%) samples. Final cytological diagnoses of
the celiac axis lymph nodes were as follows: non-small cell
carcinoma (10), small cell carcinoma (3), and negative for ma-
lignancy (7). Of the 10 cases with NSCLC metastases to celiac
axis, only 4 cases had co-existent metastases to the liver and/ or
adrenal gland. Thus, EUS-FNA upgraded the staging in 6 (7%) of
the 91 cases. There were no complications in any of the patients.
CONCLUSION: EUS-FNA identified lymph nodes at the celiac axis
345
as an important site for distant metastases. EUS – FNA is a safe
technique for the tissue acquisition from the celiac lymph nodes.
Routine cytological examination and immunohistochemistry of
the tissue obtained using EUS-FNA detects metastases that are
missed by CT scan and thus impacts the management by up-
grading the tumor to stage IV.
1
Central Texas Veterans Health Care System, Temple, Texas,
2
Scott & White Memorial Hospital, Temple, Texas
34
Esophageal Brushing Cytology in Barrett’s
Esophagus (BE)
Robert Stewart, CT(ASCP), Marek Skacel, M.D., Gary
Falk, Jennifer Brainard, M.D.
INTRODUCTION: BE is a complication of chronic gastroesopha-
geal reflux disease and is a risk factor for esophageal adenocar-
cinoma. Esophageal brushing cytology as a surveillance tool for
pts with BE is used in a minority of institutions in spite of
theoretic advantages including ability to sample a large involved
surface area, simplicity, and lower cost. We studied esophageal
brushings from pts with BE and compared results with surgical
biopsy.
METHODS: We studied 764 esophageal brushings from pts with
BE over a 4yr period. Concomitant surgical biopsy specimens
were available in all cases. Specialized columnar epithelium was
identified in all included cases. The cellularity of the cytology
preparation was recorded as low, moderate or high. Cytologic
and surgical diagnoses were compared and slides and follow up
data from discrepant cases were reviewed.
RESULTS: 9 pts with high grade dysplasia/adenocarcinoma
(HGD/AD) on biopsy had a negative esophageal brushing. Co-
lumnar cell cellularity and sampling was a factor in these cases.
4 of these 9 pts had a subsequent brushing sample obtained
between 3 wks and 4 mos showing either HGD/AD or changes
indefinite for dysplasia. The 2 pts with HGD/AD in the brushing
and negative biopsies had clinically malignant esophageal stric-
tures and were managed according to the cytology result.
CONCLUSION: In this series, esophageal brushings exactly corre-
lated with biopsy diagnoses of HGD/AD in 73%. Another 19% of
HGD/AD cases were called indefinite for dysplasia in the cytol-
ogy sample. Multiple brushing samples may improve diagnostic
yield. Addition of biomarkers may be of use in the cytologically
indefinite category to separate low grade dysplasia from reactive
atypia as this was not reliably done based on morphology alone
in our study.
The Cleveland Clinic Foundation, Cleveland, Ohio
346 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
35
The Contribution of Technology to
Cytopathology Errors
Stanley J. Geyer, M.D., Chris Campana, M.D., Brent
Brewerton, B.S., CT(ASCP), Jared Shelton, B.S., Cynthia
Pratt, CT(ASCP)
INTRODUCTION: The ASCCP recommends testing for high-risk
strains of human papillomavirus (hrHPV) in cervical cytology
specimens with an interpretation of ASC-US. In the spring of
2004 our laboratory began using the Ventana chromogenic in
situ hybridization (CISH) method for the detection of hrHPV in
all ASC-US cases. During several months of use, the cytopatholo-
gists observed cases in which the method yielded high back-
ground staining which confounded hrHPV interpretation.
METHODS: The laboratory uses ThinPrep materials and methods
for all Pap specimens. We collected 44 consecutive ASC-US cases
stained with the Ventana CISH method, in which sufficient re-
sidual liquid was available for Digene Hybrid Capture II (HCII)
testing for hrHPV. Two pathologists, neither of whom provided
the original CISH interpretation, independently reviewed the
CISH slides. A reference lab performed the HCII testing.
RESULTS: Of the 44 cases, complete disagreement (at least one
pathologist interpreted the case as positive and another called
the case negative) of CISH interpretations occurred in five cases
and partial disagreement (one or two pathologists used the term
indeterminate and the other pathologist(s) agreed the that case
was positive or negative) occurred in 4 cases. HCII results com-
pared to CISH results disclosed 11 discrepant cases: 6 were CISH
positive (according to the original sign out pathologist)/HCII
negative and 5 were CISH negative/HCII positive. Three of the 11
cases had pathologist interpretative discrepancies. Two of these
3 cases had complete disagreement and 1 had a partial disagree-
ment.
CONCLUSION: The study demonstrates that methods contribute
to discrepancies in pathology interpretations of hrHPV infectiv-
ity in Pap specimens. The variations may result from differences
in analytical sensitivity and specificity, from preanalytical fac-
tors, from variations in pathologists’ interpretations of CISH
staining, or from other undetermined circumstances. The study
illustrates that the choice of technology may contribute to pa-
thology errors and ultimately may affect patient safety. False
positive results may lead to unnecessary procedures, increased
cost of healthcare, and unwarranted anxiety for the patient.
False negative tests may delay the diagnosis and treatment of
significant intra-epithelial lesions. The resolution of the techni-
cal contribution to pathology error deserves additional study to
determine best practices for evaluating the safe and effective use
of tests to improve Pap smear and HPV interpretations. Use of
statistical tools such as receiver operator characteristics (ROC)
curves and likelihood ratios to evaluate new methods may im-
prove the prediction of risk of developing high-grade lesions and
result in improved patient care and safety.
LDS Hospital, Salt Lake City, Utah
36
Endometrial Brush Sampling and Polymer
Encapsulation Enhance the Predictive Value
of Endometrial Cytopathology for Benign
and Malignant Disease: Experience with
1,465 Tissue-Matched Cases
John Maksem, M.D.
PURPOSE: Liquid fixation and polymer encapsulation maintain
Tao-brush endometrial collections so that one can envisage
endometrial “tissue patterns” in cytology preparations. We pro-
pose a simple 4-tier scoring system for predicting endometrial
cancer risk. In addition to dyskaryosis (nuclear grade), the sys-
tem accounts for epithelial microbiopsy patterns comprising: (1)
complete differentiation, when only flat epithelial sheets and
uniform tubules are seen on the cytology slide; (2) mild dysdif-
ferentiation, commonly seen with simple or non-atypical com-
plex hyperplasia, representing presumptively hypermature but
generally benign states; (3) moderate dysdifferentiation, show-
ing epithelial clusters or tufts, pseudopapillae or short papillae
resulting from the piling-up of epithelial cells within irregular
glands; and, (4) severe dysdifferentiation, with patterns such as
cribriform structures, solid epithelial aggregates or dyshesive
single abnormal cells that are highly predictive of endometrial
hyperplasia and adenocarcinoma.
MATERIALS AND METHODS: Uterine brushings and matched hys-
terectomy (N " 1,272) or biopsy (N " 193) specimens obtained
from 1,465 women were used to show how nuclear features and
microbiopsy patterns distribute in relation to endometrial his-
tology.
RESULTS: Ninety-seven percent (97%) of endometrial carcino-
mas showed grade 3 and 4 findings; 82% of grade 3 and 4
findings comprised endometrial hyperplasia or carcinoma; and,
endometrial malignancy was vanishingly rare among grade 1
and 2 findings.
Outcomes
Grade EM1 EM2 EM3 EM4
Normal endometrium 908 249 13 0
Benign endometrial abnormality 11 98 15 3
Premalignant endometrium 0 25 22 22
Malignant endometrium 1 2 29 67
CONCLUSION: Brush cytology consistently identifies benign en-
dometrium, reliably predicts malignant and premalignant endo-
metrium, correctly ranks intermediate degrees of endometrial
pathology and has the added advantage of obtaining samples
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting 347

from atrophic endometrium more often than does endometrial MLBC TISSUE CORRELATIONS
biopsy. With the obesity epidemic ravaging developed nations,
endometrial neoplasia is expected to be on the rise. It appears that CIN 2! CIN 1 Negative Total
cytology can offer a means of risk stratification for endometrial
ASC-US, NOS 24 214 196 434
diseases, especially hyperplasia and carcinoma. These outcomes ASC-US, HPV$ 30 122 47 199
demonstrate the potential usefulness of direct endometrial sam- ASC-US, HPV- 1 36 163 200
pling by brush cytology, especially among selected populations. ASC-H 40 88 47 175
LSIL 215 1927 240 2382
Bostwick Laboratories, Orlando, Florida !HSIL/ !AIS 730 255 25 1010

CONCLUSION: MLBC using Gel Matrix Encapsulation is an alter-

native to automated LBC. One technician can be expected to
prepare about 300 cases in an 8-hour day. The cost of the

37 method (labor included) is about one order of magnitude less

than the cost of automated LBC. MLBC entails no equipment
overhead, and no proprietary fixatives are used in its execution.

An Inexpensive and Easy to Use Manual Outcomes are superior to conventional cytology.

Liquid Based Cytology (MLBC) Method: Gel
Matrix Encapsulation. Experience with
134,562 Cases with Comparison to 4,400
Adequate Cyto-histological Correlations
John Maksem, M.D.
PURPOSE: MLBC is a technique that we built upon the methods
of Ehrenreich and Kerpe (JAMA 1959; 170: 1176-1177) and Sac-
comanno et al (Acta Cytol 1963; 7: 305-310). The fixative and
coating agent are initially separated from one another, which
gives one the advantage of creating slides from cells suspended
in virtually any fixative. We have applied MLBC to 134,562 gy-
necological cytology specimens from 2002 through 2004, at
which time we received 4,400 adequate correlative biopsy exam-
inations.
MATERIALS AND METHODS: This report details: (1) fixative for-
mulation, (2) gel matrix formulation, (3) manual preparative
method, (4) outcome of cytology examinations compared to
124,786 conventional historical controls from 1996 through 1999,
and (5) comparison to 4,400 adequate colposcopic-exam histol-
ogies.
RESULTS: Since implementing MLBC, laboratory statistics
showed a 218% increase in SIL and a decreased ASC: SIL pro-
portion. Correlative adequate colposcopic-exam histologies
compare favorably with index cytological diagnoses; and there is
no evidence that the increased SIL detection is due to false-
positive cytological diagnoses. We have performed over 4,000
HPV tests of ASC-US cases using commercially available propri-
etary PCR methodologies and have shown remarkable clarifica-
tion of ASC-US by this approach.
CONVENTIONAL (‘96-99) VS. MLBC (‘02-04) CYTOLOGY OUTCOMES
Year Total Neg QNS Atypia LSIL HSIL !
1996-99 124786 117243 485 4488 1746 824
2002-04 134562 115537 1076 9222 7064 1663
Bostwick Laboratories, Orlando, Florida
38
ASC-H: A Comparison of Cytological
Preparations and Their Surgical Follow-Up
Vikram Kapoor, SCT(ASCP), Philip Galullo, CT(ASCP),
Kevin Schofield, CT(ASCP)
INTRODUCTION: The Bethesda System 2001 introduced the term
ASC-H to be used in cases which are suggestive, but not diag-
nostic of, HSIL. We performed a retrospective study to deter-
mine the utility of this diagnosis by correlating Pap smears with
biopsy results. We also compared follow-up results from Thin-
Prep, Conventional, and SurePath preparations.
MATERIALS AND METHODS: Data was obtained by performing a
retrieval search of the CoPath LIS for all Pap smears diagnosed as
ASC-H during the period of January 1, 2003–December 31, 2004.
The results were then correlated with cervical biopsy and/or endo-
cervical curettage specimens obtained within 6 months of the Pap
smear. For the purpose of this study, LSIL will encompass CIN I and
HPV infection. HSIL will encompass CIN II and CIN III.
RESULTS: A total of 227 Pap smears were diagnosed as ASC-H at
our institution. There were 176 cases with surgical follow-up
within 6 months of initial diagnosis. Of these, 26% had negative
biopsy results, 23% had LSIL on biopsy, and 51% had HSIL on
biopsy. Overall, 74% of patients with a diagnosis of ASC-H had
dysplasia on biopsy follow-up. Out of 71 ThinPrep specimens,
61% had HSIL on biopsy, 17% had LSIL, and 23% were negative.
For 15 Conventional specimens, 27% had HSIL on biopsy, 27%
had LSIL, and 47% were negative. Out of 90 SurePath specimens,
42% had HSIL on biopsy, 28% had LSIL, and 23% were negative.
CONCLUSIONS: According to current recommendations pub-
lished by The Bethesda System 2001 and the American Society
for Colposcopy and Cervical Pathology, patients with ASC-H
should be sent for colposcopy and biopsy. In our institution, 78%
348 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
of patients followed this protocol. Our HSIL detection rate of
51% is higher than that found in the ASCUS/LSIL Triage Study of
30-40%. Our findings of 74% of patients having dysplasia on
biopsy follow-up support the management recommendation of
biopsy for cases of ASC-H. ThinPrep had a higher biopsy corre-
lation rate for HSIL then SurePath (61% ThinPrep compared to
47% for SurePath). However, the overall abnormal detection rate
for both liquid-based technologies were similar (78% ThinPrep
compared to 75% for SurePath).
Yale University School of Medicine, New Haven, Connecticut
39
MarkPap® System, Current Status and
Prospective
Olivera Markovic, M.D., Ph.D., Nenad Markovic, M.D.,
Ph.D.
INTRODUCTION: MarkPap® technology was introduced as the
Cervical Acid Phosphatase—Papanicolaou (CAP-PAP) test in
1999. This is a biomarker-based technology for enhancing the
detection of abnormal cells on Papanicolaou stained back-
ground. The biomarker is visualized as a red granular deposit
flagging dysplastic (including HPV infected) cells and cancer
cells. Normal squamous epithelial cells are entirely negative. The
marker is attracting the attention of the screener reducing the
probability of their omission. Since 2003, this biomarker has
been contemplated as a surrogate end-point for colposcopy.
MATERIALS AND METHODS: The MarkPap® System comprises
MarkPap® test, a research kit, an enzyme-cytopreservative so-
lution, and Combo control slides. MarkPap Auto (automated
staining), MarkPap Digital (telecytopathology) and MarkPap Self
are under development. (www.markpap.com). The first four
products were used in three clinical trials that included 2000
subject/specimens (1500 general population and 500 high-risk,
1,250 pap smears and 750 ThinPrep thin-layers). Each trial was
conducted to show safety and efficacy of the new test in com-
parison with the control. The safety was measured as adverse
events rate, the efficacy was measured by the proportion of
detection of abnormal slides, and the accuracy (sensitivity and
specificity) of the tests validated by an optimized cytology stan-
dard. Statistical analysis of paired groups was used to measure
the degree of difference and its significance.
RESULTS: MarkPap test improved the detection of abnormal
specimens for 50% in comparison with Pap smear, and for 30%
with liquid-based Pap. The false-negative rate was below 5%.
Overall sensitivity was improved by 30% while specificity was
equivalent within 10% range. Easy automation of this test was
confirmed using a customized Varistain Gemini slide stainer
(Thermo Shandon). Due to the visual characteristics of the bi-
omarker, the electronic transfer of digital images has been ex-
cellent; biomarker-based telectyopathology could be the method
of choice for tomorrow’s cervical cancer screening. The chemical
nature of this biomarker makes it resistant to environmental
changes, maintains its presence in cervico-vaginal fluids, and
improves the accuracy of screening self-collected specimens.
CONCLUSION: The MarkPap System, a cervical acid phosphatase
biomarker-based technology has been found of significance for
more accurate, faster and less costly cervical cancer screening.
Further development is directed to meet the requirements for
developing countries that cannot afford the cost of the Pap test.
This work was supported by NIH/SBIR grants 1R- and
2R43CA80767. BioSciCon, Rockville, Maryland
40
Reporting of Endometrial Cells in
Cervicovaginal Pap Smears in Women
Between 40-50 Years of Age
Abir Tawil, M.D., Tomi Kuntzman, D.O.
OBJECTIVE: The objective of this study is to determine the sig-
nificance of the reporting of endometrial cells in pap smears of
women between 40-50 years of age, and the importance of
including a comment about the menstrual history when appro-
priate.
METHOD: This is a retrospective study of pap smears from Jan-
uary 2003 - December 2003 of women 40-50 years of age diag-
nosed with endometrial cells present. Of the 1,232 cases that
were found, 181 cases had clinical follow up (endometrial bi-
opsy, endometrial curetting or hysterectomy).
RESULTS: Of the 1,232 cases that were found, 181 (14.7%) of
women between 40-50 years of age had an endometrial study
within a year following the report of endometrial cells present in
their pap smears. Of these women, 160 (88.4%) were premeno-
pausal. In 108 (59.7%) of the women, the Pap smear was ob-
tained within the proliferative phase of the menstrual cycle. Only
21 (11.6%) of the women were post-menopausal. Only 4 (2%) of
the women showed endometrial pathology. Two postmeno-
pausal women presented with abnormal bleeding and their en-
dometrial study showed simple hyperplasia and endometrial
adenocarcinoma FIGO Grade I. One woman who was in the
luteal phase of her cycle, presented with abnormal bleeding and
her endometrial study showed simple hyperplasia. Only one
woman was in the proliferative phase of the cycle and her study
showed simple hyperplasia.
CONCLUSION: According to the 2001 Bethesda System for report-
ing cervical cytology, the presence of exfoliated endometrial cells
should be reported in all women 40 years or older. Because the
clinical history of a woman’s risk for endometrial carcinoma
(symptoms, menstrual history, hormone-therapy, etc.) is typi-
cally not known, it is felt that reporting endometrial cells in
women 40 years and older is prudent. It is left to the clinician,
who knows this information to decide on proper work up of their
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
patient. We have found, however, that mentioning of endome-
trial cells in the proliferative phase (when endometrial cells are
commonly seen) has frequently lead to further work up when it
may not be necessary. Significant endometrial pathology (com-
plex hyperplasia or carcinoma) was present in only 2 cases (1%).
In one case the patient was postmenopausal and in the other the
endometrial cells didn’t correspond with the menstrual history.
We recommend that a comment should be included each time
the endometrial cells are appropriate for the menstrual history
to help guide the clinician and to avoid unnecessary procedures
and expenses.
William Beaumont Hospital, Royal Oak, Michigan
41
Atypical Squamous Cells, Cannot Exclude
High Grade Intraepithelial Lesion (ASC-H):
The role of HPV
Michelle Reid-Nicholson, M.D., Rose Marie Gatscha, B.A.,
SCT(ASCP)CFIAC, Oscar Lin, M.D.
BACKGROUND: Atypical Squamous Cells, cannot Exclude High
Grade Intraepithelial Lesion (ASC-H) is a diagnostic category
introduced in the 2001 Bethesda System. The inclusion of this
category was justified through the ASCUS triage study (ALTS),
which had shown that 40.5% of cases diagnosed as ASC-H dem-
onstrated high-grade lesions on biopsy. Although the role of
Human Papilloma virus (HPV) testing in cases of atypical squa-
mous cells of undetermined significance (ASCUS) has been well
established, the role of HPV testing in ASC-H is not as well
recognized. We proposed to evaluate the role of HPV testing in
the diagnostic category of ASC-H, considering the fact that HPV
prevalence in ASC-H has been reported to range from 37.5% to
85.6%.
MATERIALS AND METHODS: The Cytology Service files in our
institution were searched for cases diagnosed as ASC-H from
2002 to 2004. The cases identified were evaluated for HPV status
and follow-up (FU). The positive predictive value (PPV) of ASC-H
in 1) the detection of dysplasias overall (low and high grade
squamous intraepithelial lesions) and 2) in the detection of
high-grade squamous intraepithelial lesions (HSIL) alone were
calculated. The histology in the FU biopsy was used as gold
standard.
RESULTS: Seventy-six patients with the diagnosis of ASC-H were
identified. Forty patients had biopsy FU and 28 had cytology FU.
Forty-eight of 76 cases had HPV testing performed, of which 28
were positive for high-risk (HR) and 2 for low-risk (LR) HPV.
Fourteen cases were negative for HPV and 4 cases had insuffi-
cient material. Twenty-five of the 76 cases had both HPV testing
and biopsy correlation, but 2 cases had insufficient material for
HPV analysis. The results of the remaining 23 follow-up biopsies
and HPV status are summarized in Table 1. The PPV of ASC-H for
349
dysplasia (LSIL or HSIL) is 51% and for HSIL is 31%. In the
presence of HR HPV, the PPV of ASC-H for dysplasia (LSIL and
HSIL) is 66% and for HSIL is 33%.
CORRELATION OF HPV AND HISTOLOGY ON FOLLOW-UP BIOPSIES
HPV status Negative (10) LSIL (6) HSIL (6) Atypical (1)
HR HPV 6 5 6 1
LR HPV 1
Negative 4
CONCLUSIONS: PPV for dysplasia (LSIL or HSIL) was higher in the
presence of HR HPV. However, the PPV for HSIL with or without
HR HPV were very similar. These findings suggest that HPV
testing might not be cost-effective or necessary in cases of
ASC-H. Patients with the diagnosis of ASC-H may be referred
directly to colposcopy.
Memorial Sloan-Kettering Cancer Center, New York, New York
42
Preserving the Diagnostic Validity of ASC-H:
Qualitative Cytomorphologic Features that
Distinguish ASC-H and HSIL
Farah Gaudier, M.D.1, Carey Kirkpatrick, CT(ASCP)2,
Mustafa Mahdavy, M.D.2, Odile David, M.D.1, Krzysztof
Moroz, M.D.1
PURPOSE: To determine whether there are qualitative cytomor-
phologic features that distinguish ASC-H and HSIL to increase
confidence in making a cytologic diagnosis of HSIL so as not to
diminish the clinical impact of a cytologic diagnosis of ASC-H.
MATERIALS AND METHODS: Sixty-two cases with a diagnosis of
ASC-H made between 2002 and 2004 were retrieved from the
files and were reviewed individually by 5 expert panelists blinded
to the biopsy outcome. Cases with consensus diagnosis, defined
as agreement amongst at least 3 of 5 panelists, were further
evaluated with respect to a set of cytomorphologic criteria. The
criteria included: phase of the cycle; presence of endometrial
cells; cellularity of atypical cells; cytoplasmic density; presence
of nucleoli; nuclear chromasia; chromatin coarseness; chroma-
tin distribution; and membrane irregularity. A binomial scoring
system (1 for feature present and 0 for feature absent) was
utilized for each criterion. These cases were then grouped ac-
cording to the revised cytologic diagnosis and follow-up biopsy
result. Only cases with biopsy proven HSIL (CIN2 or higher) were
selected for statistical analysis. Characteristics of the group of
cases for which the consensus diagnosis remained ASC-H were
compared to characteristics of the group of cases upgraded to
HSIL The differences between these two groups were then ana-
lyzed using Fisher’s exact probability test with p % 0.05 consid-
ered statistically significant.
RESULTS: There was consensus amongst the panelists on 51 of
350 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
the 62 cases (85%). Of those, 22 (37%) were upgraded to HSIL, 24
(40%) remained ASC-H, 2 (3%) were considered LGSIL, and 3
(5%) were considered ASC-US. Of the 22 cases upgraded to HSIL
and 24 ASC-H cases, follow-up biopsies identified 9 cases in each
group with a histologic diagnosis of CIN 2 or a more severe
lesion. Two-tailed Fisher’s exact probability test performed to
compare each cytomorphologic criterion independently showed
that non-dense cytoplasm and uneven distribution of chromatin
were statistically significant distinguishing features (p " 0.003
and 0.009, respectively). Membrane irregularity attained border-
line significance (p " 0.049). Cellularity and coarseness of chro-
matin were not significantly different between the two groups
(p " 0.205 and 0.346, respectively).
CONCLUSION: ASC-H is a reflection of some diagnostic uncer-
tainty. To preserve its diagnostic validity it should not become
an easy substitute for HSIL. Every effort should be made to
distinguish it from HSIL even if current clinical approach is
similar. We have found that quality of cytoplasm (non-dense,
delicate, fluffy) and uneven distribution of chromatin are two
features that may help to distinguish HSIL from ASC-H. Marked
membrane irregularity may prove useful in some cases although
its statistical significance in our study was borderline.
1 1
Tulane University Health Sciences Center, New Orleans, Louisi-
ana, 2Louisiana State University Health Sciences Center, New
Orleans, Louisiana
43
SurePath™ and ThinPrep® Pap Tests
Implementation: A Comparison of Abnormal
Diagnostic Outcomes
Farah Gaudier, M.D.1, Courtney C. Jackson, M.D.1,
Virganeyce Lyons-Boudreaux, M.D.1, Carey Kirkpatrick,
CT(ASCP)2, Odile David, M.D.1, Krzysztof Moroz, M.D.1
PURPOSE: To compare the diagnostic outcomes of SurePath™
(SP) and ThinPrep® (TP) tests during the initial three months of
implementation (the learning periods).
MATERIALS AND METHODS: All consecutive cases from the first
three months of SP implementation were sorted according to
Bethesda System classification and abnormal cases were com-
pared to those from the first three months of TP, which replaced
SP two years later. To account for the fact that SP was introduced
first and represented a major transition from conventional PAP
smears, we also compared abnormal SP cases from a corre-
sponding three month period one year following implementa-
tion. The differences were compared using test of proportions
with p %0.05 considered statistically significant. To evaluate
diagnostic accuracy, all HSIL cases from the implementation
periods for both tests were correlated with biopsy results when
available.
RESULTS: There were a total of 3,866 SP, (4,588 SP a year later)
and 3,430 TP with abnormal diagnoses distributed as follows:
ASC-US 280, (351) and 324, ASC-H 9, (16) and 25, LSIL 240, (380)
and 224, HSIL 51, (77) and 97, cancer 5, (6) and 3, respectively.
HSIL and ASC-H rates were increased by 114% and 213% in TP
as compared to SP (p%0.0001 and p"0.0019, respectively) during
the initial learning periods. The ASCUS rate was also found to be
significantly higher (p"0.0007) in TP. All of these differences
remained statistically significant with SP one year later. In addi-
tion, LSIL was significantly increased in the SP one year later
group when compared to both initial SP and TP (p"0.0003 and
p"0.0033, respectively). Carcinoma was not statistically different
in any of these comparisons. CIN 2 or higher was present in 24
out of 40 (60%) HSIL SP cases and in 29 out of 47 (62%) cases of
TP, difference not statistically significant (p"0.8713).
CONCLUSION: In the learning periods, significantly more cases of
HSIL, ASC-H and ASC-US were detected in TP than SP. Those
detection rates stayed significantly higher even when compared
to SP one year later. There was no difference between TP and SP
with respect to biopsy outcomes for cases of HSIL. Our study
suggests that although there is no difference in diagnostic accu-
racy of HSIL in terms of biopsy correlation, TP detects more
cervical abnormalities.
Tulane University Health Sciences Center, New Orleans, Louisi-
ana, 2Louisiana State University Health Sciences Center, New
Orleans, Louisiana
44
Morphologic, Patient and Interpreter
Profiles of High Risk HPV Positive Vs
Negative ASC-US Cases
Debora Smith, CT(ASCP), Nancy Chandler, CT(ASCP),
Bakula Dhurandhar, CT(ASCP), Liza DiFilippo, M.H.A.,
CT(ASCP), Miriam Scheiber-Pacht, CT(ASCP), Dina Mody,
M.D.
BACKGROUND: The preferred approach for management of
women with ASC-US is reflex High Risk HPV DNA (HR-HPV)
testing. This offers an objective method to monitor and assess
individual and laboratory ASC-US reporting practices. This study
was initiated to review morphological changes which can be
used to fine tune the diagnosis.
DESIGN: 150 consecutive ThinPreps with a diagnosis of ASC-US
and a reflex HR-HPV test (Hybrid Capture II) performed between
July 2002 and March 2005 were selected. Cytology slides were
retrospectively reviewed and an information grid was completed
on each case. The grid consisted of patient information, cell
morphology, cell patterns, original cytologic diagnosis by Pa-
thologist (PT), Cytotechnologist (CT) and Quality Control (QC),
review diagnosis.
RESULTS: Seventy three (49%) were HR HPV DNA positive, 75
(51%) were negative and 2 were equivocal and excluded from the
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
study. The ages ranged from 15 to 86 with an average of 32.3
years. The average age of patients with a positive HR HPV result
was 28 years and those with a negative was 39.1 years. All
patients over age 65 tested negative. Of the 25(16%) pregnant
patients, 75% were HR HPV positive. Sixty two percent of HR
HPV positive cases had few atypical cells, 15% moderate, and
24% many cells, as compared to HR HPV negative cases with
86% few cells, 14% moderate cells. The positive group had 33%
single cells, 44% groups, and 22 % both. The negative group had
75% single cells, 8% groups and 16 % both. The HR HPV negative
cases had more Trichomonas infections than the positive group.
Nuclear enlargement and hyperchromasia were observed in
both groups. The significant cytoplasmic changes in the HR HPV
positive cases were koilocytic like changes and parakeratosis. In
the negative cases the changes were cell enlargement (both
nuclear and cytoplasmic) and the small halos. Seven PT’s signed
out these cases and all were within 10% points of the labs
average HR HPV positive rate, with the exception of one PT who
was 20% higher. Six CT’s who screened these cases were within
10% points of the Laboratory average, with the exception of one
CT who was 20% lower on the positive HR HPV rate. The review
done by the CT’s would have upgraded 32% of the cases to LSIL
and downgraded 24% to NILM. Finally, 7 cases sent for HR HPV
testing were from the negative quality control rescreen and 6 of
these typed positive.
CONCLUSIONS: There are morphologic changes which can be
applied in fine tuning the ASC-US diagnosis. These are 1) Num-
ber and type of atypical cells. 2) Cell arrangements. 3) Infectious
agents. Finally performances can be monitored by comparing
the PT and the CT percentages with the laboratory HR HPV rates.
The Methodist Hospital, Houston, Texas
45
Clinical Outcome of Women with
Unsatisfactory ThinPrep Gyn Cytology
Results: A Two-Year Retrospective Follow-
Up Study
Mujtaba Husain, M.D., Rajendra Singh, M.D., Jining Feng,
Ph.D., M.D.
BACKGROUND: Liquid based cytology has increased adequacy of
the Pap test and detection of LSIL and HSIL significantly. How-
ever, still a small fraction of specimens are unsatisfactory for
interpretation due to scant squamous cellularity, often associ-
ated with blood and sometimes due to obscuration by mucus
and inflammatory cells. A recent study showed that unsatisfac-
tory conventional Pap smears were found to come from high-
risk patients and follow up of these patients showed a signifi-
cantly high detection of SIL/cancer compared to a cohort of
patients with satisfactory index Pap smears. The purpose of this
351
study was to determine clinical outcome in women with unsat-
isfactory ThinPrep cytology.
DESIGN: 117 unsatisfactory ThinPrep cervical cytology cases
were retrieved from hospital computer system from March 1 to
August 15, 2002. These women were seen in the hospital-based
clinic in a major metropolitan city. Age ranged from 15 to 61
years and only 20% were 40 or older. The patients were followed
by repeat Pap or colposcopy/biopsy. 69% of the patients had a
follow up ranging from 24-27 months and remaining 31% from
21-24 months. 112 of 117 specimens were unsatisfactory for
interpretation due to scant squamous cellularity and presence of
varying amounts of blood. The remaining 5 specimens were
obscured by inflammation or mucus.
RESULTS: 94 of 117 (80.3%) of these unsatisfactory ThinPrep
Paps had initial follow-up cytology or biopsy or both. 23 patients
had no known follow up. Most of patients had their initial follow
up within 6 months of index Pap (75 of 117; 64%). Among
patients with follow up, 67 patients (71.3%) had negative cytol-
ogy/biopsy while 8 (8.5%) continued to have unsatisfactory Paps.
LSIL/ASCUS was diagnosed in 14 patients (14.9%). HGSIL/CIN 3
was identified in 2 patients (2.1%). More significantly, two squa-
mous cell carcinomas (2.1%) and one endometrial carcinoma
(1.1%) were diagnosed in subsequent follow up.
CONCLUSION: An unsatisfactory ThinPrep Pap appears to carry a
significant risk of CIN2/3 or invasive squamous cell carcinoma
similar to the published data of unsatisfactory conventional Pap
smears. It is speculated that since blood appears to be often
associated with scant squamous cellularity, pre-treatment of
bloody ThinPrep specimens with glacial acetic acid may possibly
reduce incidence of false negative interpretation. A longitudinal
multi-institutional study is needed to address this issue.
Wayne State University, Detroit Medical Center, Detroit, Michi-
gan
46
Comparison of the Clinical Significance of
the Papanicolaou Test Interpretations LSIL
Cannot Rule Out HSIL and ASC-H
Megan DiFurio, M.D., Thomas Mailhiot, CT(ASCP), Karen
Nauscheutz, M.D.
INTRODUCTION: Despite the two-tiered classification of dysplasia
in The Bethesda System (TBS), rare cases fall into the category
squamous intraepithelial lesion (SIL) of indeterminate grade.
These Pap tests are often interpreted as “LSIL/ASC-H” or “LSIL”
with a comment indicating the presence of cells with features
approaching HSIL. Patients with LSIL/ASC-H have a significant
risk of HSIL (29-61.5%) on follow-up cervical biopsies, similar to
the risk of HSIL in patients with ASC-H Pap tests (24-68%). The
purpose of this study was to compare the distribution of biopsy
results and the positive predictive value (PPV) for HSIL in pa-
tients with ASC-H and LSIL/ASC-H Pap tests.
352 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
MATERIALS AND METHODS: A study group of 83 women with
LSIL/ASC-H and a control group of 37 women with ASC-H on
liquid-based cytology (ThinPrep®), who had tissue follow-up,
were identified. The mean length of time between Pap test and
tissue diagnosis was 3.1 and 1.1 months in the study and control
groups, respectively. The average patient age in the LSIL/ASC-H
and ASC-H groups was 29 years and 36 years, respectively. The
Fisher’s exact test was used to compare the distribution of fol-
low-up diagnoses and to determine the PPVs for a HSIL lesion on
cervical biopsy for both groups.
RESULTS: Women with LSIL/ASC-H had a lower incidence of
HSIL (PPV"40%, 95% CI: 0.292 to 0.503) on follow-up cervical
biopsy than the control ASC-H group (PPV"62%, 95% CI: 0.465
to 0.778); this difference was significant (p"0.030). In addition,
the difference in the distribution of biopsy results between the
two groups was significant (p"0.004). As expected, a follow-up
diagnosis of LSIL was seen more often in the LSIL/ASC-H group.
However, when compared to the LSIL/ASC-H group, a diagnosis
of HSIL or worse (2 cases of squamous cell carcinoma and 1 case
of adenocarcinoma in-situ), or a benign interpretation was more
common in patients with an ASC-H Pap test.
CONCLUSIONS: The current guidelines for the management of
cervical cytologic abnormalities from the American Society for
Colposcopy and Cervical Pathology (ASCCP) advocate similar
treatment algorithms for both LSIL and ASC-H. Management of
ASC-H differs only in the required review of prior material after
negative colposcopy, with revision of the diagnosis if necessary.
In our study, women with a Pap test interpretation of LSIL/
ASC-H had a significant risk of HSIL (40%) which approached
the risk of HSIL for ASC-H (62%). We recommend (1) LSIL/
ASC-H to be added to TBS classification and (2) Pap test cases of
LSIL/ASC-H need to be clinically followed in a manner similar to
ASC-H, including review of all prior material after negative col-
poscopy.
Brooke Army Medical Center, Ft. Sam Houston, Texas
47
Comparison of Manual Screening Times for
SurePath, ThinPrep and Conventional Pap
Tests
Kevin Schofield, B.S., SCT(ASCP), Brett Minger, B.S.,
CT(ASCP), Mary Chacho, M.D.
INTRODUCTION: A study was conducted to measure manual
screening times and method variations for three Pap methods
(SurePath, ThinPrep and Conventional) in a routine Hospital
laboratory practice.
METHOD: An independent process-engineering group observed
1,892 cases screened by 13 cytotechnologists in a five-day period
(42.8% SP, 43.9% TP, 13.3% C). Data was captured from time
screening started (eyes to scope) to completion. Analysis in-
cluded primary screens of adequate samples. Years of experi-
ence ranged from 3 weeks to 23 years. Daily screening habits
were not altered during the study period. Diagnostic accuracy
was assessed using current quality assurance program.
RESULTS: Screening methods varied per tech per Pap method to
include vertical, horizontal, random or a combination of each.
Average screening times indicated SurePath (3 min, 16 secs)
ThinPrep (4 min, 38 secs) and Conventional (6 minutes). Mea-
sures of central tendency (mode and median) showed a similar
relationship. Analysis of variance indicated SurePath had the
most consistent and “tightest” variation with a standard devia-
tion 22% & 41% better than the ThinPrep and Conventional
methods respectively.
CONCLUSIONS: SurePath reliably demonstrated the lowest man-
ual screening times in routine clinical practice with a 30% im-
provement over ThinPrep and 46% over Conventional regardless
of experience or method.
Yale University, New Haven, Connecticut
48
Validation of a Low-Cost Liquid-Based
Screening Method for Cervical
Intraepithelial Neoplasia
Judy M. Lee, M.D.1, Deidra Kelly, CT(ASCP)2, Zoya
Fansler, B.A.1, Patti E. Gravitt, Ph.D.3, John Maksem,
M.D.4, Douglas P. Clark, M.D.1
INTRODUCTION: Liquid-based cervical cytology offers increased
sensitivity for cervical neoplasia over conventional cervical
smears and provides the opportunity for ancillary molecular
testing, including HPV testing. Unfortunately, the cost of most
commercially-available liquid-based cytology systems is prohib-
itively expensive for resource-limited settings, such as develop-
ing countries. We hypothesize that a novel, low-cost liquid-
based method termed the Manual Membrane Method of Liquid-
Based Cytology (3MLBC) is comparable to SurePath liquid-based
cytology in both low-risk and high-risk populations.
MATERIALS AND METHODS: We have designed a retrospective,
split-sample comparison of 300 liquid-based cervical cytology
samples. The specimen cohort was chosen from a group of 150
HIV(-) and 150 HIV($) women whose SurePath specimens were
processed, screened and reviewed as part of routine clinical care.
The residual sample from each case, that would have been
discarded, was used to prepare a 3MLBC slide. Briefly, the sam-
ples were centrifuged and re-suspended in an agar-gel polymer
that was manually applied directly to a glass slide, which was
stained with the Papanicolaou stain. The patient identifiers were
removed and slides were screened by a cytotechnologist and
abnormal cases (reactive$) were reviewed by a pathologist. Fi-
nal diagnoses from the 3MLBC slides were compared with the
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting 353

original SurePath diagnoses and any available cervical biopsy fields. In 16 of 17 QC cases, abnormal cells were present in the 22
follow-up data. FOV, but were not interpreted as abnormal by the initial screen-
RESULTS: There was good overall agreement between the ing cytotechnologist. Data for the first quarter of 2005 correlates
3MLBC and SurePath cytology diagnosis (76.3% agreement; with the 2004 results.
kappa " 0.52, 95% CI 0.44 – 0.59). There was a tendency for
3MLBC to result in a more severe diagnosis than SurePath (exact First
test for symmetry, p"0.0018). When stratified by HIV status, this Quarter
2004 2004 2005
tendency was found in the HIV($) group but not in the HIV(-)
group (exact test for symmetry, p"0.0026 vs. p"0.37). Agree- Imaged Non-imaged Imaged Non-imaged
ment was somewhat lower in the HIV($) group (65.3%; kappa "
Slides 84433 22270 25479 5619
0.46. 95% CI 0.36 – 0.56). To determine the impact of 3MLBC on QC’s 21546 5937 5408 1378
clinical management, we compared the theoretical referral rates !LSIL on QC 17 (0.02%) 9 (0.04%) 6 (0.02%) 0
to colposcopy for each method. The 3MLBC method referral rate In 22 FOV 16 N/A 6 N/A
was slightly higher than SurePath overall (33.0% vs. 28.7% Not in 22 FOV 1 N/A 0 N/A
p"0.05). Using available biopsy data to determine the accuracy Conclusions: The Imager is very effective at locating abnormal
of each method to identify HSIL, the 3MLBC was found to have cells. In addition, the false negative rate has dropped by half and
a higher sensitivity (71.4% vs. 57.1%) and lower specificity (82.1% could be reduced further. The importance of evaluating the
vs. 89.7%) than SurePath. entire FOV along with ongoing education and feedback to the
cytotechnologist is being incorporated into the quality improve-
CONCLUSIONS: This pilot split-sample study suggests that the ment program.
low-cost 3MLBC cervical cytology preparation method is com-
Metropolitan Pathologists, Denver, Colorado
parable to the SurePath method. Consequently, it may be of
value for clinical trials or cervical cancer screening programs in
resource-limited settings.
50
1
The Johns Hopkins University School of Medicine, Baltimore,
Maryland, 2The Johns Hopkins Hospital, Baltimore, Maryland,
3
The Johns Hopkins University Bloomberg School of Public
HPV Digene Hybrid Capture for ASCUS
Health, Baltimore, Maryland, 4Bostwick Laboratories, Orlando, Triage in Women Over 50 in a High Risk
Florida Population
Ghazwan Sharabi, M.D., Philip Valente, M.D.
49 INTRODUCTION: Human papillomavirus (HPV) Digene Hybrid
Capture testing is now an accepted approach in the triage of
Comparison of Quality Control patients with atypical squamous cells of undetermined signifi-
Discrepancies in LSIL or Above: Imaged cance (ASCUS) for colposcopy, especially in women over 30
Versus Non-Imaged Slides years of age with a lower prevalence of HPV positivity. This study
evaluates the utility of HPV-ASCUS triage in women over 50
Fern S. Miller, M.S.M., CT(ASCP), Mary B. Kenny-
years of age in a high risk population.
Moynihan, M.D.
MATERIALS AND METHODS: Biopsy follow-up information from
INTRODUCTION: In 2004, our laboratory screened 106,703 Thin- 128 HPV positive and 93 HPV negative ASCUS patients at Uni-
Prep® Pap tests: 84,433 slides were imaged on Cytyc’s ThinPrep versity Hospital was collected from January 2003 through Janu-
Imaging System (Imager) (Cytyc Corporation, Marlborough, ary 2005. The biopsy results (HPV-CIN 1, CIN 2$) were tabulated
MA), 22,270 slides were not imaged. A retrospective comparison and stratified by age (less than 50 versus 50 or older).
was made between quality control (QC) data for imaged and RESULTS: Over a two year period 540 ASCUS cases had a con-
non-imaged cases. comitant Digene test of which 233 (43.1%) were positive. Among
MATERIALS AND METHODS: Non-imaged cases were screened women under 50 years of age, 244 of 432 (56.5%) were HPV
manually in the usual fashion. For imaged cases, 22 fields of view positive versus 35 of 108 women 50 or older (32.4%). Of the 221
(FOV) were reviewed. If no abnormality was identified in the 22 with biopsy follow-up, 128 were HPV positive and 93 were HPV
FOV, the slide was filed without further review. If abnormal cells negative. Only 14 of 93 (15.1%) HPV negative ASCUS cases had
were identified in the 22 FOV, the entire slide was screened. All positive biopsies (HPV or CIN 1 only); 63 of 128 HPV positive
negative cases, imaged and non-imaged, were candidates for cases had positive biopsies (49.2%). Of these, 24 were CIN 2$
CLIA mandated 10% QC rescreen, and those selected for QC (18.8%). For women under 50, the overall positive biopsy rate
review received a complete manual re-screen. was 52.4% (55/105) and 19.0% (20/105) for CIN 2$. For women
RESULTS: or discrepant imaged cases, a supervisor reviewed the 50 or older positive biopsies were at 34.8% (8/23) and at 17.4%
22 FOV to determine if abnormal cells were identified in those (4/23) for CIN 2$.
354 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
CONCLUSION: The incidence of HPV positivity in women 50 or
older is greater in a specifically high risk population. Although
the rate of subsequent positive biopsies is lower compared to
younger patients (34.8% versus 52.4%), the rate of biopsy con-
firmed CIN 2$ is similar (17.4% versus 19.0%). Among the AS-
CUS negative group (93 cases) 14 (15.1%) had HPV or CIN 1 on
biopsy, but no CIN 2$ was detected. While Digene in ASCUS
triage does not have as high a yield of biopsy proven HPV-CIN
overall in a high risk population, it detected CIN 2$ lesions at a
similar rate in older and younger women.
University of Texas Health Science Center at San Antonio, San
Antonio, Texas
51
High-Grade Dysplasia in Patients with
ASCUS-H PAP Smears and Correlation with
High Risk Human Papilloma Virus (HPV)
Shahnila Latif, M.D., Jan Mathews-Greer, Ph.D., Rosalina
Reyes, CT(ASCP), Elba Turbat-Herrera, M.D.
INTRODUCTION: The diagnostic category for atypical squamous
cells, cannot exclude high-grade dysplasia (ASCUS-H) is a recent
addition and was first introduced in Bethesda 2001 to differen-
tiate between immature squamous metaplasia and high-grade
squamous intra-epithelial lesions (HSIL). At present, in the USA
many institutions are performing HPV reflex testing for ASCUS;
the most common test done is for HR HPV. It is known that
young women, particularly adolescents, are more likely to get
dysplasia and rapid progression of their dysplasia as a result of
HPV infection. In our adolescent patient population the preva-
lence of cervical abnormalities, namely (ASC-US and above) is
high 24%. HPV testing is performed in our institution using the
Hybrid Capture II (HCII) assay. This study was undertaken to
establish the prevalence of HSIL in our ASCUS-H patients and
determine their high-risk HPV status.
METHODS: Thirty-six patients with ages ranging 19-79 years un-
derwent cervical biopsy for ACUS-H at our institution. Liquid-
based pap smears were prepared in the standard fashion. The
remaining sample was submitted for HCII assay for detection of
HR-HPV using a cut-off of 1.2 to prevent true positives from
being missed.
RESULTS: ut of thirty-six cervical biopsy specimens, 14 (39%)
were positive for moderate dysplasia to carcinoma in situ (HSIL),
16 (44%) for mild dysplasia with or without HPV low grade (LSIL)
and six were negative for dysplasia. All HSIL were positive for
HR-HPV. In the low-grade (LSIL) group seven cases were posi-
tive for HR- HPV.
CONCLUSION: 1:ASCUS-H is possibly being overused instead of
HSIL. 2: We believe as more cases of ASCUS-H are studied we
will understand the implications of this entity better.
Department of Pathology, Louisiana State University Health Sci-
ences Center, Shreveport, Louisiana
52
Reliable Detection of Single Copy Integrated
Human Papillomavirus in Formalin-Fixed
Paraffin-Embedded Tissue Using a Fully
Automated Assay
Peter Krein, Ph.D., Edmundo DelValle, B.S., Alma Neilsen,
M.S., Margaret Hardy, M.S., Tobin Jones, M.D., Patrick
Roche, Ph.D., Thomas Grogan, M.D., Lidija Pestic-
Dragovich, Ph.D.
INTRODUCTION: Data from a number of case-controlled studies
suggest several HPV types can be regarded as high risk for
development of cervical cancer. PCR based assays for HPV de-
tection are thought to be superior in detecting low amounts of
viral DNA in a biopsy specimen, but fail to show the nature of the
infection histologically. In situ hybridization on the other hand,
has the advantage of illuminating the infection in the context of
the tissue, but is thought to perform relatively poorly in samples
with low viral load. We have developed a fully automated assay
which can reliably detect one to two copies of integrated HPV
per cell as shown in xenograft tumors, as well as low copy
number integrated virus in human cervical lesions.
MATERIALS AND METHODS: A cocktail of DNP-labeled HPV
genomic DNA is used as probe for in situ hybridization of for-
malin fixed paraffin embedded biopsy specimens. Hybridized
probe is visualized colorimetrically. All staining procedures from
deparaffinization through cell conditioning, hybridization, and
counterstain were performed on Ventana Medical Systems, Inc.,
BenchMark® Series instruments. All tissue specimens were HPV
genotyped using common GP5$/GP6$ or PGMY09/11 PCR
primer sets. Viral integration status was determined for HPV-16
and -18 positive samples by PCR amplification of HPV E2 region,
most commonly disrupted upon viral integration.
CONCLUSIONS: In situ hybridization detection of HPV is the only
method for direct visualization of virus in the histological con-
text. We are able to discern both nuclear episomal and punctu-
ate integrated staining patterns in cervical lesions. Through the
use of integration specific PCR we were able to confirm the
punctuate staining pattern observed in the cell nuclei correlates
with virus integration. Our fully automated HPV in situ hybrid-
ization assay can detect HPV virus reliably and reproducibly in
formalin-fixed, paraffin-embedded tissue sections. Importantly,
the assay is extremely sensitive, being able to detect 1-2 copies of
integrated HPV virus in both SiHa Xenograft tumors, as well low
copy number integrated virus in cervical biopsy specimens.
Ventana Medical Systems, Inc., Tucson, Arizon
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
53
Effect of Six Week Storage of ThinPrep!
Pap Test Specimens on Specimen Adequacy
and Diagnosis
Wendy E. Swinehart, B.S.1, Daniel Courcy, B.S.1, James
Linder, M.D.1, 2, J. Lawrence Burg, Ph.D.1
INTRODUCTION: Current labeling for the ThinPrep Pap Test
(TPPT) indicates that ThinPrep" slides for cytologic evaluation
must be prepared within three weeks from the specimen collec-
tion date. This study compares specimen adequacy and cyto-
logic diagnosis from TPPT specimens stored for up to three
weeks with specimens stored for six weeks.
METHODS: Four hundred sixty-four residual TPPT specimen vials
were tested. A control slide was prepared on the ThinPrep 2000
instrument from each vial within three weeks from the sample
collection date. A portion of each remaining sample was stored
at ambient temperature for 42 days from the collection date, at
which time a test slide was prepared. Slides were randomized,
masked, and evaluated by cytotechnologists.
RESULTS: Control and test slides showed 99.6% (462/464) overall
agreement in specimen adequacy. Data showed overall diagnos-
tic agreement of 71.6% for three and six week slides. Data ana-
lyzed by detection of ASCUS$, LSIL$, and HSIL$, showed per-
cent agreement between three and six week slides of 87.9%,
87.6%, and 88.9%, respectively. The abnormal detection rates,
differences in abnormal detection rates (Control – Test), and
95% confidence intervals are: ASCUS$: Control " 57.5, Test "
55.8, difference " 1.7 (-1.7, 5.1), LSIL$: Control " 47.5, Test "
46.0, difference " 1.5 (-1.9, 4.9), and HSIL$: Control " 13.7,
Test " 11.7, difference " 2.0 (-1.3, 5.2). This indicated no statis-
tically significant difference in diagnoses after storage of speci-
mens for six weeks.
CONCLUSIONS: These data suggest that extended storage of TPPT
specimens for up to six weeks at ambient temperature does not
adversely affect specimen adequacy or cytologic diagnosis.
1
Cytyc Corporation, Marlborough, Massachusetts, 2University of
Nebraska Medical Center, Omaha, Nebraska
54
Reprocessing of ThinPrep! Pap Test
Specimens Does Not Adversely Affect
Specimen Adequacy or Diagnosis
Wendy E. Swinehart, B.S.1, Daniel Courcy, B.S.1, James
Linder, M.D.1, 2, J. Lawrence Burg, Ph.D.1
INTRODUCTION: Published studies have shown that reprocessing
Unsatisfactory ThinPrep Pap Test (TPPT) specimens using Cy-
toLyt" Solution/10% Glacial Acetic Acid (GAA) substantially im-
355
proves adequacy and allows detection of cellular abnormalities.
In addition to specimen adequacy, this study investigated
whether this reprocessing procedure adversely affected cytologic
diagnosis.
METHODS: Four hundred sixty-four residual TPPT specimens
were tested. All specimens were Satisfactory for cytological eval-
uation to allow comparisons of diagnosis before and after repro-
cessing. A control slide was prepared from each vial on the
ThinPrep 2000 instrument within three weeks from the sample
collection date. A portion of each remaining specimen was sub-
jected to a CytoLyt/10% GAA reprocessing procedure and a test
slide was prepared. All slides were randomized, masked, and
reviewed by cytotechnologists.
RESULTS: Overall agreement in specimen adequacy between
control and test slides was 99.8% (463/464) – only one repro-
cessed slide was Unsatisfactory due to scant cellularity. Overall
diagnostic agreement was 70.4%. Data analyzed by detection of
ASCUS$, LSIL$, and HSIL$ abnormalities showed diagnostic
agreement between of 85.5%, 89.6%, and 88.8%, respectively.
The abnormal detection rates, differences in abnormal detection
rates (control – test), and 95% confidence intervals are: ASCUS$:
control " 57.7, test " 57.9, difference " -0.2 (-3.9, 3.5), LSIL$:
control " 47.5, test " 48.0, difference " -0.5 (-3.6, 2.7), and
HSIL$: control " 13.6, test " 15.8, difference " -2.2 (-5.4, 1.1).
This indicated no statistically significant difference between
control and reprocessed test slides.
CONCLUSIONS: These data suggest that CytoLyt/10% GAA repro-
cessing does not adversely affect specimen adequacy or cyto-
logic diagnosis of TPPT samples.
1
Cytyc Corporation, Marlborough, Massachusetts, 2University of
Nebraska Medical Center, Omaha, Nebraska
55
Significance of Reporting “LSIL-H” on
Cervical Cytologic Specimens
Reena Jain, M.D., Mary Ann Pedigo, B.S., CT(ASCP),
Celeste N. Powers, M.D., Ph.D., William J. Frable, M.D.
INTRODUCTION/PURPOSE: The Bethesda 2001 reporting system
(TBS2001) recommends cervical cytologic diagnosis to be based
on the most abnormal cell present, regardless of number. The
objective of this study was to assess the significance of the
number of HSIL cells seen in a background of LSIL on cervical
cytology and to determine the positive predictive value (PPV) for
HSIL in follow-up cervical biopsies for these cases in our high-
risk population.
MATERIALS AND METHODS: All cervical cytology specimens di-
agnosed as HSIL and LSIL with a rare or few cells suggestive of
HSIL during the 5-year period from 1/1/2000 to 12/31/2004 were
retrieved by computer search from the cytology database of our
institution. Sixty-seven women with a cervical cytologic diagno-
356 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
sis of LSIL with a rare (1 to 2) or few (3 and above) cells
suggestive of HSIL and their follow-up cervical biopsies were
reviewed. Nineteen cases had no follow-up biopsy and were
excluded. Results were compared with a control group of sixteen
women with a cytologic diagnosis of HSIL. The predictive value
for biopsy diagnosis of HSIL for these three groups was calcu-
lated.
RESULTS: On biopsy follow-up of LSIL-H rare cell, 8/26 (31%)
cases were CIN 1 and 6/26 cases (23%) were CIN 2/3. In contrast,
6/22 cases (27%) of LSIL-H few cells were CIN 1 and 14/22 cases
(64%) were CIN 2/3 on follow-up cervical biopsy. Interestingly,
only 9/16 cases (56%) of HSIL showed CIN 2/3 on biopsy. The
remaining seven cases showed either squamous metaplasia (4)
or CIN 1 (3). Women with LSIL-H few cells had a higher inci-
dence of high grade dysplasia (PPV"64%) on follow-up cervical
biopsy than LSIL-H rare cell (PPV"23%).
CONCLUSIONS: Women with a cervical cytologic diagnosis of
LSIL-H, rare cell have a greater likelihood of a benign lesion or
CIN 1 on follow-up biopsy, similar to those with LSIL only.
However, women with LSIL-H, few cells are more likely to harbor
CIN 2/3 than those with LSIL-H, rare cell (PPV " 64% versus
23%, p % 0.01). Therefore, women with a diagnosis of LSIL-H,
few cells warrant close clinical follow-up to exclude HSIL.
Virginia Commonwealth University, Richmond, Virginia
56
Aberrant S-Phase Induction as a Molecular
Parameter for the Detection of CIN2!
Disease in Cervical Histology and Cytology
Specimens
Douglas P. Malinowski, Ph.D., Patricia L. Avissar, M.S.,
Hewei Li, M.D., Ph.D., Laura B. Hall, B.A., HTL(ASCP),
Margaret R. Parker, B.A., SCT(ASCP), Adriann J. Taylor,
B.A., Timothy J. Fisher, B.S.
OBJECTIVE: To compare Minichromosome Maintenance Pro-
tein-7 (MCM7) and Cyclin E1 (CCNE1) expression in cervical
carcinoma with other known S-phase genes as potential markers
for CIN2$ cervical disease.
METHODS: Cervical carcinoma, matching adjacent normal cervi-
cal epithelium, archival biopsy specimens and SurePathTM cer-
vical cytology samples with matching biopsies were used in this
study (N!180). Messenger RNA was quantified by TaqMan anal-
ysis. Protein expression was analyzed in biopsy and cytology
specimens using immunochemistry assays. A commercially
available antibody reagent for the detection of MCM2 and To-
poisomerase II alpha (TOP2A) was evaluated (ProExC, TriPath
Imaging, Burlington, NC).
RESULTS: The expression levels of MCM7 and CCNE1 mRNA
were elevated 3 and 4.5 fold respectively relative to normal
cervical epithelium. The mRNA for MCM7 gene displayed over-
expression in cervical squamous cell carcinoma and cervical
adenocarcinoma. Over-expression of the MCM7 protein was also
detected in 100% of the cervical carcinomas. MCM7 over-expres-
sion was specific for CIN2$ lesions with minimally detectable
MCM7 protein expression observed in CIN1 lesions or in the
parabasal layer of normal cervical epithelium. In contrast,
CCNE1 showed elevated expression in CIN1 – CIN3 dysplasia
and cervical carcinoma. As individual markers, each detected
CIN2$ disease within the ASCUS and LSIL population with the
following characteristics: sensitivity " 70%; specificity " 87%;
PPV " 70% and NPV " 93%. Over-expression of MCM2 and
TOP2A was also detected in abnormal cytology specimens using
the ProExC reagent. In this study population, the vast majority of
cytology specimens with atypical cells (ASCUS$) that over-ex-
pressed MCM2 and TOP2A correlated with biopsy-confirmed
CIN2$ lesions.
CONCLUSIONS: The over-expression of the genes for MCM7,
CCNE1 was similar to that observed previously for the S-phase
genes MCM2, MCM6 and TOP2A. Collectively, this over-expres-
sion is indicative of aberrant S-phase induction and appears to
be a characteristic of moderate-severe dysplasia and cervical
carcinoma. The over-expression of these S-phase genes repre-
sents an oncogenic alteration of the cell-cycle in CIN2$ cervical
disease and appears suitable for diagnostic applications in both
histology and cervical cytology specimens.
TriPath Oncology, Durham, North Carolina
57
Topoisomerase II alpha as a Specific
Marker for the Detection of CIN2! Cervical
Disease in Histology and Cytology
Specimens
Hewei Li, M.D., Ph.D., Patricia L. Avissar, M.S., Laura B.
Hall, B.A., HTL(ASCP), Margaret R. Parker, B.A.,
SCT(ASCP), Adriann J. Taylor, B.A., Timothy J. Fisher,
B.S., Douglas P. Malinowski, Ph.D.
OBJECTIVE: To evaluate the expression of Topoisomerase II al-
pha (TOP2A) in cervical neoplasia as a molecular marker for the
clinical diagnosis of CIN2$ cervical disease.
METHODS: Cervical carcinoma and matched adjacent normal
cervical epithelium tissues were obtained as fresh frozen speci-
mens (N"34) with corresponding paraffin biopsies and as archi-
val biopsy specimens (N"161). Cervical cytology samples were
collected in SurePathTM liquid-based media. Abnormal cytology
results were confirmed through cervical biopsies (N"181). Sam-
ples were analyzed for the presence of high-risk HPV by PCR.
Messenger RNA was quantified by TaqMan analysis. TOP2A
protein expression was determined in biopsy tissue by an im-
munohistochemistry (IHC) assay and in cytology specimens by
an immunocytochemistry (ICC) assay.
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
RESULTS: All fresh frozen cervical carcinoma specimens were
positive for high-risk HPV. TOP2A mRNA was over-expressed
3.5-fold in cervical carcinoma relative to the matched normal
cervical epithelium. TOP2A mRNA was equally over-expressed in
cervical squamous cell carcinoma and cervical adenocarcinoma.
Over-expression of the TOP2A protein was also detected in 100%
of the cervical carcinomas using IHC. The over-expression of
TOP2A protein was specific for CIN2$ lesions based upon IHC
analysis of archival biopsy specimens. Minimally detectable
TOP2A protein expression was observed in CIN1 lesions or in the
parabasal layer of normal cervical epithelium. TOP2A protein
over-expression was detected in atypical cells in cytology spec-
imens using ICC. In this study, the vast majority of cytology
specimens with atypical cells (ASCUS$) that over-expressed
TOP2A correlated with biopsy-confirmed CIN2$ lesions.
CONCLUSIONS: TOP2A over-expression appears to be a charac-
teristic of high-grade cervical dysplasia and cervical carcinoma
that arise from oncogenic HPV transformation. This over-ex-
pression is a consistent with E2F driven gene transcription re-
sulting in aberrant S-phase entry of cervical keratinocytes.
Within the population of this study, the detection of TOP2A
over-expression in ASCUS$ appears to detect cases with a high
likelihood of CIN2$ disease as determined by biopsy confirma-
tion. Thus TOP2A appears to be a promising marker for the
detection of CIN2$ cases in liquid-based cytology specimens.
TriPath Oncology, Durham, North Carolina
58
Proteomic Analysis of High-Grade
Dysplastic Cells Obtained from ThinPrep®
Cervical Slides Using Laser Capture
Microdissection
Jane Meyer, CT(ASCP)1, Ana Perez, Ph.D.2, Catherine
Diggins2, Ye Gu3, Shiaw-Lin Wu, Ph.D.3, William Hancock,
Ph.D.3, Barry Karger, Ph.D.3, David Hanlon, Ph.D.1, James
Linder, M.D.1, 4
INTRODUCTION: Laser Capture Microdissection (LCM) is a pow-
erful tool that enables the isolation of specific cell types from a
heterogeneous population. While this technology has routinely
been utilized with tissue, few studies have applied this method-
ology to investigate cytological specimens in conjunction with
protein analysis. In this study, LCM was utilized to select high-
grade dysplastic cells from ThinPrep® Pap Test ™ (Cytyc Corpo-
ration) prepared slides. Following cell capture, samples were
processed and analyzed using a highly sensitive linear ion trap
Fourier transform mass spectrometer (LTQ-FTMS). The differ-
ential expression of proteins in high-grade dysplastic cells versus
morphologically normal cervical cells may lead to the potential
identification of novel biomarkers useful in the detection of
cervical pre-neoplasia.
357
MATERIALS AND METHODS: Residual ThinPrep cervical speci-
mens having a diagnosis of HSIL (high-grade intraepithelial le-
sion) were evaluated for overall cellularity as well as the percent-
age of high-grade cells. Multiple slides were prepared from
selected cases using the ThinPrep 2000 Processor and Pap
stained. Cells selected for LCM were marked using the ThinPrep
Imaging System Review Scope. Approximately 10,000 high-grade
cells per specimen were captured via LCM using the Autopix
System® from Arcturus (Mountain View, CA). Cells were then
lysed and proteins separated via SDS-PAGE in preparation for
in-gel digestion. The resulting peptides from selected regions of
the gel were analyzed by on-line liquid chromatography with a
LTQ-FTMS. Quantitative protein differences between WNL
(within normal limits) and HSIL samples were identified by
comparing the intensities of the representative peptide ions or
using a sensitive O16/O18 differential labeling methodology.
RESULTS: Slide preparation protocols were optimized to identify
high-grade dysplastic cells from residual ThinPrep cervical sam-
ples. Selected cells were subsequently removed via LCM prior to
proteomic analysis. It was determined that PreservCyt® fixative
and ThinPrep Stain were both compatible with the subsequent
sample processing and LC-MS analysis. Several individual clin-
ical specimens were evaluated using a sensitive new LC-MS
platform, and in each case hundreds of proteins were identified
with high confidence from the capture of approximately 10,000
high-grade dysplastic cells per specimen.
CONCLUSIONS: Diagnostic cells of interest from ThinPrep cervi-
cal cytology specimens were identified, selected via LCM, and
processed for proteomic analysis using mass spectroscopy. Due
to the sensitivity of the linear ion trap MS, very few cells were
required for analysis. Protein profiles unique to high-grade dys-
plastic cells may yield promising biomarkers for molecular di-
agnostic applications.
1
Cytyc Corporation, Marlborough, Massachusetts, 2Center for
Comparative Functional Genomics, University at Albany, Albany,
New York, 3The Barnett Institute of Chemical and Biological
Analysis, Northeastern University, Boston, Massachusetts, 4Uni-
versity of Nebraska Medical Center, Omaha, Nebraska
59
Immunocytochemical Detection of MCM2
and TOP2A as Diagnostic Indicators of
High-Grade Cervical Dysplasia
Kenneth Shroyer, M.D., Ph.D., David Heinz, B.S.,
Meenakshi Singh, M.D.
INTRODUCTION: The Papanicolaou test is the most effective
screening tool for cancer that has ever been devised. Despite its
dramatic successes, however, diagnostic cytopathology has lim-
ited specificity for underlying clinically significant lesions in
cases with low-grade cytologic abnormalities. Colposcopic eval-
uation does not identify underlying clinically significant squa-
358 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
mous or glandular lesions in most cases of ASC-US or LSIL.
Although HPV testing can help identify cases of ASC-US that
have low risk for CIN 2/3, it has not proven useful for the triage
of patients with LSIL. The purpose of this study was to evaluate
the performance of a novel home-brew immunocytochemical
test (ProExTM C, TriPath Imaging, Inc.) for the detection of
aberrant S Phase induction in normal versus HSIL cervical cy-
tology specimens.
METHODS: Immunocytochemical detection of human minichro-
mosome maintenance protein 2 (MCM2) and topoisomerase IIa
(TOP2A) was performed on slides that were prepared from liq-
uid-based cytology specimens (SurePathTM, TriPath Imaging,
Inc.), using a home brew test (ProExTM C). Staining reproduc-
ibility was evaluated in a panel of 3 specimens (NIL pool, HSIL
pool, and SiHa control) by testing 2 lots on 5 consecutive days,
with 2 runs completed each day. Scoring reproducibility be-
tween 2 pathologists was compared for the classification of test
results (positive versus negative ProEx C result) in 10 NIL and 10
HSIL cases. Open vial stability of the reagents was evaluated by
comparing 2 lots of reagents that were sampled over a course of
35 days and the accuracy of the ProEx C test was tested in slides
that were prepared from NIL, SiHa-spiked NIL, and in HSIL
cases.
RESULTS: Intense nuclear staining was detected in cytologically
abnormal cells but was generally negative in normal squamous
and endocervical cells. Weak staining was also occasionally ob-
served in benign-appearing glandular cells but did not result in
misclassification of NIL versus HSIL test results. Test slides were
scored positive in 1/40 (2.5%) NIL, in 40/40 (100%) SiHa-spiked
NIL, and in 40/40 (100%) HSIL cases and there was 100% con-
cordance in test classification between 2 pathologists. No differ-
ences were observed in test performance in the open vial study
and greater than 30% of SiHa stained in 10/10 (100%) of slides.
CONCLUSIONS: The performance of this home brew test prepared
with the ProEx C reagents was consistent and showed virtually
no variability with regard to scoring and staining reproducibility
in NIL versus HSIL cytology. Further studies are indicated to
determine the potential role of the ProEx C reagent as a diag-
nostic adjunct for the triage of ASC-US/LSIL and for the detec-
tion of CIN2/3 in general screening populations.
University of Colorado at Denver and Health Sciences Center,
Aurora, Colorado
60
The Clinical Implication of Diagnosis
“Atypical Squamous Cells Cannot Exclude a
High Grade Squamous Intraepithelial Lesion
(ASC-H)” in Pregnant Women
Kazuya Onuma, Reda Saad, M.D., Anisa Kanbour, M.D.,
Amal Shakir-Kanbour, M.D., Ph.D., David Dabbs, M.D.
BACKGROUND: ASC-H was developed in the new 2001 Bethesda
System and implemented after the studies proved its clinical
outcome of high incidence of high-grade dysplasia (HSIL). How-
ever, ASC-H in pregnant women is not studied. The objective of
this study is to investigate the clinical implication and pathologic
significance of ASC-H diagnosis in pregnant women.
METHODS: Retrospective search identified 800 cases with cyto-
logic diagnosis of ASC-H at Third Tertiary Women Hospital,
during the period from January 2,2003 to June 30, 2004. Only 60
(7.5%) cases were taken during pregnancy. Cases were reviewed
and analyzed for the following cytomorphologic criteria: Smear
background, squamous maturation, atypical squamous meta-
plastic cells, cellular arrangement, nuclear size, hyperchromasia
and chromatin pattern.
RESULTS: Follow up cervical biopsies were available on 26/60
(43%) cases, 18/60 (30%) cases had only pap smears as follow-
up, 16/60 (27%) without follow-up, however, 14 of them had
HPV test. Of 26 cases with follow up biopsy, 10/26 (38%) cases
had low-grade dysplasia (LSIL), 2/16 (8%) cases showed HSIL. In
cytology follow-up patients (18 cases), only one case showed
LSIL and another case still diagnosed as ASC-H. HPV was also
performed in 39/60 (65%) cases and was positive in 23/39 (59%)
cases (5 cases with LSIL and 2 cases with HSIL) and negative in
16/39 (41%) cases (3 cases of LSIL). In total only 2/60 (3%) cases
showed HSIL, and 11/60 (18%) cases LSIL
CONCLUSION: In our study, ASC-H in pregnant women is less
likely to be associated with HSIL dysplasia than that diagnosis in
younger non-pregnant patients. Since colposcopic biopsies and
conization during pregnancy might carry a complication for
both mother and fetus, we encourage the clinician to postpone
invasive procedure till after the delivery.
Magee-Womens Hospital at University of Pittsburgh Medical Cen-
ter, Pittsburgh, Pennsylvania
61
Cytologic Features of Polyps on THINPREP
Pap Smears: An Analysis of 162 Cases
Sonya Teixeira, B.S., CT(ASCP)1, Melissa Dwyer, M.S.,
CT(ASCP)1, Latha Pisharodi, M.D.1, 2, 3
OBJECTIVE: Endocervical and endometrial polys are commonly
encountered in gynecologic pathology. The objective of this
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
study was to determine cytologic features of polyps in pap
smears prepared by the ThinPrep methodology.
METHODS: We identified 162 cases of polyps diagnosed in our
hospital during a 2-month period. Of these 86 were endometrial
polyps, 74 were endocervical polyps and 2 patients had endo-
metrial and endocervical polyps. The Pap smear immediately
prior to the diagnosis of the polyp was evaluated for cytologic
features. All pap smears were collected within five months of the
diagnosis of the polyp.
RESULTS: Four of 162 pap smears were unsatisfactory due to
inadequate cellularity. One hundred and thirty six pap smears
were diagnosed as negative for intraepithelial lesion or malig-
nancy (NIL). Nine cases were diagnosed as atypical squamous
cells of undetermined significance (ASC-US), four cases were
called atypical glandular cells, six were diagnosed as low grade
squamous intraepithelial lesion (LSIL) and three were diagnosed
as high grade intraepithelial lesion (HSIL). Abnormal cases were
followed. All cases of ASCUS had negative follow-up pap smears
or biopsies. Follow-up of atypical glandular cells yielded one
endometrial adenocarcinoma with an endocervical polyp, one
LSIL, one negative and one case without follow-up. Follow-up of
LSIL cases showed two LSILs, two negatives, and two without
follow-up. Two cases of HSIL had no follow-up, and one was
ASC-US at the subsequent Pap smear.
CONCLUSION: There are no specific cytologic features associated
with endometrial or endocervical polyps in pap smears prepared
by the ThinPrep methodology. Abnormal smears were seen in
less than 20% of all cases, the majority of which were squamous
intraepithelial lesions.
1
Women & Infants’ Hospital of Rhode Island, Providence, Rhode
Island, 2Rhode Island Hospital, Providence, Rhode Island, 3Brown
University, Providence, Rhode Island
62
Application of p16INK4a
Immunohistochemical Stain to Differentiate
Endocervical Glandular Dysplasia/Neoplasia
from Reactive Endocervical Glandular Cells
A.A.M. A. Baqui, M.D., Ph.D., Tasnuva Baqui, H.S., Lamar
Jones, HT, Elba Turbat-Herrera, M.D.
INTRODUCTION: Incidence of cervical glandular dysplasia/neo-
plasia is on the rise during recent years. Differentiation of atyp-
ical glandular cells as reactive or dysplastic may pose significant
difficulty among pathologists. Increased expression of p16INK4a
protein (p16) has been reported in dysplastic/neoplastic squa-
mous cervical lesions and also suggested but not adequately
studied for cervical glandular lesions. The purpose of this study
was to investigate the potential use of p16 immunohistochemi-
cal (IHC) stain to differentiate endocervical adenocarcinoma in
situ (AIS) and endocervical adenocarcinoma (ECA) in both sur-
359
gical and some corresponding cytological specimens, from the
reactive endocervical glandular lesions including microglandu-
lar hyperplasia (MH) and atypical glandular cells (AGC).
MATERIALS AND METHODS: Immunohistochemical staining for
p16 was performed on paraffin-embedded endocervical speci-
mens. Fifteen cases of ECA, 6 AIS, and 23 MH cases were selected
for this study. Also corresponding 7 cases of cervical Papanico-
laou (PAP) smears (2 ECA, 2 AIS and 3 MH) were also stained
with p16. The p16 immunoreactivity of AIS and ECA was com-
pared to MH. Positive for p16 is considered when !10% cells are
positive.
RESULTS: Out of 15 ECA surgical cases, 14 (93.3%) were positive
for p16. All 6 out of 6 (100%) of our AIS surgical biopsy cases were
positive for p16. All 23 (100.0%) MH cases of were negative for
p16. Four of 4 (100%) corresponding (2 ECA, 2 AIS) PAP smears,
were positive for p16 (100% sensitivity). Corresponding 3 AGC
favor reactive PAP smears from 3 MH cases were negative (100%
specificity). When positive, p16INK4a stained all cases intensely
(3$ to 4 $) and diffusely (!80% cells positive).
CONCLUSIONS: IHC for p16 showed high sensitivity and specific-
ity in differentiating endocervical dysplasia/ adenocarcinoma
(AIS, ECA) from reactive endocervical cells. This study suggests
the potential use of p16 to differentiate ambiguous cases of AGC
in PAP smears, in addition to differentiate dysplastic/neoplastic
surgical pathology specimens.
Louisiana State University Health Science Center, Shreveport,
Louisiana
63
Lack of Transformation Zone Component:
Causative Factors and Clinical Significance
Antonio Macias, M.D., John Gao, Chiara Sugrue,
SCT(ASCP)CMIAC, Patricia Wasserman, M.D.
BACKGROUND: Several studies show that squamous intraepithe-
lial lesion (SIL) cells are more prevalent in specimens in which
endocervical /transformation zone (EC/TZ) cells are present.
Other studies have found no difference in SIL detection related
to EC/TZ status. We evaluated our experience at Long Island
Jewish Medical Center with the aim of discovering factors asso-
ciated with the lack of EC/TZ cells and identifying the prevalence
of SIL cells in subsequent Pap smears.
DESIGN: A search in the computer database was performed in-
cluding specimens lacking EC/TZ cells in a period of time of 28
months (December/02-March/05) in women under the age of
40. We reviewed the Pap smear reports to obtain pertinent
clinical data, identify the presence or absence of SIL and assess
the EC/TZ component together with the likelihood of missing
SIL cells in subsequent Pap smears.
RESULTS: Of the 130,918 Paps performed during our study pe-
riod in women under the age of 40, 1425 specimens lacked
360 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
EC/TZ cells. Of these, 367 (25.7%) and 28 (1.9%) cases were
performed on pregnant women and in the postpartum period,
respectively; 69 (4.8%) cases were associated with the use of oral
contraceptives, and 71 (4.9%) of the cases were performed on
women having undergone a cervical cone biopsy. The diagnoses
were distributed as follows: 1201 (84.2%) negative for intraepi-
thelial lesion or malignancy, 121 (8.4%) low grade SIL (LSIL), 5
(0.3%) high grade SIL (HSIL), 97 (6.8%) atypical squamous cell of
undetermined significance (ASC-US), and 1 (0.07%) atypical
glandular cells (AGC). Of the 1201 negative cases, 201 cases had
at least one subsequent Pap smear with EC/TZ cells. The diag-
noses were distributed as follows: 176 (87.5%) negative, 12 (5.9%)
ASC-US, 12 (5.9%) LSIL and 1 (0.4%) HSIL.
CONCLUSIONS: Factors associated with lack of EC/TZ component
include pregnancy, use of oral contraceptives and having under-
gone a cervical cone biopsy. The percentage of negative, ASC-
US, LSIL, and HSIL cases remain approximately the same when
cases without EC/TZ cells are compared with specimens with
EC/TZ component in subsequent Paps from the same patients.
The likelihood of missing ASC-US and LSIL in women with
negative cases without EC/TZ is low (5.9%); the likelihood of
missing HSIL cases is even lower (0.4%). This retrospective study
shows no clear evidence that patients with Paps lacking EC/TZ
cells are at greater risk of concurrent or subsequent abnormal-
ities than are patients with EC/TZ cells. We conclude that the
ultimate determination of specimen adequacy should rest with
the smear taker, who must correlate the findings described in the
cytology report, with clinical knowledge of the individual pa-
tient. If the smear result is normal while lacking an EC/TZ
component, there is no need to repeat the smear earlier than the
recommended screening interval.
Division of Cytopathology, Department of Pathology, Albert Ein-
stein College of Medicine, Long Island Jewish Medical Center, New
Hyde Park, New York
64
Cervical LEEP Margin Status and Post-LEEP
Pap Smear Follow-up
Andrew Thompson, M.D., Ph.D., Mala Patel, Lynn
Sandweiss, CT(ASCP), Hongyan Li, Sharon Hirschowitz,
M.D., Jianyu Rao, M.D., Sathima Natarajan, M.D.
PURPOSE: o examine the relationships between Loop Electrosur-
gical Excision Procedure (LEEP) margin status and abnormalities
on post-LEEP Pap smears. Other factors such as pre-LEEP diag-
noses and interval to post-LEEP Pap smear were also examined.
MATERIALS AND METHODS: A retrospective study was performed
of 129 female patients (average age 33 years) with LEEP biopsies
at UCLA between October 2001 and September 2003. Each LEEP
case was analysed for pre-LEEP diagnoses, LEEP result including
margin statuses, time to follow-up Pap smear, and follow-up Pap
smear results through May 2004. Relationships were examined
between these variables.
RESULTS: 106 of the 129 LEEPs (82.2%) showed a high-grade
squamous lesion and 1 showed adenocarcinoma in situ. The
margins statuses of the LEEPs were 51.1% all margins negative
for high-grade dysplasia, 17.8% negative ectocervical margin and
positive endocervical margin, 8.5% positive ectocervical margin
and negative endocervical margin, 14.0% positive ectocervical
margin and positive endocervical margin, and 8.5% with at least
1 margin indeterminate. 79.1% of patients had at least 1 post-
LEEP follow-up Pap smear (20.9% had none) with the mean
follow-up period for the first post-LEEP Pap smear being 4.7
months. Among those patients who had post-LEEP follow-up
Pap smears, 27.5% were positive for abnormal squamous cells
(ASCUS and above) at their first satisfactory post-LEEP Pap
smear. A significantly higher proportion of such positive results
were seen in women with positive ectocervical margins than in
women with negative ectocervical margins (48.0% vs. 22.5%,
P%0.05 by Chi-Square test). However, there was no significant
difference in women with positive vs. negative endocervical
margins for their first satisfactory post-LEEP Pap smear (32.4%
and 27.4% respectively, P!0.05). Even among women with all
LEEP margins negative for high-grade dysplasia, 9.4% eventually
had at least 1 post-LEEP Pap smear read as ASC-H or HGSIL. Of
patients having their first post-LEEP Pap smear approximately 3
months after their LEEP, only 1 in 27 (3.7%) were unsatisfactory.
CONCLUSIONS: Even though post-LEEP Pap smears are required
for proper follow-up, a significant number of women do not
receive post-LEEP follow-up Pap smears. Negative margins at
LEEP biopsy do not guarantee negative post-LEEP pap smears or
an absence of high-grade squamous lesions. Women with posi-
tive ectocervical margins are more likely than those with nega-
tive ectocervical margins to have post-LEEP Pap smears positive
for abnormal squamous cells. 3 months may be an adequate
interval from LEEP biopsy to post-LEEP follow-up Pap smear.
University of California at Los Angeles, Los Angeles, California
65
A Comparison of the Yield of HGSIL and
LGSIL Diagnostic Cells Located By the
ThinPrep® Imaging System
Susan Stowell, M.A., SCT(ASCP)CMIAC, Jessica Williams,
Kamal Khurana, M.D.
INTRODUCTION: The ThinPrep® Imaging System (TIS) locates 22
fields of view (FOV) for evaluation of epithelial cell abnormali-
ties. The objective of this study was to determine and compare
the yield of diagnostic cells in cases with a previous diagnosis of
HGSIL and LGSIL based on TIS screening.
STUDY DESIGN: Twenty-five cases reported as HGSIL and 25
cases reported as LGSIL after screening by the TIS were ran-
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
domly selected. The 22 FOV selected by the Imaging System
were microscopically reviewed on a ThinPrep® Imaging review
microscope. Diagnostic cells within these fields were counted
and recorded. For LGSIL cases, mild dysplasia and HPV cyto-
pathic effect were recorded separately. Single cells and groups
were recorded in separate categories. All slides were then re-
viewed manually and diagnostic cells were counted and re-
corded in the same way. Diagnostic cells/groups located by the
imager were reported as a percentage of all diagnostic cells/
groups found manually.
RESULTS: The percent of cells/groups located by the imager
ranged from 0 to 100. The percentage of single SIL cells located
by TIS was 33.34% ($2%) and the percentage of SIL cells in
groups located by TIS was 27.06% ($2%). Cells diagnostic of
LGSIL demonstrating mild dysplasia were located more effi-
ciently by the imager at 36.90% ($2%) than cells with changes
related to HPV infection at 25.36% ($2%). The average percent of
cells/groups located by the imager was 31.70% ($2%) for HGSIL
and 27.63% ($2%) for LGSIL (p%.05). Only one case had no
diagnostic cells in any of the 22 fields located by the imager. This
case of LGSIL with HPV effect had background clues that led to
a full manual rescreen which found eight diagnostic cells/
groups.
CONCLUSION: There is not a significant difference in the yield of
diagnostic cells located by the TIS for LGSIL compared to HGSIL
diagnoses. For all categories combined, 30.37% of diagnostic
cells/groups were located and this was sufficient for the diagno-
sis.
SUNY Upstate Medical University, Syracuse, New York
66
Significance and Reproducibility of Repeat
HPV Specimens with Low RLU/CO Values
using Digene HC II Technology
Cathy Setzer, CT(ASCP), Kara Lawton, CT(ASCP), B.S.,
Mary Chacho, M.D.
INTRODUCTION: In utilizing the Digene Hybrid Capture II tech-
nology for the retesting of liquid based pap specimens, are low
RLU/CO values reproducible and is repeat testing necessary?
OBJECTIVE: To determine the significance of repeat HPV testing
by comparing ThinPrep and SurePath RLU/CO values within the
equivocal zone of " 1.0 and # 2.5 and correlating that data with
the cytological diagnosis at the time of testing and subsequent
follow up as available.
METHODS: High risk type HPV DNA retesting was performed on
129 denatured specimens (ThinPrep and SurePath) with low
RLU/CO values. Upon repeat, a positive result (! 1.0RLU/CO)
required no additional testing. A negative result (% 1.0 RLU/CO)
required a second retest. The initial RLU/CO values were com-
pared to the retest RLU/CO values to assess reproducibility and
361
correlated with Cytological diagnosis and follow up when avail-
able.
RESULTS: Of 129 samples retested for HPV DNA, 47(36%) were
reported out as positive with 39(83%) of these requiring only the
initial retesting. 30(77%) cases presented with an abnormal cy-
tology of ASCUS or greater at the time of testing and 5 had
abnormal biopsies with CIN I or greater. Of the 47 positive HPV
cases, the average RLU/CO was initially 1.8 and on retest 1.5.
CONCLUSION: Initial data has shown correlation between posi-
tive HPV reported cases following repeat testing and abnormal
cytologies. The RLU/CO results of both positive ThinPrep and
SurePath specimens upon initial retest suggests that they are
reproducible at low range RLU/CO values.
Yale University, New Haven, Connecticut
67
Aliquot Removal from ThinPrep®
Specimens: Effect on Adequacy and
Cytological Diagnosis
Charan Leng, B.S.1, Jim Linder, M.D.1, 2, Kari Linder1,
Larry Burg, Ph.D.1
OBJECTIVES: Removal of a limited-volume aliquot from Thin-
Prep® specimens, before slide preparation, would facilitate par-
allel cytological and molecular testing. However, removing a
portion of the specimen would reduce the cellular concentra-
tion. This could potentially interfere with specimen adequacy
and cytological diagnoses of subsequent ThinPrep Pap Tests
(TPPT). The objective of this study was to determine if removing
a 4-ml aliquot from gynecological specimens in PreservCyt®
Solution, before ThinPrep slide processing, adversely affected
specimen adequacy and cytological diagnosis.
METHODS: The study utilized 673 residual ThinPrep specimens
with volumes ranging from 5.6 to 18.8 ml (mean " 14.0 ml).
About 35% of the specimens (i.e., proportions reflect artificial
prevalence of disease) had non-negative clinical diagnoses (e.g.
ASCUS or greater). Specimens were divided equally and each
portion brought to 20 ml. A TPPT control slide was made from
one portion. A 4-ml aliquot was removed from the other portion,
replenished with 4 ml of unused PreservCyt Solution and a TPPT
test slide prepared. Control and test slides were masked, ran-
domized and evaluated for adequacy and cytological diagnosis
(using Bethesda 2001 criteria) by cytotechnologists (CTs) and a
panel of CTs and cytopathologists. Analysis was conducted by
partitioning the data into three groupings: Negatives vs. ASCUS
and greater (ASCUS$), % LSIL vs. LSIL and greater (LSIL$)
and % HSIL vs. HSIL and greater (HSIL$) and examining percent
agreement and abnormal cell detection rates.
RESULTS: The percent agreement between the control and test
methods for slide preparation of both CTs’ and panel CTs’ di-
agnoses for ASCUS$, LSIL$ and HSIL$, were 86%, 90% and
362 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
91%, respectively. The CTs’ diagnoses showed no significant
difference (the 95% confidence interval of the difference in-
cluded zero) in abnormal cell detection rates, which were about
47%, 33% and 17% for ASCUS$, LSIL$ and HSIL$, respectively.
The consensus diagnoses from the panel showed similar abnor-
mal detection rates of 38% (ASCUS$), 31% (LSIL$) and 10%
(HSIL$). However, the panel evaluations indicated that the test
method showed a statistically significant (p"0.0146) improve-
ment of about 3% for the detection of LSIL$ abnormalities.
Finally, there was negligible difference in slides deemed not
adequate for cytological diagnosis between the control (0.7% or
5/687) and test (0.9% or 6/687) methods.
CONCLUSIONS: The removal of a 4-ml aliquot from ThinPrep
specimens did not adversely affect cytological diagnosis. *This
method has not been reviewed or approved by the FDA.
1
Cytyc Corporation, Marlborough, Massachusetts, 2University of
Nebraska, Omaha, Nebraska
68
In House Validation of the ThinPrep Imaging
System
Brett Minger, B.S., CT(ASCP), Lorraine Savoca, A.S.,
CT(ASCP), Kevin Schofield, B.S., SCT(ASCP), Mary
Chacho, M.D.
INTRODUCTION: study was conducted to ascertain the reliability
and accuracy of Cytyc’s ThinPrep Imaging System before the
complete implementation of the system within our laboratory.
METHOD: To accomplish this, 13 cytotechnologists were asked to
use the ThinPrep Imaging System for their first 100 cases, along
with a full slide review (FSR) on each case. This gave us a total of
1230 cases (2 cytotechnologists did not complete 100 cases
each). If only the 22 fields of view ( FOV) were reviewed then
these cases were manually rescreened by the same cytotechnol-
ogist and both results were recorded.
RESULTS: Of the 1230 cases examined, 21 of the cases were called
ASC-US, 1 ASC-H, 2 cases were diagnosed as LSIL and 2 were
called AGC, after the complete manual screenings by the cyto-
technologists. These cases were then sent to the pathologist. The
pathologist, in turn, agreed with 12 of the 21 ASC-US cases, the
1 ASC-H case and 1 of the LSIL cases. The other LSIL was
downgraded to ASC-US and the remainder of the cases were
downgraded to reactive cellular changes. Of the 2 AGC cases 1
was called reactive endocervical cells and the other was called
negative with endometrial cells present. These 26 cases were
reexamined giving special attention to the 22 FOV’s. On this look
back it was determined that there were cellular entities present
that should have prompted a FSR on 23 of the cases. Three of the
cases did indeed have rare atypia - 1 cell or 1 group of a few cells
- present that was not present in a FOV. Statistically we have
shown a 0.24% false negative rate for the ThinPrep Imaging
System.
CONCLUSIONS: The ThinPrep Imaging System works as adver-
tised by the Cytyc Corporation. The 0.24% false negative rate is
well below the standard false negative rate. This review study of
these cases has helped the cytotechnologists and pathologists to
have more confidence in the system as a whole.
Yale University, New Haven, Connecticut
69
Glandular Cells in Post Hysterectomy
Vaginal Smears
Amal Shakir-Kanbour, M.D., Ph.D., Reda Saad, M.D.,
Judith Modery, B.A., SCT(ASCP), Guiliana Trucco, M.D.,
Kazyua Onuma, Anisa Kanbour, M.D.
INTRODUCTION AND PURPOSE: The presence of glandular cells in
posthysterectomy (PH) vaginal smears may represent a diagnos-
tic challenge, particularly in patients with a history of genital
carcinoma. The presence of such cells is rarely addressed in the
literature. The objective of this study is to compare the incidence
of PH vaginal smears within 2 intervals, and to evaluate and
classify these glandular cells in order to elucidate on their pos-
sible origin and significance.
MATERIALS AND METHODS: All vaginal smears in hysterectomy
patients with benign glandular cells reported at a tertiary hos-
pital during the years 1995 and 2004 were evaluated. All cases
with malignant cells were excluded. The type of hysterectomy
and original pathology, post hysterectomy interval, any patho-
logical condition of vagina, and history of radiation therapy,
were evaluated. The followings were examined: glandular cell
type (goblet cell, endocervical and ciliated), hormonal pattern,
inflammation and granulation tissue. Special stain Mucicar-
maine was performed on 8 smears.
RESULTS: The incidence of PH vaginal smears accounted for
5.1% for year 1995, as compared to 1.8% for year 2004. Of those,
115 reported as having glandular cells (0.78%). Following re-
screening, only 73 smears were qualified as being glandular cells.
Of those 28 had hysterectomy for benign lesions within 2-6 year
postoperative interval, and 45 had hysterectomy for genital can-
cer within 1-18 year post-operative interval. Postoperative radi-
ation was performed in 38/45. All the smears were reported as
negative, except 3 cases, were reported as atypical. Those in-
clude one from a prolapsed fallopian tube, second with a recto-
vaginal fistula, and the third showed granulation tissue. The
glandular cell type was mucous cells in 58/73 (80%). The mucous
cells were endocervical in 44/73 (61%) and goblet cells in 14/73
(39%). The remaining 15/73 (20%) of cases showed columnar cell
type cells in 13/73 (18%) and ciliated cells in 2/73 (2%).
CONCLUSION: Our study showed that most of the glandular cells
in PH patient vaginal smears are mucous cells (endocervical or
goblet), originating from paraurethral glands, as a result of short
vagina due to hysterectomy. Also, our study showed that the
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
incidence of reporting glandular cells in PH vaginal smears is
decreasing most probably due to the development of HPV test-
ing. Although the new Bethesda system recommend these cells
to be reported, the majority of these cases turn to be benign, and
therefore, required no further intervention.
Magee-Womens Hospital at University of Pittsburgh Medical Cen-
ter, Pittsburgh, Pennsylvania
70
Utility of Vaginal Pap Test Screening in the
Post-Hysterectomy Patient
Maryanne Hornish, CT(ASCP), M.B.A., Liron Pantanowitz,
M.D., Roxanne Florence, M.D., Robert Goulart, M.D.
INTRODUCTION: Vaginal Pap test screening in the post-hysterec-
tomy patient remains controversial, particularly when the hys-
terectomy specimen contains benign changes only, with varying
recommendations for screening frequency and termination of
such screening. The aim of this study was to retrospectively
evaluate follow-up vaginal Pap tests for a cohort of hysterectomy
patients over a 15-year period.
MATERIALS AND METHODS: he pathology computer files at Bay-
state Medical Center (BMC) were searched for all hysterectomy
specimens (excluding supracervical) accessioned in the year
1989. Hysterectomy specimens were categorized as benign (if
there was no ASCUS or greater Pap test in the three years prior
to hysterectomy, or no history of Pap tests at BMC and the
hysterectomy showed benign/negative findings), dysplasia
present (if ASCUS or greater was diagnosed on a Pap test in the
three years prior to hysterectomy or dysplasia was found in the
hysterectomy specimen), and malignant (any malignancy within
the specimen). All follow-up Pap tests (FUPs) in this cohort of
patients over a 15-year period were collated and studied.
RESULTS: In 1989, there were 354 hysterectomy specimens. The
characteristics of this study group and their FUPs are summa-
rized in the following table:
Average
Average Age of % of
Patient Patients Patients Average Negative
Hysterectomy Number of Age at with with FUPs number FUPs
Diagnosis Patients Surgery FUPs (p%0.025) of FUPs (p<0.001)
Benign 255 (72%) 45 (22-77) 44 years 50.2 3.5 99.3%
Dysplasia 16 (5%) 42 (28-74) 43 years 62.5 6.2 71.0%
Malignant 83 (23%) 58 (22-90) 56 years 67.5 6.8 96.8%
The 3 non-negative FUPs in the benign hysterectomy category
were diagnosed as ASCUS, each with negative subsequent Pap
test follow-up and no biopsies performed.
CONCLUSION: Patients with a malignancy in their hysterectomy
specimen were, on average, 10-15 years older than those who
have benign findings or only dysplasia present. Age at the time of
363
hysterectomy did not appear to influence the clinical decision to
obtain vaginal Pap test follow-up in any given hysterectomy
category. Increasing frequency of obtaining vaginal Pap tests
was seen with increasing clinical significance of hysterectomy
category (malignant ! dysplasia ! benign). The percentage of
negative follow-up Pap tests in the benign hysterectomy cate-
gory was very high (99.3%), supporting some recommendations
for decreasing the frequency (or discontinuing) Pap test screen-
ing in this subset of patients.
Baystate Medical Center, Tufts University School of Medicine,
Springfield, Massachusetts
71
Cytomorphologic Analysis of False-Positive
Squamous Cell Carcinoma in Liquid-Based
Cervical Specimens
Reda Saad, M.D., Bernardo Ruiz, M.D., Ph.D.
INTRODUCTION AND PURPOSE: Squamous intraepithelial lesions
(SIL) might mimic invasive squamous cell carcinoma in cervical
smears. Although severe dysplasia is an immediate precursor for
squamous cell carcinoma (SCC), it is important to grade accu-
rately these lesions on cervical smears. Diagnosing SCC in such
women may cause psychologic trauma and may lead to unnec-
essary surgical procedures. The objectives of this study are to
evaluate the cytomorphologic criteria of liquid-based cervical
smears falsely diagnosed as malignant and to investigate the
possible criteria which leads to an inaccurate interpretation and
false positive results.
DESIGN: omputer search for cases diagnosed as squamous cell
carcinoma in pap smears was performed and correlated with
histologic follow-up specimens. Twenty-four cases of liquid-
based cervical smears, identified as false positives, were re-
viewed. We also included 20 liquid-based cervical smears of
histologically confirmed SCC as control group. The following
cytomorphologic features were evaluated: cellularity, pleomor-
phism, fiber cells, arrangement of abnormal cells, hyperchroma-
sia, nucleoli, abnormal chromatin pattern, necrotic background,
and inflammation.
RESULTS: Review of the original cervical smears confirmed the
diagnosis of SCC, whereas, 20 cases qualified for cytologic diag-
nosis of SCC, keratinizing type, and 4 cases as nonkeratinizing
type. Pleomorphism and/or keratinizing fiber cells was found in
16/24 patients (67%), arrangement of abnormal cells: individual
in 16/24 (67%) cases, and sheets in 8/24 (33%) cases, hyperchro-
masia and abnormal chromatin pattern in all cases
(24/24,100%), prominent nucleoli in 3/24 (12.5%) cases, necrotic
background in 11/24 (46%), and inflammation in all cases (24/
24,100%). On histologic follow-up, all patients were ultimately
found to have HSIL without invasion. Cervical smears of histo-
logically confirmed SCC showed significant hypocellularity, ex-
tensive necrotic background and frequent prominent nucleoli.
364 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
CONCLUSION: High-grade dysplasia can mimic the cytologic fea-
tures of SCC on cervical pap smears, particularly when keratin-
ized. Our study showed that cytomorphological features favoring
SCC include the presence of frequent prominent nucleoli, ex-
tensive necrotic background and hypocellularity of the smear.
Other cytomorphologic features such as pleomorphism, fiber
cells, arrangements of abnormal cells, hyperchromasia, abnor-
mal chromatin pattern and inflammation, can be seen in high-
grade SIL and may lead to inaccurate interpretation.
Louisiana State University Medical Center, New Orleans, Louisi-
ana
72
Abnormal Vaginal Pap Tests in Post-
Hysterectomy Patients Versus Cervical-
Vaginal Pap Tests: A Comparison Study
Liron Pantanowitz, M.D., Roxanne Florence, M.D.,
Maryanne Hornish, CT(ASCP), M.B.A., Robert Goulart,
M.D.
INTRODUCTION: Vaginal Pap test (VPT) screening in the post-
hysterectomy patient remains controversial. To determine the
significance of an abnormal VPT result in this setting, we com-
pared the ASC-US HPV DNA positivity rate and rates of dysplasia
on subsequent biopsy for VPTs to similarly diagnosed cervical-
vaginal Pap tests (CVPTs).
MATERIALS AND METHODS: The cytopathology computer files at
Baystate Medical Center were searched for all VPT diagnoses
(NILM; Atypical including ASC-US, ASC-cannot exclude HSIL,
and Atypical glandular cells; LSIL; and HSIL/Cancer) over a
one-year period (2004). Corresponding HPV DNA positivity rates
for triaged ASC-US cases, and rates of dysplasia on follow-up
biopsy were recorded. All results were compared to a cohort of
similarly diagnosed CVPTs. Pap tests were almost entirely liquid-
based (ThinPrepTM). HPV DNA testing was performed using the
Digene® Hybrid Capture 2 High-Risk HPV DNA TestTM.
RESULTS: Of the 65,642 total Pap tests evaluated in 2004, 5,019
(7.6%) were VPTs. The greatest absolute number of VPTs was
seen in the 51-60 year patient age group, with a continually
increasing relative percentage of VPTs seen in each age decade.
The highest relative percentage (39%) of VPTs was seen in pa-
tients !80 years of age. For VPTs, there were 2.9% Atypical, 1.4%
LSIL, and 0.4% HSIL cases, compared to corresponding CVPT
diagnostic category percentages of 6.6%, 2.3%, and 0.6%, respec-
tively. Atypical/SIL ratios were 1.6 for VPTs and 2.3 for CVPTs.
Positive HPV DNA rates were 41% for VPTs and 55% for CVPTs
triaged ASC-US cases. Dysplasia was identified on follow-up
biopsy (VPTs vs. CVPTs) in 43% vs. 40% of cases for ASC-US
(HPV positive) Pap tests, 50% vs. 61% for LSIL Pap tests, and 91%
vs. 82% for HSIL Pap tests. ASC-US HPV DNA positivity rates and
rates of dysplasia on biopsy in each Pap category (VPTs vs.
CVPTs) were not statistically different.
CONCLUSION: Vaginal Pap tests (VPTs) in our hospital-based
laboratory represent a relatively small (7.6%) subset of Pap tests
evaluated annually. Nevertheless, the HPV DNA positivity rate of
tested ASC-US in these VPTs was not statistically different from
triaged ASC-US cervical-vaginal Pap tests (CVPTs). Additionally,
for all cases with ASC-US (HPV positive), LSIL, and HSIL, the
rates of dysplasia on subsequent biopsy were not statistically
different when comparing VPTs and CVPTs. Therefore, the sig-
nificance of an individual VPT for predicting dysplasia appears
to be comparable to that of a similarly diagnosed CVPT.
Baystate Medical Center, Tufts University School of Medicine,
Springfield, Massachusetts
73
Imaging-Directed CytologyTM Increases
ASCUS Detection Rate, but not hrHPV
Detection Rate
Stanley J. Geyer, M.D., Brent Brewerton, B.S., CT(ASCP),
Cynthia Pratt, CT(ASCP), Chris Campana, M.D.
INTRODUCTION: In June 2004 the Cytopathology Laboratory of LDS
Hospital implemented the use of Imaging-Directed CytologyTM
(IDC)(Cytyc, Boxborough, MA) for screening liquid-based Pap
specimens (LBPS). We undertook this study to determine if IDC
increased the rate of ASCUS detection and if IDC increased the
rate of high-risk human papillomavirus (hrHPV) identification.
METHODS: We selected for review all gynecologic cytology spec-
imens examined in January 2004 (manual screening) and all
specimens examined in January 2005 (IDC) for comparisons of
ASCUS rate and hrHPV detection. We utilized the Hybrid Cap-
ture II method (HCII) (Digene, Gaithersburg, MD) for identifi-
cation of hrHPV in ASCUS specimens.
RESULTS: In January 2004 the laboratory examined 7,194 LBPS
and identified 174 ASCUS cases (2.4%), of which 110 were tested
for hrHPV. In January 2005 the laboratory evaluated 6,987 LBPS
and identified 224 ASCUS cases (3.2%) of which 152 were tested
for hrHPV. The table depicts the results of the hrHPV testing. The
ASCUS rate increased significantly from 2.4% to 3.2% using IDC
(p"0.005, two-sided Z test), while the hrHPV detection rate in
ASCUS cases decreased significantly (p"0.003, two-sided Z test).
The overall hrHPV detection rate (# cases of positive hrHPV
cases divided by total number of cases) remained the same for
the two periods (0.70% in January 2004 and 0.59% in January
2005; p"0.483, two-sided Z test). hrHPV Identification in ASCUS
Specimens
TOTAL hrHPV Positive Negative %Positive
Manual (Jan 2004) 110 50 60 45.5%
IDC (Jan 2005) 152 41 111 27.0%
CONCLUSIONS: Because the increased number of ASCUS cases
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
identified by IDC are not associated with the increased detection
of hrHPV, these additional ASCUS cases are probably not asso-
ciated with an increased risk of developing squamous intraepi-
thelial lesions and may represent an artifact of IDC methodol-
ogy. The change in staining associated with IDC creates darker
nuclei that may account for the increased rate of ASCUS iden-
tification without the commensurate increase in hrHPV detec-
tion. By improving the sensitivity of ASCUS detection, IDC
screening reduces false-negative results and the likelihood of
missing a squamous intraepithelial lesion. Reflex HPV testing of
ASCUS cases improves specificity to avoid triage of patients to
unnecessary or costly procedures.
LDS Hospital, Salt Lake City, Utah
74
Histologic Sampling Errors as a Cause for
Discrepancy Between a Positive HPV DNA
Test and a Negative Histologic Evaluation in
Patients with ASC Pap Tests
Amy Adams, M.D., Isam Eltoum, M.D., Mary Connolly,
Jun Chen, Janie Roberson, B.S., SCT(ASCP)CMIAC, David
Chhieng, M.D.
BACKGROUND: Reflex HPV DNA testing is now a standard pro-
cedure for patients diagnosed with atypical squamous cells or
ASC. The detection of high-risk HPV DNA is often associated
with underlying squamous dysplasia or HPV infection. However,
a small but substantial number of patients with HPV positive
ASC Pap tests have a subsequent negative colposcopic biopsy
and/or endocervical curettage. The objective of the current
study is to determine what the causes are for a subsequent
negative cervical tissue sampling in patients with HPV positive
ASC Pap tests.
MATERIALS AND METHODS: A computerized search identified all
patients with ASC Pap tests and reflex HPV DNA testing by
hybrid capture between January 1, 2002, and December 31, 2003.
Patients with a positive HPV DNA test and a subsequent negative
cervical biopsy and/or endocervical curettage were included in
the study. The original histologic slides were reviewed to confirm
the original diagnosis. Three additional H-and-E-stained deeper
levels were obtained and examined if no dysplasia or evidence of
HPV infection was noted in the original sections on retrospective
review. All reviews were performed by at least two pathologists.
RESULTS: During the two-year period, we identified 102 patients
with an ASC interpretation on Pap tests, positive HPV DNA
testing, and negative cervical histology. Slides and tissue blocks
were not available in 7 cases. As a result, 95 cases were included
in the study. On retrospective review of the original histologic
sections, 4 cases (4%) were found to show histologic evidence of
focal HPV infection. When deeper levels were examined, 29
cases (31%) showed evidence of clinically significant lesions
365
cytopathic changes of HPV infection in 8 (8%) of the cases, low
grade dysplasia (CIN I) in 10 (11%), high grade dysplasia (CIN
II/III) in 8 (8%), and dysplasia not otherwise specified in 3 (3%).
CONCLUSION: Based on our study, interpretation error of the
original sections accounted for only a small percentage of cases
with a discrepancy between a positive HPV DNA result and
negative biopsy finding. However, almost a third of these cases
were due to histologic sampling error, as evidence of HPV infec-
tion or dysplasia was noted on deeper levels. Therefore, we
recommend that additional levels should be obtained to rule out
any significant lesion when patients with ASC and a positive HPV
DNA testing have an “initial negative histologic evaluation.”
University of Alabama at Birmingham, Birmingham, Alabama
75
A Model of Resource Utilization for Public
Health
Nancy Haight, CT(ASCP)1, Patricia Miller, CT(ASCP)1,
Daniel F.I. Kurtycz, M.D.1, 2, John Shalkham, M.A.,
SCT(ASCP)1, Jim Stewart III, M.D.1, 2
PURPOSE: The Public Health establishment in the United States
sees the dissemination of the Papanicolaou test as one of the
major events in the 20th century. Unfortunately, with the advent
of liquid-based and other ancillary tests, including HPV, the
resources of public health agencies that provide cervical screen-
ing have become strained. Paradoxically, these costly “improve-
ments” in Pap smear technology could act to deny access to the
very population that needs screening the most. This study was
undertaken to evaluate a cost-saving model for cervical screen-
ing and subsequently colposcopy.
MATERIALS AND METHODS: Public Health clinics with cases be-
tween November 1, 2002 and July 31, 2004, were identified.
These clinics were divided into two different groups depending
on the type of modified HPV triage used. Group One used
conventional Paps as the primary method of collection. If the
conventional Pap was ASC-US, a repeat was done using a liquid
base (ThinPrep®) preparation. If the ThinPrep® was ASC-US,
HPV in-situ hybridization (ISH) was done and positive cases
were referred to colposcopy. Group Two collected all samples in
liquid base fixative. ThinPrep® ASC-US cases had HPV (ISH)
testing done and HPV positive cases were referred to colposcopy.
Costs of conventional Pap smears, ThinPrep® Paps, HPV (ISH)
testing and colposcopy were obtained. ASC-US and HPV rates
were calculated. The total costs of each group were ascertained.
This number was divided by the primary number of patients
screened in each group. These costs were compared to a hypo-
thetical group (Group Three) that followed the previous standard
of practice of two consecutive ASC-US results on conventional
Paps, followed by colposcopy.
RESULTS: Results of this study showed a significant reduction in
366 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5

cost using the modified HPV triage of Group One. Group Two 77
had a higher ASC-US sensitivity but was also the most expensive
method. Costs for Group Three fell between Groups One and
Significance of ‘Endometrial Cells in Women
Two.
CONCLUSION: Comparison shows that using the modified HPV
>40 Years’ on PAP Smears: Correlation
triage of conventional Pap, ThinPrep® and HPV testing followed with Subsequent Endometrial Sampling
by colposcopy is a more cost effective use of public health Results
resources and provides patients with adequate follow-up care.
Narasimhan Agaram, M.D., David Dabbs, M.D., Malathy
1
Wisconsin State Laboratory of Hygiene, Madison, Wisconsin, Kapali, M.D.
2
Department of Pathology, University of Wisconsin-Madison,
Madison, Wisconsin BACKGROUND: Endometrial cells (EM) seen in Pap smears of
women ! 40 years should be reported according to the 2001
Bethesda System. The standard of care is to do a subsequent
76 endometrial sampling based on the cytologic diagnosis. Some
recent studies have argued against the utility of reporting endo-
The False-Negative Rate of the ThinPrep metrial cells based solely on the age. In this study we report our
Imaging System Compares Favorably with experience on the significance of reporting endometrial cells
Manual Screening based on correlation with the outcome on subsequent biopsy.
METHODS: We retrieved Pap tests with a diagnosis of ‘EM ! 40
David Kindelberger, Edmund Cibas, M.D. years’ that had a subsequent endometrial sampling. Clinical
INTRODUCTION: The ThinPrep Imaging System (TIS) utilizes lo- parameters like the age, last menstrual period and collection
cation-guided screening to aid a cytotechnologist (CT) in the date were recorded. All the pap smears were reviewed by 2
primary screening of gynecologic preparations. TIS identifies 22 cytopathologists independently to confirm the presence of en-
fields-of-view (FOV) based on nuclear features and cell cluster- dometrial cells. The diagnosis of the subsequent biopsy was
ing characteristics. A CT is guided through the FOV, and, if no recorded for each case, based on the surgical pathology report.
abnormalities are identified, the slide is resulted as negative. RESULTS: 410 cases of Pap tests with a diagnosis of ‘EM ! 40
Location-guided screening is a new paradigm for cytology lab- years’ and with subsequent endometrial sampling were identi-
oratories, and successful implementation depends in part on fied. In 46 cases, endometrial cells were not identified on review
trust in the instrument. This study assessed the false-negative of the Pap tests, and these cases were excluded. The findings
fraction (FNF) of TIS compared with manual screening. were as follows:
METHODS: All ThinPrep slides interpreted as negative for an
intraepithelial lesion or malignancy (NILM) pursuant to TIS re- Histologic Dx Total (%) 40-45 yrs (%) >45yrs (%)
view were manually re-screened by a second CT. Results were
compared with a historical control group of cases screened and No EM 22(6) 8(6) 14(6)
Normal EM 217(60) 71(59) 146(60)
re-screened manually. A FNF was calculated for both screening
Atrophic EM 8(2) 0(0) 8(3)
methods. Discrepancies between the primary screener’s and DPE 50(14) 20(17) 30(12)
re-screener’s diagnoses on TIS cases were evaluated to deter- EM Polyp 57(15) 19(16) 38(15)
mine whether lesional cells were present within or between the Hyperplasia without atypia 2(0.5) 0(0) 2(0.8)
FOV. Hyperplasia with atypia 3(0.8) 1(0.8) 2(0.8)
EM carcinoma 4(1) 0(0) 4(1.6)
RESULTS: One of 1776 TIS cases (0.056%) interpreted as NILM
Miscellaneous 1(0.2) 1(0.8) 0(0)
was reclassified as LSIL and none as HSIL or cancer. Review
confirmed that some lesional cells in this case were present in 207(57%) women were menstruating (45 % Day 12 and 162 !
the FOV. Two of 862 (0.23%) manually-screened cases inter- Day 12) and 157(43%) were postmenopausal (PM). The only
preted as NILM were reclassified as LSIL, and none as HSIL or significant finding between the menstruating and the PM groups
cancer. The FNFs for TIS and manual screening were 0.8 and 6.2, was in the incidence of hyperplasia with atypia (0 vs. 2%) and EM
respectively, representing a 7.75-fold decrease in the FNF with carcinoma (0.6 vs. 2%).
the implementation of the TIS. CONCLUSIONS: A significant proportion of EM ! 40 years turn
CONCLUSIONS: Implementation of the TIS results in a decrease in out to have a benign diagnosis on subsequent endometrial sam-
the FNF compared with manual screening. An understanding of pling. The significant findings of hyperplasia with atypia and EM
this effect can help CTs develop trust in location-guided screen- carcinoma, in our study, occurred in women ! 45 years and
ing instruments. predominantly in postmenopausal women. Based on the above
Department of Pathology, Brigham and Women’s Hospital and results, increasing the age limit of reporting EM cells to 45 years
Harvard Medical School, Boston, Massachusetts
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
might be cost beneficial by decreasing the number of patients
who undergo subsequent endometrial sampling.
Magee-Womens Hospital of the University of Pittsburgh Medical
Center, Pittsburgh, Pennsylvania
78
Atypical Squamous Cells Cannot Exclude
High Grade Lesion (ASC-H) Correlation with
HPV Status and Follow Up Biopsy
Robert Stumler, B.S., CT(ASCP), Lynan Moy, B.S.,
CT(ASCP), Pamela Lathrop, B.S., CT(ASCP), Jacalyn
Papillo, B.S., CT(ASCP), Abdelmonem Elhosseiny, M.D.
INTRODUCTION: There are relatively few studies of the Bethesda
System 2001 diagnostic category of ASC-H published in the
cytology literature with a large sample size. Our goal was to
evaluate the cases reported as ASC-H at our institution over a
3-year period, including follow up biopsies and HPV test results.
MATERIALS AND METHODS: We performed a retrospective review
of all Pap tests diagnosed as ASC-H from 2002-2004 at our
institution. In addition we obtained patient age, high-risk (HR)
HPV DNA test results (Digene Hybrid Capture II) and cervical
biopsy results.
RESULTS: Out of 183,375 total Pap tests, 579 cases (0.32%) re-
ceived an ASC-H cytologic diagnosis, from 537 unique patients.
Patient age ranged from 15-81 years with a median age of 28
years. Of the 537 ASC-H patients, 258 had histologic follow up at
our institution and 154 had HPV testing; 85 patients had both
adequate histologic follow up and HPV testing. Histologic follow
up on 258 patients showed 37.2% benign, 3.9% atypical (sugges-
tive of CIN), 8.5% CIN I and 48.4% CIN II or higher. HPV testing
of 154 patients showed 74% positive for HR-HPV and 26% HR-
HPV negative. In 85 patients with both histologic follow up and
HPV testing we found 30 benign biopsies (10 HPV$/20 HPV-), 7
atypical (5 HPV$/2 HPV-), 7 CIN I (5 HPV$/2 HPV-) and 41 CIN
II and higher (37 HPV$/4 HPV-).
CONCLUSIONS: The incidence of confirmed CIN II and higher in
our study was 48.4%, which is higher than several previously
reported studies and supports the ASCCP recommendation for
immediate colposcopy for an ASC-H diagnosis. Of the 85 pa-
tients with both follow up biopsy and HPV testing, 33% with a
negative biopsy had a positive HPV test, reinforcing the need for
HPV testing if the biopsy does not support the Pap diagnosis.
Incidentally, in that same subset of 85 patients, we found that
nearly 10% of ASC-H patients with CIN II or higher on biopsy
had a negative HPV result.
Fletcher Allen Health Care, Burlington, Vermont
367
79
Impact of HPV DNA Testing on a Cervical
Cancer Screening Program for HIV!
Women
Heather Sabaka, B.A., Susan M. Otell, B.A., CT(ASCP),
Monica Srodon, M.D., Jean M. Keller, P.A., Jean
Anderson, M.D., Dorothy Rosenthal, M.D., Douglas P.
Clark, M.D.
INTRODUCTION: Immunosuppressed women are at increased risk
for HPV infection and cervical intraepithelial neoplasia. The
current management guidelines recommend colposcopy for all
HIV$ women with ASC-US or SIL on cervical cytology; however,
more cost-effective management schemes, such as those incor-
porating HPV testing, may be possible. Unfortunately, there are
little available data on the utility of HPV DNA testing among
HIV$ women. We hypothesize that HPV testing may help stratify
HIV$ women for risk of CIN, similar to HIV- women.
MATERIALS AND METHODS: We performed a retrospective review
of cervical cytology reports, HPV test results, and follow-up
cervical biopsies among a cohort of 424 inner-city HIV$ women
treated at our institution between 2000-2005. Cervical cytology
samples were processed using the SurePath system and all neg-
ative and ASC-US cases were tested for high-risk HPV DNA by
Hybrid Capture 2 (Digene). A subset of LSIL and HSIL samples
were also tested for high-risk HPV DNA. Cytology reports were
correlated with HPV test results and follow-up cervical biopsies
(within 6 months of Pap).
RESULTS: Cytologic diagnoses included: negative " 49%; ASC-
US " 21%; ASC-H " 2%; LSIL " 22%; HSIL " 5%; AGUS " 0.2%.
High-risk HPV DNA was detected in the following percentage of
cases: negative " 29%; ASC-US " 60%; ASC-H " 78%; LSIL "
93%; HSIL " 95%. The prevalence of HPV positivity within each
group is similar to that found in the ALTS trial; however, the
prevalence of abnormal cytology is significantly higher in this
HIV$ cohort than the general screening population at our insti-
tution. The percentage of women with abnormal cytology who
received colposcopy was relatively low (ASC-US " 24%; ASC-
H " 44%; LSIL " 43%; HSIL " 68%), presumably due to com-
pliance issues rather than referral patterns since all women with
ASC-US$ were referred for colposcopy. Using ASC-US/HPV$ as
a cut-off for referral to colposcopy, rather than ASC-US, would
have resulted in an 8.3% reduction in the colposcopy referral
rate (n"35 fewer women referred). The sensitivity and specificity
for detection of biopsy-proven HSIL using ASC-US as a cut-off
was 96.6% and 5.8%, respectively (PPV"36.4%; NPV 75%). Rais-
ing the cut-off to ASC-US/HPV$ did not reduce the sensitivity
(96,6%), but did raise the specificity (17.3%) as well as the PPV
(39.4%) and the NPV (90%).
CONCLUSIONS: Our retrospective study revealed that the preva-
lence of abnormal cervical cytology and high-risk HPV was high
in this inner city HIV$ population. Referral of all women with
368 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
ASC-US$ cytology for colposcopy results in a high sensitivity for
detection of biopsy-proven HSIL. Our data suggest that raising
the referral cut-off to ASC-US/HPV$ would result in fewer col-
poscopy referrals while maintaining a high sensitivity for HSIL
detection. Definitive changes in management recommendations
will require further studies.
The Johns Hopkins Medical Institutions, Baltimore, Maryland
80
Collection of the SurePath Pap with a
Broom Device ! EC Brush Improves LSIL
Detection and EC Recovery when Compared
to the Broom Device Alone or the
Spatula ! EC Brush Combination
Sharon Davis-Devine, B.S., CT(ASCP)1, Sarah J. Day, B.A.,
CT(ASCP)1, Amy Anderson1, Ashley French, B.S.1, Darcy
Madison-Henness, B.S., CT(ASCP)1, Naomi Mohar, B.S.,
CT(ASCP)1, Danielle Tansy, B.S., CT(ASCP)1, Jeffrey A.
Douglas, Ph.D.2, Gregory G. Freund, M.D.2
INTRODUCTION/PURPOSE: In a previous study [BMC Women’s
Health 2004, 4:6], we identified a potential but not significant
improvement in endocervical cell (EC) recovery when multiple
Pap collection devices were used to sample the cervix during the
SurePath Liquid-Based Pap Test (SurePath Pap). Here we exam-
ine the diagnostic utility of the newly US Food And Drug Ad-
ministration approved Spatula $ EC brush combination for the
SurePath Pap and compare it to Pap collection with the broom
alone or to an off-label combination of broom $ EC brush.
MATERIALS AND METHODS: SurePath Pap vials sent to the cyto-
pathology laboratory from July, 2004 to March, 2005 were exam-
ined for the collection devices contained. The Pap collection
device combinations allowed to providers in our clinic health
care system include: Rovers Cervex-Brush (broom), Medscand
Pap Perfect Spatula $ Medscand CytoBrush Plus GT (spatula $
EC brush) or Rovers Cervex-Brush $ Surgipath C-E Brush
(broom $ EC brush). Diagnostic terminology and quality indi-
cators were derived from the 2001 revision of The Bethesda
System.
RESULTS: Examination of SurePath Paps yielded 6935 collected
with the broom alone, 2739 collected with the spatula $ EC
brush and 1739 collected with the broom $ EC brush combina-
tion. Absence of an endocervical/transformation zone was seen
in 23.29% of broom alone cases, 13.25% of spatula $ EC brush
cases (p% 0.00005 vs. broom) and 9.55% of broom $ EC cases
(p% 0.00005 vs. broom, p% 0.0001 vs. spatula $ EC brush). In
addition, significant differences were observed in the following
diagnostic categories. Negative for intraepithelial lesion or ma-
lignancy: broom alone 90.66%; spatula $ brush 92.22% (p%
0.015 vs. broom); broom $ brush 88.56% (p% 0.0083 vs. broom,
p% 0.00005 vs. spatula $ EC brush). ASC-H: broom alone
0.231%; spatula $ brush 0.073%; broom $ brush 0.403% (p%
0.016 vs. spatula $ EC brush). LSIL: broom alone 2.911%; spat-
ula $ brush 2.41%; broom $ brush 3.85% (p% 0.043 vs. broom,
p% 0.006 vs. spatula $ EC brush).
CONCLUSIONS: When Rovers Cervex-Brush $ Surgipath C-E
Brush combination is compared to the Rovers Cervex-Brush
alone or to the Medscand Pap Perfect Spatula $ Medscand
CytoBrush Plus GT combination, the broom $ EC brush better
samples the endocervical/transformation zone and increases
disease discovery by augmenting detection of ASC-H and LSIL.
1
Carle Clinic Association, Urbana, Illinois, 2University of Illinois,
Urbana, Illinois
81
Analytical Evaluation of an
Immunocytochemical Assay for the
Detection of Cervical High Grade Squamous
Intraepithelial Lesions
Deidra Kelly, B.A., CT(ASCP)1, Adriann Taylor, B.A.2,
Laura Hall, B.A., HTL(ASCP)2, Zoya Fansler, B.A.1, Erik
Kincaid, B.S., CT, MP(ASCP)1, Dorothy Rosenthal, M.D.1,
Douglas P. Clark, M.D.1
INTRODUCTION: An immunocytochemical (ICC) assay for the de-
tection of high-grade cervical squamous intraepithelial lesions
(HSIL) was integrated into the Cytology laboratory and analyti-
cally evaluated. Antibodies directed to topoisomerase IIa
(Top2A) and minichromosome maintenance protein 2 (MCM2)
(ProEx C, TriPath Imaging, Inc.) were utilized in an ICC assay
that can be used on liquid-based cervical cytology specimens to
aid in the identification of HSIL. The assay was evaluated for
scoring reproducibility, staining reproducibility, accuracy and
impact of environmental stress.
MATERIALS AND METHODS: Pre-prepared HSIL cytology pools,
no intraepithelial lesion or malignancy (NILM) pools, NILM
pools spiked with SiHa Cells and a SiHa Cell Control were
processed on the PrepStain slide processor (TriPath Imaging,
Inc.) using the Prep-Only Mode to prepare SurePath Slides. The
slides were immediately placed in a 1X dilution of the SureDetect
Slide Preparation Buffer (TriPath Imaging) for cell preparation.
Prepared slides were then stained on an autostainer (DakoCyto-
mation, Carpenteria, CA) using SureDetect Detection reagents.
Two lots of all of the reagents were assessed for reproducibility
by testing a NIL pool, a HSIL pool and control slide twice a day
over 5 consecutive days. Environmental stress testing included
open-vial testing over a 35 day period for two lots of all of the
reagents used in the assay. Accuracy was assessed by evaluating
samples that had been spiked with a relatively low number of
SiHa cells that are positive for MCM2 and Top2A. Scoring Re-
producibility was assessed for 2 cytopathologists by comparing
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting 369

the interpretation for the same set of 10 NILM and 10 positive HPV infection in women on HRT is not fully understood. Per-
samples. All slide interpretation was performed following an sistent HPV infections and/or activation of dormant HPV. Given
internally-derived scoring guide. our results, a further understanding of the underlying factors
RESULTS: Near-zero variability was demonstrated with regard to contributing to this phenomenon is indicated. Further, a review
scoring and staining reproducibility. There was no change in of Pap test recommendations calling for a decrease screening of
staining characteristics observed over the course of the study for this age group may be warranted.
the open vial testing. The Accuracy Study addressed the ability to
Fletcher Allen Health Care, Burlington, Vermont
recover and identify a small number of SiHa cells through the
processing and staining methods. The pathologists and cyto-
technologists were able to appropriately identify all of the cells of 83
interest in their preparations.
CONCLUSIONS: The ICC assay was successfully adopted in the
Comparison of Discrepancies in Current
cytology laboratory. The assay produced highly reproducible
staining and was consistently interpreted. The assay was unaf-
HSIL with Negative Look-back Cases:
fected by routine laboratory environmental factors such as re- Imaged Versus Non-Imaged Slides
agent age and climate. Fern S. Miller, M.S.M., CT(ASP), Mary B. Kenny-
1
Johns Hopkins Medical Institutions, Baltimore, Maryland, Tri- 2 Moynihan, M.D.
Path Oncology, Durham, North Carolina
INTRODUCTION: In 2004, our laboratory screened 106,703 Thin-
Prep® Pap tests: 84,433 slides were imaged on Cytyc’s ThinPrep
82 Imaging System (Imager) Cytyc Corporation, Marlborough, MA),
22,270 slides were not imaged. A retrospective comparison was
made between HSIL look-back data for current imaged and
Increased Squamous Intraepithelial Lesion non-imaged cases.
(SIL) in Women Age 60 Years and Older MATERIALS AND METHODS: Non-imaged cases were screened
Ellen Riley, B.S., CT(ASCP)IAC, Susan Warren, B.A., manually in the usual fashion. For imaged cases, 22 fields of view
SCT(ASCP)IAC, Amy Verville, B.S., CT(ASCP), Jacalyn (FOV) were reviewed. If no abnormality was identified in the 22
Papillo, B.S., CT(ASCP), Abdelmonem Elhosseiny, M.D. FOV, the slide was filed without further review. If abnormal cells
were identified in the 22 FOV, the entire slide was screened. All
INTRODUCTION: Historically, the highest incidence of Squamous new HSIL cases, imaged and non-imaged, were subject to CLIA
Intraepithelial Lesion and above (SIL$) has been in women mandated five-year look-back, and received a complete manual
under the age of 60. However, a trend towards an increase in re-screen.
SIL$ has been observed in women 60 and over (60$). A nine- RESULTS:
year retrospective review of Pap tests was undertaken to deter-
mine the incidence of SIL$ in this age group. 2004 2004 First Quarter 2005
MATERIALS AND METHODS: A retrospective review of Pap test
results from 1995-2004 was conducted. The results were divided Imaged Non-Imaged Imaged Non-Imaged
into two 54-month groups: the control group (7/95-12/99) and
Slides 84,433 22,270 25,479 5,619
the study group (7/00-12/04). Statistical analysis was done on Current HSIL 439(0.5%) 213(1.0%) 162(0.6%) 55(1.0%)
the data to determine P-values. Prior negative slides
RESULTS: available for
rescreen 102 (23.2%) 50 (23.4%) 36 (22.2%) 9 (16.4%)
Control Group Study Group Statistical Prior negatives
Women 60! 7/95-12/99 7/00-12/04 Significance discrepant on
review (non-
Total Paps 29,275 30,411 imaged) 5 0 2 0
SIL$ Paps 115 235 P"%.001 In 2004, there were 439 current HSIL imaged cases with 102
SIL$ Patients 80 122 P"%.01
(23.2%) prior negative slides available for look-back, and 213
CONCLUSION: The data shows a statistically significant increase current HSIL non-imaged cases with 50 (23.4%) prior negative
of SIL$ in women 60$. There are several possible explanations slides available for look-back. Five of the negative look-back
for this observation. Increased ‘multiple partner’ sexual activity. cases of the 439 current imaged HSIL cases had abnormal cells
This may in part be attributed to the increase usage of erectile considered to be discrepant (2-step or higher difference) versus
enhancement medications and/or increase in the rate of marital 0 of 213 current non-imaged HSIL cases. All prior negative look-
demise, both of which may contribute to an increase in multiple back cases had never been imaged. Data for the first quarter of
partner activity. Hormone Replacement Therapy. The risk of 2005 correlates with the 2004 results.
370 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
CONCLUSIONS: The Imager is very effective at locating abnormal
cells. Preliminary data suggests that current HSIL cases detected
with Imager assistance are more likely to have had a false neg-
ative smear in the past. Perhaps these current HSIL cases have
unique morphological features or paucity of abnormal cells that
evade detection on manual screen. This trend persists into 1st
quarter of 2005 and warrants additional evaluation.
Metropolitan Pathologists, Denver, Colorado
84
Effect on Abnormal Rates and Cytologic-
Histologic Correlations Using the ThinPrep
Imaging System: A Metropolitan Academic
Center’s Experience
Teri Wood, CT(ASCP), B.A., M.P.H., Diane Hamele-Bena,
M.D.
INTRODUCTION/PURPOSE: To evaluate the effects of the use of the
ThinPrep Imaging System (TPIS) on the abnormal rates of Pap
cases and cytologic-histologic correlation rates in our laboratory.
MATERIALS AND METHODS: Between March and December 2004,
42,285 Paps were screened with the TPIS. Diagnoses were com-
pared to diagnoses of conventional Pap (CP) cases (total 14,969)
and manually screened ThinPrep (MTP) cases (total 23,356) dur-
ing March and December 2003. Rates of cytologic-histologic
concordance were compared among the three groups of cases.
High risk HPV (HRHPV) testing was performed on 387 LSIL TPIS
cases and 72 LSIL TPIS cases with negative biopsies.
RESULTS: The lab’s overall abnormal (ASCUS and above) rate
increased from 10.5% pre-TPIS to 13.1% post-TPIS implementa-
tion (25% increase). When converting from CP to MTP and then
from MTP to TPIS, ASCUS/AGUS rates increased 23%, and 10%,
respectively, and LSIL rates increased 111% and 48%, respec-
tively. HSIL rates increased significantly after converting from
CP to MTP but did not increase after implementing TPIS; how-
ever, the volume of cases was relatively low in this category. For
HSIL, cytologic-histologic correlations were higher for MTP vs.
CP and remained unchanged for MTP vs. TPIS. However, cyto-
histo correlations for LSIL were lower after implementation of
TPIS. To further evaluate these LSIL cases, HRHPV testing was
performed on 387 cases diagnosed as LSIL with the TPIS. 337/
387 of these cases (87%) were HRHPV positive. In addition,
HRHPV testing was performed on 72 of LSIL TPIS cases with
negative tissue biopsies; 60/72 (83%) were HRHPV positive.
SUMMARY: Implementation of the TPIS resulted in a 25% in-
crease in the overall abnormal (ASCUS and above) rate in our
laboratory. There was no significant increase in ASCUS/AGUS or
HSIL rates. However, LSIL rates increased significantly after TPIS
implementation. Cytologic-histologic correlations did not
change significantly for HSIL cases, but tissue correlation for
LSIL was lower for TPIS cases as compared to MTP cases. The
high rate of HRHPV positivity in these TPIS LSIL cases suggests
that the TPIS is detecting increased numbers of true LSIL cases
and is a more sensitive test for detection of LSIL than MTP. The
lower rate of tissue correlation for LSIL suggests that the TPIS
may detect some smaller LSIL lesions that are not evident col-
poscopically. Whether this increased sensitivity is clinically sig-
nificant requires further investigation.
Columbia University Medical Center, New York, New York
85
Detection of Cervical High-grade Squamous
Intraepithelial Lesions from Cytologic
Samples using a Novel
Immunocytochemical Assay (ProEx C)
Deidra Kelly, B.A., CT(ASCP), Erik Kincaid, B.S., CT,
MP(ASCP), Zoya Fansler, B.A., Dorothy Rosenthal, M.D.,
Douglas P. Clark, M.D.
INTRODUCTION: Routine liquid-based cytology (LBC) provides
excellent sensitivity for the detection of cervical HSIL; however,
specificity is low. Consequently, many women with ASC-US or
LSIL cytology undergo unnecessary colposcopy. We hypothesize
that a novel immunocytochemical assay (ProEx C) that can be
performed on liquid-based cytology monolayers has a signifi-
cantly higher positive predictive value (PPV) for biopsy-proven
HSIL than routine LBC.
MATERIALS AND METHODS: We performed a retrospective anal-
ysis of 322 residual SurePath liquid-based cervical cytology sam-
ples from a specimen repository at our institution. The original
cytologic diagnoses on these specimens included: NILM, n"149;
ASC-US, n"63; ASC-H, n"26; LSIL, n"51; HSIL, n"30; Other,
n"3. Follow-up cervical biopsies were available on 172 patients
(Negative, n"99, LSIL, n"40; HSIL, n"32; SIL, n"1). An un-
stained SurePath slide was prepared from each residual speci-
men, which was then stained with the ProEx C immuno-cyto-
chemical assay. This assay utilizes a cocktail of mouse anti-
human monoclonal antibodies directed against proteins
associated with aberrant S-phase cell cycle induction (Topo-
isomerase IIa; Minichromosome maintenance protein 2), which
are visualized using a DAB chromogen and hematoxylin coun-
terstaining. Slides were then screened for positive cells (nuclear
DAB staining in any ASC-US$ cell) by a cytotechnologist and
reviewed by a cytopathologist who were blinded to the original
cytologic and biopsy diagnosis. The ProEx C assay sensitivity,
specificity, PPV, and negative predictive value (NPV) for detec-
tion of biopsy-proven HSIL within the ASC-US$ population
were determined.
RESULTS: The ProEx C assay was positive in 70 cases (22%) in
this cohort. Using biopsy-proven HSIL as an endpoint, the ProEx
C assay yielded 23 true positives, 47 false positives, 215 true
negatives, 7 false negatives, 29 unsatisfactory, and 1 not deter-
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
mined, for a sensitivity of approximately 84%, specificity of 72%,
PPV of 37% and NPV of 96%. Compared with the routine liquid-
based cytology results in this cohort, the ProEx C sensitivity for
biopsy-proven HSIL was approx. 31% greater than LSIL$ cytol-
ogy (68 vs. 84%), and specificity was approx. 30% greater (55% vs.
72%). ProEx C also showed an approx. 60% increase in PPV
relative to ASC-H (31 vs. 37%) and a 127% increase in PPV
relative to ASC-US cytology (16 vs. 37%). The NPV appears equal
to, or greater than, that of HSIL cytology (93 vs. 96%)
CONCLUSIONS: This retrospective trial suggests that ProEx C can
be used in conjunction with the Pap test to improve the sensi-
tivity and PPV for identifying women with HSIL, while maintain-
ing a high NPV in the atypical cytology population. Further
clinical trials are warranted to investigate the performance of
this test in the general screening population.
The Johns Hopkins Medical Institutions, Baltimore, Maryland
86
Detection of High Risk Human
Papillomavirus (HPV) DNA: Comparison of
Hybrid Capture 2 and INFORM™ In Situ
Hybridization
Cindy McGrath, M.D., Seth Fleisher, CT(ASCP), Terri
Pasha, B.S., Prabodh Gupta, M.D.
PURPOSE: Performance of INFORM in situ hybridization (ISH)
for detection of high risk HPV DNA as compared to Hybrid
Capture 2 (HC2) has shown varied results in the literature. The
purpose of this study is to assess the performance of ISH vs. HC2
using the SurePath liquid cytology system at a large University
Hospital laboratory.
MATERIALS AND METHODS: Consecutive cases (436) sent for HC2
testing were also evaluated using ISH for high risk (HR) HPV
subtypes over a 5 month period. Cytologic diagnoses included
normal, ASC-US, and LSIL. Additional 43 and 29 cases of cyto-
logic LSIL and HSIL, respectively, were evaluated using ISH
alone.
RESULTS: HC2 showed positive HPV HR in 6.5% (10/153) nor-
mal, 24.9% (64/257) ASC-US, and 61.5% (16/26) LSIL cases. ISH
showed positive HPV HR in 7.2% (11/153) normal, 12.5% (32/
257) ASC-US, and 46.2% (12/26) LSIL. The differences in HC2 vs.
ISH HPV HR detection for ASC-US and LSIL are statistically
significant (p%0.001), and are not significant for cytologically
normal cases. Of the additional cases of LSIL and HSIL per-
formed with ISH alone, 56.5% LSIL and 89.7% HSIL were positive
for HR HPV.
CONCLUSION: HC2 HPV HR detection was significantly improved
for ASC-US and LSIL cases as compared to ISH.
University of Pennsylvania Medical Center, Philadelphia, Penn-
sylvania
371
87
Performance of Human Papillomavirus
(HPV) High Risk (HR) DNA Testing in Daily
Clinical Practice
Cindy McGrath, M.D., Annemarie Pondo, CT(ASCP),
Prabodh Gupta, M.D.
PURPOSE: The ASC-US/LSIL Triage Study (ALTS) showed the
clinical utility of HPV HR DNA testing in patients with ASC-US
on pap smear. We evaluated the clinical usage, performance,
and biopsy follow-up of patients who had Hybrid Capture 2
(HC2) testing in our University Hospital cytopathology labora-
tory.
MATERIALS AND METHODS: Cytologic reports for 1797 cases that
had HC2 testing performed over a 16 month period were re-
viewed. Cytologic diagnosis, age, HC2 results, and subsequent
biopsy follow-up were evaluated.
RESULTS: Of 1797 cases (age range 13-83, average 36), 625, 1023,
and 149 had negative, ASC-US, and LSIL cytology respectively.
HC2 was positive for HR DNA in 15% negative, 34% ASC-US, and
62% LSIL cases. HC2 had significantly different rates for
women % or ! 30 years for negative (22% HC2$ %30 vs. 11%
HC2$ " 30; p%0.001) and ASC-US cases (45% HC2$ %30 vs.
25% HC2$ " 30; p%0.001). There was no significant difference
for any age group with LSIL. Biopsy follow-up was available for
200 cases (153 ASC-US and 47 LSIL). The majority of LSIL cases
showed low grade lesions on biopsy (72%), 82% of which were
HC2$. No high grade dysplasia was found on biopsy of LSIL. For
ASC-US cases, 95 were HC2$ with 21% negative, 67% low grade,
and 12% high grade lesions on biopsy. 51 ASC-US cases were
HC2 negative with 31% negative, 67% low grade, 0% high grade,
and 2% malignant (1 case with microinvasive carcinoma).
CONCLUSION: HC2 testing requests at our institution show dif-
ferent positivity and colposcopy referral rates for ASC-US and
LSIL as compared to ALTS data (34% vs. 54% ASC-US and 62%
vs. !83% LSIL). Though the biopsy follow-up is limited, HC2
positivity was detected in all cases with high grade dysplasia on
follow-up (5.5% overall) and was negative in one case with
subsequent microinvasive carcinoma.
University of Pennsylvania Medical Center, Philadelphia, Penn-
sylvania
88
The Utility of ASC-US with Features of
Atypical Squamous Metaplasia as a
Diagnostic Qualifier of ASC-US
David Kindelberger, Jeffrey Krane, M.D., Ph.D.
INTRODUCTION: The 2001 Bethesda System subclassifies the cat-
egory of “atypical squamous cells” into “of undetermined signif-
372 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
icance” (ASC-US) and “cannot exclude HSIL” (ASC-H). However,
when atypical immature metaplasia insufficient for the ASC-H
category is encountered, some practitioners may opt for a diag-
nosis of “ASC-US with features of atypical squamous metapla-
sia” (ASC-US/ASM). This study seeks to assess whether this
additional qualifier within the ASC-US category provides useful
risk stratification and prognostic information.
METHODS: We identified 300 cases diagnosed as ASC-US, ASC-H,
or ASC-US/ASM (100 cases in each category). Clinical follow-up,
in the form of Hybrid Capture II testing for high-risk HPV viral
types (Digene Corp., Gaithersburg, MD), cervical sampling by
biopsy, endocervical curettage and/or cone biopsy, or additional
cervical smears was recorded for each case. Cases included in
the study were given the initial “atypical” interpretation between
July 2000 and April 2002. Follow-up was monitored through
December 2004. A minimum of two follow-up procedures, oc-
curring over at least one year following the initial diagnosis, were
required for inclusion in the study.
RESULTS:
Mean Age % Developing % Developing
Category (yrs) LSIL HSIL
ASC-H 35.5 5 33
ASC-US/ASM 36.2 8 1
ASC-US 37.3 8 5
*24 ASC-H cases, 43 cases of ASC-US/ASM, and 45 cases of ASC-US were subsequently tested for HPV.
HSIL detection was significantly greater in the ASC-H category
than ASCUS or ASCUS/ASM (P%.0001), but the latter two were
not significantly different from each other
CONCLUSIONS: The practice of classifying atypical immature
metaplasia insufficient for the category of ASC-H as ASC-US is
justified, since these cases did not carry the high risk of the
ASC-H category in our study. Our data further indicate that the
risk in the ASC-US/ASM cases is not increased relative to other
squamous abnormalities in the ASC-US category. Since the qual-
ifier “with features of atypical squamous metaplasia” does not
appear to provide clinically useful information, use of this qual-
ifier with ASC-US is unnecessary and should be discouraged.
Department of Pathology, Brigham and Women’s Hospital, Bos-
ton, Massachusetts
89
Impact of the Implementation of the
ThinPrep® Imaging System on Screening
Productivity and Capacity in a Dual-Site
Commercial Laboratory Setting
Deborah Zaroor, SCT(ASCP)CMIAC, M. Lois Chaney,
CT(ASCP)IAC, Patrick James, M.D., Craig Elson, M.D.,
Blake Nestok, M.D., Dorothy Skidmore, M.S.,
MT(ASCPSBB), Connie Ballard, MT(ASCP)
INTRODUCTION/PAPER: The ThinPrep® Imaging System provides
computer-assisted guidance for screening ThinPrep® Pap Tests.
In this study we report the impact of the ThinPrep® Imaging
System on screening productivity and capacity in our laborato-
ries in Ohio and Kansas.
MATERIALS AND METHODS: LabOne, Inc. implemented the Thin-
Prep® Imaging System in December 2004 in our Ohio and Kan-
sas laboratories. We analyzed the following parameters for the
months of January-March of 2005: average number of slides per
% HPV
Positive hour by cytotechnologist, pap test TAT, and screening capacity
per FTE. All numbers were compared to a similar time frame
79.2* prior to implementation of the ThinPrep® Imaging System.
30.2*
RESULTS: We will demonstrate the improvement in productivity
60.0*
and screening capacity in our laboratories. Kansas: 99% of Pap tests
are Imaged- Directed Cytology (IDC). A gradual increase in pro-
ductivity was noted over a three-month period in January-March of
2005. Individual productivity increases varied from 1.3 sl/hr to10.8
sl/hr. Although productivity varied from individual to individual
average increases of 27% in January, 34% in February, and 42% in
March were seen. Total worked FTE’s increased during this time
from 9.7 to 9.9. Screening capacity increased 29% from 2800 to 3600
ThinPreps per week. Average TAT decreased from 6 days to 3 days.
Ohio: 76% of pap tests are IDC. An increase in productivity of 12%
was observed in January and February. The lower than anticipated
increase can be attributed to training and gradual implementation
of IDC in January as well as specimen mix.(40% of FTE’s screen
only Paps where the other 60% rotate to area hospitals to support
the non-gyn specimens and FNA procedures). In March an in-
crease of 20% was observed. Individual productivity increases var-
ied from 1.5 sl/hr to 4 sl/hr. Capacity from January through March
increased 30% to 6340 cases. FTE’s screening decreased from 15.5
in January to 13.3 in March. 92% of cases were reported in five days.
CONCLUSION: The ThinPrep® Imaging System contributed in a
positive way to capacity, workload, and turn-around-time in our
laboratories. Substantial increases in cytotechnologist produc-
tivity and laboratory capacities were observed in both Kansas
and Ohio. Turn-around-time decreased in Kansas. Productivity
and capacity can increase significantly with a specimen mix of
IDC, conventional, other liquid-based technology, and non-gyn
specimens.
LabOne, Inc., Lenexa, Kansas, LabOne of Ohio, Cincinnati, Ohio
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
90
ThinPrep® Based Tzanck Prep (TzPrep) for
the Detection of Herpes Simplex Virus
Gretchen Stuart, M.D.1, Ann Marie West, BS, CT (ASCP)2,
S. T. Gokaslan, M.D.1, M.H. Saboorian, M.D.1, Raheela
Ashfaq, M.D.1
INTRODUCTION: Tzanck smear is a valuable tool in identification
of vesiculobullous lesions of the skin such as Herpes Simplex
Virus (HSV) infection. The traditional Tzanck smear is obtained
by scraping the base of the ulcer; stained with Wright-Geimsa
stain and evaluated for the presence of multinucleated giant
cells and intranuclear inclusions. A modification of this tech-
nique includes alcohol fixed Papanicolaou stained smears. This
modified technique is used in our institution by non-dermatol-
ogists . The purpose of this study was to evaluate the feasibility
of ThinPrep® based Tzanck Prep for detection of HSV.
MATERIALS AND METHODS: The study was conducted prospec-
tively in the ICC clinic for women suspected of having HSV
infections. Each lesion was scraped twice; once with a blade and
the material smeared across a glass slide and immediately fixed
in 95% alcohol. In a second step, the base of the same lesion was
scraped again with a cytobrush and deposited in a 20-mL Pre-
servcyt® vial. The cases were alternated such that one or the
other step would occur first in an attempt to reduce bias. A single
TP slide was prepared. Both slides were stained with routine
Papanicolaou stain and concurrently reviewed by a single cyto-
pathologist.
RESULTS: Forty-two side by side modified TzS and Tz Preps were
available. Of the 42 TzS, 11 were unsatisfactory; 23 negative for
HSV and 8 positive for HSV compared to 6 unsatisfactory, 25
negative and 11 positive for HSV on TzPep. The sensitivity of
Tzanck smears vs. TzPrep was 73% vs. 91%; specificity and PPV
for both techniques was 100%. The NPV was 91% TzS vs. 97%
TzPrep. The overall preservation for TzPrep was higher com-
pared to TzSmear (48% air dried, 51% preserved).
CONCLUSION: The ThinPrep® Tzanck is a safe and effective
method for detection of HSV infection. Overall this study shows
that compared to Tzanck Smear, the Tzanck Prep showed
37.48% increased detection of HSV, and 44% reduction in un-
satisfactory rate with improvement in overall sample preserva-
tion
1
University of Texas Southwestern Medical Center, Dallas, Texas,
2
Parkland Health & Hospital System, Dallas, Texas
373
91
HPV Triage of LSIL Cases in Women Over
30 Years is Potentially Cost Effective: A
Multi-Institutional Study
Nadeem Zafar, M.D.1, Atef Hanna, M.D., Ph.D.1, Lester J.
Layfield, M.D.2, Patrick J. Ringer, B.A., CT(ASCP)3, M.
Nasar Qureshi, M.D., Ph.D.3
BACKGROUND: The prevalence of high-risk HPV types (HR-HPV)
in cervical samplings, diagnosed as LSIL, is reported to range
between 80-90% (HC2® method).1–3 This high prevalence has
lead to the current practice guidelines of recommending imme-
diate colposcopy for all LSIL cases.3 A previous study noted that
HPV triage of ASCUS cases is cost effective below HR-HPV prev-
alence of 60%.4 Furthermore, a preliminary multi-institutional
study reported HR-HPV prevalence below 60% in the subset of
LSIL patients above 30 years of age.5 We report here the HR-HPV
prevalence in LSIL cases from women over 30 years in age in 3
geographically distant populations.
DESIGN: Sixty consecutive residual samples (20 from each of the
3 participating centers) from liquid based Pap tests from women
aged 30/$ years (age range 30-66 years), routinely diagnosed as
LSIL, were evaluated for the presence and type of HPV. PCR was
performed at the Esoterix Laboratories (Calabasas Hills, Califor-
nia). DNA extracted from the specimen was amplified by PCR
using L1 specific primers for all characterized types of HPV plus
an internal control sequence. Specific PCR products were de-
tected electrophoretically and HPV was characterized for type by
restriction fragment length polymorphism (RFLP) analysis. Five
cases had insufficient material or unsatisfactory samples and
were excluded.
RESULTS: The HR-HPV prevalence in the total cohort (n"55)
averaged 54.5% (47.4%, 50% and 65% respectively for each of the
three participating centers). Similarly, non HR-HPV prevalence
(n"19) averaged 34.5% (42.1%, 31.2% and 30% respectively) and
cases negative for HPV (n"6) averaged 10.9% (10.5%, 18.8% and
5% respectively).
CONCLUSION: The prevalence of HR-HPV in women over 30 years
with LSIL varied with institution, with a mean of 54.5% (30/55)
and a range of 47.4-65%, much lower than earlier reported by
HC2® method 1,2
. An average of 45.4%, (34.5% non HR-HPV $
10.9% negative for HPV) were negative for HR-HPV cases. Co-
existence of HR-HPV and LSIL has a higher potential for con-
current presence of or progression to high grade cervical dys-
plasia, Current guidelines recommend definitive treatment of all
LSIL cases due to the reported high prevalence of HR-HPV in
LSIL cases. Our results show that the prevalence of HR-HPV in
LSIL cases in women over 30 years of age is significantly below
that previously reported. Testing for HR-HPV in LSIL cases,
therefore, is a cost-effective method of triaging cases into those
that could benefit from immediate colposcopy and those in
374 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
whom a costly and invasive follow-up procedure could be safely
avoided.
1
Diagnostic Cytopathol.2004, 30:426, 2 JNCI. 2000, 92:397,
3 between HPV positive and HPV negative ASC-US samples.
JAMA.2002, 287:2120, 4 Acta Cytol. 2004, 48:689, 5 Mod Pathol.
2005, 18:74.
1
University of Tennessee, Memphis, Tennessee 2University of
Utah, Salt Lake City, Utah, 3Lakewood Pathology Laboratory,
Lakewood, New Jersey
92
Morphologic Features of ASC-US
Interpretations in Papanicolaou Tests
Predictive of HPV Infection
Margaret Misztal, M.D., Paul Meyer, Christine Booth,
M.D., Razan Massarani-Wafai, M.D., Alia Salhadar, M.D.,
Eva Wojcik, M.D., MIAC
INTRODUCTION/PURPOSE: Analysis of Papanicolaou tests for
high-risk HPV has shown an increased incidence in samples with
squamous abnormalities. While high-risk HPV detection occurs
in the majority of samples interpreted as LSIL or HSIL, only
about half of samples interpreted as ASC-US are determined to
be high-risk HPV positive. Currently patients with ASC-US inter-
pretations may undergo analysis for HPV by one of three detec-
tion methods (PCR, Hybrid Capture, or in situ hybridization).
The aim of this study was to determine if there are any unique
cytologic features of ASC-US cells in Papanicolaou tests that are
predictive of HPV infection.
MATERIALS AND METHODS: We analyzed a total of 100 ThinPrep
Papanicolaou tests with a cytologic interpretation of ASC-US.
Fifty samples were high-risk HPV positive by in situ hybridiza-
tion (Inform HPV test, Ventana) and fifty samples were HPV
negative by in situ hybridization. Although the mean age of each
population was different (24 years old for HPV positive samples,
55 years old for HPV negative samples), it was not statistically
significant (P " 0.156). Sixteen cytologic features [parameters]
indicative of either ASC-US or HPV were analyzed including: 1)
nuclear size, 2) nuclear hyperchromasia, 3) increased N/C ratio,
4) nuclear membrane irregularity, 5) bi-nucleation, 6) coarse
chromatin, 7) irregular chromatin distribution, 8) prominent
nucleoli, 9) nuclear halo, 10) parakeratosis, 11) atypical parak-
eratosis, 12) “polka-dot” cells, 13) cytoplasmic polychromasia,
14) fungi, 15) hyperkeratosis, and 16) the presence of “other
cells” (balloon cells, kite cells, macrocytes). Each parameter was
scored in a binary fashion (e.g., present vs. absent) for each slide,
and Chi-square analysis was performed on the results.
RESULTS: Bi-nucleated squamous cells occurred significantly
more often in high-risk HPV positive ASC-US samples (P "
0.039). Increased nuclear size, nuclear hyperchromasia, in-
creased N/C ratio, nuclear membrane irregularity, coarse chro-
matin, irregular chromatin distribution, prominent nucleoli, nu-
clear halo, parakeratosis, atypical parakeratosis, “polka-dot”
cells, cytoplasmic polychromasia, fungi, hyperkeratosis, and the
presence of “other cells” did not show a significant difference
CONCLUSION: Bi-nucleated squamous cells are significantly more
likely to be present on Papanicolaou tests interpreted as ASC-US
from high-risk HPV positive patients than HPV negative patients.
Loyola University Medical Center, Maywood, Illinois
93
Detection of Adenocarcinoma Using the
ThinPrep Imaging System®
Debra Sabo, B.S., CT(ASCP), Julie Shorie, B.S., CT(ASCP),
Charles Biscotti, M.D.
INTRODUCTION: Assisted Pap test screening utilizing the Thin-
Prep Imaging System (TIS) (Cytyc Corp., Boxborough, MA) is
gaining popularity because published data suggest improved
screening accuracy and productivity. Much of the published
data involves detection of squamous lesions. Experience with
glandular abnormalities is limited. We analyzed a series of Pap
tests, from patients with adenocarcinoma involving the cervix, to
test this system’s effectiveness in detecting glandular abnormal-
ities.
METHODS: Searching the surgical pathology file identified all
cases of adenocarcinoma involving the cervix, diagnosed since
our implementation of the TIS. One of the authors (CB) con-
firmed all surgical diagnoses. Pap tests obtained within one year
prior to the surgical diagnosis formed the study group. A cyto-
technologist (CT) analyzed all Pap test slides and recorded the
presence or absence of atypical glandular cells, the number of
Imager selected fields of view (FOV) containing atypical glandu-
lar cells, and the total number of FOV containing atypical glan-
dular cells. The same CT (DS) identified a control group of 16
cases from consecutive negative slides submitted for QC review.
A second CT (JS), blinded to the original diagnoses, analyzed all
study and control slides recording a diagnosis after the 22 Im-
ager selected FOV.
RESULTS: The study group included 10 Pap tests from 10 patients
with adenocarcinoma involving the cervix (AIS and CIN-3 cases,
pure AIS-2 cases, endometrial adenocarcinoma-2 cases, and 1
case each of endocervical villoglandular adenocarcinoma and
CIN, AIS and invasive cervical adenocarcinoma, and adenosqua-
mous carcinoma). All study group slides had atypical glandular
cells. Importantly, the 22 Imager selected FOV contained atypi-
cal glandular cells in all cases, ranging from 2 to 22 (average 12)
FOV with atypical glandular cells. The 4 invasive cancer cases
had atypical glandular cells in 20 of the 22 Imager selected FOV
in one case and all 22 FOV in the other 3 cases. In contrast, the
2 pure AIS cases had atypical glandular cells in 6 and 2 of the 22
Imager selected FOV, respectively. In the blinded analysis, all
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
study group slides were interpreted as atypical. The diagnoses
included AIS-3 cases, atypical endocervical glandular cells-2
cases, atypical glandular cells NOS-2 cases, endometrial adeno-
carcinoma – 1 case, adenocarcinoma NOS–1 case, and atypical
endocervical glandular cells $ SIL–1 case. In contrast, only one
control case was interpreted as atypical (ASCUS).
CONCLUSIONS: In our series TIS assisted screening effectively
detected atypical glandular cells in Pap tests from patients with
adenocarcinoma involving the cervix. Pure AIS cases had rela-
tively few Imager selected FOV containing atypical glandular
cells compared to the invasive cancer cases.
Cleveland Clinic Foundation, Cleveland, Ohio
94
The Effect of Lubricant Jellies on the
Specimen Adequacy of ThinPrep Pap Tests
Shari McClure, CT(ASCP)1, Marianne Prey, M.D.2, Jeffrey
Chase, CT(ASCP)1
INTRODUCTION/PURPOSE: To determine the effect that specific
lubricant jellies have on the specimen adequacy of Pap tests
prepared using the ThinPrep methodology. To develop reliable
and reproducible criteria for microscopically identifying lubri-
cant jellies on ThinPrep Pap slides. To identify which, if any,
lubricant jellies are most compatible with Pap tests prepared
using the ThinPrep methodology.
MATERIALS AND METHODS: Twenty cervical cytology specimens
were collected and placed in ThinPrep vials. Each patient specimen
was split in half. The first half contained the patient specimen only.
One of nine lubricant jellies* was added to the second half of the
patient specimen. All specimens were processed on a ThinPrep
2000 instrument and the subsequent slides were stained with a
modified Papanicolaou stain. The change in specimen adequacy
was evaluated. *FemGlide, KY Jelly, Surgilube, Triad, Replens, Maxi-
lube, Walgreen’s Lubricating Jelly, AquaGel, and Aquasonic
RESULTS: All 20 vials not containing a lubricant jelly resulted in
a specimen adequacy of ‘Satisfactory for Evaluation’. 10 vials
containing a lubricant jelly (KY Jelly, Surgilube, Replens or Wal-
green’s Lubricating Jelly) resulted in a specimen adequacy of
‘Satisfactory for Evaluation’. The remaining 10 vials containing a
lubricant (FemGlide, Triad, Maxilube, Aquagel or Aquasonic)
resulted in a specimen adequacy of ‘Unsatisfactory for Evalua-
tion due to scant squamous component’.
CONCLUSIONS: The use of lubricant jellies may interfere with
slide preparation and can result in an ‘Unsatisfactory’ Pap test
based on specimen adequacy. Lubricant jellies can be micro-
scopically identified on Pap slides with a reasonable degree of
certainty. Different brands of lubricant jellies vary in the effect
which they have on the specimen adequacy of Pap tests. Our
laboratory has developed and implemented an educational
comment stating the adverse effects of lubricant jellies on Pap
375
test results. The educational comment is added to any report
with an ‘Unsatisfactory’ Pap result in which the use of a lubri-
cant jelly is suspected to have contributed.
1
Sonora-Quest Laboratories, Tempe, Arizona, 2Quest Diagnostics,
St. Louis, Missouri
95
p16 Immunostain in Conjunction with
Cytomorphology for Triage of Women with
ASC-US: A Comparison Study with HPV DNA
Testing
Ming Guo, M.D., Nour Sneige, M.D., Yee Jee Jan, M.D.,
Bramara Mutyala, B.S., MT, CT(ASCP), Shobha Patel,
B.S., CT(ASCP), Valerie Dunmire, B.S., M.S., David
Cogdell, B.S., M.S.
INTRODUCTION: p16 immunocytochemistry assay has been re-
ported to have a potential utility in triage of women with mild
abnormal Pap results. However, non-specific positive p16 stain
in non-neoplastic cells may cause a high p16 positive rate lead-
ing to low specificity of the test. In this study, we evaluated a
combined p16 immunocytochemistry and cytomorphology
strategy in triage of women with ASC-US Pap results and com-
pared the findings with HPV DNA testing results.
MATERIALS AND METHODS: We selected 39 cases of SurePath Pap
tests with ASC-US and follow-up biopsies from M.D. Anderson
Cancer Center between November 2004 and March 2005. The
mean age of the women is 40.6 year old with a median of 41 year
old. Duplicated slides were stained with p16 (Novocastra, En-
gland). The residual material was tested for HPV DNA by PCR
using GP5$/GP6$ primer. The specimens containing only p16
positively stained dysplastic cells were classified as p16 positive
cases. The results of p16 and HPV DNA testing were compared
with the follow-up biopsies.
RESULTS: p16 was positive in 33.3% (13/39), whereas HPV DNA
was positive in 48.7% (19/39) of women with ASC-US. All 7 cases
of CIN 2/3 were positive for p16. Five out of seven CIN 2/3 cases
were positive for HPV DNA. p16 immunocytochemistry assay
showed higher sensitivity, specificity, positive and negative pre-
dictive values than those of HPV DNA testing to detect CIN 2/3.
Biopsy Diagnoses
Negative/CIN 1 CIN2/3 Total
HPV$; p16$ 4 5 9
HPV-; p16$ 2 2 4
HPV$; p16 - 10 0 10
HPV-; p16- 16 0 16
Total 32 7 39
376 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
% Sensitivity % Specificity % PPV % NPV
P16 100.0 (7/7) 81.3 (26/32) 53.9 (7/13) 100.0 (26/26)
HPV 71.4 (5/7) 56.3 (18/32) 26.3 (5/19) 95.0 (19/20)
CONCLUSIONS: p16 immunocytochemistry assay in conjunction
with cytomorphology is highly sensitive and specific to detect
CIN 2/3 from women with ASC-US Pap results and may be more
efficient than HPV DNA testing as a triage tool for women with
ASC-US Pap results.
The University of Texas, M.D. Anderson Cancer Center, Houston,
Texas
96
An Alternative Approach to Ten Percent
Rescreen of Gynecologic Specimens as a
Quality Control Method: Is 100%
Prescreening a Solution?
Marsha G. Unverferth, M.Ed., SCT(ASCP), Afsoon Moktar,
M.B.A., B.H.S., CT(ASCP)
OBJECTIVE: The effectiveness of ten percent rescreen of negative
gynecologic specimens has been questioned by some experts.
This study explores the value of performing 100 percent pre-
screen of gynecologic specimens as an alternative method of
quality control.
MATERIALS AND METHODS: During a period of 23 weeks, 10,917
gynecologic smears were examined in the study and consisted of
both thin layer and conventional types of specimens. Each spec-
imen was submitted to a one minute, 10X evaluation and des-
ignated either R (a suspected abnormal specimen) or N (a sus-
pected negative specimen) by a prescreening cytotechnologist.
The specimens were then evaluated by the routine cytotechnol-
ogist in the usual manner. Any discrepancies between the pre-
screening and routine cytotechnologist were evaluated by a third
cytotechnologist. The confirmed abnormal designations by the
prescreener were submitted to the pathologist for final diagno-
sis.
RESULTS: Out of the 10,884 gynecologic specimens evaluated,
733 (6.73%) discrepant cases were identified. Of the 733 discrep-
ant cases, 349 (47.61%) were overcalled, while 327 (44.61%) were
undercalled by the prescreener. Fifty seven (7.78%) designated
as R by the prescreener and confirmed by the third cytotechnol-
ogist were submitted to the pathologists for review. Of the fifty
seven cases, 31 (54.39%) were called ASC-US, 7 (12.28%) were
called LSIL, 2 (3.51%) were called HSIL and 17 (29.82%) were
called reactive by the pathologist.
CONCLUSIONS: The pick-up of 40 abnormal cases (ASC-US and
above) through the 100% prescreen method of quality control is
note-worthy; although these results do not statistically (0.37%)
fully justify the implementation of this method.
Bellarmine University, Louisville, Kentucky, Medical Laboratory
Consultants, Louisville, Kentucky
97
Does HPV Genotyping Help to Predict the
Presence of CIN2/3 in Women with Mild
Abnormal Pap Results?
Ming Guo, M.D., Nour Sneige, M.D., Yee Jee Jan, M.D.,
David Cogdell, B.S., M.S., Valerie Dunmire, B.S., M.S.
INTRODUCTION: HPV DNA testing has been widely used as a
triage tool for women with ASC-US or co-screening with Pap test
for women of 30 years and older. However, the role of HPV
genotyping to predict the presence of CIN 2/3 in women with
mild abnormal Pap results is still unclear. It has been reported
that 8 types of high-risk HPV (16, 18, 31, 33, 35, 45, 52 and 58) are
responsible for more than 80% of cervical cancer. We conducted
HPV genotyping for the above 8 HPV types using real-time PCR
in SurePath Pap specimens and compared the HPV genotyping
results with the follow-up biopsy.
MATERIALS AND METHODS: We selected 83 cases of SurePath Pap
tests (20 cases of HSIL, 23 cases of LSIL and 40 cases of ASC-US)
with follow up biopsies from M.D. Anderson Cancer Center
between November 2004 and April 2005. The mean age of
women was 40 years old with median of 41 years old. HPV DNA
was tested by PCR using GP5$/GP6$ consensus primer. HPV
DNA positive cases were further genotyped using real-time PCR
for 8 types of high-risk HPV (types 16, 18, 31, 33, 35, 45, 52 and
58). HPV DNA testing results were stratified with cytology diag-
noses and compared with biopsy results.
RESULTS: CIN 2/3 was diagnosed in all of the HSIL (20/20,
100%), 7 out of 23 (30.4%) LSIL and 7 out of 40 (17.5%) ASC-US.
HPV DNA positivity increased significantly from ASC-US to
LSIL/HSIL groups (Table). The positivity of the major HPV types
(type 16, 18, 31, 33, 35, 45, 52 and 58) also increased with the
severity of Pap results compared to other HPV types. The asso-
ciation of the major HPV types with HPV positive CIN 2/3 was
high and consistent in ASC-US (80.0 %, 4/5), LSIL (85.7 %, 6/7)
and HSIL (82.4 %, 14/17). HPV type 16 was observed in 66.7% of
CIN 2/3 (16/24). The chance of having CIN 2/3 was 50% (4/8) in
ASC-US group and 46.2% (6/13) in LSIL group when HPV type 16
was tested positive.
% ASC-US % LSIL %HSIL
GP5$/GP6$ 47.5 (19/40) 82.6 (19/23) 85.0 (17/20)
Major HPV Types 42.1 (8/19) 68.4 (13/19) 82.4 (14/17)
Other HPV Types 57.9 (11/19) 31.6 (6/19) 17.6 (3/17)
CONCLUSIONS: The major high-risk HPV types (types 16, 18, 31,
33, 35, 45, 52 and 58) are closely associated with CIN 2/3 regard-
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
less of Pap test classification. Positive results of HPV type 16 in
cytology specimens are highly specific to predict CIN 2/3 from
women with LSIL and ASC-US Pap results. HPV genotyping is
useful in cytology to predict the presence of CIN 2/3 in women
with mild abnormal Pap results.
The University of Texas, M.D. Anderson Cancer Center, Houston,
Texas
98
ASC-H: Post Implementation Correlation of
the ThinPrep® Imaging System with Biopsy
Findings
Ann Marie West, B.S., CT (ASCP)2, S.T. Gokaslan, M.D.1,
M.H. Saboorian, M.D.1, Raheela Ashfaq, M.D.1
BACKGROUND: Clinical trials for the ThinPrep® Imaging Sys-
tem (TIS) have indicated an increased sensitivity for the de-
tection of high-grade lesions. It has been previously demon-
strated that the ASC-H diagnostic category leads to a better
detection of cervical intraepithelial neoplasia and is a predic-
tor of underlying HSIL. To determine if the TIS shows in-
creased sensitivity for ASC-H, we examined TIS ASC-H rate in
TPPT, correlated to surgical biopsy and compared these to a
historical cohort of ASC-H cases examined prior to implemen-
tation of the TIS.
DESIGN: The study period was 11 months (Post-Imager) in
duration (May 2004-March 2005) Post TIS implementation
ThinPrep® Pap Test (TPPT) interpreted as atypical squamous
cells, cannot exclude HSIL (ASC-H) constituted the cases for
the study (116 cases). The slides were reviewed by a cytotech-
nologist (CT) using the TIS and referred to a pathologist for
final interpretation. The classification of specimens as ASC-H
was made according to the 2001 Bethesda System (TBS) for
reporting cervical cytology. Prospective data was collected for
the cohort of all ASC-H Paps and correlated with subsequent
surgical biopsy results. The data was analyzed to evaluate the
screening rates of ASC-H utilizing the TIS for the detection of
histologic HSIL (CIN 2 or worse) in terms of sensitivity, spec-
ificity, positive predictive value (PPV), and negative predictive
value (NPV). These results were compared to ASC-H data
collated in the 11 months prior to implementation of the TIS
(Pre-Imager).
RESULTS: The pre-imager ASC-H rate was 0.25% (117/2609) vs.
0.24% (114/2486) post imager. Overall 83.5% of ASC-H cases in
pre-imager correlated with an abnormal biopsy of CIN I $and
with 31.7 % (27/85) ASC-H cases correlating with a biopsy diag-
nosis of CIN II$. Post imager ASC-H correlated 68% (51/75) with
an abnormal biopsy and 34.6% (26/75) with a biopsy diagnosis of
CIN II$. Post imager, a larger cohort of ASC-H on biopsy were
negative 32% vs. 16% pre-imager. Also the numbers of LSIL on
biopsy were reduced 51% pre-imager vs. 33.3% post imager.
377
CONCLUSIONS: This study shows that the detection of ASC-H
cases by TIS is similar to manual with the same degree of
correlation with underlying high grade lesions on biopsy. The
higher percentage of negative biopsies on ASC-H post-imager
may be due to overcalls or may be related to biopsy sampling
and require further follow-up. The reduction in post imager LSIL
correlation may be due to improved detection of LSIL in the post
imager era.
1
University of Texas Southwestern Medical Center, Dallas, Texas,
2
Parkland Health & Hospital System, Dallas, Texas
99
Atypical Squamous Cells, Cannot Exclude a
High Grade Squamous intraepithelial lesion
(ASC-H) – A Two Year Experience and
Follow-up
Lakshmy Parameswaran, M.D., Nidhi Kumar, M.D., Vinod
Shidham, M.D., Zainab Basir, M.D.
INTRODUCTION: A newly introduced category in the 2001 Be-
thesda system is ASC-H. The clinical significance and manage-
ment of ASC-H diagnosis in gynecological smears is still under
investigation. In this study, we evaluated the follow-up diagnosis
of ASC-H gynecological smears in our laboratory over a period of
two years.
MATERIALS AND METHODS: Data was obtained from the archived
files of our laboratory which processes approximately 87,000
gynecological smears a year, including 70,000 liquid based cy-
tology smears (SurePath™) and 17,000 conventional smears.
During the two year period considered in the study, 243 cases
were interpreted as ASC-H. Follow up either by tissue biopsy or
gynecologic smear was available for 156 (64.2%) cases. Since the
laboratory also processes specimens from outside the hospital
clients, some of these patients have had biopsies at other insti-
tutions, results of which were not available in our laboratory
database.
RESULTS: Among the 156 patients with clinicopathologic follow
up, 106 (68%) cases had follow up tissue biopsies and 50 (32%)
cases had follow up gynecologic smears.
LSIL (30.2%) HSIL (34.9%)
NEGATIVE
(34.9%) CIN1/ HPV CIN2 CIN3/CIS/AIS DYSPLASIA NOS
37 32 16 18 3
Table 1. Follow up Biopsy Results
In cases with both follow up tissue biopsy and smears, the tissue
biopsy was given preference. In the absence of tissue biopsy, the
subsequent gynecologic smear was considered follow up.
378 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
POSITIVE (42%)
NEGATIVE
(58%) ASCUS LSIL LSIL! ASC-H ASC-H HSIL CA
29 11 3 0 2 5 0 2/23 (9%) of cases, screening errors for 4/23 (18%) and interpre-
Table 2. Follow up Gynecologic Smear Results
CONCLUSIONS: Patients with ASC-H interpretation on gyneco-
logic smears should have further clinical follow up in light of the
observation of high index of clinically significant cervical intra-
epithelial lesions (65.1% by biopsy and 42% by gynecologic
smears) among them.
Medical College of Wisconsin, Milwaukee, Wisconsin
100
Screening, Diagnostic and Sampling Errors
in the Diagnosis of Endocervical
Adenocarcinoma in situ (AIS) in Liquid-
Based Pap Tests (LBPT, SurePath)
Dan McKeon, SCT(ASCP)1, Brian Berg, M.D.2, Stuart
Cameron, M.D.2, Klint Kjeldahl, CT(ASCP)1, Evin Gulbahce,
M.D.1, Stefan Pambuccian, M.D.1
INTRODUCTION/PURPOSE: Cytologic screening for cervical cancer
and its precursors (squamous intraepithelial lesions, SIL) by the
Pap test has led to a dramatic decrease in incidence of squamous
cell carcinomas of the cervix, but had little impact on the inci-
dence of adenocarcinomas. This suggests that the cytologic di-
agnosis of the cervical adenocarcinoma precursors, AIS, is more
difficult than that of SILs. The current study addresses the ques-
tion whether this is due to more difficult sampling because of the
location of the lesions in the endocervical canal (sampling er-
rors), failure to detect the abnormal cells (screening errors), or
failure to recognize the cells as glandular or as dysplastic (inter-
pretative errors).
MATERIALS AND METHODS: LBPT obtained within 12 months of
a histologic diagnosis of AIS or AIS$SIL made during a 4-year
period (2001–2004) were reviewed, admixed with contemporary
control slides to reduce bias and reclassified according to Be-
thesda 2001 criteria. In cases where no diagnosis of a glandular
abnormality had been originally made, the abnormal glandular
cells that were found on review were considered screening errors
if originally not dotted and interpretative errors if they had been
dotted. A sampling error was assumed if abnormal glandular
cells were not found on review.
RESULTS: During this interval we identified 51 cases with a
histologic diagnosis of AIS or AIS$SIL. Of these, 28 had a LBPT
preceding the diagnosis of AIS within our system, 23 of which
(from 21 women aged 19-61, mean 35) could be retrieved. These
were all satisfactory (21 screening and 2 diagnostic) SurePath
LBPT with adequate transformation zone component. The orig-
inal diagnosis documented the presence of a glandular abnor-
mality in 10/23 (43.5%)(6 AGC, 2 AIS and 2 AGC$SIL), while 11
were diagnosed as squamous abnormalities (4 HSIL, 3 ASC-US,
3 LSIL, 1 ASC-H) and 2 as NILM. Sampling errors accounted for
tative errors for 6/23 (26%). There was no difference between the
original glandular abnormality detection rate between cases
with and without coexistent squamous abnormalities on histol-
ogy.
CONCLUSIONS: Unlike a recent conventional Pap test study
where sampling errors were the main cause of failure to report
the glandular abnormality in cases of AIS and AIS$SIL we found
that interpretative errors (mostly the misidentification of glan-
dular abnormalities as squamous) and screening errors were
common especially in the presence of a squamous abnormality.
This is possibly due to the “satisfaction of search” phenomenon.
Our findings suggest the need for more attention to glandular
lesions in LBPT and possibly the need to develop more effective
criteria, specifically designed for use in LBPT.
1
Fairview University Medical Center, Minneapolis, Minnesota,
2
University of Minnesota, Minneapolis, Minnesota
101
Effect of Reflex HPV Testing on the ASC-US
Rate: Our First Year Experience
Edina Paal, Katherine Berezowski, M.D., Mary K. Sidawy,
M.D.
PURPOSE: eflex testing for high-risk HPV DNA is the preferred
management for women with ASC-US when liquid-based prep-
aration (LBP) is used. Concerns have been raised that the im-
plementation of HPV reflex testing may result in an increase in
the ASC-US rate. The aim of this study is to compare our ASC-US
rate before and after the implementation of HPV reflex testing,
and to evaluate the detection rate of high-risk HPV DNA in our
patient population.
MATERIALS AND METHODS: All ASC-US cases reported on LBP
were retrieved from the computer files of our institution since
the introduction of the reflex HPV testing (April 2004 to April
2005). Reflex HPV testing was performed in a reference labora-
tory using the Digene Hybrid Capture methodology. The rate of
ASC-US cases, as well as the ASC/SIL ratio for the study period
was determined and compared to the data of the two previous
years. Although the number of cervical smears slightly decreased
during the study period, our patient population did not signifi-
cantly change during this three-year period.
RESULTS: Out of 2281 satisfactory LPB, 98 cases reported as
ASC-US were referred to a reference laboratory for reflex HPV
testing during the study period. In forty eight cases (49%) one or
more strains of high- risk HPV DNA was detected. The following
table compares the ASC-US rate, the SIL rate and the ASC/SIL
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
ratio of the study period with the preceding two years.
2002 2003 Study period
(2553 cases) (2581 cases) (2281 cases)
ASC-US 93 (3.8%) 107 (4.1%) 98 (4.3%)
SIL 63 (2.5%) 88 (3.4%) 56 (2.5%)
ASC/SIL 1.5 1.2 1.7
CONCLUSIONS: Our results show that the implementation of HPV
reflex testing did not alter the ASC-US rate in our laboratory.
High-risk HPV DNA was detected in 49% of ASC-US cases. The
latter rate is in keeping with the reported rate of 50% of the ALTS
trial
Department of Pathology, George Washington University, Wash-
ington, DC
102
HPV Results Influence the Management of
Patients Diagnosed as Atypical Squamous
Cells of Undetermined Significance (ASC-
US)
Joy Sen, Harvey Cramer, M.D.
INTRODUCTION/PURPOSE: eflex HPV testing for ASC-US cases
using the Hybrid Capture® II DNA test was recently introduced
at our hospital. In this study, we determine the effect of HPV
testing results on the clinical management of patients diagnosed
with ASC-US.
MATERIALS AND METHODS: All of the cytology cases diagnosed as
ASC-US over the 18 month period since HPV testing began at our
institution were identified. The follow-up cytology and surgical
pathology (SP) findings were correlated with the high-risk HPV
(HR-HPV) findings.
RESULTS: Of the 225 cases diagnosed as ASC-US, 28 (12%) were
conventional smears (CS) and 197 were ThinPrep (TP) Pap tests
(88%). HR-HPV testing was performed for 113 of 197 TPs (57%).
Of the 84 TPs in which HPV testing was not performed, follow-up
cytology was obtained in 38 cases (45%) and follow-up SP was
obtained in 14 cases (17%). Follow-up LSIL was identified in 3 of
38 cases (8%) and HSIL was identified in 2 of 38 cases (5%). Of
the 14 cases with SP follow-up, there were 10 cases of mild
dysplasia (71%) and 1 case of moderate dysplasia (7%). HR-HPV
was positive in 51 cases (45%), negative in 52 cases (46%), equiv-
ocal in 7 cases (6%) and unsatisfactory in 3 cases (3%). Cytologic
follow-up was obtained in 29 of 51 HR-HPV-positive cases (57%),
15 of 52 HR-HPV-negative cases (29%), and 6 of 10 HR-HPV-
equivocal/unsatisfactory cases (60%). SP follow-up was obtained
in 26 of 51 HR-HPV-positive cases (51%), 3 of 52 HR-HPV-
negative cases (6%) and 3 of 10 HR-HPV-equivocal/unsatisfac-
tory cases (30%). Among the 29 HR-HPV-positive cases with
cytologic follow-up, there were 9 cases of LSIL (31%), and 1 case
of HSIL (3%). Among the 26 HR-HPV-positive cases with SP
379
follow-up, there were 18 cases of mild dysplasia (69%) and 6
cases of moderate (5) or severe (1) dysplasia (23%). Follow-up of
the 10 HPV-equivocal/unsatisfactory cases identified 2 cases of
LSIL, no cases of HSIL, 2 cases of mild dysplasia and one case of
moderate dysplasia. Among the HR-HPV-negative group, fol-
low-up disclosed no cases of SIL or dysplasia.
CONCLUSION: HPV results influence the clinical management of
ASC-US patients. Follow-up rates were highest for HR-HPV-
positive patients. No cases of SIL/dysplasia were noted on
follow-up of patients who were negative for HR-HPV while fol-
low-up SIL/dysplasia was noted for the majority of ASC-US/HR-
HPV-positive patients. Continued follow-up of ASC-US/HR-
HPV-negative patients is unnecessary while colposcopy and
biopsy is recommended for all ASC-US patients who are positive
for HR-HPV.
Indiana University School of Medicine, Indianapolis, Indiana
103
Long-term Follow-up of Unsatisfactory
Cervical Smears
Ben Coleman1, Angela Moman1, Terry Bilyeu, CT(ASCP)2,
Harvey Cramer, M.D.1
INTRODUCTION/PURPOSE: Unsatisfactory cervical smears can be
caused by a number of factors including excessive inflammation
and blood which may be associated with underlying malignancy.
In this study, long-term follow-up of conventional cervical cy-
tology cases designated as unsatisfactory for evaluation was
performed.
MATERIALS AND METHODS: A computerized search of the cytol-
ogy records was performed at our institution for the 14 year
period extending from March 1988 through February 2002. All
cervical cytology cases designated as unsatisfactory for evalua-
tion were identified and the follow-up cytologic and histologic
diagnoses were recorded.
RESULTS: During the 14 year period encompassed by this study,
a total of 578 cervical cytology cases were diagnosed as unsatis-
factory for evaluation. In 188 cases (33%), there was neither
cytologic nor histologic follow-up. In 390 cases (67%), there was
either follow-up cytology (329 cases, 84%) or surgical biopsy (61
cases, 61%). The follow-up time for the patients undergoing
cytology follow-up alone arranged from 1 to 4,675 days with a
standard deviation of 865 days. The follow-up time for patients
undergoing surgical pathology follow-up ranged from 12 to 3905
days with a standard deviation of 719 days. The follow-up cyto-
logic diagnoses were as follows: negative (266 cases, 81%),
ASC-US (32 cases, 10%), LSIL (26 cases, 8%), HSIL (3 cases, 1%)
and AGC (2 cases, % 1%). The follow-up histologic diagnoses
were: endometrial or endocervical polyp (5 cases, 8%), inflam-
matory, benign or atypical (10 cases, 16%), mild dysplasia/HPV
effect (22 cases, 36%), dysplasia NOS (3 cases, 5%), high grade
380 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5

dysplasia (15 cases, 25%) and carcinoma (6 cases, 10%). Among by cervical biopsy. The corresponding HPV type groups are
the 6 cases of carcinoma, there were 4 adenocarcinomas and 2 tabulated below.
squamous cell carcinomas.
CONCLUSION: The long-term risk of developing carcinoma fol- Multiple
lowing an unsatisfactory cervical smear was only 1.5% among Cases DigHR! AddHR! UR! LR! Types
the patients subjected to follow-up. An unexpected large per-
HSIL Pap 58 51(87.9%) 3(5.2%) 3(5.2%) 1(1.7%) 8.6%
centage of patients with unsatisfactory cervical cytology findings LSIL Pap 314 187(59.6%) 81(25.8%) 20(6.4%) 26(8.3%) 24.5%
lacked both cytologic and histologic follow-up. Although ac- HSIL Biopsy 115 100(87.0%) 10(8.7%) 4(3.5%) 1(0.9%) 15.7%
counting for only 16% of patients who had undergone follow-up LSIL Biopsy 331 210(63.4%) 69(20.8%) 25(7.6%) 27(8.1%) 18.0%
examinations, surgical pathology identified all of the carcinomas There were 83 cases with sequentially changing HPV types. Of
while no carcinomas were identified among the patients who these, 73.5% had a positive test point without a Digene HCII
were followed by cytology only. type. In addition, 3 cases were diagnosed as squamous cell
1
Indiana University School of Medicine, Indianapolis, Indiana, carcinoma (SCC) by cervical biopsy. One was positive for a
2
Wishard Memorial Hospital, Indianapolis, Indiana Digene high risk type, one positive for a non-Digene additional
high risk type (type 53), and one positive for both a Digene and
a non-Digene additional high risk type.
104 CONCLUSION: The results show that a significant number of cases
of SIL diagnosed by Pap test (31%) or biopsy (25.5%) are positive
Withdrawn only for high/intermediate or unknown risk HPV types not found
in the Digene panel. Of these, 10.4% of Pap tests and 12.2% of
biopsies were HSIL. In addition, 73.5% of cases with sequentially
105 changing HPV types had a test point that would have been
undetected by the Digene panel. Finally, one case of SCC was
Analysis of HPV Types by PCR Testing in positive for one of the additional high risk HPV types not found

Squamous Intraepithelial Lesions (SIL) in the Digene panel.

Diagnosed by Pap Test and Cervical Biopsy Caritas St. Elizabeth’s Medical Center, Boston, Massachusetts

Brenda J. Sweeney, B.S., SCT(ASCP), Shelly E. Kenyon,

M.S. CT(ASCP), Leanne M. Barber, B.S., CT(ASCP), James
F. Happel, B.S., DLM(ASCP)HTL, Douglas R. Schneider,
M.D.
INTRODUCTION: Digene’s HCII high/intermediate risk HPV test
panel is widely used for detection of HPV infection in abnormal
Pap tests and detects 13 types. At least 16 additional types have
been put forward for inclusion in the high/intermediate risk
category. Others are of unknown risk. This study compares HPV
types detected by PCR in SIL diagnosed by Pap test or cervical
biopsy.
METHODS: Cases of SIL found by Pap test or cervical biopsy from
a series of 1,012 Pap tests were divided into high grade (HSIL)
and low grade (LSIL) lesions and correlated with HPV PCR test
results. The HPV types were grouped into the Digene HCII high/
intermediate risk types (DigHR), additional high/intermediate
risk types as determined by the reference laboratory (AddHR),
unknown risk types (UR), and low risk types (LR). Cases with
multiple HPV types were included in the HCII group if one HCII
HPV type was present. Otherwise they were grouped in the
additional high/intermediate or unknown risk categories as ap-
propriate. Finally, cases of sequentially changing HPV infection
were analysed with respect to the Digene panel types versus
other types.
RESULTS: Of the 1,012 pap tests analysed for HPV, 372 cases were
diagnosed as SIL by Pap test and 451 cases were diagnosed as SIL
106
Cytologic Features of Endocervical
Adenocarcinoma in situ (AIS) in Liquid-
Based Pap Tests (LBPT, SurePath)
Stuart Cameron, M.D.1, Brian Berg, M.D.1, Dan McKeon,
SCT(ASCP)2, Samy Amirouche, CT(ASCP)2, Klint Kjeldahl,
CT(ASCP)2, Evin Gulbahce, M.D.1, Stefan Pambuccian,
M.D.1
INTRODUCTION/PURPOSE: A number of recent studies have in-
vestigated the validity and applicability to LBPT of diagnostic
criteria for endocervical glandular lesions as described in con-
ventional cervical smears. However, since these studies have
typically included cases diagnosed as atypical glandular cells on
Pap tests, where the likelihood of a glandular endocervical lesion
on follow-up is very low and squamous lesions predominate, we
have undertaken a study to assess the presence of the described
cytologic features of endocervical glandular lesions in LBPT pre-
ceding a histologic diagnosis of AIS.
MATERIALS AND METHODS: LBPT obtained within 12 months of
a histologic diagnosis of AIS or AIS$SIL made during a 4-year
period (2001–2004) were retrieved, admixed with contemporary
control slides to reduce bias and reviewed by two observers over
a multi-headed microscope. Forty cytologic features relating to
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
cellularity, background, cytoarchitecture and cytoplasmic, nu-
clear or nucleolar features were either recorded as present/
absent or semi-quantitated.
RESULTS: 23 LBPT from 21 women aged 19-61 (mean 35) con-
stituted the study set. Some of the diagnostic criteria for AIS were
seen in 21/23 cases allowing the diagnosis of AGC in 11 and of
AIS in 10 cases. The background did not show diathesis, blood or
significant inflammation. The architectural features that were
most commonly encountered were crowding (20/21) and
pseudostratification (17/21), while rosettes (3/21), atypical single
cells (4/21) and feathering (4/21) were less commonly encoun-
tered. Cytologic features such as slight nuclear hyperchromasia
(21/21), coarse chromatin (21/21), high nuclear to cytoplasmic
ratio (11/21), and prominent nucleoli (9/21) were seen in a
significant number of cases. None of the controls showed any of
these features. Mitoses and apoptotic debris were rarely seen.
CONCLUSIONS: Our study suggests that the cytologic criteria for
the diagnosis of endocervical glandular lesions in LBPT are sim-
ilar to those described in conventional Pap smears. However,
feathering and atypical single cells were less commonly encoun-
tered in our study than described in the conventional Pap smear.
A comparative study between conventional Pap smears and
LBPT preceding a histologic diagnosis of AIS is, however, needed
to detect any subtle differences that may exist.
1
University of Minnesota, Minneapolis, Minnesota, 2
Fairview
University Medical Center, Minneapolis, Minnesota
107
LSIL with ASC-H in Cervical Smears -
Should it be an Independent Category for
Proper Management and QA Statistics?
N. Kumar, L Parameswaran, M.D., A. Cafaro, Z Basir,
M.D., B. Behmaram, M.D., A.R. Chavan, M.D., V.B.
Shidham, M.D., FIAC, MRCPath
INTRODUCTION: In practice, cervical smears exhibiting definite
‘low grade squamous intraepithelial lesion’ (LSIL) occasionally
also contain admixed cells of ‘atypical squamous cells, cannot
exclude high grade squamous intraepithelial lesion’ (ASC-H).
Unfortunately the reporting of this concurrence is not standard-
ized. While some laboratories interpret this as LSIL and com-
ment on the possibility of high grade lesion, others essentially
report the opposite. A standardized method of reporting would
help to guide subsequent therapy and better avail itself for
monitoring and quality assurance.
MATERIALS AND METHODS: To evaluate the significance of ‘low
grade squamous intraepithelial lesion, cannot exclude high
grade squamous intraepithelial lesion’ (LSIL-H) as a distinct
category, we compared follow up cervical biopsy findings in
cases with cervical smears originally interpreted as LSIL, LSIL-H,
and ASC-H. Total 77,979 cases reported during one year period
381
by five cytopathologists were analyzed. Of these, 1,778 cases
(ages 16 to 65 years) were interpreted as LSIL, LSIL-H, and
ASC-H. Concurrent biopsy results (performed at the time of
cytology or within 3 months of cytology) were available in 683
cases (557 LSIL, 88 LSIL-H, and 38 ASCH). Chi-square test was
applied for statistical analysis.
RESULTS: Table: 1 The pattern of biopsy results in 683 cases.
Biopsy results LSIL* LSIL-H* ASCH*
Negative 32% (179) 24% a (21) 32% (12)
Low grade lesions
(HPV & CIN-1) 58% (323) 43% c (38) 37% c (14)
High grade
lesions (CIN-2
& CIN-3) 10% b (55) 33% b (29) 31% b (12)
Total 100% (557) 100% (88) 100% (38)
*Degrees of freedom: 4, Chi-square " 44.6970046011447, p # 0.001. CIN, cervical intraepithelial
neoplasia.
The differences in the pattern of biopsy results amongst the
three categories* were statistically significant (p value # 0.001).
As compared with those called LSIL b; LSIL-H and ASC-H showed
greater association with high grade dysplasia. Compared to LSIL
and ASC-H; negative biopsy results were least common with
a
LSIL-H . LSIL-H c had higher association with low grade lesion
than ASC-H c.
CONCLUSION: Distinct biopsy result pattern, different from LSIL
and ASC-H, justifies a separate category for LSIL-H.
Department of Pathology, Medical College of Wisconsin, Milwau-
kee, Wisconsin
108
A Subset of ASC-H is Associated with HPV
Results in Biopsy
N. Kumar, L. Parameswaran, M.D, A. Cafaro, Z. Basir,
M.D., B. Behmaram, A.R. Chavan, M.D., V.B. Shidham,
M.D., FIAC, MRCPath
INTRODUCTION: Cytomorphological spectrum of human papil-
loma virus (HPV) in cervical smears is broad. As reported previ-
ously, groups of smaller atypical parakeratotic cells with high
nuclear to cytoplasmic ratio seen as hyperchromatic crowded
groups may sometimes be difficult to interpret. Such cells in the
absence of unequivocal features of ‘low grade squamous intra-
epithelial lesion’ (LSIL) may be interpreted as atypical squamous
cells: cannot exclude high grade lesion (ASC-H).
MATERIALS AND METHODS: To evaluate the association of ASC-H
and HPV, we compared the biopsy results in cases with LSIL,
LSIL with ASC-H (LSIL-H), and ASC-H in cervical smears. 1,778
cases (ages 16 to 65 years) out of total 77,979 cases reported
during one year period were analyzed by five cytopathologists.
Results of biopsy performed at the time of cytology or within 3
382 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
months of cytology were available in 683 cases (557 LSIL, 88
LSIL-H, and 38 ASC-H) (634 liquid based- SurePathTM Preps, 49
conventional smears). Chi-square test was applied for statistical
analysis (Table 1).
Biopsy results % (n)
Low grade lesions
Cytopathology HPV with
(n) HPV CIN-1 CIN-1
LSIL* (557) 13% (70) 33% (184) 12% b (69)
LSIL-H* (88) 10%a (9) 17% a (15) 16% b (14)
!ASC-H* (38) 13% a (5) 21% a (8) 3% c (1)
*Degrees of freedom: 6, Chi-square " 46.1553235864449, p # 0.001.
n, number of cases; CIN, cervical intraepithelial neoplasia
RESULTS: The pattern of biopsy results in 683 cases (Table 1).
Overall, the differences in the pattern of biopsy results observed
in each of the three categories* were statistically significant ( p
value # 0.001). CIN-1 c was less frequent with ASC-H alone, as
compared to HPV a and HPV with CIN-1 a. The frequency of
b
CIN-1 in LSIL-H was comparable to that with LSIL alone (p
value 1). However, the frequency of HPV a and HPV with CIN-1 a in
LSIL-H were comparable to those with ASC-H alone (p value 1).
CONCLUSIONS: A subset of ASC-H (either alone or in association
with LSIL) has overlapping cytomorphology resembling HSIL
and is associated with HPV. Proper understanding of the cyto-
morphological spectrum of HPV cytopathic effects in addition to
widely recognized koilocytes may prevent the interpretation of
this subset as ASC-H.
Department of Pathology, Medical College of Wisconsin, Milwau-
kee, Wisconsin
109
Effects of Selected Lubricants on the
ThinPrepTM Pap Test: Cytomorphology and
Unsatisfactory Rate
Rukmini Modem, M.D., Liron Pantanowitz, M.D., Roxanne
Florence, M.D., Maryanne Hornish, CT(ASCP), M.B.A.,
Karen Russett, CT(ASCP), Robert Goulart, M.D.
INTRODUCTION: ACOG guidelines recommend lubricants be
avoided when obtaining a Pap test. However, lubricants may be
used with plastic specula or to minimize patient discomfort.
Prior studies of lubricant effects on cervical cytology have been
largely limited to conventional Pap smears. Our aim was to study
the effects of lubricant on the morphology and cellularity of
liquid-based (ThinPrepTM) Pap tests, as identified within a large
hospital-based laboratory setting.
MATERIALS AND METHODS: A total of 100 consecutive unsatis-
factory ThinPrep Pap tests were tracked (5-month period) in the
division of cytopathology at Baystate Medical Center (BMC).
Half (n"50) of the tests were received after the mailing of Cytyc
Corporation and BMC-to-physician educational letters, request-
ing lubricants be avoided if possible while obtaining a Pap test.
In addition, the rate of all Pap tests with lubricant present
(partially obscuring lubricant and unsatisfactory squamous cel-
lularity secondary to lubricant) were compared in the pre-mail-
ing (group 1) versus post-mailing (group 2) patients.
RESULTS: Vials with lubricant contained clusters of white pre-
CIN-2 &/or 3 cipitate, which on Papanicolaou-stained ThinPrepTM slides was
pink-purple and granular, frequently adherent to clumped epi-
10% (55) thelial cells. Glacial acetic acid wash had no beneficial effect.
33% (29)
Lubricant was detected in 59 cases, representing 0.35% of group
31% (12)
1 tests and 0.21% of group 2 tests. Unsatisfactory Pap tests in
Group 1 accounted for 0.73% of 6,843 Pap tests evaluated, in
which 20% were unsatisfactory due to lubricant. Group 2 unsat-
isfactory Pap tests represented 0.31% of 16,342 Pap tests, in
which 16% were unsatisfactory due to lubricant. The slight re-
duction in unsatisfactory cases due to lubricant was significant
(p%0.025). Lubricant was observed in Pap tests of older patients
(mean 55, range 17-90 years), when compared to the age of all
patients (mean 40, range 12-95 years) evaluated in the 5-month
study period (p %0.001).
CONCLUSION: Lubricant material may adversely interfere with
the cytologic preparation and interpretation of ThinPrep Pap
tests, leading to an unsatisfactory specimen in a subset of these
patients. Although lubricant may mimic blood, glacial acetic
acid wash has no beneficial effect. On average, contamination of
Pap tests with lubricant was seen in older patients, and in our
experience, clinician education did appear to reduce the preva-
lence of this finding. As corroborated in letters distributed by the
Cytyc Corporation in August 2004 and April 2005, the interfer-
ence is associated with a subset of lubricant products on the
market. Examples of non-interfering lubricants currently avail-
able are also listed, with their respective medical supply distrib-
utors.
Baystate Medical Center, Tufts University School of Medicine,
Springfield, Massachusetts
110
HPV Testing (HCII) Helps Triage ASC-US and
Higher Grade Pap Smears: A Correlation
with Surgical Biopsies
Rosalina Reyes, BSMT, SCT(ASCP)IAC, Elba Turbat-
Herrera, M.D.
BACKGROUND: Since High-Risk HPV is the main causative agent
in the pathogenesis of cervical cancer, a number of tests have
been used as adjuncts to the Pap smear for objective assessment
of women at risk of neoplasia. One of these is Hybrid Capture II
(HCII) from Digene that utilizes a DNA binding assay.
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
OBJECTIVE: The reported high sensitivity of HCII poses a prob-
lem in the diagnosis of women with little or no immediate risk of
disease. Due to recent reports limiting its clinical utility, this
retrospective study was conducted to estimate HCII’s effective-
ness to predict HSIL by correlation with Pap smears and avail-
able biopsy samples. The study also addresses the question of
abnormal Paps and HPV DNA results with negative biopsies.
METHODS:Pap smears with corresponding HPV HCII (n"918)
were retrieved from LSUHSC files from March 2004 to February
2005. 838 (91%) had an ASC-US on cytology and 625 (68%) were
positive for High Risk HPV HCII. All available biopsies (283) were
correlated with cytology and HPV results (2 cases were excluded
due to incorrect sampling). Discordant results were reviewed
blindly by the authors.
RESULTS:A total of 283(31%) biopsies were available, 53(19%) of
which are Negative, 166(59%) LSIL (CINI) and 62(22%) HSIL
(CIN II-III). HCII Positive cases: 134/166(81%) were LSIL and
58/62(94%) HSIL. LSIL biopsies resulted from 151/166(91%) LSIL
(Mild Dysplasia) and 7/166(4%) HSIL (Moderate Dysplasia to
CIS). HSIL biopsies resulted from ASC-US on 48/62(77%) and
HSIL in 11/62(18%), with no LSIL detected. Of the 53 Negative
biopsies reviewed, 25 were modified to CIN I (20 HPV$ and 5
HPV-), 28 were Negative (16 HPV$, 11 HPV- and 1 Insufficient),
and no CIN II-III. 26/29 Negative biopsies that showed lack of
squamo-columnar junction represent inadequate sampling.
CONCLUSIONS:1) The high proportion of abnormal Pap smears
with HPV positivity in women with abnormal biopsies confirms
the usefulness of HCII in women with HR-HPV. 2) Review of
originally negative biopsy reports 55/238(19%) reduced the Neg-
ative cases to 28/283(10%). Squamo-columnar junction is not
observed in 49% of these biopsies, therefore, we recommend
re-biopsy for better detection of High-grade lesions in women
with abnormal Pap results. 3) Endocervical curettage is not ad-
equate to verify abnormal Pap results. 4) ASC-US, suggestive of
HPV with negative HPV DNA correlates with post-menopausal
pseudokoilocytosis. 5) ASC-US, cannot rule out HSIL, with HPV
negativity, can originate from metaplastic epithelium with
atypia.
Louisiana State University Health Sciences Center, Shreveport,
Louisiana
111
Comparison of the Effects of Vortexing and
Lysing Procedures on the Unsatisfactory
Rate in Reprocessed ThinPrep® Pap Test
Specimens
Leslie Cloar, CT(ASCP), Sandra Baker, CT(ASCP), Marie
Hawkins, Murat Gokden, M.D.
INTRODUCTION/PURPOSE: Although liquid-based methods have
many advantages over conventional pap test, the unsatisfactory
383
rate remains to be a significant problem for the cytology labo-
ratory, as well as for the patient. Reprocessing the remaining
specimen in the PreservCyt™ (Cytyc Corp., Marlborough, MA)
vial using the glacial acetic acid lysing procedure has been
shown to eliminate the unsatisfactory diagnosis in a group of
cases. Here, we describe and evaluate a vortexing method we use
to reprocess unsatisfactory ThinPrep® (TP; Cytyc Corp., Marl-
borough, MA) pap tests, and compare the effectiveness of vor-
texing and lysing procedures on unsatisfactory TP specimens.
MATERIALS AND METHODS: In our laboratory, 8852 TP pap test
specimens have been processed over a fourteen month period
(01/01/04-02/28/05), using ThinPrep 2000 Processor (Cytyc
Corp., Marlborough, MA). Of these, 130 were diagnosed as un-
satisfactory according to 2001 Bethesda System. Each specimen
with an unsatisfactory diagnosis on initial TP slide was randomly
assigned to one of the three reprocessing methods: 1.Vortex
method, by vortexing the TP vial contents at medium speed for
30 minutes (65 cases), 2. Previously described glacial acetic acid
lysing procedure (60 cases), 3. A combination of both (5 cases).
In each case, the second slide, prepared through reprocessing of
the specimen, was also screened by a cytotechnologist, reviewed
by a cytopathologist, and a final diagnosis was rendered.
RESULTS: Eighty of 130 cases (61.5%) were satisfactory after
reprocessing. Vortexing, lysing, and combination of both meth-
ods had comparable success rates at 61.5%, 61.6%, and 60%,
respectively. Approximately 7% of the initially unsatisfactory
cases were found to be abnormal after reprocessing (atypical
squamous cells of undetermined significance: 5 cases, atypical
glandular cells of undetermined significance: 2 of the cases,
positive for carcinoma: 2 cases).
CONCLUSIONS: Reprocessing has proven to be a satisfactory so-
lution to unsatisfactory diagnoses in TP pap tests. For a signifi-
cant group of patients (61.5%), whose pap test would have oth-
erwise been diagnosed as unsatisfactory, return visits for a
repeat pap test, as well as additional costs to the patients and to
the healthcare system were prevented. A subpopulation of these
patients also had significant abnormalities. Of the methods used,
vortexing yields comparably effective results. In addition, it is a
simple, one-step procedure, which is more time- and cost-effec-
tive than lysing procedure. Therefore, it should be considered as
the procedure of choice to reduce the number of unsatisfactory
diagnoses in TP specimens.
Department of Pathology, University of Arkansas for Medical
Sciences, Little Rock, Arkansas
384 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
112
HPV Prevalence and Genotype in Atypical
Glandular Cells (AGC) Diagnosed by 2001
Bethesda (2001B) Criteria
Brian Berg, M.D.1, Stuart Cameron, M.D.1, Dan McKeon,
SCT(ASCP)2, XingJing Wang, Ph.D.1, Deniz Aslan, M.D.1,
Evin Gulbahce, M.D.1, Stefan Pambuccian, M.D.1
INTRODUCTION/PURPOSE: While the value of HPV DNA testing in
women with atypical squamous cells (ASC) is well documented
and established, few studies have investigated the role of HPV
testing and genotyping in women diagnosed as AGC according
to the 2001 Bethesda criteria. We have reviewed our experience
with HPV DNA testing by a PCR-based method in AGC to deter-
mine its utility in various sub-categories of AGC.
MATERIALS AND METHODS: The Cytopathology and Molecular
Diagnostics databases of our institution were searched for all
liquid-based Pap tests (LBPT) that had a diagnosis of AGC (either
alone or in combination with a squamous abnormality) from
4/1/01 to 12/31/04. Cases diagnosed before the introduction of
the 2001B were reviewed and diagnosed by these criteria. Patient
characteristics (physiologic state, contraception, hormonal ther-
apy, hysterectomy, previous abnormal Paps, history of gyneco-
logic malignancy) and any cytologic or histologic follow-up
within 6 months were recorded. HPV DNA tests were performed
on the residual fluids by a PCR-based method using MY09/11
consensus primers and RFLP for typing.
RESULTS: During this period, we identified 63 nonconsecutive
LBPT with a diagnosis of AGC that had HPV DNA testing per-
formed. During this interval our laboratory processed over
250,000 Pap tests and performed over 10,000 HPV tests. Overall,
12 of 63 (19.05%) of LBPTs were HPV positive for 7 high-risk HPV
types, the most common of which was HPV 16 (4 cases). Of
these, 5/36 cases of AGUS-NOS (13.89%), 4/8 cases of atypical
endocervical cells (50%), and 2/2 cases of ASCUS with AGC
(100%) were hr-HPV p-positive. Follow-up biopsies were avail-
able in 33 (52%) cases and showed 2 cases of CIN2/3 and 3 cases
of AIS, while one case was diagnosed as endometrial hyperpla-
sia. Of the significant squamous or glandular cervical lesions 4/5
were positive for hr-HPV. The 2/3 AIS cases that were positive for
hr-HPV harbored HPV 16 and 18. Of the 27 cases with negative
biopsy follow-up, 3 were positive for hr-HPV.
CONCLUSIONS:We found a relatively low overall HPV positive rate
in our population of AGC. This may be due to the fact that our
laboratory screens an older population with a low incidence of
SIL, where AGC is more commonly of endometrial origin. The
subcategory of AGC most likely to be HPV positive was AGC-
endocervical. HPV positivity predicted most significant cervical
lesions within this population.
1
University of Minnesota, Minneapolis, Minnesota, 2
Fairview
University Medical Center, Minneapolis, Minnesota
113
Predictive Value of Borderline HPV DNA
Test Results for the Presence of Squamous
Intraepithelial Lesions (SIL)
Maura Pieretti, Ph.D.2, Elliot Carter, M.D.1, John Webster,
M.D.1 Anita B. O’Connor, MT(ASCP)1, Sally Holmes,
CT(ASCP)CMIAC1, Carole W. Boudreaux, M.D.
Reflex HPV DNA testing for oncogenic HPV types is the preferred
follow-up for a liquid based ASC-US Pap interpretation. This
testing is commonly performed using Digene’s Hybrid Capture II
assay. However, several laboratories including ours have ob-
served problems with low-end reproducibility. To address this
issue, Digene has recently modified the test procedure. The
purpose of our study (initiated before the methodology change)
was to assess the predictive value of borderline HPV DNA results
for the presence of SIL in a series of 120 SurePath™ samples. Of
these 120 cases, HPV DNA testing (oncogenic types only) was
repeated for an initial group of 58 samples with an RLU ratio
range of 0.75-1.5. In 16 of the 58 cases (27%) the results changed
from positive to negative or from negative to positive. The
change in the overall rate of our HPV borderline results prior to
and after modification of the Digene procedure has not been
significant (2.7% pre-modification vs. 3.8% post-modification).
Seventy-five of 120 patients had cytologic and/or histologic fol-
low-up studies within 6 to17 months. Of these, 8 had mild
dysplasia and 1 had severe dysplasia on biopsy while 7 had LSIL
and 1 had HSIL on repeat Pap. Significantly, a total of 17 of 75
patients (22.7%) with RLU ratios of 0.75-2.5 had SIL on anatomic
follow-up. Given the large number of patients with HPV DNA
testing results in the borderline range and significant findings on
cytologic or histologic follow-up, our laboratory’s policy is to
forego repeat testing and report all RLU values of 1.0 or greater
as positive.
1
University of South Alabama, Mobile, Alabama, 2BayCare Lab-
oratories, Clearwater, Florida
114
How Age Affects Results of Two HPV
Testing Methodologies
Razan Massarani-Wafai, M.D., Alia Salhadar, M.D.,
Christine Booth, M.D., Mike Laudermith, Paul Pioro, Eva
Wojcik, M.D.
BACKGROUND: HPV testing in cervical cytology is recommended
as the primary ancillary test used in conjunction with atypical
squamous (ASC-US) Pap test results. The performance standard
for use of the HPV test in cytologic specimens was set by the
2001 Bethesda System, ALTS and the ASCCP guidelines using the
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting 385

Hybrid capture (HC) HPV DNA signal amplification (Digene) 115

methodology. In this study, we compare the performance of the
HC II test to the slide-based chromogenic in situ hybridization Improved Detection and Classification of
(ISH) methodology (Ventana) and compared the distribution of
results according to patient age.
Cervical Glandular Lesions by the ThinPrep
DESIGN: In our institution, 541 ThinPrep® samples were tested
Pap Test: A Systematic Review and Meta-
for high-risk HPV by HC II and 435 samples were tested with ISH. Analysis
The test was performed per physician request or as a reflex test James Linder, M.D.,1, 3 Nancy Chenette1, Ellen Sheets,
for an ASC-US interpretation. Results were designated as nega-
M.D.1, I. Elaine Allen, Ph.D.2, Rhonda Estok, B.S., R.N.2,
tive, positive or equivocal for high risk HPV. HC II tests were sent
Katie Cella, B.A.2, Luba Nalysnyk, M.D., M.P.H.2
out whereas ISH was performed in house and interpreted by
cytopathologists. HPV results using the two methodologies were INTRODUCTION: The optimal detection and classification of ade-
correlated with Pap test results and patient age. nocarcinoma and related glandular lesions is an important fea-
RESULTS: A total of 205 cases (38%) were positive for high-risk ture of Pap Testing. This study compares this attribute of the
HPV by HC II, whereas only 87 cases (20%) were positive by ISH. ThinPrep Pap test and the conventional Pap smear in a compre-
Table 1 correlates the HPV results of both HC II and ISH with the hensive meta-analysis.
Pap test results. Correlation with patient age is shown in Table 2. METHODS: A meta-analysis of the published literature of glandu-
TABLE 1: COMPARISON OF HC II AND ISH ACCORDING TO CYTO- lar disease detection by the ThinPrep Pap test was conducted.
LOGIC DIAGNOSIS The source of data was limited to comparative studies of Thin-
Prep Pap test vs. conventional Pap smear published in English
Pap Dx HC II ISH between 1996 and 2003. A comprehensive literature search of
electronic databases was conducted (MEDLINE, Current Con-
NILM 50/208 (24%) 6/132 (4.5%)
tents™, and Cochrane Library). Data from 20 studies meeting
ASC-US 74/224 (33%) 53/230 (23%)
LSIL 69/93 (74%) 25/68 (37%) protocol-specified inclusion criteria were extracted by two re-
HSIL 12/13 (92%) 3/5 (60%) viewers, then logged into a relational database for statistical

TABLE 2: COMPARISON OF HC II AND ISH BY AGE analysis. Meta-analyses of odds ratios (OR) were performed
comparing the detection rates of adenocarcinoma, AGUS, and
Age HC II ISH all glandular abnormalities for the ThinPrep Pap test and con-
ventional Pap smears. All comparisons were calculated using
122/236 (52%) 57/211 (27%) Random Effects Models (REM) to control for heterogeneity
30-40 40/140 (29%) 16/108 (15%)
40-50 27/109 (25%) 10/76 (13%) which was examined using Cochran’s Q statistic. All calculations
!50 16/56 (29%) 3/40 (7.5%) were performed using Comprehensive Meta-Analysis version 1.0
and SPSS software version 11.2.
CONCLUSIONS: The percentage of patients positive for high-risk
RESULTS: The ThinPrep Pap test had a significantly higher de-
HPV in the cytologically benign group (NILM) was much less
tection rate of adenocarcinoma than conventional Pap smear in
using the ISH methodology. Unlike HC II, ISH shows a signifi-
the direct-to-vial studies (OR"2.13; 95% CI: 1.17, 3.86, p%0.01).
cant decrease in HPV positivity in older patients. High-risk HPV
Additionally, meta-analysis of ORs for histologically confirmed
positive patients by ISH in the LSIL group is also much lower
adenocarcinoma rates over all cytologic samples with glandular
than that in the group tested by HC II. These results suggest that
abnormalities showed that ThinPrep Pap test was significantly
the recommended follow-up of patients with a LSIL interpreta-
more predictive of true adenocarcinoma diagnosis when com-
tion by the 2001 ASCCP guidelines may need to be re-evaluated.
pared to conventional Pap smear (OR"1.54; 95% CI: 1.04, 2.28,
Loyola University Medical Center, Maywood, Illinois p%0.05).
CONCLUSIONS: This is the first systematic review regarding the
effectiveness of the ThinPrep Pap test vs. conventional Pap
smears in the detection of cervical glandular abnormalities. In
the aggregate, data are based on 20 studies evaluating more than
1,400,000 Pap smears. The results of these meta-analyses suggest
that there is a significant increase in the detection of adenocar-
cinoma by the ThinPrep Pap method compared to the conven-
tional Pap smear. Although the conventional Pap smear appears
to better detect AGUS, data suggest that ThinPrep Pap test might
be more effective than conventional Pap smear in correctly
386 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
classifying glandular abnormalities and detecting true adenocar-
cinoma.
1 1
Cytyc Corporation, Marlborough Massachusetts, 2Metaworks
Corporation, Medford, Massachusetts, 3University of Nebraska
Medical Center, Omaha, Nebraska
116
Lessons Learned by a Provider of Glass-
slide Based Continuing Education Quality
Assessment Programs in Gyn Cytology
Roger W. Wall, M.S., CT(ASCP)1, 2, Gary W. Gill,
CT(ASCP)CFIAC1, 2, Rhonda L. Metzler, SCT(ASCP)1,
Jennifer J. Clark, SCT(ASCP)1, Michael D. Glant, M.D.1, 2,
Carol C. Eisenhut, M.D.2
INTRODUCTION/PURPOSE: Glass-slide based gynecologic cytology
continuing education and quality assessment programs can
identify performance needs that may help participants improve
their interpretive skills. The lessons learned are relevant to the
administration of CMS-approved nationwide cytological profi-
ciency testing that began in 2005.
MATERIALS AND METHODS: Midwest Institute for Medical Edu-
cation (formerly known as Current Education in Cytology
(CEIC)) introduced in 1994 a conventional Pap test education/
quality assessment program, its first among a family of glass-
slide based cytology programs marketed as CytoQuest®. Over
the past 11 years, over 3000 field-validated Pap test slides have
been reviewed by nearly 7000 cytotechnologists and pathologists
distributed among more than 700 laboratories. There are 3 dif-
ficulty levels with each balanced CytoQuest program: routine,
moderately difficult, and very difficult. All routine cases require
an ongoing diagnostic agreement rate of 90% or better. Moder-
ately difficult cases must maintain a diagnostic agreement of
80% or better. Very difficult cases or bonus cases are any case
performing at less that 80% diagnostic agreement among the
participants. Data collected are broken down into years of ex-
perience for cytotechnologists (less than 5 years or greater than
5 years) as well as whether or not the pathologists are board
certified in AP only, or AP and Cytopathology. Participants
whose answers fall within an acceptable diagnostic range are
graded correct. Statistical analyses that compare a laboratory’s
performance with that of other laboratories overall are provided
to the laboratory.
RESULTS: Comparison results will be presented such as: Cyto-
technologists trained within 5 years of taking CytoQuest identify
certain diagnostic entities (e.g., adenocarcinoma in situ) more
accurately. Cytopathologists perform better overall than pathol-
ogists without cytopathology certification. There is consistently a
greater degree of diagnostic agreement with liquid based prep-
arations than conventional GYN preparations. The diagnostic
category of least agreement, regardless of preparation type, is
“Unsatisfactory”.
Midwest Institute for Medical Education, Indianapolis Indiana,
2
DCL Medical Laboratories, Indianapolis, Indiana
117
Liquid-Based Pap Tests (LBPT) Diagnosed
as Unsatisfactory by 2001 Bethesda
Criteria: Should We Worry?
Mariam Alsharif, M.D.1, Evin Gulbahce, M.D.1, Dan
McKeon, CT(ASCP)2, Klint Kjeldahl, CT(ASCP)2, Stefan
Pambuccian, M.D.1
INTRODUCTION/PURPOSE: The introduction of LBPT and the use
of Bethesda 2001 criteria of adequacy have reduced the inci-
dence of unsatisfactory Pap tests (UPT) in most laboratories. A
performed on conventional Pap smears (CPS), using Bethesda II
criteria of adequacy (Ransdell JR, Cancer Cytopathology 1997;
81:139-143) has found that UPT were more often from high risk
patients, and had significant lesions on follow-up than satisfac-
tory Paps. However, since the presence of obscuring inflamma-
tion that may “hide” abnormal cells was frequently a cause of
UPT in CPS and is rarely encountered in LBPT, we undertook
this study to assess the main categories, patient correlates and
cytologic or histologic follow-up of UPT in LBPT.
MATERIALS AND METHODS: A search of the cytology database
was performed to find all unsatisfactory LBPT from 01/01/03 to
12/31/04. Patient characteristics (physiologic state, contracep-
tion, hormonal therapy, hysterectomy, previous abnormal Paps,
history of gynecologic malignancy), LBPT characteristics
(screening versus diagnostic, type of LBPT-ThinPrep (TP) versus
SurePath (SP)), reason for UPT, and any cytologic or histologic
follow-up with 6 months were recorded.
RESULTS: A total of 190 Paps (26 CPT, 132 SP, 32 TP) from
women aged 16-890 (mean 47) were signed out as unsatisfactory
during this interval, while over 123,000 were considered satis-
factory and yielded a diagnosis of ASC in 5% and SIL in 2.2% in
our institution. Of the women with UPT 37 were hysterecto-
mized, 4 had received radiation for gynecologic malignancy, an
additional 34 were postmenopausal, 17 were postpartum, 5 were
pregnant, 5 on oral contraceptives and 3 on Depo-Provera. The
reason for UPT was: scant cellularity (148), acellularity (17),
obscuring inflammation (5), unprocessed or not collected (11),
too thick (1)., presence of obscuring foreign material (1) and
unknown (7). There were 29 diagnostic and 158 screening Paps.
Follow-up was available in 126 cases (cytologic in 103 and his-
tologic in 23) Of the cases with cytologic follow-up, 90 were
negative, and 13 were signed out as abnormal (6 ASC, 6 LSIL, 1
HSIL). Of the cases with histologic follow-up, 9 where diagnosed
as CIN (6 CIN1, 3 CIN2/3), and 3 had endometrial hyperplasia (2)
or carcinoma (1).
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
CONCLUSIONS: The main cause of UPT in LBPT is low cellularity;
this was, in our experience, more frequently found in TP than in
SP. Our results suggest that women with UPT should be fol-
lowed-up; however, the prevalence of abnormalities in follow-up
Paps in these women is only slightly higher than in the general
screening population, due to the presence of a higher percentage
of diagnostic Paps.
1 2
University of Minnesota, Minneapolis, Minnesota, Fairview
University Medical Center, Minneapolis, Minnesota
118
Human Papillomavirus Testing Detects the
Same Ratio of High Grade Lesions in
Adolescents as in the Non-Adolescent Age
Group
Regina S. Meara, M.D., David Chhieng, M.D., M.B.A.,
Janie Roberson, CT(ASCP), Isam Eltoum, M.D., M.B.A.
INTRODUCTION: The American Society of Colposcopy and Cervi-
cal Pathology (ASCCP) recently recommended the use of HPV-
DNA reflex testing for cases with ASC-US. However, these guide-
lines were based almost entirely on the ASCUS/LSIL Triage
Study (ALTS) which did not include women younger than 18
years old. Currently, we have little data on the outcome of HPV
triage in this age group. The objective of this study is to deter-
mine the rate of high-grade lesions detected through HPV triage
in adolescents and to compare it to the rate detected through
cytologic triage. In addition, these rates are compared to those
found in the non-adolescent age group.
METHODS: We reviewed all liquid-based Pap tests submitted for
reflex HPV testing to our institution during the period of January
2002 to January 2004. Women were triaged according to ASCCP
guidelines. Digene HPV tests were performed when requested by
a physician. Surgical pathology results were correlated with cy-
tologic and virology findings. As a measure of test performance,
the proportion of high-grade lesions confirmed on biopsy
through reflex testing were compared to high-grade lesions de-
tected through cytologic triage. Finally, these rates were com-
pared to those found in the non-adolescent age group.
RESULTS: The total number of ThinPrep tests performed during
the study period shown to have ASC-US was 2565. In the ado-
lescent age group, HPV testing was positive in 53 of 85 (62%)
samples, while 734 of 2222 (33%) samples were positive in non-
adolescents. Biopsy results were available for 23 (43%) adoles-
cents, of which 4 (17%) showed high-grade dysplasia. Biopsy
results were available for 504 (69%) non-adolescents, of which 38
(8%) showed a high-grade lesion. An additional 151 adolescents
and 1414 non-adolescents, with lesions of LGSIL or higher on
Pap smear, underwent biopsy. Thirty-seven (25%) adolescents
had a high-grade lesion on biopsy as compared to 467 (33%) of
non-adolescents. Finally, in adolescents, 4 of 41 (10%) high-
387
grade lesions detected on biopsy were determined via the HPV
testing arm, while in non-adolescents, 38 of 505 (8%) high-grade
lesions detected on biopsy were detected via the HPV testing
arm.
CONCLUSION: HPV infection in patients with ASC-US is signifi-
cantly higher in the adolescent age group. Non-adolescents were
more likely to have a high-grade lesion detected via the cytologic
referral arm. Finally, when comparing the detection of high-
grade lesions found on biopsy in patients who underwent HPV
triage versus those who had cytologic referral, HPV testing de-
tects the same ratio of high grade lesions in adolescents as in the
non-adolescent age group.
University of Alabama at Birmingham, Birmingham, Alabama
119
ASC-US with Insufficient Residual Sample
for PCR-Based HPV DNA Reflex Testing:
Should We Worry?
Deniz Aslan, M.D., Xinjing Wang, Ph.D., Stefan
Pambuccian, M.D.
INTRODUCTION/PURPOSE: When HPV DNA testing is used to
manage women with ASC-US diagnosed on a liquid-based Pap
test (LBPT) sample, a small percentage of samples (%1% for PCR,
%2% for ISH and !3% for HC2) have insufficient residual fluid
(QNS) after processing for HPV DNA testing. Little is known
about the patient, follow-up Pap and histologic correlates of
such samples. Knowledge about the factors contributing to sam-
ples QNS for HPV testing will become more important in light of
the proposed guidelines of screening women over 30 by a com-
bination of Pap test and HPV DNA test. We review our experi-
ence with SurePath samples QNS for HPV testing by PCR.
MATERIALS AND METHODS: The files of our institution over a 3
year period were searched for HPV DNA tests that were QNS
(defined as, uninterpretable due to no human genomic internal
control amplified). During this period, 10,780 HPV DNA tests
were performed in our institution on residual liquid-based Pap
test samples by PCR using MY09/011 primers and genotyping by
restriction enzyme digestion. Follow-up diagnostic tests, includ-
ing LBPT, HPV DNA, and colposcopic biopsy results were re-
trieved.
RESULTS: The study included 70 patients, between ages 15 and
70 (mean 37). Mean follow up period was 15.5 months (range
0-27). The corresponding Pap test diagnoses of the samples of
the residua of which were QNS by PCR testing were, 31 negative
for intraepithelial lesion (mean age 40), 33 ASCUS, 3 ASC-H, 1
LSIL, 1 AGC, and 1 unsatisfactory for evaluation. There were a
total of 10 patients with abnormal follow results. All but 2 ab-
normal follow-up results were in the ASCUS group. 1 abnormal
result was in the negative group (CIN I biopsy at 26 months), and
1 in the LSIL group. There were 2 high grade lesions (CIN II),
388 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
both of which were in the ASCUS group. Other abnormal fol-
low-up included 2 LSILs, 3 ASCUSes, 3 HPV PCR positives (high
risk), and two CIN I biopsies in 6 patients.
CONCLUSIONS: Women with ASCUS, and technically failed HPV
PCR should be managed with immediate repeat Pap and/or PCR.
Women with a negative Pap result, however are at low risk for
any squamous abnormality, and can be safely managed by a
repeat Pap test and/or HPV DNA testing as recommended by
guidelines.
University of Minnesota, Minneapolis, Minnesota
120
Atypical Glandular Cells (AGC) in Human
Immunodeficiency Virus (HIV) infected
women (HIV!) Compared to AGC in HIV-
Negative Women
Gabriela Oprea-Ilies, M.D.1, Talaat Tadros, M.D.1, Stefan
Pambuccian, M.D.2, George Birdsong, M.D.1, Steven Lau,
M.D.1
INTRODUCTION: AGC is a category used to report cytologic
changes in glandular cells present in the Papanicolaou (Pap)
cervical cytology. They are predominantly of endocervical or
endometrial origin. Studies have shown that the category of
AGUS carries a high risk for squamous intraepithelial lesions and
for glandular, endocervical and endometrial lesions. Human
Papilloma virus (HPV) changes and cervical cancer are diag-
nosed with increased frequency in HIV$ women. To date there
is no study dedicated to the significance of the cytologic and
histologic preceding lesions to the diagnosis of AGUS and the
follow-up in HIV$ women in comparison with HIV- women.
MATERIALS AND METHODS: Our files were searched for the AGC
diagnoses on Pap smears: 18 HIV$ (Western Blot) women were
found; 18 HIV- by ELISA women matched in a close age range
formed the control group. The average age at the time of AGC
diagnosis was 41.3 (range 31-60) for HIV$ and 40.3 (range 28-51)
for HIV(-) women. All of these women had at least one histologic
specimen as follow-up.
RESULTS: A previous abnormal cytology/histology specimen was
present in 15/17 HIV$ and 10/15 HIV- women. This difference
was not statistically significant. The AGUS diagnosis was fol-
lowed by biopsy in all cases studied. The follow-up is repre-
sented by a total of 222 Paps (12 /patient), 39 biopsies (2/
patient), 5 conization and 2 hysterectomies in the HIV$ group
and 142 Paps (7.88/patient), 44 biopsies (2.44/patient), 3 cone
biopsies and 6 hysterectomies in the HIV(-) group, statistically
similar.The benign follow-up histologic diagnosis was repre-
sented by endocervical and endometrial polyps, microglandular
hyperplasia and tubal metaplasia. Three HIV$ and 4 HIV(-)
women over 40-years of age had benign endometrial follow-up.
One HIV(-) patient had endometrial hyperplasia on biopsy fol-
low-up. The Pap cytology and tissue follow-up, analysed by
chi-square, are presented in Table 1.
TABLE 1. PAP AND HISTOLOGIC FOLLOW-UP IN HIV! AND HIV-
WOMEN
HIV
status Atypical LSIL HSIL p value CIN Benign p value
HIV$ 15 7 7 11 4
HIV- 13 1 0 %0.05 8 25 %0.01
CONCLUSIONS: HIV($) women diagnosed with AGC on screening
Paps are more likely to have an abnormal cytologic and/or
histologic mostly squamous follow-up. The HIV(-) women were
more likely to have a benign follow-up. AGC requires an appro-
priate management according to current guidelines to detect
squamous or glandular lesions, with special attention to HIV($)
women.
1
Grady Memorial Hospital, Atlanta, Georgia, 2University of Min-
nesota, Minneapolis, Minnesota
121
Causes of Atypical Glandular Cells on
Cytology: A Comparison of Conventional
and Liquid-based Preparations
Jason Kendelhardt, M.D., Karen Nauschuetz, M.D.
INTRODUCTION: Atypical Glandular Cells (AGC) is a poorly un-
derstood interpretation on cervical cytology. Determining what
histologic lesions can cause AGC on a Pap smear is invaluable for
clinicians and cytologists in assessing the treatment of patients.
Knowing whether the lesions are the same for conventional
smear (CS) or ThinPrep (TP) is also important.
MATERIALS AND METHODS: All Pap smears from 1999, 2002, and
2003 were reviewed for the terms “atypical glandular”, “endo-
metrial”, and “endocervical” in the diagnosis line. All cases with
histologic follow-up within one year were pulled and compared.
A total of 407 CS cases and 187 TP cases were reviewed.
RESULTS: The most commonly occurring histologic diagnosis
following a Pap smear interpretation with AGC was a squamous
intraepithelial lesion (SIL). High Grade SIL was identified on
follow-up of 11% of CS and 10% of TP Pap smears. Low grade SIL
was found after 26% of CS and 30% of TP Pap smears. A plethora
of benign diagnoses were found on histologic follow-up as well.
No significant difference between CS and TP was identified
except in the rate of adenocarcinoma in-situ (AIS) on follow-up,
whereby 1% of CS and 4% of TP cases showed AIS on subsequent
histology. Also significant was the decreased rate of squamous
metaplasia seen on follow-up after TP. While a common finding
on histology after CS (38% of all cases), it was found substantially
less often after TP (17% of all cases).
CONCLUSIONS: AGC, while frequently associated with benign
lesions, have a significant chance of predicting high grade SIL or
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
carcinoma, glandular or squamous. ThinPrep appears to be at
least as good as conventional smear in detecting these lesions,
and may surpass conventional smear in detecting adenocarci-
noma in situ.
Brooke Army Medical Center, San Antonio, Texas
122
Detection of HSV-1 and HSV-2 from Liquid
Cytology Medias Using the Roche
LightCycler
Jane Gibson, Ph.D., David Vinson
Herpes Simplex Virus (HSV) is the most prevalent sexually trans-
mitted disease (STDs) in the United Sates. There are nearly 1
million new cases diagnosed annually1. Detection of micro-or-
ganisms from liquid cytology specimens has been demonstrated.
The ability to include HSV-1 and HSV-2 detection from such
specimens allows additional diagnostic opportunities without
specimen re-collection. The use of Real-time PCR instruments
such as the Roche LightCycler combine PCR detection capability
with rapid thermocycling making it a desirable platform for the
clinical laboratory.
We have documented detection of HSV-1 and HSV-2 from Pre-
servCyt" and SurePath" liquid cytology medias using the Roche
LightCycler HSV 1/2 primer/hybridization probe ASR reagents.
Triplicate samples of HSV-1 and HSV-2 dilutions ranging from
5-640 copies per reaction were prepared from viral standards
obtained from Advanced Biotechnologies, Inc. (ABI).
PreservCyt" media studies demonstrated the presence of HSV-1
in all replicates at each dilution with melting temperatures rang-
ing from 52.50-53.53° C ( x " 52.68 & 0.42°C) with a lower limit
of detection (LLD) of 5-50 copies of HSV-1 per PCR reaction.
HSV-2 was also detected in all replicates at each dilution with
melting temperatures ranging from 65.74-67.07°C (x "66.34 &
0.40°C), with a lower limit of detection (LLD) of 5-50 copies of
HSV-2 per reaction. HSV-1 and HSV-2 dilutions in Sure-Path"
media were evaluated in an identical fashion. HSV-1 was de-
tected in all triplicate reactions at each dilution, with an LLD of
5-50 copies of HSV-1 per PCR reaction, and melting tempera-
tures ranging from 52.42-53.43°C (x "52.80&0.37°C). HSV-2 was
also detected in all triplicate reactions at each dilution with an
LLD of 5-50 copies of HSV-2 per reaction, and melting temper-
atures ranging from 65.80-66.99°C (x "66.33&0.41°C). Statistical
analyses of both data sets indicate no significant differences
between melting temperatures found for PreservCyt" and Sure-
Path" medias for HSV-1 (p0.05) and HSV-2 detection (p0.05).
Specificity studies using HSV-related virus standards VZV/
HHV-3, EBV/HHV-4, CMV/HHV-5, HHV-6, HHV-7, and KSV/
HHV-8 were also performed to confirm detection specificity in
PreservCyt" and SurePath" medias.
These results demonstrate LightCycler detection of HSV-1 and
389
HSV-2 from liquid cytology medias with a LLD of 5-50 copies per
reaction. reaction. .
AmeriPath, Orlando, Florida
123
Automated Imaging - The Impact on
Cervical Pap Test Screening: A Single
Institution Experience
Esther Elishaev, M.D., Reda Saad, M.D., Rose Crane,
CT(ASCP), Mamatha Chivukula, M.D., Nancy Mauser,
M.P.M, CT(ASCP), David Dabbs, M.D.
INTRODUCTION: The Papanicolaou cervical test is the most effec-
tive screening tool developed for prevention, early diagnosis and
timely recognition of neoplastic changes preceding invasive cer-
vical carcinoma, with consequent dramatic effect on mortality.
The long used conventional Pap smear was gradually replaced
by a liquid based alternative in the last decade. The advantages
of this method are the examination of a small surface area and
the opportunity to screen the sample for presence of high risk
HPV. Although screening with an automated cervical imaging
system is increasing, the diagnostic rate of this new technique is
not well established. In addition there are no available data on
HPV detection rates with this system.
AIM OF THE STUDY: To investigate possible differences of diag-
nostic rates and HPV results between assisted screening with the
automated cervical imaging system (Cytyc) and manually
screening by cytotechnologists. The Bethesda 2001 Nomencla-
ture guidelines for the following parameters were measured:
adequacy of the sample, rate of UNSAT and presence of endo-
metrial cells, diagnosis rate of ASC-US, AGC, ASC-H, LSIL, HSIL
and positive/suspicious and the percentage of HPV tests or-
dered.
MATERIALS AND METHODS: A total of 21,336 Pap tests (group I)
were screened over three consecutive months using the auto-
mated cervical imaging system (Cytyc), and were compared with
the total of 27,287 Pap tests (group II) that were screened by
cytotechnologists over a three month period prior to image
implementation.
RESULTS: There was an increase in the diagnostic rate of ASC-
US, ASC-H, LSIL and HSIL in-group I compared to group II. The
rate of diagnosis in group I for ASC-US was 8.80%, ASC-H 0.64%,
LSIL 4.84%, HSIL 0.78%, compared to group II where the diag-
nosis of ASC-US was 6.22%, ASC-H 0.36%, LSIL 4.12%, HSIL
0.58%. DiGene HPV testing was performed on group I at the rate
of 12.49% compared to the 9.36% in group II. Despite the differ-
ence in the percentage of HPV ordering between the two groups
the incidence of HPV positive cases was the same.
CONCLUSION: The current study demonstrates that there is a
difference in the diagnostic rates between the automated image
assisted screening of cervical samples compared to the manual
390 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
method. Since the use of the automated screening method is
increasing, further studies are needed to investigate the sensi-
tivity and cost effectiveness of this technique.
Magee-Womens Hospital, University of Pittsburgh Medical Cen-
ter, Pittsburgh, Pennsylvania
124
ThinPrep Imaging System™ (TIS)
Performance of Sensitivity and Specificity:
The Magee Experience
Mamatha Chivukula, M.D., Reda Saad, M.D., Esther
Elishaev, M.D., Susan White, B.S., SCT(ASCP), Nancy
Mauser, M.P.M., SCT(ASCP), David Dabbs, M.D.
INTRODUCTION: Many cytology laboratories across the country
are adopting liquid-based cytology for Pap test screening. With
the success of ThinPrep, the Imaging System (TIS) was intro-
duced to improve the accuracy of screening diagnoses. We re-
port our initial experience with TIS at Magee-Womens Hospital.
MATERIALS AND METHODS: We introduced the ThinPrep Imag-
ing System ™(TIS), (Cytyc Corp, Marlborough, MA), in Decem-
ber 2004. The imager diagnoses over the first four months (De-
cember 2004 to March 2005) of its implementation were
reviewed and analyzed. Our protocol included each cytotechnol-
ogist manually rescreening 200 negative slides to get acquainted
to the imager slides as well as a quality check for the TIS. We
rescreened 3400 slides manually which were initially determined
to be negative using the TIS.
RESULTS: 29,146 Pap tests were imaged on the TIS. The mean
ASC-US rate with the automated imager is 8.70% (2535/29,146).
The mean LSIL rate is 4.47% (1300/ 29,146). The imager during
these months did not miss any high-grade lesion with a mean
detection rate of 0.72% (210/ 29,146). The missed rate on TIS is
0.06% (2/3400). The TIS missed two LSIL during the months of
December and March. The cytotechnologist when performing
the quality control (QC) evaluation procedure for the TIS de-
tected both of these cases. The subsequent biopsies revealed
CIN1.
CONCLUSIONS:The overall performance of the TIS in our lab
appears to be highly satisfactory in terms of improving sensitiv-
ity as well as specificity of screening cervical precursor lesions.
The two cases of LSIL missed by TIS appear to be random. The
increased accuracy of detection of HSIL shows a positive impact
of TIS in our lab.
Magee-Womens Hospital of University of Pittsburgh Medical Cen-
ter, Pittsburgh, Pennsylvania
125
Aspiration Cytopathology of Peripancreatic
Space: A Clinicoradiologic and
Cytopathologic Analysis of 42 Cases
Wei Zhang2, Sheila Sheth1, Olga Ioffe, M.D.2, Syed Ali,
M.D.1
INTRODUCTION: The pancreas is surrounded by a soft tissue zone
referred to as “peripancreatic space” (PPS), which contains
lymph nodes and neurovascular structures. Pathologic lesions of
PPS are extremely rare and have never been reported in the
cytology literature.
MATERIALS AND METHODS: Retrospective review of cytopathol-
ogy files at two large institutions revealed 42 cases of PPS in a
16-year period (1989 to 2004). Clinico-radiologic findings and
follow-up information were additionally reviewed. Specimens
were obtained by transabdominal fine needle aspiration (FNA)
(33) or endoscopic ultrasound-guided FNA (9). Cases were also
sent for flow cytometry (FC) (7) for suspected non-Hodgkin
lymphoma (NHL) and immunoperoxidase (IPOX) staining (10).
RESULTS: Patients ranged in age from 23-83 yrs (mean 60) with
an equal gender distribution. Eleven patients (26%) had prior
history of malignancy. The major clinical presentations included
abdominal pain, jaundice, nausea/vomiting, and abnormal liver
function tests. Radiographic characteristics included diffuse
lymphadenopathy, cystic/solid soft tissue masses with a size
range of 1.5 to 8 cm. Cytologically, 4/42 cases (10%) were non-
diagnostic, 9/42 (21%) were diagnosed as benign (pseudocysts,
inflammation/reactive changes), 4/42 (10%) were designated
atypical/suspicious for cancer (of which 2 were confirmed later
as adenocarcinoma on tissue biopsies), and 25/42 (60%) were
deemed cancers (summarized in table below):
Cytopathologic Diagnosis Number of cases Incidence (%)
Metastatic adenocarcinoma,
pancreatic 4 16
Metastatic adenocarcinoma,
other sites 12 48
NHL 6 24
Metastatic islet cell tumor 2 8
Metastatic melanoma 1 4
(24%) patients had metastasis of prior known malignancy from
colon, kidney, liver and skin. Two cases diagnosed as metastatic
pancreatic adenocarcinoma on FNA were later found to be of
colonic and gynecological tract in origin in the subsequent sur-
gical specimens. Two cases diagnosed as benign on FNA turned
out to be NHL and anaplastic carcinoma on tissue studies.
CONCLUSIONS: 1) Aspiration of PPS masses is a rare occurrence.
2) Majority of lesions are metastatic carcinomas (16/42, 38%)
most commonly of pancreatic origin (4/42, 10%). 3). Rarely, PPS
mass is an initial presentation of an occult cancer (5/25, 20%). 4)
FC and IPOX are useful adjuncts. 4) The sensitivity of PPS aspi-
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
ration for a cancer diagnosis is 93% with a positive predictive
value of 100%.
1
The Johns Hopkins Hospital, Baltimore, Maryland, 2University of
Maryland Medical Center, Baltimore, Maryland
126
Pancreatic Mucinous Lesions: A
Retrospective Analysis with Cyto-histologic
Correlation
Jing Zhai, M.D., Ranjit Sarkar, CT(ASCP), Lourdes Ylagan,
M.D.
INTRODUCTION: The diagnosis of mucinous pancreatic lesions
which include: mucinous adenocarcinoma (MAd), intraductal
papillary mucinous neoplasm (IPMN), mucinous cystic neo-
plasm (MCN), and mucinous metaplasia (MM) is critical given
different clinical management and prognosis. A multidisci-
plinary approach is adopted, including clinical, radiological, lab-
oratory and cytological evaluation before a definitive procedure
is done. This retrospective study is done to assess the cytological
features, limitations and pitfalls associated with these entities in
cytologic samples.
MATERIALS AND METHODS: A search for pancreatic cytology
specimens with histologic confirmation of the various pancreatic
mucinous lesions was done: 10 MAd, 14 IPMN, 11 MCN, and 3
MM. The majority had been endoscopically detected and ultra-
sound guided fine needle aspirations. The cellularity, extracel-
lular mucin, epithelial architecture, mucinous nature of the ep-
ithelium, cell shape and nuclear features were evaluated on the
cytology material. Twenty-three cytologic features were blindly
evaluated without knowledge of the surgical pathology diag-
noses by two cytopathologists. A Fisher’s exact test was used for
analysis.
RESULTS: Of the 23 cytologic features evaluated, the presence of
three dimensional cluster and nuclear atypia which includes:
nuclear crowding, increased N/C ratio, anisonucleosis, nuclear
membrane irregularity, coarse granular chromatin and promi-
nent nucleoli, were found to be consistently associated with
mucinous adenocarcinomas. There were no statistically signifi-
cant cytologic features which helped in differentiating IPMN,
MCN, and MM. There was a relatively high false positive (FP)
rate in the IPMN group (5/14, 36%). Review of the histologic
specimen showed intraepithelial atypia which could be inter-
preted as carcinoma-in-situ. The histologic specimen of one FP
case of MM (1/3, 33%) showed marked intraepithelial acute
inflammation. One false negative case of adenocarcinoma was
due to sampling error.
CONCLUSIONS: The cytological diagnosis of mucinous pancreatic
lesions remains challenging except for mucinous adenocarci-
noma. The findings were largely non specific in the differentia-
tion between IPMN, MCN and MM. FP diagnosis of adenocar-
391
cinoma occurs not infrequently in the setting of IPMN with
severe atypia or carcinoma-in-situ and in lesions with associated
intraepithelial acute inflammation and can be a pitfall in the
diagnosis of these lesions.
Washington University, St. Louis, Missouri
127
Evaluation of Performance of EUS- FNA in
Preoperative Node Staging of Cancers of
Esophagus, Lung and Pancreas
Hong Q. Peng, M.D.1, Fabio Tavora, M.D.1, Mary Eline
Kling, M.D.1, Bruce Greenwald, M.D.2, Peter Darwin,
M.D.2, Anna Berry, M.D.1
BACKGROUND: Lymph node status is an important predictor of
outcome and management in cancers. Endoscopic ultrasonog-
raphy (EUS) is considered the most accurate modality for local
staging of esophageal cancer and also applied to lung and pan-
creatic cancers. Fine needle aspiration cytology has been shown
to increase the accuracy of nodal staging by EUS. However, few
studies have examined the correlations between EUS parameters
and FNA findings.
DESIGN: We reviewed the cytology diagnosis and EUS report of
consecutive patients who had undergone EUS- FNA preopera-
tive staging for esophageal, lung and pancreatic cancers at our
institution from January 2002 to August 2004. When available,
histologic follow-up was also reviewed. Eight morphologic EUS
features of the lymph node were compared with corresponding
cytological diagnoses.
RESULTS: A total of 122 EUS-FNA lymph nodes were identified
from 77 patients. There were 55 esophageal cancers, 2 esopha-
geal dysplasias, 5 lung cancers and 15 pancreatic cancers. On
site assessment was performed in all cases. Number of passes
ranged from 1 to 5. About one third of cases had surgical follow-
up. Cytologically diagnostic categories included: positive for ma-
lignancy 42 (34%), suspicious 2 (1.6%), atypical 2 (1.6%), benign
62 (51%), and non-diagnostic 14 (11.5%). We compared cytolog-
ically malignant and benign lymph node groups with eight EUS
parameters including total number of lymph nodes found by
EUS, the shape, margin, long axis, short axis, echogenicity and
location of the lymph node, as well as EUS tumor staging re-
spectively. Among these parameters, the total number of lymph
nodes, short axis, margin and the location of lymph node were
statistically significant different (P% 0.05) between nodes posi-
tive and nodes negative groups. Using multiple logistical regres-
sion analysis, short axis is the strongest predictor for positivity
with an odds ratio of 1.6 and 95% CI of 1.05 and 2.55 (P%0.0001).
For lymph nodes short axis !5mm, sensitivity was 91%, but
specificity was 10%. For short axis !12mm, specificity rose to
93%, but sensitivity dropped to 36%. Abdominal lymph nodes
(level 16-20) in esophageal and lung cancers are more likely to be
392 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
positive. Conclusions: The short axis of the lymph node is the
best EUS feature to predict a positive lymph node. Short axis !5
mm should always be sampled for FNA. If the short axis is !12
mm, the node is highly likely to be malignant. Five or more
lymph nodes found by EUS are more likely to have positive
staging. Lymph nodes found in an abdominal location in esoph-
ageal and lung cancers are likely malignant.
1
Department of Pathology, 2Department of Medicine Division of
Gastroenterology, University of Maryland Medical System, Balti-
more, Maryland
128
Aspiration Cytopathology of Neuroendocrine
Neoplasms in the Liver: A Clinico-
Radiologic and Cytopathologic Analysis of
189 Cases
Stacy Wang, Julie Wu, Sheila Sheth, Syed Ali, M.D.
INTRODUCTION: Neuroendocrine tumors (NT) in the liver are
uncommon and range in spectrum from benign/low-grade neo-
plasms to highly aggressive cancers. An accurate interpretation
on fine needle aspiration (FNA) is essential for optimum patient
management and disease prognosis.
MATERIALS AND METHODS: A retrospective search of the cyto-
pathology archives at a major teaching institution revealed 189
cases of NTs out of a total of 2629 liver FNAs performed in a
17-year period (1989 –2005). Immunoperoxidase (IPOX) stains,
histologic material (where available) and clinical/radiologic in-
formation was additionally reviewed.
RESULTS: Patients ranged in age from 13– 89 years (mean 60),
with a M:F ratio of 1.5:1. The most common clinical presentation
was a liver mass (74%), followed by abdominal pain (33%) and
weight loss (20%). Radiology disclosed solitary to several nodules
(average number-2.5) with a largest mean size of 4.9 cm. Of the
189 cases, cytopathologic evaluation revealed; no benign and
189 (100%) malignant NTs. Of these, 183 (97%) cases were met-
astatic and 6 (3%) primary in the liver. Neuroendocrine carci-
noma (NEC), NOS was the most prevalent diagnosis (65, 34%),
followed by small cell carcinoma [SCC] (46, 24%), islet cell tumor
(23, 12%), carcinoid tumor (22, 11%) and others. Only one case
called NT was later reclassified as a non-neuroendocrine neo-
plasm (angiosarcoma). 31 (16%) tumors had focal features of
neuroendocrine (NE) differentiation and all but three of those,
on follow-up turned out to be non-NE tumors- adenocarcinoma
[Aca] (20), hepatocellular carcinoma (5) and others. At the time
of diagnosis, only 53 (29%) of the metastatic cases had a known
history of a primary tumor from the lung (31, 58%), pancreas (28,
53%), gastrointestinal tract [GIT] (10, 19%), and skin (4, 8%). 145
(77%) of cases received special stains at the time of primary
diagnosis. The number of stains used ranged from 1–15 (mean 5)
with chromogranin (130, 90%), synaptophysin (95, 66%), CD56
(24, 17%), and NSE (23, 16%), the most commonly used markers.
Positivity was reported for; chromogranin-91 (70%), synapto-
physin-71 (75%), CD56-16 (67%) and NSE-12 (52%). Of the 95
cases for which mortality information was available, the mean
survival was 17.8 months, ranging from 1 day to 120 months.
CONCLUSIONS: Hepatic NTs are rare (189/2629, 7%). NEC, NOS is
the most common subtype followed by SCC. Primary NTs are
extremely rare with most of the tumors being metastatic in
nature. Lung is the most common source of metastasis followed
by pancreas and GIT. Tumors with focal neuroendocrine mor-
phology on follow-up turn out to be mostly metastatic Aca. IPOX
stains are extremely helpful for proper identification of NE phe-
notype.
The Johns Hopkins Hospital, Baltimore, Maryland
129
The Cytology of Pancreatic Acinar Cell
Carcinoma Sampled by Endoscopic
Ultrasound-Guided Fine Needle Aspiration
Alison Presley, M.D.1, Michael Stanley, M.D.2, Vanessa
Shami, M.D.1, Edward Stelow, M.D.1
INTRODUCTION: Endoscopic ultrasound (EUS)-guided fine nee-
dle aspiration (FNA) has proved itself a useful and accurate
method for the sampling of lesions of the pancreas. Although the
cytologic features of pancreatic ductal adenocarcinoma have
been well described, the cytology of some of the less common
neoplasms of the pancreas may present the pathologist with
numerous pitfalls. Acinar cell carcinoma (ACC) of the pancreas is
rare and when sampled can show cytologic features that may
overlap both with non-neoplastic pancreatic acinar tissue and
with pancreatic endocrine tumors. This study reviews the clini-
cal and cytologic features of 4 cases of ACC sampled by EUS-
guided FNA.
MATERIALS AND METHODS: The cytology files at two institutions
were reviewed for all cases of pancreatic ACC sampled between
1/1/00 and 12/31/04. Follow-up data was obtained for all cases.
Clinical and sonographic findings were pursued. Cytologic find-
ings including overall cellularity, cytologic architecture and co-
hesion, and cytoplasmic and nuclear features were evaluated.
Original diagnoses and use of cell block material were also
recorded.
RESULTS: Four cases of pancreatic ACC were identified, all of
which had surgical follow-up (3 pancreatoduodenectomy spec-
imens and 1 core biopsy of an hepatic metastasis). The patients
were 2 men and 2 women and ages were 49, 55, 64, and 85 years.
One patient presented with jaundice, 2 patients presented with
abdominal pain and 1 patient was noted to have a pancreatic
mass incidentally by radiography. All masses were hypoechoic
and located in the head of the pancreas. Tumor size ranged from
3-15 cm in greatest dimension. Original FNA diagnoses were as
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
follows: (1) non-diagnostic, normal pancreatic acinar tissue only,
(2) pancreatic endocrine tumor, (3) favor neuroendocrine tumor,
low-grade, and (4) ACC. Smears showed abundant cellularity in
3 cases and low cellularity in 1 case. All cases showed admixed
loose clusters and single cells. Acinar formations were present in
all cases. All cases showed low nuclear to cytoplasmic ratios with
abundant granular cytoplasm and indistinct cell borders. Nuclei
had minimally irregular contours and were centrally and eccen-
trically located, sometimes giving a plasmacytoid appearance to
the cells. Rare cells were binucleated. The chromatin was coarse
and prominent nucleoli were seen in all cases. Mitoses were not
seen. Only one case had abundant material for cell block and
that was the case correctly diagnosed as acinar cell carcinoma.
CONCLUSIONS: ACC is a rare pancreatic neoplasm that may
easily be misinterpreted as benign acinar tissue or pancreatic
endocrine tumor when sampled by FNA. Although cytologic
features are helpful, a good cell block may be key for the correct
prospective diagnosis.
1 INTRODUCTION: Bile duct brushing cytology has become a rela-
University of Virginia, Charlottesville, Virginia, 2 Abbott North-
western Hospital, Minneapolis, Minnesota
130
Cystic Islet Cell Tumors: Clinical,
Radiologic, Cytologic, and Histologic
Features
Benjamin Coleman, Robert Emerson
INTRODUCTION: Both nonneoplastic lesions and a variety of neo-
plasms may present as cystic masses in the pancreas. While islet
cell tumors are cystic in a minority of cases, islet cell tumors are
a rare cause of a cystic pancreatic mass.
MATERIALS AND METHODS: We performed a computerized
search for islet cell tumors of the pancreas diagnosed by fine
needle aspiration (FNA) cytology or histology for a 14-year pe-
riod. Reports were reviewed and cases with radiologic or gross
evidence of cystic change composed the study population. The
clinical and radiologic features of these cases were reviewed and
follow-up information was obtained. The cytologic and histo-
logic material was reviewed.
RESULTS: From a series of 79 histologically confirmed islet cell
tumors, 12 cases with gross cystic change were identified (15 %).
Three of these had preceding FNA cytology specimens. An ad-
ditional 3 cases of radiologically cystic tumors diagnosed only by
FNA cytology as islet cell tumors were identified. The diagnosis
was confirmed in these 3 cases by immunoreactivity with syn-
aptophysin and chromogranin on cell block sections. The mean
patient age was 53 years (range: 23-77). One patient had von
Hippel-Lindau syndrome and 3 patients had multiple endocrine
neoplasia syndrome type I. Tumor location was pancreatic head
(3 cases), body (1 case), tail (10 cases), or not specified (1 case).
Four tumors were multifocal. Mean tumor size was 3.4 cm (range
393
0.1 to 10 cm). Review of the 7 cases diagnosed by FNA demon-
strated the usual cytologic features of islet cell tumors: moder-
ately cohesive plasmacytoid cells with coarse chromatin and
moderately abundant granular cytoplasm.
CONCLUSIONS: Islet cell tumors account for a small portion of
cystic pancreatic masses, but should be included in the differ-
ential diagnosis of cystic lesions of the pancreas.
Indiana University School of Medicine, Indianapolis, Indiana
131
In Situ Neoplasia of the Bile Duct:
Morphologic Features in Brushing Cytology
Jennifer Jones, Carolyn Brunnemer, CT(ASCP), Robert
Emerson
tively routine component of the evaluation of patients present-
ing with jaundice and suspected obstruction of the common bile
duct. Brushing cytology has demonstrated high specificity but
only moderate sensitivity for invasive adenocarcinomas of the
bile ducts. Rarely, noninvasive lesions of the bile ducts may be
sampled, but the morphologic features of these lesions, in par-
ticular those that might differ from invasive tumors, have not
been well described.
MATERIALS AND METHODS: We performed a computerized
search for a 14 year period for in situ lesions of the common bile
duct and ampulla of Vater diagnosed by histology. Cases with an
invasive component, even if minor, were excluded. For each of
these cases, a search was made for preceding bile duct brushing
cytology specimens. The original reports, slides, radiographic
reports, and patient histories were reviewed.
RESULTS: We identified 8 patients with bile duct brushing cytol-
ogy specimens which were followed by histologic diagnosis of an
in situ epithelial lesion. There were 3 cases of adenocarcinoma
in situ of the common bile duct, 4 cases of papillary (noninva-
sive) adenocarcinoma of the common bile duct, and 1 case of
tubulovillous adenoma with high grade dysplasia of the ampulla
of Vater. The mean patient age was 58 years (range 43-69). Six
patients were male and 2 were female. Seven were jaundiced at
presentation and the eighth presented with pruritus and in-
creased serum hepatic transaminases. On radiologic evaluation,
3 had a stricture, 3 had filling defects, 1 had dilation of the duct,
and 1 had a mass. The preceding bile duct brush cytology spec-
imens were diagnosed as negative in 1 case, atypical cells in 5
cases, and atypical cells consistent with adenocarcinoma in 2
cases. Review of the brushing cytology specimens revealed pap-
illary groups in 7 cases. Atypical glandular groups with a feath-
ered pattern were present in 3 cases, similar to those described
in endocervical adenocarcinoma in situ in Papanicolaou smears.
CONCLUSIONS: In situ lesions of the bile ducts lack some of the
features seen in bile duct adenocarcinomas but have some mor-
394 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
phologic features which may be useful in their recognition in
bile duct brushing cytology.
Indiana University School of Medicine, Indianapolis, Indiana
132
Aspiration Cytopathology of Cancers
Metastatic to the Kidney
Banafsheh Rashidi2, Olga Ioffe, M.D.2, Syed Ali, M.D.1
INTRODUCTION: Renal metastases are rare, often clinically silent
and have been reported in up to 7% of all patients dying of
cancer. Since secondary tumors in the kidney are approached
non-surgically, fine needle aspiration (FNA) appears to be an
ideal diagnostic tool when dealing with such a clinical scenario.
MATERIALS AND METHODS: We retrospectively reviewed renal
metastases diagnosed by FNA in a 15 year period (1989-2004)
from the cytopathology archives of two large tertiary care cen-
ters. All cases of locally invasive tumors in the kidney and he-
matologic neoplasms were excluded from the study. Material
was obtained via ultrasound-guided FNA. Smears were stained
with Diff Quik and Papanicolaou stains. Patients’ clinical history
and histologic diagnoses, where available, were additionally re-
viewed.
RESULTS: Of the 518 renal FNA evaluated, 202 (39%) were diag-
nosed as primary renal cell carcinoma (RCC), whereas 17 (3%)
were interpreted as cancers metastatic to the kidney. The pa-
tients range in age from 26-75 years (mean age 52) with a M:F
ratio of 1.8:1. Clinical presentation at the time of diagnosis was
available in 10 cases; eight were incidental findings at the time of
follow-up radiologic studies in patients with known cancer, one
presented with hematuria, and one with abdominal fullness. The
location (side) of metastasis was; left kidney in 10 (59%), right
kidney in 5 (29%) and bilateral in 2 (12%) cases. Of the bilateral
cases, one had two masses in the same (right) kidney. Size of the
mass was known in 10 cases and ranged from 0.7 to 15 cm (mean
5.4 cm). Information about metastasis to the other organs was
available in 9 cases; 5 had metastasis to other organs including
lung and liver at the time of diagnosis of the kidney metastasis.
FNA diagnoses included adenocarcinoma-4 (23%), squamous
cell carcinoma-3 (18%), malignant melanoma -2 (12%), sarco-
ma-2 (12%) and others-6 (35%). The primary site of the meta-
static tumor was known in 14/17 (82%) cases with lung (6) being
the most common followed by esophagus (2), skin (2), breast (2),
testis (1) and uterus (1).The time interval between the initial
diagnosis of the primary tumor to the diagnosis of the renal
metastasis was known in 10 patients and ranged from 0 (primary
tumor and renal metastasis diagnosed simultaneously) to 11
years (mean 3.6 years). Cytomorphologic features in each tumor
type displayed the expected findings.
CONCLUSION: Metastasis to the kidney is rare. Lung carcinoma is
the most common of the metastatic tumors. There can be a long
clinical latency necessitating careful evaluation of the patients’
history when interpreting such FNA. An accurate characteriza-
tion of the neoplastic process is vital since clinical approach is
different from primary RCC.
1
The Johns Hopkins Hospital, Baltimore, Maryland, 2University of
Maryland Medical Center, Baltimore, Maryland
133
The Causes of False Negative Pancreatic
Cytology
Mark Williams, M.D., Helen Cathro, M.D., Kristen Atkins,
M.D., Henry Frierson, M.D., Mark Stoler, M.D., Venessa
Shami, M.D., Edward Stelow, M.D.
INTRODUCTION: The location of the pancreas may sometimes
render cytologic sampling difficult. Furthermore, the cytomor-
phologic profiles of some pancreatic lesions may be difficult to
prospectively diagnose. Because of these factors, false negative
(FN) pancreatic cytology specimens are not uncommon. These
cases can then present difficulties for clinicians who may be
wary of instituting treatment, which often may entail resection.
We investigated our experience with FN pancreatic cytology and
some possible causes.
MATERIALS AND METHODS: All pancreata that were resected over
a 14 year period at our institution were reviewed. Our cytology
database was searched for all preceding cytologic specimens
from these cases. Cytology diagnoses were reviewed and judged
to be FNs or true positives based on whether they were sufficient
to warrant resection. These results were then compared for the
different methods of collection, tumor sites, tumor sizes and
tumor types.
RESULTS: Between 7/1/91 and 12/31/05, 131 pancreata were
resected at our institution. The overall sensitivity of pancreatic
cytology was 74%. Brush cytology had a sensitivity of 46%, in-
traductal aspiration had a sensitivity of 66%, and fine needle
aspiration (FNA) had a sensitivity of 85%. Sampling of lesions in
the pancreatic head showed a sensitivity of 79%, whereas sam-
pling of pancreatic body and tail lesions showed a sensitivity of
54%. Sampling of lesions less than 1 cm in size showed a sensi-
tivity of 67%, sampling of lesions 1-3 cm in size showed a
sensitivity of 82% and sampling of lesions greater than 3 cm in
size showed a sensitivity of 65%. The sampling of various neo-
plasms showed the following sensitivities: pancreatic ductal ad-
enocarcinomas: 79%; pancreatic endocrine tumors: 75%; intra-
ductal papillary mucinous neoplasms (IPMNs): 70%; acinar cell
carcinomas: 100%; and mucinous cystic neoplasms (MCNs):
42%.
CONCLUSIONS: The pancreas may present difficulties both for
those who attempt cytologic sampling and those who must
interpret those samples. It appears that FNA is much more
sensitive than brush cytology and intraductal aspiration for the
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
diagnosis of pancreatic lesions. Lesions sized 1-3 cm and those
located in the pancreatic head may be easier to sample than
smaller or larger lesions or those of the pancreatic body and tail.
Finally, the correct prospective diagnosis of pancreatic MCNs
appears to be more difficult for cytopathologists than the diag-
nosis of solid pancreatic neoplasms and even IPMNs.
University of Virginia, Charlottesville, Virginia
134
Distinct Cytomorphologic Features of Solid-
Pseudopapillary Tumor of the Pancreas
Mai Gu, Ph.D., MIAC, May Myint, SCT(ASCP)
INTRODUCTION: olid-pseudopapillary tumor (SPT) is an uncom-
mon neoplasm of the pancreas, accounting for approximately
1-2% of all pancreatic exocrine tumors. Although the etiology is
not known, the majority of SPT has a favorable prognosis and is
cured by surgical resection. Therefore, an accurate diagnosis
prior to surgery is crucial in clinical management of these pa-
tients. Endoscopic ultrasound (EUS)-guided fine needle aspira-
tion (FNA) is frequently used for such pre-operative evaluation
of pancreatic tumors.
MATERIALS AND METHODS: From October 2003 to November
2004, SPT of the pancreas diagnosed by EUS-guided FNA was
retrieved through a computer query. The EUS findings and the
number of aspiration passes necessary for an adequate speci-
men were documented in each case. Immunohistochemical
(IHC) studies were performed in cell block material. Cytology
slides and surgical resection specimens were retrospectively re-
viewed.
RESULTS: Four patients were found during this period, three
women and one man. The average age was 33 years-old (range
30 – 36 years). The average size of the tumor by EUS was 2.4 cm
(1.8 – 3.0 cm). The tumor, characterized by EUS as either hypo-
echoic or mixed echo texture with or without a cyst component,
was located in the pancreatic head in one patient, head/neck in
one, body in one, and body/tail in one, respectively. One pass
was sufficient for an accurate diagnosis in all patients. The
cytologic features were characterized by high cellularity (4/4),
long thin delicate papillae (4/4), predominantly discohesive
round to oval uniform cells with indistinct borders (4/4), and
abundant vacuolated basophilic cytoplasm (3/4). The nuclei
were small, uniform, round (1/4) or round to oval (3/4), some of
which were eccentrically located (2/4). Chromatin was fine and
evenly distributed (4/4) . Micronucleoli and smooth nuclear
membranes were seen in all cases. The background was clean
(4/4) with occasional foamy histiocytes (2/4) and hyaline glob-
ules (3/4). The main differential diagnosis included endocrine
tumor and acinar carcinoma. IHC studies revealed that tumor
cells were positive for AE1/AE3 (4/4), CAM 5.2 (2/4), vimentin
(2/4), !1-antichymotrypsin (2/4), !1-antitrypsin (1/4), CA19-9
395
(1/4), weakly and focally positive for synaptophysin (2/4). Tumor
cells were negative for PAS (4/4) and chromogranin (4/4). All
patients had subsequent surgical resection and the cytologic
diagnosis was confirmed in all.
CONCLUSIONS: SPT of the pancreas has distinct cytologic fea-
tures and can be accurately diagnosed by cytology. EUS-guided
FNA is a cost-effective and highly efficient means for evaluating
these tumors.
University of California Irvine Medical Center, Orange, California
135
NQO1 Expression in Cytologic Samples of
Pancreatic Carcinoma Obtained by
Endoscopic Ultrasound (EUS) Guided Fine-
Needle Aspiration (FNA)
Nida Awadallah, B.S., Donna Dehn, Ph.D., Raj J. Shah,
M.D., David Ross, Ph.D., Yang K. Chen, M.D., Kenneth R.
Shroyer, M.D., Ph.D.
BACKGROUND: The evaluation of EUS-FNA specimens from pan-
creatic mass lesions may be limited by subtle cytologic features
of malignancy, sparse cellularity, or extensive necrosis leading to
false negative test results. Conversely, false positive diagnoses
may result from reactive cellular features associated with acute
or chronic inflammation. We evaluated the immunocytochemi-
cal detection of NAD(P)H:quinone oxidoreductase (NQO1), a
two-electron reductase that is elevated in many solid tumors, as
a potential adjunct for detecting pancreatic carcinoma in EUS-
FNA samples.
METHODS: NQO1 immunocytochemistry was performed by an
indirect immunoperoxidase method on archival EUS-FNA cell
block sections from 15 patients who were evaluated for sus-
pected pancreatic carcinoma. NQO1 stain intensity was scored
on a scale of 0 to 3$ and the proportion of positive cells was
scored on a scale of 0 to 100%.
RESULTS: NQO1 nuclear and cytoplasmic staining was detected
in 7/7 (100%) cases that had cytologic features of pancreatic
adenocarcinoma on H&E, in 1/6 (17%) cases that were negative
on initial cytologic diagnosis, and in 1/2 (50%) cases that were
initially classified as atypical but not diagnostic for adenocarci-
noma. In the seven cases that were originally classified as ade-
nocarcinomas, median stain intensity in lesional epithelial cells
was 3$ (range 2-3$) and the median number of stained tumor
cells was 60% (range 10-100%). Endothelial cells and duodenal
surface mucosal epithelial cells were also positive for NQO1 but
were readily differentiated from tumor cells on a morphologic
basis. The NQO1 positive case that was originally classified as
negative for carcinoma showed strong (3$) NQO1 staining of
necrotic tumor cells. Rare viable tumor cells were detected on
recut sections from this case but viable tumor cells were not
represented in the cell block section that was available at the
396 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
time of initial diagnosis. Subsequent resection demonstrated
metastatic neuroendocrine carcinoma. NQO1 staining in the
atypical case was restricted to cytologically abnormal cells and
this case was subsequently confirmed to be malignant by lapa-
roscopic biopsy. The atypical case that was negative for NQO1
was from a patient with well differentiated pancreatic carcinoma
but lesional cells were not present in the cell block sections.
CONCLUSION: NQO1 is a highly sensitive and specific marker of
pancreatic adenocarcinoma in EUS-FNA cell block sections.
Large scale prospective studies will determine if NQO1 will im-
prove the yield of EUS-FNA cytology for detecting pancreatic
carcinoma.
University of Colorado at Denver and Health Sciences Center,
Aurora, Colorado
136
A Comparative Study of Pancreatic Fine
Needle Aspirations by Percutaneous CT-
guidance and Endoscopic Ultrasound
Mohiedean Ghofrani, M.D., David L. Rimm, M.D., Ph.D.,
Diane P. Kowalski, M.D.
INTRODUCTION: In the past few years percutaneous CT-guided
fine needle aspiration (FNA) biopsy of the pancreas has given
way to endoscopic ultrasound (EUS)-guided FNA. In this study
we compared the sensitivity, specificity, number of non-diag-
nostic aspirates, and number of definite cytologic diagnoses for
each of these two methods.
MATERIALS AND METHODS: A search through the Pathology De-
partment computer system was performed to construct a data-
base of all percutaneous CT-guided FNA biopsies of the pan-
creas during the 5 year period of 1994 to 1998. All EUS-guided
pancreatic FNAs performed in 2003 were also included in the
database, as well as any pertinent surgical follow-up available for
cases in either of the two groups. Final diagnoses reported for
each case were categorized into one of five preset categories:
negative, atypical, suspicious, positive, or non-diagnostic. Neg-
ative and positive diagnoses were considered “definite” as op-
posed to the “indeterminate” diagnoses, namely atypical and
suspicious. Sensitivity and specificity calculations were per-
formed by correlating definite cytologic diagnoses with their
surgical follow-up.
RESULTS: During the 5-year period of 1994 to 1998, 90 CT-guided
pancreatic FNAs from 82 unique patients were identified. On the
other hand in 2003 alone a total of 137 EUS-guided FNAs from
131 unique patients were recorded. Of the 90 CT-guided FNAs,
13 (14.4%) were non-diagnostic for cytologic evaluation, whereas
of the 137 EUS-guided FNAs only 9 (6.6%) were non-diagnostic.
Chi-square testing showed this greater than twofold difference
to be statistically significant (p%0.05). On the other hand among
CT-guided FNAs the number of definite diagnoses (negative or
positive) was significantly higher compared to EUS-guided FNAs
(75.6% vs. 64.2% of diagnostic specimens, p%0.002). Docu-
mented surgical follow-up was available for 34 patients (41.5%)
in the CT-guided group and 37 patients (28.2%) in the EUS-
guided group. Specificity of pancreatic FNA was 100% in both
groups, while the sensitivity of EUS-guided aspirates was higher
than CT-guided specimens (90% vs. 63.6%).
CONCLUSIONS: The sensitivity of EUS-guided FNA of the pan-
creas is higher than percutaneous CT-guided FNA. The specific-
ity of pancreatic FNA for diagnosing carcinoma is very high,
either by percutaneous approach or EUS-guided biopsy. EUS-
guided FNAs are less likely to be non-diagnostic, but they are
also more likely to yield an indeterminate rather than definite
cytologic diagnosis compared to the CT-guided approach.
Yale University School of Medicine, Department of Pathology,
Cytology Division, New Haven, Connecticut
137
Pancreatic Small Cell Neoplasms Diagnosed
by Endoscopic Ultrasound Guided Fine
Needle Aspiration
Allyson Baker, Sarah Bean, M.D., Mohammad Eloubeidi,
M.D., M.P.H., Isam Eltoum, M.D., Nirag Jhala, M.D., MIAC,
Ralph Crowe, M.D., Darhsana Jhala, M.D., David Chhieng,
M.D.
INTRODUCTION: Endoscopic ultrasound guided fine needle aspi-
ration (EUS-FNA) has been accepted in the diagnosis of pancre-
atic lesions in adult patients. This study was designed to review
our experience of small cell neoplasms evaluated by EUS-FNA
and the usefulness of ancillary studies in their differential diag-
nosis. The term ‘small cell” refers to the relatively small size of
the neoplastic cells, i.e. about 2 to 4 times the size of that of small
round lymphoctye.
MATERIALS AND METHODS: A computerized search retrieved all
cases of adult small cell neoplasms encountered in pancreatic
EUS-FNA from the years 2000 to 2004. The cytology and the
results of the ancillary studies, which included immunocyto-
chemistry and flow cytometry, were reviewed. Follow up in-
cluded review of current and prior relevant surgical materials
and clinical records.
RESULTS: Of all the pancreatic EUS-FNAs performed in our
institute during the study period, 32 cases were classified as
small cell neoplasms, accounting for 3% of all pancreatic EUS-
FNA. They are from 32 patients, 17 males and 15 females, with
ages ranged from 38 to 94 with a median age of 54. A history of
prior malignancy was present at the time of FNA in 3 patients.
Fourteen patients had concurrent or prior pathologic samples
for comparison. The remaining patients were followed up clin-
ically for at least 6 months. The final diagnoses were as follows:
26 neuroendocrine tumors (islet cell tumors), 1 solid and cystic
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
papillary neoplasm, 3 non-Hodgkin’s lymphomas, 1 small cell
variant of squamous cell carcinoma of the uterine cervix small
cell variant, and I Ewing’s sarcoma. Five were metastatic lesions
and the remaining were primary pancreatic neoplasms. In 25
cases, ancillary studies, immunocytochemistry (22) and flow
cytometry (3) were performed to assist in the differential diag-
nosis. Cytologically, 29 cases were cases were correctly diag-
nosed and two cases were classified as suspicious for a neoplasm
or malignancy. The case of solid and cystic papillary neoplasm
was initially interpreted as neuroendorcine neoplasm cytologi-
cally. There was no false-negative diagnosis.
CONCLUSION: Neoplasms with small cell morphology accounted
for a small but significant number of pancreatic EUS-FNAs per-
formed in an academic institute. A variety of neoplasms can
enter into the differential diagnosis. Careful attention to the
morphology, clinical correlation, and ancillary studies are help-
ful in arriving at the correct diagnosis.
University of Alabama at Birmingham, Birmingham, Alabama
138
Pancreatic Duct Aspiration and Brushing
Cytology Diagnosis of Pancreatic
Intraductal Papillary Mucinous Neoplasm is
Highly Specific
Robert Emerson, Joyashree Sen, Harvey Cramer, M.D.
INTRODUCTION: Intraductal papillary mucinous neoplasms
(IPMN) of the pancreas have been considered difficult to diag-
nose by cytologic examination. We previously have documented
the ability of endoscopic ultrasound-guided fine needle aspira-
tion (EUS-guided FNA) cytology to diagnose IPMN with a sen-
sitivity of 71% and a specificity of 90%, but the reliability of
diagnoses made by pancreatic duct aspiration and brushing
cytology has not been well documented.
MATERIALS AND METHODS: We performed a computerized search
for IPMN diagnoses made on pancreatic duct aspiration and brush-
ing specimens for a 4 year period and then searched for histologic
follow-up. A second search was performed for cases of histologi-
cally proven IPMN without coexisting adenocarcinoma that had a
preceding pancreatic duct aspiration or brushing specimen.
RESULTS: We identified 45 cases diagnosed as IPMN or consis-
tent with IPMN by pancreatic duct aspiration. Histologic fol-
low-up was available for 16 patients (36%). The histologic diag-
noses were IPMN (13 cases), pancreatic intraductal neoplasia (1
case), invasive adenocarcinoma (1 case), and IPMN with invasive
adenocarcinoma (1 case). Five cases were diagnosed as IPMN or
consistent with IPMN by pancreatic duct brushing cytology.
Histologic follow-up was available for 3 patients (60%). The
histologic diagnoses were IPMN (2 cases) and IPMN with inva-
sive adenocarcinoma (1 case). Forty-one cases of histologically-
proven IPMN without coexisting adenocarcinoma were diag-
397
nosed by histology during this time period. Of these, 29 (71%)
had a preceding pancreatic duct aspirate and 6 (15%) had a
preceding brushing cytology. The diagnosis made by pancreatic
duct aspiration in the 29 cases was negative or nondiagnostic (15
cases) or IPMN or consistent with IPMN (14 cases). Among the 6
cases of histologically-proven IPMN that had a preceding pan-
creatic duct brushing, the cytologic diagnosis was negative or
nondiagnostic (5 cases) or IPMN (1 case).
CONCLUSIONS: In this series, pancreatic duct aspiration cytology
has a sensitivity of 48%, a specificity for IPMN of 88%, and a
specificity for pancreatic neoplasia of 100%. While pancreatic
duct brushing cytology has a sensitivity of 17% and a specificity
of 100%. The specificity of these techniques compares favorably
with EUS-guided FNA, but the sensitivity is significantly lower.
Indiana University School of Medicine, Indianapolis, Indiana
139
Fine Needle Aspiration Cytology of
Lymphoplasmacytic Sclerosing Pancreatitis
Heather Peterman, M.D.1, Tamar Giorgadze, M.D., Ph.D.1,
2 1
3, Anjali Saqi, M.D. , Natsuko Shiina, CT(ASCP)IAC ,
1 1
Prabodh Gupta, M.D., FIAC , Tunde Farkas, M.D.
INTRODUCTION: Lymphoplasmacytic sclerosing pancreatitis
(LPSP) is a rare form of chronic pancreatitis (CP) characterized
by dense lymphoplasmacytic infiltrates involving pancreatic lob-
ules and ducts, acinar atrophy, marked interstitial fibrosis,
and/or obliterative phlebitis of small to medium sized vessels.
This lesion most often presents with clinical and radiographic
findings that are suggestive of malignancy. The cytological fea-
tures of LPSP have not previously been described. With the
increasing use of endoscopic ultrasound guided fine needle as-
pirations (EUS-FNAs) of pancreas, our purpose was to charac-
terize salient cytological features that would aid in distinguish-
ing LPSP from CP.
MATERIALS AND METHODS: Retrospective review of 76 cases of
pancreatic EUS-FNA specimens was performed after a Whipple
or distal pancreatectomy final diagnosis of lymphoplasmacytic
sclerosing pancreatitis was made.. All theses cases eventually
underwent a Whipple’s or distal pancreatectomy procedure with
a final diagnosis of lymphoplasmacytic sclerosing pancreatitis.
The These se aspirates were reevaluated in order to identify
possible cytomorphological features that would be helpful in
distinguishing LPSP from CP.: These features included epithelial
atypia, presence and predominant population of the inflamma-
tory infiltrate, their association with the pancreatic epithelium,
histiocytes and/or macrophages, and occurrence of fibrosis
and/or necrotic debris.
RESULTS: The histopathological features seen in the resected
surgical specimens were typical for LPSP in all cases. Cytomor-
phological features of LPSP included fragments of dense fibrous
398 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
tissue and a prominent lymphoplasmacytic infiltrate intimately
associated with the ductal epithelium that had no or minimal
cytological atypia. No pronounced ductal epithelial atypia or,
presence of degenerated cells and/or macrophages in the back-
ground of necrotic debris was noted.
CONCLUSIONS: While there is a significant overlap in the cytomor-
phology of LPSP and CP, distinction between these two entities
would be useful as LPSP is more likely to be mistaken clinically and
radiographically for malignancy and to lead to surgical intervention
for the patient. Salient features that would aid in distinguishing
these histopathologically different forms of chronic pancreatitis are
the intimate association of a predominantly lymphoplasmacytic
infiltrate with the ductal epithelium and absence of degenerated
cells, pronounced ductal epithelial atypia, macrophages, and/or
necrotic debris in the cases of LPSP.
1
University of Pennsylvania, Philadelphia, Pennsylvania, 2New York
Presbyterian Hospital, Columbia Medical Center, New York, New
York, 3East Tennessee State University, Johnson City, Tennessee
140
Fine Needle Aspiration of Papillary Renal
Cell Carcinomas – Is Cytologic Diagnosis
and Subtyping Possible?
P. Iyengar, M.D., H. Al-Ahmadie, M.D., V.E. Reuter, M.D.,
S. Olgac, M.D., O. Lin, M.D., Ph.D.
INTRODUCTION: Papillary renal cell carcinoma (PRCC) is a sub-
type of renal cell carcinomas recognized by the current WHO
classification. Most recently, a sub-classification of types 1 and 2
PRCC has been advocated. These 2 subtypes share a papillary
architecture, but differ in regards to cytomorphology, cytogenet-
ics and prognosis. The ability of fine needle aspiration (FNA)
biopsies to accurately diagnose and subtype PRCC has not been
previously studied. This distinction is of value not only for its
prognostic implication but also for tailoring future treatment
with targeted therapies. We described the morphologic findings
of both subtypes and evaluated the ability of FNA in correctly
identifying PRCC and its 2 subtypes.
MATERIALS AND METHODS: A series of 136 consecutively re-
sected renal tumors, including 13 cases of PRCC, were submitted
to bench FNA. The cytologic evaluation was performed without
knowledge of the histologic diagnosis. We assessed the morpho-
logic features of both types 1 and 2 PRCC, as well as, the sensi-
tivity, specificity, positive predictive value (PPV) and negative
predictive value (NPV) of renal FNAs in correctly identifying
PRCC and its 2 histologic subtypes.
RESULTS: Cytomorphologically, both types 1 and 2 showed clusters
with papillary architecture. In general, the cell population in type 1
PRCC was homogeneous with small to moderate amount of dense
cytoplasm, round nuclei with inconspicuous nucleoli, and promi-
nent nuclear grooves. Type 2 PRCC contained larger cells with
abundant cytoplasm, pleomorphic nuclei and distinct prominent
nucleoli. Correlation of the original cytologic diagnosis with the
histologic diagnosis revealed that only 8 of the 13 cases were cor-
rectly diagnosed (6 type 1 and 2 type 2 PRCC). The remaining 5
PRCC cases were cytologically misdiagnosed as other forms of RCC.
The accuracy of renal FNAs in the diagnosis and subclassification
of PRCC is summarized in Table 1.
CONCLUSIONS: Cytology showed low sensitivity and PPV in both
the diagnosis and sub-classification of PRCC, although the spec-
ificity was high. Ancillary studies may play a future role in
improving the accuracy of FNA in the diagnosis and sub-classi-
fication of PRCC.
Memorial Sloan-Kettering Cancer Center, New York, New York
141
Endoscopic Ultrasound-guided Biopsies of
Pancreatic Masses: Comparison Between
Fine Needle Aspirations and Needle Core
Biopsies
Shine Yun, M.D.1, Helen Remotti, M.D.1, Anjali Saqi, M.D.2
INTRODUCTION/PURPOSE: The literature has shown that endo-
scopic ultrasound-guided fine needle aspiration (EUS-guided
FNA) is a highly sensitive and specific method for diagnosing
pancreatic masses. In our institution, we often receive concur-
rent pancreatic FNAs and needle core biopsies (NCB) obtained
by endoscopic ultrasound-guidance. Our aim was to determine
the efficacies of pancreatic EUS-guided FNAs and NCBs.
MATERIALS AND METHOD: We retrospectively reviewed 252 con-
secutive pancreatic EUS-guided FNAs received over a 24 month
period. Cases with concurrent NCBs were selected for the study.
The diagnoses from the two modalities were compared and
designated concordant (CC) or discordant (DC).
RESULT: Fifty-six of 252 (22.2%) cases had concurrent NCBs. The
final diagnoses included adenocarcinoma, pancreatic endocrine
neoplasm, cystic mucinous neoplasm, solid pseudopapillary tu-
mor, renal cell carcinoma, breast carcinoma, and hepatocellular
carcinoma. Of the 56 cases, 33/56 (58.9%) were CC and 23/56
(41.1%) were DC.
TABLE 1: DISCORDANT CASES
FNA NCB Number of cases
P N 11
N P 3
P AS 4
AS P 2
AS N 2
N AS 1
P " Positive for neoplastic cells
N " Negative for neoplastic cells
AS " Atypical – favor neoplasm/ suspicious for neoplasm
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
The sensitivities for FNAs and NCBs were 86% and 63%, respec-
tively. There was no correlation between CC/DC cases and type
of neoplasm.
CONCLUSION: Direct comparison of concurrent EUS-guided
FNAs and NCBs show that the former have a greater sensitivity
for diagnosing pancreatic neoplasms, both primary and meta-
static. Furthermore, the smaller gauge of FNA needles make
FNAs intrinsically safer while providing greater material.
1
Columbia Presbyterian Medical Center, New York, New York,
2 ferentiation between GI contamination and benign and malig-
New York Presbyterian Hospital, Weill Cornell Medical Center,
New York, New York
142
Cytomorphology and B72.3 Labeling of
Benign and Malignant Ductal Epithelium in
Pancreatic Lesions Compared to GI
Contamination
Ranjana Nawgiri, M.D.1, Julie Nagle, M.D.2, David Wilbur,
M.D.2, Martha Pitman, M.D.2
BACKGROUND: EUS guided pancreatic FNAB has become a
widely used method for the diagnosis of pancreatic lesions. The
procedure, however, introduces GI contamination (GIC) result-
ing in a potential diagnostic pitfall. It is important to distinguish
both benign and malignant pancreatic epithelial cells from GI C.
We studied the cytomorphology and B72.3 immunoreactivity of
lesional epithelium from benign and malignant ductal lesions of
the pancreas by direct lesional sampling and compared the
findings to our previously established template of GIC (Acta
Cytol 2004; 48:718; Diagn Cytopathol, in press).
DESIGN: Air-dried smears, fixed smears and ThinPrep® speci-
mens were obtained using a cytobrush directly from benign and
malignant ductal lesions of 18 Whipple specimens. Smear back-
ground, cell architecture, and cellular features were analyzed.
Immunocytochemical staining with B72.3 was performed in 14
cases by routine immunoperoxidase methods.
RESULTS: There were 12 ductal carcinomas and 6 cases of pan-
creatitis. Large monolayered sheets were only seen in GIC. Lu-
minal edges, brush borders and goblet cells separated duodenal
GIC (luminal edges only in gastric GIC) from benign and malig-
nant ductal groups, and 3D groups, glands and acini distin-
guished malignant from benign ductal groups and GIC. High NC
ratio, vacuolated cytoplasm, irregular nuclei and nucleoli sepa-
rated malignant from benign ductal and GIC. GI and benign
ductal epithelium were not distinguished by cellular features.
Differences in cytomorphology by specimen preparation were
noncontributory to distinguishing these groups. B72.3 previ-
ously shown to strongly stain goblet cells and demonstrate a fine
punctate perinuclear staining pattern in GIC, failed to stain all
benign ductal epithelium and showed strong cytoplasmic stain-
ing at least focally in all 10 malignancies stained, although a
399
similar perinuclear staining pattern or no staining was also
noted in some epithelial groups in all cases as well.
CONCLUSIONS: Pancreatic carcinomas were distinguished from
benign ductal epithelium in pancreatitis and GI contamination
primarily by 3D architecture and atypical cellular features in-
cluding high NC ratio, irregular nuclei, nucleoli and vacuolated
cytoplasm. A background of necrosis and cellular debris sup-
ports a malignancy. Benign ductal and GI epithelium were only
distinguished by architecture. B72.3 shows promise in the dif-
nant ductal epithelium with no staining supporting ductal cells,
fine punctate perinuclear staining correlating with GI contami-
nation and strong cytoplasmic staining supporting malignancy.
Heterogeneous staining of malignant epithelium, however,
could present a pitfall of under diagnosis in malignancy.
1
Brigham and Women’s Hospital, Harvard Medical School, Bos-
ton, Massachusetts, 2Massachusetts General Hospital, Harvard
Medical School, Boston, Massachusetts
143
Endoscopic Ultrasound Guided Fine Needle
Aspiration of Adrenal Glands for Detection
of Metastatic Lung Carcinomas – A Large
Prospective Study
Shanthi Gopal, M.D.1, Renu Khode, M.D.1, Eric Rachut,
M.D.1, Edwin Johnson, M.D.1, Robert Astarita, M.D.1,
Douglas Toler, Ph.D., CT(ASCP)1, Yashodeep Jadhav,
M.D.1, Phalguni Mukhopadhyay, M.D.1, Brian Camazine,
M.D.1, Richard Erickson, M.D.2, Shyam Narayana, M.D.1,
Pankaj Singh, M.D.1
BACKGROUND: Lung cancer with metastases to adrenal gland
carries a grave prognosis and precludes curative surgical resec-
tion.
AIM: he aim of this study was to determine the utility of endo-
scopic ultrasound guided fine needle aspiration (EUS-FNA) in
the diagnosis of adrenal lesions and its impact on the staging of
lung carcinoma.
METHODS: Prospective study was conducted in a tertiary care
center. Consecutive patients with diagnosis of lung carcinoma
were included for the evaluation of the metastases to the adrenal
glands. Adrenal gland examination was performed using CT scan
and EUS. EUS-FNA was performed based on the endosono-
graphic appearance of left adrenal gland. On site evaluation for
adequacy of samples was provided by a pathologist. Air-dried
and alcohol-fixed smears were made from each pass. Cell blocks
were made from the remaining aspirated material from all
passes by pooling in 95% alcohol. Immediate evaluation was
provided using air-dried, Diff-Quik stained smears. The final
diagnosis was rendered after reviewing alcohol-fixed, Papanico-
laou stained smears and cell block.
400 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
RESULTS: Ninety-one patients (mean age 65 years) with con-
firmed tissue diagnosis of lung carcinoma were enrolled. En-
dosonographic examination using linear array echoendoscope
showed enlarged/deformed left adrenal gland in 34/91 cases. CT
scan showed enlarged left and/or both adrenal glands in 13/91
cases. EUS-FNA was performed in 34 cases, based on the en-
dosonographic appearance of left adrenal gland. The median
number of needle passes was 3 (range 1-6). On site evaluation
was reported as “adequate” in 31/34 cases and “scant” in the
remaining 3. Final diagnosis was reported as “positive for ma-
lignant cells” in 13/34 and “negative for malignant cells” in 21/34
cases. Four of the 13 malignant cases (as identified by EUS) were
missed on CT scan. The malignant cases were subclassified as
“non small cell carcinoma (9/ 13)” and “small cell carcinoma (4/
13).” Immunohistochemistry (CK7, TTF-1, synaptophysin and
chromogranin) was used to support the cytological diagnosis.
CONCLUSION: This large prospective study emphasizes the value
of EUS-FNA in the detection of left adrenal metastatic lesions
and its impact on staging. It also demonstrates that EUS-FNA
can detect occult metastatic lesions in the left adrenal gland that
appear normal on CT scan.
1
Central Texas Veterans Health Care System, Temple, Texas,
2 sults suggest that high resolution 3-D optical tomography may
Scott & White Memorial Hospital, Temple, Texas
144
True 3-D Cell Nuclear Structure Imaging to
Detect Cancer
Florence W. Patten, B.S., CFIAC, Michael G. Meyer, M.S.,
Alan C. Nelson, Ph.D., Thomas Neumann, M.D.
INTRODUCTION: isionGate, Inc. (Gig Harbor, WA, USA), in collab-
oration with the University of Washington (Seattle, WA, USA), is
developing an automated screening system for high resolution
cellular imaging in true 3-D using optical projection tomography
to detect early lung cancer. The UVisionTM System is capable of
analyzing cellular morphology in 3-D to assess nuclear aberra-
tions with visualization of submicron-sized features. Lung cells
from normal and cancer patients have been examined, and are
illustrated here.
METHODS: In addition to sputa and FNAs, cell culture lines
CCD-34Lu (lung fibroblasts) and A549 (Bronchioloalveolar Can-
cer) are used in this preliminary study. Cells are collected in 50%
ethanol, stained with hematoxylin and embedded in a clear gel
medium, then injected into a microcapillary tube and rotated
through 360 degrees. As the cells are rotated, high-resolution
extended depth-of-field optical images are acquired via automa-
tion. These images, or pseudoprojections, are then used to com-
pute 3-D images using a filtered back-projection algorithm. The
tomographic 3-D reconstructions are computed and visualized
using VolView software.
RESULTS: Initial studies have focused upon identification of
sub-micron nuclear features. Remarkable differences in nuclear
membrane contours and chromatin texture are observed in
CCD-34Lu and A-549 cells. Prominent nuclear membrane in-
vaginations and chromatin channels, partitions, and tubular
structures, not readily seen in conventional 2-D microscopy,
yield clearly visible discrimination between these cell lines. Fur-
ther, to provide an instructive demonstration of an inherently
3-D analysis, we have examined the inactivated X-chromosome
(Xi), or Barr body, having a constant size and physical location
within the nucleus, to compare its 3-D appearance under the
UVisionTM System vs., conventional 2-D microscopy.
CONCLUSION: The UVisionTM System is capable of resolving
minute nuclear structures. 3-D imaging facilitates the identifi-
cation of a wide variety of cellular structures not readily appar-
ent in routine 2-D preparations. It may well be that 3-D images
provide a more direct measure of so-called malignancy associ-
ated changes (MACs) that have been observed in conventional
microscopy. The power of optical projection tomography lies in
its potential to create a quantitative 3-D image via absorption-
based contrast, which yields true densitometric information.
Complimentary methods, such as specific fluorescent or other
biomarkers, can be employed on the same system. Current re-
permit detection of nuclear features predictive of early, preinva-
sive lung cancer.
VisionGate, Inc., Gig Harbor, Washington
145
Expression Patterns of hMSH2, hMLH1 and
PTEN in Primary Lung Carcinomas
Ekaterini Politi, M.D., Ph.D.1, George Kanellis, M.D.1,
Antonios Gouliamos, M.D., Ph.D.2, Ioulia Chatzistamou,
M.D., Ph.D.1, Vasiliki Kyriakidou, M.D.1, Helen Koutselini,
M.D., Ph.D., FIAC1
PURPOSE: Derangements in the tumor suppressor gene PTEN
and the mismatch repair genes hMSH2 and hMLH1 have an
important role in carcinogenesis. The purpose of our study was
to assess immunocytochemically the pattern of protein expres-
sion for these genes in the different histological types of primary
lung carcinomas.
MATERIALS AND METHODS: FNA specimens from 45 patients
aged from 42-81 years (mean age 65,4), with primary lung car-
cinomas (15 adenocarcinomas, 19 squamous cell carcinomas
and 11 small cell carcinomas) were included in our study. We
performed a biotin-streptavidin-peroxidase immunocytochemi-
cal method using specific monoclonal antibodies for hMSH2,
hMLH1 and PTEN (clones FE11, G168-15 and 28H6 respectively).
RESULTS: Nine of 15 adenocarcinomas (60%) and 12 of 19 squa-
mous cell carcinomas (63.16%) had reduced expression levels of
the MSH2 protein. The percentage of MSH2 positive cells and
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
the intensity of staining tended to be higher in the better differ-
entiated tumors. In contrast, all the small cell carcinomas (100%)
expressed MSH2. The majority of small cell lung carcinomas
(10/11) showed loss of PTEN expression and the same pattern
was observed in the poorly differentiated non-small cell tumors
(14/20). All the carcinomas studied were hMLH1 negative.
CONCLUSION: The current study indicates that mismatch repair
deficiency as far as it concerns the hMSH2 gene is largely not
associated with the pathogenesis of small cell lung carcinoma,
while it is a common event in the development of adenocarci-
nomas and squamous cell lung carcinomas. Loss of PTEN ex-
pression seems to be a frequent event in small cell lung carci-
nomas and in poorly differentiated NSCLCs. Our results also
suggest that hMLH1 is the major altered mismatch repair gene
involved in lung cancer tumorigenesis.
1
Cytopathology Department, Aretaieio University Hospital, Ath-
ens, Greece, 2Radiology Department, Aretaieio University Hospi-
tal, Athens, Greece
146
Adequacy of Retrieval of Pulmonary Fine
Needle Biopsies: A Report Card for
Radiologists
Jeanne Jax, B.S., CT(ASCP), Donna Cary, B.S., CT(ASCP),
Edward Bernacki, Jr., M.D.
INTRODUCTION/PURPOSE: Fine needle biopsy (FNB) retrieval of
lesional cells from a space occupying pulmonary lesion requires
both competent manual technique and radiologic localizational
skills. The purpose of this study is to assess the adequacy rates of
the radiologists performing pulmonary FNBs in our institution
and to identify factors that impact adequacy rates.
MATERIALS AND METHODS: From January 1, 2003 to September
30, 2004, 225 pulmonary FNBs were performed at our institution.
196 (87%) of these biopsies were performed by a core group of 9
radiologists. Each radiologist performed a minimum of 12 FNBs.
The adequacy and non-diagnostic rates for each of the 9 radiol-
ogists was determined. The number of passes for each of the
adequate cases was compared to the number of passes for the
inadequate non-diagnostic cases. Utilization of the “rush for
adequacy” service by individual radiologists was determined.
RESULTS: 159/196 (81%) pulmonary FNBs were adequate for
diagnosis and 37/196 (19%) were inadequate for diagnosis. The
individual adequacy rates for 9 radiologists ranged from 65%-
100%. Three radiologists had adequacy rates below 70%. These 3
radiologists performed 31% of the pulmonary FNBs. The remain-
ing 6 radiologists had adequacy rates ranging from 81%-100%.
The total number of passes for adequate cases was 3.7 and the
total number of passes for inadequate cases was 3.6. In 42/196
(21%) FNBs, a “rush for adequacy” was requested by the radiol-
ogist. The rate of radiologist’s request for adequacy assessment
vary from 0-67%.
401
CONCLUSION: Our results indicate that there is a spectrum of
success between radiologists retrieving pulmonary FNBs. This
radiologist report card can be used to improve the radiologic
retrieval service. Possible improvements include: radiologist re-
training, supervision and participation by a successful retriever,
small core group of radiologists performing pulmonary FNBs,
and increased utilization of the “rush for adequacy service”
provided by the Department of Cytopathology.
William Beaumont Hospital, Royal Oak, Michigan
147
Needle Biopsy of Pulmonary Lesions: Fine
Needle Biopsy Versus Core Biopsy
Donna Cary, B.S., CT(ASCP), Nassim Banisaeed, M.D.,
Edward Bernacki, Jr., M.D.
INTRODUCTION/PURPOSE: Accurate unequivocal diagnoses of be-
nign and malignant pulmonary lesions can be made by utilizing
the fine needle biopsy (FNB) or core biopsy technique. The
purpose of this study is to compare the diagnostic results of FNB
to core biopsy and to determine whether one technique is su-
perior to the other.
MATERIALS AND METHODS: From January 1, 2003 to September
30, 2004, 225 pulmonary FNBs were performed by the radiolo-
gists under computed tomography guidance at our institution.
52/225 (23%) had concurrent or subsequent core biopsies of the
same target lesion by the radiologist. The cytologic diagnoses
from the FNB were compared to the surgical pathologic diag-
noses from the core biopsies. In an attempt to explain the
discordant cases, the non-diagnostic FNBs and core biopsies
were analyzed by evaluating variable data including size and
location of the pulmonary target lesions and experience of the
radiologist.
RESULTS: 34/52 (65%) cases were diagnostic with both FNB and
core biopsy. 6/52 (12%) cases were non-diagnostic with both
FNB and core biopsy. 7/52 (13%) cases were diagnostic with FNB
and non-diagnostic with core biopsy. 5/52 (10%) cases were
diagnostic with core biopsy and non-diagnostic with FNB. The
non-diagnostic rate for FNB was 21% and the non-diagnostic
rate for core biopsy was 25%. The combination of FNB and core
biopsy yielded a diagnostic rate of 88%.
CONCLUSION: The diagnostic rate for FNB was 79% and the
diagnostic rate for core biopsy was 75%. When utilized together
the diagnostic rate of both of these needle biopsy procedures
increased to 88%. Our comparative study of 52 patients with fine
needle biopsy and core biopsy of pulmonary lesions demon-
strates that fine needle biopsy is comparable and not inferior to
core biopsy. In our institution there was a perception by some
clinicians and pathologists that core biopsy of pulmonary le-
402 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
sions was a superior diagnostic test when compared to fine
needle biopsy. Our results do not confirm that impression.
William Beaumont Hospital, Royal Oak, Michigan
148
Incidence of Malignancy in Pericardial,
Peritoneal, and Pleural Effusions in Patients
40 Years Old and Younger
Sophie Aalaei, Vijaya Reddy, M.D., Larry Kluskens, M.D.,
Ph.D., Paolo Gattuso, M.D.
PURPOSE: To examine the frequency of malignant effusions and
their origin in pericardial, peritoneal and pleural body effusions
in patients 40 years old and younger.
MATERIALS AND METHODS: Between January 1990 and Decem-
ber 2004, a total of 697 pericardial, peritoneal, and pleural fluids
were recorded in patients 40 years and younger. The pertinent
clinical history and cytologic reports were reviewed.
RESULTS: There were 76 (11%) pericardial fluids, 268 (38%) peri-
toneal fluids and 353 (51%) pleural fluids. Overall incidence of
malignant effusions was 98 (14%), which could further be sub-
divided into pericardial 5 (5%), peritoneal 27(28%), and pleural
66 (67%) fluid collections. The most common malignancy en-
countered was adenocarcinoma 55 (56%), followed by malignant
lymphoma 23 (24%), carcinoma NOS 15 (15%), squamous cell
carcinoma 2 (2%), and one case each (1%) of neuroendocrine
carcinoma, rhabdomyosarcoma, as well as neuroblastoma.
Among the patients with malignant effusions, there were 21
(21%) males and 77 (79%) females. Their ages ranged between 1
and 40 years with the mean of 35.3. The most common primary
site encountered was the breast 29 (30%), followed by lymphoid
23 (24%), gynecologic tract 16 (16%), lung 11(11%), gastrointes-
tinal tract 4 (4%), and other sites 15 (15%). The remaining cases
were: 15 (2.0%) suspicious for malignancy and 584 (84%) benign
effusions.
CONCLUSIONS: The most common malignancy encountered was
breast carcinoma followed by lymphoid neoplasms. The most
common malignant effusion was pleural. The majority of our
patients with malignant effusions were females. The high num-
bers of breast carcinomas in our study may reflect the large
volume of breast cancer patient treated at our institution.
Rush University Medical Center, Chicago, Illinois
149
The Diagnostic Utility of the Differential Cell
Count of the Broncho-Alveolar Lavage (BAL)
Fluid in Patients with Interstitial Lung
Disease
Jennifer Laudadio, Lydia Christiansen, Mostafa Fraig,
M.D.
BACKGROUND: The role of BAL fluid cellular analysis in investi-
gating interstitial lung disease is well recognized. However, the
clinical correlation of the BAL cellular profiles from these pa-
tients is not well defined. The purpose of this study is to evaluate
the utility of the differential cell count of BAL fluid in distin-
guishing the subtypes of interstitial lung disease or in narrowing
the differential diagnosis of such entities.
DESIGN: A computer-based search of the files of the Divisions of
Cytopathology and Surgical Pathology revealed 65 cases of bi-
opsy- proven interstitial lung disease spanning the period 2000-
2003. These included 22 cases of idiopathic interstitial pneumo-
nia (ILD group), divided into 4 cases of Usual Interstitial
Pneumonia (UIP), 11 cases of Non Specific Interstitial Pneumo-
nia (NSIP) and 7 cases of Respiratory Bronchiolitis-Interstitial
Lung Disease RB-ILD/Desquamative Interstitial Pneumonia
(DIP). Twenty eight cases of Acute Lung Injury pattern (ALI
group) were included, which were divided into 15 cases of Dif-
fuse Alveolar Damage (DAD) and 13 cases of Bronchiolitis oblit-
erans/Organizing Pneumonia (BOOP). In addition, 15 cases of
sarcoidosis were also included. The cell counts, surgical pathol-
ogy and cytology material were reviewed and correlated with the
clinical outcome whenever it was pertinent.
RESULTS: The cellular composition of the BAL fluid from the ILD
as a group was not significantly different from that of the ALI
pattern group. Within each group, the cell count was not signif-
icantly different to differentiate the subtypes from each other.
However, Sarcoidosis demonstrated a significantly different cel-
lular profile from that of the ILD and ALI groups in both the
neutrophilic and lymphocytic counts but not the macrophage
counts. Relative eosinophilia (!5%) was present in a total of 9/65
(14%) of cases and correlated with short survival in UIP and good
response to steroid therapy in the other disease entities.
CONCLUSION: Differential Cell count of BAL fluid could be help-
ful in the differential diagnosis of sarcoidosis versus other inter-
stitial lung disease. Increased eosinophilic count correlates with
bad prognosis in UIP and better prognosis in other interstitial
lung disease entities. Larger studies are needed to validate such
findings and to control for other variables such as smoking and
environmental exposures.
Medical University of South Carolina, Charleston, South Carolina
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
150
Reflex Gomori Methenamine Silver Staining
for Pneumocystis Jirovecii on
Bronchoalveolar Lavage Cytologic
Specimens: A Review
Mauricio Zapata, M.D., James Little, M.D., Aziza Nassar,
M.D., Melinda Lewis, M.D., Sanjay Logani, M.D., Momin
Siddiqui, M.D., FIAC
INTRODUCTION/PURPOSE: Pneumocystis jirovecii (formerly Pneu-
mocystis carinii) is an opportunistic pathogen causing life threat-
ening pneumonia (PCP) in immunosuppressed individuals.
Traditionally, its diagnosis has been dependent on direct dem-
onstration of the organism on bronchoalveolar lavage (BAL)
specimens. In addition, the use of special stains such as Gomo-
ri’s methenamine silver (GMS) stain has been advocated to
highlight the organisms in BAL specimens. The purpose of this
study was to determine the utility of reflex GMS staining on all
BAL specimens for identification of P. jirovecii.
MATERIALS AND METHODS: At our institution all BAL specimens
have routinely had a reflex GMS stains performed. A retrospec-
tive review of all BAL specimens for the years 2000-2004 was
performed. BAL samples were processed as cytospins, which
were stained with Papanicolaou and GMS stains. These slides
were reviewed and the data was tabulated.
RESULTS: A total of 2984 BAL specimens (males, 1961 and fe-
males, 1023) were identified over a five year period (2000-2004).
The age ranged from16 to 92 years (average age, 36 years). A total
of 116 (3.8 % of total BAL) BAL specimens (males, 97 and fe-
males, 19) were diagnostic of P. jirovecii. The age ranged from 19
to 44 years (average age, 31 years) in this group. P.jirovecii was
identified on both Papanicolaou stained and GMS stained slides
in 103 specimens (88.7 % of total positive BAL), Papanicolaou
stained slide alone, in 11 specimens (9.4% of total positive BAL),
and GMS stained slide alone, in 2 specimens (1.7 % of total
positive BAL). The cost of each GMS stain was assessed at $50.
CONCLUSIONS: P. Jirovecii in BAL specimens in the last five years
has been seen in only 3.8 % of the specimens. This may be
attributed to improved management of immunocompromised
patients. Performing reflex GMS stain on all BAL specimens does
not improve the diagnostic identification of P. jirovecii since
majority of diagnosis can be rendered on Papanicolaou stained
slides. A cost analysis was conducted, which estimated that GMS
staining on 2,868 specimens, with no demonstration of P. jirove-
cii cost $143,400. Thus, from a cost-benefit perspective GMS
staining can be performed, when Papanicolaou stain is either
negative or suggestive of P. jirovecii, however, the clinical pre-
sentation is consistent with PCP.
Emory University Hospital, Atlanta, Georgia
403
151
EUS-FNA of Mediastinal Lesions: Diagnostic
Performance and Potential Pitfalls
Isam Eltoum, M.D.1, Mohit Mehra2, David Chhieng, M.D.1,
Darshana Jhala, M.D.1, Nirag Jhala, M.D., MIAC1, Ralph
Crowe, M.D.1, Mohamad Eloubeidi, M.D., M.P.H.2
INTRODUCTION: Endoscopic ultrasound guided fine needle aspi-
ration (EUS-FNA) has being increasingly performed to evaluate
primary mediastinal lesions as well as to stage patients with
known primary cancer, particularly lung carcinoma. In this
study, we assessed the diagnostic accuracy of EUS-FNA and
reviewed the potential pitfalls associated with EUS-FNA of me-
diastinal lesions.
MATERIALS AND METHODS: The study included all consecutive
patients coming to EUS-FNA program in the period August 2000
to Dec 2004 for staging of lung cancer or for evaluating medias-
tinal mass. Based on surgical resection, pathologic findings, or
close follow-up, the final outcome was considered the reference
standard. Diagnostic accuracy was determined using sensitivity,
specificity, likelihood ratios and predictive values. The false neg-
ative/positive cases were re-evaluated to classify the source of
errors and the potential pitfalls were reviewed.
RESULTS: 286 EUS-FNA were performed on 157 consecutive
patients (151 one site, 51 two sites and 15 ! 2 sites). Cytological
diagnoses in these cases as fellows: insufficient 2, benign 178,
atypical 1, suspicious 4, and malignant 101. 35 lesions were
excluded from further analysis because of lack of adequate fol-
low up. 161 lesions were detected in patients (mean age, SD, 63,
9.6) presented for staging for non-small cell carcinoma. 90 le-
sions were detected in patients (mean age, SD, 55, 16.2) who
presented primarily for mediastinal mass workup. Overall sen-
sitivity/specificity, positive/negative likelihood ratio and posi-
tive/negative predictive values were 88%/100%, 235/0.12, 100%/
90%, respectively. There is no significant difference in diagnostic
performance of EUS-FNA between the two groups. Error in all 14
false negative cases was due to sampling. Potential pitfalls in-
cludes: (a) epithelioid histiocytes with dense cytoplasm, seen in
almost all cases (anthracotic macrophages vs. metastatic mela-
noma (n"2) vs. carcinoma) (b) necrotic debris (histoplasmosis
(n"6) vs. necrotic carcinoma (c) spindle-epithelioid clusters
(granuloma n" 18) vs. mesenchymal tumor (n"1) (d) mature
squames seen in almost all cases (esophageal contaminants vs.
well-differentiated squamous cell carcinoma (n"10) (e) cyst
content (unsatisfactory specimen (n" 2) vs. foregut cyst (n"9) )
(f) small blue cells (small cell carcinoma (n"5) vs. lymphoma
(n"5) vs. benign lymph node).
CONCLUSIONS: EUS-FNA is valuable tool for assessing mediasti-
nal lesions and demonstrated a high diagnostic performance.
Mediastinal lesions showed a wide spectrum of cytodiagnosis.
With increasing use of EUS-FNA technique, cytologist need to be
404 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
familiar with errors and potential pitfalls associated with these
lesions.
1
Division of Anatomic Pathology, Department of Pathology, Uni-
versity of Alabama at Birmingham, Birmingham, Alabama,
2
Division of Gastroenterology and Hepatology, Department of
Medicine, University of Alabama at Birmingham, Birmingham,
Alabama
152
Clinical Utility of Immunostains in the
Differential Diagnosis of Lung Malignancies
by FNA Biopsies
Ming Guo, M.D., Yee Jee Jan, M.D., Yun Gong, M.D.,
Betsy Jacob, B.S., M.S., CT(ASCP), Nour Sneige, M.D.
INTRODUCTION: Differential diagnosis of metastatic adenocar-
cinoma from primary lung carcinoma on the basis of cyto-
morphology may be difficult, especially in patients with a
history of more than one malignancy. Thyroid transcription
factor 1 (TTF-1), cytokeratin 7/20 and other immuno-markers
have been used in FNA lung biopsies to help with diagnoses.
Although the role of TTF-1 and other immunomarkers in
diagnosis of lung malignancies have been reported, published
data of clinical efficacy of these markers in lung FNA biopsies
are limited. This study was conducted to evaluate the clinical
utility of TTF-1 and other immunomarkers in FNA lung biop-
sies.
MATERIALS AND METHODS: From 640 cases of FNA lung biopsies
performed at M.D. Anderson Cancer Center in 2004, we retro-
spectively reviewed 93 cases with diagnostic difficulties. Of 93
patients, 71 (76.4 %) have a history of one (60 cases) or more
than one malignancies (11 cases). TTF-1 immunostain was per-
formed in all 93 cases. CK7/20 and other immunostains were
performed in 43 % and 46 % cases, respectively. Core biopsies or
excisional lung specimens were available in 41 cases. The con-
tributory rates of TTF-1, CK7/20 and other immunomarkers
toward cytology diagnoses were determined by a comparison of
our cytology diagnoses with the clinical/radiological/pathologi-
cal diagnoses.
RESULTS: Of 93 patients, 71 patients with a history of malig-
nancy, 30 (42.2 %) were confirmed to have primary lung carci-
noma. A high cytology diagnostic accuracy was observed in
correlation with clinical/radiological/pathological findings (Ta-
ble 1). TTF-1 was positive in 72.0 % (36/50) of primary lung
carcinoma and 5.6 % (2/36) of metastatic carcinoma of the lung.
The contributory rates of TTF-1, CK 7/20 and other immunos-
tains for diagnoses of FNA lung biopsies are 69.9%, 18.3 % and
16.1%, respectively. The combined contributory rate of immu-
nostains for cytology diagnoses is 86.0 %.
Clinic/Radiology/Pathology
FNA
Dianoses Primary Lung Metastatic Undetermined Total
Primary Lung 48 0 1 49
Metastatic 1 31 0 32
Undetermined 1 5 6 12
Total 50 36 7 93
CONCLUSIONS: Immunocytochemistry assay of TTF-1 is highly
contributory for the diagnoses of FNA lung biopsy (69.9 %). If
combined with CK7/20 and/or other immunostains, the immu-
nocytochemistry contributes to 86.0 % of the diagnoses to dif-
ferentiate primary lung from metastatic carcinoma.
The University of Texas, M.D. Anderson Cancer Center, Houston,
Texas
153
EGFR Abnormalities in Lung Cytology
Specimens: FISH and
Immunohistochemistry (IHC) Correlation
Eric Wei, M.D., Ph.D., Elba Turbat-Herrera, M.D., Sherry
Martin, MT, M.H.S., Mary Lowery-Nordberg, Ph.D.
INTRODUCTION: Epidermal Growth Factor Receptor (EGFR) is a
transmembrane receptor overexpressed in various epithelial tu-
mors, including 34% to 84% of lung carcinomas. It contributes to
tumor progression and portends shorter patient survival. Bron-
chial washing/brushing and fine needle aspiration are often
used for cytological diagnoses of lung cancer. Morphological
examinations to differentiate reactive from malignant cells
sometimes can be difficult. Assessing EGFR expression levels by
fluorescence in situ hybridization (FISH) and IHC may provide
critical information of chromosomal and tumor marker abnor-
malities, and assist cytological diagnoses.
MATERIALS AND METHODS: Fifty patients with bronchial wash-
ing/brushing or fine needle aspiration specimens, and corre-
sponding histologically confirmed lung biopsies were studied for
EGFR expression with FISH and IHC. FISH analyses were initi-
ated using an EGFR locus specific probe for the EGFR locus on
chromosome 7 at 7p12 (LSI® EGFR SpectrumOrangeTM / CEP® 7
SpectrumGreenTM). Copy numbers of the EGFR gene locus were
studied. EGFR FISH results were compared with our previous
FISH results with combined EGFR, c-myc, 5p15.2 and chromo-
some 6 probes. Cell blocks, if available, and tissue biopsy sec-
tions were used for evaluating EGFR protein expression (EGFR
pharmDxTM, Dakocytomation). Normal epithelium and stromal
cells provided a negative internal control. Tumor cells were
either positive or negative for EGFR expression. Quantitation of
EGFR expression was performed using an automated cellular
imaging system (ACIS, Chromavision, Inc.).
RESULTS: Aneuploidy and/or gene amplification of EGFR was
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
identified by FISH. The chromosomal abnormalities of EGFR
were often accompanied by other chromosomal irregularities
exhibited with c-myc, 5p15.2 or chromosome 6, suggesting gen-
eral genomic instability. Gene amplification of EGFR was found
mainly in tumor cells with high protein expression. Atypical and
suspicious cases by cytology had 60% positivity confirmed for
malignancy by biopsy. EGFR tended to show gene amplification
or aneuploidy in those confirmed specimens with malignancy.
CONCLUSIONS: EGFR is overexpressed in a certain percentage of
lung carcinomas by both gene amplification and aneuploidy.
This may be accompanied by other chromosomal abnormalities.
FISH analysis of EGFR may be a useful adjunctive method to
stratify controversial cases and provide important prognostic
information.
Louisiana State University Health Sciences Center, Shreveport,
Louisiana
154
Causes of Lack of Correlation Between
Cytologic Examination of GMS-Stained
Bronchoalveolar Lavage Specimens (BALs)
and Culture Results
Anna Marie Carley, M.D.1, Mackenzie Cooper, CT(ASCP)2,
Dan McKeon, SCT(ASCP)2, Patricia Ferrieri, M.D.1, David
Carley1, Janis Thurston, MT2, Jennifer Ellison2, Stefan
Pambuccian, M.D.1
INTRODUCTION/PURPOSE: Fungal infections are a leading cause
of morbidity and mortality in solid organ and bone marrow
transplant patients. Cytological evaluation of GMS-stained BALs
offers a means of rapid diagnosis of such infections. We under-
took this study to try to elucidate the causes of discrepancy
between cytology and culture results.
MATERIALS AND METHODS: Our institution’s cytology and mi-
crobiology databases were reviewed for all consecutive BALs
from 1/2/2003 to 6/27/2003. Cytology results were available
same day, while culture results took up to 28 days. The following
features were evaluated semi-quantitatively on GMS and Papa-
nicolaou-stained slides from BALs: alveolar macrophages, mu-
cus, respiratory epithelium, squamous cells, fungal organisms,
RBCs, PMNs and volume of fluid submitted.
RESULTS: 376 BALs from 225 predominantly lung and bone
marrow transplant patients were evaluated. 93 (25%) cases were
positive for fungi on cytology (Cy$)(19 ‘consistent with Aspergil-
lus,’ 64 ‘pseudohyphae’ and 5 ‘small budding yeast’) while 250
(66.5%) cases had fungi on culture (Cu$), of which 86 Aspergil-
lus spp., 145 Candida spp and 1 Histoplasma spp. Cytology and
culture correlated as follows: 114 cases Cy-/Cu-, 169 cases Cy-/
Cu$, 81 cases Cy$/Cu$ and 12 cases Cy$/Cu-. The cytologic
species prediction was confirmed by culture in 13 cases of As-
pergillus and 52 cases of Candida. In 17 cases in the Cy$/Cu$
405
group, the fungus species prediction on cytology did not corre-
late with culture results. 7 cases called ‘pseudohyphae/budding
yeast’ cultured for non-Candida fungi (4 Aspergillus spp, 3 other);
3 cases reported as ‘consistent with Aspergillus’ cultured for
other fungi (2 Candida spp., 1 other). All 5 cases reported as
‘small budding yeast’, where a suspicion for Histoplasma was
raised cultured for Candida spp. Blind review of the Cy$/Cu$
non-correlating cases showed an improvement in the correla-
tion, with exact speciation in 4 additional cases. Mean fungal
growth on culture was less in the Cy$/Cu$ non-correlated
group versus the Cy$/Cu$ correlated group. No significant
differences were found between cases selected from the Cy$/
Cu$ (16 cases) and Cy-/Cu$ (33 cases) groups in the semi-
quantitated features studied. On rescreening 2/33 cases in the
Cy-/Cu$ group were found to have a ‘rare hyphal fragment’
giving a false negative rate of 11.1%.
CONCLUSIONS: GMS cytological evaluation of BALs provides fast,
relatively specific means for detecting fungi in immunocompro-
mised patients, though the exact identification of the fungal
species may be difficult, especially in cases with rare organisms.
Single or light colony growth on culture may explain the non-
correlated cases. Criteria for specimen adequacy have yet to be
determined.
1 2
University of Minnesota, Minneapolis, Minnesota, Fairview
University Medical Center, Minneapolis, Minnesota
155
Role of On Site Cytopathology Assessment
of Endoscopic Ultrasound Guided Fine
Needle Aspiration in Lung Tumors
Renu Khode, M.D.1, Robert Astarita, M.D.1, Eric Rachut,
M.D.1, Shanthi Gopal, M.D.1, Edwin Johnson, M.D.1,
Douglas Toler, Ph.D., CT(ASCP)1, Yashodeep Jadhav,
M.D.1, Phalguni Mukhopadhyay, M.D.1, Brian Camazine,
M.D.1, Richard Erickson, M.D.2, Shyam Narayana, M.D.1,
Pankaj Singh, M.D.1
BACKGROUND: Endoscopic ultrasound guided fine needle aspi-
ration (EUS – FNA) is being increasingly used as means of
obtaining cytological material in patients with lung carcinoma.
Since multiple sites are sampled for complete staging, the on site
information plays a very important role in further sampling.
AIM: To assess the accuracy of “on site cytopathology assess-
ment” of EUS – FNA.
METHODS: Data was collected from an ongoing large prospective
study. All consecutive patients with newly diagnosed lung mass,
underwent EUS examination in the following order: liver, adre-
nal gland, celiac axis lymph nodes, mediastinal lymph nodes (
para-esophageal, subcarinal, aorto-pulmonary window, para-
tracheal) and mediastinal lung masses. EUS - FNA was per-
formed of the suspicious lesions or lymph nodes. Air-dried, Diff
406 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
Quik stained smears were assessed for adequacy and cytology
diagnosis. Additional passes were requested if the initial speci-
men was hypocellular, or for additional material for the cell
block or ancillary studies like immunohistochemistry (IHC). The
final diagnosis was rendered after reviewing alcohol-fixed, Pa-
panicolaou stained smears and the cell block.
RESULTS: A total of 236 sites (liver-21; adrenal-43; celiac axis-19;
mediastinal mass-30; mediastinal lymph nodes-123) were sam-
pled from 116 patients with lung mass. On site evaluation for
adequacy was done in all 236 samples. On site diagnosis was
provided in 151 samples. Of the 236 samples, the cellularity of
the aspirates was graded as: adequate (223), scant (9) and insuf-
ficient (4).The diagnostic terminology used was: malignant (109),
atypia (0), suspicious for malignancy (2), negative for malig-
nancy (115) and non-diagnostic (10). In a total of 17 /151 sam-
ples (11%), the on site diagnosis was different from the final
diagnosis. Contamination of the aspirate by normal gastrointes-
tinal mucosa leading to over interpretation and presence of
diagnostic material in the cell block or the Pap stained smears,
were the reasons for these discrepancies. Overall, the on site
evaluation, had a sensitivity of 98%, specificity of 84%, positive
predictive value of 97%, negative predictive value of 88% and an
accuracy of 97%.
CONCLUSIONS: This extensive study of 236 samples showed high
accuracy (97%) of onsite cytopathology assessment. It helps to
determine the presence of adequate diagnostic material, and
renders a preliminary diagnosis. It guides the endoscopist, in
decision making for obtaining the sample from other sites. Thus
diagnosis and staging of the lung tumor is established in a single
procedure. On site evaluation has a high positive predictive
value for diagnosis of malignancy, but it has a low specificity
(84%) and therefore treatment plans should be based upon the
final report.
1
Central Texas Veterans Health Care System, Temple, Texas,
2 recipients. All (7/7) patients with ASP documented by cultures/
Scott & White Memorial Hospital, Temple, Texas
156
Bronchoalveolar Lavage as Predictor of
Pulmonary Pathology in Bone Marrow
Transplant Recipients: Results From a
Large Transplant Center
Monika Roychowdhury, M.D.1, Mariam Alsharif, M.D.1,
Dan McKeon, CT(ASCP)2, Stephanie Hart-Gouleau, M.D.1
Stefan Pambuccian, M.D.1, Evin Gulbahce, M.D.1
INTRODUCTION/PURPOSE: Pulmonary complications are a com-
mon cause of morbidity and mortality and are seen in 30-60% of
bone marrow transplant(BMT) recipients. Because of poor gen-
eral health and cytopenias, these patients are not candidates for
transbronchial (TBB) or open lung biopsy (OLB) and frequently
undergo bronchoalveolar lavage (BAL) to identify the pulmonary
pathology. The aim of this study is to determine the diagnostic
yield of BAL in BMT recipients.
MATERIALS AND METHODS: We identified BMT recipients with
BAL followed by TBB, OLB, or autopsy performed within two
weeks from 6/96-6/04. The age, disease, type of BMT, lung/
blood cultures were recorded. BAL were reviewed for adequacy
(!10 alveolar macrophages/10hpf), background, viral inclu-
sions, fungi (silver stains), and a differential cell count was done.
Histological slides and special stains were reviewed when avail-
able.
RESULTS: We had 36 BAL from 28 patients with age range:16mo-
68yr; ave:34yr; F/M:11/17. Underlying disease requiring BMT
were: Hematopoietic malignancy(15), Fanconi s/aplastic ane-
mia(6), Myelodysplastic Syndrome(4), congenital/metabolic dis-
ease(3). 25 patients has allogeneic, 3 autologous BMT. None of
the patients had OLB or TBB within 2 weeks of BAL. 4 patients
had only blood in BAL and 17 BAL from 16 patients had bloody
background or moderate amount of blood. Hemosiderin laden
macrophages(HLM) were identified in 30 BAL from 24 patients
(ave:12%; range 0-52%). Significant number of HLM (!20%) was
present in 6 BAL from 5 patients of which 3 had diffuse alveolar
hemorrhage (DAH), 1 diffuse alveolar damage (DAD), and 1
Aspergillus (ASP) identified in tissue. 9 patients had inadequate
(IA) BAL. 8/9 patients with IA BAL had only blood, or bloody
background 7 of which had one or more of DAD, DAH, and ASP.
Sequential increase in HLM and/or blood in the background was
associated with DAH and DAD in 4/5 patients who had consec-
utive BALs. 0/7 patients with ASP in tissue(5) or culture(2) had
fungal organisms identified on BAL. 6/7 patients with atypical/
reactive cells in BAL had DAD or DAH.
CONCLUSIONS: 1- Inadequate BAL with a bloody background
may indicate a serious lung injury in BMT recipients. 3/7 pa-
tients with DAH had bloody background which were diagnosed
as IA. 2- BAL is not sensitive for detection of fungi in BMT
histology had negative prior BAL. 3-Sequential increase in HLM
and blood in BAL are also commonly associated with DAD and
DAH.
1 2
University of Minnesota, Minneapolis, Minnesota, Fairview
University Medical Center, Minneapolis, Minnesota
157
Utility of Cell Block Preparations in
Cytologic Specimens Diagnostic of
Lymphoma
Liron Pantanowitz, M.D., Jonathon Freeman, M.D., John
Hunt, M.D., Roxanne Florence, M.D., Maryanne Hornish,
CT(ASCP), M.B.A., Robert Goulart, M.D.
INTRODUCTION: The ability to diagnose lymphoma by cytologic
preparations has expanded with the use of ancillary studies.
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
Although cell blocks are not currently a widely used adjunct in
the evaluation of lymphoid lesions, they are routinely prepared
in our laboratory. The aim of this study was to evaluate the
benefit of cell block preparations in cytologic specimens that
were diagnostic of lymphoma.
MATERIALS AND METHODS: We performed a 10-year retrospec-
tive review of all cytologic specimens, including fine needle
aspiration biopsies (FNAB) and body cavity fluids, that were
diagnostic of lymphoma and in which a cell block was prepared.
71 (42%) of these cases were liquid-based (ThinPrepTM) speci-
mens. The success of immunophenotyping by immunohisto-
chemistry (B-cell vs. T-cell) on cell block material to help estab-
lish clonality, and deferment to flow cytometry and/or
concurrent biopsy material in these cases, were recorded. Cases
were stratified as to whether or not a clinically meaningful (ad-
equate for patient treatment) diagnostic lymphoma category was
reached.
RESULTS: We studied 171 specimens from 160 patients (72 male;
88 female) with an age range of 4-92 years (mean 64 years).
Specimens included 138 FNAB and 33 body cavity fluids (24
pleural, 7 peritoneal, and 2 pericardial). Cell block morphology
was contributory in 162 cases (95%). Immunophenotyping on
cell blocks was performed in 91 cases (53%), and yielded inter-
pretable results in 84 (92%) of these cases. Clinically meaningful
lymphoma diagnoses, based solely on cytomorphology and in
some cases cell block immunohistochemistry, were made in 145
cases (85%), with these diagnostic groups including large cell
(n"112; 77%), low grade (n"21; 14%), Hodgkin (n"8; 5%), lym-
phoblastic (n"3; 2%), and anaplastic lymphoma (n"1; 1%).
CONCLUSION: Cell blocks provided contributory morphology
(95%) and, when attempted, sufficient material for immunophe-
notyping (92%). Evaluation of cell block material thereby con-
tributed to a clinically meaningful diagnosis of lymphoma in a
high percentage (85%) of our cases. The number of diagnostic
cases may have been greater if, in a subset of cases, ancillary
studies were not deferred to concurrent flow cytometry or bi-
opsy material. Therefore, we believe that cell block preparations
are a valuable adjunct in the cytologic diagnosis of lymphoma
and may reduce the need for surgical excision and/or supple-
mentary ancillary studies, such as flow cytometry.
Baystate Medical Center, Tufts University School of Medicine,
Springfield, Massachusetts
158
Utility of Fine Needle Aspiration in
Diagnosis of Burkitt’s Lymphoma
Julie Baird, M.D., Aziza Nassar, M.D., Melinda Lewis,
M.D., Sanjay Logani, M.D., Momin Siddiqui, M.D., FIAC
INTRODUCTION/PURPOSE: Fine needle aspiration (FNA) is rou-
tinely used in diagnosing and typing malignant lymphoma.
407
However, diagnosis of Burkitt’s lymphoma requires correlation
of morphology with immunophenotypic and cytogenetic data.
This study illustrates the utility of fine needle aspiration in
obtaining material for both morphologic and additional ancillary
studies.
MATERIALS AND METHODS: A total of 21 patients who were
diagnosed with Burkitt’s lymphoma in our institution in the last
5 years, were selected for this study. FNA was used as the initial
diagnostic modality in all cases. Smears prepared by DiffQuik
and Papanicolaou stain were reviewed, in addition cell blocks
with diagnostic material was available in all cases. A portion of
the specimen was also submitted for flow cytometric and cyto-
genetic studies.
RESULTS: All 21 cases (17 males and 4 females) morphologically
showed classic features of Burkitt’s lymphoma. The age range
was 6 to 19 years (average age, 9 years). The FNA aspiration sites
were as follows: Abdominal mass (ileo-cecal region)-14 cases,
mesenteric lymph nodes-4 cases, spleen-2 cases and liver-1 case.
The cells were monotonous and of medium size. Scant cyto-
plasm was present in all 21 cases, and small cytoplasmic vacu-
oles were noted in 19 of 21 cases. The nuclei were oval to round,
noncleaved and had blastic chromatin. Multiple small but prom-
inent nucleoli were also uniformly present in all 21 cases. All 21
cases, had 8q24 c-myc breakpoint gene rearrangement by FISH
on cell block preparations. In addition, CD10 positivity was seen
in 21 of 21 patients, CD20 positivity in 20 of 21 patients, CD77 in
19 of 21 patients, and Ki-67 positivity in 21 of 21 patients,
ranging from 95-100% expression.
CONCLUSION: This study documents the utility of FNA in the
diagnosis of Burkitt’s lymphoma. The morphologic features of
Burkitt’s lymphoma were studied, in detail. Also, it shows that
the FNA diagnosis of Burkitt’s lymphoma is reliant on flow
cytometry and c-myc rearrangement by FISH on cell block prep-
aration. Thus FNA is helpful in making provisional assessment of
the nature of the specimen and then collecting appropriate
specimens, which should include adequate material for flow
cytometry and cytogenetic studies.
Emory University Hospital, Atlanta, Georgia
159
Grading Follicular Lymphomas in Fine-
Needle Aspiration Biopsies: The role of
ThinPrep® Slides and Flow Cytometry
Guilherme Brandao, M.D., Oscar Lin, M.D., Susan
McKenzie, Ruth Rose, B.S.
INTRODUCTION: Follicular lymphomas (FL) are treated according
to their grade, with grades 1 and 2 managed as low grade lym-
phomas, while some groups have advocated a more aggressive
management of grade 3 FL. The criterion recommended by the
WHO in the grading of follicular lymphomas is based on the
408 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
absolute number of centroblasts counted in 10 neoplastic folli-
cles. Due to the lack of architecture to evaluate, grading of
follicular lymphomas remains one of the limitations of fine
needle aspiration (FNA). In this study, we evaluated the feasibil-
ity of grading FL using ThinPrep® (TP) slides from fine needle
aspiration biopsies (FNA) specimens. The rationale to use TP
slides are 1) better fixation; 2) excellent nuclear morphology; 3)
clean background; 4) random distribution of cells allowing for
standardized counting of centroblasts. We also analyzed the data
generated by flow cytometry, when available. The percentage of
small and large CD10$ cells was calculated and correlated with
the histologic findings.
METHODS: We searched our files and retrospectively reviewed 54
cases of lymph node FNA from patients with histologically con-
firmed follicular lymphoma (21 grade 1, 17 grade 2 and 16 grade
3). Two pathologists (OL and GAB) independently counted the
number of centroblasts present in 300 lymphoid cells and the
number of centroblasts present in 10 high/power fields (HPF) in
TP Papanicolaou slides. The percentage of CD10$ small and
large cells was calculated using the flow cytometry results.
RESULTS: The number of centroblasts/300 lymphoid cells
ranged from 1 to 12 in grade 1 FL (mean"4.6); 4 to 44 in grade
2 FL (mean"15.5) and 62 to 266 in grade 3 FL(mean"151.4). The
number of centroblasts/ 10 HPF ranged from 1 to 10 (mean"3.4)
in grade 1 FL, 4 to 38 (mean"16.7) in grade 2 FL, and 42 to over
100 (mean"78.7) in grade 3 FL. Thirty-four of 54 cases (62.9%)
had concomitant flow cytometry (18 grade 1, 7 grade 2 and 9
grade 3). In cases where two populations of CD10$ B-cells could
be identified and quantified, the percentage of small cells varied
from 67 to 92% (mean" 83.7%) in grade 1 FL; 8 to 91%
(mean"61.9%) in grade 2 FL and 11 to 88% (mean" 55.8%) in
grade 3 FL.
CONCLUSION: The count of number of centroblasts either in 300
lymphoid cells or per 10 HPF in TP slides is a reliable and
reproducible method to separate grade 1 and 2 FL from grade 3
FL in FNA samples. Analyses of cell size by flow cytometry can be
useful in a selected number of cases but the overlap of results in
different grades hinders its usefulness.
Memorial Sloan-Kettering Cancer Center, New York, New York
160
T-Cell Lymphoma: Is an Accurate
Cytopathologic Diagnosis Possible Based on
Morphology Alone?
Christopher Owens, Edward Weir, M.D., Yener Erozan,
M.D., Dorothy Rosenthal, M.D., FIAC, Syed Ali, M.D.
INTRODUCTION: Differentiating T-cell lymphoma (TCL) from re-
active lymphoid hyperplasia (RLH) on fine needle aspiration
(FNA) is often extremely difficult due to significant morphologic
overlap. Immunophenotypic information is critical for accurate
diagnosis and flow cytometric (FC) analysis has greatly increased
the diagnostic yield in this setting. Our study retrospectively
analyzed the sensitivity of an FNA diagnosis for TCL through a
blinded review by four pathologists without FC data. The
blinded diagnoses were compared to the original cytopathologic
diagnoses, rendered with the help of FC data, and the sensitivity
of the two were compared.
MATERIALS AND METHODS: The cytopathology archives at a
major teaching hospital identified 20 cases of TCL and RLH in a
10-year period (1995-2004) that had FC data as well as histologic
and/or prolonged clinical follow-up. Smears were stained with
Diff Quik and Papanicolaou stains. The slides were blinded and
the study participants (three cytopathologists and one hemato-
pathologist) with a brief clinical history, reviewed key slides of
each case categorizing them as benign (BLH) or malignant
(TCL). Their diagnoses as well as the original cytopathologic
diagnoses rendered with help from FC analysis were compared
to the final reference diagnoses based on histologic material
and/or prolonged clinical follow-up.
RESULTS: The combined sensitivity, specificity, positive predic-
tive value, and negative predictive value for the four pathologists
blinded to FC data was 78%, 75%, 76%, and 77% respectively.
The sensitivity, specificity, positive predictive value, and nega-
tive predictive value of the original cytopathologic diagnoses
rendered with help from FC data were 100%, 90%, 91%, and
100% respectively. Anaplastic large cell lymphoma (ALCL) was
the T-cell entity most often correctly identified as malignant
(15/16 or 94% of occasions) and was specifically diagnosed the
most (3/16 or 19% of occasions) based on cytomorphology
alone. Participants in general found cellular monotony, marked
irregularity of nuclear membrane, multinucleation (in ALCL)
and cellular necrosis to be helpful for the diagnosis of TCL.
CONCLUSIONS: Immunophenotypic data from FC analysis en-
hances the ability to differentiate TCL from RLH in FNA. Making
specific diagnoses for TCL based only on cytomorphology alone
is often difficult due to significant morphologic overlap. ALCL is
the only T-cell entity with specific cytomorphologic features. FC
analysis greatly increases the sensitivity, specificity, PPV and
NPV of a cytopathologic diagnosis in such setting. On-site eval-
uation by a cytopathologist is recommended to ensure suspi-
cious cases are triaged for FC analysis.
The Johns Hopkins Hospital, Baltimore, Maryland
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting 409

161 CONCLUSIONS: Access to expert hematopathologist review

yielded accurate FNA diagnosis of lymphoma as a general diag-

Initial Diagnosis of Hematopoietic nosis. However, specific diagnoses of lymphoma subtype were
usually rendered on the subsequent surgical biopsy rather than
Neoplasms on FNA: Access to Expert FNA even when Flow was done.
Hematopathologists Facilitates Triaging of
University of New Mexico School of Medicine, Albuquerque, New
Patients Requiring Further Study but Does Mexico
Not Result in Specific FNA Diagnoses in
Most Cases
Fatma Midani, Sanjay Lahiri, Therese Bocklage, M.D.
162
INTRODUCTION/PURPOSE: In response to a recent article in the Initial Diagnosis of Lymphoma on FNA:
medical oncology literature reporting a high number of anti-
Which are the Cases that Present the Most
quated and incorrect diagnoses on FNA biopsies of lymphomas,
we evaluated all 2002- first quarter 2005 FNA’s performed at our
Challenge Even with Consultation by Expert
institution in which clinicians were concerned about the diag- Hematopathologists?
nosis of lymphoma. Immediate access to expert hematopatholo- Fatma Midani, Sanjay Lahiri, Therese Bocklage, M.D.
gists to help triage specimens was a routine part of FNA evalu-
INTRODUCTION/PURPOSE: Even with expanded immunocyto-
ation at our hospital; this feature contrasts with the data
chemistry panels, sophisticated flow cytometric analysis of sur-
published in the article by Hehn et al (Journal of Clinical Oncol-
face markers, and consultation by expert hematopathologists,
ogy 22:3046-3052; 2004). We sought to determine if expert he-
some hematopoietic lesions remain difficult to categorize as
matopathologist consultation improves upon this recently re-
benign or malignant on FNA. We reviewed the FNA cases we
ported rate of inaccurate lymphoma diagnoses rendered on
diagnosed as “atypical/suspicious” (n"40) over the last 3.25
FNA.
years. These cases derived from patients for whom the clinician
MATERIALS AND METHODS: For the years 12/1/2002-3/31/2005
was concerned about a possible lymphoma. We sought to de-
(3.25 years), anatomic pathology software files were searched for
termine why these cases were difficult to specifically diagnose on
all fine needle aspiration biopsies performed because clinicians
FNA.
were concerned about lymphoma/leukemia. FNA results and
MATERIALS/METHODS: After appropriate IRB approval, the ana-
follow up surgical pathology and bone marrow biopsies were
tomic pathology software files from two hospitals were searched
reviewed, with review of patient medical records for cases in
for FNA’s performed to ‘rule out lymphoma’. The time frame
which no follow-up surgical biopsy was performed. Additional
extended 3.25 years (1/1/02-3/31/05). Patient medical records
parameters recorded included percent of unsatisfactory cases,
and follow-up diagnoses were reviewed for all atypical/suspi-
percent of cases in which flow cytometric analysis was per-
cious cases. All slides and flow results of the atypical/suspicious
formed, and percent of cases in which expert hematopathologist cases were re-reviewed with additional review by an expert he-
consultation was sought. False negative and false positive rates matopathologist as deemed desirable.
were calculated (excluding atypical/suspicious cases). RESULTS: Three hundred thirty eight FNA’s were performed on
RESULTS: A total of 338 FNA’s performed for ‘rule out lymphoma’ 286 patients. Seventy five percent of FNA’s comprised biopsies of
were reviewed. These cases were performed on 286 patients, 57% lymph nodes with 25% performed at other sites. Twelve percent
female, 43% male. Seventy five percent comprised FNA’s of lymph (40 cases) were diagnosed on FNA as “atypical or suspicious”.
nodes with 25% comprising FNA’s of other body sites. Twenty two of the patients (55%) subsequently underwent fur-
ther surgical biopsy or bone marrow biopsy. Follow up diag-
Number of noses on these 22 cases comprised: three benign lymph nodes of
Percent of Percent of Cases
which one consisted of HIV toxoplasmosis adenitis, one Castle-
Cases with Cases with (Percent of
FNA Flow Expert Total man’s disease, one atypical follicular hyperplasia, four
Diagnosis Final Patient Diagnosis Performed Consultation FNA.s) Hodgkin’s lymphoma, two carcinomas (one breast carcinoma
and one poorly differentiated adenocarcinoma, NOS, of the pa-
Unsat. None rendered 8% 8% 40 (12%) rotid) and 10 malignant lymphomas (five diffuse large B-cell
FNA
lymphoma, three follicular lymphoma, one T-cell lymphoma,
Benign Benign 48% 79% 162 (48%)
Atypical 58% Malignant 45% 96% 40 (12%) and one sclerosing mediastinal lymphoma), and one patient was
Malignant Malignant Lymphoma 78% 87% 88 (26%) found to have CLL on a bone marrow biopsy. One patient
Lymphoma showed a lambda restriction of B-cells on flow cytometric study
Malignant Malignant Nonlymphoma 0% 0% 8 (2.4%)
with minimally atypical lymphocytes on the smears. Seventeen
Neoplasm
patients had no follow-up biopsies and by further clinical eval-
410 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
uation were considered to have benign adenopathy (follow-up
time maximum of 3.2 years). The number of patients with a
malignant hematopoietic process comprised seventeen individ-
uals (42%). One case remained atypical after surgical biopsy.
CONCLUSIONS: Review of slides and flow studies (performed on
45% of cases) indicate that outright diagnosis of a malignant
hematopoietic process was most frequently prevented by lack of
flow cytometric results or lack of corroborating flow cytometric
results in the setting of scattered atypical lymphoid-appearing
cells. In addition, cases with a marked number of intermixed
benign lymphocytes were difficult to interpret as conclusively
malignant. In these settings, caution is advised in order to avoid
a false positive diagnosis.
University of New Mexico School of Medicine, Department of
Pathology, Albuquerque, New Mexico
163
Cytoplasmic Vacuoles is a Good Diagnostic
Clue of Malignant Melanoma in Fine Needle
Aspiration Cytology
Lian-Song Chen, M.D., Anwar Raza, M.D., Camilla Cobb,
M.D.
Malignant melanoma is a highly aggressive malignancy with a
high risk of metastasis. Multiple morphologic features of mela-
noma have been described in Fine Needle Aspiration smears. To
efficiently triage the patient’s specimen based on one or two
Diff-Quik stained smears in the FNA clinic is very challenging,
especially when the number of cells are limited. Morphologic
clues for diagnosis should be consistent and easy to appreciate
in Diff-Quik stained smears. A total 43 FNA diagnosed malignant
melanoma cases were searched by Copath software search pro-
gram from the years 2000 to 2004, at our Institution. We re-
viewed 29 cases with available Diff-Quik and Papanicolaou slides
and adequate cellularity. Six morphologic features were included
in this study: pigmentation, plasmacytoid appearance, cytoplas-
mic vacuoles, macronuclei/binucleation, intranuclear inclu-
sions, and prominent nucleoli. Pigment was identified in 20
cases (71%), plasmacytoid appearance in 21 cases (75%), cyto-
plasmic vacuoles in 23 cases (82%). The remaining three features
related to the nucleus or nucleolus was present in almost all the
cases, although they are not always readily identified in Diff-
Quik smears. Also, these latter features do not indicate the ex-
pression of the cell phenotype; instead they are good clues for
atypia/malignancy. Cytoplasmic features reflect the differentia-
tion of the cells as well as their lineage/origin. Our study indi-
cates that the cytoplasmic vacuoles in Diff-Quik stained smears
are the most consistent cytoplasmic cytological features of ma-
lignant melanoma. The vacuoles are well-defined, round, clear
spaces in the cytoplasm of the malignant cells. The number of
the vacuoles usually are variable from case to case, but are
almost always multiple (more than 60). The size usually is uni-
form in the same cell, but varies in different cases. The cause of
these vacuoles is not clear, earlier reports in the literature have
postulated abnormal melanogenesis as a possible mechanism.
The other non-melanoma cells in the same smears served as an
internal negative control. These vacuoles are also faintly visible
in Papanicolaou-stained smears. The common finding of multi-
ple cytoplasmic vacuoles as a diagnostic feature is not really
emphasized in the literature. Our conclusion of this study is that
multiple cytoplasmic vacuoles can be a good diagnostic clue for
malignant melanoma in FNA cytology.
Los Angeles County Hospital and University of Southern Califor-
nia, Department of Pathology, Los Angeles, California
164
Endoscopic Ultrasound (EUS)-Guided Fine-
Needle Aspiration (FNA) of Celiac Lymph
Nodes/Celiac Plexus: Review of 64 Cases
Sarah Navina, M.D., Mary Lucido, M.D., Rebecca Lai,
M.D., Shawn Mallery, M.D., Ricardo Bardales, M.D.
INTRODUCTION: EUS guided-FNA of celiac lymph nodes is indi-
cated for evaluating nodal cancer staging. It is particularly useful
for diagnosing M1a disease in esophageal carcinoma, and is
accepted as superior to other imaging modalities. Additionally, it
is used for staging lung, gastric and pancreatic carcinomas, and
evaluating adenopathy of unknown cause. We report our find-
ings in the cytological evaluation of celiac lymph nodes with
emphasis on EUS correlation, cytomorphology, and potential
pitfalls for cytologists.
METHODS: 66 EUS-guided FNA of celiac lymph nodes were per-
formed at our institution over a period of 61 months (January
2000 to February 2005). 64 cases were available for review. Gas-
troenterologists performed the EUS-guided FNA via a transgas-
tric approach by using the Pentax FG38UX and Olympus UM130
videoechoendoscope and a 22- or 25-gauge needle. DiffQuik®-
and Papanicolaou-stained smears and cell blocks (when avail-
able) were reviewed and cytomorphologic findings recorded.
EUS correlation was noted when available.
RESULTS: After review EUS-guided FNA diagnoses included pos-
itive (n " 34) and negative (n " 28) for malignancy and non-
diagnostic (n " 2). Two of 4 cases originally reported as non-
diagnostic, were subsequently categorized as ‘negative’ for
metastasis. Primary sources for malignancy included esophagus
(n " 13), GE junction (n " 2), stomach (n " 1), pancreas (n "
10), lung (n " 3), bile duct (n " 1), and non-Hodgkin’s lym-
phoma (n " 4). Cytomorphologic diagnosis was accurate with
100% sensitivity and 100% specificity. In addition to metastatic
carcinoma and lymphoma, other remarkable diagnosis included
granulomatous lymphadenitis (n " 1). Two of the 28 benign
cases showed normal celiac plexus tissue and no evidence of
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
malignancy. Celiac plexus was indistinguishable from lymphad-
enopathy by EUS in both cases. Patients evidenced noticeable
discomfort at the time of needle aspiration in both cases. Aspi-
rate smears showed cell aggregates, often arranged in whorls of
variable cellularity, placed in a fibrillary background. The aggre-
gates exhibited two cell types: numerous cohesive medium-sized
cuboidal cells mixed with rare large ganglion cells. The latter
cells were best appreciated in the Papanicolaou-stained smears.
Rare isolated ganglion cells were also present in the background
smear. 73% of lymph nodes positive for malignancy and 44% of
negative lymph nodes showed " 2 of 4 EUS criteria for malig-
nant lymph nodes respectively.
CONCLUSIONS: EUS-guided FNA evaluation of celiac lymph node
is minimally invasive and provides sufficient material for cyto-
logic diagnosis. Cytologic diagnosis is highly accurate, sensitive,
and specific for the diagnosis of malignancy and proves better
than EUS diagnosis alone. Although present in a small percent-
age of cases (1.3%), celiac plexus tissue can morphologically
mimic metastatic carcinoma. Clinically, patient discomfort at
time of aspiration might indicate celiac plexus sampling.
Department of Laboratory Medicine and Pathology, University of
Minnesota, Minneapolis, Minnesota, Division of Gastroenterol-
ogy, Department of Internal Medicine, Hennepin County Medical
Center, University of Minnesota, Minneapolis, Minnesota
165
Monitoring Support Staff Personnel: A Plan
to Improve Quality in the Cytopathology
Laboratory
Vikram Kapoor, SCT(ASCP), Kevin Schofield, CT(ASCP)
INTRODUCTION: One area often overlooked in quality improve-
ment of the cytopathology laboratory is monitoring the accuracy
of support staff personnel. By identifying and tracking errors,
appropriate corrective measures can be taken.
METHODS: The method used to assess the performance of cler-
ical staff depends on the job function. Accessioning errors can
include, but are not limited to: incorrect patient demographics,
clinical history, and specimens accessioned to an incorrect pa-
tient. Accessioning errors are recorded in an Excel spreadsheet.
These are then compiled on a monthly basis and reviewed with
the individual responsible. In our laboratory, data entry person-
nel input the cytotechnologists’ diagnosis’ and billing codes into
the LIS. All cases selected for random quality control re-screen-
ing, along with those undergoing targeted (high risk) re-screen-
ing, are examined for accuracy by the QC cytotechnologist for
proper diagnosis, fee codes, and clinical history. Accessioning
errors are handled as described above. If data entry errors are
found, they are corrected and noted in the LIS. A retrieval report
is run monthly to compile these errors. They are broken down by
data entry personnel, documented in an Excel spreadsheet, and
reviewed with the appropriate person. The final component of
411
this QA program is a random retrospective review of 25 gyneco-
logic and 10 non-gynecologic cases. Requisitions and working
drafts are verified with data entered in the LIS for accuracy.
Slides are reviewed for correct number of slides, name, and
accession number. In addition, for non-gynecologic specimens
the gross description in the LIS is verified with the initial gross
record. All material is checked for proper filing.
RESULTS: This part of the laboratory quality improvement plan
has recently been implemented. More time is needed to deter-
mine the its utility. Our goal is that support staff personnel will
receive monthly feedback on their performance and know that
their work has the potential to be reviewed for errors.
CONCLUSION: Often errors made during accessioning or data
entry are overlooked, or they are corrected without the person
responsible ever knowing about the mistake. This plan brings
errors to their attention and can identify problematic areas to be
rectified in order to minimize the likelihood of future errors..
Yale University School of Medicine, New Haven, Connecticut
166
Cytology-Histology Correlation of Cervical
Cones: Important QA Parameters That
Identify Specific Targets for Continuing
Education
Samantha J. Witt, B.S., CT(ASCP), Jana R. Johnson, B.S.,
CT(ASCP), Jill L. Caudill, M.Ed., SCT(ASCP), Amy C.
Clayton, M.D.
INTRODUCTION: Cytology-histology correlation is a standard
quality assurance (QA) process. In our laboratory classifying the
discrepant cases has been limited to a few broad categories. The
purpose of our study was to evaluate correlations from cervical
conization specimens and classify Pap under and overcalls spe-
cifically into morphology based categories. This scheme can
then be used to target differential diagnostic difficulties for con-
tinuing education purposes.
MATERIALS AND METHODS: A computer search of pathology files
was performed to identify all cervical conization specimens re-
ceived in the frozen section lab (enriched for nonvisualized
lesions by colposcopy, or prior negative biopsy specimens) from
a one year period (2004). Cases were correlated with results from
three prior Pap specimens, and any intervening biopsy speci-
mens. Discrepant (one step difference) cervical cytology cases
were reviewed and categorized (tissue or cytology sample failure,
tissue over or undercall, cytology over or undercall). Cytology
undercalls were subcategorized into screening or interpretation
errors. The interpretation errors and cytology overcalls were
then divided into errors related to hyperchromatic groups
(HCG), squamous metaplasia (SM) or atrophy (A). An atypical
squamous/glandular cell (ASG) “justified” category was also
scored in the overcall group.
412 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
RESULTS: Eighty-four cervical conization specimens were iden-
tified by the computer search. Sixteen cases were eliminated
because previous cytology specimens were not available for re-
view. Histology outcome included benign (10), CIN I(11) and
CIN II and greater(63). Forty-three cases demonstrated cytology
and histology correlation. Twenty-three cases were discrepant.
Analysis results revealed cytology sample failure (8), tissue sam-
ple failure (2), tissue overcalls (1), cytology overcalls (7) and
cytology undercalls (5). Pap undercalls were subcategorized into
screening error (0) and interpretation error (5). Interpretation
error was further divided into hyperchromatic groups (HCG) (0),
squamous metaplasia (4), and atrophy (1). Pap overcalls were
subcategorized into HCG (2), squamous metaplasia (2), atrophy
(0), and ASCUS/AGUS justified (4). One case with overlapping
morphologic criteria was assigned to two categories.
CONCLUSION: Our study identifies several trends that lead to
potential discrepancies between conization and cytology diag-
noses. These findings can aid in targeting continuing education
in the cytology laboratory. This model for categorizing the mor-
phological features of discrepant cases will be incorporated into
our cytology-histology correlation process to monitor trends for
these problem areas.
Mayo Clinic, Rochester, Minnesota
167
Preliminary Versus Final Diagnosis in Fine
Needle Aspiration- How Often and Why are
Diagnoses Changed
Ola El-Zammar, M.D., Kamal Khurana, M.D.
BACKGROUND: Rapid assessment of specimen and preliminary
diagnosis are often requested by radiologists and clinicians to
ensure optimal diagnostic yield and triage patients for manage-
ment of disease process. However, review of limited material
without ancillary studies may occasionally result in a different
final diagnosis. The objective of our study was to review the
discrepancies between the preliminary and final diagnoses.
DESIGN: All cytology reports from 1/1/01 to12/31/03 that had
preliminary diagnosis were reviewed. Cases with discrepant final
diagnosis were identified and subcategorized based on type of
change. Reasons for changed diagnosis were also analyzed.
RESULTS: Between 1/1/01 and 12/31/03 there were 738 FNAs
with preliminary diagnoses. Of these 94 (12.8%) had discrepan-
cies between the preliminary and final diagnoses. Of the 31
specimens initially considered to be benign/non-neoplastic
(BNN), 8, 4, 6 and 13 cases were found to be different BNN,
benign neoplasms (BN), atypical/suspicious (AS), and malignant
neoplasm (MN), respectively. Final diagnosis on three initially
diagnosed BN was found to be a different BN (1), and MN (2). 21
specimens with preliminary diagnosis of AS were found to be
BNN (8), and MN (13). A single case deemed MN was found to be
AS. Thirty-four cases with preliminary diagnosis of MN were
found to be different MN. Seventy-nine cases had additional
slides and/or cellblock at the time of final diagnosis. 42 cases had
ancillary studies. Cases were referred to consultant pathologists
in 3 cases. Different pathologists interpreted the preliminary and
final diagnosis in 15 cases.
CONCLUSIONS: At our institution discrepancies between prelim-
inary and final diagnoses occur 12.8% of the time. Most com-
monly, these involve a change from MN to a different type of MN
and change of AS or BNN to MN. Availability of additional slides,
cellblock material and results of ancillary studies at the time of
final diagnosis most likely contribute to discrepancies between
preliminary and final diagnosis.
State University of New York-Upstate Medical University, Syra-
cuse, New York
168
Automated Monitoring Cytotechnologist-
Cytopathologist Discrepancy in Non-
Gynecologic Cytopathology
Peggy Whigham, CT(ASCP), Marius John-Marc Ilario,
M.D., Melina B. Flanagan, M.D., M.S., Ph.D., Nancy
Mauser, SCT(ASCP), Anthony L. Piccoli, Stephen S. Raab,
M.D., N. Paul Ohori, M.D.
INTRODUCTION/PURPOSE: At our institution we have been mon-
itoring Cytotechnologist-Cytopathologist discrepancy on Non-
Gynecologic cases for approximately 18 months. Since the CT
and Pathologist-review all non-gynecologic cytopathology cases,
we developed a method at our institution utilizing our Labora-
tory Information System (LIS) to compare the CT’s interpreta-
tion to the Pathologist’s diagnosis as an indicator for monitoring
one aspect of CT performance. Recently we programmed and
automated our LIS to generate a discrepancy statistical report
summarizing this data with select criteria.
MATERIALS AND METHODS: Using our LIS, we generate a daily
report that lists all cases with discrepancy in diagnoses between
the CT and Pathologist. The General Supervisor reviews this
report for diagnostic discrepancy in each case and disperses to
each CT as feedback. In order to determine the degree of dis-
crepancy, numerical values are assigned to each primary inter-
pretation. Bi-annually/annually an automated report is gener-
ated that summarizes all cases reviewed, documenting the
number of cases where there is concordance and the degree of
discrepancy for each CT. This data is used to determine the rate
of concordance and degree of discrepancy for each CT and the
entire Laboratory.
RESULTS: For the entire laboratory, the overall percentage of
concordant cases was consistently above 82% for the 12 months
analyzed. Regarding the monthly discrepancies, the proportion
of minor discrepancies ranged from 12.7% to 17.2% and the
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
proportion of major discrepancies ranged from 2.2% to 2.9%. At
bi-monthly intervals, the General Supervisor conducts a slide
review session comprised of cases where there were major dis-
crepancies, which has proven useful for educational enhance-
ment.
CONCLUSIONS: The frequency distribution of discrepancies by
degree approximates a natural (Gaussian) curve serving as base-
line information when comparing changes in practice or per-
sonnel. The details derived from reviewing discrepant cases are
useful in educational enhancement and identifying areas for
quality improvement.
Department of Pathology, University of Pittsburgh Medical Cen-
ter, Pittsburgh, Pennsylvania
169
Fine-Needle Aspiration Cytology in Medical
Missions
1 2
Cesar V. Reyes, M.D. , Elisa A. Reyes, R.N.
INTRODUCTION AND OBJECTIVES: In medical-surgical missions,
healthcare professionals travel to developing countries with a
goal to provide free short-term services to the less fortunate local
citizens. Laboratory testing is limited and minimal. Pre-, intra-,
or postoperative histological evaluation of excised/ resected tis-
sue is generally not performed for lack of facilities and appara-
tus. Thus, diagnoses and treatments are largely based on clinical
impression. The application of fine-needle aspiration cytology
(FNAC) in these situations is useful and practical, and heretofore
has not been reported in the literature. This paper relates a
five-year experience of utilizing FNAC during missions in the
impoverished areas of the Philippines.
MATERIALS AND METHODS, AND RESULTS: The locales served
were the FEU Hospital in Manila in 1992 and 1993, Orani
(Bataan) District Hospital 1994, Pasig (Rizal) General Hospital
1995, and Ilocos Regional Training Center 2004. The volunteers
were of multi-specialties, numbering around 50 physicians and
paramedical professionals. The FNAC paraphernalia was usual
and simple; and a microscope was brought along and donated at
mission’s end. Ten percent of the work loads [average during
each four-day mission 60 surgical and 450 non-surgical cases]
were FNAC-evaluated. FNAC results consisted of colloid goiter in
74 cases, lymphnodal tuberculosis (n"37), epidermal inclusion
cysts (n"25), lipomas (n"21), reactive lymphadenopathy
(n"10), non-diagnostic cytologies (n"7), Hashimoto’s thyroid-
itis (n"4), breast ductal carcinomas (n"3), parotid mixed tu-
mors (n"3), salivary gland adenocarcinoma (n"1), and postpar-
tum mastitis (n"1). On-site diagnoses strongly influenced the
treatment options of patients and were respectively affirmed
with postoperative FNAC and/or scrape cytology (n"127) of
excised/resected tissues. Cancer patients were assisted to get
transferred to metropolitan/ university institutions for further
413
therapy; otherwise, follow-up of cases was relegated to the local
practitioners.
CONCLUSION: While the volunteers gained extremely rewarding
experience in these limited works, FNAC proved to be a very
useful adjunct in the delivery of short-term healthcare during
medical-surgical missions to the impoverished areas.
1
Department of Pathology, Hines VA Hospital, Hines, Illinois and
Morris Hospital, Morris, Illinois, 2Westlake Community Hospital,
Melrose Park, Illinois
170
Cost Efficiency Analysis for Fine Needle
Aspiration in the Work-up of Parotid and
Submandibular Salivary Gland Nodules
Lester Layfield, M.D., Evelyn Gopez, M.D.
INTRODUCTION: The utility and cost effectiveness of salivary
gland fine-needle aspiration (FNA) is controversial. Some au-
thorities argue FNA has no added value over clinico-radio-
graphic study because most salivary gland nodules occur in the
parotid and the tumor’s relationship to the facial nerve deter-
mines the operative procedure rather than the histology. Other
experts contend FNA is of value by reducing the overall number
of operative procedures performed.
MATERIALS AND METHODS: We studied 306 salivary gland nod-
ules (214 parotid and 92 submandibular gland) undergoing FNA.
171 were subsequently surgically resected and the remaining 135
followed clinically. A 17% error rate was associated with the
non-operative group necessitating later surgical resection. The
costs of the FNAs and surgical resections (when performed) were
calculated based on Medicare reimbursement rates. Costs were
based on all patients undergoing initial FNA. The expense of
initial resection was based on the observed percentage of pa-
tients undergoing resection in our series. The costs of resections
related to erroneous FNA diagnoses were based on the error rate
associated with FNA diagnoses most often clinically followed
(i.e. chronic sialadenitis). The costs of FNA, initial resection and
subsequent resection related to FNA errors were summed and
compared to the costs which would have occurred if all nodules
had been resected.
RESULTS: FNA reduced the number of operative procedures by
approximately 65% for submandibular nodules and 35% for pa-
rotid nodules. Diagnoses which resulted in non-surgical man-
agement included: chronic radiation induced sialadenitis; in-
traparotid lymph node; recurrent lymphoma; and accessory
nodules or lobes in the parotid gland. Pure surgical management
was associated with an expenditure of $275,750 per 100 patients.
FNA management was associated with an expenditure of
$204,052 per 100 patients, representing a savings of $71,698 (26%
savings from surgical management alone).
CONCLUSIONS: Based on these data FNA appears to be cost
414 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
effective in addition to supplying pre-operative diagnoses help-
ful in counseling, operative planning and allaying patient anxi-
ety.
University of Utah Health Sciences Center, Salt Lake City, Utah
171
Salivary Gland Fine Needle Aspiration: 15
Years Experience
John Gao, Chiara Sugrue, SCT(ASCP)CMIAC, Antonio
Macias, M.D., Yan Shi, Patricia Wasserman, M.D.
BACKGROUND: To evaluate the accuracy of Fine Needle Aspira-
tion (FNA) in salivary glands at Long Island Jewish Medical
Center.
DESIGN: We reviewed all salivary gland FNAs over the 1990 –2004
period. Histology follow-up was compared to the cytology find-
ings, when available.
RESULTS: During the period examined, 857 patients had salivary
gland FNAs: (509 female, 348 male; age range 2 months old to
100 years old). Out of these, 340 (39.7%) had histological follow-
up, which is the main focus of our study. 67 of 340 (19.7%) were
cytologically interpreted as primary malignant tumors (10 ade-
nocarcinomas, 19 lymphomas, 8 adenoid cystic carcinomas, 15
mucoepidermoid carcinomas, 15 squamous cell carcinomas),
and 10 (2.9%) were metastatic carcinomas. Five of these malig-
nant FNAs were histologically benign lesions (2 reactive lymph
nodes, 1 cyst, 1 pleomorphic adenoma and 1 myxoid neuroma).
223 FNAs (65.6%) were cytologically diagnosed as benign lesions
(80 pleomorphic or monomorphic adenomas, 63 Warthin’s tu-
mors, 22 reactive lymph nodes, 16 benign parotid tissues, 10
oncocytomas, 7 cysts, 4 lipomas, and 21 others). 17 of these
benign FNAs were histologically malignant (9 lymphomas, 2
squamous cell carcinomas, 2 adenocarcinomas, 2 mucoepider-
moid carcinomas, 1 adenoid cystic carcinoma and 1 basal cell
carcinoma). 19 FNAs (5.6%) were cytologically suspicious for
malignancy. The histology follow-up proved that 9 of these sus-
picious lesions were malignant (5 lymphomas, 1 squamous cell
carcinoma, 1 adenocarcinoma, 1 adenoid cystic carcinoma and
1 metastatic carcinoma,) and 10 were benign (5 reactive lymph
nodes, 2 chronic sialadenitis, 1 cyst, 1 monomorphic adenoma
and 1 Mikulicz’s disease). Of 21 non-diagnostic FNAs (6.2%) due
to scanty cellularity, 2 (9.5%) were histologically recognized to be
malignant tumors (1 squamous cell carcinoma and 1 adenocar-
cinoma), and 19 were identified as benign lesions.
CONCLUSION: The sensitivity and specificity for FNA of the sali-
vary glands in this study were 93.5% and 92.4% respectively. Our
data show that FNA is a reliable and effective technique to
evaluate salivary gland lesions. However, the potential false-
positive and false-negative interpretations may occur in salivary
gland FNAs due to overlapping cytomorphological features.
Cytopathology Division, Department of Pathology, Albert Einstein
College of Medicine, New Hyde Park, New York
172
FNA Diagnosis of Myxoid Liposarcoma: A
17 Year Experience in a Cancer Center
Christina V. Fanning, M.D., Esmeralda C. Marginean,
M.D., Jon Trent, M.D.
BACKGROUND: FNA plays an important role in the preoperative
diagnosis and management of patients seen at our institution for
treatment of soft tissue sarcomas. In our experience using FNA
in mesenchymal tumors, we have recognized myxoid liposar-
coma (MLS) as a sarcoma with distinctive cytologic features. The
purpose of our study was to review and report our experience in
diagnosing myxoid liposarcomas on FNA.
METHODS: We performed a search of the pathology and sarcoma
databases for all patients who had a diagnosis of MLS made on
FNA and/or surgical excisional biopsy between 1987 - 2004. Our
search identified 64 patients in which the diagnosis of MLS was
made on FNA. We reviewed the aspirates, surgical material, and
clinical records of all these cases.
RESULTS: The 64 MLS cases comprised primary tumors (38
cases), distant metastases (22 cases) and local recurrences (4
cases). Primary tumors were mainly in the thigh (25) and lower
leg (10) with the remainder in the buttock (3). The follow-up of
the primary tumors ranged between 1-17 years (mean 5.5 years).
No follow-up was available in 7 cases. The FNA diagnosis of MLS
was confirmed by histological examination of the subsequent
surgical resection in all primary tumors, all locally recurrent
tumors and 14 (64 %) of distant metastases. Eight patients with
distant metastases did not undergo resection and received ad-
juvant therapy based on the cytological diagnosis only. Based on
FNA, there were no false positive results or misclassifications of
MLS with other myxoid soft tissue sarcomas. We identified one
false negative case diagnosed as a myxoma on FNA, which
proved to be MLS by surgical biopsy. This case yielded a pauci-
cellular aspirate lacking definitive criteria for MLS. Cytologic
features of MLS were (1) extracellular myxoid matrix (2) plexi-
form vascular pattern (3) tumor cells with uniform oval to round
nuclei (4) signet ring lipoblasts. The first three features were
required for diagnosis while the presence of lipoblasts was con-
sidered an optional feature. Smears from conventional MLS
showed uniform small oval nuclei dispersed loosely in an abun-
dant filmy myxoid background substance. The chromatin was
evenly distributed, nucleoli were absent or indistinct and cyto-
plasm was inapparent. Arborizing capillary-like vessels were
present. Cellular MLS had rounder, closely arranged cells with
distinct nucleoli and thin rims of cytoplasm. The vascular pat-
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
tern was less obvious due to compression by the increased
cellularity.
CONCLUSION: Our 17 year experience shows that FNA is an
accurate and valuable tool for diagnosis of MLS and in most
cases, neoadjuvant chemotherapy and preoperative radiation
therapy can be started without an additional confirmatory bi-
opsy. A diagnosis of MLS was correct in all cases diagnosed as
such by FNA. Only one false negative result was identified. MLS
cannot be excluded on paucicellular myxoid samples lacking
definitive criteria. Such cases need additional sampling if sar-
coma is suspected on clinical-radiological grounds.
M.D. Anderson Cancer Center, Houston, Texas
173
Cytopathologic Findings of Soft Tissue and
Bone Lesions in the Pediatric Population
Jeffrey Iding1, Laurentia Nodit2, Uma Rao2, Anil Parwani,
M.D., Ph.D.2, Syed Ali, M.D.1
INTRODUCTION: Fine needle aspiration (FNA) of soft tissue and
bone lesions is seldom performed in the pediatric population.
Hence, the clinical utility of the modality in this patient group
has not been adequately addressed in the cytopathology litera-
ture.
DESIGN: A 16-year (1989-2004) retrospective review of the cyto-
pathology archives at two large teaching institutions revealed 66
cases of soft tissue and bone FNA in patients 21 years or younger.
Material was obtained via FNA with or without radiologic guid-
ance. All cases had on-site evaluation by a cytopathologist.
Smears were stained with Diff Quik and Papanicolaou stains.
The histopathologic material (where available) and the medical
records of all the cases were additionally reviewed.
RESULTS: The patients ranged in age from 2 to 21 years (mean
12) with a M:F ratio of 1.2:1. Indications for FNA were most often
worsening pain, soft tissue mass, or abnormal radiographic find-
ings, typically a solitary lytic bone lesion. Of the 66 FNA, 31 (47%)
had a cytopathologic diagnosis of neoplasm or suspicious for
neoplasm, 25 (38%) were non-neoplastic, and 10 (15%) were
unsatisfactory for evaluation due to lack of adequate cellularity.
The majority of the diagnosed neoplasms were malignant (28/
31, 90%), usually sarcomas (18/28, 64%, PNET/Ewing sarcoma
the most common), though a variety of other cancers were
identified including a metastatic squamous cell carcinoma. In 11
of the 66 total cases (17%), FNA was able to establish a diagnosis
of a new malignancy, while in another 3 cases a new benign
neoplasm was diagnosed. When restricted to neoplasm with
subsequent histologic evaluation of tissue taken from the site of
aspiration, FNA was diagnostic in 14/17 cases yielding a sensi-
tivity of 82%. Of the three cases in which FNA was unable to
establish a diagnosis, only one proved malignant on subsequent
surgical resection (mucoepidermoid carcinoma presenting as
415
soft tissue neck mass). In another subset of patients with known
malignancies presenting for evaluation of recurrence or metas-
tases, FNA yielded technically satisfactory, diagnostic specimens
in 18/20 cases (90%).
CONCLUSIONS: FNA is a useful adjunct to the management of
soft tissue and bone lesions in the pediatric population. Nearly
half of the patients referred for FNA warrant a concerning diag-
nosis, either suspicious or consistent with a range of cancers, of
which approximately 1/3rd are first time diagnoses. FNA is ca-
pable of establishing new diagnoses of neoplasm over 80% of the
time, and, in cases of new malignancies, failed to establish the
diagnosis in only 1/12 cases. For patients with known malignan-
cies being evaluated for disease progression, FNA often obviates
the need for more invasive diagnostic procedures.
1
The Johns Hopkins Hospital, Baltimore, Maryland, 2 University
of Pittsburgh Medical Center, Pittsburgh, Pennsylvania
174
Cytologic Features of Primary Cartilaginous
Neoplasms of the Bone in Crush
Preparations
David Cimbaluk, M.D., Deepa Kasuganti, M.D., Vijaya
Reddy, M.D., Victor Gould, M.D., Paolo Gattuso, M.D.
PURPOSE: Chondroid lesions of the bone are rarely subjected to
fine needle aspiration. However, since some of these lesions may
mimic a metastatic epithelial tumor to the bone, it is important
to recognize them as such to avoid misdiagnosis. The aim of this
study was to examine the cytologic features of a spectrum of
cartilaginous neoplasms in crush preparations prepared at the
time of frozen section.
MATERIALS AND METHODS: From 2000 to 2004, a total of 46 cases
of chondroid lesions were submitted for frozen section evalua-
tion at our institution. The concomitant crush preparation was
prepared in each case and reviewed retrospectively.
RESULTS: The patient population included 18 females and 28
males with a mean age of 37 years. Twelve cases (26.0%) were
located in the femur, 9 (19.6%) in the pelvis, and 5 (10.9%) in the
humerus. Twenty cases (43.5%) occurred in diverse sites (4 hand,
4 foot, 3 radius, 2 skull base, 2 tibia, 2 shoulder, 2 knee, and 1
clavicle). Histological diagnosis included 16 enchondromas, 11
chondroblastomas, 11 chondrosarcomas, 6 chondromyxoid fi-
bromas, and 2 synovial chondromatosis. The cytologic features
are summarized as follows: Enchondromas displayed large
amounts of myxoid material containing sparse, uniform chon-
drocytes. Chondroblastomas showed single mononuclear cells
with round to oval nuclei, a moderate amount of dense blue
cytoplasm, abundant multinucleated osteoclast-like giant cells
and chondroid matrix. Chondrosarcomas showed single cells
with a plasmacytoid appearance with enlarged nuclei, promi-
nent nucleoli, and dense blue cytoplasm containing both small
416 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
and large cytoplasmic vacuoles. Cellular myxoid material was
prominent. Chondromyxoid fibromas displayed abundant frag-
ments of myxoid and fibrous stroma, small groups of oval cells
with uniform nuclei containing longitudinal nuclear grooves,
and scattered osteoclast-like giant cells. Synovial chondromato-
sis showed single chondrocytes with eccentrically located nuclei
and dense blue cytoplasm in a background of myxoid material.
CONCLUSIONS: (1) Benign chondroid lesions can be easily sepa-
rated on cytologic material from malignant chondroid neo-
plasms mainly by the lack of cytologic atypia, hypocellular/
acellular myxoid matrix, and absence of prominent nucleoli. (2)
Chondromyxoid fibroma can be separated from chondroblas-
toma by the paucity of osteoclast-like giant cells, abundant frag-
ments of myxoid and fibrous tissue, and longitudinal nuclear
grooves. (3) Enchondroma and synovial chondromatosis share
similar cytologic features. (4) Chondrosarcoma may mimic met-
astatic carcinoma/adenocarcinoma due to the presence of single
cells showing a plasmacytoid appearance with vacuolated cyto-
plasm.
Department of Pathology, Rush University Medical Center, Chi-
cago, Illinois
175
On-Site Gross Specimen Adequacy
Evaluation of 292 EUS-FNA Specimens
Performed by a Cytotechnologist in the
Community Hospital Setting
W. Fore, B.A., L. DeCavallas, CT(ASCP), B. Gumustop,
M.D.
INTRODUCTION: Endoscopic ultrasound fine needle aspiration
(EUS-FNA) is an essential tool in the evaluation and diagnosis of
suspected neoplastic lesions. The standard procedure includes
on-site microscopic specimen evaluation by a cytopathologist, a
step believed to be essential for optimizing adequate specimen
yield and diagnostic accuracy. In procedures incorporating this
step, literature reports diagnostic specimen yields ranging from
86-98% but with an average time commitment of 56.2 minutes
required of the cytopathologist. As a result, the procedure is time
consuming, expensive, and places great demands on the cyto-
pathology laboratory. This study hopes to demonstrate that us-
ing only on-site gross specimen evaluation performed by an
experienced cytotechnologist is less time consuming, more cost
effective, and does not increase non-diagnostic specimens.
MATERIALS AND METHODS: Records were reviewed of 292 EUS-
FNA procedures and specimens over a two and a half year period
at a community hospital. During each procedure, the attending
cytotechnologist followed a specific protocol for obtaining an
adequate specimen using gross evaluation only. The gross spec-
imen adequacy criteria and protocol were documented and de-
scribed. The duration, site of lesion, number of FNA passes,
cytologic microscopic diagnosis, and any subsequent surgical
follow up was recorded for each procedure.
RESULTS: The average time required of the cytotechnologist per
procedure was approximately 20 minutes. The average number
of FNA passes per procedure was 2.27 with a range of one to five
passes per procedure. Overall, 275 specimens (94.2%) were ad-
equate for a cytologic diagnosis and 17 specimens (5.8%) were
non-diagnostic. Cytologic diagnosis included 145 negative, 28
inconclusive, 17 suspicious, and 85 positive results. Surgical
follow up was obtained on 57 cases.
CONCLUSIONS: Using only on-site gross specimen adequacy
evaluation by an experienced cytotechnologist during an EUS-
FNA procedure, diagnostic cellular yields comparable to those
obtained using on-site microscopic specimen evaluation by a
cytopathologist can be achieved. As a result, using only on-site
gross specimen evaluation by the attending cytotechnologist,
EUS-FNA technology can be made more readily available and
more cost effective in a community hospital.
Albany College of Pharmacy, Albany, New York, St. Peter’s Hos-
pital, Albany, New York
176
Use of a Collodion Bag Technique for
Collection of Cells from Fine Needle
Aspiration Biopsy Samples with Low
Cellularity
Surayya Siddiqui, Nour Sneige, M.D., Savitri
Krishnamurthy, M.D.
BACKGROUND: Reports have described several techniques for
enhancing the collection of cytologic material that are insuffi-
cient for preparing routine cell blocks. These include entrapping
the material in different bases, such as agar, plasma thrombin,
and collodion. We studied the feasibility of a collodion bag
technique for collection of cells from fine needle aspiration
biopsy (FNAB) samples with varying cell counts. We also ex-
plored the use of this technique for ancillary immunohistochem-
ical staining of FNAB samples with low cellularity.
MATERIALS AND METHODS: Sixteen FNAB cases in which, the
sediment of the aspirate, which was collected in 10 ml of hepa-
rinized RPMI, was insufficient for preparing routine cell blocks
because of the absence of a significant cell pellet on centrifuga-
tion were selected. A cell count of the RPMI soln with the
aspirate was performed in 10 of the 16 cases by using a Coulter
counter. The RPMI soln with the aspirate was centrifuged, fixed
in 10% buffered formalin mixed with 95% alcohol, and intro-
duced into glass tubes coated with collodion. Collodion bags
containing the cellular material were prepared and subsequently
cut into 5 $m sections, and stained with hematoxylin and eosin.
Immunostaining was performed in three cases with antibodies
against estrogen receptor, cytokeratin, leukocyte common anti-
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
gen by using the modified avidin, biotin complex method in a
DAKO autostainer with diaminobenzidine as the chromogen.
RESULTS: The 10 aspirates in which the cells were counted prior
to preparing the collodion bags showed counts ranging from
82,000/ml to 853,000 ml/ (mean " 349200/ml). Six of the eight
cases with counts greater than 100,000/ml showed good cellular
material on the sections of the collodion-bag cell blocks. Two
cases with a count less than 100,000/ml and two cases with a
count greater than 100,000/ml were essentially acellular. Cell
preservation was excellent in eight cases that were processed
soon after collection in comparison with six cases that were
processed 1-3 days after collection. Successful immunostaining
was performed in three selected cases.
CONCLUSIONS: 1. The collodion bag technique is feasible for
speedy recovery of cells from FNAB samples with low cellularity.
2. Using cell count of 100,000/ml or more can increase the
probability of successful cell recovery by using this technique. 3.
Cellular preservation can be optimal if processed soon after
collection. 4. Collodion bag technique can be particularly valu-
able for performing ancillary immunostaining of aspirates with
low cellularity.
University of Texas, M.D. Anderson Cancer Center, Houston,
Texas
177
Detection of RNA from SiHa Cells Stored in
SurePath™ Preservative Fluid for Gene
Expression Analysis
Hewei Li, M.D., Ph.D., Patricia Avissar, M.S., Douglas
Malinowski, Ph.D.
OBJECTIVE: To demonstrate the adequacy of SiHa cells stored in
SurePath™ (TriPath Imaging, Inc.) for RNA isolation and gene
expression analysis.
MATERIALS AND METHODS: Freshly cultured SiHa cells (HPV16-
positive, cervical carcinoma cell line, ATCC #HTB-35) were
washed with phosphate buffered saline and stored in SurePath™
Preservative Fluid at room temperature for 1 hour, 15 hours, 1
day, 2 days, 7 days and 21 days. Total RNA was isolated from
SurePath™-preserved SiHa cells using the MasterPure RNA Pu-
rification Kit (Epicentre). The yield of the RNA extracts was
measured by optical density (OD), and the quality of the ex-
tracted RNA was assessed by formaldehyde-denatured agarose
gel electrophoresis. Beta-Actin and HPV16-E6 mRNA were re-
verse transcripted (RT-PCR) into cDNA using High Capacity
cDNA Archive kit (Applied Biosystems, Inc.). Detection of the
RT-cDNAs of the beta-Actin and HPV16-E6 genes was demon-
strated by end-point PCR (Ambion Inc.), using PCR primer sets
designed to amplify three smaller internal regions of the full-
length cDNA of the beta-Actin, with amplicon sizes ranging from
77 bp to 950 bp, and for two fragments of HPV16-E6.
417
RESULTS: Optimal recovery of total RNA was demonstrated by
OD measurement. Extracts from SiHa cells stored in SurePath™
between 15 hours and 21 days, showed similar RNA electro-
phoresis patterns to freshly cultured SiHa cells with detectable
18S rRNA bands ('2,000 nucleotides in length) as analyzed by
agarose gel electrophoresis. The 28S rRNA band ('4,700 nucle-
otides) was not detectable at 21 days. SiHa cells stored in Sure-
Path™ at ambient temperature up to 21 days permitted the
detection of beta-Actin mRNA by RT-PCR and end-point PCR.
Specific amplification of beta-Actin cDNA regions between 78-87
bp and 950 bp were successfully detected. Similarly, mRNA for
HPV16-E6 gene was detected by PCR amplification of cDNA up
to 21 days storage. No degradation in the yield of PCR amplicons
for beta-Actin or HPV16-E6 mRNA were observed during the
21-day storage at room temperature.
CONCLUSIONS: Our results demonstrate the adequacy of cells
collected and stored in SurePath™ for RNA isolation, RT-PCR
detection of mRNA and the specific detection of HPV E6 mRNA
using RT-PCR based methods.
TriPath Oncology, Inc, Durham, North Carolina
178
Comparison of Liqui-PREP™ and the
ThinPrep® Pap Test for Liquid-Based
Cervical Cytology Preparation Time
Jacob Ronnen, M.D.1, James Geyer, Ph.D.2
INTRODUCTION/PURPOSE: In a laboratory processing approxi-
mately 200 liquid-based cervical specimens per day, studies
were preformed to compare the processing times of ThinPrep®
Pap Test (Cytyc Corp, MA) and Liqui-PREP™ Cytology System
(LGM International Inc., FL).
MATERIALS AND METHODS: Workflow analysis demonstrated
that for one ThinPrep 2000 Processor, the mean processing time
per specimen was 5 minutes. In order to maximize time and
efficiency two ThinPrep 2000 Processors were used to justify a
single full time equivalent (FTE) dedicated to ThinPrep® prep-
aration. Processing time included accessioning (labeling etc.)
and hands-on preparation (up to staining and cover slipping).
This approach allowed one technologist to efficiently alternate
ThinPrep® processing between two instruments. The Liqui-
PREP™ liquid-based cytology system was similarly optimized
for workflow efficiency and throughput. Briefly, Liqui-PREP™
technology is based on centrifuging cervical cells collected in a
buffered alcohol fixative into a pellet, which is re-suspended in a
semi-liquid gel (Cellular Base Solution) and plated onto a clean
microscope slide. This simple procedure was batched to take
advantage of economies of scale and to reduce individual spec-
imen manipulation. Following the manufacturer’s package in-
sert, we customized the procedure to fit work flow through our
laboratory. Basically, specimen-handling steps were reduced
418 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
with an appropriately sized centrifuge, test tube racks, pipettors
and disposable pipettes for sample preparation, organization,
reagent additions and cell transfer. Operationally, it was decided
to treat all specimens with the manufacture’s optional sample
“Cleaning Solution” to further reduce handling steps.
RESULTS:
Slides/hour
(Slides/min) Slides/6 hours FTE
ThinPrep® 20 (3.0min) 120 1
Liqui-PREP™ 40 (1.5min) 240 1 RESULTS: 210 slides (105 cases) were scored as positive or neg-
CONCLUSION: The Liqui-PREP™ system has substantially less
overall preparation time then the Cytyc ThinPrep® Pap Test.
The Liqui-PREP™ Cytology System produces liquid-based slides
of high diagnostic quality. In addition, Liqui-PREP™ fixative
lyses red blood cells eliminating the ancillary specimen handling
used to remove excess blood in the Cytyc process (e.g. acetic
acid treatment).
1
Care-Med Clinic, Ramat Hasharon, Israel, 2LGM International,
Inc., Ft. Lauderdale, Florida
179
Automation of Molecular Testing in the
Cytology Laboratory for the Screening of
Cervical Disease in an
Immunocytochemistry Assay Format
Nikki Prpic, HT(ASCP), Peggy Parker, B.A., SCT(ASCP),
Adriann Taylor, B.A., Timothy Fischer, B.S.
INTRODUCTION: Immunocytochemistry (ICC) staining is a rela-
tively new assay technique that is based on Immunohistochem-
istry (IHC) assay principles. Ideally ICC testing should be per-
formed in the cytology laboratory. However, the cytology
laboratory has many constraints with regards to space, technol-
ogy and automation. ICC assays that are to be conducted in the
cytology laboratory should be designed to be reproducible and
robust. Additionally the laboratory requires a high level of auto-
mation to facilitate the addition of new assays in an already busy
environment. An ICC assay was designed and evaluated using
ProExTM C antibodies and SureDetect Reagents (TriPath Imag-
ing) on an automated stainer, SMS3600 (TriPath Imaging) in a
routine cytology laboratory.
MATERIALS AND METHODS: Performance and reproducibility
studies were conducted on the SMS3600. 50 NIL and 55 abnor-
mal (ASCUS$) residual SurePath cytology samples were selected
for performance studies and compared to the reference method.
Two slides were prepared from these 105 cases on the PrepStain
System using the Slide Preparation Only mode. One set of slides
was stained on the SMS3600 while the other was stained using a
reference staining method. All slides were stained with Pro Ex C
antibody cocktail using SureDetect Counterstain and Detection
Reagents. Positive and negative controls were evaluated for each
staining batch. Pretreatment of the slides was performed by
soaking the slides in Slide Pretreatment Buffer (TriPath Imaging)
for at least 1 hour and then heating the slides for 15 minutes at
95 C prior to the execution of the ICC assay. Reproducibility
studies were performed on three SMS3600 stainers twice a day
for three days with two independent operators. A panel of three
samples, a NIL pool, a HSIL pool and SiHa positive control was
tested in duplicate for each run.
ative for immunoreactivity for the performance study. All slides
were reviewed for specimen adequacy using the TBS 2001 crite-
ria. Three cases were determined to be unsatisfactory due to lack
of sufficient cells and were eliminated from the study. 82%
(41/50 cases) of the NIL cases were negative for the ProEx C
antibody and 18% (9/50 cases) were positive. 19% (10/52 cases)
of the abnormal cases were negative for the ProEx C antibody,
while 80% (42/52 cases) were positive for the ProEx C antibody.
There was 100% concordance between the test method and the
reference method for immunoreactivity. Analysis for the repro-
ducibility slides consisted of run to run, operator to operator,
and instrument to instrument variability. All slides produced
appropriate antibody staining with minimal or no staining vari-
ability. 100% (216 slides) concordance was achieved between all
parameters tested.
CONCLUSION: The ICC assay was automated on the SMS 3600
reducing assay complexity and turn around time. The auto-
mated assay produced highly accurate and reproducible results.
TriPath Oncology, Durham, North Carolina
180
Evaluation of the ThinPrep! UroCyte™
System as a Specimen Processing and
Slide Preparation Method for UroVysion™
Erin Coffman, M.S.1, Steve Bioren, B.S.1, Shannon
Browne, MT(ASCP), NCA1, Robyn Kressin, CT(ASCP)1,
Jeffrey Panella, M.S.2
INTRODUCTION AND PURPOSE: The UroVysion™ assay is a sen-
sitive and specific slide-based molecular assay that uses Fluo-
rescence In-Situ Hybridization (FISH) technology to monitor for
recurrent bladder cancer. However, the currently approved
specimen processing technique is labor intensive and the
method of slide preparation lacks standardization. In order to
overcome these limitations, UroCyte™, an optimized specimen
processing and slide preparation system was developed. Uro-
Cyte uses the automated ThinPrep 2000 Processor and a spe-
cially designed filter to prepare a thin-layer of cells onto a slide.
This study compared UroCyte and the recommended UroVysion
sample processing technique.
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
MATERIALS AND METHODS: A total of 120 urine specimens were
collected into PreservCyt Solution using ThinPrep UroCyte
Urine Collection Kits. Each specimen was split into two equal
volumes and one portion processed using the UroCyte system
while the other portion was processed according to the proce-
dure described in the UroVysion Bladder Cancer Recurrence Kit
package insert (i.e., Carnoy’s fixed manually dropped Shandon
slides). 105 of the 120 paired slides were Papanicolaou stained
and total cell counts determined. The remaining 15 pairs were
processed by UroVysion and analyzed for DAPI and FISH chro-
mosomal signal quality, according to the rating guidelines pro-
vided by Vysis. To eliminate review bias, all slides were random-
ized and blinded. The time required to perform each of the two
preparation techniques was also compared.
RESULTS: UroCyte exhibited greater cellularity than the Shandon
manual drop method in 95% of the 105 slide pairs. The average
counts of squamous and urothelial cells obtained with UroCyte
was 4,603 cells, compared to 1,224 cells for the control. All
UroCyte slides processed by the UroVysion assay yielded better
chromosomal signal scores and equivalent DAPI signal quality
compared to the control method (P % 0.01) when analyzed using
a paired t-Test. Furthermore, the UroCyte System demonstrated
a 59% overall reduction in processing time compared to the
control specimen preparation method.
CONCLUSIONS: The UroCyte System demonstrated increased cell
coverage, improved FISH signal quality, and a reduction in spec-
imen processing time compared to the currently approved
UroVysion sample preparation method. The findings of this
study suggest that this technology can help simplify and stan-
dardize sample preparation and evaluation for UroVysion as well
as other slide-based assays used in the field of molecular and
cytological diagnostics.
1 2
Cytyc Corporation, Marlborough, Massachusetts, Vysis, Inc.,
Downers Grove, Illinois
181
Non-diagnostic Thyroid Fine-Needle
Aspiration Cytology: Is Not to Be
Considered a Benign Diagnosis.
Mouhammad Sharaf El-Dean, M.D., Mohamed El-
Fakharany, M.D., Basim Al-Khafaji, M.D.
INTRODUCTION: Fine-needle aspiration biopsy (FNAB) of the thy-
roid gland is considered the cornerstone of clinical evaluation of
thyroid nodules. However, the medical literature states that 5-25
% of thyroid FNABs can be nondiagnostic. Furthermore, clinical/
pathological follow-up of these nondiagnostic FNABs is associ-
ated with a 10% rate of identifying a neoplastic lesion. Clinicians’
frustration with non-diagnostic FNAs and the implication of this
diagnosis are a continuing clinical dilemma.
METHODS: We reviewed 273 consecutive cases of thyroid FNABs
419
that were identified from the computer files of our Department
of Pathology from July 2002 to June 2003. Histological correla-
tion and clinical follow-up were also reviewed.
RESULTS: On site evaluation for FNABs by a cytopathologist is
not a requisite by clinicians at our institute. Of the total 273 cases
reviewed, 78 cases (29%) were nondiagnostic. Of which, 36 cases
(N"36) were further evaluated by either repeat FNABs (23 cases)
or surgical intervention (13 cases). Eight cases (23%) were diag-
nosed as neoplasm (7 papillary carcinoma, and 1 Follicular
adenoma), while 18 cases (50%) were diagnosed as non-neoplas-
tic this included: 14 (39%) colloid nodules, 2 (5%) lymphocytic
thyroiditis, and 2 (5%) revealed no pathological abnormality on
surgical resection. Meanwhile, 10 cases (28%) were again non-
diagnostic on repeat FNABs.
CONCLUSION: The twenty nine percent rate of non-diagnostic
FNABs at our institution falls at the upper end of reported rates
in the medical literature. This may be related to the lack of
on-sight cytopathological evaluation. Nondiagnostic thyroid
FNABs should not be considered a “benign” diagnosis, as clini-
cal/pathological follow-up in our study shows that as many as
23% of cases can harbor a neoplastic lesion, a percentage which
is double that previously reported.
St. John Hospital and Medical Center, Detroit, Michigan
182
Multinucleated Giant Cells in Thyroid Fine
Needle Aspiration: Pathologic Significance
and Clinical Correlates
Danielle Wehle, Syed Ali, M.D.
INTRODUCTION: Non-neoplastic, multinucleated giant cells
(MGCs) of probable histiocytic origin are sometimes encoun-
tered in thyroid fine needle aspiration (FNA). Although their
presence is well known, the exact pathologic significance in
relationship to the clinical outcome has rarely been studied in a
large series.
MATERIALS AND METHODS: A retrospective review of the cyto-
pathology archives at a major teaching institution revealed 141
cases with significant amount of MGCs (!10 per smear) out of a
total of 7038 thyroid FNAs performed in a 16-year period (1989-
2005). Cytopathologic material was obtained by FNA with or
without radiologic-guidance. Follow-up surgical pathology diag-
noses and medical records of all the cases were additionally
reviewed.
RESULTS: Patients ranged in age from 12 to 85 years (mean 49)
with a M:F ratio of 1: 6.4. Ten of the 141 cases were excluded
from the study due to lack of adequate tissue or clinical follow-
up. Of the remaining 131 cases, the cytopathologic diagnoses
included; non-neoplastic lesions (86, 66%), atypical/suspicious
lesions (19, 14%) and neoplasm (26, 20%). The non-neoplastic
category consisted of; adenomatoid nodule (AN) (40, 47%), lym-
420 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
phocytic thyroiditis NOS (13, 15%), Hashimoto thyroiditis [HT]
(9, 10%) and other lesions. The neoplastic category included;
benign tumors (9, 35%) [follicular adenoma- 5, 19% and others],
and malignant tumors (17, 65%) [papillary carcinoma- 12, 46%,
anaplastic carcinoma- 3, 12%, and others]. In the atypical/sus-
picious category, the tissue and clinical follow-up showed; non-
neoplastic lesions (7, 37%- HT and others) and malignant neo-
plasms (12, 63%- papillary carcinoma [10], Hürthle cell
carcinoma [1] and medullary carcinoma [1]). If a neoplastic or
atypical lesion contained MGCs, the lesion most often repre-
sented a malignant neoplasm (64%) rather than a benign tumor.
The most common malignant diagnosis with MGCs was papil-
lary carcinoma (76%) 3 of which were the follicular variants.
Others were medullary carcinoma (7%), anaplastic carcinoma
(10%), Hürthle cell carcinoma (3%), and squamous cell carci-
noma (3%).
CONCLUSIONS: MGCs are uncommonly noted on thyroid FNA
(141/7038, 2%). Thyroid FNA containing MGCs predominantly
signify a non-neoplastic lesion (66%). When a neoplastic or
atypical lesion is seen on FNA with MGCs, the diagnosis is most
often malignant (64%) rather than a benign tumor. Malignant
neoplasms containing MGCs are most commonly papillary car-
cinomas (76%). Of the 26 neoplastic (20%) and 19 atypical (15%)
lesions, 29 (22% of total cases) were malignant by either cyto-
pathologic or follow-up evaluation or both.
The Johns Hopkins Hospital, Baltimore, Maryland
183
Inter-observer Variability with the
Interpretation of Thyroid FNA Specimens
Showing Predominately Hürthle Cells
Edward Stelow, M.D.1, Carolyn Woon, M.D.2, Kristin
Atkins, M.D.1, Ricardo Bardales, M.D.3, Helen Cathro,
M.D.1, Henry Frierson, M.D.1, Michael Stanley, M.D.4, Kay
Savik, M.S.2, Stefan Pambuccian, M.D.2
INTRODUCTION: Fine Needle Aspiration (FNA) is a widely used
technique for the diagnosis and triaging of thyroid lesions. Re-
cently, it has been shown that the pathologic interpretation of
selected thyroid surgical specimens can show a high degree of
inter-observer variability (IV). We have also recently shown that
the interpretation of some thyroid FNA specimens also shows a
moderate degree of IV. As Hürthle cells may be seen as the
predominant cell type in both Hürthle cell neoplasms and
chronic thyroiditis and as the therapy for each diagnosis may
vary greatly, we chose to investigate whether pathologists con-
sistently interpret FNA specimens from these lesions.
MATERIALS AND METHODS: The cytology files of a single cyto-
pathologist who reviews over 600 thyroid FNA samples a year
were reviewed and 24 consecutive cases which showed Hürthle
cells as the predominant cell type were selected. Five other
observers then reviewed the cases blinded of the original diag-
noses. A single cytopathologist also reviewed the cases and as-
sessed cytologic features semiquantitatively. IV was calculated
and individual case diagnoses were compared with cytologic
features.
RESULTS: IV was extremely high before diagnoses were collapsed
into triage recommendations (thyroiditis" medical treatment
versus Hürthle cell lesion/neoplasm" surgical treatment). Even
after such a collapse, inter-observer agreement remained only
fair (kappa"0.39). Six cases showed complete agreement regard-
ing triage, 12 cases showed 5 of 6 observers in agreement re-
garding triage, 5 cases showed 4 of 6 observers in agreement
regarding triage, and a single case showed a split of 3 to 3
observers regarding triage. The quantity of slides received,
Hürthle cells, microfollicles and lymphocytes all correlated with
the overall triage recommendations.
CONCLUSIONS: Pathologists reviewing thyroid FNAs showing
predominately Hürthle cells show little inter-observer agree-
ment for the final rendered interpretation. Even when the inter-
pretations are collapsed into triage recommendations, agree-
ment remains only fair as 25% of the cases we reviewed showed
only 3 or 4 of the 6 observers in agreement. Further investigation
into poorly performing cases may help to define more stringent
criteria for the diagnosis of these lesions.
1
University of Virginia, Charlottesville, Virginia, 2University of
Minnesota, Minneapolis, Minnesota, 3Hennepin County Medical
Center, Minneapolis, Minnesota, 4Abbott Northwestern Hospital,
Minneapolis, Minnesota
184
Predictive Value of Quantitative Analysis of
Microfollicles in Fine Needle Aspiration
Cytology of Follicular Lesions of the Thyroid
Beth Ujevich, B.A., SCT(ASCP)H, Liu Yulin, M.D., Ph.D.,
Telma Pereira, M.D., Gologan Olga, M.D., Odeta Lapkus,
M.D., Marino Leon, M.D., Jan Silverman, M.D., CL Kim,
M.D.
BACKGROUND: Fine needle aspiration cytology of thyroid (FNAC)
has been widely used as a fast, accurate, sensitive and cost-
effective diagnostic procedure in evaluating nodular thyroid le-
sions. However, in the follicular lesion category of thyroid FNAC,
separating a benign hyperplastic nodule from a follicular neo-
plasm can be very difficult. Previous studies have indicated that
microfollicle formation is a predictive feature for follicular neo-
plasm in thyroid FNAC, but microfollicles can also be observed
in FNA of benign nodular hyperplasia or even in the normal
thyroid. In addition, follicular neoplasm may not always exhibit
a predominant microfollicular pattern. However, the predictive
value of percentage/number of microfollicles for follicular neo-
plasm in thyroid FNA cytology has not been studied. We exam-
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
ined the percentage as well as cytomorphologic features of mi-
crofollicles in thyroid FNAC and correlated the cytology results
with histologic findings.
DESIGN: 40 resected thyroids consisting of 20 follicular neo-
plasms (follicular adenoma and follicular carcinoma) and 20
benign nodular hyperplasia with the previous FNA were re-
trieved. Cytology cases with insufficient material for diagnosis
were excluded. Percentage, cytologic cellular overlapping and
presence of large flat groups of follicular cells were blindly re-
viewed by two cytopathologists. The statistic analysis was per-
formed with Chi-Square method.
RESULTS: The cytomorphologic findings are summarized in the
table below.
CONCLUSION: 1) Our results demonstrated that more than 50% of
microfollicle formation with cellular overlapping is always seen
in a follicular neoplasm. 2) Less than 25% of microfollicles with-
out overlapping features is often seen in benign nodular hyper-
plasia. 3) The presence of large flat follicular group is also a good
predictive cytomorphologic feature for a benign non-neoplastic
follicular lesion.
50– Large
>75% 75% 25-50% <25% MF with flat
MF MF MF MF overlapping group
Follicular
neoplasm 16/20 20–Feb 20–Jan 20–Jan 20/20 20–Feb
Non–
neoplastic 0/20 0/20 20–Mar 17/20 2/20 (focally) 18/20
Allegheny General Hospital, Pittsburgh, Pennsylvania
185
The Utility of GLUT-1 in Cell Blocks: An
Adjunct to Fine Needle Aspiration Diagnosis
of Squamous Lesions of the Neck.
Vishal Chandan, M.D.1, William Faquin, M.D., Ph.D.2,
David Wilbur, M.D.2, Kamal Khurana, M.D.1
Glucose transporter-1 (GLUT-1), a facilitative cell surface glu-
cose transport protein is normally expressed in erythrocytes,
perineurium, blood-brain barrier and aberrantly expressed in a
wide spectrum of epithelial malignancies. The purpose of this
study was to determine the utility of GLUT-1 immunostaining as
an adjunct to the diagnosis of malignancy in fine-needle aspi-
rations of squamous lesions of the neck.
Using a monoclonal antibody to GLUT-1 and a standard avidin-
biotin complex technique, immunostaining was performed on
paraffin-embedded cell blocks of 28 cases with the following
cytologic diagnoses: (1) metastatic squamous cell carcinoma
(SCC) (11 cases); (2) atypical squamous cells, SCC cannot be
excluded (6 cases); and (3) cytologic findings consistent with
branchial cleft cyst (11 cases). Tissue or clinical follow-up was
421
available in all cases. GLUT-1 expression was considered posi-
tive when at least 10% of squamous cells exhibited distinct cell
membrane reactivity.
All 11 (100%) cases with an unequivocal cytologic diagnosis of
metastatic SCC were positive for GLUT-1. Tissue follow-up con-
firmed metastatic SCC in all of these 11 cases. The squamous
cells in all 11 (100%) cases with cytologic findings consistent with
branchial cleft cyst were negative for GLUT-1; tissue follow-up
confirmed the diagnoses of branchial cleft cyst in 8 cases. In the
remaining 3 cases excision was not performed, as the cystic
lesion was completely decompressed and, clinically, no recur-
rences were identified at 28, 20 and 16 months of follow-up. Of
the 6 cases with cytologic diagnosis of atypical squamous cells, 3
(50%) were negative and 3 (50%) were positive for GLUT-1.
Subsequent excisional biopsies in these cases revealed 4 cases of
metastatic SCC (3 were positive and 1 was negative for GLUT-1),
1 case of branchial cleft cyst (negative for GLUT-1) and 1 case of
thyroglossal duct cyst (negative for GLUT-1).
Our findings suggest that GLUT-1 immunostaining is a useful
adjunct in differentiating benign and malignant squamous le-
sions in cell block material. While negative staining for GLUT-1
does not exclude malignancy, positive staining may aid in accu-
rate diagnosis of malignancy in cytomorphologically equivocal
squamous lesions of the neck.
1
Department of Pathology, State University of New York-Upstate
Medical University, Syracuse, New York, 2Department of Pathol-
ogy, Massachusetts General Hospital, Harvard Medical School,
Boston, Massachusetts
186
Use of Ck-19 Immunostain in FNA
Diagnosis of Pseudopapillary Hyperplasia of
the Thyroid
Quintus Chess, M.D., FIAC, Olcay Cubukcu-Dimopulo,
M.D., Stephanie W. Signer, CT(ASCP)CFIAC
INTRODUCTION: Cytokeratin-19 (CK-19) immunostain has been
shown to be a useful adjunct for the FNA diagnosis of papillary
thyroid carcinoma (PTC). In our lab, this marker has been used
routinely to confirm this diagnosis, and has been a sensitive
indicator for differentiated papillary carcinoma. Pseudopapillary
hyperplasia (PPH) is a benign condition, usually associated with
chronic goiter, which mimics papillary carcinoma. In PPH, com-
plex branched fibroepithelial fronds are identified, but at high
magnification the nuclear changes of papillary carcinoma are
not present. Nuclear changes such as optical clarity, chromatin
grooves and intranuclear inclusions are necessary for the diag-
nosis of PTC. A two-year retrospective study was done, to deter-
mine the diagnostic role of CK-19 in the distinction between PTC
and PPH in thyroid FNA samples.
MATERIALS AND METHODS: During the study period (2003-2005)
the laboratory received 8,897 thyroid FNA samples. Among these
422 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
there were 262 malignant diagnoses (3%). Among malignant
diagnoses, PTC accounted for the overwhelming majority of
cases. During this same time interval, one of our pathologists
(QC) collected 25 cases with the cytomorphologic findings of
PPH. CK-19 stain was done on these cytologically benign cases,
utilizing both direct smear samples fixed in 95% ethanol for
pap-stain and de-colorized, and cell block material fixed in
formalin.
RESULTS: CK-19 immunostain was negative in 23 out of 25 cases
of PPH. (92%). One case had an indeterminate reaction and one
case had a positive staining reaction. The FNA case with the
positive CK-19 reaction was reviewed and reclassified from “neg-
ative” to “significant atypia” on the basis of combined morpho-
logic/immunophenotypic findings. Surgical pathology study
confirmed PTC. The FNA case with the indeterminate CK-19
stain was also reviewed and was still classified as negative. This
was a cystic lesion. Repeat FNA one year later remains negative
and follow-up continues. One of the 23 patients with negative
CK-19 underwent thyroid surgery based on clinical indications.
Surgical pathology showed a large goiter with a microscopic
PTC; slide review confirmed that the negative FNA result was
due to a sampling discrepancy. Since the FNA diagnosis was
negative in the cases selected for this study, most of these cases
have no surgical pathology follow-up to-date. Rather, clinical
observation is ongoing.
CONCLUSION: In this study, CK-19 immunostain is a specific
marker for PTC, and a negative CK-19 stain is a reliable diagnos-
tic adjunct to confirm the benign character of PPH, which mim-
ics PTC in FNA samples. Patients with PPH confirmed by a
negative CK-19 reaction can be safely followed without surgery.
CBL Path, Mamaroneck, New York
187
Suspicious for Hürthle Cell Neoplasm by
Fine Needle Aspiration (FNA) of the Thyroid,
Cytological Review with Surgical Pathology
Follow Up
Diva Saloma! o, M.D., Thomas Sebo, M.D., Marie Passow,
CT(ASCP), Amy Wendel, CT(ASCP), Mandi Lingren,
CT(ASCP)
INTRODUCTION: FNA of the thyroid gland is an important screen-
ing tool for patients with nodules. With the use of more sensitive
and refined radiographic techniques there has been an increase
in the number of non-palpable nodules found, leading to FNA.
“Suspicious for Hürthle cell neoplasm” (SHCN) is a diagnostic
category that is followed by surgical resection. We reviewed the
follow-up in all cases with this FNA diagnosis with emphasis on
cases that only showed Hashimoto’s thyroiditis (HT) or papillary
carcinoma (PapCa)at surgery.
MATERIALS AND METHODS: A computer-based search was per-
formed for all thyroid FNAs with diagnosis of SHCN followed by
surgical resection, from January 1999 to December 2004. These
cases were further categorized on the basis of surgical diagnosis
of adenoma, Hürthle cell carcinoma (HCC), PapCa and HT. The
HT and PapCa cases were re-reviewed for the presence or ab-
sence of cytological criteria, which would have allowed us to
separate these cases from SHCN.
RESULTS: A total of 165 cases were identified (126 women and 39
men, average age 53.9 years, range 18 to 85 years). Surgical
resection of these cases identified benign adenomas (135; 82 %),
Hürthle cell carcinoma (13; 8%), papillary carcinomas(12, 7%)
and HT (5; 3%). Five cases had an incidental microscopic focus
of papillary carcinoma (% 1 cm) in addition to the adenoma.
Cytological review of the smears available in 4 cases with HT at
surgery showed lymphocytes in all 4 cases, with minimal
amounts (1 field) in 2 cases, and moderate amounts (! 1 field) in
the other 2 cases. None of the HT smears showed transgressing
vessels, a feature described as highly suggestive of Hürthle cell
neoplasm. The cytological review of the smears available in 6
cases with PapCa showed nuclear contour irregularity and
grooves in 5 cases, rare intranuclear inclusions in 4 cases, and
papillary groups in 2 cases. Transgressing vessels were seen in 4
of these cases.
CONCLUSIONS: SHCN is a diagnosis rendered when in a thyroid
FNA specimen shows the presence of a pure population of
Hürthle cells. Our tissue follow-up showed that the majority of
these cases were benign, mostly adenomas and a few Hürthle
cell carcinomas. In the few cases with Pap Ca at surgery, cyto-
logical features for PapCa (intranuclear inclusion or papillary
groups) were seen on re-review of the smears. HT was uncom-
monly identified at surgery. The presence of lymphocytes, even
in small numbers and the absence of transgressing vessels are
features more indicative of HT. Therefore, thyroid FNA smears
with these findings should be interpreted cautiously.
Mayo Clinic, Rochester, Minnesota
188
The Value of On-Site Adequacy Assessment
of Thyroid Fine Needle Aspirations is a
Function of Operator Experience
Danita Beckman, CT(ASCP), Mohiedean Ghofrani, M.D.,
David L. Rimm, M.D., Ph.D.
INTRODUCTION: Cytotechnologists and pathologists often per-
form on-site adequacy assessment (AA) of thyroid fine needle
aspirations (FNAs) to provide immediate feedback as to whether
adequate material has been obtained for cytologic diagnosis.
This study was designed to determine whether on-site AA in fact
results in a significant decrease in non-diagnostic specimens
among ultrasound (US)-guided needle aspirations of the thyroid
as well as those performed by palpation alone.
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
MATERIALS AND METHODS: A search was performed to identify
in-house thyroid FNAs from January 1, 2000 through December
31, 2003 that were obtained under US guidance or by palpation
only. It was then recorded whether on-site AA was performed.
The submitting physician and final diagnosis were also recorded
for each case. Contingency tables were constructed and evalu-
ated by Chi-squared analysis.
RESULTS: Out of 1,502 in-house thyroid FNAs, 981 (65.3%) were
performed under US guidance and 521 (34.7%) were performed
by palpation alone. On-site AA of the aspirated material was
performed in 323 cases (21.5%), while 1,179 cases (78.5%) were
performed without AA. Of the 418 palpation-guided FNAs that
were performed without AA, 70 (16.7%) were reported as non-
diagnostic, while of the 103 palpation-guided FNAs with AA, only
7 (6.8%) were inadequate for diagnosis. This difference was
statistically significant (p%0.025). Among 761 US-guided FNAs
without AA, 54 (7.1%) were non-diagnostic, which was not sta-
tistically different from the 10 out of 220 (4.5%) non-diagnostic
rate of US-guided FNAs with AA. However, when these US-
guided FNAs were further divided into two groups based on the
experience of the radiologist performing the FNA, the non-diag-
nostic rate in the experienced radiologist group was only 5.4%,
or 32 of 592 US-guided FNAs, even though AA was not per-
formed. Among radiologists with less experience, AA signifi-
cantly reduced the non-diagnostic rate from 13.0% (22 of 169
FNAs without AA), to 4.5% (10 of 220 FNAs with AA) (p%0.01).
CONCLUSIONS: On-site AA of thyroid FNAs significantly reduces
the number of non-diagnostic aspirates in cases performed by
palpation only. However, the benefit of AA for US-guided FNAs
depends on the experience of the radiologist. Experienced radi-
ologists with a relatively low non-diagnostic rate do not appear
to benefit from on-site AA. However, if the radiologist does not
have extensive experience in performing thyroid FNAs, on-site
AA leads to a significant reduction in non-diagnostic aspirates.
Yale University School of Medicine, Department of Pathology,
Cytology Division, New Haven, Connecticut
189
Onsite Cytologic Evaluation of Papillary
Thyroid Carcinomas: Ultrasonographic
Predictors with Histologic Correlation.
William J. Klump, M.D., Sean Cote, D.O., Christopher
Merritt, M.D., Marluce Bibbo, M.D., Sc.D., FIAC
INTRODUCTION: Onsite cytologic and radiologic evaluation of
thyroid nodules is an effective method for detecting papillary
thyroid carcinomas, though false negative diagnoses do occur.
Histologic and ultrasonographic correlation with cytologic diag-
noses might further elucidate worrisome radiologic features, an
awareness of which would allow the cytologist to request addi-
tional sampling if necessary.
423
OBJECTIVE: To correlate cytologic diagnoses with ultrasono-
graphic features and histologic findings of papillary thyroid car-
cinomas.
MATERIALS AND METHODS: Histologic findings of 54 thyroid
resection specimens (31 papillary carcinomas and 23 goiters)
were reviewed by two pathologists. These features included des-
moplasia, margin characteristics, presence and type of calcifica-
tions, and size. The ultrasonographic features of the correspond-
ing specimens included echogenicity, homogeneity, margin
characteristics, and presence and type of calcifications and were
reviewed in a blinded fashion by a radiologist. Based on the
above criteria, the radiologist then classified the nodule as be-
nign, indeterminate, or suspicious. Finally, the ultrasonographic
impressions were compared with the cytologic diagnoses, which
were classified as benign, indeterminate, and suspicious/posi-
tive.
RESULTS: Compared to goiters, papillary carcinomas were asso-
ciated with a significantly higher percentage of calcifications
(52.0% vs. 9.0%), irregular margins (61.3% vs. 26.0%), hypoechoic
matrix (51.6% vs. 26.1%), and a lower frequency of cystic pat-
terns (0.0% vs. 13.6%). Fine, stippled calcifications accounted for
69.0% of radiographically detected calcifications. Histologically,
45.0% of these stippled calcifications were intratumoral. Tissue
desmoplasia was strongly correlated with suspicious findings on
ultrasound (53.9% of suspicious lesions having desmoplasia
compared to 0.0% of sonographically benign lesions). However,
desmoplasia did not correlate with lesion echogenicity or matrix.
Respectively, ultrasound and cytology diagnosed papillary car-
cinomas as benign in 6.5% and 8.0%, indeterminate in 29.0% and
23.0%, and suspicious in 64.5% and suspicious/positive in 69.0%
of cases.
CONCLUSION: Ultrasonographic evidence of irregular margins,
stippled calcifications, and hypoechogenicity should raise the
cytologist’s suspicion for papillary carcinoma. Although strongly
associated with sonographically suspicious lesions, desmoplasia
did not appear to have a specific ultrasonographic correlate.
Cytologic detection of papillary carcinomas increased with tu-
mor size. In contrast, radiologic detection increased as tumor
size decreased. Overall, cytology and radiology showed close
correlation in diagnosing papillary thyroid carcinomas.
Thomas Jefferson University Hospital, Philadelphia, Pennsylva-
nia
424 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
190
Vitreous Fluid Cytology: A Correlation Study
Between the Benign Cytomorphology and
Clinical Diagnosis/Follow Up
Jing Zhai, M.D., Ph.D.1, J. William Harbour2, Morton E.
Smith2, Rosa M. Davila, M.D.1
INTRODUCTION: Vitreous samples can be obtained as part of a
therapeutic procedure or to establish a diagnosis. In diagnostic
vitrectomy, the main goal is usually to distinguish inflammatory
from neoplastic processes. Our current interest is in assessing
the significance of a cytologically benign vitreous sample and in
trying to identify cellular patterns that may correspond to spe-
cific clinical/laboratory entities.
MATERIALS AND METHODS: Vitreous fluids with a benign cyto-
logic diagnosis were identified retrospectively from the pathol-
ogy department files. The slides were reviewed and clinical in-
formation and follow up (F/U) was obtained from the patients’
medical records.
RESULTS: Ninety one cytologically benign vitreous cases were
identified from 1994 to 2004. Clinical F/U was available in 54/91
cases. The mean age of patients with clinical F/U was 58.6 years
(range: 9-90) and the M:F ratio was 0.61. Most patients had
unilateral disease (OD 14, OS 19, OU 21) and most samples
(92.5%) were obtained to establish a diagnosis. The mean clinical
F/U was 2 years (range: 7 days to 9.3 years). Cytologically, most
samples had low cellularity (30/54; 56%) and lacked significant
blood (39/54; 72%). Macrophages and/or lymphocytes were the
predominant cell types in 75.9% (41/54) of cases. Their corre-
sponding final clinical diagnoses were malignant lymphoma (1),
chronic uveitis/vitritis of undetermined etiology (21), viral uve-
itis/vitritis (4), hemorrhage (3), autoimmune uveitis (2), toxo-
plasma (3), retinal detachment (1), epiretinal membrane (1), and
unstated (5). The case of malignant lymphoma was diagnosed by
cytologic evaluation of vitreous fluid obtained from the opposite
eye. Neutrophils accompanied by macrophages and/or lympho-
cytes were identified in 14.8 % (8/54) of cases. The correspond-
ing final clinical diagnoses were: macular pucker (2), fungal
endophthalmitis (1), staph endophthalmitis (1), endogenous en-
dophthalmitis (1), endophthalmitis (1), panuveitis of uncertain
etiology (1), hemorrhage (1). Three cases had blood only, and
were consistent with clinical diagnosis of hemorrhage. One case
had only lens fragments, consistent with the clinical diagnosis.
One case had abundant eosinophils and a final clinical diagnosis
of toxocara.
CONCLUSIONS: With few exceptions, the cytologic features of
benign vitreous fluids don’t correspond to specific clinical/lab-
oratory diagnoses. However, the presence of abundant eosino-
phils was suggestive of a parasitic infection, such as toxocara,
and the almost exclusive presence of blood was indicative of
hemorrhage. Based on our cases with clinical F/U, the negative
predictive value of a vitreous sample is 98.2%.
1
Department of Pathology & Immunology, Washington University
Medical Center, St. Louis, Missouri, 2Department of Ophthalmol-
ogy & Visual Sciences, Washington University Medical Center, St.
Louis, Missouri
191
Fine Needle Aspiration Biopsy of Positron
Emission Tomography Positive Thyroid
Nodules from Multiple Myeloma Patients
Luis De Las Casas, M.D.1, Leslie Cloar, CT(ASCP)1,
Donald Bodenner, M.D.1, Soheila Korourian, M.D.1,
Roberto Miranda, M.D.2, Maria Thomas, M.D.1
INTRODUCTION: Extramedullary plasmacytomas are rare tumors
identified in less than 5% of patients with multiple myeloma;
however, 75-90% of these neoplasms occur in the head and neck
region. Recently, the availability of functional imaging tech-
niques such positron emission tomography (PET), that relies on
in vivo visualization of the increased glucose metabolism exhib-
ited in malignant and inflammatory processes, has increased the
detection of previously undiscovered lesions. Whole body PET
plays an important role in the evaluation of patients with cancer
and is an integral part of the metastatic evaluation in various
tumors. PET has become a standard modality for the metastatic
work up for multiple myeloma patients in our institution.
MATERIALS AND METHODS: All cases of ultrasound guided fine
needle aspiration biopsy (FNAB) of PET positive (standardized
uptake values !2) thyroid nodules from multiple myeloma pa-
tients during a 3 year period (2003-2005) at the University of
Arkansas for Medical Sciences were retrieved.
RESULTS: Ten aspirates were identified and reviewed. The ren-
dered cytologic diagnoses were positive for neoplasm in 2 cases
(one follicular neoplasm, and one possible medullary thyroid
carcinoma), atypical in 4 cases (hyperplastic thyroid nodule
versus follicular neoplasm, recommending surgical excision),
negative in 3 cases (two consistent with benign colloid nodules,
and one Hashimoto’s thyroiditis), and non-diagnostic in 1 case
(due to scant cellularity). None of the ten patients (0%) have
evidence of a thyroid plasmacytoma (involvement by multiple
myeloma) on cytologic preparations. All cytologic diagnoses
were reviewed by two board certified cytopathologists. Seven out
of the ten cases (70%) had recommendation for thyroidectomy
for primary thyroid processes. Only one of the patients under-
went a total thyroidectomy with bilateral neck dissection for
medullary carcinoma of the thyroid. The other patients have
either postponed the thyroidectomy due to their critical medical
condition or been scheduled for surgery.
CONCLUSION: The results of this study calls attention to the
importance of investigating PET positive thyroid nodules in mul-
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
tiple myeloma patients by FNAB, and not simply assuming that
these are sites of metastatic disease. Furthermore, our results
indicate that PET positive thyroid nodules in multiple myeloma
patients are more likely to be primary thyroid processes rather
than metastatic multiple myeloma.
1
University of Arkansas for Medical Sciences, Little Rock, Arkan-
sas, 2Truman Medical Center, Kansas City, Missouri
192
Molecular Detection of the BRAF (V600E)
Activating Gene Mutation in Fine Needle
Aspiration of Papillary Thyroid Carcinoma
Using LightCycler PCR and Fluorescent
Melting Curve Analysis
Joel S. Bentz, M.D.1, Leslie R. Rowe, M.S., CT(ASCP)2,
Brandon G. Bentz, M.D.3, Joseph A. Holden, M.D., Ph.D.1
INTRODUCTION: Fine needle aspiration (FNA) is widely utilized
for preoperative evaluation of patients with suspicion of thyroid
malignancy. Recently, a point mutation in exon 15 (T1799A/
V600E) of the BRAF gene has been reported to be the most
common genetic event in papillary thyroid carcinoma (PTC). In
this retrospective study, we report a molecular assay to detect
the BRAF V600E gene mutation in PTC specimens.
MATERIALS AND METHODS: The thyroid cancer database at the
study institution was searched for patients with preoperative
FNA and surgical thyroidectomy showing PTC. A total of 25
archival thyroid FNAs and corresponding formalin-fixed, paraf-
fin-embedded (FFPE) surgical samples were identified. Five
matched cases with a benign diagnosis, and 20 matched pairs of
PTC were evaluated for the BRAF V600E mutation using Light-
Cycler PCR and fluorescent melting curve analysis (LCPCR).
Prior to analysis, percent tumor cell content was noted for each
FNA sample. DNA was extracted from FNA stained smears using
a slide scape lysate (SSL) technique. For surgical samples, DNA
was extracted from 5 $ paraffin sections using manual micro-
dissection of the area of interest. Primers were designed to
amplify a 250 bp region of exon 15 of the BRAF gene. Positive
(mutant) controls were developed using extracted DNA from the
NPA PTC cell line. Genomic DNA was used for wild type (WT)
controls. All mutation results identified by LCPCR were con-
firmed by direct DNA sequencing.
RESULTS: A total of 18 cases of PTC with sufficient DNA in
matched samples were included in the final data analysis. Two
FNA cases had a PCR amplification failure. Using LCPCR, the
BRAF V600E mutation was identified in 14/18 (78%) FNAs and
14/18 (78%) surgical resection samples. There was 100% concor-
dance between preoperative FNA and FFPE tissue, and each
mutation detected was confirmed by direct DNA sequencing.
The BRAF V600E mutation was not detected in FNAB or FFPE
samples from 5 benign thyroid nodules.
425
CONCLUSIONS: LCPCR showed high sensitivity and specificity for
detection of the BRAF V600E gene mutation in thyroid FNA.
Analysis of BRAF gene status with this technique may be useful
in PTC, particularly indeterminate cases, and potentially helpful
for selecting patients for RAF-kinase inhibitor (e.g. BAY 43-9006)
therapy.
1
Department of Pathology, University of Utah, Salt Lake City,
Utah, 2Institute for Clinical and Experimental Pathology, ARUP
Laboratories, Salt Lake City, Utah, 3Division of Otolaryngology -
Head & Neck Surgery, Department of Surgery, University of Utah,
Salt Lake City, Utah
193
Utility of BRAF V600E Mutation Detection in
Cytologically Indeterminate Thyroid Nodules
Leslie R. Rowe, M.S., CT(ASCP)1, Brandon G. Bentz,
M.D.3, Joseph A. Holden, M.D., Ph.D.2, Joel S. Bentz,
M.D.2
INTRODUCTION: Fine needle aspiration (FNA) is widely utilized
for preoperative evaluation of patients with thyroid nodules.
However, approximately 20% of all thyroid FNAs are indetermi-
nate for malignancy. Recently, a mutation in exon 15 (T1799A/
V600E) of the BRAF gene has been reported to be the most
frequent genetic event in papillary thyroid carcinoma (PTC), and
found to be highly specific for the most common type of thyroid
malignancy. In this retrospective study, we assessed the utility of
BRAF V600E mutation detection for refining indeterminate cy-
tologic diagnoses in thyroid FNA samples.
MATERIALS AND METHODS: The thyroid cancer database at the
study institution was searched for patients with indeterminate
preoperative thyroid FNA and surgical thyroidectomy showing
PTC. A total of 19 archival thyroid FNAs and corresponding
formalin-fixed, paraffin-embedded (FFPE) surgical samples were
identified. All cases were evaluated for the BRAF V600E mutation
using LightCycler PCR and fluorescent melting curve analysis
(LCPCR). Prior to analysis, percent tumor cell content was noted
for each FNA sample. DNA was extracted from FNA stained
smears using a slide scrape lysate (SSL) technique. For surgical
samples, DNA was extracted from 5 $ paraffin sections using
manual microdissection of the area of interest. Primers were
designed to amplify a 250 bp region of exon 15 of the BRAF gene.
Positive (mutant) controls were developed using extracted DNA
from the NPA thyroid cancer cell line. Genomic DNA was used
for wild type (WT) controls. All mutation results identified by
LCPCR were confirmed by direct DNA sequencing.
RESULTS: Of the preoperative indeterminate thyroid FNAs, 3/19
(15.8%) were positive on both FNA and FFPE samples, 10/19
(52.6%) were negative on both, while 6 were negative on FNA but
positive on FFPE. The overall concordance between FNA and
FFPE was 68%. Of the discordant pairs, 5/6 FNAs contained less
than 50% tumor cells.
426 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
CONCLUSIONS: Using BRAF mutation analysis, the preoperative
diagnosis of PTC was confirmed in 15.8% of indeterminate FNA
samples that could not be conclusively diagnosed on cytology
alone. When used with indeterminate FNA samples, BRAF mu-
tation analysis may be a useful adjunct technique for confirming
the diagnosis of malignancy in an otherwise equivocal case.
However, overall tumor cell content of the FNA sample may be
a limiting factor for mutation detection.
1
Institute for Clinical and Experimental Pathology, ARUP Labo-
ratories, Salt Lake City, Utah, 2Department of Pathology, Univer-
sity of Utah, Salt Lake City, Utah, 3Division of Otolaryngology -
Head & Neck Surgery, Department of Surgery, University of Utah,
Salt Lake City, Utah
194
Fine Needle Aspiration Biopsy (FNAB) – A
Screening or a Diagnostic Tool for Thyroid
Neoplasm?
Maria Thomas, M.D., Donald Simpson, M.P.H., CT(ASCP),
Luis De Las Casas, M.D., Soheila Korourian, M.D.
INTRODUCTION/PURPOSE: Fine needle aspiration biopsy (FNAB)
is a reliable modality for initial evaluation of thyroid nodules.
False-positive, false-negative and indeterminate FNAB results
complicate patient management decisions. The purpose of this
study was to evaluate our accuracy in diagnosing thyroid neo-
plasm by FNAB.
MATERIALS AND METHODS: All FNABs of the thyroid were iden-
tified from our files for a five year period (2000-2004). These
cases were reviewed by two Cytopathologists and correlated with
surgical excisions.
RESULTS: A total of Five hundred and eighty-eight diagnostic
FNABs were performed during that time period. Fifty-two cases
revealed a thyroid neoplasm on excision (24 papillary thyroid
carcinomas, 22 follicular adenomas / carcinoma, 2 medullary
carcinomas, 2 anaplastic thyroid carcinomas, 1 neuroendocrine
carcinoma and 1 insular carcinoma). Thirty-nine of these were
called positive or suspicious on FNA (39/52) with a sensitivity of
75%. Four hundred and ninety-eight cases were diagnosed as
negative for malignancy; however thirteen of these cases re-
vealed a neoplasm on excision (13/498; 4%) resulting in a spec-
ificity of 98%. Nine of these thirteen cases showed low cellular-
ity/ sampling error on FNAB; eight of which revealed follicular
neoplasms and one a microscopic focus of papillary thyroid
carcinoma on excision, which was inadequately sampled on
FNAB. The four cases that were adequately sampled on FNAB
revealed 2 follicular variant of papillary thyroid carcinomas, 1
papillary thyroid carcinoma and 1 follicular adenoma on exci-
sion. The positive and negative predictive values of FNAB in the
evaluation of thyroid neoplasm in our laboratory are 81% and
97% respectively. Thirty-seven cases were diagnosed as atypical
on FNAB and were excised; 43% of these showed a neoplastic
process (8 follicular neoplasms, 5 follicular variant of papillary
thyroid carcinomas and 3 papillary thyroid carcinomas).
CONCLUSION: FNAB of thyroid has a high specificity and negative
predictive value, making it a very valuable diagnostic as well as
screening tool in excluding thyroid neoplasm. The sensitivity
which is dependent on sampling and interpretive errors, stresses
the importance of repeat FNAB in clinically worrisome lesions.
University of Arkansas for Medical Sciences, Little Rock, Arkansas
195
Outcomes of Thyroid FNA Diagnoses of
Follicular Neoplasm/Lesion vs. Hürthle Cell
Neoplasm/Lesion: A Two-Institution
Experience
Robert Pu, M.D., Ph.D.1, Jack Yang, M.D.2,3, Patricia
Wasserman, M.D.2, Tawfiqul Bhuiya, M.D.2, Kent Griffith,
M.P.H., M.S.1, Claire Michael, M.D.1
INTRODUCTION: Thyroid fine needle aspiration (FNA) is a stan-
dard procedure in the triage of thyroid nodules. The diagnosis of
an adequately sampled thyroid FNA is generally divided into
three categories: benign, malignant, and indeterminate. The
third group usually includes follicular neoplasm/lesion, and
sometimes a more specific diagnosis, such as Hürthle cell neo-
plasm or follicular neoplasm/lesion with Hürthle cell changes.
Whether FNA diagnosis of Hürthle cell neoplasm/lesion denotes
a worse outcome is controversial.
MATERIALS AND METHODS: A cohort of 303 cases of indetermi-
nate thyroid FNA cases with a diagnosis of either follicular neo-
plasm/lesion or Hürthle cell neoplasm/lesion was identified,
and follow up diagnoses were correlated in order to assess the
clinical significance of the more specific Hürthle cell category.
RESULTS: Of this cohort, 216 cases were interpreted as follicular
neoplasm/lesion while 87 cases were diagnosed as Hürthle cell
neoplasm/lesion. There was no age or gender difference be-
tween Hürthle cell neoplasm/lesion and follicular neoplasm/
lesion groups (age, 50 vs. 47; male, 18% vs. 20%). Upon excision,
the FNA diagnosis of Hürthle cell neoplasm/lesion group had 14
(16%) benign cases including 46 cases of adenoma (12 (14%)
follicular adenoma, and 34 (39%) Hürthle cell adenoma), and 27
(31%) malignant cases, including 12 papillary carcinoma (PC)
and 15 Hürthle cell carcinoma (HC). The follicular neoplasm/
lesion group had 82 (38%) benign cases including 8 cases of
Hashimoto’s thyroiditis, 73 cases of follicular adenoma (33.8%),
one case of granular cell tumor, and 60 (27.8%) malignant cases,
including 46 cases of PC, 12 cases of follicular carcinoma (FC), 1
case of HC, and 1 case of parathyroid carcinoma. Although there
was a slightly higher percentage of carcinomas in the Hürthle
cell neoplasm group than in the follicular neoplasm group (31%
vs. 27.8%), the difference was not statistically significant be-
tween these two FNA diagnostic groups (p"0.5771). When com-
 American Society of Cytopathology Abstracts: 53rd Annual Scientific Meeting
paring the cases by cancer versus non-cancer surgical diagnostic
categories, the groups were comparable for age, but were signif-
icantly different for their distributions of gender, with the cancer
group having the higher proportion of male patients (28.7%)
than the non-cancer group (16.7%, p " 0.0259).
CONCLUSIONS: Based on our results from two institutions and
with over more than 5 years follow-up, the FNA diagnosis of
Hürthle cell neoplasm/lesion does not impart a worse outcome
than the FNA diagnosis of follicular lesion/neoplasm. Male pa-
tients with FNAs categorized as Hürthle cell neoplasm/lesion
have a higher likelihood of malignancy in surgical follow-up.
1
University of Michigan, Ann Arbor Michigan, 2Long Island Jew-
ish Medical Center, New Hyde Park, New York, 3University of
Texas Medical Branch at Galveston, Galveston, Texas
196
Thyroid FNA Error Rates are Highly Variable
Lori Eichelberger, Harvey Cramer, M.D.
INTRODUCTION/PURPOSE: Thyroid FNA is the diagnostic test of
first choice for the investigation of patients who present with
thyroid nodules. Skills and styles of thyroid FNA cytologic inter-
pretation are highly variable. Inappropriate use of the atypical or
suspicious diagnostic categories may lead to unnecessary thy-
roid surgery.
MATERIALS AND METHODS: A computerized search of the FNA
database was performed for a 3.5 year period. All thyroid FNA
cases with correlating surgical pathology were identified and all
cytology and surgical pathology reports and selected cytology
and surgical pathology slides were retrospectively reviewed.
RESULTS: During the 42 month period of this study, a total of 152
thyroid FNAs with correlating surgical pathology were identified.
The cytologic diagnosis was correct for 100 of 152 cases (66
percent). There were no false positive cases among any of the 19
cases diagnosed definitively as malignant by FNA (15 papillary
carcinomas, 1 squamous cell carcinoma, 1 carcinoma NOS, 2
malignant lymphomas). Of 37 cases diagnosed as negative/col-
loid nodule, there were only 3 cases in which follow-up histo-
pathology disclosed a follicular adenoma; the cytologic diagnosis
was confirmed in the other 34 cases (92%). Among the cases
diagnosed as atypical or suspicious, there were 20 undercalls
and 32 overcalls. The cytologic diagnoses for the 152 cases were
performed by 9 different pathologists. The number of cases
diagnosed by each of the pathologists and their corresponding
combined undercall and overcall error rates were as follows:
1(0%), 2 (100%), 3 (33%), 5 (60%), 7 (14%), 13 (46%), 14 (43%), 22
(18%) and 85 (33%). The error rate for the 3 best-performing
pathologists was 17% compared to an error rate of 60% for the 6
poorest-performing pathologists. The type of error also varied
considerably among the pathologists. For 3 of the pathologists,
all of the errors were undercalls while for one of the pathologists,
all of the errors were overcalls. There were 3 pathologists with an
427
equal distribution of undercalls and overcalls and for the pathol-
ogist who was responsible for the largest number of errors,
overcalls accounted for 68% of the errors.
CONCLUSIONS: Error rates among pathologists were highly vari-
able as were the types of errors encountered. Experience did not
correlate with the error rate. In 32 of the 152 cases (21%) diag-
nosed as atypical or suspicious for malignancy, overcall errors
were committed, arguably resulting in unnecessary thyroid sur-
gery. Overutilization of the atypical and suspicious categories
should be strongly discouraged and interpretation of thyroid
FNAs should be restricted to only those pathologists who dem-
onstrate sufficient diagnostic expertise.
Indiana University School of Medicine, Indianapolis, Indiana
197
Detection of Methylation Profiles of Thyroid
Stimulating Hormone Receptor and 13 Other
Tumor Suppressor Genes in Thyroid
Neoplasm
Thomas S. Scheidemantle, CT(ASCP), Lin Wang, M.S.,
Dawn O’Brien, CT(ASCP), Jennifer Brainard, M.D., Charles
Biscotti, M.D., Bin Yang, M.D., Ph.D.
BACKGROUND: Fine needle aspiration (FNA) cytology is currently
the best diagnostic tool in the differential diagnosis of a thyroid
nodule. However, in approximately 20% of FNAs, results are
indeterminate and fail to differentiate benign from malignant.
Cancer-specific molecular markers are needed to supplement
the cytopathologic assessment of thyroid nodules. Recent stud-
ies indicate that thyroid tumorigenesis involves both genetic and
epigenetic alterations. To test whether DNA methylation profil-
ing can be useful in distinguishing benign from malignant thy-
roid lesions, we have studied methylation profiles of thyroid
stimulating hormone receptor (TSHR) and 13 other tumor sup-
pressor genes in 30 thyroid FNA samples.
DESIGN: Thirty thyroid FNAs include 10 cytopathologically diag-
nosed colloid nodules, 7 papillary thyroid carcinomas, and 13
hypercellular follicular nodules. Genomic DNA was extracted
using Gentra system’s Puregene kit and chemically converted by
bisulfite before amplification. Promoter methylation of TSHR
and 13 candidate tumor suppressor genes (RAR-beta, p14, p15,
p16, p73, RASSF1a, FHIT, DAPK, MGMT, BRCA1, SOCS1, GSTP
and VHL) was analyzed by methylation-specific PCR. Methyl-
ation profile was correlated with clinicopathologic follow-up
data.
RESULTS: Based on clinical follow up data, 30 cases include 14
CN, 8 follicular adenoma, 8 thyroid carcinoma, including 6 pap-
illary thyroid carcinoma, 1 anaplastic thyroid carcinoma and 1
follicular carcinoma. Although methylation of all genes, except
VHL, was seen in thyroid tumors, the most frequently methyl-
ated genes were p16 (90%), RASSF1a (87%), p14 and
428 CANCER (CANCER CYTOPATHOLOGY) SUPPLEMENT October 25, 2005 / Volume 105 / Issue 5
BRCA1(60%). However, methylation of none of these tumor sup-
pressor genes differentiated colloid nodule from follicular ade-
noma or from thyroid carcinoma. In contrast, there was a sig-
nificant difference in methylation of TSHR between benign and
malignant thyroid lesions (p%0.05). Methylation of TSHR was
seen in 10/14 (71%) of colloid nodule and 5/8 (63%) of follicular
adenoma, but only in 2/8 (25%) of thyroid carcinoma. Both cases
of TSHR-methylated thyroid carcinoma were follicular variant of
papillary thyroid carcinoma. The other striking finding was the
methylation status of VHL gene in thyroid carcinoma. Methyl-
ation of VHL was not identified in any thyroid specimen ana-
lyzed. However, unmethylated bands were present in all (100%)
colloid nodule and follicular adenoma, but only detected in 3/8
(38%). Previous study has reported that loss of heterozeigosity of
3p25.3 (VHL resides) was seen in 67% of thyroid carcinoma. Our
study here suggests that biallelic loss of VHL gene may be in-
volved in thyroid carcinogenesis.
CONCLUSIONS: Our study with limited genes suggests that meth-
ylation of majority of these genes, commonly altered in other
malignancies, does not distinguish benign from malignant cells
in thyroid. Loss of TSHR methylation observed in this study
could explain the mechanism of increased TSHR transcripts
detected in papillary thyroid carcinoma. Further genetic analysis
should confirm whether biallelic loss of VHL gene plays impor-
tant role in development of thyroid carcinoma.
Cleveland Clinic Foundation, Cleveland, Ohio