Chapter 6 The Bethesda System For Reporting Cervical Cytology

6
The Bethesda System for Reporting

Cervical Cytology
RITU NAYAR | DAVID C. WILBUR
CONTENTS develop a uniform descriptive terminology for cervical/vaginal

cytologic interpretation. The format that emerged became
The Bethesda System: Historical Perspective known as The Bethesda System (TBS).1
The 2001 Bethesda System Approximately 2 years later, a second meeting was convened
Report Format to critique and refine the terminology based on experience with
the use of the system in actual laboratory practice. Minor modi-
Specimen Adequacy fications were incorporated into the 1991 Bethesda System that
Bethesda 2001 Specimen Adequacy Categories streamlined the terminology.2 In addition, an ad hoc committee
Squamous Cellularity developed criteria for specimen adequacy and Bethesda inter-
Quality Indicators pretive categories, culminating in the first TBS atlas that out-
Management Guidelines lined and illustrated the morphologic features.3 By the mid- to
Impact on Laboratory Practice late 1990s, there was a significant penetration of TBS into
General Categorization cytopathology practice with approximately 90% of laboratories
Interpretation/Result in the USA using the Bethesda terminology for reporting of
Negative for Intraepithelial Lesion or Malignancy (NILM) cervical/vaginal cytology.4 Articles published since the release of
Endometrial Cells the second manual have shown increased consistency of termi-
Epithelial Cell Abnormalities: Squamous Cell nology across countries, which, has greatly facilitated commu-
Epithelial Cell Abnormalities: Glandular Cell nication between systems which until this time, had little hope
of sharing gynecologic screening data.
Educational Notes/Suggestions The fundamental aim of TBS has been to communicate
Ancillary Testing clinically relevant information from the laboratory to the
Automated Review
patient’s healthcare provider, using uniform, reasonably repro-
ducible terminology, which reflects the most current under-
Interobserver Reproducibility in Cervical Cytology standing of the biology of cervical neoplasia. Advances in the
The Bethesda System and Reporting Anal–Rectal understanding of the biology of cervical cancer, results from
Cytology clinical trials, the introduction of liquid-based cytology, human
Concluding Remarks
papillomavirus (HPV) testing, and automated screening devices
for cervical cytology led to the decision to convene the third
Bethesda workshop in April 2001.
THE 2001 BETHESDA SYSTEM

The Bethesda System: TBS 2001 Process
Historical Perspective Approximately 8 months prior to the workshop, nine forum
Terminology forms the basis for effective communication groups, consisting of 6–10 individuals with a breadth of exper-
between the laboratory and clinician. The clinician is expected tise in the area of cervical cancer, were organized under the
to provide relevant patient information to the laboratory. It is sponsorship of the NCI to formulate draft recommendations.
the laboratory’s responsibility to report results using terminol- Internet bulletin boards were open to the worldwide cytology
ogy that clearly conveys the diagnostic interpretation of the community for 6 months during the pre-conference process of
morphologic findings. The use of a uniform diagnostic termi- review and discussion. Over 1000 comments were considered
nology facilitates communication by establishing a common in revising the pre-workshop drafts. The 2001 Bethesda work-
language that, in theory, does not vary significantly from cytolo- shop was co-sponsored by 44 international professional organi-
gist to cytologist or laboratory to laboratory. However, termi- zations and attended by over 400 individuals, including
nology is not static over time; rather, it evolves in parallel with pathologists, cytotechnologists, gynecologists, attorneys, patient
increased understanding of the pathogenesis and biology of advocates, and other healthcare workers involved in women’s
disease. The framework, therefore, must be flexible enough to health initiatives. The revised draft recommendations were pre-
incorporate advances in scientific knowledge without creating sented by each forum group and, after open discussions and
undue confusion or complexity. voting by all participants, the 2001 Bethesda consensus termi-
In 1988, the National Cancer Institute (NCI) sponsored an nology was finalized and published in 2002.5
open workshop – including cytotechnologists, pathologists, Following the Bethesda workshop, the American Society
clinicians, and representatives of professional organizations – to for Colposcopy and Cervical Pathology (ASCCP) held a
70
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6 The Bethesda System for Reporting Cervical Cytology 71
comparable consensus workshop on patient management in and experience obtained in the last decade regarding HPV
September 2001. This was also preceded by an internet discus- biology and the operating characteristics of liquid-based cytol-
sion, and resulted in the development of evidence-based man- ogy. In addition, the many management changes made based
agement guidelines for abnormal cervical cytology corresponding on long-term follow-up data from TBS categories will form a
to the 2001 Bethesda reporting format.6 The ASCCP manage- new section in the monograph. No substantive changes in the
ment guidelines were subsequently updated at consensus con- terminology are planned, however subtle refinements in several
ferences held in 2006, 2009, and 2012.7 After the initial categories will be forthcoming.
publication of TBS 2001 terminology (Box 6-1), the NCI
approached the American Society of Cytopathology (ASC) to
collaborate on publication of the second edition of the Bethesda
Report Format
Atlas8 and the development of an accompanying Bethesda The basic structure of TBS includes three elements, based on
System educational website.9 Images chosen for the atlas and communication needs germane, but not limited, to gynecologic
website underwent an extensive selection/validation process, cytology: (1) statement of specimen adequacy, (2) general cat-
and included classic as well as morphologically difficult and egorization, and (3) descriptive terminology. The specimen
“borderline” images, illustrated on both conventional prepara- type – conventional smear, LBP, or other – should also be stated
tions and liquid-based preparations (LBPs). A subset of images in the report (Box 6-1).
chosen for the Bethesda Atlas were used to assess interobserver
reproducibility in gynecologic cytology – the details of this
Bethesda interobserver reproducibility project are described
Specimen Adequacy
below.10 Reporting of adequacy was an important quality assurance
Planning for a third edition of the Bethesda Atlas is currently measure introduced by TBS. The 1988 Bethesda System incor-
underway. This work will focus on the increasing knowledge porated a classification of three categories of specimen
BOX 6-1 THE 2001 BETHESDA SYSTEM

SPECIMEN TYPE Other
Indicate conventional smear vs. liquid-based preparation vs. other • Endometrial cells (in a woman >40 years of age) (specify if
“negative for squamous intraepithelial lesion”)
ADEQUACY OF THE SPECIMEN
• Satisfactory for evaluation (describe presence or absence Epithelial Cell Abnormalities
of endocervical/transformation zone component and any Squamous Cell
other quality indicators, e.g., partially obscuring blood, • Atypical squamous cells
inflammation) • of undetermined significance (ASC-US)
• Unsatisfactory for evaluation … (specify reason) • cannot exclude HSIL (ASC-H)
• Specimen rejected/not processed (specify reason) • Low-grade squamous intraepithelial lesion (LSIL) (encompass-
• Specimen processed and examined, but unsatisfactory for ing: HPV/mild dysplasia/CIN 1)
evaluation of epithelial abnormality because of (specify • High-grade squamous intraepithelial lesion (HSIL) (encompass-
reason) ing: moderate and severe dysplasia, CIS/CIN 2 and CIN 3)
• with features suspicious for invasion
GENERAL CATEGORIZATION (OPTIONAL) • Squamous cell carcinoma
• Negative for intraepithelial lesion or malignancy
Glandular Cell
• Other
• Atypical
• Epithelial cell abnormality: see Interpretation/Result (specify
• endocervical cells (NOS or specify in comments)
squamous or glandular as appropriate)
• endometrial cells (NOS or specify in comments)
INTERPRETATION/RESULT • glandular cells (NOS or specify in comments)
• Atypical
Negative for Intraepithelial Lesion or Malignancy • endocervical cells, favor neoplastic
(when there is no cellular evidence of neoplasia, state this in the • glandular cells, favor neoplastic
General Categorization above and/or in the Interpretation/Result • Endocervical adenocarcinoma in situ
section of the report – whether or not there are organisms or other • Adenocarcinoma
non-neoplastic findings) • endocervical
• endometrial
Organisms
• extrauterine adenocarcinoma
• Trichomonas vaginalis
• not otherwise specified (NOS)
• Fungal organisms morphologically consistent with Candida
• Other Malignant Neoplasms (Specify)
spp.
• Shift in flora suggestive of bacterial vaginosis ANCILLARY TESTING
• Bacteria morphologically consistent with Actinomyces spp.
Provide a brief description of the test method(s) and report result so
• Cellular changes associated with herpes simplex virus
that it is easily understood by the clinician.
Other Non-neoplastic Findings (Optional to Report;
List not Inclusive) AUTOMATED REVIEW
• Reactive cellular changes associated with: If case is examined by automated device, specify device and result.
• Inflammation (includes typical repair)
• Radiation EDUCATIONAL NOTES AND SUGGESTIONS (OPTIONAL)
• Intrauterine contraceptive device (IUD) Suggestions should be concise and consistent with follow-up guide-
• Glandular cells status post-hysterectomy lines published by professional organizations (references to relevant
• Atrophy publications may be included).
72 PART 2 Diagnostic Cytology
adequacy – satisfactory, less than optimal, and unsatisfactory SQUAMOUS CELLULARITY

– into the format of the report but did not outline specific
morphologic criteria for evaluation of adequacy. Participants TBS 1991 required that well-preserved and well-visualized
at the 1991 Second Workshop, and others in the cytopathology squamous epithelial cells should cover more than 10% of the
community,11,12 voiced the need for developing consensus slide surface. In order to address adequacy on conventional as
guidelines. In response, following the second workshop, a Cri- well as LBPs, and to improve interobserver reproducibility, TBS
teria Committee formulated the definitions for adequacy, 2001 went further to provide numerical estimates of what con-
based on a combination of experience and review of an admit- stitutes adequacy for squamous cellularity in cervical cytology
tedly sparse scientific database. Three categories – “satisfac- preparations.
tory,” “satisfactory but limited by …,” and “unsatisfactory” • Conventional smears. An adequate conventional prepara-
– based on estimates of overall squamous cellularity, assess- tion should have a minimum of approximately 8000–12 000
ment of the transformation zone component, and the presence/ well-preserved and well-visualized squamous cells. This
extent of obscuring or limiting factors, were suggested in an minimum cell count should be estimated, not counted.
initial attempt to develop a more standardized approach to The count includes both nucleated mature and metaplas-
the evaluation of adequacy.13 It was emphasized that the indi- tic squamous cells. The percentage of hypocellular areas,
cated percentages should be used as general ranges, not strict if present, should be estimated and the fields counted
numerical cut-offs and that patient-related clinical factors and should reflect this proportion. The Bethesda Atlas8 and
previous cytologic findings should always be taken into website9 provide “reference images” of known cellularity
consideration. at low (×4) magnification as a resource for cytologists to
compare with the specimen being assessed.
BETHESDA 2001 SPECIMEN • Liquid-based preparations. An adequate LBP should have
an estimated minimum of at least 5000 well-visualized,
ADEQUACY CATEGORIES
well-preserved squamous cells.8,15 Estimation of cellularity
In 2001, substantial changes were made to the adequacy com- is suggested in borderline cases by performing representa-
ponent of TBS. The previously used borderline adequacy cat- tive field counts. A minimum of 10 fields (usually at ×40)
egory of “less than optimal” (1988)/“satisfactory but limited by are assessed along a diameter that includes the center of
…” (1991) was deleted in order to provide the clinician a clearer the preparation. The average number of squamous cells
and more reproducible indication of the adequacy of the speci- per field is thus estimated. One preliminary study sug-
men.14 The classification recommended in TBS 2001 is either as gested that LBPs containing 5000–20 000 squamous cells
“satisfactory” or “unsatisfactory”: should be considered as “borderline” cellularity.16
• Satisfactory8 – Satisfactory for evaluation (describe pres- The reader is referred to the Bethesda Atlas for cellularity
ence or absence of endocervical/transformation zone tables and figures.8 TBS numeric criteria for cellularity may not
component and any other quality indicators, e.g., partially be applicable to vaginal specimens, cases with extensive cytoly-
obscuring blood, inflammation, etc.). For “satisfactory” sis, cell clustering, and some cases of atrophy. Cytologists should
specimens, including information on transformation zone utilize clinical information and their best judgment when inter-
sampling and other adequacy qualifiers (obscuring ele- preting such cases. At present, there are no published studies
ments, poor preservation, etc.) encourages specimen specifically addressing the relationship between low cellularity
takers to pay greater attention to specimen procurement and false-negative rate.
and handling. Any factors that compromise specimen
quality can be mentioned in a note. QUALITY INDICATORS
• Unsatisfactory8 – For unsatisfactory specimens, the report Patient/Specimen Identification
should indicate whether the laboratory processed/
evaluated the slide. Suggested wording is: and Technical Interpretability
• Rejected specimen – specimen rejected/not processed Correct specimen identification is essential for evaluation and
because (specify reason: specimen not labelled, broken is required in the USA by the Clinical Laboratory Improve-
slide, etc.) ment Amendments of 1988 (CLIA 88). In addition to ensuring
• Fully evaluated, unsatisfactory specimen – specimen that the specimen corresponds to the correct patient, proper
processed and examined, but unsatisfactory for evalua- identification allows the laboratory to locate prior records
tion of epithelial abnormality because of (specify reason: and slides from the patient that may influence the current
inadequate squamous component, obscuring blood, evaluation.
etc.). The cellular material must be well fixed and unobscured for
Additional comments/recommendations may be made as interpretation. Minimal data regarding how obscuring factors
deemed appropriate. affect the interpretive reliability of a cervical specimen are avail-
While unsatisfactory specimens which are processed and able. In order to be considered obscuring, the epithelial cell
evaluated are not suitable for excluding an intraepithelial lesion morphology must be uninterpretable. For example, although
or malignancy, the presence of endometrial cells in a women 40 most cervical samples contain inflammatory cells, moderate
years or older, or the presence of organisms, can be reported in numbers do not generally obscure the nuclei of squamous cells.
this context, since this information may prove to be clinically Even a large amount of inflammation or blood may be accept-
relevant for patient management. able if it is spread thinly such that the intermixed epithelial cells
As in prior Bethesda adequacy guidelines, if abnormal can be easily visualized. In general, specimens with more than
cells are detected, the specimen cannot be categorized as 75% of epithelial cells obscured are considered unsatisfactory.
“unsatisfactory.” A variety of factors may compromise visualization of the cells.
Heavy inflammation, blood, and extensive cytolysis are patient- high-risk patients and have significantly more squamous
related and independent of the sample taker. However, presence intraepithelial lesions (SIL)/cancer on follow-up than patients
of air-drying, thick uneven smears, or lubricant is often inversely with satisfactory index Pap tests.25 In the USA, the majority of
correlated with the skill and experience of the clinician. With Pap tests are now liquid-based, which decreases unsatisfactory
LBPs a number of these factors become less significant; however, results from air-drying and obscuring factors, making insuffi-
appropriate collection/rinsing techniques need to be utilized. cient squamous cells the major cause of unsatisfactory results.
Clinicians who repeatedly obtain technically poor quality speci- For quality assurance, it may be prudent to have the pathologist
mens may benefit from constructive feedback provided in the review unsatisfactory cases prior to final sign-out because of the
written report, by telephone, or in a summary format compar- clinical implications of such a report and the association of
ing adequacy rates of peer group clinicians. obscuring blood/inflammation with invasive cancers.26
A recent meta-analysis found that negative cytology had
Clinical Information good specificity and negative predictive value, despite absent or
Providing pertinent clinical information may increase the sen- insufficient EC/TZ component.7 HPV testing appears to be
sitivity and reliability of the evaluation by directing attention independent of transformation zone sampling7 and offers an
to a clinical question or by clarifying otherwise uncertain cyto- added margin of safety for women who are co-tested. There are
logic findings. At a minimum, age and date of last menstrual minimal data regarding the management of women with an
period should be provided. Absence of this information does unsatisfactory Pap test that is obtained as part of co-testing for
not, however, preclude evaluation; therefore, the specimen may HPV, although risk for high-grade lesions in women with nega-
remain categorized as “satisfactory” in these circumstances. tive HPV tests appears to be low.7 Some of the currently avail-
able HPV tests lack a control for epithelial cellularity, so, when
Sampling of the Transformation Zone using these methods, a negative HPV test cannot always be
Presence of endocervical or squamous metaplastic cells forms relied upon, as it may be falsely negative because of an insuf-
the microscopic basis for the assumption that the transforma- ficient sample. Other HPV detection tests include a cellular
tion zone has been sampled. The numeric criterion for a trans- DNA control to ensure that an adequate cell sample has been
formation zone component, at least 10 well-preserved obtained in order to ensure the validity of a negative result.
endocervical or squamous metaplastic cells, did not change The ASCCP preferred management guideline for unsatisfac-
from TBS 1991; however, due to the widespread utilization of tory Pap tests is a repeat test within a short interval of 2–4
LBPs, single endocervical/metaplastic cells are acceptable and months; triage by HPV testing is not recommended. For women
clusters are no longer required. This definition applies to speci- aged 30 years and older who are co-tested and have an unsatis-
mens from both premenopausal and postmenopausal women factory cytology and a positive HPV test, either a repeat cytol-
having a cervix. In the situation of marked atrophy, where meta- ogy in 2–4 months or colposcopy is acceptable. Colposcopy is
plastic and endocervical cells often cannot be distinguished recommended for women with two consecutive unsatisfactory
from parabasal cells, the laboratory has the option of making a cytology tests. Management of women with negative cytology
comment regarding the difficulty in assessing the transforma- results but with an absent or insufficient EC/TZ component is
tion zone component. Patient factors, such as location of the also detailed in the ASCCP guidelines.7 For women aged 21–29
transformation zone, age, pregnancy, and previous therapy, may years with negative cytology and absent or insufficient EC/TZ
limit the clinician’s ability to obtain an endocervical sample, component, routine screening is recommended. HPV testing is
despite optimal collection technique. unacceptable in this age group because of the high prevalence
Numerous cross-sectional studies have demonstrated that of background HPV infection in this age range. For women
smears with endocervical and/or metaplastic cells have a signifi- aged 30 years and older with cytology reported as negative and
cantly higher frequency and higher grade of squamous epithe- with absent or insufficient EC/TZ component and no or
lial abnormality detected than do smears without such cells.17–20 unknown HPV test result, HPV testing is preferred but repeat
Paradoxically, short-term longitudinal studies of women whose cytology in 3 years is acceptable if HPV testing is not performed.
initial negative smears lacked an endocervical component have If the HPV test is done and is negative, return to routine screen-
shown no increase in abnormalities on repeat, satisfactory ing is recommended; if the HPV test is positive, repeating both
smears (as might be expected if the initial smears had a higher tests in 1 year is acceptable.
false-negative rate).21,22
Based on the above studies, TBS 2001 did not require the IMPACT ON LABORATORY PRACTICE
presence of a transformation zone component to categorize a
specimen as satisfactory – adequate squamous cellularity is the The incorporation of specimen adequacy as an integral part
only criterion. The absence of a transformation zone compo- of the cervical cytology report has been acknowledged as one
nent is considered to be a quality indicator. With the reported of the most important contributions of TBS. The impact on
increase in endocervical carcinoma,23,24 the importance of the laboratory practice has been dramatic. Surveys conducted by
transformation zone component may undergo further evalua- the College of American Pathologists (CAP) revealed that, in
tion in the future, in order to ensure optimized screening per- 1990, only 35% of responding laboratories routinely reported
formance in the setting of endocervical neoplasia. specimen adequacy; by 1992, this figure increased to 85%.26 A
1991 CAP survey found that most laboratories reported unsat-
isfactory specimen rates of 0.5–1.0%. By the year 2003, a CAP
MANAGEMENT GUIDELINES
survey assessing Bethesda implementation and reporting rates27
Unsatisfactory cases are unreliable for detection of an epithelial found that 73.6% of responding laboratories had eliminated the
abnormality; furthermore, a longitudinal study found that use of the “satisfactory but limited by …” category and the 2001
unsatisfactory Papanicolaou (Pap) tests are more often from TBS minimum squamous cellularity criteria had been adopted
by 85.3%. Experts had predicted an increase in unsatisfactory two prior Bethesda categories of “within normal limits” (WNL)
rates with the use of TBS 2001 criteria. While some studies have and “benign cellular changes” (BCC). The basis of this change
indeed reported this (up to a 10-fold increase on conventional was to clearly communicate to the physician that, despite any
smears28), other reports from the USA29 and Europe30 did not other “benign” changes reported, the Pap test is “negative” or
show an increase in the unsatisfactory rate after conversion to without evidence of cervical intraepithelial neoplasia (CIN) or
TBS 2001 adequacy criteria. Possibilities suggested by the malignancy.
authors include improved sampling and preparation methods, Clearly, the main purpose of cervical cytology screening is
related predominantly to liquid-based methodology, or, alter- the detection of cervical squamous cell carcinoma and its pre-
natively, lack of attention to the TBS 2001 criteria.26 The CAP cursors; however, reporting the findings of organisms or reac-
survey from 2006 showed that use of TBS criteria for unsatisfac- tive conditions can make an important contribution to patient
tory specimens is widespread, with 94.5% of US laboratories care. This documentation can facilitate patient triage, provide
using TBS minimum cellularity criteria.27 Unsatisfactory rates clinical–cytologic correlation, and focus attention on cytomor-
have increased since 1996; however, the median rates are 1.1% phologic criteria during microscopic screening and interpreta-
or less for all preparations.31 tion of cervical cytology.
The category of “infections” was changed to “organisms” in
TBS 2001, since the presence of some organisms represents
General Categorization colonization, rather than a clinically significant infection. Excel-
The general categorization is a clerical component of the report lent specificity and reproducibility can be achieved for the
to aid clinicians and their office staff in triaging patients/ cytopathologic interpretation of fungal elements, Trichomonas
prioritizing cases for review and to assist laboratories in compil- vaginalis, Actinomyces, and herpes simplex virus, by application
ing statistical information. of reproducible morphologic criteria. The interpretation of
There are three headings used under the general category: Chlamydia spp. is not listed in TBS because of the acknowl-
1. “Negative for intraepithelial lesion or malignancy” for edged low diagnostic accuracy of routine cytology for this
specimens in which an epithelial abnormality is not iden- organism and because of the availability of other, more accurate
tified. Organisms and other benign/reactive cellular detection methods. TBS lists the organisms that should be
changes can be reported in the Interpretation under this reported; however, the laboratory is advised to discuss the rel-
category evance of reporting organisms and other non-neoplastic find-
2. “Other” may be utilized for cases in which there is no clear ings with their clinicians and come to a decision about what to
cytologic abnormality but the findings may warrant report under the NILM category.
follow-up/investigation based on patient risk, for example Cells manifest reactive morphologic changes in response to
endometrial cells in a woman 40 years of age or older a variety of traumatic insults such as infection, inflammation,
3. “Epithelial cell abnormality” may be utilized for squa- and radiation. Reparative processes, radiation, atrophy, and
mous or glandular epithelial abnormalities. Specify, as far intrauterine contraceptive devices are examples of entities that
as possible, which type of abnormality is present. induce cellular changes that may mimic intraepithelial lesions
If more than one diagnostic entity is present – for example, or even cancer. Severe reactive/reparative changes are difficult
an infectious process and an epithelial abnormality – the speci- to distinguish from neoplastic changes and such interpretations
men should be categorized according to the most clinically are well known to have lower reproducibility than classic
significant lesion; in this example, epithelial cell abnormality. repair.32 It is, however, important to recognize benign reactive
However, the general category should not replace narrative features in order to avoid over-interpretation and resulting
(descriptive) terminology for communicating the interpretation/ false-positive interpretations. A CAP report indicates that
result. Some laboratories also extend the concept of a general reparative changes tend to be easier to recognize on LBP, yield-
or summary categorization to non-gynecologic specimens. ing fewer false-positives than on conventional smears.33
Keratotic cellular changes – hyperkeratosis, parakeratosis,
and dyskeratosis – are descriptive terms that do not clearly com-
Interpretation/Result municate a diagnostic interpretation and are not included in
The prior Bethesda System use of the term “diagnosis” was TBS. The classification of such changes as benign/reactive or
replaced by “interpretation/result” in 2001. Workshop partici- dysplastic should be based on the cytoplasmic and nuclear
pants felt that cervical cytology is a screening, not a diagnostic, alterations present and reported under the appropriate general
test, which provides the clinician with information on morpho- category/interpretation.
logic findings that need to be integrated with the patient’s Occasionally, benign-appearing glandular cells may be seen
other clinical findings for a final diagnosis and subsequent in post-hysterectomy patients that can have a wide variety of
management.5 sources, including adenosis, metaplasia, and secondary to
surgery (residual cervix after supracervical hysterectomy, tra-
chelectomy and prolapse of the remaining fallopian tube after
NEGATIVE FOR INTRAEPITHELIAL LESION
a simple hysterectomy).34,35 This finding can be communicated
OR MALIGNANCY (NILM)
to the clinician under the NILM category and, as noted in the
The Bethesda 2001 category of NILM is used to report non- 2012 ASCCP guidelines, post-hysterectomy patients with a
neoplastic findings in the absence of an intraepithelial lesion or cytologic report of benign glandular cells do not require further
malignancy. This term is used both as a general categorization follow-up.7 Other non-neoplastic changes that may be reported
and as an interpretation and incorporates the reporting of under NILM include atrophy and tubal metaplasia. Details
organisms and other non-neoplastic findings, such as reactive regarding the morphology of these entities are discussed else-
cellular changes (Box 6-1). The NILM category replaces the where in this book.
ENDOMETRIAL CELLS EPITHELIAL CELL ABNORMALITIES:

SQUAMOUS CELL
TBS 1991 recommended that benign-appearing endometrial
cells in postmenopausal women be reported as an “epithelial Squamous intraepithelial lesion (SIL) encompasses the mor-
cell abnormality,” based on the increased risk for endometrial phologic spectrum of non-invasive squamous epithelial
adenocarcinoma (6%) and endometrial hyperplasia (12%) on abnormalities associated with HPV infection. Since TBS was
a meta-analysis.3,36,37 introduced in 1988, this spectrum has always been divided into
In TBS 2001, a new category was included to report the pres- low-grade (LSIL) and high-grade (HSIL) categories. LSIL
ence of benign-appearing endometrial cells in women aged 40 encompasses changes referred to as “HPV effect,” “koilocytosis,”
years or older.5,8 The basis for including this new category in and mild dysplasia/cervical intraepithelial neoplasia (CIN 1).
TBS 2001 was two-fold: (1) review of the published literature HSIL includes moderate dysplasia (CIN 2) and severe dysplasia/
showed an exceedingly low rate of significant lesions in anyone carcinoma in situ (CIN 3). The basis for this bipartite classifica-
less than 40 years of age and (2) pathologists may lack clinical tion of SIL in TBS is based on the principles that this division
information on menstrual dates/menopausal status, hormone (a) better reflects natural history and clinical management and
therapy/tamoxifen, abnormal bleeding, and other endometrial (b) has better intra- and interobserver reproducibility than does
carcinoma risk factors. It is important to include in the inter- a three-tiered reporting system.
pretation whether the cytology is “negative for squamous
intraepithelial lesion.” Atypical Squamous Cells (ASC)
Only exfoliated, intact endometrial cells should be reported The term ASCUS was initially introduced into the earliest
under the “other” category. As described in Bethesda 2001, the version of TBS to reflect the reality and limitations of light
exfoliated groups of endometrial cells may be of epithelial and/ microscopy in classifying borderline cytologic changes. The
or stromal origin; morphologic distinction of these two cell use of multiple ASCUS qualifiers such as “not otherwise speci-
types is usually not possible. Directly sampled lower uterine fied” (NOS), “favor reactive,” and “favor SIL/dysplasia” led to
segment or abraded stromal cells/histiocytes, when present overuse of this category and, by 1996, ASCUS interpretations
alone, should not be reported under this category. Atypical accounted for a mean of 5.2% of all cervical cytology reports
endometrial cells should be reported as an epithelial glandular in the USA.38 ASCUS interpretations caused dilemmas for clini-
cell abnormality.1,8 cians due to the lack of standardized follow-up and variability
The prevalence of benign-appearing endometrial cells in of outcomes.
Pap tests from women aged 40 years or older is difficult to With advances in the understanding of the biology of HPV
assess due to differences in study designs, but has been esti- infections and results from various natural history studies,39 as
mated to range from 1–3/100 to 1/1600 or less.36 This TBS well as from the NCI ALTS trial,40 the focus of cervical cancer
category has been controversial for clinicians and initially screening has shifted from detecting and treating any CIN to
resulted in an increase in endometrial biopsies. After adoption focusing on treating high-grade CIN. Based on this concept, in
of the TBS 2001, there have been many reports in the cytology TBS 2001, the term ASCUS was replaced by ASC, which has a
literature that have shown minimal risk associated with this narrower definition and only two qualifiers: atypical squamous
interpretation, especially in premenopausal women.37 Based on cells of undetermined significance (ASC-US) and atypical squa-
the data, some laboratories have made the decision not to mous cells, cannot exclude HSIL (ASC-H).5 A subclassification
report endometrial cells in a woman over 40 years, if there is a was aimed at having greater clinical utility by clearly separating
given history indicating that a patient is premenopausal and equivocal findings into those that are worrisome for HSIL in
the endometrial cells are not present out of cycle. This category distinction from other types of ASC. As a general guide, the
is thus reserved for women older than 40 who are out of phase, majority of ASC interpretations should fall into the ASC-
postmenopausal, or for whom an adequate menstrual history US qualifier (90–95%) with only 5–10% into the ASC-H
is not available. It is likely that this Bethesda category will be category.5,8
re-evaluated and possibly modified in the third edition of the ASC is not a single biologic or interpretive entity: it encom-
Bethesda Atlas. passes a spectrum of cellular changes reflecting a variety of
When using this general category and interpretation, it may pathologic processes that for one reason or another cannot be
be useful to add an educational note to this interpretation, in more definitively categorized. Specifically, ASC should be used
order to clearly communicate to clinicians that this interpreta- for changes suggestive of SIL, which are either quantitatively or
tion has an increased risk of neoplasia, but the risk is low, qualitatively insufficient for a definitive interpretation. For a cell
especially in premenopausal women and those without endome- to be classified as ASC, it should show, at a minimum, squa-
trial carcinoma risk factors, and that clinical correlation with mous differentiation, an increase in nuclear–cytoplasmic ratio,
other risk factors and symptoms is necessary. Examples of edu- and minimal nuclear changes.8 In each case of ASC, the
cational notes for this interpretation can be found in the second cytopathologist must consider the summation of the morpho-
edition of the Bethesda Atlas.8 logic abnormalities in terms of quantity and severity within the
The 2006 ASCCP guidelines provided subsequent guidance context of the clinical information provided.
and suggest that for asymptomatic women who are docu-
mented by clinical history to be premenopausal, with benign Atypical Squamous Cells of Undetermined Significance
appearing endometrial cells, endometrial stromal cells, or his- (ASC-US). Most often, ASC-US involves non-inflammatory
tiocytes, no further evaluation is required. For documented changes in squamous cells with mature, superficial/
postmenopausal women with endometrial cells, on the other intermediate-type cytoplasm. Nuclear enlargement is approxi-
hand, endometrial assessment is suggested, regardless of mately two-and-a-half to three times the area of a normal inter-
symptoms.7 mediate squamous nucleus, but the chromatin remains evenly
distributed without significant hyperchromasia. Nuclear out- criteria, 61% correlated with colposcopic biopsies positive for
lines are smooth and regular, although there may be variation CIN.42
in nuclear size. The differential diagnosis is usually between a Laboratory rates of ASC will vary depending on the patient
reactive change and LSIL but the change(s) quantitatively or population, the diagnostic criteria used, and the experience and
qualitatively fall short of establishing a definitive interpretation skill of the microscopist(s). If used appropriately, ASC should
of LSIL. Round or ovoid cells that resemble large metaplastic or be an infrequent designation employed only when cellular
small intermediate cells may also be classified as ASC-US. In changes elude a more definitive interpretation. Although there
LBPs, the cells may appear smaller and rounder than in conven- is no “correct” percentage rate of ASC, benchmarks were pro-
tional smears. The cells in question should always be compared vided when ASCUS was introduced in the Bethesda terminol-
with “normal”-appearing intermediate cells on the same slide. ogy. In a low-risk population, it was suggested that the rate of
In distinguishing reactive changes, cells that demonstrate pale ASCUS should be less than 5%. For laboratories that serve high-
round nuclei and even chromatin distribution favor an inter- risk populations (e.g., sexually transmitted disease clinics or
pretation as NILM rather than ASC. colposcopy clinics), the rate of ASCUS could be higher, but, by
1991 guidelines, should not exceed two to three times the rate
Atypical Squamous Cells, cannot Exclude High-grade Squa- of SIL; thus the ratio of ASCUS/SIL suggested was in the range
mous Intraepithelial Lesions (ASC-H). The ASC-H category of 2–3 : 1. A 1993 CAP survey focusing on laboratory utilization
is useful for changes suggestive of, but fall short of, a definite of ASCUS found that 86% of responding laboratories used the
interpretation of HSIL. The differential includes HSIL and term ASCUS and the median ASCUS rate was 2.8%, with 90%
mimics of HSIL. A variety of patterns can be recognized: of laboratories reporting rates of less than 9%. The median
1. Small cells with a high nuclear to cytoplasmic ratio or ASCUS/SIL ratio was 1.7; for 90% of laboratories, the calculated
“atypical (immature) metaplasia.” Nuclear abnormalities ratio was <3.6.38 In 2003, a follow-up CAP survey on Bethesda
such as abnormal shapes, hyperchromasia, and chromatin 2001 implementation and reporting rates showed a decrease in
irregularity favor HSIL over benign metaplasia. the average ASC/SIL ratio (from a median of 2.0 in 1996 to 1.4
2. Crowded sheet pattern or so-called hyperchromatic cell in 2002). This can be explained by increased LSIL detection on
groups. Dense cytoplasm, polygonal cell shape, and dis- LBP and also possibly by using Bethesda 2001 criteria more
tinct cell borders favor squamous over endocervical cells. stringently.27 In the 2006 CAP survey on practice patterns in
This cell pattern includes a broad differential from normal cervicovaginal cytology, there was an increase in LBP LSIL rates
(atrophy, endometrial cells, endocervical) to neoplastic when compared with 2003 data (2.5% vs. 2.1%). LBPs had
(endocervical adenocarcinoma, HSIL, or HSIL involving higher median SIL and ASC rates. Most ASC cases were subclas-
glands) changes. sified as undetermined significance (median, 4.3%). The median
3. Atypical cells in the setting of atrophy, atypia seen follow- ratio of ASC to SIL/carcinomas for all specimen types com-
ing radiation therapy, poorly preserved endometrial cells bined was 1.5, similar to the 2003 median ratio of 1.4.43
or histiocytes, and intrauterine device users may all show Sherman and colleagues, in a study correlating cytopatho-
cellular changes that are difficult to distinguish from logic diagnoses with detection of HPV DNA, also found that
HSIL. In such situations, a designation as ASC-H may be use of TBS criteria reduced the percentage of inconclusive
appropriate. “atypical” smears. Overall, a consistent relationship between
high-risk HPV detection and TBS diagnostic categories was
evident. High-risk HPV types were detected in 10% of negative
Laboratory Reporting of ASC smears, 30% of ASCUS, and 60% of SIL specimens. Based on
Subsequent to the publication and dissemination of TBS these data, the authors proposed using high-risk HPV testing
1988/1991, many clinicians felt overwhelmed by ASCUS inter- as an objective quality assurance measure to assess the perform-
pretations in their patient practices. This phenomenon was not ance of a cytopathology laboratory.44 These results were sub-
limited to the USA or to TBS; greatly increased rates of minor stantiated by ALTS.40,45
degrees of abnormality have been observed in countries that do It is well established that ASC-US is one of the least repro-
not use TBS.41 The reasons underlying this real or perceived ducible cytologic interpretations.10,46 Various quality assurance
ASCUS explosion were two-fold: (1) the constant specter of monitors may be utilized to evaluate the laboratory’s utilization
medical-legal litigation has lowered the threshold for diagnosis of ASC. These include the following:
of cellular abnormalities in many laboratories7,27 and (2) atypi- 1. Correlation of ASC-US cases with high-risk HPV positiv-
cal cases historically may have been camouflaged in vague terms ity rates; results from ALTS indicated that this should be
such as “inflammatory atypia,” “benign atypia,” “borderline in the range of 40–60%, or in essence that ASC-US is a
HPV,” and “koilocytotic atypia.” The aggregation of all such 50–50 proposition between SIL (usually LSIL) and cel-
equivocal cases under one heading highlighted the subjective, lular changes unrelated to HPV;47,48 however, later esti-
interpretative nature of cytopathologic diagnosis, something mates from CAP surveys showed the median high-risk
long understood by laboratorians but not always recognized by HPV reported by responding laboratories to be 36.6% in
clinicians. Some contend that, in TBS 1991, ASCUS merely ASC-US cases43
replaced the old Pap class 2 or “inflammatory atypia” designa- 2. Correlation of ASC cases with results of colposcopically
tions. However, a study by Sidawy and Tabbara demonstrated directed biopsy
that, by using criteria similar to those outlined above, almost 3. Review of ASC cases by a second cytopathologist
two-thirds of 88 smears previously interpreted as “inflamma- 4. Calculation of ASC/SIL ratio.
tory atypia” could be reclassified as reactive; only three out of Juskevicius and colleagues showed that measuring the ASC/
57 cases (5%) had CIN (all low grade) on follow-up colposcopic SIL ratio of individual cytopathologists and providing it to
biopsies. In contrast, among the smears that fulfilled ASCUS them periodically in a confidential manner is a useful quality
control measure.49,50 After implementation of LBPs, many labo- ASC-H on the other hand needs more aggressive follow-up,
ratories have reported an increase in SIL rates over the increase with colposcopic evaluation recommended at the first interpre-
in ASC rates, such that lower ASC/SIL ratios are being seen in tation, irrespective of an HPV co-test result. HPV testing is not
many laboratories.27 The prior 2–3 : 1 suggested ratio for ASC/ recommended for triage of ASC-H. However, if a CIN 2/3 lesion
SIL may undergo revision as future benchmarking results are is not identified at colposcopy, follow-up with either HPV
gathered. The ASC-HPV+/ASC ratio closely mirrors the ASC/ testing at 12 months or cytology testing at 6 and 12 months is
SIL ratio. However, the ASC-HPV+/ASC ratio offers the addi- recommended.7
tional advantage of identification of aberrant trends where ASC
and SIL are both being misinterpreted, which may allow ASC/ Squamous Intraepithelial Lesions (SIL)
SIL ratios to remain within “acceptable” ranges despite the erro- In TBS, LSIL and HSIL encompass the spectrum of precursors
neous trend.51,52 to squamous carcinoma of the cervix. Unlike CIN and dysplasia
classifications that maintain HPV as a separate diagnostic cat-
Clinical Management of ASC egory, low-grade SIL incorporates changes of HPV as well as
Among all the changes introduced by the implementation of mild dysplasia/CIN 1. High-grade SIL includes moderate
TBS terminology into practice, none was more controversial dysplasia/CIN 2, severe dysplasia/CIN 3, and carcinoma in situ/
than the category of ASCUS. At that time, the majority of CIN 3. Cytologists are of course free to append degrees of dys-
abnormal Pap tests reported annually in the USA, approxi- plasia or CIN classifications to a SIL interpretation.
mately 2.5 million, were interpreted as ASCUS and had highly
variable management at considerable cost to the healthcare Conceptual Basis for Two-tiered Terminology of SIL. Previ-
system. Another 1.2 million were interpreted as LSIL.45 In an ous terminology classifications – degrees of dysplasia and grades
effort to determine the best management strategy (effective as of CIN 1–3 – have emphasized the morphologic continuum of
well as cost-effective) for women with these equivocal and low- squamous lesions that was thought to reflect a continuous
grade abnormalities, the NCI sponsored the ASCUS/LSIL Triage process in the development of cervical cancer. Natural history
Study (ALTS), which was completed in 2001.40 ALTS allowed for studies39 and HPV research have since established that HPV
a data-driven approach to management of these prevalent cer- infection is a necessary cause for cervical carcinogenesis;59,60
vical cytologic abnormalities. In 2003, the USA adopted HPV however, even most oncogenic or high-risk HPV types cause
co-testing at 3-year intervals for screening women between 30 transient low-risk lesions that regress, and cervical carcinoma
and 65 years and, in 2012, this screening interval was extended develops in a small subset of persistent/progressive HPV infec-
to 5 years. Since then additional data from other clinical trials, tions.61 It is estimated that approximately 70% of cervical
especially that derived from approximately 1.4 million women cancers are associated with HPV 16 or 18.62
receiving care at Kaiser Permanente Northern California The two-tiered LSIL/HSIL Bethesda approach attempts to
(KPNC) was used to formulate the 2012 ASCCP management morphologically distinguish minor from significant lesions;
guidelines.7,53 however, morphology is an imperfect reflection of biologic
ASC-US is the most common cytologic abnormality, but it potential. Low-grade lesions, particularly those that persist, may
carries the lowest risk of CIN 3+, partly because one-third progress or be associated with the development of high-grade
to two-thirds are not HPV-associated and thus have a low lesions, and some high-grade lesions may regress.61 Some have
prevalence of invasive cancer, estimated at 0.1–0.2%.54 The questioned setting TBS division of LSIL and HSIL at the break-
prevalence of CIN 2/3 is substantially higher in women with point of CIN 1–2, or mild/moderate dysplasia, arguing that
ASC-H (37–40%)55,56 than in those with ASC-US (11.6%).54 some CIN 2/moderate dysplasias should be considered low-
ASC-US/high-risk HPV-positive cases over 2-year follow-up in grade lesions. However, because some CIN 2 lesions represent
ALTS have the same cumulative risk of CIN 2/3, about 27–28%, high-grade disease processes, conservatism dictated its inclu-
as a cytologic LSIL.47 In contrast, women in the ALTS who were sion into the more severe TBS category to ensure maximal
ASC-US/high-risk HPV negative showed a very low (1.4%) sensitivity of the process.
absolute risk of subsequently detected CIN 3 or worse and no More recently, a CAP-ASCCP co-sponsored consensus
cancers were detected in the 2-year study period, similar to effort led to the recommendation for use of a two-tiered
women with negative cytology in the absence of HPV terminology for the histopathology of HPV-associated lesions
testing.57,58 of the lower anogenital tract (the Lower Anogenital Squa-
The ASCCP 2001 consensus guidelines6 for ASC follow-up, mous Terminology (LAST) project). The reasons for this rec-
namely the use of reflex HPV testing, saw widespread penetra- ommendation was similar to TBS and included better
tion into US practice. In the 2012 ASCCP guidelines,7 HPV correlation with HPV biology and improved reproducibility,
DNA testing, repeat cytologic testing, and colposcopy are all especially with the recommended use of p16 immunohisto-
acceptable methods for managing ASC-US in women over 20 chemistry in equivocal cases of CIN 2.63 This recommenda-
years of age; however, when liquid-based cytology is used, reflex tion also allowed for the classification of like disease processes
oncogenic or high-risk HPV DNA testing is the preferred man- together – thus bringing together cytologic and histologic ter-
agement approach. ASC-US/high-risk HPV-positive women minology for the same entities. The World Health Organiza-
should be managed in a fashion similar to those with LSIL. In tion (WHO) is also expected to release a similar two-tier
adolescents (20 years and younger), follow-up with annual histopathologic terminology in its gynecologic atlas to be
cytology is suggested due to the high prevalence of HPV in this published in 2014.
age group and the low risk of persistence.7 Women who are
co-tested and found to be ASC-US/HPV negative should be Morphologic Features. An interpretation of LSIL based on
followed up at 3 years rather than a 5-year interval, since their cellular changes associated with HPV requires nuclear as well
risk is similar to that of a negative Pap alone.7 as cytoplasmic abnormalities. Nuclear changes may include
enlargement with hyperchromasia or pyknosis, and chromatin Management of SIL

smudging and wrinkling of nuclear contours. Cytoplasmic Low-Grade SIL. Based on natural history studies of HPV
changes consist of a well-defined perinuclear cavity, associated infection, it is clear that the majority of cytologically detected
with peripheral thickening of the cytoplasm or cytoplasmic LSIL regress within an average of 2 years.39 After implementa-
orangeophilia, and rounding of cellular contours. Specimens tion of liquid-based cervical cytology, there has been a steady
with subtle changes that fall short of definitive LSIL may be increase in the rate of LSIL in the USA – in 2006 the median
categorized as ASC-US. Cytoplasmic vacuolization (pseu- rate was 2.4%.27 Anecdotal experiences suggest that this has
dokoilocytosis) alone, in the absence of any nuclear atypia, is further increased with the use of location-guided screening.
considered a benign change and should not be classified as LSIL Initial colposcopy identifies prevalent CIN 2 or greater in 18%
or ASC-US. of women with LSIL; subsequent follow-up over 2 years identi-
Intraepithelial precursors of squamous cell carcinoma fied another approximately 10% CIN 2/3, irrespective of
present a spectrum of morphologic changes within which one whether the initial colposcopy was negative or showed histo-
is able, in most cases, to classify lesions as LSIL or HSIL; however, logic CIN 1.58,67
occasional “borderline” cases occur. In the CAP Interlaboratory The ASCUS-LSIL Triage Study showed that the natural
Comparison Program in Cervicovaginal Cytology (PAP), the history of LSIL approximates that of HPV-positive ASCUS,45,47
discrepant rate between low- and high-grade lesions ranged suggesting that women with either should be managed simi-
from 9.8% to 15% for cytotechnologist, pathologist, laboratory, larly. Analysis of the KPNC dataset confirmed that women
and all responses.64 Cytology and histology may also be discrep- with LSIL at ages 21–24 years carry a lower risk of CIN 3+ than
ant; 15–25% of women with LSIL cytology are found to have older women.68 In ALTS, the HPV positivity rate in LSIL was
histologic CIN 2/3 on further workup.48,65 Features that favor a 83%; a meta-analysis published in 2006 reported a 76.6% posi-
high-grade lesion include increased numbers of abnormal cells, tivity rate.61 Thus HPV DNA testing is not suggested for initial
higher nucleus to cytoplasmic ratios, greater irregularities in the triage of LSIL. However, when co-testing is performed in
outline of the nuclear envelope and nuclear chromatin distribu- women 30 years of age and older, some women will have HPV-
tion, and increased number of chromocenters. The appearance negative LSIL. In the KPNC cohort, the risk of CIN 3+ in
of the cytoplasm may also assist in determining whether a bor- HPV-negative women with LSIL was low, similar to that of
derline case is low- or high-grade SIL. LSIL changes typically ASC-US alone.53
involve “mature,” intermediate, or superficial type cytoplasm For women with LSIL cytology and no HPV test or with a
with well-defined polygonal borders. Cells of HSIL have a more positive HPV test, colposcopy is recommended. If co-testing
immature type of cytoplasm, either delicate and lacy or dense/ shows HPV-negative LSIL, repeat co-testing at 1 year is pre-
metaplastic, with rounded cell borders. In a minority of LSIL ferred, but colposcopy is acceptable. If repeat co-testing at 1
cases, there can be the presence of equivocal immature cells for year is elected, and if the cytology is ASC-US or worse or
which a high-grade lesion cannot be excluded. Some laborato- the HPV test is positive (i.e., if the co-testing result is other
ries have utilized the term LSIL-H (low-grade squamous than HPV negative, cytology negative), colposcopy is recom-
intraepithelial lesion – a high-grade process cannot be excluded) mended. If the co-testing result at 1 year is HPV negative and
to report such lesions and have studied the outcome. As one cytology negative, repeat co-testing after an additional 3 years
might expect, the follow-up percentage of HSIL on biopsy is is recommended.
intermediate to outright LSIL or HSIL cases.63 TBS does not If all tests are negative at that time, routine screening is
advocate the use of this terminology, although it may have a appropriate. In addition, the current management guidelines
place as a slightly increased risk category that could prompt also allow for HPV triage of postmenopausal women with LSIL.
more active surveillance in the future. Lesions previously A negative HPV test in this circumstance allows a deferral of
termed “pleomorphic dysplasia,” “keratinizing dysplasia,” or colposcopic examination with repeat cytology in 1 year. Details
“atypical condyloma,” which are composed of single cells or of recommendations for managing LSIL in adolescents, post-
clusters of cells with enlarged hyperchromatic nuclei and abun- menopausal, and pregnant women can be found in the 2012
dant but abnormally keratinized cytoplasm, are always consid- ASCCP management guidelines.7
ered HSIL. High-Grade SIL. The median percentile reporting rate of
HSIL in the USA is estimated at 0.5%,27 and approximately 2%
HSIL cannot Exclude Invasion. In rare cases of HSIL, invasive of women with HSIL cytology have invasive carcinoma.68
carcinoma may be difficult to exclude. Examples include atypi- Follow-up of cytologic HSIL carries a significant risk of a CIN
cal keratinized cells without diathesis/necrosis in the back- 2/3 – a single colposcopy identifies 53–66% of prevalent CIN
ground or cases in which the background is suspicious but 2/3; and CIN 2/3 is found in 84–97% of women who proceed
malignant cells are not seen.8 This terminology may be used in to a loop electrosurgical procedure (LEEP).64 Thus, both col-
such cases to communicate the increased concern to the poscopy and LEEP are acceptable for management of cytologic
clinician. HSIL.
Squamous Cell Carcinoma. Squamous cell carcinoma is EPITHELIAL CELL ABNORMALITIES:

defined as an invasive malignant tumor with squamous differ- GLANDULAR CELL
entiation. TBS does not subdivide squamous cell carcinoma
into keratinizing and non-keratinizing types, although the atlas Background
does discuss the morphology separately. On LBPs, tumor dia- Cervical cytology is primarily a screening test for cervical squa-
thesis may be more difficult to recognize; and as such, in the mous intraepithelial lesions and squamous cell carcinoma;
USA, a trend toward undercalling squamous cell carcinoma as cytology may have lower sensitivity for detection of glandular
HSIL, especially on LBPs, has been noted.66 lesions due to limitations in sampling and interpretation.
to the smooth communal border typical of glandular frag-

Reporting Glandular Cells in TBS 2001 ments. In LBPs, cells are more rounded and 3-dimensional. In
In TBS 2001, the term atypical glandular cells of undetermined this category, cellular changes, while suspicious for in situ or
significance (AGUS) has been eliminated to avoid confusion, invasive adenocarcinoma, are quantitatively or qualitatively
particularly among clinical staff, with ASC-US. Abnormal glan- insufficient for an outright interpretation as such.
dular cells should be subclassified when possible as endocervical
or endometrial; otherwise the generic term “atypical glandular Atypical Endometrial Cells. These are usually small groups of
cells” should be used. It is also advisable to use the qualifiers cells with slightly enlarged nuclei, and variable prominence of
“not otherwise specified” or “favor neoplastic” for endocervical nucleoli and nuclear hyperchromasia. Their distinction from
and glandular cells to convey the level of concern about any cytologically benign endometrial cells is based primarily on the
abnormality identified. The qualifier “favor reactive” from TBS criterion of increased nuclear size. When dealing with LBPs, it
1991 has been eliminated, as follow-up studies show that results is important to keep in mind that menstrual/shed endometrium
are similar to those in the NOS category, and, as such, this quali- is often well preserved and may show nuclear size and shape
fier provides no useful predictive value. Atypical endometrial pleomorphism and the presence of nucleoli. The differential of
cells are not further qualified due to difficulty in doing so and atypical endometrial cells is broad and may include endome-
lack of reproducibility of the morphologic criteria. Adenocar- trial polyps, endometritis, IUD-associated changes, hyperplasia,
cinoma in situ was established as a separate interpretive entity and carcinoma.
in TBS 2001, having been well described and shown to have
moderately good reproducibility since the 1991 TBS version.69 Endocervical Adenocarcinoma (In Situ and Invasive).
Endocervical AIS is a high-grade endocervical neoplastic lesion
Atypical Glandular Cells (AGC) that cytologically demonstrates nuclear enlargement, hyper-
As with its squamous ASC counterpart, this designation applies chromasia, stratification, and mitotic activity. Invasive carci-
to glandular cells that demonstrate changes beyond those noma overlaps cytologically with AIS, but may show features of
encountered in benign reactive processes, yet which are insuf- invasion, including prominent nucleoli and tumor diathesis.
ficient for an interpretation of in situ or invasive adenocarci- The possibility of a coexisting squamous lesion should always
noma. This interpretation should be further qualified, where be carefully assessed when a glandular lesion is detected, due to
possible, to indicate whether the cells are thought to be of the high rate of coexistence of SIL in cases with AIS.70–72
endocervical or endometrial origin. This category includes a
broad morphologic spectrum ranging from atypical-appearing, Endometrial Adenocarcinoma. The cytologic features are
reactive processes all the way to adenocarcinoma in situ (AIS). directly related to the histologic grade of the tumor, with well-
Therefore, lesions falling into this category should be further differentiated cases yielding malignant cells with minimal
subclassified, if possible, according to whether a neoplastic atypia and poorly differentiated tumors being obviously malig-
process is favored or the changes are nonspecific (NOS). Spe- nant. Tumor diathesis is often difficult to appreciate, particu-
cific comments may be added to the interpretation if pertinent larly in LBPs. In general, endometrial lesions yield fewer cells
clinical findings and/or history are available and relevant than do directly sampled endocervical lesions.
(polyps, IUD, etc.).
Extrauterine Adenocarcinoma. A clean background and
Atypical Endocervical Cells, NOS. Endocervical cells can show tumors whose cytologic features are not characteristic of
a variety of changes associated with benign/reactive processes uterine/cervical tumors should raise the possibility of metas-
in the endocervical canal. Reactive endocervical cells can show tasis. Diathesis is usually not seen unless there is direct exten-
some pleomorphism of cell size as well as nuclear enlargement, sion from the rectum or bladder with associated tissue
multinucleation, and prominent nucleoli; however, there is destruction.
usually a honeycomb or sheet-like pattern and nuclei remain
round and the chromatin bland. Such changes are usually rec- Diagnostic Difficulties
ognized as NILM and not included in the AGC category. Cells Criteria indicating invasion, tumor diathesis, and macronucle-
that show cytologic changes beyond those recognized easily as oli may be absent in the majority of well-differentiated, early
reactive, such as significant nuclear enlargement/crowding, adenocarcinomas. It also can be difficult to differentiate SIL
hyperchromasia, loss of mucin, and loss of polarity, should be with gland involvement from AIS. HSIL/CIS involving endocer-
considered for inclusion in the atypical endocervical cells, NOS vical glands may yield round cell clusters with smooth periph-
category. Such changes may be seen in conditions such as tubal eral contours showing group polarity and “columnar” shape of
metaplasia, radiation therapy, endocervical polyps, and micro- individual cells, thus mimicking a glandular abnormality.73
glandular hyperplasia, and in IUD users, but also in neoplastic Additionally, SIL and AIS may coexist in up to 50% of cases,
conditions in a small percentage of cases. This category there- and, at conization, a high proportion of AIS specimens demon-
fore includes changes that are in excess of those attributable to strate concurrent SIL.72,73
a reactive/reparative condition but which fall short of those seen Benign entities such as tubal metaplasia, directly sampled
in glandular neoplasia. lower uterine segment (LUS) endometrial cells, and cervical
endometriosis may all morphologically mimic AIS. Fragments
Atypical Endocervical Cells, Favor Neoplastic. These cells are of tubal metaplasia may demonstrate crowded sheets of glan-
characterized by cellular strips and rosettes demonstrating dular cells with enlarged nuclei as well as cell fragments with
elongated, overlapping nuclei with moderately coarse chroma- nuclear palisading and nuclear overlap, mimicking some of the
tin and hyperchromasia. The peripheral border of the glandular morphologic features of AIS.74 However, rosette formation is
clusters may be “feathered,” with protruding nuclei, in contrast uncommon in tubal metaplasia, and the nuclear chromatin
tends to be more finely granular. The most helpful findings with colposcopy acceptable either at the initial evaluation or
though, when present, are cytoplasmic terminal bars and deferred until the results of endometrial and endocervical sam-
cilia. pling are known.7
Directly sampled endometrial tissue may mimic AGC or
glandular neoplasia. Inadvertent sampling of the LUS may
occur because of closer approximation of the LUS to the cervi-
Educational Notes/Suggestions
cal os following cone biopsy75 or with aggressive use of endocer- The use of educational notes/comments is optional. If these are
vical brushes. In contrast to spontaneously exfoliated used by the pathologist/laboratory, it is suggested that they be
endometrial cells, which typically shed as tight ball-like clusters, concise, be phrased in the form of a suggestion, not a directive,
direct brushing of endometrial tissue yields large cellular frag- and be substantiated by published guidelines from professional
ments. These fragments often recapitulate their native organizations.5 Examples can be found in the second edition of
3-dimensional architecture with branching tubular glands the Bethesda Atlas.8
enmeshed in stroma composed of round to spindle-shaped
cells.76 Glandular cells show crowding with overlapping round
nuclei and scant cytoplasm. Peripheral palisading may be
Ancillary Testing
evident. The low-power recognition of branching glands and If ancillary testing, such as high-risk HPV, has been per-
glandular–stromal complexes is an important clue to avoid con- formed, whether the report is issued concurrently with the
fusion with AGC or glandular neoplasia. cervical cytology result or as an addendum/separate report
Conventional smears with a diagnosis of adenocarcinoma will depend on the laboratory’s information system, turn
consistently identified correctly by CAP interlaboratory glass around time for such testing, and clinical expectations. The
slide program participants were significantly more likely to have methodology utilized for the ancillary test should be specified.
more abnormal cells, larger abnormal cells, larger nuclei, Suggestions for reporting of molecular tests are provided in
marked atypia, and hyperchromasia than cases that performed the Bethesda Atlas.8
poorly.77 Glandular lesions have a slightly different morphology The 2006 and 2012 ASCCP guidelines provide guidance on
on LBPs; specifically the cells may be flatter, feathering less HPV genotyping, and, if performed, these results should also
prominent, and diathesis more difficult to appreciate.8 Details be reported with reference to, or in conjunction with, the cervi-
are discussed elsewhere in this book. However, as for squamous cal cytology result.
lesions, there have been reports showing increased detection of
glandular abnormalities on LBPs compared with conventional
smears.72,78,79
Automated Review
For cervical cytology preparations that undergo computer-only
Management or computer-assisted review, the type of instrument used and
An AGC interpretation is poorly reproducible80 and un any result should be included in the report. In addition, if there
common.81 estimated to be reported in only 0.2% of cervical was no “human” review of the slide, this should be made clear
cytology tests in the USA.27 While AGC may be associated in the report. Details of various automated devices used for
with benign and reactive conditions such as endocervical/ screening Pap tests are discussed elsewhere in this book.
endometrial polyps, it is clear from several studies that AGC is
a “high-risk” interpretation compared with ASC; the reported Interobserver Reproducibility
rate of neoplasia in follow-up of AGC ranges from 9% to
38%.71,72,80,81
in Cervical Cytology
As has been well documented, follow-up of AGC is more In an effort to improve standardization, clarity, and reproduc-
often associated with squamous lesions than glandular lesions. ibility of cervical cytology reporting, the second edition of the
HPV is associated with most endocervical, but not endometrial, Bethesda Atlas8 emphasized more detailed morphologic criteria
cancers, and as such a negative HPV test can be useful in iden- and had many more images, which were complemented by
tifying women at greater risk for endometrial rather than cervi- additional images on the Bethesda website.9 In addition, as part
cal disease.77 There are rare types of endocervical adenocarcinoma of the ASC-NCI Bethesda Project, a web-based interobserver
(gastric, minimal deviation) that are HPV negative, and are reproducibility study was designed to gauge cervical cytology
therefore also not ruled out in the presence of a negative reproducibility prior to publication of the atlas and website. A
HPV test. range of classic and borderline images74 is included for inter-
For women with all subcategories of AGC and AIS except pretation; approximately 651 cytotechnologists and patholo-
atypical endometrial cells, colposcopy with endocervical sam- gists worldwide participated in the study. It was apparent from
pling is recommended regardless of HPV result; triage by reflex the results that the morphology presented was more important
HPV testing is not recommended, and triage using repeat cervi- in classifying images correctly than were professional or aca-
cal cytology is unacceptable. HPV testing, if obtained, can be demic degrees, or other variables assessed. In this study, exact
useful in the downstream management following the initial agreement with the TBS panel was relatively low (57%),
colposcopy. Endometrial sampling is recommended in con- although agreement was 84.1% at the threshold of distinguish-
junction with colposcopy and endocervical sampling for women ing NILM from non-negative. Participants achieved a higher
35 years of age and older with all subcategories of AGC and AIS sensitivity for correctly classifying high-grade squamous lesions
and also for women younger than 35 years, with clinical indica- than that for high-grade glandular lesions. The details of this
tions suggesting they may be at risk for endometrial neoplasia. study have been published10 and all the images and associated
For women with atypical endometrial cells, initial evaluation histograms of participants’ responses are available for review on
limited to endometrial and endocervical sampling is preferred, the Bethesda website.9
The Bethesda System and Reporting and, more recently, dual testing, which combines cytology
Anal–Rectal Cytology screening with HPV testing. HPV vaccines entered the market
in 2006, and are likely to further decrease the incidence of inva-
Anal cancer is considered an appropriate target for cytologic sive squamous cell carcinoma of the cervix and its precursor
screening in selected high-risk populations. The anatomic com- lesions. Cervical cancer screening guidelines have undergone
monality of the anal–rectal canal and the cervical mucosa is significant changes after implementation of LBPs and HPV
reflected in that both have a transformation zone. HPV is a testing and with advances in the understanding of cervical neo-
common risk/etiologic factor for cancers of the anus and cervix plasia.84 It is predicted that the number of cervical cytology tests
and subsequently the morphology of cytology samples from performed will decrease significantly in the future if there is
both sites is comparable. It follows that sampling devices, prep- compliance with these guidelines.85
aration techniques, and morphologic interpretation using TBS TBS has met the goals that were conceived at the time of its
terminology utilized for cervical cytology can readily be applied implementation in 1988. It has seen successful penetration into
for anal–rectal cytology screening. cervical cytology reporting worldwide, allowing laboratories to
Adequacy criteria for anal–rectal cytology are based, at use consistent terminology in conveying results to clinicians
present, on limited personal experiences. As a guide, minimum and thus enabling comparison of studies across many countries
adequacy cellularity should be in the range of 2000–3000 nucle- and healthcare systems. The use of the Bethesda ASC-US ter-
ated squamous cells for conventional smears and for LBP minology prompted the NCI-sponsored ALTS trial, the results
samples 1–2 nucleated cells/high-power field for ThinPrep of which have significantly impacted the management of equiv-
(20 mm diameter) preparations and 3–6 nucleated squamous ocal and low-grade cervical cytologic abnormalities. The
cells/high-power field for SurePath (13 mm diameter) Bethesda terminology has been updated twice – in 1991 and
preparations.8 2001 – since its inception in 1988 in order to keep pace with
Normal elements seen in anal–rectal specimens include the advances in our understanding of cervical cancer and evolv-
nucleated, anucleate, and metaplastic squamous cells, rectal ing technologies in cervical cancer screening and prevention.
columnar cells, fecal matter, and mucus. A comment should be TBS also provided the basis for the ASCCP to develop con
included in the report about the presence of a transformation sensus guidelines for management of cervical cytologic abnor-
zone component. Cytomorphologic criteria are quite similar to malities as defined by TBS. Continuous quality assurance for
those utilized for cervical cytologic interpretation; however, gynecologic cytology86 is aided by having a consistently used
there is a higher incidence of poor preservation, cellular degen- reporting system such as Bethesda.
eration, and cytoplasmic keratinization/parakeratosis, and In 2012, a two-tier terminology (LSIL/HSIL) has also been
classic koilocytes are less frequently identified.8,82 When target- recommended for reporting the histopathology of HPV-
ing high-risk groups, the rate of epithelial abnormalities noted associated lesions of the lower anogenital tract. The reasons
is far higher than that reported for cervical cytology.82,83 Reports for this recommendation were similar to those of TBS for
from the USA suggest that anal–rectal cytology screening is cervical cytology, including improved reproducibility, better
sensitive but has low specificity for predicting the grade of the communication with clinicians and colleagues, better under-
lesion, with a tendency to under-represent the grade of squa- standing of HPV biology, and harmonization of nomenclature
mous abnormality. While it has been shown that screening for like entities across multiple specimen types.63
high-risk patients by cytology is effective, the present impedi- TBS has also served as a model for development of addi-
ments to the success of early detection of anal cancer by this tional standardized reporting systems such as for thyroid cytol-
method include limited clinical expertise and means for the ogy.87 The process of re-evaluation and revision for reporting
subsequent treatment/follow-up of these patients, and the high cervical cytology will continue in the future in order to provide
risk of complications associated with excisional procedures at the most accurate, reproducible, and relevant terminology. A
this site. third edition of the TBS manual is currently underway with a
planned release in 2015. Optimal communication and ulti-
mately patient care outcomes will therefore ensue from these
Concluding Remarks efforts.
Cervical cytology has seen many changes since its introduction
in the 1960s – liquid-based sampling techniques, automated Access the references list online at http://www.expert
preparation, computer-assisted screening, HPV DNA testing, consult.com
6 The Bethesda System for Reporting Cervical Cytology 81.e1
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Chapter 6 The Bethesda System For Reporting Cervical Cytology

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Chapter 6 The Bethesda System For Reporting Cervical Cytology

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6

The Bethesda System for Reporting

CONTENTS develop a uniform descriptive terminology for cervical/vaginal

THE 2001 BETHESDA SYSTEM

BOX 6-1 THE 2001 BETHESDA SYSTEM

adequacy – satisfactory, less than optimal, and unsatisfactory SQUAMOUS CELLULARITY

ENDOMETRIAL CELLS EPITHELIAL CELL ABNORMALITIES:

enlargement with hyperchromasia or pyknosis, and chromatin Management of SIL

Squamous Cell Carcinoma. Squamous cell carcinoma is EPITHELIAL CELL ABNORMALITIES:

to the smooth communal border typical of glandular frag-

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