CE UPDATE—IMMUNOLOGY IV

Antony C. Bakke, PhD

Clinical Applications
of Flow Cytometry

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During the last 20 years, flow cytometry has
become a routine clinical method in many labora-
tories.1 This article discusses current clinical uses
of flow cytometry, emphasizing immunopheno-
typing, reticulocyte and reticulated platelet enu-
meration, multiple drug resistance assays, cell
function assays, and apoptosis. A partial list of the
standard clinical instruments and additional
instruments with features of automated cytome-
try is given in the Table. Recent advances are
briefly discussed; advanced research uses are not.
All of the assays described are used by clinical lab-
oratories for patient care.
ABSTRACT Flow cytometry is an important analytical tool
that has moved from research to the clinical laboratory in the
past 20 years. Although still important in research, many
applications have become routine clinical tests required for
diagnosis, estimation of prognosis, and monitoring of
treatment. In addition, new applications, such as minimal
residual disease detection and multiple-drug resistance
monitoring, are being evaluated for clinical testing.
This is the final article in a 4-part continuing education series on immunology. On
completion of this article, the reader will be able to describe current clinical uses of flow
cytometry, identify some of the monoclonal antibodies used in immunophenotyping,
and identify areas for future clinical applications of this technology.

DNA Analysis

Scientific Communications
Laser-equipped flow cytometers were originally flow cytometry. Immunophenotyping, or charac-
developed to provide a rapid screening method forterization of cell subpopulations based on differen-
From the Department
cervical specimens using a method to measure tiation antigens, is probably the most well of Pathology, and
altered DNA content. The instrument needed to recognized area in flow cytometry. These marker Immunology and
antigens are detected with antibodies and either
precisely analyze both intrinsic and extrinsic cellu- Flow Cytometry
flow cytometry, fluorescence microscopy, or
lar features on a cell by cell basis. Intrinsic features Laboratory, Oregon
Health Sciences
included size, granularity, and autofluorescence.immunohistochemistry. Immunophenotyping is
University,
Extrinsic measurements required the addition of aused clinically for diagnosis and subclassification Portland, OR.
of leukemia and lymphoma, diagnosis of primary
fluorescent probe to detect the feature of interest. Reprint requests to
For DNA content, this probe was a stoichiometric immunodeficiency disorders, enumeration of stem Dr Bakke, Oregon

4
stain for DNA. Both determination of ploidy and cells in peripheral blood, diagnosis of paroxysmal Health Sciences

Section
nocturnal hemoglobinuria (PNH), monitoring of
analysis of cell division kinetics (eg, S-phase frac- University,
tion, doubling time, mitotic index) have impor- immune status in patients with HIV infection, Department of
Pathology–L471,
tant implications in areas such as clinical tumormonitoring of immunosuppressive therapy with 3181 SW Sam
prognosis.2 Although the goal of detecting cells OKT3 and other monoclonal antibodies, T-cell Jackson Park Rd,
with abnormal DNA content was reached, the fact cross-matching for transplantation, monitoring Portland, OR 97201.
for posttransplantation rejection episodes, detec-
that many cancers, especially in early stages of dis-
tion of minimal residual disease in cancer, and tis-
ease, do not have significant, quantitative changes
in DNA content has limited use of flow cytometry sue typing for HLA-B27. All of these procedures
for this purpose.3 require the use of 1 or more antibodies (usually
monoclonal) to detect the antigens.
Immunophenotyping Antibodies are carefully standardized by a
Developments in immunology and other areas of series of international leukocyte differentiation
cancer research have greatly expanded the use of workshops and are given CD (cluster of differenti-
ation) designations to unify terminology. The CD

F E B R U A RY 2 0 0 0 VO L U M E 3 1 , N U M B E R 2 L A B O R ATO RY M E D I C I N E 97
 Instruments Used for Flow Cytometry
Instrument Manufacturer
FACS Calibur Becton Dickinson, San Jose, CA
XL Beckman-Coulter, Miami, FL
PAS Partec, Munster, Germany
BRYTE HS BioRad, Hercules, CA
IMAGN 2000 Biometric Imaging &
Becton Dickinson, San Jose, CA
LaserScanning Cytometer CompuCyte, Cambridge, MA
CellDyn 4000 Abbott, North Chicago, IL
number can refer to both the antibody and the
antigen. Therefore, FITC-CD3 indicates a mono-
clonal antibody against the CD3 antigen, which is
conjugated to fluorescein isothiocyanate (FITC) to
make it fluoresce green when excited with blue
light. On the other hand, CD3+ T lymphocytes are
the subpopulation of lymphocytes bearing the
CD3 antigen, which is positive when stained with
FITC-CD3.
The latest advances in flow cytometry involve
multiple-color analysis, enabling evaluation of
coexpression of several markers while reducing
the total amounts of antibody and specimen
required for analysis. With the availability of mul-
tiple-color analysis, strategies have evolved that
enable more refined gating of populations, such as
CD45 gating for all WBCs or CD19 gating for B
lymphocytes. This type of gating has enhanced or
replaced the traditional scatter gating (Fig 1), with
applications in immunodeficiency disorders, such
as HIV infection,4 and hematologic neoplasia.5 In
HIV infection, enumeration of CD4+ T cells has
become a standard surrogate for estimating stage
of disease. Of importance, a CD4 count <200
cells/mL together with a positive antibody test for
HIV is diagnostic of AIDS, enabling proper diag-
nosis and treatment in many patients.6 An addi-
tional method of assessing stage of disease for HIV
and response to therapy has been developed using
quantitative expression of CD38 on CD8+ T cells.
This evaluation is not yet used widely in clinical
laboratories, but may be in the future because it
98 L A B O R ATO RY M E D I C I N E VO L U M E 3 1 , N U M B E R 2 F E B R U A RY 2 0 0 0
Uses
Immunophenotyping, DNA, reticulocytes,
platelets, multiple drug resistance, apoptosis,
cell function
Same as above
Same as above
DNA, bacterial drug sensitivity, reticulocytes,
platelets, some immunophenotyping
CD4, CD8, CD34, residual WBCs, platelet
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activation
Immunophenotyping, DNA, reticulocytes,
platelets, multiple drug resistance, apoptosis
Immunophenotyping for CD3, CD4, CD8,
CD34, CD61
gives additional prognostic information indepen-
dent of viral load by polymerase chain reaction
(PCR) or CD4 T-cell count.7,8
Immunophenotyping is required for accurate
diagnosis of primary immune deficiency disorders.
Many have characteristic defects that are easily
detected.9 For example, X-linked (Bruton’s) agam-
maglobulinemia is due to a mutation in a specific
tyrosine kinase gene required for B-lymphocyte
maturation. Patients completely lack B lympho-
cytes in the peripheral blood, which can be shown
by lack of staining with CD19 antibodies. Another
example, hyperimmunoglobulinemia M syn-
drome, is due to a mutation in the CD40 ligand. It
is required for immunoglobulin isotype switching,
but is lacking on stimulated T lymphocytes.
In the area of hematologic neoplasia, immuno-
phenotyping is considered the standard of med-
ical care. Hematopoietic malignancies represent
specific stages of differentiation and may be lym-
phoid, myeloid, or biphenotypic. Routine mor-
phology and cytochemistry are absolutely
necessary for diagnosis but cannot differentiate
many subtypes of these diseases, such as pre-B-cell
acute lymphoblastic leukemia. Each subtype
expresses different biologic behavior, as reflected
in different prognoses and effective treatments.
Recent advances have added intracellular
staining for antigens such as CD3, CD19, CD79a,
and myeloperoxidase, enabling identification of
the earliest stages of lymphoid and myeloid dif-
ferentiation.10 Exact classification cannot be
done without immunophenotyping, either with
 Fig 1. Multicolor
A B staining of a lymph
104 104 node fine-needle
aspirate with anti-
R4 (CD 19+)
kappa-fluorescein
isothiocyanate
103 103 (FITC), anti-lambda-
phycoerythrin (PE),
and CD19-peridinin
CD 19-PerCP

Lambda-PE
chlorophyll protein
102 (PerCP). Cells are
102
stained, washed, and
analyzed. FITC
fluoresces green, PE
101 is yellow, and PerCP

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101
(alternately, PE-Cy5)
is orange. A, Dot plot
of side, or 90-degree,
100 light scatter on the
100 100 101 102 103 104
0 200 400 600 800 1,000 X-axis vs CD19-
Kappa-FITC
SSC Height PerCP staining on
the Y-axis. CD19-
positive, low side
scatter cells (SSC)
flow cytometry or immunohistochemistry, which cytometry. Lack of CD55 and CD59 confers sensi- are gated in region
are complementary techniques. With either tivity to activated complement on the cells. This R4. B, Kappa and
method, fewer than 50,000 cells from difficult defect begins in a hematopoietic clone of cells in lambda staining of
the CD19-gated cells
specimens, such as fine-needle aspirate, cere- the bone marrow. In any patient, the percentage of in A. Monoclonal,
brospinal fluid, or vitreous fluid, can be stained PNH cells may vary from 1% to 100%. Of impor- kappa-positive,
and analyzed for B-lymphocyte monoclonality. A tance, patients with PNH may have near normal presumptive
typical example of flow cytometry is shown in expression on RBCs, due to the shortened life span neoplastic, B-cell
Figure 1. Flow cytometry has the advantage of of PNH RBCs or to transfusions. In addition, population is
present.
speed and simpler methods, but lacks the ability PNH cells may be either completely negative (type
to visualize malignant cells. III) or bear decreased amounts of these GPI-
Many solid tumors are currently being treated anchored proteins (type II). Inasmuch as the

with marrow ablative chemotherapy followed by
either autologous or allogeneic stem cell trans-
plantation. Colony-stimulating factors induce
production and release of more stem cells into the
blood, where they are collected along with other
WBCs. However, the timing of release of these
cells from the bone marrow varies among
patients. Since the peripheral WBC is an unreli-
able surrogate, stem cells in the blood must be
enumerated with CD34 staining.11 Hematopoi-
Scientific Communications
abnormal PNH clone usually increases over time,
repeated testing may be necessary.
Other flow cytometry assays involving
immunophenotyping include monitoring of
OKT3 antibody immunosuppression and tissue
typing for HLA-B27, which is important in diag-
nosing spondylarthritides. OKT3 monoclonal
antibody is directed against the CD3 antigen on T
lymphocytes. After binding to CD3, the antibody
induces T-lymphocyte activation and removal of

etic stem cell evaluation requires detection of
small numbers of cells (1 in 1,000) and often
needs to be done in real time to avert unnecessary
and expensive additional collections.
Defects in the production of normal
hematopoietic cells by the bone marrow can also
be detected with flow cytometry. For example,
patients with PNH lack specific glycosylphos-
phatidylinositol (GPI)–anchored proteins on
hematopoietic cells, because of a defect in the
PIGA (phosphatidylinositol glycan A gene).12
Many GPI-anchored proteins (eg, CD14, CD16,
CD55, CD59, CD66b) are easily detected with flow
4
CD3 plus the T-lymphocyte antigen receptor from
Section
the cell surface. As a result, the T lymphocyte is
effectively “blind” and cannot identify and attack
any target (ie, transplanted tissues), resulting in
immunosuppression. Monitoring effective
removal of CD3 from the T lymphocyte is used to
determine effective treatment.13
RBCs and Platelets
RBCs and platelets can be evaluated with flow
cytometry. Reticulocytes can easily be measured
using a stain for the residual RNA in these cells.

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 Similar to the methylene blue staining method, a
percentage of reticulocytes is measured and used
to calculate the absolute number. In addition, flow
cytometry provides a measure of reticulocyte
maturity, the immature reticulocyte fraction (pre-
viously termed the reticulocyte maturity index).14
An analogous measurement for platelets pro-
vides an estimate of young, “reticulated” platelets
by counting platelets that stain with an RNA dye
(eg, thiazole orange, coriphosphine-O). The
platelets in whole blood are also labeled with phy-
coerythrin-conjugated CD41 antibody to differ-
entiate them from other small particles. The RBCs
in the specimen are used as an internal negative
control for staining intensity, and the CD41-posi-
tive platelets are evaluated for RNA content (Fig
2).15 An increase in the number of reticulated
platelets indicates increased bone marrow produc-
tion of platelets and is consistent with a diagnosis
of idiopathic thrombocytopenic purpura.
Cell Function
Cell function assays are performed by only a few
reference laboratories, but can be helpful in cer-
tain patients. Perhaps the most clinically relevant
assays of cell function are those for oxidative burst
and phagocytosis by neutrophils. Defects are char-
acteristic of chronic granulomatous disease. Flow
cytometric assays are simpler and easier to per-
form than complex bactericidal assays. In the case
of oxidative burst assays, the neutrophils are
loaded with a nonfluorescent dye that becomes
fluorescent when it is oxidized by stimulated
cells.16 This is analogous to the nitroblue tetra-
zolium test. For phagocytosis, fluorescence-tagged
bacteria can be incubated with viable neutrophils,
and internalization measured.
Other functional assays for lymphocytes include
proliferation and stimulation by mitogens and the
natural killer cytotoxicity assay. Proliferation is
measured using fluorescent antibodies that detect
bromodeoxyuridine incorporation, analogous to
tritiated thymidine incorporation. Cytotoxicity
assays use target cells, which are viably labeled with
a fluorescent marker. Once they are killed, the tar-
get cells lose their fluorescence and are no longer
counted in the assay. Percent cytotoxicity is calcu-
lated by comparing the remaining viable targets
with a control lacking natural killer cells.
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Future Clinical Uses
Often patients with idiopathic thrombocytopenic
purpura have antiplatelet antibodies that can be
measured with flow cytometry. Previous studies
showed that platelet-associated IgG could be of
some use clinically, but specificity has been low,
primarily because of nonspecific binding of IgG to
Fc and complement receptors on platelets. Gating
of platelets with flow cytometry and determination
of the specific site of antibody binding with fluo-
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rescence resonance energy transfer has increased
the specificity of the assay by eliminating the con-
tribution of non-specifically–bound IgG.17
Platelet hyperactivity and circulating activated
platelets are associated with unstable angina, acute
myocardial infarction, cardiovascular accident,
cardiopulmonary bypass, diabetes mellitus, emo-
tional stress, and blood bank storage of platelets,
among other conditions.18 Several assays have
been developed to measure either activated
platelets or platelet microparticles using anti-
CD62 and anti-CD41, and reagents that detect
externalized phosphatidylserine on platelets. In
addition, non–flow cytometric assays are avail-
able. Because of the importance of platelet activity
in many clinical conditions, some assay of platelet
activation will eventually be widely utilized.
The role of flow cytometry as an aid in deter-
mining prognosis in patients with cancer has
become increasingly important with the advent of
assays for detection of minimal residual disease
and assessment of multiple drug resistance.
Although not so sensitive as PCR methods,
observing phenotypic markers is often the only
method for determining the presence of minimal
residual disease without known or consistent
genetic aberrations. Although the methods are dif-
ficult to establish, a number of laboratories have
reported that it is possible to routinely identify 1
malignant cell among 10,000 normal cells with
flow cytometry.19 In some studies, this level of
detection was clinically relevant, whereas the more
sensitive PCR did not predict clinical response.20
Drug resistance is a principal reason for failure
of cancer response to chemotherapy. Multiple drug
resistance is mediated by several mechanisms in
cancer cells, including the p-glycoprotein (MDR1)
and other pumps (eg, lung resistance protein
[LRP], multiple drug resistance–related protein
[MRP]), the glutathione transferase antioxidant
system, increased resistance to apoptosis due to
 A B
104 400

320

103
RBC-G2 240

Counts
Platelets-G1
FSC-H

Marker at inflection
160
102

80

101

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0
100 101 102 103 104
FL1-H
100
100 101 102 103 104
FL2-H

C
Fig 2. Whole blood is stained with thiazole orange (RNA 50
stain) and CD41-phycoerythrin (PE) antibody. The blood
is diluted and analyzed, without washing, on the flow
cytometer. A, Dot plot of log forward scatter (FSC) on 40

the Y-axis vs PE-CD41 (FL2) staining. Medium forward

scatter, CD41-positive platelets (G1), and high forward 30
Counts

scatter, CD41-negative RBCs (G2) are gated. B, Thiazole

orange staining (FL1) of gated RBCs. Reticulocytes and
WBCs appear positive. A marker is set at the inflection 20

point on the curve and is used to calculate a platelet

setting. C, Thiazole orange staining (FL1) of gated 10
platelets. Based on biochemical studies of RNA content
in reticulocytes and reticulated platelets, the marker is
set at 3 times the value from the reticulocytes in B. This 0
100 101 102 103 104

Scientific Communications
is a normal individual with <1% reticulated platelets. D,
FL1-H
Thiazole orange staining (FL1) of gated platelets from a
patient with idiopathic thrombocytopenic purpura. Fifty
percent of the platelets contain sufficient RNA to be
considered young platelets.
D

50
bcl-2 overexpression, p53 mutations, and other
factors. Since MDR1 inhibitors are available for
clinical use, they have been a natural candidate for 40

study. Accumulation or efflux of several fluores-

4
cent dyes or antibody staining for MDR1 (p-glyco- 30
Counts

Section
protein) has been used to measure the level of
MDR1. Both measurements correlate with clinical 20

outcome in acute myeloid leukemia.21

Analysis of apoptosis, or programmed cell 10
death, is a major research application of flow
cytometry and may become a new clinical applica- 0
100 101 102 103 104
tion for determining the efficacy of chemotherapy
FL1-H
in patients with leukemia and other types of malig-
nancies.22 During this important biologic process,
there are changes in the plasma membrane and the
mitochondria, a cascade of intracellular proteases

F E B R U A RY 2 0 0 0 VO L U M E 3 1 , N U M B E R 2 L A B O R ATO RY M E D I C I N E 101
 104

Necrotic

Consensus Documents, Laboratory

103 Standards, and Regulations
Propidium lodide

The complexity of multiparameter flow cytome-

try and its use in clinical medicine necessitate
standards and regulation for flow cytometry to
102
achieve interlaboratory reproducibility and
ensure quality patient care. Several groups
Viable
around the world have met to establish consen-
101 Apoptotic sus protocols and guidelines for current methods

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in flow cytometry, including proficiency testing,
accreditation, and training.23
100 0
10 101 102 103 104 Conclusion
Annexin-V–FITC
Flow cytometry is a powerful tool with many appli-
cations in pathology. Although it is currently used
Fig 3. T-cell line (Jurkat) is cultured with camptothecin for 2 hours to
for many standard tests, new applications are con-
induce apoptosis. Cells are then stained with fluorescein isothiocyanate tinually being developed, including assays for min-
(FITC)–annexin-V and propidium iodide. Viable cells are negative for both imal residual disease detection and multiple drug
stains, cells that have recently entered the apoptosis cascade are positive resistance assessment. As a tool, flow cytometry
for annexin only (apoptotic), and cells that are late in apoptosis and have
does not stand alone but enhances other methods,
permeable membranes are positive for both annexin and propidium
iodide (necrotic).
from morphologic to molecular analysis.l

(caspases) are activated, and, finally, cellular DNA References

becomes fragmented. Apoptosis is different from 1. McCoy JP Jr, Keren DF. Current practices in clinical flow
cytometry. Am J Clin Pathol. 1999;111:161–168.
necrosis. Morphologically, apoptosis is character- 2. Hedley DW, Clark GM, Cornelisse CJ, et al. Consensus
ized by cell shrinkage, chromatin condensation, review of the clinical utility of DNA cytometry in carcinoma of
and formation of apoptotic bodies, which are the breast. Cytometry. 1993;14:482–485.
3. Koss LG, Czerniak B, Herz F, et al. Flow cytometric mea-
phagocytized, inducing little inflammation. Con- surements of DNA and other cell components in human
versely, necrosis is characterized by cellular tumors: a critical appraisal. Human Pathol. 1989;20:528–548.
swelling, is induced by hypoxia and toxins, and 4. Mandy FF, Bergeron M, Minkus T. Evolution of leukocyte
immunophenotyping as influenced by the HIV/AIDS pan-
results in extensive inflammation. demic: a short history of the development of gating strategies
Cellular changes characteristic of apoptosis or for CD4+ T-cell enumeration. Cytometry. 1997;30:157–165.
necrosis can be measured with flow cytometric 5. Lacombe F, Durrieu F, Dumain P, et al. Flow cytometry
CD45 gating for immunophenotyping of acute myeloid
techniques such as annexin-V binding to phos- leukemia. Leukemia. 1997;11:1878–1886.
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MMWR. 1992;41:119.
enzyme-based assays of the active caspases. Newly 7. Giorgi JV, Hultin LE, McKeating JA, et al. Shorter survival
apoptotic cells are positive for annexin-V but not in advanced human immunodeficiency virus type 1 infection is
propidium iodide (Fig 3). In cells that are farther more closely associated with T lymphocyte activation than
with plasma virus burden or virus chemokine coreceptor
in apoptosis or have died by necrosis, holes usage. J Infect Dis. 1999;179:859–870.
develop in the plasma membrane and become 8. Burgisser P, Hammann C, Kaufman D, et al. Expression of
positive for propidium iodide, which enters and CD28 and CD38 by CD8+ T lymphocytes in HIV-1 infection
correlates with markers of disease severity and changes towards
stains DNA. Similar results can be obtained with normalization under treatment: the Swiss HIV cohort study.
patient specimens, although apoptotic cells exist Clin Exp Immunol. 1999;115:458–463.
for only a short time in the body before being 9. Ten RM. Primary immunodeficiencies. Mayo Clin Proc.
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102 L A B O R ATO RY M E D I C I N E VO L U M E 3 1 , N U M B E R 2 F E B R U A RY 2 0 0 0
 Internet
Resources
Here are some Internet sites that
offer more information on topics
discussed in this issue of Laboratory Medicine.
Coagulation
“Coagulation Diseases” is available on the
Community Outreach Health Information System
(COHIS) Web site. COHIS was developed by Boston
University to provide information that is easy to read
and understand to students, children, and educators.
http://www.bu.edu/cohis/cardvasc/blood/coag.htm
“Urbana Atlas of Pathology,” available on the
University of Illinois College of Medicine at Urbana-
Champaign Web site, contains pathologic images
due to disseminated intravascular coagulation.
http://www.med.uiuc.edu/PathAtlasf/CVAtlas039.html
http://www.med.uiuc.edu/PathAtlasf/Atlas101.html
http://www.med.uiuc.edu/PathAtlasf/Atlas102.html
Diabetes
“Conquering Diabetes: A Strategic Plan for the 21st
Century (1999; NIH Publication No. 99-4398),” a report
of the Congressionally-established Diabetes Research
Working Group, National Institutes of Health (NIH), is
available on the National Institute of Diabetes and
Digestive and Kidney Diseases, NIH Web site
http://www.ep.niddk.nih.gov/dwg/fr.pdf
“Diabetes Info” is available on the American
Diabetes Association Web site
http://www.diabetes.org/ada/diabetesinfo.asp
Flow Cytometry
The Clinical Cytometry Society Web site includes a
directory of individuals working in the field of cytom-
etry and a vendor directory, in addition to numerous
links to related journals, organizations, and products.
http://www.cytometry.org
Consensus documents and guidelines are available
on the CytoRelay (Martinsried, Germany) Home page
http://www.biochem.mpg.de/research-groups/
valet/cytorel.html
Purdue University Cytometry Laboratories (West
Lafayette, IN) Web site
http://flowcyt.cyto.purdue.edu
The tutorial “Conjugation of Monoclonal Antibodies”
by Mario Roederer, PhD, is available on his Home page
http://www.drmr.com/abcon/index.html
“Which Statistic When? A tutorial on the statistics
commonly used when analyzing flow cytometry
data” by Geoffrey Osborne is available on the John
Curtin School of Medical Research (Canberra City,
Australia) Web site.
http://jcsmr.anu.edu.au/facslab/statistics.html
These sites were accessed December 21, 1999, and
are offered for reader information only. A site’s
presence on this list does not constitute an
endorsement by the ASCP.
13. Henell KR, Bakke A, Kenny TA, et al. Degree of modula-
tion of cell-surface CD3 by anti-lymphocyte therapies. Trans-
plant Proc. 1991;23:1070–1071.
14. Davis BH, Bigelow NC. Reticulocyte analysis and reticu-
locyte maturity index. Methods Cell Biol. 1994;42:263–273.
15. Bonan JL, Rinder HM, Smith BR. Determination of the
percentage of thiazole orange (TO)–positive, “reticulated”
platelets using autologus erythrocyte TO fluorescence as an
internal standard. Cytometry. 1993;14:690–694.
16. O’Gorman MRG, Corrochano V. Rapid whole-blood flow
cytometry assay for diagnosis of chronic granulomatous dis-
ease. Clin Diagn Lab Immunol. 1995;2:227–232.
17. Koksch M, Rothe G, Kiefel V, et al. Fluorescence reso-
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nance energy transfer as a new method for the epitope-specific
characterization of anti-platelet antibodies. J Immunol Meth-
ods. 1995;187:53–67.
18. Michelson AD. Flow cytometry: a clinical test of platelet
function. Blood. 1996;12:4925–4936.
19. Coustan-Smith E, Behm FG, Sanchez J, et al. Immuno-
logical detection of minimal residual disease in children with
acute lymphoblastic leukemia. Lancet. 1998;351:550–554. Test Time!
20. Campana D, Pui C. Detection of minimal residual disease Look for the CE
in acute leukemia: methodological advances and clinical signif- Update exam on
icance. Blood. 1995;85:1416–1434. Immunology (001) in
21. Willman CL. Immunophenotyping and cytogenetics in
this issue of
older adults with acute myeloid leukemia: significance of
Laboratory Medicine.
expression of the multidrug resistance gene-1 (MDR1).
Leukemia. 1996;10:S33–S35. Participants will earn
22. Au JL, Panchal N, Li D, et al. Apoptosis: a new pharmaco- 4 CMLE credit hours.
dynamic endpoint. Pharm Res. 1997;14:1659–1671.
23. Stelzer GT, Marti G, Hurley A, et al. U.S.-Canadian con-
sensus recommendations on the immunophenotypic analysis
of hematologic neoplasia by flow cytometry: standardization
and validation of laboratory procedures. Cytometry.
1997;30:214–230.
Scientific Communications
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 CONTINUING EDUCATION UPDATE EXAM Date Completed (Required)
Immunology (001)
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Answers: : Please Multiple-Choice Questions 6. What is the most common use of flow cytometry in
select the one best clinical medicine?
answer for each 1. Which of the following thyroid profiles is A. Enzyme immunoassays for autoantibodies
item by placing an recommended by the American Thyroid Association? B. Immunophenotyping of cell populations
A. Thyroid uptake followed by thyroid stimulating hormone C. Multiple drug resistance analysis
X in the box. D. Bacteria detection
(TSH)
B. TSH plus thyroid uptake
A B C D E 7. Detection of IgM antibodies specific for a
C. TSH followed by free thyroxine index (FT4)
1 D. Thyroid binding globulin plus T4 particular pathogen in serum from a neonate
indicates
2. Which of the following investigators was the first A. exposure of the neonate to the pathogen.
2 to use a proteolytic enzyme to digest tissue for B. maternal-fetal transfer of antibody in utero.
immunochemical staining? C. production of maternal IgM.
A. Huang D. an anamnestic response due to secondary exposure.
3 B. Sternberger
C. Guesdon 8. Programmed cell death is a normal process
D. Coons occurring in the body both naturally and after
4 chemotherapy for malignancies. What is another
3. The phrase “analyte specific antigen” was term for programmed cell death?
designated by which of the following regulatory A. Lysis
5 B. Necrosis
groups to replace the earlier designation “research
use only”? C. Apoptosis
A. Clinical Laboratory Improvement Amendment Committee D. Inflammation
6
B. College of American Pathologists
C. US Food and Drug Administration 9. Which cells are required for transplantation to
D. National Committee for Clinical Laboratory Standards reconstitute bone marrow after intensive
7 chemotherapy?
4. Which cardiac marker appears first following an A. Lymphocytes
acute myocardial infarction? B. RBCs
8 C. Monocytes
A. Creatine kinase (CK)
B. CK-MB D. CD34-positive stem cells
9 C. Homocysteine
D. Myoglobin 10. What common feature of reticulocytes and
reticulated platelets can be measured with flow
10 5. Which of the following microbial antigens can be cytometry using a single stain?
detected with direct microbial methods? A. DNA content
A. Capsular proteins B. Cell surface markers
B. Viral cell wall C. RNA content
C. Cell wall components D. Hemoglobin content
D. Inert polysaccharide
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