CE UPDATEIMMUNOLOGY IV

Antony C. Bakke, PhD

Clinical Applications
of Flow Cytometry
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

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From the Department

of Pathology, and
Immunology and
Flow Cytometry
Laboratory, Oregon
Health Sciences
University,
Portland, OR.
Reprint requests to
Dr Bakke, Oregon
Health Sciences
University,
Department of
PathologyL471,
3181 SW Sam
Jackson Park Rd,
Portland, OR 97201.

L A B O R ATO RY M E D I C I N E

flow cytometry. Immunophenotyping, or characterization of cell subpopulations based on differentiation antigens, is probably the most well
recognized area in flow cytometry. These marker
antigens are detected with antibodies and either
flow cytometry, fluorescence microscopy, or
immunohistochemistry. Immunophenotyping is
used clinically for diagnosis and subclassification
of leukemia and lymphoma, diagnosis of primary
immunodeficiency disorders, enumeration of stem
cells in peripheral blood, diagnosis of paroxysmal
nocturnal hemoglobinuria (PNH), monitoring of
immune status in patients with HIV infection,
monitoring of immunosuppressive therapy with
OKT3 and other monoclonal antibodies, T-cell
cross-matching for transplantation, monitoring
for posttransplantation rejection episodes, detection of minimal residual disease in cancer, and tissue typing for HLA-B27. All of these procedures
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 differentiation) designations to unify terminology. The CD

Section

Laser-equipped flow cytometers were originally

developed to provide a rapid screening method for
cervical specimens using a method to measure
altered DNA content. The instrument needed to
precisely analyze both intrinsic and extrinsic cellular features on a cell by cell basis. Intrinsic features
included size, granularity, and autofluorescence.
Extrinsic measurements required the addition of a
fluorescent probe to detect the feature of interest.
For DNA content, this probe was a stoichiometric
stain for DNA. Both determination of ploidy and
analysis of cell division kinetics (eg, S-phase fraction, doubling time, mitotic index) have important implications in areas such as clinical tumor
prognosis.2 Although the goal of detecting cells
with abnormal DNA content was reached, the fact
that many cancers, especially in early stages of disease, do not have significant, quantitative changes
in DNA content has limited use of flow cytometry
for this purpose.3

Scientific Communications

During the last 20 years, flow cytometry has

become a routine clinical method in many laboratories.1 This article discusses current clinical uses
of flow cytometry, emphasizing immunophenotyping, reticulocyte and reticulated platelet enumeration, multiple drug resistance assays, cell
function assays, and apoptosis. A partial list of the
standard clinical instruments and additional
instruments with features of automated cytometry is given in the Table. Recent advances are
briefly discussed; advanced research uses are not.
All of the assays described are used by clinical laboratories for patient care.

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Instruments Used for Flow Cytometry

Instrument

Manufacturer

Uses

FACS Calibur

Becton Dickinson, San Jose, CA

Immunophenotyping, DNA, reticulocytes,

platelets, multiple drug resistance, apoptosis,
cell function

XL

Beckman-Coulter, Miami, FL

Same as above

PAS

Partec, Munster, Germany

Same as above

BRYTE HS

BioRad, Hercules, CA

DNA, bacterial drug sensitivity, reticulocytes,

platelets, some immunophenotyping

IMAGN 2000

Biometric Imaging &

Becton Dickinson, San Jose, CA

CD4, CD8, CD34, residual WBCs, platelet

activation

LaserScanning Cytometer

CompuCyte, Cambridge, MA

Immunophenotyping, DNA, reticulocytes,

platelets, multiple drug resistance, apoptosis

CellDyn 4000

Abbott, North Chicago, IL

Immunophenotyping for CD3, CD4, CD8,

CD34, CD61

number can refer to both the antibody and the

antigen. Therefore, FITC-CD3 indicates a monoclonal 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 multiple-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 diagnosis and treatment in many patients.6 An additional 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

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gives additional prognostic information independent 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 (Brutons) agammaglobulinemia is due to a mutation in a specific
tyrosine kinase gene required for B-lymphocyte
maturation. Patients completely lack B lymphocytes in the peripheral blood, which can be shown
by lack of staining with CD19 antibodies. Another
example, hyperimmunoglobulinemia M syndrome, 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, immunophenotyping is considered the standard of medical care. Hematopoietic malignancies represent
specific stages of differentiation and may be lymphoid, myeloid, or biphenotypic. Routine morphology 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 differentiation.10 Exact classification cannot be
done without immunophenotyping, either with

R4 (CD 19+)
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Lambda-PE

CD 19-PerCP

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SSC Height

flow cytometry or immunohistochemistry, which

are complementary techniques. With either
method, fewer than 50,000 cells from difficult
specimens, such as fine-needle aspirate, cerebrospinal fluid, or vitreous fluid, can be stained
and analyzed for B-lymphocyte monoclonality. A
typical example of flow cytometry is shown in
Figure 1. Flow cytometry has the advantage of
speed and simpler methods, but lacks the ability
to visualize malignant cells.
Many solid tumors are currently being treated
with marrow ablative chemotherapy followed by
either autologous or allogeneic stem cell transplantation. 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 unreliable surrogate, stem cells in the blood must be
enumerated with CD34 staining.11 Hematopoietic 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 glycosylphosphatidylinositol (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

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Kappa-FITC

cytometry. Lack of CD55 and CD59 confers sensitivity to activated complement on the cells. This
defect begins in a hematopoietic clone of cells in
the bone marrow. In any patient, the percentage of
PNH cells may vary from 1% to 100%. Of importance, patients with PNH may have near normal
expression on RBCs, due to the shortened life span
of PNH RBCs or to transfusions. In addition,
PNH cells may be either completely negative (type
III) or bear decreased amounts of these GPIanchored proteins (type II). Inasmuch as the
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 diagnosing 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
CD3 plus the T-lymphocyte antigen receptor from
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

Scientific Communications

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Fig 1. Multicolor
staining of a lymph
node fine-needle
aspirate with antikappa-fluorescein
isothiocyanate
(FITC), anti-lambdaphycoerythrin (PE),
and CD19-peridinin
chlorophyll protein
(PerCP). Cells are
stained, washed, and
analyzed. FITC
fluoresces green, PE
is yellow, and PerCP
(alternately, PE-Cy5)
is orange. A, Dot plot
of side, or 90-degree,
light scatter on the
X-axis vs CD19PerCP staining on
the Y-axis. CD19positive, low side
scatter cells (SSC)
are gated in region
R4. B, Kappa and
lambda staining of
the CD19-gated cells
in A. Monoclonal,
kappa-positive,
presumptive
neoplastic, B-cell
population is
present.

Section

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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99

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 (previously termed the reticulocyte maturity index).14
An analogous measurement for platelets provides 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 phycoerythrin-conjugated CD41 antibody to differentiate them from other small particles. The RBCs
in the specimen are used as an internal negative
control for staining intensity, and the CD41-positive platelets are evaluated for RNA content (Fig
2).15 An increase in the number of reticulated
platelets indicates increased bone marrow production 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 certain patients. Perhaps the most clinically relevant
assays of cell function are those for oxidative burst
and phagocytosis by neutrophils. Defects are characteristic of chronic granulomatous disease. Flow
cytometric assays are simpler and easier to perform 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 tetrazolium 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 target cells lose their fluorescence and are no longer
counted in the assay. Percent cytotoxicity is calculated 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 fluorescence resonance energy transfer has increased
the specificity of the assay by eliminating the contribution of non-specificallybound IgG.17
Platelet hyperactivity and circulating activated
platelets are associated with unstable angina, acute
myocardial infarction, cardiovascular accident,
cardiopulmonary bypass, diabetes mellitus, emotional 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 antiCD62 and anti-CD41, and reagents that detect
externalized phosphatidylserine on platelets. In
addition, nonflow cytometric assays are available. 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 determining 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 difficult 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 resistancerelated protein
[MRP]), the glutathione transferase antioxidant
system, increased resistance to apoptosis due to

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320
103

Counts

RBC-G2

FSC-H

Platelets-G1
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240

Marker at inflection

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80
101
0

100

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104

FL1-H
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FL2-H

C
50

40

Counts

30

20

10

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101

102

103

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FL1-H

D
50

40

30

Section

Counts

bcl-2 overexpression, p53 mutations, and other

factors. Since MDR1 inhibitors are available for
clinical use, they have been a natural candidate for
study. Accumulation or efflux of several fluorescent dyes or antibody staining for MDR1 (p-glycoprotein) has been used to measure the level of
MDR1. Both measurements correlate with clinical
outcome in acute myeloid leukemia.21
Analysis of apoptosis, or programmed cell
death, is a major research application of flow
cytometry and may become a new clinical application for determining the efficacy of chemotherapy
in patients with leukemia and other types of malignancies.22 During this important biologic process,
there are changes in the plasma membrane and the
mitochondria, a cascade of intracellular proteases

Scientific Communications

Fig 2. Whole blood is stained with thiazole orange (RNA

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
the Y-axis vs PE-CD41 (FL2) staining. Medium forward
scatter, CD41-positive platelets (G1), and high forward
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
point on the curve and is used to calculate a platelet
setting. C, Thiazole orange staining (FL1) of gated
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
is a normal individual with <1% reticulated platelets. D,
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.

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Propidium lodide

Necrotic

Consensus Documents, Laboratory

Standards, and Regulations

103

The complexity of multiparameter flow cytometry and its use in clinical medicine necessitate
standards and regulation for flow cytometry to
achieve interlaboratory reproducibility and
ensure quality patient care. Several groups
around the world have met to establish consensus protocols and guidelines for current methods
in flow cytometry, including proficiency testing,
accreditation, and training.23

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10

Viable

Apoptotic

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Annexin-VFITC

Fig 3. T-cell line (Jurkat) is cultured with camptothecin for 2 hours to

induce apoptosis. Cells are then stained with fluorescein isothiocyanate
(FITC)annexin-V and propidium iodide. Viable cells are negative for both
stains, cells that have recently entered the apoptosis cascade are positive
for annexin only (apoptotic), and cells that are late in apoptosis and have
permeable membranes are positive for both annexin and propidium
iodide (necrotic).

Conclusion

Flow cytometry is a powerful tool with many applications in pathology. Although it is currently used
for many standard tests, new applications are continually being developed, including assays for minimal residual disease detection and multiple drug
resistance assessment. As a tool, flow cytometry
does not stand alone but enhances other methods,
from morphologic to molecular analysis.l

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

1. McCoy JP Jr, Keren DF. Current practices in clinical flow
becomes fragmented. Apoptosis is different from
cytometry. Am J Clin Pathol. 1999;111:161168.
necrosis. Morphologically, apoptosis is character2. 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;14:482485.
3. Koss LG, Czerniak B, Herz F, et al. Flow cytometric meaphagocytized, 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;20:528548.
4. Mandy FF, Bergeron M, Minkus T. Evolution of leukocyte
swelling, is induced by hypoxia and toxins, and
immunophenotyping as influenced by the HIV/AIDS panresults 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;30:157165.
5. Lacombe F, Durrieu F, Dumain P, et al. Flow cytometry
necrosis can be measured with flow cytometric
CD45 gating for immunophenotyping of acute myeloid
techniques such as annexin-V binding to phos- leukemia. Leukemia. 1997;11:18781886.
phatidylserine on the cell surface, DNA nick label6. Centers for Disease Control and Prevention. 1993
ing, permeability dyes, and antibody-based or Expanded Surveillance Case Definition for AIDS. No. RR-17.
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;179:859870.
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;115:458463.
9. Ten RM. Primary immunodeficiencies. Mayo Clin Proc.
for only a short time in the body before being
1998;73:865872.
removed by mononuclear phagocytes. This
10. Knapp W, Strobl H, Majdic O. Flow cytometric analysis of
important phenomenon has implications for cell-surface and intracellular antigens in leukemia diagnosis.
many areas of biology, including cell regulation Cytometry. 1994;18:187198.
11. Chin-Lee I, Anderson L, Keeney M, et al. Quality assurand tumor proliferation.
ance of stem cell enumeration by flow cytometry. Cytometry.
1996;30:296303.
12. Rosse WF. Paroxysmal nocturnal hemoglobinuria as a
molecular disease. Medicine. 1997;76:6393.

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Internet
Resources

Urbana Atlas of Pathology, available on the

University of Illinois College of Medicine at UrbanaChampaign 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

Test Time!
Look for the CE
Update exam on
Immunology (001) in
this issue of
Laboratory Medicine.
Participants will earn
4 CMLE credit hours.

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

Scientific Communications

Flow Cytometry
The Clinical Cytometry Society Web site includes a
directory of individuals working in the field of cytometry and a vendor directory, in addition to numerous
links to related journals, organizations, and products.
http://www.cytometry.org

Please let us know your opinion of the Immunology (001)

series. Place an X in one box for each question. Return
this form (or a photocopy) by fax to: (312) 850-8817; or,
mail to: ASCP Press Administration, 2100 W Harrison St,
Chicago, IL 60612-3798. Thank you for your input.
Excell
Defici
1 The series met the objectives stat

2 The series provided useful technical d

3 The information provided in the series was new and tim

4 Technical points were explained clearly and were easy

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

5 The text was organized

6. Illustrations, charts, and tables helped explain text

Comments: (Attach additional pages, if necessary.)

18689

These sites were accessed December 21, 1999, and

are offered for reader information only. A sites
presence on this list does not constitute an
endorsement by the ASCP.
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103

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

13. Henell KR, Bakke A, Kenny TA, et al. Degree of modulation of cell-surface CD3 by anti-lymphocyte therapies. Transplant Proc. 1991;23:10701071.
14. Davis BH, Bigelow NC. Reticulocyte analysis and reticulocyte maturity index. Methods Cell Biol. 1994;42:263273.
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;14:690694.
16. OGorman MRG, Corrochano V. Rapid whole-blood flow
cytometry assay for diagnosis of chronic granulomatous disease. Clin Diagn Lab Immunol. 1995;2:227232.
17. Koksch M, Rothe G, Kiefel V, et al. Fluorescence resonance energy transfer as a new method for the epitope-specific
characterization of anti-platelet antibodies. J Immunol Methods. 1995;187:5367.
18. Michelson AD. Flow cytometry: a clinical test of platelet
function. Blood. 1996;12:49254936.
19. Coustan-Smith E, Behm FG, Sanchez J, et al. Immunological detection of minimal residual disease in children with
acute lymphoblastic leukemia. Lancet. 1998;351:550554.
20. Campana D, Pui C. Detection of minimal residual disease
in acute leukemia: methodological advances and clinical significance. Blood. 1995;85:14161434.
21. Willman CL. Immunophenotyping and cytogenetics in
older adults with acute myeloid leukemia: significance of
expression of the multidrug resistance gene-1 (MDR1).
Leukemia. 1996;10:S33S35.
22. Au JL, Panchal N, Li D, et al. Apoptosis: a new pharmacodynamic endpoint. Pharm Res. 1997;14:16591671.
23. Stelzer GT, Marti G, Hurley A, et al. U.S.-Canadian consensus recommendations on the immunophenotypic analysis
of hematologic neoplasia by flow cytometry: standardization
and validation of laboratory procedures. Cytometry.
1997;30:214230.

Section

Here are some Internet sites that

offer more information on topics
discussed in this issue of Laboratory Medicine.

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Multiple-Choice Questions

6. What is the most common use of flow cytometry in

clinical medicine?

1. Which of the following thyroid profiles is

recommended by the American Thyroid Association?

A. Enzyme immunoassays for autoantibodies

B. Immunophenotyping of cell populations
C. Multiple drug resistance analysis
D. Bacteria detection

A. Thyroid uptake followed by thyroid stimulating hormone

(TSH)
B. TSH plus thyroid uptake
C. TSH followed by free thyroxine index (FT4)
D. Thyroid binding globulin plus T4

2. Which of the following investigators was the first

to use a proteolytic enzyme to digest tissue for
immunochemical staining?
A. Huang
B. Sternberger
C. Guesdon
D. Coons

3. The phrase analyte specific antigen was

designated by which of the following regulatory
groups to replace the earlier designation research
use only?
A. Clinical Laboratory Improvement Amendment Committee
B. College of American Pathologists
C. US Food and Drug Administration
D. National Committee for Clinical Laboratory Standards

4. Which cardiac marker appears first following an

acute myocardial infarction?
A. Creatine kinase (CK)
B. CK-MB
C. Homocysteine
D. Myoglobin

5. Which of the following microbial antigens can be

detected with direct microbial methods?
A. Capsular proteins
B. Viral cell wall
C. Cell wall components
D. Inert polysaccharide

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7. Detection of IgM antibodies specific for a

particular pathogen in serum from a neonate
indicates
A. exposure of the neonate to the pathogen.
B. maternal-fetal transfer of antibody in utero.
C. production of maternal IgM.
D. an anamnestic response due to secondary exposure.

8. Programmed cell death is a normal process

occurring in the body both naturally and after
chemotherapy for malignancies. What is another
term for programmed cell death?
A. Lysis
B. Necrosis
C. Apoptosis
D. Inflammation

9. Which cells are required for transplantation to

reconstitute bone marrow after intensive
chemotherapy?
A. Lymphocytes
B. RBCs
C. Monocytes
D. CD34-positive stem cells

10. What common feature of reticulocytes and

reticulated platelets can be measured with flow
cytometry using a single stain?
A. DNA content
B. Cell surface markers
C. RNA content
D. Hemoglobin content

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