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During the last 20 years, flow cytometry hasbecome a routine clinical method in many labora-tories.1 This article discusses current clinical usesof flow cytometry, emphasizing immunopheno-typing, reticulocyte and reticulated platelet enu-meration, multiple drug resistance assays, cellfunction assays, and apoptosis. A partial list of thestandard clinical instruments and additionalinstruments with features of automated cytome-try is given in the Table. Recent advances arebriefly discussed; advanced research uses are not.All of the assays described are used by clinical lab-oratories for patient care.

DDNNAA AAnnaallyyssiissLaser-equipped flow cytometers were originallydeveloped to provide a rapid screening method forcervical specimens using a method to measurealtered DNA content. The instrument needed toprecisely analyze both intrinsic and extrinsic cellu-lar features on a cell by cell basis. Intrinsic featuresincluded size, granularity, and autofluorescence.Extrinsic measurements required the addition of afluorescent probe to detect the feature of interest.For DNA content, this probe was a stoichiometricstain for DNA. Both determination of ploidy andanalysis of cell division kinetics (eg, S-phase frac-tion, doubling time, mitotic index) have impor-tant implications in areas such as clinical tumorprognosis.2 Although the goal of detecting cellswith abnormal DNA content was reached, the factthat many cancers, especially in early stages of dis-ease, do not have significant, quantitative changesin DNA content has limited use of flow cytometryfor this purpose.3

IImmmmuunnoopphheennoottyyppiinnggDevelopments in immunology and other areas ofcancer research have greatly expanded the use of

flow cytometry. Immunophenotyping, or charac-terization of cell subpopulations based on differen-tiation antigens, is probably the most wellrecognized area in flow cytometry. These markerantigens are detected with antibodies and eitherflow cytometry, fluorescence microscopy, orimmunohistochemistry. Immunophenotyping isused clinically for diagnosis and subclassificationof leukemia and lymphoma, diagnosis of primaryimmunodeficiency disorders, enumeration of stemcells in peripheral blood, diagnosis of paroxysmalnocturnal hemoglobinuria (PNH), monitoring ofimmune status in patients with HIV infection,monitoring of immunosuppressive therapy withOKT3 and other monoclonal antibodies, T-cellcross-matching for transplantation, monitoringfor posttransplantation rejection episodes, detec-tion of minimal residual disease in cancer, and tis-sue typing for HLA-B27. All of these proceduresrequire the use of 1 or more antibodies (usuallymonoclonal) to detect the antigens.

Antibodies are carefully standardized by aseries of international leukocyte differentiationworkshops and are given CD (cluster of differenti-ation) designations to unify terminology. The CD

From the Departmentof Pathology, andImmunology andFlow CytometryLaboratory, OregonHealth SciencesUniversity, Portland, OR.

Reprint requests toDr Bakke, OregonHealth SciencesUniversity,Department ofPathology–L471,3181 SW SamJackson Park Rd,Portland, OR 97201.

Antony C. Bakke, PhDCC EE UU PP DD AA TT EE —— II MM MM UU NN OO LL OO GG YY II VV

Flow cytometry is an important analytical toolthat has moved from research to the clinical laboratory in thepast 20 years. Although still important in research, manyapplications have become routine clinical tests required fordiagnosis, estimation of prognosis, and monitoring oftreatment. In addition, new applications, such as minimalresidual disease detection and multiple-drug resistancemonitoring, 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.

ABSTRACT

Clinical Applications of Flow Cytometry

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number can refer to both the antibody and theantigen. Therefore, FITC-CD3 indicates a mono-clonal antibody against the CD3 antigen, which isconjugated to fluorescein isothiocyanate (FITC) tomake it fluoresce green when excited with bluelight. On the other hand, CD3+ T lymphocytes arethe subpopulation of lymphocytes bearing theCD3 antigen, which is positive when stained withFITC-CD3.

The latest advances in flow cytometry involvemultiple-color analysis, enabling evaluation ofcoexpression of several markers while reducingthe total amounts of antibody and specimenrequired for analysis. With the availability of mul-tiple-color analysis, strategies have evolved thatenable more refined gating of populations, such asCD45 gating for all WBCs or CD19 gating for Blymphocytes. This type of gating has enhanced orreplaced the traditional scatter gating (Fig 1), withapplications in immunodeficiency disorders, suchas HIV infection,4 and hematologic neoplasia.5 InHIV infection, enumeration of CD4+ T cells hasbecome a standard surrogate for estimating stageof disease. Of importance, a CD4 count <200cells/mL together with a positive antibody test forHIV is diagnostic of AIDS, enabling proper diag-nosis and treatment in many patients.6 An addi-tional method of assessing stage of disease for HIVand response to therapy has been developed usingquantitative expression of CD38 on CD8+ T cells.This evaluation is not yet used widely in clinicallaboratories, but may be in the future because it

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 accuratediagnosis of primary immune deficiency disorders.Many have characteristic defects that are easilydetected.9 For example, X-linked (Bruton’s) agam-maglobulinemia is due to a mutation in a specifictyrosine kinase gene required for B-lymphocytematuration. Patients completely lack B lympho-cytes in the peripheral blood, which can be shownby lack of staining with CD19 antibodies. Anotherexample, hyperimmunoglobulinemia M syn-drome, is due to a mutation in the CD40 ligand. Itis 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 representspecific stages of differentiation and may be lym-phoid, myeloid, or biphenotypic. Routine mor-phology and cytochemistry are absolutelynecessary for diagnosis but cannot differentiatemany subtypes of these diseases, such as pre-B-cellacute lymphoblastic leukemia. Each subtypeexpresses different biologic behavior, as reflectedin different prognoses and effective treatments.

Recent advances have added intracellularstaining for antigens such as CD3, CD19, CD79a,and myeloperoxidase, enabling identification ofthe earliest stages of lymphoid and myeloid dif-ferentiation.10 Exact classification cannot bedone without immunophenotyping, either with

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 & CD4, CD8, CD34, residual WBCs, platelet Becton Dickinson, San Jose, CA 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

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flow cytometry or immunohistochemistry, whichare complementary techniques. With eithermethod, fewer than 50,000 cells from difficultspecimens, such as fine-needle aspirate, cere-brospinal fluid, or vitreous fluid, can be stainedand analyzed for B-lymphocyte monoclonality. Atypical example of flow cytometry is shown inFigure 1. Flow cytometry has the advantage ofspeed and simpler methods, but lacks the abilityto visualize malignant cells.

Many solid tumors are currently being treatedwith marrow ablative chemotherapy followed byeither autologous or allogeneic stem cell trans-plantation. Colony-stimulating factors induceproduction and release of more stem cells into theblood, where they are collected along with otherWBCs. However, the timing of release of thesecells from the bone marrow varies amongpatients. Since the peripheral WBC is an unreli-able surrogate, stem cells in the blood must beenumerated with CD34 staining.11 Hematopoi-etic stem cell evaluation requires detection ofsmall numbers of cells (1 in 1,000) and oftenneeds to be done in real time to avert unnecessaryand expensive additional collections.

Defects in the production of normalhematopoietic cells by the bone marrow can alsobe detected with flow cytometry. For example,patients with PNH lack specific glycosylphos-phatidylinositol (GPI)–anchored proteins onhematopoietic cells, because of a defect in thePIGA (phosphatidylinositol glycan A gene).12

Many GPI-anchored proteins (eg, CD14, CD16,CD55, CD59, CD66b) are easily detected with flow

cytometry. Lack of CD55 and CD59 confers sensi-tivity to activated complement on the cells. Thisdefect begins in a hematopoietic clone of cells inthe bone marrow. In any patient, the percentage ofPNH cells may vary from 1% to 100%. Of impor-tance, patients with PNH may have near normalexpression on RBCs, due to the shortened life spanof PNH RBCs or to transfusions. In addition,PNH cells may be either completely negative (typeIII) or bear decreased amounts of these GPI-anchored proteins (type II). Inasmuch as theabnormal PNH clone usually increases over time,repeated testing may be necessary.

Other flow cytometry assays involvingimmunophenotyping include monitoring ofOKT3 antibody immunosuppression and tissuetyping for HLA-B27, which is important in diag-nosing spondylarthritides. OKT3 monoclonalantibody is directed against the CD3 antigen on Tlymphocytes. After binding to CD3, the antibodyinduces T-lymphocyte activation and removal ofCD3 plus the T-lymphocyte antigen receptor fromthe cell surface. As a result, the T lymphocyte iseffectively “blind” and cannot identify and attackany target (ie, transplanted tissues), resulting inimmunosuppression. Monitoring effectiveremoval of CD3 from the T lymphocyte is used todetermine effective treatment.13

RRBBCCss aanndd PPllaatteelleettssRBCs and platelets can be evaluated with flowcytometry. Reticulocytes can easily be measuredusing a stain for the residual RNA in these cells.

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Fig 1. Multicolorstaining of a lymphnode fine-needleaspirate with anti-kappa-fluoresceinisothiocyanate(FITC), anti-lambda-phycoerythrin (PE),and CD19-peridininchlorophyll protein(PerCP). Cells arestained, washed, andanalyzed. FITCfluoresces green, PEis yellow, and PerCP(alternately, PE-Cy5)is orange. A, Dot plotof side, or 90-degree,light scatter on theX-axis vs CD19-PerCP staining onthe Y-axis. CD19-positive, low sidescatter cells (SSC)are gated in regionR4. B, Kappa andlambda staining ofthe CD19-gated cellsin A. Monoclonal,kappa-positive,presumptiveneoplastic, B-cellpopulation ispresent.

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Similar to the methylene blue staining method, apercentage of reticulocytes is measured and usedto calculate the absolute number. In addition, flowcytometry provides a measure of reticulocytematurity, the immature reticulocyte fraction (pre-viously termed the reticulocyte maturity index).14

An analogous measurement for platelets pro-vides an estimate of young, “reticulated” plateletsby counting platelets that stain with an RNA dye(eg, thiazole orange, coriphosphine-O). Theplatelets in whole blood are also labeled with phy-coerythrin-conjugated CD41 antibody to differ-entiate them from other small particles. The RBCsin the specimen are used as an internal negativecontrol for staining intensity, and the CD41-posi-tive platelets are evaluated for RNA content (Fig2).15 An increase in the number of reticulatedplatelets indicates increased bone marrow produc-tion of platelets and is consistent with a diagnosisof idiopathic thrombocytopenic purpura.

CCeellll FFuunnccttiioonnCell function assays are performed by only a fewreference laboratories, but can be helpful in cer-tain patients. Perhaps the most clinically relevantassays of cell function are those for oxidative burstand phagocytosis by neutrophils. Defects are char-acteristic of chronic granulomatous disease. Flowcytometric assays are simpler and easier to per-form than complex bactericidal assays. In the caseof oxidative burst assays, the neutrophils areloaded with a nonfluorescent dye that becomesfluorescent when it is oxidized by stimulatedcells.16 This is analogous to the nitroblue tetra-zolium test. For phagocytosis, fluorescence-taggedbacteria can be incubated with viable neutrophils,and internalization measured.

Other functional assays for lymphocytes includeproliferation and stimulation by mitogens and thenatural killer cytotoxicity assay. Proliferation ismeasured using fluorescent antibodies that detectbromodeoxyuridine incorporation, analogous totritiated thymidine incorporation. Cytotoxicityassays use target cells, which are viably labeled witha fluorescent marker. Once they are killed, the tar-get cells lose their fluorescence and are no longercounted in the assay. Percent cytotoxicity is calcu-lated by comparing the remaining viable targetswith a control lacking natural killer cells.

FFuuttuurree CClliinniiccaall UUsseessOften patients with idiopathic thrombocytopenicpurpura have antiplatelet antibodies that can bemeasured with flow cytometry. Previous studiesshowed that platelet-associated IgG could be ofsome use clinically, but specificity has been low,primarily because of nonspecific binding of IgG toFc and complement receptors on platelets. Gatingof platelets with flow cytometry and determinationof the specific site of antibody binding with fluo-rescence resonance energy transfer has increasedthe specificity of the assay by eliminating the con-tribution of non-specifically–bound IgG.17

Platelet hyperactivity and circulating activatedplatelets are associated with unstable angina, acutemyocardial infarction, cardiovascular accident,cardiopulmonary bypass, diabetes mellitus, emo-tional stress, and blood bank storage of platelets,among other conditions.18 Several assays havebeen developed to measure either activatedplatelets or platelet microparticles using anti-CD62 and anti-CD41, and reagents that detectexternalized phosphatidylserine on platelets. Inaddition, non–flow cytometric assays are avail-able. Because of the importance of platelet activityin many clinical conditions, some assay of plateletactivation will eventually be widely utilized.

The role of flow cytometry as an aid in deter-mining prognosis in patients with cancer hasbecome increasingly important with the advent ofassays for detection of minimal residual diseaseand assessment of multiple drug resistance.Although not so sensitive as PCR methods,observing phenotypic markers is often the onlymethod for determining the presence of minimalresidual disease without known or consistentgenetic aberrations. Although the methods are dif-ficult to establish, a number of laboratories havereported that it is possible to routinely identify 1malignant cell among 10,000 normal cells withflow cytometry.19 In some studies, this level ofdetection was clinically relevant, whereas the moresensitive PCR did not predict clinical response.20

Drug resistance is a principal reason for failureof cancer response to chemotherapy. Multiple drugresistance is mediated by several mechanisms incancer cells, including the p-glycoprotein (MDR1)and other pumps (eg, lung resistance protein[LRP], multiple drug resistance–related protein[MRP]), the glutathione transferase antioxidantsystem, increased resistance to apoptosis due to

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bcl-2 overexpression, p53 mutations, and otherfactors. Since MDR1 inhibitors are available forclinical use, they have been a natural candidate forstudy. Accumulation or efflux of several fluores-cent dyes or antibody staining for MDR1 (p-glyco-protein) has been used to measure the level ofMDR1. Both measurements correlate with clinicaloutcome in acute myeloid leukemia.21

Analysis of apoptosis, or programmed celldeath, is a major research application of flowcytometry and may become a new clinical applica-tion for determining the efficacy of chemotherapyin patients with leukemia and other types of malig-nancies.22 During this important biologic process,there are changes in the plasma membrane and themitochondria, a cascade of intracellular proteases

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Fig 2. Whole blood is stained with thiazole orange (RNAstain) and CD41-phycoerythrin (PE) antibody. The bloodis diluted and analyzed, without washing, on the flowcytometer. A, Dot plot of log forward scatter (FSC) onthe Y-axis vs PE-CD41 (FL2) staining. Medium forwardscatter, CD41-positive platelets (G1), and high forwardscatter, CD41-negative RBCs (G2) are gated. B, Thiazoleorange staining (FL1) of gated RBCs. Reticulocytes andWBCs appear positive. A marker is set at the inflectionpoint on the curve and is used to calculate a plateletsetting. C, Thiazole orange staining (FL1) of gatedplatelets. Based on biochemical studies of RNA contentin reticulocytes and reticulated platelets, the marker isset at 3 times the value from the reticulocytes in B. Thisis a normal individual with <1% reticulated platelets. D,Thiazole orange staining (FL1) of gated platelets from apatient with idiopathic thrombocytopenic purpura. Fiftypercent of the platelets contain sufficient RNA to beconsidered young platelets.

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(caspases) are activated, and, finally, cellular DNAbecomes fragmented. Apoptosis is different fromnecrosis. Morphologically, apoptosis is character-ized by cell shrinkage, chromatin condensation,and formation of apoptotic bodies, which arephagocytized, inducing little inflammation. Con-versely, necrosis is characterized by cellularswelling, is induced by hypoxia and toxins, andresults in extensive inflammation.

Cellular changes characteristic of apoptosis ornecrosis can be measured with flow cytometrictechniques such as annexin-V binding to phos-phatidylserine on the cell surface, DNA nick label-ing, permeability dyes, and antibody-based orenzyme-based assays of the active caspases. Newlyapoptotic cells are positive for annexin-V but notpropidium iodide (Fig 3). In cells that are fartherin apoptosis or have died by necrosis, holesdevelop in the plasma membrane and becomepositive for propidium iodide, which enters andstains DNA. Similar results can be obtained withpatient specimens, although apoptotic cells existfor only a short time in the body before beingremoved by mononuclear phagocytes. Thisimportant phenomenon has implications formany areas of biology, including cell regulationand tumor proliferation.

CCoonnsseennssuuss DDooccuummeennttss,, LLaabboorraattoorryySSttaannddaarrddss,, aanndd RReegguullaattiioonnssThe complexity of multiparameter flow cytome-try and its use in clinical medicine necessitatestandards and regulation for flow cytometry toachieve interlaboratory reproducibility andensure quality patient care. Several groupsaround the world have met to establish consen-sus protocols and guidelines for current methodsin flow cytometry, including proficiency testing,accreditation, and training.23

CCoonncclluussiioonnFlow cytometry is a powerful tool with many appli-cations in pathology. Although it is currently usedfor many standard tests, new applications are con-tinually being developed, including assays for min-imal residual disease detection and multiple drugresistance assessment. As a tool, flow cytometrydoes not stand alone but enhances other methods,from morphologic to molecular analysis.l

References1. McCoy JP Jr, Keren DF. Current practices in clinical flow

cytometry. Am J Clin Pathol. 1999;111:161–168.2. Hedley DW, Clark GM, Cornelisse CJ, et al. Consensus

review of the clinical utility of DNA cytometry in carcinoma ofthe breast. Cytometry. 1993;14:482–485.

3. Koss LG, Czerniak B, Herz F, et al. Flow cytometric mea-surements of DNA and other cell components in humantumors: a critical appraisal. Human Pathol. 1989;20:528–548.

4. Mandy FF, Bergeron M, Minkus T. Evolution of leukocyteimmunophenotyping as influenced by the HIV/AIDS pan-demic: a short history of the development of gating strategiesfor CD4+ T-cell enumeration. Cytometry. 1997;30:157–165.

5. Lacombe F, Durrieu F, Dumain P, et al. Flow cytometryCD45 gating for immunophenotyping of acute myeloidleukemia. Leukemia. 1997;11:1878–1886.

6. Centers for Disease Control and Prevention. 1993Expanded Surveillance Case Definition for AIDS. No. RR-17.MMWR. 1992;41:119.

7. Giorgi JV, Hultin LE, McKeating JA, et al. Shorter survivalin advanced human immunodeficiency virus type 1 infection ismore closely associated with T lymphocyte activation thanwith plasma virus burden or virus chemokine coreceptorusage. J Infect Dis. 1999;179:859–870.

8. Burgisser P, Hammann C, Kaufman D, et al. Expression ofCD28 and CD38 by CD8+ T lymphocytes in HIV-1 infectioncorrelates with markers of disease severity and changes towardsnormalization under treatment: the Swiss HIV cohort study.Clin Exp Immunol. 1999;115:458–463.

9. Ten RM. Primary immunodeficiencies. Mayo Clin Proc.1998;73:865–872.

10. Knapp W, Strobl H, Majdic O. Flow cytometric analysis ofcell-surface and intracellular antigens in leukemia diagnosis.Cytometry. 1994;18:187–198.

11. Chin-Lee I, Anderson L, Keeney M, et al. Quality assur-ance of stem cell enumeration by flow cytometry. Cytometry.1996;30:296–303.

12. Rosse WF. Paroxysmal nocturnal hemoglobinuria as amolecular disease. Medicine. 1997;76:63–93.

Fig 3. T-cell line (Jurkat) is cultured with camptothecin for 2 hours toinduce apoptosis. Cells are then stained with fluorescein isothiocyanate(FITC)–annexin-V and propidium iodide. Viable cells are negative for bothstains, cells that have recently entered the apoptosis cascade are positivefor annexin only (apoptotic), and cells that are late in apoptosis and havepermeable membranes are positive for both annexin and propidiumiodide (necrotic).

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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 thepercentage of thiazole orange (TO)–positive, “reticulated”platelets using autologus erythrocyte TO fluorescence as aninternal standard. Cytometry. 1993;14:690–694.

16. O’Gorman MRG, Corrochano V. Rapid whole-blood flowcytometry 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-nance energy transfer as a new method for the epitope-specificcharacterization of anti-platelet antibodies. J Immunol Meth-ods. 1995;187:53–67.

18. Michelson AD. Flow cytometry: a clinical test of plateletfunction. Blood. 1996;12:4925–4936.

19. Coustan-Smith E, Behm FG, Sanchez J, et al. Immuno-logical detection of minimal residual disease in children withacute lymphoblastic leukemia. Lancet. 1998;351:550–554.

20. Campana D, Pui C. Detection of minimal residual diseasein acute leukemia: methodological advances and clinical signif-icance. Blood. 1995;85:1416–1434.

21. Willman CL. Immunophenotyping and cytogenetics inolder adults with acute myeloid leukemia: significance ofexpression of the multidrug resistance gene-1 (MDR1).Leukemia. 1996;10:S33–S35.

22. Au JL, Panchal N, Li D, et al. Apoptosis: a new pharmaco-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 analysisof hematologic neoplasia by flow cytometry: standardizationand validation of laboratory procedures. Cytometry.1997;30:214–230.

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Test Time!Look for the CEUpdate exam on Immunology (001) inthis issue ofLaboratory Medicine.Participants will earn4 CMLE credit hours.

Please let us know your opinion of the Immunology (001)series. Place an X in one box for each question. Returnthis 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.

The series met the objectives stat1

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3 The information provided in the series was new and tim

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4 Technical points were explained clearly and were easy

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The text was organized

1 2 3 4 56. Illustrations, charts, and tables helped explain text

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18689

IInntteerrnneettRReessoouurrcceessHere are some Internet sites thatoffer more information on topics

discussed in this issue of Laboratory Medicine.

CCooaagguullaattiioonn“Coagulation Diseases” is available on the Community Outreach Health Information System(COHIS) Web site. COHIS was developed by BostonUniversity to provide information that is easy to readand understand to students, children, and educators.

http://www.bu.edu/cohis/cardvasc/blood/coag.htm

“Urbana Atlas of Pathology,” available on theUniversity of Illinois College of Medicine at Urbana-Champaign Web site, contains pathologic imagesdue to disseminated intravascular coagulation.

http://www.med.uiuc.edu/PathAtlasf/CVAtlas039.htmlhttp://www.med.uiuc.edu/PathAtlasf/Atlas101.htmlhttp://www.med.uiuc.edu/PathAtlasf/Atlas102.html

DDiiaabbeetteess“Conquering Diabetes: A Strategic Plan for the 21stCentury (1999; NIH Publication No. 99-4398),” a reportof the Congressionally-established Diabetes ResearchWorking Group, National Institutes of Health (NIH), isavailable on the National Institute of Diabetes andDigestive and Kidney Diseases, NIH Web site

http://www.ep.niddk.nih.gov/dwg/fr.pdf

“Diabetes Info” is available on the AmericanDiabetes Association Web site

http://www.diabetes.org/ada/diabetesinfo.asp

FFllooww CCyyttoommeettrryyThe Clinical Cytometry Society Web site includes adirectory of individuals working in the field of cytom-etry and a vendor directory, in addition to numerouslinks to related journals, organizations, and products.

http://www.cytometry.org

Consensus documents and guidelines are availableon the CytoRelay (Martinsried, Germany) Home page

http://www.biochem.mpg.de/research-groups/ valet/cytorel.html

Purdue University Cytometry Laboratories (WestLafayette, 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 statisticscommonly used when analyzing flow cytometrydata” by Geoffrey Osborne is available on the JohnCurtin School of Medical Research (Canberra City,Australia) Web site.

http://jcsmr.anu.edu.au/facslab/statistics.html

These sites were accessed December 21, 1999, andare offered for reader information only. A site’spresence on this list does not constitute anendorsement by the ASCP.

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Multiple-Choice Questions

1. Which of the following thyroid profiles isrecommended by the American Thyroid Association?A. Thyroid uptake followed by thyroid stimulating hormone(TSH)B. TSH plus thyroid uptakeC. TSH followed by free thyroxine index (FT4)D. Thyroid binding globulin plus T4

2. Which of the following investigators was the firstto use a proteolytic enzyme to digest tissue forimmunochemical staining?A. HuangB. SternbergerC. GuesdonD. Coons

3. The phrase “analyte specific antigen” wasdesignated by which of the following regulatorygroups to replace the earlier designation “researchuse only”?A. Clinical Laboratory Improvement Amendment CommitteeB. College of American PathologistsC. US Food and Drug AdministrationD. National Committee for Clinical Laboratory Standards

4. Which cardiac marker appears first following anacute myocardial infarction?A. Creatine kinase (CK)B. CK-MBC. HomocysteineD. Myoglobin

5. Which of the following microbial antigens can bedetected with direct microbial methods?A. Capsular proteinsB. Viral cell wallC. Cell wall componentsD. Inert polysaccharide

6. What is the most common use of flow cytometry inclinical medicine?A. Enzyme immunoassays for autoantibodiesB. Immunophenotyping of cell populationsC. Multiple drug resistance analysisD. Bacteria detection

7. Detection of IgM antibodies specific for aparticular pathogen in serum from a neonateindicatesA. 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 processoccurring in the body both naturally and afterchemotherapy for malignancies. What is anotherterm for programmed cell death?A. LysisB. NecrosisC. ApoptosisD. Inflammation

9. Which cells are required for transplantation toreconstitute bone marrow after intensivechemotherapy?A. LymphocytesB. RBCsC. MonocytesD. CD34-positive stem cells

10. What common feature of reticulocytes andreticulated platelets can be measured with flowcytometry using a single stain?A. DNA contentB. Cell surface markersC. RNA contentD. Hemoglobin content

CONTINUING EDUCATION UPDATE EXAMImmunology (001)

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