Flow cytometry: Principles and clinical applications in hematology.
Flow cytometry measures optical and fluorescence characteristics of single cells (or any other particle, including nuclei, microorganisms, chromosome preparations, and latex beads). Physical properties, such as size (represented by forward angle light scatter) and internal complexity (represented by right-angle scatter) can resolve certain cell populations. Fluorescent dyes may bind or intercalate with different cellular components such as DNA or RNA. Additionally, antibodies conjugated to fluorescent dyes can bind specific proteins on cell membranes or inside cells. When labeled cells are passed by a light source, the fluorescent molecules are excited to a higher energy state. Upon returning to their resting states, the fluorochromes emit light energy at higher wavelengths. The use of multiple fluorochromes, each with similar excitation wavelengths and different emission wavelengths (or "colors"), allows several cell properties to be measured simultaneously. Commonly used dyes include propidium iodide, phycoerythrin, and fluorescein, although many other dyes are available. Tandem dyes with internal fluorescence resonance energy transfer can create even longer wavelengths and more colors. Table 1 lists clinical applications and cellular characteristics that are commonly measured. Several excellent texts and reviews are available (1-6).
Inside a flow cytometer, cells in suspension are drawn into a stream created by a surrounding sheath of isotonic fluid that creates laminar flow, allowing the cells to pass individually through an interrogation point. At the interrogation point, a beam of monochromatic light, usually from a laser, intersects the cells. Emitted light is given off in all directions and is collected via optics that direct the light to a series of filters and dichroic mirrors that isolate particular wavelength bands. The light signals are detected by photomultiplier tubes and digitized for computer analysis. Fig. 1 is a schematic diagram of the fluidic and optical components of a flow cytometer. The resulting information usually is displayed in histogram or two-dimensional dot-plot formats.
DNA Content Analysis
The measurement of cellular DNA content by flow cytometry uses fluorescent dyes, such as propidium iodide, that intercalate into the DNA helical structure. The fluorescent signal is directly proportional to the amount of DNA in the nucleus and can identify gross gains or losses in DNA. Abnormal DNA content, also known as "DNA content aneuploidy'", can be determined in a tumor cell population. DNA aneuploidy generally is associated with malignancy; however, certain benign conditions may appear aneuploid (7-12). DNA aneuploidy correlates with a worse prognosis in many types of cancer but is associated with improved survival in rhabdomyosarcoma, neuroblastoma, multiple myeloma, and childhood acute lymphoblastic leukemia (ALL)  (11,13-16). In multiple myeloma, ALL, and myelodysplastic syndromes, hypodiploid tumors cells portend a poor prognosis. In contrast, hyperdiploid cells in ALL have a better prognosis (11,13). For many hematologic malignancies, there are conflicting reports regarding the independent prognostic value of DNA content analysis. Although conventional cytogenetics can detect smaller DNA content differences, flow cytometry allows more rapid analysis of a larger number of cells.
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Immunophenotyping Applications in Hematology
The distributed nature of the hematopoietic system makes it amenable to flow cytometric analysis. Many surface proteins and glycoproteins on erythrocytes, leukocytes, and platelets have been studied in great detail. The availability of monoclonal antibodies directed against these surface proteins permits flow cytometric analysis of erythrocytes, leukocytes, and platelets. Antibodies against intracellular proteins such as myeloperoxidase and terminal deoxynucleotidyl transferase are also commercially available and permit analysis of an increasing number of intracellular markers.
The use of flow cytometry for the detection and quantification of fetal red cells in maternal blood has increased in recent years. Currently in the United States, rhesus D-negative women receive prophylactic Rh-immune globulin at 28 weeks and also within 72 h of delivery (17). The standard single dose is enough to prevent alloimmunization from ~15 mL of fetal rhesus D+ red cells. If fetomaternal hemorrhage is suspected, the mother's blood is tested for the presence and quantity of fetal red cells, and an appropriate amount of Rh-immune globulin is administered. The quantitative test most frequently used in clinical laboratories is the Kleihauer-Betke acid-elution test. This test is fraught with interobserver and interlaboratory variability, and is tedious and time-consuming (18). The use of flow cytometry for the detection of fetal cells is much more objective, reproducible, and sensitive than the Kleihauer-Betke test (19-21). Fluorescently labeled antibodies to the rhesus (D) antigen can be used, or more recently, antibodies directed against hemoglobin F (19-27). This intracellular approach, which uses permeabilization of the red cell membrane and an antibody to the [gamma] chain of human hemoglobin, is precise and sensitive (21). This method has the ability to distinguish fetal cells from F-cells (adult red cells with small amounts of hemoglobin F). Fig. 2 is a histogram of a positive test for feto-maternal hemorrhage. Although the flow cytometry method is technically superior to the Kleihauer-Betke test, cost, instrument availability, and stat access may limit its practical utility.
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder that leads to intravascular hemolysis with associated thrombotic and infectious complications. PNH can arise in the setting of aplastic anemia and may be followed by acute leukemia (28). The disease is caused by deficient biosynthesis of a glycosylphosphatidylinositol linker that anchors several complement and immunoregulatory surface proteins on erythrocytes, monocytes, neutrophils, lymphocytes, and platelets (28-31). On erythrocytes, deficiencies of decay-accelerating factor and membrane-inhibitor of reactive lysis render red cells susceptible to complement-mediated lysis (30, 31). Conventional laboratory tests for the diagnosis of PNH include the sugar water test and the Ham's acid hemolysis test (32). Problems associated with these tests include stringent specimen requirements and limited specificity. Antibodies to CD55 and CD59 are specific for decay-accelerating factor and membrane-inhibitor of reactive lysis, respectively, and can be analyzed by flow cytometry to make a definitive diagnosis of PNH (29, 3335). In affected patients, two or more populations of erythrocytes can be readily identified, with different degrees of expression of CD55 and CD59 (Fig. 3)
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Reticulocyte counts are based on identification of residual ribosomes and RNA in immature nonnucleated red blood cells (RBCs). Traditionally, a blood smear is stained with a dye that precipitates the nucleic acid, and the cells are counted manually (36). This method is subjective, imprecise, labor-intensive, and tedious. The flow cytometric enumeration of reticulocytes uses fluorescent dyes that bind the residual RNA, such as thiazole orange (37, 38). This method provides excellent discrimination between reticulocytes and mature RBCs, with greater precision, sensitivity, and reproducibility than the traditional method (37, 38). However, Howell-Jolley bodies (a remnant of nuclear DNA) are not distinguished from reticulocytes (39). Because the fluorescence intensity is directly proportional to the amount of RNA and related to the immaturity of the RBC, a reticulocyte maturity index has been used clinically to assess bone marrow engraftment and erythropoietic activity and to help classify anemas (34, 38, 40, 41). Some current automated cell counters use similar technology to estimate reticulocyte counts (42).
In the blood bank, flow cytometry can be used as a complementary or replacement test for red cell immunology, including RBC-bound immunoglobulins and red cell antigens (43). In multiply transfused patients, determining the recipient's blood type can be very difficult. Flow cytometry has been used to accurately identify and phenotype the recipient's red cells (44). Flow cytometry is being used increasingly in the blood bank to assess leukocyte contamination in leukocyte-reduced blood products (45, 46).
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Immunologic monitoring of HIV-infected patients is a mainstay of the clinical flow cytometry laboratory. HIV infects helper/inducer T lymphocytes via the CD4 antigen. Infected lymphocytes may be lysed when new virions are released or may be removed by the cellular immune system. As HIV disease progresses, CD4-positive T lymphocytes decrease in total number. The absolute CD4 count provides a powerful laboratory measurement for predicting, staging, and monitoring disease progression and response to treatment in HIV-infected individuals. Quantitative viral load testing is a complementary test for clinical monitoring of disease and is correlated inversely to CD4 counts (47, 48). However, CD4 counts directly assess the patient's immune status and not just the amount of virus. It is likely that both CD4 T-cell enumeration and HIV viral load will continue to be used for diagnosis, prognosis, and therapeutic management of HIV-infected persons.
Perhaps the best example of simultaneous analysis of multiple characteristics by flow cytometry involves the immunophenotyping of leukemias and lymphomas. Immunophenotyping as part of the diagnostic work-up of hematologic malignancies offers a rapid and effective means of providing a diagnosis. The ability to analyze multiple cellular characteristics, along with new antibodies and gating strategies, has substantially enhanced the utility of flow cytometry in the diagnosis of leukemias and lymphomas. Different leukemias and lymphomas often have subtle differences in their antigen profiles that make them ideal for analysis by flow cytometry. Diagnostic interpretations depend on a combination of antigen patterns and fluorescence intensity. Several recent review articles are available (49-60). Flow cytometry is very effective in distinguishing myeloid and lymphoid lineages in acute leukemias and minimally differentiated leukemias. Additionally, CD45/side scatter gating often can better isolate the blast population for more definitive phenotyping than is possible with forward scatter/side scatter gating. Fig. 4 is an example of CD45/side scatter gating for an acute myeloid leukemia. Although most acute myeloid leukemias are difficult to classify by phenotype alone, flow cytometry can be useful in distinguishing certain acute myeloid leukemias, such as acute promyelocytic leukemia (61, 62). Flow cytometry can also be used to identify leukemias that may be resistant to therapy (63). In ALL, phenotype has been shown to correlate strongly with outcome (64, 65).
The B-cell lymphoproliferative disorders often have specific antigen patterns. The use of a wide range of antibodies allows clinicians to make specific diagnoses based on patterns of antigen expression. Table 2 lists some of the common phenotypes expressed by various B-cell lymphoproliferative disorders. Not only is the presence or absence of antigens useful in making specific diagnoses, the strength of antigen expression can also aid in diagnosis. One example is the weak expression of CD20 and immunoglobulin light chains commonly seen in chronic lymphocytic leukemia. Flow cytometry is particularly good at identifying clonality in B-cell populations. Although T-cell neoplasms may exhibit a predominance of antigens CD4 or CD8, these antigens should not be considered as surrogate markers of clonality. The use of antibodies to the T-cell receptor family may occasionally be helpful in a small percentage of cases; however, many reactive processes can show expansion of particular T-cell receptor clones (66-69). Antigen deletions are common in T-cell lymphomas and may suggest neoplasia, but the only way to definitively diagnose T-cell clonality is by molecular methods. Flow cytometry can be used for lymphoma phenotyping of fine needle aspirates, and is a powerful adjunct to cytologic diagnosis (70). The high sensitivity and capacity for simultaneous analysis of multiple characteristics make flow cytometry useful for the detection of minimal residual disease, especially if abnormal patterns of antigen expression are present (71-75). Flow cytometry is not recommended for the diagnosis of Hodgkin lymphoma, chronic myelogenous leukemia, or myelodysplastic syndrome, although disease progression in the latter two conditions can often be monitored.
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Neutropenia may be immune or nonimmune in nature. The work-up frequently entails a bone marrow examination. Immune neutropenia may result from granulocyte-specific autoantibodies, granulocyte-specific alloantibodies, or transfusion-related anti-HLA antibodies. Flow cytometry can readily identify anti-neutrophil antibodies that are either bound to granulocytes or free in plasma (76). Autoimmune neutropenias may develop in patients with autoimmune disorders such as Felty syndrome, systemic lupus erythematosus, and Hashimoto thyroiditis. When immune-related, flow cytometry can detect anti-neutrophil antibodies and confirm the origin of neutropenia, possibly eliminating the need for a bone marrow procedure. Conversely, the absence of anti-neutrophil antibodies narrows the differential diagnosis to nonimmune causes such as bone marrow failure, myelodysplasia, or marrow-infiltrative processes.
Functional deficiencies of leukocytes can be assessed by flow cytometry. Assays for oxidative burst, phagocytosis, opsonization, adhesion, and structure are available (77). One clinical example measures neutrophil adhesion molecules central to a diagnosis of leukocyte adhesion deficiency syndrome type I (78). This syndrome is characterized by an immunodeficiency related to defective neutrophil and monocyte migration to sites of inflammation. The disorder is caused by a congenital deficiency of the leukocyte [beta]2 integrin receptor complex (CD11/CD18 antigen complex) on the myeloid cell surface. This receptor complex binds endothelial cell ligands such as intercellular adhesion molecule-1 (CD54 antigen), which is necessary for neutrophil adherence and transendothelial migration (78, 79). Flow cytometry can be used to identify neutrophils that lack the CD11/CD18 antigen complex to establish a diagnosis that is otherwise difficult to make.
The analysis of platelets by flow cytometry is becoming more common in both research and clinical laboratories. Platelet-associated immunoglobulin assays by flow cytometry can be direct or indirect assays, similar to other platelet-associated immunoglobulin immunoassays. In autoimmune thrombocytopenic purpura, free serum antibodies are not found as frequently as platelet-bound antibodies (80-83). In contrast, in cases of alloantibody formation, serum antibodies may be detected without evidence of platelet-associated antibodies (84). Flow cytometry is an excellent method for direct analysis of platelet-bound antibodies, and it has also been shown to be of benefit in detection of free plasma antibodies (81, 85).
The use of thiazole orange, a fluorescent dye that binds RNA, allows immature platelets (also referred to as reticulated platelets) to be quantified (86-88). The reticulated platelet count can be used to determine the rate of thrombopoiesis. This measurement can separate unexplained thrombocytopenias into those with increased destruction and those with defects in platelet production.
The pathogenesis and molecular defects of many primary thrombocytopathies are well known and relate to defects in structural or functional glycoproteins, such as the abnormal expression of gpIIb/IIIa in Glanzmann thrombasthenia and gpIb in Bernard-Soulier disease (8994). Flow cytometry is a rapid and useful method of obtaining a diagnosis.
Until recently, functional analysis of platelet activation was used primarily in research. Many immunological markers of platelet activation have been described, and the commercial availability of antibodies permits flow cytometric determination of platelet activation (95-97). Platelet activation may be clinically important in stored blood components, after cardiopulmonary bypass and renal dialysis, and in the treatment of patients with myocardial infarction or thrombotic events.
Qualification of Soluble Molecules
Soluble antigens or antibodies can be quantified by flow cytometry if standard cells or beads are used. For example, OKT3 is a mouse anti-human antibody useful in treating transplant rejection. Circulating concentrations of OKT3 can be quantified by incubating with normal CD3positive lymphocytes, followed by a fluorescently labeled anti-mouse antibody (98). Fluorescence values are compared to a calibration curve generated with known amounts of OKT3. Recently, multiplex assays for several antigens have become possible by the use of beads indexed by incorporating two different dyes (99-102).
Flow cytometry is a powerful technique for correlating multiple characteristics on single cells. This qualitative and quantitative technique has made the transition from a research tool to standard clinical testing. Applications in hematology include DNA content analysis, leukemia and lymphoma phenotyping, immunologic monitoring of HIV-infected individuals, and assessment of structural and functional properties of erythrocytes, leukocytes, and platelets. Smaller, less expensive instruments and an increasing number of clinically useful antibodies are creating more opportunities for routine clinical laboratories to use flow cytometry in the diagnosis and management of disease.
(1.) Keren DF, Hanson CA, Hurtubise PE. Flow cytometry and clinical diagnosis. Chicago: ASCP Press, 1994:664 pp.
(2.) Shapiro H. Practical flow cytometry, 3rd ed. New York: Wiley-Liss, 1995:542 pp.
(3.) Orfao A, Ruiz-Arguelles A, Lacombe F, Ault K, Basso G, Danova M. Flow cytometry: its applications in hematology. Haematologica 1995;80:69-81.
(4.) Recktenwald DJ. Introduction to flow cytometry: principles, fluorochromes, instrument set-up, calibration. J Hematother 1993; 2:387-94.
(5.) Watson JV. The early fluidic and optical physics of cytometry. Cytometry 1999;38:1-14.
(6.) Mandy FF, Bergeron M, Minkus T. Principles of flow cytometry. Transfus Sci 1995;16:303-14.
(7.) Joensuu H, Klemi PJ. DNA aneuploidy in adenomas of endocrine organs. Am J Pathol 1988;132:145-51.
(8.) Joensuu H, Klemi P, Eerola E. DNA aneuploidy in follicular adenomas of the thyroid gland. Am J Pathol 1986;124:373-6.
(9.) Hedley DW, Shankey TV, Wheeless LL. DNA cytometry consensus conference. Cytometry 1993;14:471.
(10.) Friedlander ML, Hedley DW, Taylor IW. Clinical and biological significance of aneuploidy in human tumours. J Clin Pathol 1984;37:961-74.
(11.) Duque RE, Andreeff M, Braylan RC, Diamond LW, Peiper SC. Consensus review of the clinical utility of DNA flow cytometry in neoplastic hematopathology. Cytometry 1993;14:492-6.
(12.) Albro J, Bauer KD, Hitchcock CL, Wittwer CT. Improved DNA content histograms from formalin-fixed, paraffin-embedded liver tissue by proteinase K digestion. Cytometry 1993;14:673-8.
(13.) Look AT, Roberson PK, Williams DL, Rivera G, Bowman WP, Pui CH, et al. Prognostic importance of blast cell DNA content in childhood acute lymphoblastic leukemia. Blood 1985;65:1079-86.
(14.) Dressier LG, Bartow SA. DNA flow cytometry in solid tumors: practical aspects and clinical applications. Semin Diagn Pathol 1989;6:55-82.
(15.) Gunawan B, Fuzesi L, Granzen B, Keller U, Mertens R, Steinau G, et al. Clinical aspects of alveolar rhabdomyosarcoma with translocation t(1;13)(p36;g14) and hypotetraploidy. Pathol Oncol Res 1999; 5:211-3.
(16.) Gollin SM, Janecka IP. Cytogenetics of cranial base tumors. J Neurooncol 1994;20:241-54.
(17.) Hartwell EA. Use of Rh immune globulin: ASCP practice parameter. American Society of Clinical Pathologists [see comments]. Am J Clin Pathol 1998;110:281-92.
(18.) Polesky HF, Sebring ES. Evaluation of methods for detection and quantitation of fetal cells and their effect on RhIgG usage. Am J Clin Pathol 1981;76:525-9.
(19.) Bayliss KM, Kueck BD, Johnson ST, Fueger JT, McFadden PW, Mikulski D, et al. Detecting fetomaternal hemorrhage: a comparison of five methods [see comments]. Transfusion 1991;31: 303-7.
(20.) Bromilow IM, Duguid JK. Measurement of feto-maternal haemorrhage: a comparative study of three Kleihauer techniques and two flow cytometry methods. Clin Lab Haematol 1997;19:13742.
(21.) Davis BH, Olsen S, Bigelow NC, Chen JC. Detection of fetal red cells in fetomaternal hemorrhage using a fetal hemoglobin monoclonal antibody by flow cytometry. Transfusion 1998;38: 749-56.
(22.) Navenot JM, Merghoub T, Ducrocq R, Muller JY, Krishnamoorthy R, Blanchard D. New method for quantitative determination of fetal hemoglobin-containing red blood cells by flow cytometry: application to sickle-cell disease. Cytometry 1998;32:186-90.
(23.) Campbell TA, Ware RE, Mason M. Detection of hemoglobin variants in erythrocytes by flow cytometry. Cytometry 1999;35: 242-8.
(24.) Johnson PR, Tait RC, Austin EB, Shwe KH, Lee D. Flow cytometry in diagnosis and management of large fetomaternal haemorrhage [see comments]. J Clin Pathol 1995;48:1005-8.
(25.) Oosterwijk JC, Knepfle CF, Mesker WE, Vrolijk H, Sloos WC, Pattenier H, et al. Strategies for rare-event detection: an approach for automated fetal cell detection in maternal blood. Am J Hum Genet 1998;63:1783-92.
(26.) Oostenvijk JC, Mesker WE, Ouwerkerk-van Velzen MC, Knepfle CF, Wiesmeijer KC, Beverstock GC, et al. Fetal cell detection in maternal blood: a study in 236 samples using erythroblast morphology, DAB and HbFstaining, and FISH analysis. Cytometry 1998;32:178-85.
(27.) Nance SJ, Nelson JM, Arndt PA, Lam HC, Garratty G. Quantitation of fetal-maternal hemorrhage by flow cytometry. A simple and accurate method. Am J Clin Pathol 1989;91:288-92.
(28.) Fores R, Alcocer M, Diez-Martin JL, Fernandez MN. Flow cytometric analysis of decay-accelerating factor (CD55) on neutrophils from aplastic anaemia patients. Br J Haematol 1995;90:728-30.
(29.) Hall SE, Rosse WF. The use of monoclonal antibodies and flow cytometry in the diagnosis of paroxysmal nocturnal hemoglobinuria. Blood 1996;87:5332-40.
(30.) Yamada N, Miyata T, Maeda K, Kitani T, Takeda J, Kinoshita T. Somatic mutations of the PIG-A gene found in Japanese patients with paroxysmal nocturnal hemoglobinuria. Blood 1995;85:885-92.
(31.) Rotoli B, Boccuni P. The PIG-A gene somatic mutation responsible for paroxysmal nocturnal hemoglobinuria. Haematologica 1995;80:539-45.
(32.) Elghetany M, Davey FR. Erythrocytic disorders. In: Henry JB, ed. Clinical diagnosis and management by laboratory methods, 19th ed. Philadelphia: WB Saunders, 1996:617-63.
(33.) Iwamoto N, Kawaguchi T, Nagakura S, Hidaka M, Horikawa K, Kagimoto T, et al. Markedly high population of affected reticulocytes negative for decay-accelerating factor and CD59 in paroxysmal nocturnal hemoglobinuria. Blood 1995;85:2228-32.
(34.) Ware RE, Rosse WF, Hall SE. Immunophenotypic analysis of reticulocytes in paroxysmal nocturnal hemoglobinuria. Blood 1995;86:1586-9.
(35.) Alfinito F, Del Vecchio L, Rocco S, Boccuni P, Musto P, Rotoli B. Blood cell flow cytometry in paroxysmal nocturnal hemoglobinuria: a tool for measuring the extent of the PNH clone. Leukemia 1996;10:1326-30.
(36.) Hutchison RE, Davey FR. Hematopoiesis. In: Henry JB, ed. Clinical diagnosis and management by laboratory methods, 19th ed. Philadelphia: WB Saunders, 1996:594-616.
(37.) Davis BH, Bigelow NC. Flow cytometric reticulocyte quantification using thiazole orange provides clinically useful reticulocyte maturity index. Arch Pathol Lab Med 1989;113:684-9.
(38.) Davis BH, Ornvold K, Bigelow NC. Flow cytometric reticulocyte maturity index: a useful laboratory parameter of erythropoietic activity in anemia. Cytometry 1995;22:35-9.
(39.) Corash L, Rheinschmidt M, Lieu S, Meers P, Brew E. Enumeration of reticulocytes using fluorescence-activated flow cytometry. Pathol Immunopathol Res 1988;7:381-94.
(40.) Lazarus HM, Chahine A, Lacerna K, Wamble A, laffaldano C, Straight M, et al. Kinetics of erythrogenesis after bone marrow transplantation. Am J Clin Pathol 1992;97:574-83.
(41.) Davis BH, Bigelow N, Ball ED, Mills L, Cornwell GG. Utility of flow cytometric reticulocyte quantification as a predictor of engraftment in autologous bone marrow transplantation. Am J Hematol 1989;32:81-7.
(42.) Kickler TS. Clinical analyzers. Advances in automated cell counting. Anal Chem 1999;71:3638-5R.
(43.) Garratty G, Arndt PA. Applications of flow cytofluorometry to red blood cell immunology [In Process Citation]. Cytometry 1999;38: 259-67.
(44.) Griffin GD, Lippert LE, Dow NS, Berger TA, Hickman MR, Salata KF. A flow cytometric method for phenotyping recipient red cells following transfusion [see comments]. Transfusion 1994;34: 233-7.
(45.) Lombardo JF, Cusack NA, Rajagopalan C, Sangaline RJ, Ambruso DR. Flow cytometric analysis of residual white blood cell concentration and platelet activation antigens in double filtered platelet concentrates. J Lab Clin Med 1993;122:557-66.
(46.) Barclay R, Walker B, Allan R, Reid C, Duffin E, Kane E, et al. Flow cytometric determination of residual leucocytes in filter-depleted blood products: an evaluation of Becton-Dickinson's LeucoCOUNT system. Transfus Sci 1998;19:399-403.
(47.) Saag MS, Holodniy M, Kuritzkes DR, O'Brien WA, Coombs R, Poscher ME, et al. HIV viral load markers in clinical practice. Nat Med 1996;2:625-9.
(48.) Mellors JW, Rinaldo CR Jr, Gupta P, White RM, Todd JA, Kingsley LA. Prognosis in HIV-1 infection predicted by the quantity of virus in plasma [published erratum appears in Science 1997;275:14]. Science 1996;272:1167-70.
(49.) Khalidi HS, Medeiros U, Chang KL, Brynes RK, Slovak ML, Arber DA. The immunophenotype of adult acute myeloid leukemia: high frequency of lymphoid antigen expression and comparison of immunophenotype, French-American-British classification, and karyotypic abnormalities. Am J Clin Pathol 1998;109:211-20.
(50.) Deegan MJ. Membrane antigen analysis in the diagnosis of lymphoid leukemias and lymphomas. Differential diagnosis, prognosis as related to immunophenotype, and recommendations for testing. Arch Pathol Lab Med 1989;113:606-18.
(51.) OrFao A, Schmitz G, Brando B, Ruiz-Arguelles A, Basso G, Braylan R, et al. Clinically useful information provided by the flow cytometric immunophenotyping of hematological malignancies: current status and future directions. Clin Chem 1999;45:1708-17.
(52.) Dunphy CH. Comprehensive review of adult acute myelogenous leukemia: cytomorphological, enzyme cytochemical, flow cytometric immunophenotypic, and cytogenetic findings. J Clin Lab Anal 1999;13:19-26.
(53.) Ruiz-Arguelles A, Duque RE, OrFao A. Report on the first Latin American Consensus Conference for Flow Cytometric Immunophenotyping of Leukemia. Cytometry 1998;34:39-42.
(54.) DiGiuseppe JA, Borowitz MJ. Clinical utility of flow cytometry in the chronic lymphoid leukemias. Semin Oncol 1998;25:6-10.
(55.) Davis BH, Foucar K, Szczarkowski W, Ball E, Witzig T, Foon KA, et al. U.S.-Canadian Consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry: medical indications. Cytometry 1997;30:249-63.
(56.) Braylan RC, Atwater SK, Diamond L, Hassett JM, Johnson M, Kidd PG, et al. U.S.-Canadian Consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry: data reporting. Cytometry 1997;30:245-8.
(57.) Borowitz MJ, Bray R, Gascoyne R, Melnick S, Parker JW, Picker L, et al. U.S.-Canadian Consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry: data analysis and interpretation. Cytometry 1997;30:23644.
(58.) Stewart CC, Behm FG, Carey JL, Cornbleet J, Duque RE, Hudnall SD, et al. U.S.-Canadian Consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry: selection of antibody combinations. Cytometry 1997; 30:231-5.
(59.) Stelzer GT, Marti G, Hurley A, McCoy P Jr, Lovett EJ, Schwartz A. U.S.-Canadian Consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry: standardization and validation of laboratory procedures. Cytometry 1997;30:214-30.
(60.) Jennings CD, Foon KA. Recent advances in flow cytometry: application to the diagnosis of hematologic malignancy. Blood 1997;90:2863-92.
(61.) Erber WN, Asbahr H, Rule SA, Scott CS. Unique immunophenotype of acute promyelocytic leukaemia as defined by CD9 and CD68 antibodies. Br J Haematol 1994;88:101-4.
(62.) Stone RM, Mayer RJ. The unique aspects of acute promyelocytic leukemia. J Clin Oncol 1990;8:1913-21.
(63.) Maslak P, Hegewisch-Becker S, Godfrey L, Andreeff M. Flow cytometric determination of the multidrug-resistant phenotype in acute leukemia. Cytometry 1994;17:84-93.
(64.) Farhi DC, Rosenthal NS. Acute lymphoblastic leukemia. Clin Lab Med 2000;20:17-28,vii.
(65.) Bene MC, Bernier M, Castoldi G, Faure GC, Knapp W, Ludwig WD, et al. Impact of immunophenotyping on management of acute leukemias. Haematologica 1999;84:1024-34.
(66.) Tbakhi A, Edinger M, Myles J, Pohlman B, Tubbs RR. Flow cytometric immunophenotyping of non-Hodgkin's lymphomas and related disorders. Cytometry 1996;25:113-24.
(67.) Sallah S, Smith SV, Lony LC, Woodard P, Schmitz JL, Folds JD. Gamma/delta T-cell hepatosplenic lymphoma: review of the literature, diagnosis by flow cytometry and concomitant autoimmune hemolytic anemia. Ann Hematol 1997;74:139-42.
(68.) Soudeyns H, Champagne P, Holloway CL, Silvestri GU, Ringuette N, Samson J, et al. Transient T cell receptor beta-chain variable region-specific expansions of CD4+ and CD8+ T cells during the early phase of pediatric human immunodeficiency virus infection: characterization of expanded cell populations by T cell receptor phenotyping. J Infect Dis 2000;181:107-20.
(69.) Martins EB, Graham AK, Chapman RW, Fleming KA. Elevation of gamma delta T lymphocytes in peripheral blood and livers of patients with primary sclerosing cholangitis and other autoimmune liver diseases. Hepatology 1996;23:988-93.
(70.) Darzynkiewicz Z. Flow cytometry in cytopathology. Overview and perspectives. Anal Quant Cytol Histol 1988;10:459-61.
(71.) Baer MR. Assessment of minimal residual disease in patients with acute leukemia. Curr Opin Oncol 1998;10:17-22.
(72.) Campana D, Coustan-Smith E, Janossy G. The immunologic detection of minimal residual disease in acute leukemia [published erratum appears in Blood 1990;76:1901]. Blood 1990; 76:163-71.
(73.) Nagler A, Condiotti R, Rabinowitz R, Schlesinger M, Nguyen M, Terstappen LW. Detection of minimal residual disease (MRD) after bone marrow transplantation (BMT) by multi-parameter flow cytometry (MPFC). Med Oncol 1999;16:177-87.
(74.) OrFao A, Ciudad J, Lopez-Berges MC, Lopez A, Vidriales B, Caballero MD, et al. Acute lymphoblastic leukemia (ALL): detection of minimal residual disease (MRD) at flow cytometry. Leuk Lymphoma 1994;13:87-90.
(75.) Yin JA, Tobal K. Detection of minimal residual disease in acute myeloid leukaemia: methodologies, clinical and biological significance. Br J Haematol 1999;106:578-90.
(76.) Rubinstein DB, Farrington GK, O'Donnell C, Hartman KR, Wright DG. Autoantibodies to leukocyte alphaMbeta2 integrin glycoproteins in HIV infection. Clin Immunol 1999;90:352-9.
(77.) van Eeden SF, Klut ME, Walker BA, Hogg JC. The use of flow cytometry to measure neutrophil function. J Immunol Methods 1999;232:23-43.
(78.) Todd RFd, Freyer DR. The CD11/CD18 leukocyte glycoprotein deficiency. Hematol Oncol Clin N Am 1988;2:13-31.
(79.) Mazzone A, Ricevuti G. Leukocyte CD11/CD18 integrins: biological and clinical relevance. Haematologica 1995;80:161-75.
(80.) Kokawa T, Nomura S, Yanabu M, Yasunaga K. Detection of platelet antigen for antiplatelet antibodies in idiopathic thrombocytopenic purpura by flow cytometry, antigen-capture ELISA, and immunoblotting: a comparative study. Eur J Haematol 1993;50: 74-80.
(81.) Rosenfeld CS, Bodensteiner DC. Detection of platelet alloantibodies by flow cytometry. Characterization and clinical significance. Am J Clin Pathol 1986;85:207-12.
(82.) Stockelberg D, Hou M, Jacobsson S, Kutti J, Wadenvik H. Detection of platelet antibodies in chronic idiopathic thrombocytopenic purpura (ITP). A comparative study using flow cytometry, a whole platelet ELISA, and an antigen capture ELISA. Eur J Haematol 1996; 56:72-7.
(83.) Tazzari PL, Ricci F, Vianelli N, Tassi C, Belletti D, Pierri I, et al. Detection of platelet-associated antibodies by flow cytometry in hematological autoimmune disorders. Ann Hematol 1995;70: 267-72.
(84.) Legler TJ, Fischer I, Dittmann J, Samson G, Lynen R, Humpe A, et al. Frequency and causes of refractoriness in multiply transfused patients. Ann Hematol 1997;74:185-9.
(85.) Gonzalez-Conejero R, Rivera J, Rosillo MC, Lozano ML, Garcia VV. Comparative study of three methods to detect free plasma antiplatelet antibodies. Acta Haematol 1996;96:135-9.
(86.) Bonan JL, Rinder HM, Smith BR. Determination of the percentage of thiazole orange (TO)-positive, "reticulated" platelets using autologous erythrocyte TO fluorescence as an internal standard. Cytometry 1993;14:690-4.
(87.) Kienast J, Schmitz G. Flow cytometric analysis of thiazole orange uptake by platelets: a diagnostic aid in the evaluation of thrombocytopenic disorders. Blood 1990;75:116-21.
(88.) Rinder HM, Munz UJ, Ault KA, Bonan JL, Smith BR. Reticulated platelets in the evaluation of thrombopoietic disorders. Arch Pathol Lab Med 1993;117:606-10.
(89.) Sharp WJ, Khanduri UD, Christie BS. Rapid heterozygote detection in Glanzmann's thrombasthenia. Br J Haematol 1998;101: 66-9.
(90.) Ginsberg MH, Frelinger AL, Lam SC, Forsyth J, McMillan R, Plow EF, et al. Analysis of platelet aggregation disorders based on flow cytometric analysis of membrane glycoprotein Ilb-Illa with confor mation-specific monoclonal antibodies. Blood 1990;76:2017-23.
(91.) Cohn RJ, Sherman GG, Glencross DK. Flow cytometric analysis of platelet surface glycoproteins in the diagnosis of Bernard-Soulier syndrome. Pediatr Hematol Oncol 1997;14:43-50.
(92.) Yesilipek MA, Karadogan I, Undar L. Bernard-Soulier syndrome: a flow cytometric analysis of membrane GP-Ib expression. Turk J Pediatr 1996;38:375-9.
(93.) Bunescu A, Lindahl T, Solum N0, Schulman S, Larsson A, Lundahl J, et al. Partial expression of GP Ib measured by flow cytometry in two patients with Bernard-Soulier syndrome. Thromb Res 1994;76:441-50.
(94.) Tomer A, ScharF RE, McMillan R, Ruggeri ZM, Harker LA. Bernard-Soulier syndrome: quantitative characterization of megakaryocytes and platelets by flow cytometric and platelet kinetic measurements. Eur J Haematol 1994;52:193-200.
(95.) Michelson AD. Flow cytometry: a clinical test of platelet function. Blood 1996;87:4925-36.
(96.) Michelson AD, Furman MI. Laboratory markers of platelet activation and their clinical significance. Curr Opin Hematol 1999;6: 342-8.
(97.) Fanelli A, Bergamini C, Rapi S, Caldini A, Spinelli A, Buggiani A, et al. Flow cytometric detection of circulating activated platelets in primary antiphospholipid syndrome. Correlation with thrombo cytopenia and anticardiolipin antibodies. Lupus 1997;6:261-7.
(98.) Wittwer CT, Knape WA, Bristow MR, Gilbert EM, Renlund DG, O'Connell JB, et al. The quantitative flow cytometric plasma OKT3 assay. Its potential application in cardiac transplantation. Transplantation 1989;48:533-5.
(99.) Oliver KG, Kettman JR, Fulton RJ. Multiplexed analysis of human cytokines by use of the FIowMetrix system. Clin Chem 1998;44: 2057-60.
(100.) Smith PL, WaIkerPeach CR, Fulton RJ, DuBois DB. A rapid, sensitive, multiplexed assay for detection of viral nucleic acids using the FIowMetrix system. Clin Chem 1998;44:2054-6.
(101.) Gordon RF, McDade RL. Multiplexed quantification of human IgG, IgA, and IgM with the FlowMetrix system. Clin Chem 1997;43: 1799-801.
(102.) Fulton RJ, McDade RL, Smith PL, Kienker U, Kettman JR Jr. Advanced multiplexed analysis with the FlowMetrix system. Clin Chem 1997;43:1749-56.
MICHAEL BROWN and CARL WTTTWER *
Department of Pathology, University of Utah, ARUP Laboratories, Inc., Salt Lake City, UT 84132.
 Nonstandard abbreviations: ALL, acute lymphoblastic leukemia; PNH, paroxysmal nocturnal hemoglobinuria; and RBC, red blood cell.
* Address correspondence to this author at: Department of Pathology, University of Utah, 50 North Medical Dr., Salt Lake City, UT 84132. E-mail email@example.com.
Received February 15, 2000; accepted April 10, 2000.
Table 1. Common clinical uses of flow cytometry. Field Clinical application Common characteristic measured Immunology Histocompatibility IgG, IgM cross-matching Transplantation rejection CD3, circulating OKT3 HLA-B27 detection HLA-B27 Immunodeficiency studies CD4, CD8 Oncology DNA content and S phase of DNA tumors Measurement of Ki-67, PCNA (a) proliferation markers Hematology Leukemia and lymphoma Leukocyte surface phenotyping antigens Identification of prognostically important subgroups TdT, MPO Hematopoietic progenitor CD34 cell enumeration Diagnosis of systemic CD25, CD69 mastocytosis Reticulocyte enumeration RNA Autoimmune and alloimmune disorders Anti-platelet antibodies IgG, IgM Anti-neutrophil IgG antibodies Immune complexes Complement, IgG Feto-maternal hemorrhage Hemoglobin F, quantification rhesus D Blood banking Immunohematology Erythrocyte surface antigens Assessment of leukocyte Forward and side contamination of blood scatter, leukocyte products surface antigens Genetic disorders PNH CD55, CD59 Leukocyte adhesion CD11/CD18 complex deficiency (a) PCNA, proliferating cell nuclear antigen; TdT, terminal deoxynucleotidyltransferase; MPO, myeloperoxidase. Table 2. Common phenotypes of &cell lymphoproliferative disorders. Diagnosis CD5 CD10 CD19 CD20 CD23 SLL/CLL (a) + - + +(w) + Mantle cell lymphoma + - + + - Follicle center lymphoma - + + + -/+ Marginal zone lymphoma - - + + - Hairy cell leukemia - - + + - Diagnosis CD79b FMC-7 CD25 CD11C CD103 SLL/CLL (a) - - -/+ +/_ - Mantle cell lymphoma + + - - - Follicle center lymphoma +/- +/- - - - Marginal zone lymphoma +/- +/- -/+ + - Hairy cell leukemia +/- +/- +/- + + (a) SLL/CLL, small lymphocytic lymphoma/chronic lymphocytic leukemia; +, positive; -, negative; +/-, often positive; -/+, occasionally positive; w, weak expression.
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|Title Annotation:||Beckman Conference|
|Author:||Brown, Michael; Wtttwer, Carl|
|Date:||Aug 1, 2000|
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