Flow Cytometric Analysis of Hematologic Neoplasia

  • Raul C. Braylan
  • John B. Anderson
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 55)


Flow cytometry (FCM) is a powerful technology that allows the rapid analysis of cellular components such as surface and intracellular antigens, or DNA content. The measurements are fast and are based on optical signals emitted by cells labeled with fluorochromes as they flow suspended in a liquid medium through an intense laser beam. The signals include scattered light, which provides information on cell size and granularity, and fluorescence derived from dyes or fluorochrome-labeled antibodies bound to specific cell components.


Propidium Iodide Neoplastic Cell Newborn Calf Serum Nonviable Cell Intracellular Antigen 
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  1. 1.
    Braylan, R. C. (1993) Flow-cytometric DNA analysis in the diagnosis and prognosis of lymphoma. Am. J. Clin. Pathol. 99, 374–380.PubMedGoogle Scholar
  2. 2.
    Look, A. T., Roberson, P. K., Williams, D. L., Rivera, G., Bowman, W. P., Pui, C. H., Ochs, J., Abromowitch, M., Kalwinsky, D., and Dahl, G. V. (1985) Prognostic importance of blast cell DNA content in childhood acute lymphoblastic leukemia. Clin. Lab. Med. 65, 1079–1086.Google Scholar
  3. 3.
    Trueworthy, R., Shuster, J., Look, T., Crist, W., Borowitz, M., Carroll, A., Frankel, L., Harris, M., Wagner, H., and Haggard, M. (1992) Ploidy of lympho-blasts is the strongest predictor of treatment outcome in B-progenitor cell acute lymphoblastic leukemia of childhood: aPediatric Oncology Group study. J. Clin. Oncol. 10, 606–613.PubMedGoogle Scholar
  4. 4.
    Freedman, A. S. and Nadler, L. M. (1991) Immunologic markers in non-Hodgkin’s lymphoma. Hematol. Oncol. Clin. North Am. 5, 871–889.PubMedGoogle Scholar
  5. 5.
    Hurwitz, C. A., Loken, M. R., Graham, M. L., Karp, J. E., Borowitz, M. J., Pullen, D. J., and Civin, C. I. (1988) Asynchronous antigen expression in B lineage acute lymphoblastic leukemia. Clin. Lab Med. 72, 299–307.Google Scholar
  6. 6.
    Terstappen, L. W., Safford, M., Unterhalt, M., Konemann, S., Zurlutter, K., Piechotka, K., Drescher, M., Aul, C., Buchner, T., Hiddemann, W., et al. (1992) Flow cytometric characterization of acute myeloid leukemia: IV. Comparison to the differentiation pathway of normal hematopoietic progenitor cells. Leukemia 6, 993–1000.PubMedGoogle Scholar
  7. 7.
    Jennings, C. D. and Foon, K. A. (1997) Recent advances in flow cytometry: application to the diagnosis of hematologic malignancy. Clin. Lab. Med. 90, 2863–2892.Google Scholar
  8. 8.
    Cabezudo, E., Matutes, E., Ramrattan, M., Morilla, R., and Catovsky, D. (1997) Analysis of residual disease in chronic lymphocytic leukemia by flow cytometry. Leukemia 11, 1909–1914.CrossRefPubMedGoogle Scholar
  9. 9.
    Campana, D. (1994) Monitoring minimal residual disease in acute leukemia: expectations, possibilities and initial clinical results. Int. J. Clin. Lab. Res. 24, 132–138.CrossRefPubMedGoogle Scholar
  10. 10.
    Ciudad, J., San Miguel, J. F., Lopez-Berges, M. C., Garcia Marcos, M. A., Gonzalez, M., Vazquez, L., del Canizo, M. C., Lopez, A., van Dongen, J. J., and Orfao, A. (1999) Detection of abnormalities in B-cell differentiation pattern is a useful tool to predict relapse in precursor-B-ALL. Br. J. Haematol. 104, 695–705.Google Scholar
  11. 11.
    Drach, J., Gattringer, C., and Huber, H. (1991) Combined flow cytometric assessment of cell surface antigens and nuclear TdT for the detection of minimal residual disease in acute leukaemia. Br. J. Haematol. 77, 37–42.CrossRefPubMedGoogle Scholar
  12. 12.
    Dworzak, M. N., Stolz, F., Froschl, G., Printz, D., Henn, T., Fischer, S., Fleischer, C., Haas, O. A., Fritsch, G., Gadner, H., and Panzer-Grumayer, E. R. (1999) Detection of residual disease in pediatric B-cell precursor acute lymphoblastic leukemia by comparative phenotype mapping: a study of five cases controlled by genetic methods. Exp. Hematol. 27, 673–681.CrossRefPubMedGoogle Scholar
  13. 13.
    Farahat, N., Morilla, A., Owusu-Ankomah, K., Morilla, R., Pinkerton, C. R., Treleaven, J. G., Matutes, E., Powles, R. L., and Catovsky, D. (1998) Detection of minimal residual disease in B-lineage acute lymphoblastic leukaemia by quantitative flow cytometry. Br. J. Haematol. 101, 158–164.CrossRefPubMedGoogle Scholar
  14. 14.
    Letwin, B. W., Wallace, P. K., Muirhead, K. A., Hensler, G. L., Kashatus, W. H., and Horan, P. K. (1990) An improved clonal excess assay using flow cytometry and B-cell gating. Clin. Lab. Med. 75, 1178–1185.Google Scholar
  15. 15.
    Weir, E. G., Cowan, K., LeBeau, P., and Borowitz, M. J. (1999) A limited antibody panel can distinguish B-precursor acute lymphoblastic leukemia from normal B precursors with four color flow cytometry: implications for residual disease detection. Leukemia 13, 558–567.CrossRefPubMedGoogle Scholar
  16. 16.
    Warzynski, M. J., Podgurski, A. E., Boldt, D. M., and Otto, R. N. (1990) An automated method to prepare cell suspensions from human biopsy samples for immunophenotyping by flow cytometry. Am. J. Clin. Pathol. 93, 104–108.PubMedGoogle Scholar
  17. 17.
    Koistinen, P. (1987) Human peripheral blood and bone marrow cell separation using density gradient centrifugation on Lymphoprep and Percoll in haema-tological diseases. Scand. J. Clin. Lab. Invest. 47, 709–714.PubMedGoogle Scholar
  18. 18.
    Boyum, A. (1968) Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand. J. Clin. Lab. Invest. Suppl. 97, 77–89.PubMedGoogle Scholar
  19. 19.
    Renzi, P. and Ginns, L. C. (1987) Analysis of T cell subsets in normal adults. Comparison of whole blood lysis technique to Ficoll-Hypaque separation by flow cytometry. J. Immunol. Methods 98, 53–56.CrossRefPubMedGoogle Scholar
  20. 20.
    Tamul, K. R., Schmitz, J. L., Kane, K., and Folds, J. D. (1995) Comparison of the effects of Ficoll-Hypaque separation and whole blood lysis on results of immunophenotypic analysis of blood and bone marrow samples from patients with hematologic malignancies. Clin. Diagn. Lab. Immunol. 2, 337–342.PubMedGoogle Scholar
  21. 21.
    Ritchie, A. W., Gray, R. A., and Micklem, H. S. (1983) Right angle light scatter: a necessary parameter in flow cytofluorimetric analysis of human peripheral blood mononuclear cells. J. Immunol. Methods 64, 109–117.CrossRefPubMedGoogle Scholar
  22. 22.
    Wing, M. G., Montgomery, A. M., Songsivilai, S., and Watson, J. V. (1990) An improved method for the detection of cell surface antigens in samples of low viability using flow cytometry. J. Immunol. Methods 126, 21–27.CrossRefPubMedGoogle Scholar
  23. 23.
    Hassett, J. and Parker, J. (1995) Laboratory practices in reporting flow cytometry phenotyping results for leukemia/lymphoma specimens: results of a survey. Cytometry 22, 264–281.CrossRefPubMedGoogle Scholar
  24. 24.
    Stelzer, G. T., Marti, G., Hurley, A., McCoy, P., Jr., Lovett, E. J., and Schwartz, A. (1997) U.S.-Canadian Consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry: standardization and validation of laboratory procedures. Cytometry 30, 214–230.CrossRefPubMedGoogle Scholar
  25. 25.
    Rothe, G. and Schmitz, G. (1996) Consensus protocol for the flow cytometric immunophenotyping of hematopoietic malignancies. Working Group on Flow Cytometry and Image Analysis. Leukemia 10, 877–895.PubMedGoogle Scholar
  26. 26.
    Stewart, C. C., Behm, F. G., Carey, J. L., Cornbleet, J., Duque, R. E., Hudnall, S. D., Hurtubise, P. E., Loken, M., Tubbs, R. R., and Wormsley, S. (1997) U.S.-Canadian Consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry: selection of antibody combinations. Cytometry 30, 231–235.CrossRefPubMedGoogle Scholar
  27. 27.
    Lanza, F., Latorraca, A., Moretti, S., Castagnari, B., Ferrari, L., and Castoldi, G. (1997) Comparative analysis of different permeabilization methods for the flow cytometry measurement of cytoplasmic myeloperoxidase and lysozyme in normal and leukemic cells. Cytometry 30, 134–144.CrossRefPubMedGoogle Scholar
  28. 28.
    Knapp, W., Strobl, H., and Majdic, O. (1994) Flow cytometric analysis of cell-surface and intracellular antigens in leukemia diagnosis. Cytometry 18, 187–198.CrossRefPubMedGoogle Scholar
  29. 29.
    Groeneveld, K., te Marvelde, J. G., van den Beemd, M. W., Hooijkaas, H., and van Dongen, J. J. (1996) Flow cytometric detection of intracellular antigens for immunophenotyping of normal and malignant leukocytes. Leukemia 10, 1383–1389.PubMedGoogle Scholar
  30. 30.
    Janossy, G., Coustan Smith, E., and Campana, D. (1989) The reliability of cytoplasmic CD3 and CD22 antigen expression in the immunodiagnosis of acute leukemia: a study of 500 cases. Leukemia 3, 170–181.PubMedGoogle Scholar
  31. 31.
    Smets, L. A., Slater, R., van Wering, E. R., Does-van den Berg, A., Hart, A. A., Veerman, A. J., and Kamps, W. A. (1995) DNA index and %S-phase cells determined in acute lymphoblastic leukemia of children: a report from studies ALL V, ALL VI, and ALL VII (1979-1991) of the Dutch Childhood Leukemia Study Group and The Netherlands Workgroup on Cancer Genetics and Cytogenetics. Med. Pediatr. Oncol. 25, 437–444.CrossRefPubMedGoogle Scholar
  32. 32.
    Braylan, R. C., Benson, N. A., Nourse, V., and Kruth, H. S. (1982) Correlated analysis of cellular DNA, membrane antigens and light scatter of human lym-phoid cells. Cytometry 2, 337–343.CrossRefPubMedGoogle Scholar
  33. 33.
    Nowak, R., Oelschlaegel, U., Schuler, U., Zengler, H., Hofmann, R., Ehninger, G., and Andreeff, M. (1997) Sensitivity of combined DNA/immunophenotype flow cytometry for the detection of low levels of aneuploid lymphoblastic leukemia cells in bone marrow. Cytometry 30, 47–53.CrossRefPubMedGoogle Scholar
  34. 34.
    Pollice, A. A., McCoy, J. P., Jr., Shackney, S. E., Smith, C. A., Agarwal, J., Burholt, D. R., Janocko, L. E., Hornicek, F. J., Singh, S. G., and Hartsock, R. J. (1992) Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins. Cytometry 13, 432–444.CrossRefPubMedGoogle Scholar
  35. 35.
    San Miguel, J. F., Garcia-Sanz, R., Gonzalez, M., and Orfao, A. (1995) Immunophenotype and DNA cell content in multiple myeloma. Baillieres Clin. Haematol. 8, 735–759.CrossRefPubMedGoogle Scholar
  36. 36.
    Braylan, R. C., Benson, N. A., and Iturraspe, J. (1993) Analysis of lymphomas by flow cytometry. Current and emerging strategies. Ann. N. Y. Acad. Sci. 677, 364–378.CrossRefPubMedGoogle Scholar
  37. 37.
    Stelzer, G. T., Shults, K. E., and Loken, M. R. (1993) CD45 gating for routine flow cytometric analysis of human bone marrow specimens. Ann. N. Y. Acad. Sci. 677, 265–280.CrossRefPubMedGoogle Scholar
  38. 38.
    Borowitz, M. J., Guenther, K. L., Shults, K. E., and Stelzer, G. T. (1993) Immunophenotyping of acute leukemia by flow cytometric analysis. Use of CD45 and right-angle light scatter to gate on leukemic blasts in three-color analysis. Am. J. Clin. Pathol. 100, 534–540.PubMedGoogle Scholar
  39. 39.
    Lacombe, F., Durrieu, F., Briais, A., Dumain, P., Belloc, F., Bascans, E., Reiffers, J., Boisseau, M. R., and Bernard, P. (1997) Flow cytometry CD45 gating for immunophenotyping of acute myeloid leukemia. Leukemia 11, 1878–1886.CrossRefPubMedGoogle Scholar
  40. 40.
    Sun, T., Sangaline, R., Ryder, J., Gibbens, K., Rollo, C., Stewart, S., and Rajagopalan, C. (1997) Gating strategy for immunophenotyping of leukemia and lymphoma. Am. J. Clin. Pathol. 108, 152–157.PubMedGoogle Scholar
  41. 41.
    Rainer, R. O., Hodges, L., and Seltzer, G. T. (1995) CD 45 gating correlates with bone marrow differential. Cytometry 22, 139–145.CrossRefPubMedGoogle Scholar
  42. 42.
    Duque, R. E., Andreeff, M., Braylan, R. C., Diamond, L. W., and Peiper, S. C. (1993) Consensus review of the clinical utility of DNA flow cytometry in neo-plastic hematopathology. Cytometry 14, 492–496.CrossRefPubMedGoogle Scholar
  43. 43.
    Braylan, R. C., Benson, N. A., and Nourse, V. A. (1984) Cellular DNA of human neoplastic B-cells measured by flow cytometry. Cancer Res. 44, 5010–5016.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2001

Authors and Affiliations

  • Raul C. Braylan
    • 1
  • John B. Anderson
    • 1
  1. 1.Shands Hospital at the University of Florida College of MedicineGainesville

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