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How Flow Cytometry Can Work for You: Fundamentals of Flow Cytometry in Neoplastic Hematolymphoid Cell Populations

Posted September 4, 2012

Nichole L. Steidler, MD
CAP Surgical Pathology Committee

Flow cytometry is a powerful tool that rapidly interrogates entire cell populations for expression of cell surface or cytoplasmic antigens. Cells are labeled with multiple fluorescently tagged antibodies and are analyzed in single-file by a laser within the flow cytometer. Up to eight different fluorescent antibodies are used routinely in clinical laboratories to determine the immunophenotype of a cell population. Characterization by flow cytometry depends on cell size, complexity, and a combination of antigen patterns and intensities, which together define an immunophenotypic profile. Neoplastic cell populations of leukemias and lymphomas have subtle differences in their immunophenotypic profile, which make them ideal for flow cytometry. Specimens for flow cytometry include peripheral blood, bone marrow aspirates and cores, fine-needle aspirates, and fresh tissue biopsies, such as lymph nodes and body fluids. Submitted aspirates and tissues must be sterile and viable, and submission in culture media, such as RPMI, is best.

In acute leukemia, flow cytometric analysis is invaluable in the determination of blast lineage, myeloid or lymphoid. Recurrent genetic abnormalities in acute myeloid leukemias can sometimes be predicted by certain flow cytometric patterns, although cytogenetic and/or molecular studies must be performed for confirmation. Further, the immunophenotypic profile of an acute leukemia obtained at the time of diagnosis can act as a “fingerprint” and may be useful for detection of minimal residual disease after treatment. With adequate sample quality and viable cells, flow cytometry can detect leukemic cells at a lower detection limit of one in 10,000.

The role of flow cytometry in the evaluation of lymphoproliferative disorders is to differentiate mature B-cells and T-cells. Both the antigen expression pattern and intensity of markers are useful in establishing a diagnosis. Clonal proliferations are confirmed frequently by molecular studies, such as B-cell immunoglobulin or T-cell receptor gene rearrangements. Since T-cell clonality can occasionally be seen in some reactive processes, flow cytometry and molecular results must be correlated with morphology, immunohistochemistry, and clinical findings.

In the evaluation of plasma cell neoplasms, flow cytometry is used to differentiate plasma cells from lymphoid cells with plasmacytic differentiation. Plasma cells are fragile, and they often do not withstand the sample preparation process required to perform flow cytometry. Thus, enumeration of plasma cells by flow cytometry is frequently underestimated by up to 50% when compared to plasma cells identified on bone marrow review. As a result, flow cytometric analysis may miss small abnormal plasma cell populations.

Flow cytometry is not indicated typically in mature neutrophilia, polycythemia, basophilia, or thrombocytosis. It is not typically useful for the diagnosis of classical Hodgkin lymphoma, a myelodysplastic syndrome (MDS) without increased blasts, or chronic myelogenous leukemia (CML). Flow cytometry, however, may be useful in these entities with disease progression and increasing blast populations.

Flow cytometry is indicated in the setting of cytopenia or leukocytosis or in the presence of atypical cells or blasts. Limitations of flow cytometric analysis include a practical limit to the number of cell markers that can be detected, and fragile cell types may be lost during processing (eg, plasma cells). Finally, flow cytometry is susceptible to sampling issues. Bone marrow involvement by lymphoma may be focal, fibrosis may hinder aspiration of neoplastic cells, and lymph nodes specimens may have extensive necrosis. Thus, when flow cytometry results are negative, sampling issues must be considered, and morphologic evaluation of the bone marrow or lymph node is essential.

The most accurate diagnosis for any hematolymphoid neoplasm requires the integration of all available data, which typically includes clinical information, morphology, immunohistochemistry, flow cytometry, and molecular studies.

References

  1. Brown M, Wittwer C. Flow cytometry: principles and clinical applications in hematology. Clin Chem. 2000;46(8 Pt 2):1221–1229.
  2. Craig FE, Foon KA. Flow cytometric immunophenotyping for hematologic neoplasms. Blood. 2008;111(8):3941–3967.
  3. Dunphy CH. Applications of flow cytometry and immunohistochemistry to diagnostic hematopathology. Arch Pathol Lab Med. 2004;128(9):1004–1022.
  4. Peters JM, Ansari MQ. Multiparameter flow cytometry in the diagnosis and management of acute leukemia. Arch Pathol Lab Med. 2011;135(1):44–54.
  5. Smock KJ, Perkins SL, Bahler DW. Quantitation of plasma cells in bone marrow aspirates by flow cytometric analysis compared with morphologic assessment. Arch Pathol Lab Med. 2007;131(6):951–955.

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NewsPath® Editor: Kyle L. Eskue, MD
This newsletter is produced in cooperation with the College of American Pathologists Public Affairs Committee and the NewsPath Editorial Board and may be reproduced in whole or in part as a service to the medical community. Copyright © 2012 by the College of American Pathologists.
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