Preferred Tissues And Methodologies For Immunophenotying

Multiparameter flow cytometry90,91 is the favored methodology when establishing a leukemia immunophenotype from antico-agulated peripheral blood or bone marrow aspirate (Figure 15-2). This technique incorporates structural cell characteristics, such as size and granularity, as well as antigenic characteristics, whereby most commonly two to four antigens are captured simultaneously by using various fluorochromes (fluorescein isothiocyanate [FITC], phycoerythrin [PE], etc.) The choice of antibodies and thus the size of antibody panels will vary with the suspected disease, the state of disease (presentation versus remission), and the purpose of the typing. If the purpose is solely to establish a diagnosis, the panel could be stripped to the bare minimum of lineage-specific antigens (myeloperoxidase, cytoplasmic CD3, cytoplasmic CD22). Given that clinically important subtypes would be missed when testing is extremely limited, a minimum antibody panel should be developed in any clinical flow cytometry laboratory that ensures that established prognostic leukemia subtypes are detected (CD11b+ AML, intracytoplasmic mu chain+ Pre-B ALL, etc.) It is recommended to streamline the choice of antibodies. In order to obtain an indication for the lineage association of a leukemic cell population (lymphoid versus myeloid) and to select a marker most suitable as a gating antibody to be added to each test tube, we pretest every leukemia patient on whom no previous immunophenotype information is available. Pretesting is done with a small series of antibody combinations: CD45/CD14, CD33/CD34, CD13/CD115, HLA-DR/CD11b, CD10/CD19, CD7/CD2, CD3/CD5. With the number of available antibodies and their potential diagnostic and prognostic significance continuously increasing, strategies for targeted testing are cost-efficient without compromising the quality of the analysis.

Comparative immunophenotypic analyses in peripheral blood and bone marrow have not shown significant differences in antigen profiles.92 Staining whole blood or bone marrow is preferred over isolating mononuclear cells in order to avoid selective loss of cell populations and potential effects on antibody binding.93,94 The results of antibody binding have been found to vary with cell manipulations, such as density gradient centrifugation or preincubation at 37°C, in neutrophils owing to the presence of intracellular storage pools of many of the myelomonocytic antigens (CD35, CD16, etc.).94 In either case, the white cell count should be adjusted to the amounts of antibody added and antibodies should be incubated with the cells in the presence of human AB serum to block Fc receptors. Removal of erythroid cells is accomplished by erythrocyte lysis, preferably after staining with antibodies directed to surface antigens.

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