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Immunophenotyping
of Leukemias/ Lymphomas
Clinical Significance and Methodology:
Immunophenotyping by flow cytometry categorizes leukemias and lymphomas by the expression of cell surface antigens, cytoplasmic and nuclear antigens
(1, 2).
Flow cytometry is performed on cells in liquid suspension (i.e. blood, bone marrow, body fluids or tissue cell suspensions) that have been incubated with fluorescently labeled antibodies directed against specific cellular proteins. The relative fluorescence intensity of the positive cells indicates the amount of antibody bound to specific binding sites on the cell, and therefore provides a relative measure of antigen expression
(3).
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instrument used for routine clinical specimens is
capable of examining up to 3 different antigens
simultaneously, in addition to forward and orthogonal
light scatter, thereby providing highly detailed
information about cell size and immunophenotype.
Because erythrocytes interfere with the flow
cytometric evaluation of the leukocytes, all but the
most immature erythroid cells are lysed prior to
analysis. Assuming the sample is sufficiently
cellular, approximately 20,000 viable white cells are
routinely analyzed with each antibody combination.
Evaluating such a large number of cells enables one to
identify very small populations of abnormal cells,
which may represent as few as 0.1% of the cells in the
specimen
(4).
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The specific panel of antibodies used is selected based on the morphologic appearance of the cells present in the specimen, as well as the available clinical information
(5, 6).
The referring physician may request antibodies of particular interest. The attending pathologist issues a written report and interpretation for each case; summary figures of the flow cytometric histograms are routinely included in these reports.
References:
1) General Haematology Task Force of the BCSH (1994) J. Clin. Pathol. 47, 871.
2) Bene M.C., Castoldi G., Knapp W. et al (1995) Leukemia 9, 1783.
3) Deegan M.J. (1989) Arch. Pathol. Lab. Med. 113, 606.
4) Drach J., Drach D., Glassl H. et al (1992) Cytometry 13, 893.
5) Greaves M.F., Janossy G., Peto J. et al (1981) Br. J. Haematol. 48, 179.
6) Koziner B., Gebhard D., Denny T. et al (1982) Am. J. Med. 73, 802.
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