General considerations for the immunophenotyping of leukaemias and lymphomas

Immunophenotypic analysis of hematological specimens is a useful laboratory adjunct to surgical pathology and cytology to confirm or characterise further diagnoses of leukaemia or lymphoma (Duque and Braylan, 1991; Knowles et al., 1992; Sun, 1993; Willman, 1992). In a generic sense, immunophenotypic analysis can pertain to any type of tumour or tissue; but for the purposes of this chapter, discussion will be confined to hematological and lymphoreticular specimens. Methods of immunophenotyping vary most significantly in the type of detection system used to ascertain the occurrence of antigen–antibody binding, and the instrumentation used to observe or quantify that binding. The most common detection systems used for immunophenotyping are based on fluorescence and enzymatic histochemistry. Instrumentation ranges from flow cytometry to laser scanning cytometry, to epifluorescence microscopy, to immunohistochemistry and bright-field light microscopy. Before describing the techniques of laser scanning cytometric immunophenotyping, we will first examine certain technical aspects of the various existing methodologies, so that the reader may choose the method best suited to the clinical or research purpose at hand.

The optimal method should be capable of assessing multiple antigens simultaneously. Simultaneous assessment of at least two and, better yet, even three or four antigens on individual cells permits definitive identification of different populations and subpopulations of cells within a particular specimen. For example, in the quantitative analysis of helper and suppressor T-cells, it is mandatory to have first defined the population of Tlymphocytes in general, apart from all other cell types.