Immunophenotyping of leukemia cells plays the crucial role in identification of proliferating cell line, what is very useful for individual treatment monitoring and detection of residual disease. The purpose of this study was to develop standard laboratory procedure for immunophenotyping of leukemia cells in mature B cell non-Hodgkin lymphomas (NHL) to establish the kit of necessary antibodies and to work out strategy of typing in CLL/SLL, MCL and MZL.
Leukocytes from 115 patients with B-cell NHL and 15 blood donors were tested. Immunophenotype determination was carried out by direct antibody staining, three-color immunophenotyping with CD19 gating and analysis with FACS Canto flow cytometer.
Flow cytometry analysis revealed that the first step for proper determination of phenotype of leukemic cells is evaluation of expression of CD5, CD20, CD23, CD79b, CD22, CD10, FMC7, kappa/lambda immunoglobulin light chains, and IgM, IgD as well as the antigen density. We established useful screening kits for three-color flow cytometry. Proposed three color kit are: CD45/CD14, CD5/CD20/CD19, CD23/CD79b/CD19, CD22/CD10/CD19, FMC7, kappa/lambda/CD19, IgM/ IgD/CD19.
Our data indicate that short panel (including markers for CD5, CD19, CD23, CD79b, CD20, kappa, lambda) is sufficient for typical CLL/SLL phenotyping and the panel extended by markers for CD22, FMC7, CD10, IgM and IgD allows for CLL/PLL, MCL and MZL differentiation. Intensity of antigen expression determination, especially CD20, CD79b and light chains, should be integral part of immunophenotyping analysis.