Analysis of surface and cytoplasmic immunoglobulin light/heavy chains by flow cytometry using a lysed-whole-blood technique: Implications for the differential diagnosis of B-cell malignancies.

The study was aimed at the proper detection of surface and cytoplasmic clonal Ig light/heavy chains in the frame of multiparameter flow cytometry analysis of some B-cell malignancies. An exact direct evidence has been obtained that the leukemia cells following staining by antibodies to immunoglobulins will need to be washed to eliminate free plasma Igs. The results of proper Ig detection with simultaneous unaltered staining of further 2-3 markers on the cell surface after elimination of free plasma Ig in the whole blood sample are described. In differential diagnosis of some chronic B-cell malignancies and subclassification of some acute B-leukemias the detection of intracytoplasmic light/heavy chain Igs is required. The unique phenotypic structures of multiple myeloma (MM) cells have been utilized in our approach to detect cytoplasmic Ig light and heavy chains. A modified 2-step method for analysis of cytoplasmic immunoglobulin light chains by flow cytometry in MM patients was used and the method was extended for measurement of IgM heavy chain in B-ALL. For membrane staining in MM patients cells the combination of CD45-FITC and CD138-PE was used; the CD138 was found to be more specific than CD38 for MM cells. The whole blood cells were lysed, acquired on flow cytometry (first acquisition), then permeabilized by paraformaldehyde and saponin, and incubated with anti-kappa-FITC and anti-lambda-FITC antibodies and acquired again (second acquisition). In B-ALL patients cells in first step the combinations of CD45-FITC or CD22-FITC and CD10-PE have been successfully applied and after RBC lysis, acquisition and membrane permeabilization anti-IgA-FITC and anti-IgM-FITC were applied and cells were acquired again. The FITC fluorescence intensity of the second measurement was equal to the sum of surface CD45 or CD22 marker expression during the first step, and cytoplasmic clonal light or heavy chains expression during the second acquisition in both, MM and pre-B ALL patients, as well.