Concomitant delineation of surface Ig, B-cell differentiation antigens, and HLADR on lymphoid proliferations using three-color immunocytometry.

Accurate and consistent enumeration of B-cell subpopulations in lymphoid tissue was achieved through multiparameter three-color immunofluorescence and flow cytometric analysis (FCM). Phycoerythrin (PE)-anti-CD19 (Leu12) and biotinylated anti-HLADr/streptavidin-Duochrome (PE/Texas Red), used in conjunction with polyclonal fluorescein isothiocyanate (FITC) conjugated anti-surface immunoglobulin (SIg) antibodies, effectively separated non-specific binding and background fluorescence from true B-cell surface FITC immunofluorescence, while concomitantly analyzing for HLADr and CD19 phenotypic expression/deletion. Autofluorescence was measured to establish a fluorescence threshold. A second control measured non-specific binding of isotypic control mouse Ig and non-immune rabbit IgG. Cell suspensions from 128 samples of various lymphoid proliferations were studied. In 116 of the 128 samples, kappa/lambda ratios determined by flow cytometry correlated well with immunocytology results obtained using cytospins from the same cell suspension and with histopathologically established diagnosis. Clonality and lineage as defined immunotypically by flow cytometry was concordant with genotypic results in 64 of the 67 cases evaluated. SIg, HLADr, and CD19 deletions were demonstrated by flow cytometry in 8, 4, and 1 case(s), respectively. Discordance was usually attributable to selective loss of large neoplastic cells in flow cytometry specimens or absent expression of SIg by some cytoplasmic Ig (CIg+) lymphomas.