Double-marker analysis of B lymphocyte subsets using a combination of immunofluorescence and rosetting with immunomagnetic beads.

Double-marker assays were developed for enumerating CD5+ B-cells and CD25+ activated B-cells. To measure CD5+ B-cells, CD19+ (pan B-specific) cells were first isolated from peripheral blood mononuclear cells using anti-Leu12-coated immunomagnetic beads (Dynabeads). Positively selected cells were stained for surface immunoglobulin (sIg) and then incubated with anti-Leu1-coated Dynabeads. Fluorescent cells which also bound Dynabeads could be readily enumerated. The proportion of CD5+ B-cells was found to be significantly elevated in 9 of 20 patients with definite rheumatoid arthritis. B-cells bearing the interleukin-2 receptor (IL-2-R, CD25) were identified in pokeweed mitogen-stimulated PBM by sIg fluorescence and rosetting with anti-Tac-labelled Dynabeads. The kinetics of CD25 expression following activation were found to be different for B- and non-B-cells.