BACKGROUND AND OBJECTIVE: Distinction between B-cell chronic leukemias can be difficult due to overlap in cell morphology and immunologic features. We investigated, by quantitative flow cytometry, the expression of CD79b, CD5 and CD19 in cells from a variety of B-cell disorders to see whether this analysis adds further information useful to the diagnosis and characterization of these diseases. DESIGN AND METHODS: Peripheral blood cells from 6 normal individuals were used as reference controls. The diseases of the 63 patients investigated comprised: 29 chronic lymphocytic leukemia (CLL), six of them with atypical morphology, 6 B-cell prolymphocytic leukemia (PLL), 12 splenic lymphoma with villous lymphocytes (SLVL) and 16 mantle-cell (Mc) lymphoma in leukemic phase. The study was carried out by triple immunostaining with directly conjugated monoclonal antibodies (MoAb) against CD79b, CD5 and CD19 and quantitative estimation of the antigens per cell assessed with standard microbeads (Quantum Simply Cellular). RESULTS: Compared to normal B-cells, the number of CD19 molecules was significantly lower in cells from all of the B-cell disorders except PLL. The intensity of CD5 in leukemic B-cells was significantly higher in CLL cells, including atypical cases, and Mc lymphoma than in normal B-cells, whilst PLL and SLVL had values similar to those of normal B-lymphocytes. CD79b was expressed at lower levels in all types of leukemic cells compared to normal B-lymphocytes but differences were statistically significant in CLL, Mc lymphoma and SLVL. The number of CD79b molecules per cell was significantly lower in typical CLL than in the remaining B-cell diseases whilst the comparison of CD5 and CD19 intensity between CLL and non-CLL samples failed to show any statistically significant difference. INTERPRETATION AND CONCLUSIONS: Distinct antigen density patterns for the various conditions emerged from this analysis: Typical CLL was characterized by moderate CD5 and weak or negative CD79b expression. Mc lymphoma showed an homogeneous pattern, characterized by similar expression of CD5 than CLL but significantly stronger expression of CD79b whilst PLL and SLVL had weak CD5 and moderate CD79b expression. Atypical CLL had an intermediate pattern of CD79b antigen expression ranging from weak to moderate with bright CD5. Unlike CD5 and CD79b, CD19 did not discriminate the various B-cell disorders but only between normal and leukemic cells.