Antigen levels in normal B cells and B cell malignancies established by a multicolor quantitative flow cytometry assay

Antigen density on B cells may influence the efficacy of targeted therapies, yet current diagnostic tools, such as immunohistochemistry (IHC), offer limited quantitation. We developed a standardized, quantitative flow cytometry assay to assess CD19, CD20, and CD22 expression and established reference ranges in non-malignant and malignant B-cell populations.A 10-color flow cytometry panel was designed with saturating antibody concentrations and calibrated fluorochrome-conjugated quantitation beads to determine antigen molecules per cell. Instrument calibration, antibody titration, and antigen stability were validated per clinical laboratory guidelines. Antigen density was evaluated in peripheral blood (PB), bone marrow (BM), and lymph node (LN) samples from non-malignant and untreated B-cell lymphoma cases.CD19, CD20, and CD22 expression levels were highest in peripheral blood B cells and significantly lower in bone marrow and lymph nodes. In B-cell lymphomas, antigen density was reduced relative to non-malignant controls, particularly CD19 in follicular and mantle cell lymphomas. In DLBCL, recent rituximab exposure (within 6 months) was associated with significant CD20 downregulation and modest reductions in CD19 and CD22. Quantitative flow cytometry demonstrated greater sensitivity than IHC and could detect mixed abnormal populations and antigen downregulation not captured by standard IHC and flow cytometry methods.This quantitative flow cytometry assay enables robust assessment of B-cell antigen density across disease states and treatment timelines. The assay provides critical data to inform therapeutic selection and potential response prediction, with broader applicability to biomarker development in immunotherapy.