Title: Development of B-cell reference materials for comparable and quantitative cytometric expression analysis

Abstract

Objectives: Cell-based therapies have emerged as a novel approach to treat cancer and other conditions. Several studies highlighted the crucial role of quantifying surface CD19 using flow cytometry in providing guidance for proper selection of targeted Immunotherapy in B- cell malignancies. However, the lack of adequate reference materials and the complexity of the cytometer instrumentation have resulted in few reference standards to ensure comparable and quantitative CD19 expression analysis. This study was designed to evaluate CD19 expression in potential biological cell reference materials and provide an assessment of their suitability to support the development of CD19 reference standards. Methods: CD19 expression measurements were made using QuantiBrite PE and CD4 based quantification schemes. Additionally, CD19 expression on a selected human peripheral blood mononuclear cells (PBMC) was evaluated using mass cytometry to verify result comparability between two orthogonal measurement techniques. Three commercial PBMC-A, PBMC-B, and PBMC-C made by three different manufacturers and one synthetic CD19 B cell material were tested. Variables potentially contributing to the differences in CD19 expression, PBMC manufacturing process, number of healthy donors used for each PBMC lot, antibody reagent, operators, and experimental days were considered in the evaluation. Results & Conclusions: Mean of CD19 ABC (antibodies bound per cell) is 7700 with a range from 4700 to 11300 at a 95% confidence level was obtained for PBMC-A, 10900 with a range from 7400 to 14900 for PBMC-B, and 14000 with a range from 7200 to 22000 for PBMC-C. Full understanding of the sources of uncertainty, their relative contributions and areas of improvement will lead to production of high-quality and robust reference material for quantitative expression measurement for many application fields not limited to flow cytometry.