Establishing CD19 B-cell reference control materials for comparable and quantitative cytometric expression analysis

Abstract

In the field of cell-based therapeutics, there is a great need for high-quality, robust, and validated measurements for cell characterization. Flow cytometry has emerged as a critically important platform due to its high-throughput capability and its ability to simultaneously measure multiple parameters in the same sample. However, to assure the confidence in measurement, well characterized biological reference materials are needed for standardizing clinical assays and harmonizing flow cytometric results between laboratories. To date, the lack of adequate reference materials, and the complexity of the cytometer instrumentation have resulted in few standards. This study was designed to evaluate CD19 expression in three potential biological cell reference materials and provide a preliminary assessment of their suitability to support future development of CD19 reference standards. Three commercially available human peripheral blood mononuclear cells (PBMCs) obtained from three different manufacturers were tested. Variables that could potentially contribute to the differences in the CD19 expression, such as PBMCs manufacturing process, number of healthy donors used in manufacturing each PBMC lot, antibody reagent, operators, and experimental days were included in our evaluation. CD19 antibodies bound per cell (ABC) values were measured using two flow cytometry-based quantification schemes with two independent calibration methods, a single point calibration using a CD4 reference cell and QuantiBrite PE bead calibration. Three lots of PBMC from three different manufacturers were obtained. Each lot of PBMC was tested on three different experimental days by three operators using three different lots of unimolar anti-CD19PE conjugates. CD19 ABC values were obtained in parallel on a selected lot of the PBMC samples using mass spectrometry (CyTOF) with two independent calibration methods, EQ4 and bead-based calibration were evaluated with CyTOF-technology. Including all studied variabilities such as PBMC lot, antibody reagent lot, and operator, the averaged mean values of CD19 ABC for the three PBMC manufacturers (A,B, and C) obtained by flow cytometry were found to be: 7953 with a %CV of 9.0 for PBMC-A, 10535 with a %CV of 7.8 for PBMC-B, and 12384 with a %CV of 16 for PBMC-C. These CD19 ABC values agree closely with the findings using CyTOF. The averaged mean values of CD19 ABC for the tested PBMCs is 9295 using flow cytometry-based method and 9699 using CyTOF. The relative contributions from various sources of uncertainty in CD19 ABC values were quantified for the flow cytometry-based measurement scheme. This uncertainty analysis suggests that the number of antigens or ligand binding sites per cell in each PBMC preparation is the largest source of variability. On the other hand, the calibration method does not add significant uncertainty to the expression estimates. Our preliminary assessment showed the suitability of the tested materials to serve as PBMC-based CD19+ reference control materials for use in quantifying relevant B cell markers in B cell lymphoproliferative disorders and immunotherapy. However, users should consider the variabilities resulting from different lots of PBMC and antibody reagent when utilizing cell-based reference materials for quantification purposes and perform bridging studies to ensure harmonization between the results before switching to a new lot.

Fig 1. Gating Strategy to identify CD19 positive populations from PBMC-A, PBMC-B, and PBMC-C.

Representative dot plots of FSC-A vs. FSC-H were used for doublets exclusion (left). The dot plots of FSC-A vs. SSC-A were then utilized to gate on lymphocyte population (middle), followed by subsequent gating on CD19 positive cells in the respective histograms (right).

Fig 2. MedFI values and associated standard deviations obtained for the three PBMC preparations.

MedFI values and associated standard deviations obtained for the three PBMC preparations, PBMC-A (top row), PBMC-B (middle row), and PBMC-C (bottom row). Variables contributing to variability of MedFI values include different lots of each PBMC preparation (left column), CD19 PE 1:1 antibody reagent (middle column), and experimental days with different operators (right column).

Fig 3. CD19 expression levels in unit of ABC and associated standard deviations at ~95% confidence level.

CD19 expression levels in unit of ABC and associated standard deviations at ~95% confidence level for the three PBMCs preparations (PBMC-A, PBMC-B, and PBMC-C) using the computational method described in the method section for the two quantification approaches of flow cytometry, QuantiBrite PE calibration (light grey bar) and CD4 cell reference calibration (grey bar), and the two flow spectrometry method combined uncertainty analysis (red bar). The CyTOF quantification was conducted on a selected subset of the PBMC samples. The CD19 expression levels and associated standard deviations obtained using two CyTOF quantification methods, EQ4 calibration (blue bar) and ABC bead calibration (green bar) are also displayed within the flow cytometric results from the combined uncertainty analysis.

Fig 4. Relative variation in CD19 measurement.

Relative variation (i.e. total variance divided by the mean squared) in typical CD19 measurement by PBMC manufacturer and calibration method. The relative contributions of each source of uncertainty for each PBMC preparation are indicated by the different colors.