CD19 Expression Analysis Using Two Flow cytometric Quantitative Schemes: An approach for Identifying Robust B-cell Reference Control Materials

Published

March 19, 2021

Author(s)

Hao-Wei Wang, Paul Patrone, Anthony Kearsley, Jessica Watson, Liang Lim, Jothir Pichaandi, Olga Ornatsky, Daniel Majonis, Rukmini Bhardwaj, Howard Mostowski, Steven Bauer, Heba Degheidy, Lili Wang

Abstract

Cell-based therapies have emerged as a novel approach to treat cancer and other conditions. Quantitative flow cytometry plays an increasingly important role in predicting therapy outcome in a wide range of hematologic malignancies. Several studies highlighted the crucial role of quantifying surface CD19 in providing guidance for proper selection of targeted Immunotherapy in B- cell malignancies. We designed this study to quantify the expression levels of CD19 on B cells using the antibodies bound per cell approach (ABC) and to explore associated measurement uncertainties in three commercial lyophilized PBMC preparations. The mean of CD19 ABC obtained 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. Our results indicated that cell marker number per cell in each PBMC preparation is by far the largest source of variability in the measurement, accounting for between 50% and 70%. The calibration method does not significantly affect the estimates for the other sources of variation, suggesting that the likelihood analysis is able to isolate their individual contributions. In addition, we noted that the relative contribution from random effects is relatively large in one PBMC cell preparation. A 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 marker measurement for many application fields that are not only limited to flow cytometry.

Citation

PLoS One

Pub Type

Journals

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Keywords

Quantitative flow cytometry, CD19 expression, B-cell reference materials, Antibodies bound per cell, human peripheral blood mononuclear cells (PBMCs), Mass cytometry (CyTOF), CD4 reference marker, Quantibrite PE calibration, Combined uncertainties, sources of uncertainty

Citation

Wang, H. , Patrone, P. , Kearsley, A. , Watson, J. , Lim, L. , Pichaandi, J. , Ornatsky, O. , Majonis, D. , Bhardwaj, R. , Mostowski, H. , Bauer, S. , Degheidy, H. and Wang, L. (2021), CD19 Expression Analysis Using Two Flow cytometric Quantitative Schemes: An approach for Identifying Robust B-cell Reference Control Materials, PLoS One, [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=929983 (Accessed October 9, 2025)