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Related Experiment Video

Updated: Nov 12, 2025

Flow Cytometric Characterization of Murine B Cell Development

Flow Cytometric Characterization of Murine B Cell Development

Published on: January 22, 2021

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

Lili Wang¹, Rukmini Bhardwaj², Howard Mostowski²

¹Biosystems and Biomaterials Division, National Institute of Standards and Technology (NIST), Gaithersburg, Maryland, United States of America.

|March 19, 2021

Summary

This summary is machine-generated.

This study evaluated CD19 expression in human peripheral blood mononuclear cells (PBMCs) to develop reference standards for flow cytometry. Results show variability in CD19 antigen sites, highlighting the need for lot-specific assessments in cell-based therapeutics.

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Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

Published on: May 8, 2018

Area of Science:

Biotechnology
Immunology
Analytical Chemistry

Background:

Cell-based therapeutics require validated measurements for cell characterization.
Flow cytometry is crucial for high-throughput analysis but lacks standardized reference materials.
Standardization is needed to harmonize flow cytometric results across laboratories.

Purpose of the Study:

To evaluate CD19 expression in three commercial peripheral blood mononuclear cell (PBMC) lots from different manufacturers.
To assess the suitability of these PBMCs as biological reference materials for CD19 quantification.
To identify sources of variability in CD19 expression measurements.

Main Methods:

Flow cytometry was used to measure CD19 antibodies bound per cell (ABC) values.
Two calibration methods were employed: single-point calibration with CD4 reference cells and QuantiBrite PE bead calibration.
Mass spectrometry (CyTOF) was used in parallel for comparison, with EQ4 and bead-based calibrations.

Main Results:

Averaged mean CD19 ABC values by flow cytometry were 7953 (PBMC-A, %CV 9.0), 10535 (PBMC-B, %CV 7.8), and 12384 (PBMC-C, %CV 16).
Flow cytometry and CyTOF results showed close agreement, with overall averaged mean CD19 ABC values of 9295 and 9699, respectively.
Uncertainty analysis indicated that the number of antigens per cell is the largest source of variability, not the calibration method.

Conclusions:

The tested PBMCs show potential as CD19+ reference control materials for quantifying B cell markers.
Variability in PBMC and antibody reagent lots must be considered when using cell-based reference materials.
Bridging studies are recommended before switching to new lots to ensure result harmonization.