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Consistent, multi-instrument single tube quantification of CD20 in antibody bound per cell based on CD4 reference
Heba Degheidy1, Fatima Abbasi1, Howard Mostowski1
1Center for Biologics Evaluation and Research, U.S. Food and Drug Administration (FDA), Silver Spring, Maryland, 20993.
Cytometry. Part B, Clinical Cytometry
|May 28, 2015
Summary
Standardizing flow cytometry measurements is crucial for disease diagnosis and monitoring. This study demonstrates robust CD20 quantification using a single-tube assay, ensuring consistent results across multiple instruments and fluorochromes.
Area of Science:
- Immunology
- Biotechnology
- Medical Diagnostics
Background:
- Flow cytometry is essential for diagnosing diseases like leukemia and monitoring treatments.
- Ensuring consistent, reproducible flow cytometry results across different labs and instruments remains a significant challenge.
- Previous work established instrument standardization and CD19 quantification using antibody bound per cell (ABC) standards.
Purpose of the Study:
- To develop and validate a robust single-tube assay for quantifying CD20 expression relative to CD4.
- To assess the consistency and reproducibility of CD20 quantification across multiple flow cytometer platforms.
- To demonstrate the utility of cell surface marker quantification independent of fluorochrome choice.
Main Methods:
- Implemented a single-tube assay for simultaneous CD20 and CD4 quantification.
- Utilized anti-CD20 antibody (clone L27) and anti-CD4 antibody (clone SK3) in three different fluorochromes.
- Applied novel gating strategies for robust cell surface marker quantification.
- Measured CD20 expression levels relative to CD4 using antibody bound per cell (ABC) standards.
Main Results:
- Achieved robust cell surface marker quantification using the single-tube assay format.
- Demonstrated consistent CD20 expression levels (ABC values) across five different instrument platforms.
- Confirmed that quantification results were independent of the fluorochrome used (PE, APC, or PerCP Cy5.5).
Conclusions:
- Cell surface marker quantification, specifically CD20, can be reliably performed using a single-tube assay.
- The developed method provides consistent and reproducible results across diverse instrument platforms.
- This approach enhances the reliability of flow cytometry for clinical applications, including disease diagnosis and monitoring.
