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Consistent, Multi-Instrument Single Tube Quantification of CD20 in Antibody Bound per Cell Based on CD4 Reference



Heba Degheidy, Fatima Abbasi, Howard Mostowski, Adolfas Gaigalas, Gerald Marti, Steven Bauer, Lili Wang


Detecting changes in the expression levels of cell antigens could provide critical information for the diagnosis of many diseases, e.g. leukemia, lymphoma and immunodeficiency diseases, detecting minimal residual disease, monitoring immunotherapies and discovery of meaningful clinical disease markers. One of the most significant challenges in flow cytometry is how to best ensure measurement quality and generate consistent and reproducible inter-laboratory and intra-laboratory results across multiple cytometer platforms and locations longitudinally over time. In a previous study, we developed a procedure for instrument standardization across four different flow cytometer platforms from the same vendor (reference). CD19 quantification was performed on standardized instruments relative to CD4 expression on T lymphocytes with a known amount of antibody bound per cell (ABC) as a quantification standard. Consistent and reliable measures of CD19 expression were obtained independent of fluorochrome used demonstrating the utility of this approach. In the present investigation, quantification of CD20 relative to CD4 reference marker was implemented within a single tube containing both antibodies. Relative quantification of CD20 was performed using anti- CD20 antibody clone (L27) in three different fluorochromes relative to anti-CD4 antibody (clone SK3). Our results demonstrated that cell surface marker quantification can be performed robustly using the single tube assay format with novel gating strategies. The ABC values obtained for CD20 expression levels using PE, APC or PerCP Cy5.5 are consistent over the 5 different instrument platforms for any given normal donor independent of the fluorochrome used.
Cytometry Part B-Clinical Cytometry


Flow Cytometry, CD20 quantification, instrument characterization and standardization, Mean/Geometric mean fluorescence intensity (MFI), Antibody bound per cell (ABC).


Degheidy, H. , Abbasi, F. , Mostowski, H. , Gaigalas, A. , Marti, G. , Bauer, S. and Wang, L. (2015), Consistent, Multi-Instrument Single Tube Quantification of CD20 in Antibody Bound per Cell Based on CD4 Reference, Cytometry Part B-Clinical Cytometry, [online], (Accessed April 19, 2024)
Created July 29, 2015, Updated October 12, 2021