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Cytometry B Clin Cytom 2016 Mar;90(2):159-67

Consistent, multi-instrument single tube quantification of CD20 in antibody bound per cell based on CD4 reference.

Degheidy H, Abbasi F, Mostowski H, Gaigalas AK, Marti G, Bauer S, Wang L


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 manufacturer. CD19 quantification was performed on three of the 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 apparently healthy donor independent of the fluorochrome used.

Category: Journal Article
PubMed ID: #26013593 DOI: 10.1002/cyto.b.21253
Includes FDA Authors from Scientific Area(s): Biologics Medical Devices
Entry Created: 2014-12-02 Entry Last Modified: 2018-07-01