Standardization of Flow Cytometric Detection of Antigen Expression

Published

January 25, 2024

Author(s)

Linhua Tian, Aaron Nelson, Tyler Lowe, Linda Weaver, Constance Yuan, Paul C. DeRose, Maryalice Stetler-Stevenson, Lili Wang

Abstract

Background: While response to antigen-based immunotherapy relies upon the level of antigen expression by tumor cells and as decreasing levels of antigen expression can be an early indicator of developing resistance to therapy, we have developed an antigen quantification assay using ABC values. Use of ABC antigen quantification as a clinical assay requires methods for quality control and for interlaboratory and inter-cytometer platform standardization. We report on an assay to assess variability of flow cytometric quantification, including inter-instrument, inter-laboratory, and antibody lot-to-lot variability. Methods: A single lot of Cytotrol™ Lyophilized Control Cells (Beckman Coulter) used for all studies was stained with single antibodies or antibody cocktails. The variability in antigen quantification across BD FACSCanto II, BD Lyric, Attune NxT and CytoFlex LX instrument platforms in 2 separate laboratories was evaluated. In addition, the effect of the antibody clone utilized and the importance of a custom antibody preparation of a 1:1 molar ratio (fluorophore to protein, F/P) compared to a commercial off-the-shelf, 1:1 antibody-fluorophore conjugate were determined. The two antigen quantification methods, QuantiBrite PE calibration and a linearity calibration combined with a single point scale transformation with CD4 as the reference marker were compared. Results: Use of single lot control cells allowed validation of reproducibility across time and between flow cytometer platforms and laboratories. Single lot control cells also allowed assessment of different antibody lots, cocktail preparation, and different antibody clones. Off the shelf antibody preparations with good controls of fluorophore to protein ratios were reasonably comparable to the custom 1:1 unimolar antibody preparation. Geometric Mean fluorescent Intensity (GeoMFI) was not, however, comparable across instruments and inter-laboratory. The use of CD4 as the reference marker can minimize variability in the determination of ABC values. Conclusion: Because of the importance of known target antigen expression in antigen-based immunotherapy, comparable and reliable antigen quantification is vital in managing these patients. An antigen quantification assay using ABC values was previously developed to provide vital information on patients receiving antigen directed immunotherapy. If this assay is to be utilized in a clinical setting, quality control methods have to be instituted to assure reproducibility and allow validation across laboratories. We have demonstrated that use of a lyophilized cell control is highly valuable in achieving these goals. We further determined that off the shelf standard antibodies can be employed, but GFI is not useful for this purpose.

Citation

Cytometry Part B-Clinical Cytometry

Pub Type

Journals

Download Paper

https://doi.org/10.1002/cyto.b.22155

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Citation

Tian, L. , Nelson, A. , Lowe, T. , Weaver, L. , Yuan, C. , DeRose, P. , Stetler-Stevenson, M. and Wang, L. (2024), Standardization of Flow Cytometric Detection of Antigen Expression, Cytometry Part B-Clinical Cytometry, [online], https://doi.org/10.1002/cyto.b.22155, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=936014 (Accessed January 17, 2025)