Human CD4+ Lymphocytes for Antigen Quantification: Characterization Using Conventional Flow Cytometry and Mass Cytometry
To transform the linear fluorescence intensity scale obtained with fluorescent microspheres to an antibody bound per cell (ABC) scale, a biological cell reference material is needed. Optimally, this material should have a reproducible and tight ABC value for the expression of a known clinical reference biomarker. In the present study, we characterized commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and two lyophilized PBMC preparations, Cyto-Trol and PBMCNIBSC relative to freshly prepared PBMC and whole blood samples. It was found that the ABC values for CD4 expression on cryopreserved PBMC were consistent with those of freshly obtained PBMC and whole blood samples. By comparison, the ABC value for CD4 expression on Cyto-Trol is lower and the value on PBMCNIBSC is much lower than those of freshly prepared cell samples using both conventional flow cytometry and CyTOFTM mass cytometry. By performing simultaneous surface and intracellular staining measurements on these two cell samples, we found that both cell membranes are mostly intact. Moreover, CD4+ cell diameters from both lyophilized cell preparations are smaller than those of PBMC and whole blood. This could result in steric interference in antibody binding to the lyophilized cells. Further investigation of the fixation effect on the detected CD4 expression suggests that the very low ABC value obtained for CD4+ cells from lyophilized PBMCNIBSC is largely due to paraformaldehyde fixation; this significantly decreases available antibody binding sites. This study provides confirmation that the results obtained from the newly developed mass cytometry are directly comparable to the results from conventional flow cytometry when both methods are standardized using the same ABC approach.
Human CD4+ Lymphocytes for Antigen Quantification: Characterization Using Conventional Flow Cytometry and Mass Cytometry, Cytometry Part A, [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=909406
(Accessed June 6, 2023)