This article is a US government work and, as such, is in the public domain in the United States of America.
Human CD4+ lymphocytes for antigen quantification: Characterization using conventional flow cytometry and mass cytometry†
Article first published online: 26 APR 2012
Published 2012 Wiley Periodicals, Inc.
Cytometry Part A
Volume 81A, Issue 7, pages 567–575, July 2012
How to Cite
Wang, L., Abbasi, F., Ornatsky, O., Cole, K. D., Misakian, M., Gaigalas, A. K., He, H.-J., Marti, G. E., Tanner, S. and Stebbings, R. (2012), Human CD4+ lymphocytes for antigen quantification: Characterization using conventional flow cytometry and mass cytometry. Cytometry, 81A: 567–575. doi: 10.1002/cyto.a.22060
- Issue published online: 22 JUN 2012
- Article first published online: 26 APR 2012
- Manuscript Accepted: 28 MAR 2012
- Manuscript Revised: 12 MAR 2012
- Manuscript Received: 7 SEP 2011
- quantitative multiparameter flow cytometry;
- CyTOF™ mass cytometry;
- antibody bound per cell;
- cryopreserved peripheral blood mononuclear cells;
- whole blood;
- lyophilized peripheral blood mononuclear cell;
- CD4 expression;
- cell membrane intactness;
- cell diameter;
- fixation effect
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 this study, we characterized commercially available cryopreserved peripheral blood mononuclear cells (PBMCs) and two lyophilized PBMC preparations, Cyto-Trol and PBMC–National Institute for Biological Standard and Control (NIBSC) 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 PBMC–NIBSC is much lower than those of freshly prepared cell samples using both conventional flow cytometry and CyTOF™ 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 PBMC–NIBSC 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. Published 2012 Wiley Periodicals, Inc.