The accurate quantification of T cell subtypes and their proportions is of great significance in cell-based biomanufacturing, diagnosis, and advanced therapy. The development and application of a cell reference material (RM) provide a solid foundation for reliable and consistent T cell quantification worldwide. However, creating a cell RM that is both accurate and practical remains a challenge. In this study, we have developed a series of T cell RMs with a certified subtype proportion based on traceable accurate quantification and stable long-term preservation. We developed a quantitative flow cytometry method for the ratio of T cell subtypes with improved accuracy by using the calibration of certified reference materials of polystyrene beads. The relative standard deviation (RSD) for the quantification of CD3, CD4, and CD8 subtypes was 0.43%, 0.64%, and 1.31%, respectively. To ensure long-term stability, an innovative lyophilization preservation technique was developed for our T cell RMs. The morphology and surface antigens (CD45, CD3, CD4, and CD8) of T cell RMs were characterized after lyophilization using immunofluorescence, demonstrating their equally good integrity compared with fresh cells. Their stability at 4 °C was demonstrated by continuous monitoring over 12 months. The final value assignment of the RMs was performed through quantification using flow cytometry in different laboratories. One of our RMs has been applied for the calibration of 54 different flow cytometry instruments. The T cell RMs have outstanding potential in the quality control of multiparameter flow cytometry measurements, and we believe they have great application prospects for the establishment and validation of T cell assays.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC11635674PMC
http://dx.doi.org/10.1021/acsomega.4c06867DOI Listing

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