W186 - Optimizing CD45RA/CD45RO Co-staining on Human Peripheral Blood Samples

Abstract Text: CD45RA and CD45RO are widely used markers to differentiate between naïve and activated/memory T cells in humans. Monoclonal antibodies HI100 and UCHL1 are two commonly used reagents to detect CD45RA and CD45RO, respectively. The change of CD45 variants expression during naïve to memory T cell transition results in a distinctive "curved" staining profile of CD45RA and CD45RO in flow cytometric analysis. However, we observed that co-staining using HI100 and certain tandem dyes, such as PE/Cyanine7, conjugated UCHL1 on human whole blood samples can distort this characteristic “curved” profile. This phenomenon is donor-dependent and can be alleviated by analyzing pre-washed (or pre-lysed) whole blood or peripheral blood mononuclear cells (PBMCs). Sequential staining of UCHL1 followed by HI100 also overcomes the issue, while staining HI100 first did not. These findings suggest that plasma components might play a role in the interaction between HI100 and UCHL1 PE/Cyanine7 conjugates. Further investigation revealed that complement component 1q (C1q) was one of the key factors contributing to the atypical co-staining profile. Finally, we developed a novel anti-human CD45RA monoclonal antibody, clone S23030B, which does not exhibit this interaction with UCHL1 conjugates.