AbstractHigh-dimensional mass cytometry (CyTOF) phenotyping allows for the routine measurement of over 40 parameters and is increasingly being utilized across a wide range of studies. However, CyTOF-specific panel design and optimization represent challenges to wider adoption and standardization of immune profiling with CyTOF. To address this, Fluidigm recently commercialized its MaxPar Direct Immune Profiling Assay (MDIPA), which comprises a lyophilized 30-marker antibody panel that is able to identify all major circulating immune cell subsets and offers a streamlined solution for standardized human immune monitoring. However, in the course of applying the MDIPA to characterize large numbers of whole blood samples, we observed several instances of unusual aberrant staining patterns, most notably CD19 expression on non-B cells, which can potentially confound data analysis and lead to erroneous interpretation of results when using this assay. Here, we report that this complex phenomenon is mediated by donor-specific plasma factors that mediate non-specific interactions between specific antibodies in the MDIPA panel. Our findings additionally suggest specific strategies that can be used to mitigate the issue, including the use of PBMCs or lysed/washed whole blood to remove endogenous plasma prior to staining, or blocking specific antibodies in the MDIPA panel.
Longitudinal immune profiling reveals key myeloid signatures associated with COVID-19