Massively parallel flow-cytometry-based screening of hematopoietic lineage cell populations from up to 25 donors simultaneously

This study aimed to develop a method allowing high-dimensional and technically uniform screening of surface markers on cells of hematopoietic origin. High-dimensional screening of cell phenotypes is primarily the domain of single-cell RNA sequencing (RNAseq), which allows simultaneous analysis of the expression of thousands of genes in several thousands of cells. However, rare cell populations can often substantially impact tissue homeostasis or disease pathogenesis, and dysregulation of rare populations can easily be missed when only a few thousand cells are analyzed. With the presented methodological approach, it is possible to screen hundreds of markers on millions of cells in a technically uniform manner and thus identify and characterize changes in rare populations. We utilize the highly expressed markers CD45 on immune cells and CD71 on erythroid progenitors to create unique fluorescent barcodes on each of the 25 samples. Double-barcoded samples are co-stained with a broad immunophenotyping panel. The panel is designed in such a way that allows the addition of PE-labelled antibody, which was used for screening purposes. Multiplexed samples are divided into hundreds of aliquots and co-stained, each aliquot with a different PE-labelled antibody. Utilizing a broad immunophenotyping panel and machine-learning algorithms, we can predict the co-expression of hundreds of screened markers with a high degree of precision. This technique is suitable for screening immune cells in bone marrow from different locations, blood specimens, or any tissue with a substantial presence of immune cells, such as tumors or inflamed tissue areas in autoimmune conditions. It represents an approach that can significantly improve our ability to recognize dysregulated immune cell populations and, if needed, precisely target subsequent experiments covering lower cell counts such as RNAseq.