A prospective, international, multisite comparison of platelet isolation techniques for genome-wide transcriptomics: Communication from the SSC of the ISTH
Genome-wide platelet transcriptomics is increasingly being used to uncover new aspects of platelet biology and as a diagnostic and prognostic tool. Nevertheless, platelet isolation methods for transcriptomic studies are not standardized, introducing challenges for cross-study comparisons, data integration, and replication. In this prospective multicenter study, we assessed how three of the most commonly-used platelet isolation protocols influence metrics from next-generation bulk RNA sequencing and functional assays. Compared with washing alone, more stringent removal of leukocytes by anti-CD45+ beads or PALL filters resulted in sufficient quantity of RNA for next-generation sequencing and similar quality of RNA sequencing metrics. The more stringent removal of leukocytes resulted in lower relative expression of known leukocyte-specific genes and higher relative expression of known platelet-specific genes. The results were consistent across enrolling sites, suggesting that the techniques are transferrable and reproducible. The use of anti-CD45+ beads reduced integrin aIIbb3 activation to PAR-1 activating peptide (SFLLRN-TRAP) but not ADP, compared to washing alone, while the isolation method had no influence on basal platelet reactivity. In conclusion, genome-wide transcriptional and functional assays in platelets are influenced by isolation technique. These results should help the research community make informed choices about platelet isolation techniques in their own platelet studies. Overall design: To investigate the effects of platelet isolation on the quality of genome-wide transcriptomics, blood was collected from healthy donors across 4 international sites (University of Utah; Camera, Milan site, Italy; University of Debrecen, Hungary; Perugia, Italy) and split into three modes of isolation, washed platelets, leuckocyte-bead depletion (LDPs), or through PALL-filtration, to isolate platelets. RNA was extracted from isolated platelets. Then, gene expression analysis using data obtained from RNA-seq of platelets from 3 different isolation techniques across 4 sites was performed.