A whole-blood cryopreservation method streamlines clinical sample collection for multimodal single-cell immune profiling in sepsis.

Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) has enhanced our understanding of host immune mechanisms in small cohorts, particularly in diseases with complex and heterogeneous immune responses such as sepsis. However, standard PBMC isolation from blood requires technical expertise and over 2 hours of on-site processing using Ficoll density gradient separation ("Ficoll") for scRNA-seq compatibility, precluding large-scale sample collection at most clinical sites. To minimize on-site processing, we developed cryopreservation with PBMC recovery offsite (Cryo-PRO), a method of immediate on-site whole-blood cryopreservation and subsequent batched PBMC isolation in a central laboratory prior to sequencing. We compared multimodal single-cell immune profiling results from samples processed using Cryo-PRO versus standard on-site Ficoll separation in 23 patients with sepsis. Key outputs, including cell substate fractions, marker genes, and surface protein expression were similar for each method across multiple cell types and substates, including an important monocyte substate enriched in patients with sepsis. Capture of T cell receptor transcripts was also comparable across both methods. Cryo-PRO reduced on-site sample processing time from more than 2 hours to less than 15 minutes and was reproducible across 2 enrollment sites, thus demonstrating potential for expanding multimodal single-cell analyses in multicenter studies of sepsis and other diseases.