Cryo-PRO facilitates whole blood cryopreservation for single-cell RNA sequencing of immune cells from clinical samples

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, PBMC isolation from blood requires technical expertise, training, and approximately two hours of onsite processing using Ficoll density gradient separation ('Ficoll') for scRNA-seq compatibility, precluding large-scale sample collection at most clinical sites. To minimize onsite processing, we developed Cryo-PRO (Cryopreservation with PBMC Recovery Offsite), a method of PBMC isolation from cryopreserved whole blood that allows immediate onsite sample cryopreservation and subsequent PBMC isolation in a central laboratory prior to sequencing. We compared scRNA-seq results from samples processed using Cryo-PRO versus standard onsite Ficoll separation in 23 patients with sepsis. Critical scRNA-seq outputs including cell substate fractions and marker genes were similar for each method across multiple cell types and substates, including an important monocyte substate enriched in patients with sepsis (Pearson correlation 0.78, p<0.001; 70% of top marker genes shared). Cryo-PRO reduced onsite sample processing time from >2 hours to <15 minutes and was reproducible across two enrollment sites, thus demonstrating potential for expanding scRNA-seq in multicenter studies of sepsis and other diseases.