Blood Preservation Workflows for Single Cell Technologies Using Off-the-Shelf Reagents

Blood is a valuable and easily accessible sample type for clinical research that provides a snapshot of the organ systems at the time of collection. Readouts from blood help inform clinical treatments, investigate disease mechanisms, evaluate drug responses, and monitor outcomes. The application of single cell technologies to blood cells has driven many recent advancements in biomedical research, but logistical challenges in blood processing hinder broad adoption of single cell methods by the clinical research community. Here, we detail workflows for preserving whole blood and fixing PBMCs for Chromium Single Cell assays using off-the-shelf reagents. We use scRNA-seq to demonstrate that these methods yield comparable performance in the number of genes detected, cell type proportions, and clonotype discovery and frequency to unfixed samples. Coupled with high throughput immunomagnetic bead-based cell enrichment that is scalable and automatable, these workflows enable flexibility in the design and execution of clinical studies.

Method 1 Whole blood cryopreservation: Direct cryopreservation of whole blood enables storage without the burden of immediate PBMC isolation. scRNA-seq demonstrates similar gene detection, cell proportions, and clonotype discovery and frequency between cryopreserved whole blood and PBMCs. PBMCs from cryopreserved blood can be used in all applications suitable for cryopreserved PBMCs, including Chromium Single Cell assays and functional assays. Whole blood cryopreservation enables the establishment of long-term blood banks for a wide range of downstream applications requiring viable cells

Method 2 PBMC fixation: We have developed a streamlined fixation protocol for freshly isolated or thawed PBMCs, compatible with the reverse transcription-based Chromium GEM-X Single Cell 3’ v4 and Chromium GEM-X Single Cell 5’ v3 assays. PBMCs are fixed in a solution containing methanol and the reversible protein crosslinker DSP. Concurrent dehydration by methanol and crosslinking by DSP enables storage of the samples for at least one month at -80°C in the quenched fixation buffer without reducing performance metrics. The high alcohol content inhibits nucleases and ice crystal formation, minimizing both enzymatic and physical degradation of RNA and cell morphology. DSP crosslinks are reversed by reducing agents in the GEM-X emulsion droplets, ensuring stability until individual cells have been partitioned. scRNA-seq shows that this method offers sample stability and compatibility with reverse transcription-based assays and V(D)J sequencing. Fixation yields comparable gene detection, cell type proportions, and clonotype discovery and frequency to unfixed PBMCs. Averaged across multiple donors, at least 87% of V(D)J cells in fixed samples yield full length V-J spanning contigs compared to 95% of unfixed samples.

Method 3 Whole blood fixation: Using the whole transcriptome, probe-based Gene Expression Flex scRNA-seq assay, we demonstrate whole blood can be directly fixed with formaldehyde and stored at -80°C in the fixation buffer itself before enrichment of PBMCs or total leukocytes. This method does not reduce gene complexity or cell type proportions compared to PBMCs and leukocytes enriched prior to fixation. Notably, this workflow preserves highly fragile neutrophils in the leukocyte fraction, which are a notoriously challenging cell type to maintain with cryopreservation. Fixed blood remains stable in storage for at least 3 months.

Single cell genomics tools are transformative for biomedical research, but logistical challenges with blood transportation and sample processing have hindered their practicality in clinical research. These workflows improve accessibility of single cell technologies to clinical research programs by enabling long-term sample storage, batch processing, and simplification of the blood supply chain for decentralized clinical studies. We have developed multiple workflows with off-the-shelf reagents for compatibility with various Chromium single cell solutions to empower clinical researchers under diverse experimental design constraints. Validated using scRNA-seq on blood samples from individuals with a range of cancers, autoimmune disorders, and no reported conditions, these workflows are ready for application in clinical research.