96P - A novel strategy for single-nucleus RNA-seq of frozen PAXgene blood: A clinical alternative to single-cell RNA-seq of cryopreserved PBMCs

Background

Archival storage of blood for single-cell (sc) RNA-Seq involves cryopreservation of peripheral blood mononuclear cells (PBMCs). However, this method excludes granulocytes and requires isolation of viable cells, whose transcriptomes may change due to processing time and technician handling. Hence, this method might overlook important data from temporal collection of clinical specimens. By contrast, blood collection in Qiagen PAXgene Blood RNA tubes is routinely practiced in medical sites without any special processing, and is completed with highly stabilized transcriptomes. Here, we present a novel method for single-nucleus (sn) RNA-Seq nuclei from frozen PAXgene blood as an alternative to PBMCs.

Methods

Fresh blood from healthy donors was collected, frozen in PAXgene tubes and processed by our novel method, which utilizes single-nucleus isolation and CRISPR-based strategy, yielding 3’RNA-Seq data via 10x Genomics’ Chromium platform. PBMCs from the same bloods were processed in parallel.

Results

Data quality evaluation revealed that PBMC transcript detection was higher than PAXgene, yet PAXgene RNA quality might exceed that of PBMCs. Unsupervised clustering was used to define cells and marker gene detection was used to assign cell types in PBMC and PAXgene data. Cell type subpopulations could be identified in PAXgene data, including central- and effector memory cells, and CD14⁺/16⁺ monocytes. Importantly, cells clustered based on cell type, indicating that sample biology was not masked by collection method. However, PAXgene specimens were distinct due to robust detection of granulocytes, including neutrophils (30-50% of blood cells). Proportions of PAXgene mononuclear cell populations were not significantly different from donor matched PBMCs. Results indicate that frozen PAXgene blood can be used to generate snRNA-Seq data that is comparable to PBMC scRNA-Seq, while also enabling transcriptomic characterization of granulocytes.

Conclusions

These findings provide preliminary evidence that PAXgene blood collection represents a simple, stable alternative to PBMC cryopreservation that could facilitate high quality, reproducible single-cell genomic profiling in clinical studies.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

AstraZeneca.

Funding

AsatraZeneca.

Disclosure

A. Rotem, O. Chaudhary, G. Duclos, P. Gathungu, M. Rao, R. Aguilar, L. Amir-Zilberstein, V. Shankarappa, C. Rands, X. Chen, E. Galery Harbolick, R. Halpin, M. Steinberg, J. Boland, M. Scaltriti, B. Dougherty: Financial Interests, Full or part-time Employment: AstraZeneca.