Blood Collection Workflow for Interrogation of Translational Samples with Single Cell Technologies

Blood is a valuable and easily accessible sample type for clinical researchers that provides a snapshot of the organ systems at the time of sample collection. Readouts obtained from blood help inform clinical treatments, investigate disease mechanisms, evaluate drug response, and monitor outcomes. However, the impact of existing blood collection workflows on data output from new and high resolution technologies, like single cell RNA sequencing (scRNA-seq) remains a challenge. Recent scRNA-seq studies have shown that changes in gene expression can occur in blood cells in as little as a few hours post collection1. While bulk measurements rely on sample fixation to preserve the sample during transport, this approach does not preserve intact cells and hence, is not compatible with single cell technologies. With the recent availability of fixation compatible scRNA-seq assays comes the need to preserve blood while retaining single cell information. In this study, we evaluated various PBMC isolation and blood preservation methods which preserve single cell information while easing sample logistical constraints.

Our initial experiments compared various PBMC isolation methods. scRNA seq analysis of these samples demonstrated that all isolation methods captured single cell gene expression with some variability in cell types along with user-variability. Changes in cell type proportions were observed in as little as 4 hrs post blood draw, emphasizing the need for an effective blood preservation method to retain single cells during transport and blood cell isolation.

Next, we compared different methods of blood preservation including cryopreservation and fixation. Healthy and diseased whole blood samples were collected, preserved, stored followed by isolation of blood samples at different time points. scRNA-seq analysis of the samples demonstrated that blood can be preserved, transported, and stored (weeks to months) before blood cell isolation. Blood cells isolated from these cells can then be stored long term for scRNA-seq analysis. This blood collection and preparation workflow also eases blood transportation logistical constraints, allowing for batch shipping of samples from distributed collection sites. Coupled with automatable cell isolation methods and multiplexing single cell sequencing solutions, these workflows can be adapted for large scale translational research studies.

References:

• Massoni-Badosa, R., Iacono, G., Moutinho, C. et al. Sampling time-dependent artifacts in single-cell genomics studies. Genome Biol 21, 112 (2020).

Keywords: single cell RNA-seq, blood preservation