Blood handling and leukocyte isolation methods impact the global transcriptome of immune cells

BACKGROUND: Transcriptional profiling with ultra-low input methods can yield valuable insights into disease, particularly when applied to the study of immune cells using RNA-sequencing. The advent of these methods has allowed for their use in profiling cells collected in clinical trials and other studies that involve the coordination of human-derived material. To date, few studies have sought to quantify what effects that collection and handling of this material can have on resulting data. RESULTS: We characterized the global effects of blood handling, methods for leukocyte isolation, and preservation media on low numbers of immune cells isolated from blood. We found overall that storage/shipping temperature of blood prior to leukocyte isolation and sorting led to global changes in both CD8(+) T cells and monocytes, including alterations in immune-related gene sets. We found that the use of a leukocyte filtration system minimized these alterations and we applied this method to generate high-quality transcriptional data from sorted immune cells isolated from the blood of intracerebral hemorrhage patients and matched healthy controls. CONCLUSIONS: Our data underscore the necessity of processing samples with comparably defined protocols prior to transcriptional profiling and demonstrate that a filtration method can be applied to quickly isolate immune cells of interest while minimizing transcriptional bias. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12865-018-0268-6) contains supplementary material, which is available to authorized users.