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

BMC Immunol. 2018 Oct 30;19(1):30. doi: 10.1186/s12865-018-0268-6.

Abstract

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.

Keywords: Immune profiling; Peripheral blood mononuclear cells; RNA-seq; Transcriptome.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • CD8-Positive T-Lymphocytes / cytology
  • CD8-Positive T-Lymphocytes / immunology*
  • Gene Expression Profiling
  • Humans
  • Leukocyte Reduction Procedures / methods*
  • Leukocytes, Mononuclear / cytology
  • Leukocytes, Mononuclear / immunology*
  • Sequence Analysis, RNA / methods
  • Transcriptome*