Evaluation of the effect of storage condition on cell extraction and flow cytometric analysis from intestinal biopsies

J Immunol Methods. 2018 Aug;459:50-54. doi: 10.1016/j.jim.2018.05.004. Epub 2018 Jun 1.

Abstract

Background: Flow cytometric (FC) analysis of intestinal tissue biopsies requires prompt cell isolation and processing to prevent cell death and generate valid data. We examined the effect of storage conditions prior to cell isolation and FC on viable cell yield and the proportions of immune cell phenotypes from intestinal biopsies.

Methods: Biopsies (N = 224) from inflamed or non-inflamed ileal and/or colonic tissue from three patients with Crohn's disease were processed and analyzed immediately in duplicate, or stored under different conditions. Cells were isolated and stained for specific markers, followed by FC.

Results: Decreased mean live CD45+ cell counts were observed after storage of biopsies at -80 °C dimethyl sulfoxide (DMSO)/citrate buffer compared with immediate processing (1794.3 vs. 19,672.7; p = 0.006]). A non-significant decrease in CD45+ live cell count occurred after storage at -20 °C in DMSO/citrate buffer and cell yield was adequate for subsequent analysis. CD3+ cell proportions were significantly lower after storage at 4 °C in complete medium for 48 h compared with immediate analysis. Mean CD14+ cell proportions were significantly higher after storage of biopsies at -80 °C in DMSO/citrate buffer compared with immediate analysis (2.61% vs. 1.31%, p = 0.007). CD4+, CD8+ and CD4+/CD8+ cell proportions were unaffected by storage condition.

Conclusion: Storage of intestinal tissue biopsies at -20 °C in DMSO/citrate buffer for up to 48 h resulted in sufficient viable cell yield for FC analysis without affecting subsequent marker-positive cell proportions. These findings support the potential shipping and storage of intestinal biopsies for centralized FC analysis in multicenter clinical trials.

Keywords: Central analysis; Flow cytometry; Inflammatory bowel disease; Multicenter clinical trial; Translational research.

Publication types

  • Evaluation Study

MeSH terms

  • Adult
  • Biomarkers
  • Biopsy
  • Cell Count / standards
  • Cell Separation / standards*
  • Crohn Disease / diagnosis
  • Dimethyl Sulfoxide
  • Female
  • Flow Cytometry / standards*
  • Freezing
  • Humans
  • Intestinal Mucosa / cytology*
  • Intestines / pathology*
  • Prospective Studies
  • Specimen Handling / standards*
  • Young Adult

Substances

  • Biomarkers
  • Dimethyl Sulfoxide