Delayed processing of blood increases the frequency of activated CD11b+ CD15+ granulocytes which inhibit T cell function

J Immunol Methods. 2009 Feb 28;341(1-2):68-75. doi: 10.1016/j.jim.2008.10.019. Epub 2008 Nov 27.


We tested whether granulocytes, which contaminate PBMC isolates after prolonged blood storage at room temperature, are responsible for inhibited T cell function in aged blood. We extend previous observations by characterizing these contaminating granulocytes as CD11b+ CD15+ cells comparable to activated CD11b+ CD15+ granulocytes induced by incubation of blood with FMLP. Granulocyte contamination of PBMC was observed within 6-8 h after venipuncture and room temperature storage (2.3 fold increase), and increased 11.3-fold by 24-26 h in comparison to PBMC from fresh blood. Refrigerated 22-26 hour storage of blood exacerbated granulocyte contamination (84-fold increase). In contrast, granulocyte contamination was markedly reduced if blood was diluted in RPMI-1640 medium (3.9-fold increase) or PBS (1.8-fold increase) prior to 22-26 hour room temperature storage. Granulocyte contamination significantly correlated with reduced CD3zeta chain expression, a marker of T cell dysfunction. Correspondingly, T cell proliferation following PHA stimulation was significantly decreased in PBMC with contaminating granulocytes from aged blood (77% of control) or FMLP treated blood (44% of control). Minimizing granulocyte contamination in PBMC of aged blood by cell sorting, or by reducing granulocyte activation by diluting blood in PBS prior to storage, increased CD3zeta chain expression and increased T cell proliferation following stimulation. These data indicate that granulocytes inhibit T cell function in aged blood. Therefore, preventing granulocyte activation in blood specimens is critical to maintain optimal T cell function. This may be accomplished by limiting the time from venipuncture to PBMC isolation to <8 h and may be extended to 26 h by simply diluting blood in PBS prior to room temperature storage.

Publication types

  • Comparative Study

MeSH terms

  • Blood Component Removal / methods*
  • CD11b Antigen*
  • CD3 Complex / immunology*
  • Cell Proliferation
  • Female
  • Flow Cytometry
  • Gene Expression Regulation / immunology*
  • Granulocytes / cytology
  • Granulocytes / immunology*
  • Humans
  • Lewis X Antigen*
  • Male
  • Preservation, Biological
  • T-Lymphocytes / cytology
  • T-Lymphocytes / immunology*
  • Time Factors


  • CD11b Antigen
  • CD3 Complex
  • ITGAM protein, human
  • Lewis X Antigen