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

Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA 15213, United States.
Journal of Immunological Methods (Impact Factor: 2.01). 12/2008; 341(1-2):68-75. DOI: 10.1016/j.jim.2008.10.019
Source: PubMed

ABSTRACT 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.

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    • "It is not known whether there were negative effects from storing or shipping at 4°C. Our data show that there is better viability, cell yield, and function when cells are shipped at room temperature (22°C) or 30°C than at 15°C, and it is generally accepted that storage of whole blood at 4°C negatively impacts cell viability [5], function [29], and population recovery [6,7,43,44]. Acknowledging the range of data in the literature, in a separate study, we are also evaluating the function of PBMC processed the same day (< 8 h) or after overnight shipping or storage (manuscript in preparation). "
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