Delayed processing of blood increases the frequency of activated CD11b+ CD15+ granulocytes which inhibit T cell function.
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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ABSTRACT: In vitro production of bovine interferon gamma (BoIFN-γ) cytokine from bovine peripheral blood mononuclear cells (PBMCs) can be detected using the most sensitive enzyme-linked immunosorbent spot (ELISPOT) assay. ELISPOT assays are dependent on the quantity and quality of PBMC preparations and hence contribute significantly to the performance of this assay. In order to standardise the methods for isolation of PBMCs, we compared two methods for the processing of bovine blood which included aliquots of blood that were stored in a horizontal position without dilution or agitation and aliquots of blood that were immediately diluted 1:1 with complete Rosewell Park Memorial Institute (RPMI) 1640 medium and stored in a horizontal position with gentle agitation. PBMCs were isolated at 2, 4, 6, 8 and 24 h and at 4°C and at 22°C ± 2°C. They were stimulated using tuberculosis-specific antigens, after which the ELISPOT assay was performed. Quantities of spot-forming cells (SFC) created by the release of BoIFN-γ in ELISPOT assays were significantly greater in the samples stored at 22°C ± 2°C than those held +4°C and the intensity of the signals dropped following processing after 6 h. A further drop in SFC was observed in those samples that had been stored undiluted and without agitation. These findings demonstrated that optimisation of PBMC isolation procedures can lead to increased sensitivity in the detection of BoIFN-γ using the ELISPOT assay, thus contributing to an enhanced diagnosis of bovine tuberculosis.Veterinaria italiana 47(1):25-34. · 0.68 Impact Factor
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ABSTRACT: To determine whether activated CD11b(+) CD15(+) granulocytes increase in the blood of patients with uveal melanoma. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation from the blood of patients with primary choroidal/ciliochoroidal uveal melanomas (six women, four men; age range, 46-91 years) and healthy control donors (14 women, 10 men; age range, 50-81 years). The expression of CD15 and CD68 on CD11b(+) myeloid cells within PBMCs and primary uveal melanomas was evaluated by flow cytometry. CD3zeta chain expression by CD3epsilon(+) T cells in PBMCs and within primary uveal melanomas was measured as an indirect indication of T-cell function. The percentage of CD11b(+) cells in PBMCs of patients with uveal melanoma increased 1.8-fold in comparison to healthy donors and comprised three subsets: CD68 negative CD15(+) granulocytes, which increased 4.1-fold; CD68(-) CD15(-) cells, which increased threefold; and CD68(+) CD15(low) cells, which were unchanged. A significant (2.7-fold) reduction in CD3zeta chain expression on CD3epsilon(+) T cells, a marker of T-cell dysfunction, was observed in PBMCs of patients with uveal melanoma in comparison with healthy control subjects and correlated significantly with the percentage of CD11b(+) cells in PBMCs. CD3zeta chain expression on T cells within primary tumors was equivalent to CD3zeta expression in PBMCs of the same patient in four of five patients analyzed. Activated CD11b(+) CD15(+) granulocytes expand in the blood of patients with uveal melanoma and may contribute to immune evasion by ocular tumors by inhibiting T-cell function via decreasing CD3zeta chain expression.Investigative ophthalmology & visual science 05/2009; 50(9):4295-303. DOI:10.1167/iovs.08-3012 · 3.66 Impact Factor
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ABSTRACT: Flexibility in sample processing may improve test utility of the quantitative antigen-specific T cell assay (T-SPOT.TB). We investigated whether delayed sample processing with and without the use of T-Cell Xtend, a proprietary reagent, impacted upon test accuracy. Blood samples obtained from 363 sequentially recruited tuberculosis suspects or treated patients were processed immediately (day 0) and at different times after receipt of the sample [approximately 24-h (day 1) or approximately 32-h (day 2)] with and without adding T-Cell Xtend. T-Cell-Xtend-independent median ELISPOT counts (spot forming cells per million peripheral blood mononuclear cells) were significantly higher at day 1 versus day 0 (114 vs. 100; n=66; p=0.03); inter-time-point agreement between the results was 95.45% and the conversion/reversion rate was 4.55%. By contrast, counts on day 0 without T-Cell Xtend versus day 1 with T-Cell Xtend were similar (56 vs. 56; n=215), inter-time-point agreement between the results was 97.17%, and the conversion/reversion rate was 2.83%. Counts performed at day 2 with T-Cell Xtend were not significantly different from day 0. These findings were independent of HIV status. There was high agreement between results when samples were processed immediately and after a 24-h delay. However, although the use of T-Cell Xtend appeared to reduce the number of conversions/reversions this reduction was not statistically significant. Larger studies are required to clarify these findings.The Journal of infection 02/2010; 60(5):344-50. DOI:10.1016/j.jinf.2010.01.012 · 4.02 Impact Factor