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: 1.82). 12/2008; 341(1-2):68-75. DOI: 10.1016/j.jim.2008.10.019
Source: PubMed


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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    • "Previously, it was shown that the length of time from venipuncture to cryopreservation was the most important parameter influencing T-cell performance in cellular immune assays, affecting subsequent cell recovery and function (Bull et al., 2007). Recent observations indicate that several other parameters involved in blood processing as well as antigen-stimulation can impact cell viability and the measured T-cell responses (Owen et al., 2007; Jeurink et al., 2008; McKenna et al., 2009; Weinberg et al., 2009; Afonso et al., 2010; Mallone et al., 2011; Kutscher et al., 2013). Moreover, the sensitivity of whole blood versus PBMC assays is still under debate, with different studies reaching opposite conclusions (Suni et al., 1998; Hoffmeister et al., 2003). "
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    ABSTRACT: Intracellular cytokine staining (ICS) assay is increasingly used in vaccine clinical trials to measure antigen-specific T-cell mediated immune (CMI) responses in cryopreserved peripheral blood mononuclear cells (PBMCs) and whole blood. However, recent observations indicate that several parameters involved in blood processing can impact PBMC viability and CMI responses, especially in antiretroviral therapy (ART)-naïve HIV-1-infected individuals. In this phase I study (NCT01610427), we collected blood samples from 22 ART-naïve HIV-1-infected adults. PBMCs were isolated and processed for ICS assay. The individual and combined effects of the following parameters were investigated: time between blood collection and PBMC processing (time-to-process: 2, 7 or 24hours); time between PBMC thawing and initiation of in vitro stimulation with HIV-1 antigens (resting-time: 0, 2, 6 and 18hours); and duration of antigen-stimulation in PBMC cultures (stimulation-time: 6hours or overnight). The cell recovery after thawing, cell viability after ICS and magnitude of HIV-specific CD8(+) T-cell responses were considered to determine the optimal combination of process conditions. The impact of time-to-process (2 or 4hours) on HIV-specific CD8(+) T-cell responses was also assessed in a whole blood ICS assay. A higher quality of cells in terms of recovery and viability (up to 81% and >80% respectively) was obtained with shorter time-to-process (less than 7hours) and resting-time (less than 2hours) intervals. Longer (overnight) rather than shorter (6hours) stimulation-time intervals increased the frequency of CD8(+)-specific T-cell responses using ICS in PBMCs without change of the functionality. The CD8(+) specific T-cell responses detected using fresh whole blood showed a good correlation with the responses detected using frozen PBMCs. Our results support the need of standardized procedures for the evaluation of CMI responses, especially in HIV-1-infected, ART-naïve patients.
    Journal of Immunological Methods 09/2014; 414. DOI:10.1016/j.jim.2014.09.001 · 1.82 Impact Factor
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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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    ABSTRACT: Clinical trials of immunologic therapies provide opportunities to study the cellular and molecular effects of those therapies and may permit identification of biomarkers of response. When the trials are performed at multiple centers, transport and storage of clinical specimens become important variables that may affect lymphocyte viability and function in blood and tissue specimens. The effect of temperature during storage and shipment of peripheral blood on subsequent processing, recovery, and function of lymphocytes is understudied and represents the focus of this study. Peripheral blood samples (n = 285) from patients enrolled in 2 clinical trials of a melanoma vaccine were shipped from clinical centers 250 or 1100 miles to a central laboratory at the sponsoring institution. The yield of peripheral blood mononuclear cells (PBMC) collected before and after cryostorage was correlated with temperatures encountered during shipment. Also, to simulate shipping of whole blood, heparinized blood from healthy donors was collected and stored at 15 °C, 22 °C, 30 °C, or 40 °C, for varied intervals before isolation of PBMC. Specimen integrity was assessed by measures of yield, recovery, viability, and function of isolated lymphocytes. Several packaging systems were also evaluated during simulated shipping for the ability to maintain the internal temperature in adverse temperatures over time. Blood specimen containers experienced temperatures during shipment ranging from -1 to 35 °C. Exposure to temperatures above room temperature (22 °C) resulted in greater yields of PBMC. Reduced cell recovery following cryo-preservation as well as decreased viability and immune function were observed in specimens exposed to 15 °C or 40 °C for greater than 8 hours when compared to storage at 22 °C. There was a trend toward improved preservation of blood specimen integrity stored at 30 °C prior to processing for all time points tested. Internal temperatures of blood shipping containers were maintained longer in an acceptable range when warm packs were included. Blood packages shipped overnight by commercial carrier may encounter extreme seasonal temperatures. Therefore, considerations in the design of shipping containers should include protecting against extreme ambient temperature deviations and maintaining specimen temperature above 22 °C or preferably near 30 °C.
    Journal of Translational Medicine 03/2011; 9(1):26. DOI:10.1186/1479-5876-9-26 · 3.93 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 · 3.40 Impact Factor
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