Quality Monitoring of HIV-1-Infected and Uninfected Peripheral Blood Mononuclear Cell Samples in a Resource-Limited Setting

Abstract

Human immunodeficiency virus type 1 (HIV-1) vaccine and natural history studies are critically dependent on the ability to isolate, cryopreserve, and thaw peripheral blood mononuclear cell (PBMC) samples with a high level of quality and reproducibility. Here we characterize the yield, viability, phenotype, and function of PBMC from HIV-1-infected and uninfected Ugandans and describe measures to ascertain reproducibility and sample quality at the sites that perform cryopreservation. We have developed a comprehensive internal quality control program to monitor processing, including components of method validation. Quality indicators for real-time performance assessment included the time from venipuncture to cryopreservation, time for PBMC processing, yield of PBMC from whole blood, and viability of the PBMC before cryopreservation. Immune phenotype analysis indicated lowered B-cell frequencies following processing and cryopreservation for both HIV-1-infected and uninfected subjects (P < 0.007), but all other major lymphocyte subsets were unchanged. Long-term cryopreservation did not impact function, as unstimulated specimens exhibited low background and all specimens responded to staphylococcal enterotoxin B (SEB) by gamma interferon and interleukin-2 production, as measured by intracellular cytokine staining. Samples stored for more than 3 years did not decay with regard to yield or viability, regardless of HIV-1 infection status. These results demonstrate that it is possible to achieve the high level of quality necessary for vaccine trials and natural history studies in a resource-limited setting and provide strategies for laboratories to monitor PBMC processing performance.Cryopreserved peripheral blood mononuclear cells (PBMC) are critical to studies of HIV-1 infection and for the development of an effective HIV vaccine. High-quality cryopreservation is particularly challenging and important in resource-limited settings, where natural history, therapeutic, and preventative protocols are being developed. Cryopreservation and batch testing allow for simultaneous assessment of critical samples, thereby reducing interassay variability. Since standard method validation measures, such as accuracy, precision, linearity, and reportable ranges, are not easily obtained for PBMC processing, more studies are needed to understand the parameters influencing the reproducibility of processing and quality of cryopreserved PBMC samples in resource-limited settings.Separation of PBMC should yield a pure population of mononuclear cells (95% Ϯ 5%) (18), with minimal

Human Immunodeficiency Virus Type 1 Infection Is Associated with Increased NK Cell Polyfunctionality and Higher Levels of KIR3DL1+NK Cells in Ugandans Carrying the HLA-B Bw4 Motif

“…Study participants aged 19 to 48 years were from a community-based cohort in the Kayunga district, Uganda (26). Peripheral blood mononuclear cells (PBMCs) were isolated from acid citrate dextrose (ACD)-anticoagulated whole blood within 6 h of collection by centrifugation through Ficoll-Hypaque (Pharmacia, Sweden) using Leucosep tubes (Greiner Bio-One, Germany) at 800 ϫ g for 15 min and cryopreserved as previously described (39). The study was approved by institutional review boards both in the United States and Uganda.…”

The Center for HIV/AIDS Vaccine Immunology (CHAVI) multi-site quality assurance program for cryopreserved Human Peripheral Blood Mononuclear Cells

“…Quality control (QC) indicators of initial PBMC integrity include viability, PBMC yield/mL of whole blood, and the time elapsed between blood collection and cryopreservation (Bull et al, 2007; Dyer et al, 2007; Kierstead et al, 2007; Olemukan et al, 2010). Previous studies have highlighted the importance of obtaining post-thaw PBMC viabilities of ≥70% since the results of functional and phenotypic studies are strictly dependent on the viability of the cryopreserved PBMC (Cox et al, 2005; Kreher et al, 2003; Reimann et al, 2000; Sleasman et al, 1997; Weinberg et al, 2000; Weinberg et al, 2009; Weinberg et al, 2010).…”

Establishment and maintenance of a PBMC repository for functional cellular studies in support of clinical vaccine trials

“…It should be noted that most of the studies performed in support to vaccine clinical trials are utilizing cryopreserved cellular samples. The testing of cryopreserved T and NK cells has already been bridged to that of samples collected from fresh blood (Horton et al, 2007; Pollara et al, 2011; Russell et al, 2003), whereas there is still unclear notions on the effect of cryopreservation on the recovery of the functional B cell populations (Olemukan et al, 2010; Reimann et al, 2000). This clinical trial shed new light on the type and specificity of immune responses that correlated with lower risk of HIV-1 infection.…”