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Optimization of a whole blood phenotyping assay for enumeration of peripheral blood leukocyte populations in multicenter clinical trials.

ABSTRACT: Vaccination with viral vectors or adjuvants can induce early changes in circulating peripheral blood leukocytes that are predictive of a protective immune response. In this study, we define an 11-color whole blood antibody staining Trucount Panel (TP1) to enumerate and phenotype the major leukocyte populations in a human vaccine experimental medicine trial setting. TP1 can be prepared up to 8weeks prior to use, enabling bulk preparation at a central laboratory and distribution to clinical sites. Cells in whole blood must be stained within 4h of draw to accurately detect the major cell populations. Staining of cells with TP1 followed by storage and shipping at -80°C to a central laboratory has little to no effect on the cell concentrations observed. We also present data from an HIV vaccine multicenter clinical trial obtained using the optimized TP1 assay protocol and show that the data produced accurately correlates with complete blood count (CBC) data. Taken together, these data indicate the optimized TP1 panel assay can be used in a multicenter clinical trial setting to increase our understanding of systemic responses to vaccination or disease.

SUBMITTER: Hensley-McBain T

PROVIDER: S-EPMC4171197 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Optimization of a whole blood phenotyping assay for enumeration of peripheral blood leukocyte populations in multicenter clinical trials.

Hensley-McBain Tiffany T Heit Antje A De Rosa Stephen C SC McElrath M Juliana MJ Andersen-Nissen Erica E

Journal of immunological methods 20140609

Vaccination with viral vectors or adjuvants can induce early changes in circulating peripheral blood leukocytes that are predictive of a protective immune response. In this study, we define an 11-color whole blood antibody staining Trucount Panel (TP1) to enumerate and phenotype the major leukocyte populations in a human vaccine experimental medicine trial setting. TP1 can be prepared up to 8weeks prior to use, enabling bulk preparation at a central laboratory and distribution to clinical sites. ...[more]

PMID: 24925805

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Project description:BackgroundEnvironmental exposures including air pollutants, toxic metals, and psychosocial stress have been associated with shorter telomere length (TL) in newborns. These exposures have in turn been linked to an enhanced inflammatory immune response. Increased inflammation during pregnancy may be a central biological pathway linking environmental factors with reduced TL at birth. Approaches that more comprehensively characterize the prenatal inflammatory milieu rather than targeting specific individual cytokines in relation to newborn TL may better elucidate inflammatory mechanisms.MethodsAnalyses included 129 mother-child dyads enrolled in the PRogramming of Intergenerational Stress Mechanisms (PRISM) pregnancy cohort. We measured 92 inflammation related proteins during pregnancy in maternal serum using the Olink protein array and quantified cord blood relative leukocyte TL (rLTL) via qPCR. We leveraged a tree-based machine learning algorithm to select the most important inflammatory related proteins jointly associated with rLTL. We then evaluated the combined association between the selected proteins with rLTL using Bayesian Weighted Quantile Sum (BWQS) Regression. Analyses were adjusted for gestational week of serum collection, maternal race/ethnicity, age, and education, and fetal sex. We evaluated major biological function of the identified proteins by using the UniProtKB, a centralized repository of curated functional information.ResultsThree proteins were negatively and linearly associated with rLTL (CASP8 β: -0.22 p = 0.008, BNGF β: -0.43 p = 0.033, TRANCE β: 0.38 p = 0.004). Results from BWQS regression showed a significant overall decrease in rLTL (β: -0.26 95%CrI: -0.43, -0.07) per quartile increase of the mixture, with CASP8 contributing the greatest weight (CASP8 50%; BNGF 27%, and TRANCE 23%). The identified proteins were involved in the regulation of apoptotic processes and cell proliferation.ConclusionsThis proteomics approach identifies novel maternal prenatal inflammatory protein biomarkers associated with shortened rLTL in newborns.

Project description:DNA damage occurs constantly in every cell triggered by endogenous processes of replication and metabolism, and external influences such as ionizing radiation and intercalating chemicals. Large sets of proteins are involved in sensing, stabilizing and repairing this damage including control of cell cycle and proliferation. Some of these factors are phosphorylated upon activation and can be used as biomarkers of DNA damage response (DDR) by flow and mass cytometry. Differential survival rates of lymphocyte subsets in response to DNA damage are well established, characterizing NK cells as most resistant and B cells as most sensitive to DNA damage. We investigated DDR to low dose gamma radiation (2Gy) in peripheral blood lymphocytes of 26 healthy donors and 3 patients with ataxia telangiectasia (AT) using mass cytometry. γH2AX, p-CHK2, p-ATM and p53 were analyzed as specific DDR biomarkers for functional readouts of DNA repair efficiency in combination with cell cycle and T, B and NK cell populations characterized by 20 surface markers. We identified significant differences in DDR among lymphocyte populations in healthy individuals. Whereas CD56+CD16+ NK cells showed a strong γH2AX response to low dose ionizing radiation, a reduced response rate could be observed in CD19+CD20+ B cells that was associated with reduced survival. Interestingly, γH2AX induction level correlated inversely with ATM-dependent p-CHK2 and p53 responses. Differential DDR could be further noticed in naïve compared to memory T and B cell subsets, characterized by reduced γH2AX, but increased p53 induction in naïve T cells. In contrast, DDR was abrogated in all lymphocyte populations of AT patients. Our results demonstrate differential DDR capacities in lymphocyte subsets that depend on maturation and correlate inversely with DNA damage-related survival. Importantly, DDR analysis of peripheral blood cells for diagnostic purposes should be stratified to lymphocyte subsets.

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Optimization of a whole blood phenotyping assay for enumeration of peripheral blood leukocyte populations in multicenter clinical trials.

Publications

Optimization of a whole blood phenotyping assay for enumeration of peripheral blood leukocyte populations in multicenter clinical trials.

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