Journal of immunological methods · 2025
From phlebotomy to peripheral blood mononuclear cell isolation; effect of time and early-life HIV/ART exposure on cell yield and viability in paediatric samples in a high HIV prevalence setting.
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Mataramvura Hope, Ncube Patience, Duri Kerina
Аннотация
BACKGROUND: Viral infection/exposure and time from phlebotomy to peripheral blood mononuclear cell (PBMC) isolation (TPP) may affect PBMC yield and viability. We investigated the impact of TPP and early-life HIV/ART exposure on PBMC, including NK cells yield and viability.
METHODS: Blood in EDTA tubes with varying TPP was used to isolate PBMCs via density-gradient centrifugation, counted under a microscope, and assessed for viability with Trypan-Blue. Antibody staining (CD14, CD3, and CD19) excluded monocytes and T/B lymphocytes, while CD16 and CD56 staining with a LIVE/DEAD marker identified viable NK cells. Comparisons were made among HIV-exposed uninfected (HEU) children (long-term [HEULT] and medium-term [HEUMT] ART exposure), HIV-exposed infected (HEI), and HIV-unexposed uninfected (HUU) children.
RESULTS: We enrolled 112 children (50 % female) with a median age of 5.1 years [interquartile range (IQR):4.8-6.0], categorized as 37-HUU, 36-HEULT, 34-HEUMT, and 5-HEI children. Median blood volume was 7.5 ml (IQR:7.5-8.0), with HEI children showing slightly higher PBMC yields/ml than HUU and HEU (p = 0.092). Median TPP was 90 min (IQR:64.8-112.0), with viability remaining high (>95 %) up to 3 h, after this the yields/ml decreased compared to TPP ≤ 1 h (1.3 vs. 2.8 × 106 cells/ml) (p = 0.010) for all children. Overall, TPP negatively correlated with yields/ml and viability (r = -0.35, p < 0.001; r = -0.47, p < 0.001 respectively). Median frequency of viable CD56 + CD16+/- NK cells was 8.2 %, unaffected by neither TPP nor HIV/ART exposure. Total lymphocytes were lower in males [18.6 % (IQR:11.3-22.2)] than in females [23.8 % (IQR:14.3-29.9)] (p = 0.018).
CONCLUSION: HIV infection, not exposure, may increase PBMC yields. TPP under 3 h is ideal for optimal yields.
METHODS: Blood in EDTA tubes with varying TPP was used to isolate PBMCs via density-gradient centrifugation, counted under a microscope, and assessed for viability with Trypan-Blue. Antibody staining (CD14, CD3, and CD19) excluded monocytes and T/B lymphocytes, while CD16 and CD56 staining with a LIVE/DEAD marker identified viable NK cells. Comparisons were made among HIV-exposed uninfected (HEU) children (long-term [HEULT] and medium-term [HEUMT] ART exposure), HIV-exposed infected (HEI), and HIV-unexposed uninfected (HUU) children.
RESULTS: We enrolled 112 children (50 % female) with a median age of 5.1 years [interquartile range (IQR):4.8-6.0], categorized as 37-HUU, 36-HEULT, 34-HEUMT, and 5-HEI children. Median blood volume was 7.5 ml (IQR:7.5-8.0), with HEI children showing slightly higher PBMC yields/ml than HUU and HEU (p = 0.092). Median TPP was 90 min (IQR:64.8-112.0), with viability remaining high (>95 %) up to 3 h, after this the yields/ml decreased compared to TPP ≤ 1 h (1.3 vs. 2.8 × 106 cells/ml) (p = 0.010) for all children. Overall, TPP negatively correlated with yields/ml and viability (r = -0.35, p < 0.001; r = -0.47, p < 0.001 respectively). Median frequency of viable CD56 + CD16+/- NK cells was 8.2 %, unaffected by neither TPP nor HIV/ART exposure. Total lymphocytes were lower in males [18.6 % (IQR:11.3-22.2)] than in females [23.8 % (IQR:14.3-29.9)] (p = 0.018).
CONCLUSION: HIV infection, not exposure, may increase PBMC yields. TPP under 3 h is ideal for optimal yields.