Quantitative and Functional Assessment of the Influence of Routinely Used Cryopreservation Media on Mononuclear Leukocytes for Medical Research.

Quantitative and functional analysis of mononuclear leukocyte populations is an invaluable tool to understand the role of the immune system in the pathogenesis of a disease. Cryopreservation of mononuclear cells (MNCs) is routinely used to guarantee similar experimental conditions. Immune cells react differently to cryopreservation, and populations and functions of immune cells change during the process of freeze-thawing. To allow for a setup that preserves cell number and function optimally, we tested four different cryopreservation media. MNCs from 15 human individuals were analyzed. Before freezing and after thawing, the distribution of leukocytes was quantified by flow cytometry. Cultured cells were stimulated using lipopolysaccharide, and their immune response was quantified by flow cytometry, quantitative polymerase chain reaction (qPCR), and enzyme-linked immunosorbent assay (ELISA). Ultimately, the performance of the cryopreservation media was ranked. Cell recovery and viability were different between the media. Cryopreservation led to changes in the relative number of monocytes, T cells, B cells, and their subsets. The inflammatory response of MNCs was altered by cryopreservation, enhancing the basal production of inflammatory cytokines. Different cryopreservation media induce biases, which needs to be considered when designing a study relying on cryopreservation. Here, we provide an overview of four different cryopreservation media for choosing the optimal medium for a specific task.

Decreased Platelet Inhibition by Thienopyridines in Hyperuricemia.

PURPOSE: Hyperuricemia carries an increased risk of atherothrombotic events in acute coronary syndrome (ACS) patients undergoing percutaneous coronary intervention (PCI). This may at least in part be due to inadequate P2Y12 inhibition. The aim of this study was to prospectively investigate the potential association between hyperuricemia and decreased platelet inhibition by P2Y12 antagonists. METHODS: Levels of uric acid as well as on-treatment residual platelet reactivity in response to adenosine diphosphate (ADP) were assessed in 301 clopidogrel-treated patients undergoing elective angioplasty and stenting, and in 206 prasugrel- (n = 118) or ticagrelor-treated (n = 88) ACS patients following acute PCI. Cut-off values for high on-treatment residual ADP-inducible platelet reactivity (HRPR) were based on previous studies showing an association of test results with clinical outcomes. RESULTS: Hyperuricemia was significantly associated with increased on-treatment residual ADP-inducible platelet reactivity in clopidogrel- and prasugrel-treated patients in univariate analyses and after adjustment for differences in patient characteristics by multivariate regression analyses. In contrast, ticagrelor-treated patients without and with hyperuricemia showed similar levels of on-treatment residual platelet reactivity to ADP. HRPR occurred more frequently in clopidogrel- and prasugrel-treated patients with hyperuricemia than in those with normal uric acid levels. In contrast, hyperuricemic patients receiving ticagrelor did not have a higher risk of HRPR compared with those with normal uric acid levels. CONCLUSION: Hyperuricemia is associated with decreased platelet inhibition by thienopyridines but a normal response to ticagrelor. It remains to be established if lowering uric acid increases the antiplatelet effects of clopidogrel and prasugrel in hyperuricemic patients with HRPR.

Platelet-to-lymphocyte and Neutrophil-to-lymphocyte Ratios Predict Target Vessel Restenosis after Infrainguinal Angioplasty with Stent Implantation.

Platelet-to-lymphocyte (PLR), neutrophil-to-lymphocyte (NLR) and lymphocyte-to-monocyte (LMR) ratios are associated with the occurrence of critical limb ischemia in peripheral artery disease (PAD). We therefore investigated whether PLR, NLR or LMR are linked to target vessel restenosis (TVR) following infrainguinal angioplasty and stenting. Moreover, we studied on-treatment platelet reactivity and neutrophil-platelet aggregate (NPA) formation as potential underlying mechanisms. Platelet, neutrophil, lymphocyte and monocyte counts were determined one day after angioplasty and stenting in 95 stable PAD patients. Platelet reactivity and NPA formation in response to protease-activated receptor-1 stimulation were measured by light transmission aggregometry (LTA) and flow cytometry, respectively. PLR and NLR were significantly higher in patients who subsequently developed TVR (both p < 0.05). In contrast, LMR did not differ significantly between patients without and with TVR (p = 0.28). A PLR ≥ 91 and NLR ≥2.75 were identified as the best thresholds to predict TVR, providing sensitivities of 87.5% and 81.3%, and specificities of 34.9% and 50.8%, respectively, and were therefore defined as high PLR and high NLR. TVR occurred significantly more often in patients with high PLR and high NLR than in those with lower ratios (both p < 0.05). Patients with high PLR and high NLR exhibited significantly increased on-treatment platelet aggregation compared to those with lower ratios, and patients with high PLR had higher levels of NPA formation (all p < 0.01). In conclusion, PLR and NLR predict TVR after infrainguinal angioplasty with stent implantation. Platelet activation and neutrophil-platelet interaction may be involved in the underlying pathomechanisms.