RESUMO
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.
Assuntos
Técnicas de Cultura de Células/métodos , Criopreservação/métodos , Leucócitos Mononucleares/citologia , Sobrevivência Celular , Células Cultivadas , Feminino , Citometria de Fluxo , Humanos , Contagem de Leucócitos , Leucócitos Mononucleares/metabolismo , MasculinoRESUMO
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.
Assuntos
Síndrome Coronariana Aguda/cirurgia , Hiperuricemia/epidemiologia , Inibidores da Agregação Plaquetária/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Tienopiridinas/farmacologia , Difosfato de Adenosina/farmacologia , Idoso , Angioplastia/efeitos adversos , Angioplastia/métodos , Clopidogrel/farmacologia , Comorbidade , Citocromo P-450 CYP2C9/genética , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Ativação Plaquetária/efeitos dos fármacos , Estudos Prospectivos , Stents/efeitos adversos , Ticagrelor/farmacologia , Ticlopidina/farmacologia , Ácido Úrico/sangueRESUMO
BACKGROUND: Patients with venous thromboembolism (VTE) are at risk for psychological consequences. However, as opposed to physical sequelae of VTE, mental health issues are understudied. OBJECTIVES: To assess anxiety after VTE and investigate associated clinical characteristics. PATIENTS/METHODS: We conducted a prospective cohort study, including patients with acute deep vein thrombosis (DVT) and/or pulmonary embolism (PE). Patients with cancer, pregnancy, or puerperium were excluded. Anxiety was assessed with the PROMIS short form 8a. Standardized T-scores were calculated (reference: 50, SD 10), with higher values indicating more anxiety. We associated clinical characteristics at baseline with T-scores at three-month follow-up in a multivariable linear regression model. Patient clusters depending on anxiety trajectories were explored. RESULTS: We included 257 patients (38.5% women) with a median (IQR) age of 54.1 (42.2-63.5) years. While mean (SD) T-scores decreased from baseline to follow-up (51.03 (9.18) to 46.74 (8.89), p<0.001), we observed an increase in 23.7%. Female sex (T-score change: 3.09, 95% confidence interval (CI), 0.96-5.22), older age until 45 years, and anxiety at baseline were associated with increased T-scores at follow-up. VTE history (-1.55, 95%CI, -3.62 to 0.52) and PE (-1.23, 95%CI, -3.16 to 0.69) were associated with reduced T-scores, albeit not reaching statistical significance. In a cluster of older, female patients with DVT, anxiety tended to increase over time. CONCLUSIONS: While most patients with VTE reported reduced anxiety over time, some patients experienced worsening. Female sex, older age, more anxiety at baseline, no VTE history, and DVT were associated with increased anxiety three months after VTE.
RESUMO
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.