BackgroundImmune platelet refractoriness is mainly caused by human leukocyte antigen antibodies (80-90% of cases) and, to a lesser extent, by human platelet antigen antibodies. Refractoriness can be diagnosed by laboratory tests and patients should receive compatible platelet transfusions. A fast, effective and low cost antibody-screening method which detects platelet human leukocyte/platelet antigen antibodies is essential in the management of immune platelet refractoriness. ObjectiveThe aim of this study was to evaluate the efficiency of the flow cytometry platelet immunofluorescence test to screen for immune platelet refractoriness. MethodsA group of prospective hematologic patients with clinically suspected platelet refractoriness treated in a referral center in Campinas, SP during July 2006 and July 2011 was enrolled in this study. Platelet antibodies were screened using the flow cytometry platelet immunofluorescence test. Anti-human leukocyte antigen antibodies were detected by commercially available methods. The sensitivity, specificity and predictive values of the immunofluorescence test were determined taking into account that the majority of antiplatelet antibodies presented human leukocyte antigen specificity. ResultsSeventy-six samples from 32 female and 38 male patients with a median age of 43.5 years (range: 5-84 years) were analyzed. The sensitivity of the test was 86.11% and specificity 75.00% with a positive predictive value of 75.61% and a negative predictive value of 85.71%. The accuracy of the method was 80.26%. ConclusionThis study shows that the flow cytometry platelet immunofluorescence test has a high correlation with the anti-human leukocyte antigen antibodies. Despite a few limitations, the method seems to be efficient, fast and feasible as the initial screening for platelet antibody detection and a useful tool to crossmatch platelets for the transfusional support of patients with immune platelet refractoriness.
Fetal alloimmune thrombocytopenia is a potentially lethal condition, but early detection and prevention lead to successful outcome in most cases.
Introduction: Deep venous thrombosis (DVT) is a common disease and post-thrombotic syndrome (PTS) is a complication present in about 20-50% of patients after the thrombotic episode (Kahn, 2016 - Hematology Am Soc Hematol Educ Program). The presence of residual venous thrombosis (RVT) can contribute to DVT recurrence and its role in SPT is controversial (Pradoni et al., 2009 - Annals of Internal Medicine; Cosmi et al., 2010 - Eur J Vasc Endovasc Surg). Fibrinolysis is the mechanism responsible for blood clot dissolution and decreased plasma fibrinolytic potential (DPFP), leads to an increased risk of DVT and appears to be associated with PTS, but not with recurrence(Lisman, 2017 - Semin Thromb Hemost; Meltzer et al., 2008 - PLoS Med). Furthermore, hypercoagulability and hypofibrinolysis synergistically enhance the risk of a first venous event, driven probably by elevated plasma levels of TAFI and PAI-1(Bombardier et al., 2012 - Thromb Res; Lisman, 2017 - Semin Thromb Hemost). Relationship between fibrinolysis and DVT has been focused on plasma levels of proteins or protease-inhibitor complexes, and not on plasma fibrinolytic potential (PFP). CloFAL is a turbidimetric assay that calculates the coagulation index (CI) and fibrinolytic index (FI), and allows the evaluation of global fibrinolysis based on clot lysis, with good analytical sensitivity for hypercoagulability and hypofibrinolysis. Preliminary studies of our group demonstrated decreased median fibrinolytic index in patients with lower limb DVT when compared to controls (98% vs 146%, p = 0.003). With the objective to increase scientific knowledge, we evaluated PFP in DVT patients and its association with PTS, RVT, and PAI-1 levels. Methods: PFP was determined by CloFAL assay and PAI-1 by ELISA method. The reference values of CI and FI were calculated using the median values obtained in plasma samples of 20 healthy subjects, along with the percentiles 25th and 75th (for CI, median was 104%, interquartile range (IQR) = 70-140%; for F, median = 97%; IQR = 80-180%). PTS and RVT were evaluated by Villalta scale and doppler ultrasound (US), respectively. Outpatients with DVT of lower limbs up to two years after the acute episode attended at Hemostasis Clinic of Hemocentro (University of Campinas - UNICAMP, SP Brazil) from 2014 to 2019 were included. Healthy controls were workers from Hemocentro. Exclusion criteria were cancer, anticoagulation, renal or hepatic disease, and infection. Continuous variables among groups were compared with Mann-Whitney test. A multiple regression analysis was performed by to evaluate the effect of age, sex, type of thrombosis (spontaneous or provoked), PAI-1, RVT, time between DVT and blood collection and CI on the observed values of FI. Results: Seventy-eight patients and 42 controls were included. The samples were collected 16 months (range = 1-27) after acute DVT. Forty-three (55%) patients presented PTS and 41 (71%) showed RVT on US. Our results corroborate our previous study, as patients demonstrated a significant increase of CI (p = 0.0009) and decrease of FI (p = 0.0009) (Table 1). However, when patients were analyzed according to the presence of SPT or RVT, no difference was observed. Median PAI-1 levels corrected by BMI were higher in patients compared to controls (8.5 vs 4.8, p< 0.0001). No difference was observed in PAI-1 levels when we compared patients according to SPT and RVT status. When multiple variables were analyzed with a multiple regression analysis, only CI statistically significantly predicted FI (R2= 0.278; p=0.003). Conclusions: Patients with DVT, even in the chronic phase, presented hypofibrinolysis, when compared to controls. We initially found higher PAI-1 levels in patients when compared to controls but with a multivariate analysis, CI was the only variable that predicted FI. Therefore, other factors not included in this study may contribute to the lower FI values observed in our cohort. The presence of RVT or PTS did not induce modifications of global fibrinolysis. Disclosures No relevant conflicts of interest to declare.
Venous thromboembolism (VTE) is a common multifactorial disease, and the knowledge in the pathophysiology can improve prophylaxis and treatment. The myeloid lineage, especially neutrophils, have been implied in the pathophysiology of VTE, but it is still unclear whether its activation is persistent after the acute phase of the disease. In addition to phagocytosis, neutrophils participate of the innate immunity through the release of NETs (Neutrophil Extracellular Traps): Reactive Oxygen Species (ROS)-triggered antimicrobial structures composed of DNA lined with granular components (Brinkmann et al., 2004). NETs also contribute to the coagulation, as they facilitate the interaction between erythrocytes, leukocytes, platelets and endothelium, besides potentially inhibit anticoagulant pathways (Fuchs et al., 2012). The molecular mechanisms underlying the "NETosis" are still being characterized, but it is known that following inflammatory stimuli, the generation of ROS leads to the dissolution of intracellular membranes and translocation of Myeloperoxidase (MPO) and Neutrophil Elastase (ELANE) to nucleus. Histones are then citrullinated by Peptidyl Arginine Deiminase 4 (PADI4) and degraded by ELANE, promoting chromatin decondensation, plasma membrane rupture and NET's release. Previously we showed increased neutrophil activation, adhesive properties and the presence of NETs serum markers in patients with VTE in the chronic phase (Zapponi et al., 2014; Zapponi, manuscript in preparation). However, assessment of NETs activity is challenging and, until now, there is no consensus in markers or gold standards measurements. We decided to investigate if the expression of the genes PADI4, ELANE, MPO, could provide additional information regarding NETosis activity, through an indirect analysis of the process by Real-Time PCR. We analyzed samples from 20 controls and 19 patients previously included in the NET study, respecting the time window in which the NETs plasma markers were assessed (between 3 and 43 months after the TVP event). RNA extraction was performed from peripheral blood leukocytes using Trizol reagent, according to the manufacturer's protocol. Real time-PCR reactions were performed with qPCRBIO SyGreen mix (PCRBio), on RotorGene Q (Qiagen) with a reaction efficiency above 0.99. The relative fold changes (Fc) of the genes PADI4, ELANE, MPO were calculated using the Pfaffl method (Pfaffl, 2001 - Nucleic Acids Res.) and the significance level of Mann-Whitney test was considered if p <0.05. The 39 cDNA samples were amplified in duplicates and a standard sample was included for normalization between different runs. The threshold values (Ct) from PADI4, ELANE, MPO were adjusted for the individual's neutrophil proportion and the geometric means of the housekeeping genes (Actin Beta and Glyceraldehyde-3-Phosphate Dehydrogenase) were used for normalization between samples. The standard deviation was 0.30 between runs and ranged between duplicates from 0 to 1.16 (0.096 on average). The patients showed significant increased expression of PADI4 (p <0.01), MPO and ELANE (p <0.05) when compared to controls (Figure 1). Neutrophils emerges fully differentiated from the bone marrow and ranges from 40 to 70% of circulating leukocytes (Cowland and Borregaard, 2016). Isolating these cells can be challenging and the process itself may influence expression profile. The use of total leukocytes would not be the most appropriate process but considering that the analyzed genes are predominantly expressed in neutrophils, the Ct values were weighted according to the neutrophil to leucocytes proportions of each individual. Our results indicate that the increased neutrophils activation in patients can be represented in PADI4, ELANE and MPO gene expression of peripheral blood leukocytes, which could better represent NETotic activity in these patients. We cannot rule out that the expression of these genes in myeloid precursors could better represent NETotic activity, but our results indicate that this analysis may also be viable in peripheral blood. Disclosures No relevant conflicts of interest to declare.
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