Five new F10 variants in hereditary factor x deficiency detected by high-throughput sequencing

International audienceFactor X deficiency is a rare inherited bleeding disorder. To date, 181 variants are reported in the recently updated F10-gene variant database.This study aimed to describe new F10 variants.The gene was analysed 16 consecutive families with FX by targeted high-throughput sequencing approach, including F10, F9, F8 genes, and 78 genes dedicated haematological malignancies.We identified 19 (17 missense, one nonsense frameshift) two copy number variations. Two patients presenting combined FVII-FX showed loss of (del13q34) associated missense on remaining allele, leading FX:C significantly lower than FVII:C level explaining their unusual history. We five novel variants. Three (p.Glu22Val affecting signal peptide cleavage site, p.Cys342Tyr removing disulphide bond between heavy light chains, p.Val385Met located peptidase S1 domain) were detected at compound heterozygosis status three severe symptoms below 10 IU/dL. truncating p.Tyr279* p.Thr434Aspfs*13 an altered protein found heterozygous state mild history.This feasibility interest approach for disorders, enabling report screening 3-weeks delay, suitable clinical use. The description may contribute better understanding phenotype-genotype correlation deficiency

A performance evaluation of sthemO 301 coagulation analyzer and associated reagents

International audienceAIM: The study objective was to evaluate the performance of sthemO 301 system and to compare it with the analyzer used in our university hospital laboratory (STA R Max® 2), for a selection of hemostasis parameters. METHODS: Method comparison (according to CLSI EP09-A3), carryover (according to CLSI H57-A), APTT sensitivity to heparin (according to CLSI H47-A2), HIL level assessment, and productivity were performed using leftover samples from our laboratory (n > 1000). Commercial quality control materials were used to evaluate precision (according to CLSI EP15-A3) and accuracy. The assays tested on sthemO 301 were: PT, APTT (silica and kaolin activators), fibrinogen (Fib), thrombin time (TT), chromogenic and clotting protein C (PC) activity, and von Willebrand factor antigen (VWF:Ag) levels. RESULTS: All intra-assay and inter-assay precision CVs were below the maximal precision limit proposed by the French Group for Hemostasis and Thrombosis (GFHT). Accuracy was verified with bias below GFHT criteria and most Z-scores were between -2 and +2. No clinically relevant carryover was detected. Silica APTT reagent sensitivity to unfractionated heparin was moderate, as expected. Productivity results were consistent over the 10 repeats performed. The overall agreement between the two systems was excellent for all assays, with Spearman rank correlation coefficient all above 0.9 and slopes of Passing-Bablok correlation near 1 and intercepts close to 0. CONCLUSION: For the methods tested, sthemO 301 system met all the criteria to implement a novel coagulation analyzer in the laboratory and result comparability with STA R Max® 2 was good

Platelet Functions During Extracorporeal Membrane Oxygenation. Platelet-Leukocyte Aggregates Analyzed by Flow Cytometry as a Promising Tool to Monitor Platelet Activation

International audienceExtracorporeal membrane oxygenation (ECMO) is an extracorporeal circulation used to manage patients with severe circulatory or respiratory failure. It is associated with both high bleeding and thrombosis risks, mainly as a result of biomaterial/blood interface phenomena, high shear stress, and complex inflammatory response involving the activation of coagulation and complement systems, endothelial cells, leukocytes, and platelets. Besides their critical role in hemostasis, platelets are important players in inflammatory reactions, especially due to their ability to bind and activate leukocytes. Hence, we reviewed studies on platelet function of ECMO patients. Moreover, we addressed the issue of platelet-leukocyte aggregates (PLAs), which is a key step in both platelet and leukocyte activation, and deserves to be investigated in these patients. A reduced expression of GPIb and GPVI was found under ECMO therapy, due to the shedding processes. However, defective platelet aggregation is inconsistently reported and is still not clearly defined. Due to the high susceptibility of PLAs to pre-analytical conditions, defining and strictly adhering to a rigorous laboratory methodology is essential for reliable and reproducible results, especially in the setting of complex inflammatory situations like ECMO. We provide results on sample preparation and flow cytometric whole blood evaluation of circulating PLAs

Impact of age on in vitro metabolism of clopidogrel: a potential explanation for high on-treatment platelet reactivity in the elderly?

International audienceBACKGROUND: High on-treatment platelet reactivity has been reported in 30% of patients on clopidogrel and 50% in elderly patients; however, little is known about the mechanisms of this biological resistance. One hypothesis is an age-related impaired hepatic metabolism of the prodrug clopidogrel, leading to a lower formation of its active metabolite (clopidogrel-AM). OBJECTIVES: To compare the levels of clopidogrel-AM formed in vitro using "old" and "young" human liver microsomes (HLMs) and their consequences on platelet functions. METHODS: We developed an in vitro model using "old" (73.6 ± 2.3 years) and "young" (51.2 ± 8.5 years) HLMs, added to platelet-rich plasma from 21 healthy donors with or without clopidogrel (50 μM) and incubated at 37 °C for 30 (T30) and 45 minutes (T45). Clopidogrel-AM was quantified by liquid chromatography-mass spectrometry/mass spectrometry method. Platelet aggregation was performed by light transmission aggregometry. RESULTS: The generation of clopidogrel-AM increased over time and reached concentrations comparable with those reported in treated patients. At T30, mean clopidogrel-AM concentrations were significantly higher with "young" (8.56 μg/L; 95% CI, 5.87-11.24) than with "old" HLMs (7.64 μg/L; 95% CI, 5.14-10.14; P = .002); and at T45, 11.40 μg/L; 95% CI (7.57-15.22) vs 10.63 μg/L, 95% CI (7.10-14.15), P = .02 (n = 21). Despite a significant inhibition of platelet aggregation, no significant difference was found in light transmission aggregometry (adenosine diphosphate, 10 μM) after clopidogrel metabolism by "old" or "young" HLMs, probably because of low sensitivity of the method to small variations of clopidogrel-AM. CONCLUSION: In this original model combining metabolic and functional approaches, less clopidogrel-AM was produced with HLMs from older patients. This provides support for a decreased CYP450 activity that may contribute to high on-treatment platelet reactivity in elderly patients

Apixaban and rivaroxaban in obese patients treated for venous thromboembolism: Drug levels and clinical outcomes

International audienceBACKGROUND: Direct oral anticoagulants (DOAC) use remains challenging in obese patients treated for Venous-Thrombo-Embolism (VTE) due to the paucity of prospective and dedicated studies. OBJECTIVE: To assess rivaroxaban and apixaban concentrations at different time-points after intake, in obese patients followed at a thrombosis center and treated for VTE; to define factors associated with DOAC levels outside the on-therapy ranges; and to evaluate bleeding and thrombosis rates during follow-up. METHODS: Observational prospective study in two French University hospitals. Apixaban or rivaroxaban concentrations were measured after the first visit, regardless of last intake in obese patients receiving DOAC for VTE. Concentrations were compared to published reference values for non-obese patients. Demographic, clinical, biological and therapeutic data were collected. Univariate and multivariate analyses were performed to identify factors associated to DOAC concentrations outside the on-therapy ranges. RESULTS: Out of the 146 patients included, 22 (15%) had DOAC concentrations outside the on-therapy ranges, mainly in the rivaroxaban group (n = 17). Age ≤ 63 years, use of rivaroxaban and time since last intake ≤8 h were associated with DOAC concentrations outside the on-therapy ranges, in multivariable analysis. During the median follow-up of 16 months, two (1%) patients receiving apixaban had recurrent VTE. No patient had major bleeding, 11 (8%) patients had minor bleeding. CONCLUSION: In this specific prospective bi-centric study dedicated to VTE obese patients, use of DOACs at fixed doses led to concentrations similar to those of non-obese patients in a high proportion of patients, without any effect of the BMI, and with risk-benefit profile comparable to non-obese patients