Antithrombin Levels and Heparin Responsiveness during Venoarterial Extracorporeal Membrane Oxygenation: A Prospective Single-center Cohort Study.

BACKGROUND: Unfractionated heparin, administered during venoarterial extracorporeal membrane oxygenation to prevent thromboembolic events, largely depends on plasma antithrombin for its antithrombotic effects. Decreased heparin responsiveness seems frequent on extracorporeal membrane oxygenation; however, its association with acquired antithrombin deficiency is poorly understood. The objective of this study was to describe longitudinal changes in plasma antithrombin levels during extracorporeal membrane oxygenation support and evaluate the association between antithrombin levels and heparin responsiveness. The hypothesis was that extracorporeal membrane oxygenation support would be associated with acquired antithrombin deficiency and related decreased heparin responsiveness. METHODS: Adults receiving venoarterial extracorporeal membrane oxygenation were prospectively included. All patients received continuous intravenous unfractionated heparin using a standardized protocol (target anti-Xa 0.3 to 0.5 IU/ml). For each patient, arterial blood was withdrawn into citrate-containing tubes at 11 time points (from hour 0 up to day 7). Anti-Xa (without dextran or antithrombin added) and antithrombin levels were measured. The primary outcome was the antithrombin plasma level. In the absence of consensus, antithrombin deficiency was defined as a time-weighted average of antithrombin less than or equal to 70%. Data regarding clinical management and heparin dosage were collected. RESULTS: Fifty patients, including 42% postcardiotomy, were included between April 2020 and May 2021, with a total of 447 samples. Median extracorporeal membrane oxygenation duration was 7 (interquartile range, 4 to 12) days. Median antithrombin level was 48% (37 to 60%) at baseline. Antithrombin levels significantly increased throughout the follow-up. Time-weighted average of antithrombin levels was 63% (57 to 73%) and was less than or equal to 70% in 32 (64%) of patients. Overall, 45 (90%) patients had at least one antithrombin value less than 70%, and 35 (70%) had at least one antithrombin value less than 50%. Antithrombin levels were not significantly associated with heparin responsiveness evaluated by anti-Xa assay or heparin dosage. CONCLUSIONS: Venoarterial extracorporeal membrane oxygenation support was associated with a moderate acquired antithrombin deficiency, mainly during the first 72 h, that did not correlate with heparin responsiveness.

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

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

A performance evaluation of sthemO 301 coagulation analyzer and associated reagents.

AIM: 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.

Factors Influencing Unfractionated Heparin Pharmacokinetics and Pharmacodynamics During a Cardiopulmonary Bypass.

BACKGROUND: Unfractionated heparin (UFH) is commonly used during cardiac surgery with a cardiopulmonary bypass to prevent blood clotting. However, empirical administration of UFH leads to variable responses. Pharmacokinetic and pharmacodynamic modeling can be used to optimize UFH dosing and perform real-time individualization. In previous studies, many factors that could influence UFH pharmacokinetics/pharmacodynamics had not been taken into account such as hemodilution or the type of UFH. Few covariates were identified probably owing to a lack of statistical power. This study aims to address these limitations through a meta-analysis of individual data from two studies. METHODS: An individual patient data meta-analysis was conducted using data from two single-center prospective observational studies, where different UFH types were used for anticoagulation. A pharmacodynamic/pharmacodynamic model of UFH was developed using a non-linear mixed-effects approach. Time-varying covariates such as hemodilution and fluid infusions during a cardiopulmonary bypass were considered. RESULTS: Activities of UFH's anti-activated factor/anti-thrombin were best described by a two-compartment model. Unfractionated heparin clearance was influenced by body weight and the specific UFH type. Volume of distribution was influenced by body weight and pre-operative fibrinogen levels. Pharmacodynamic data followed a log-linear model, accounting for the effect of hemodilution and the pre-operative fibrinogen level. Equations were derived from the model to personalize UFH dosing based on the targeted activated clotting time level and patient covariates. CONCLUSIONS: The population model effectively characterized UFH's pharmacokinetics/pharmacodynamics in cardiopulmonary bypass patients. This meta-analysis incorporated new covariates related to UFH's pharmacokinetics/pharmacodynamics, enabling personalized dosing regimens. The proposed model holds potential for individualization using a Bayesian estimation.

Phenotypic and proteomic analysis of plasma extracellular vesicles highlights them as potential biomarkers of primary Sjögren syndrome.

Sjögren syndrome (SjS) is an autoimmune disease characterized by the destruction of the exocrine gland epithelia, causing a dryness of mucosa called sicca symptoms, and whose main life-threatening complication is lymphoma. There is a need for new biomarkers in this disease, notably diagnostic biomarkers for patients with genuine sicca symptoms that do not meet current criteria, and prognostic biomarkers for patients at risk of lymphoma. Plasma extracellular vesicles (EVs) are promising biomarker candidates in several diseases, but their potential has not yet been explored in SjS. In this proof-of-concept study, we characterized EVs from primary SjS patients (pSS, n=12) at the phenotypic and proteomic levels, compared to EVs from healthy donor (HD, n=8) and systemic lupus erythematosus patients (SLE, n=12). Specific plasma EVs subpopulations, derived from neutrophils, endothelial, and epithelial cells, were found increased in pSS. We also identified a pSS proteomic signature in plasma EVs, including neutrophil-, epithelial-, and endothelial-related proteins, such as integrin alpha M (ITGAM), olfactomedin-4 (OLFM4), Ras-related protein RAB10, and CD36. Overall, our results support the relevance of plasma EVs as biomarkers in SjS.

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

Background: 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.

Factors Influencing Anti-Xa Assays: A Multicenter Prospective Study in Critically Ill and Noncritically Ill Patients Receiving Unfractionated Heparin.

BACKGROUND: The presence of dextran sulfate (DS) in reagents and the type of blood collection tube (citrate/citrated-theophylline-adenosine-dipyridamole [CTAD]) can lead to discrepancies between unfractionated heparin (UFH) anti-Xa levels. OBJECTIVES: To evaluate the extent of the effect (1) of different reagents containing or not containing DS and (2) of the blood collection tubes, on UFH anti-Xa levels, in various clinical situations (NCT04700670). METHODS: We prospectively included patients from eight centers: group (G)1, cardiopulmonary bypass (CPB) after heparin neutralization (n = 39); G2, cardiothoracic intensive care unit (ICU) after CPB (n = 35); G3, medical ICU (n = 53); G4, other medical inpatients (n = 38). Blood was collected into citrated and CTAD tubes. Chromogenic anti-Xa assays were centrally performed, using seven reagent/analyzer combinations including two without DS. The association between anti-Xa levels and covariates was tested using a linear mixed-effects model. RESULTS: We analyzed 4,546 anti-Xa values from 165 patients. Median anti-Xa levels were systematically higher with reagents containing DS, whatever the patient group, with the greatest effect observed in G1 (0.32 vs. 0.05 IU/mL). Anti-Xa levels were slightly higher in CTAD than in citrate samples, irrespective of the assay. The model showed: (1) a significant dextran-patient group interaction (p < 0.0001), the effect of DS on anti-Xa levels varying from 30.9% in G4 to 296% in G1, and (2) a significant effect of CTAD, varying between patient groups (p = 0.0302). CONCLUSION: The variability of anti-Xa levels with a great overestimation of the values, using a reagent containing DS, can lead to different treatment decisions, especially after heparin neutralization by protamine. Clinical consequences of these differences remain to be demonstrated.

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

BACKGROUND: 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.

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

Extracorporeal 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.