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.

[Interference of oral anti-Xa anticoagulants during monitoring of unfractionated heparin treatments: practical and future attitudes]. / Interférence des anticoagulants oraux directs anti-Xa lors de la surveillance des traitements par héparine non-fractionnée : attitudes pratiques et à venir.

Contrary to direct oral anticoagulants (DOAC), unfractionated heparin (UFH) requires daily monitoring when administered at therapeutic dose. At present, UFH monitoring is preferably carried out by measuring plasma anti-Xa activity, however, in patients previously treated with an anti-Xa DOAC and switched to UFH, there is a high risk of DOAC interfering with the measurement of UFH anti-Xa activity. Residual anti-Xa DOAC in the sample can lead to an overestimation of the anticoagulant activity attributed to heparin and thus to incorrect anticoagulation. This risk of interference should not be overlooked because interference may occur even at concentration of DOAC below the hemostatic safety threshold and can last several days. To overcome this issue, several alternatives are being studied. This note provides an update on anti-Xa DOAC interference and different strategies available in current practice. It also underlines the importance of communication between biologists and clinicians on anticoagulant treatments received by patients.

Tinzaparin, an alternative to subcutaneous unfractionated heparin, in patients with severe and end-stage renal impairment: a retrospective observational single-center study.

BACKGROUND: Tinzaparin could be easier to manage than unfractionated heparin, in patients with severe renal impairment. However, clinical and pharmacological data regarding its use in such patients are lacking. The aims of this study were to determine, in patients with eGFR<30 mL.min-1: tinzaparin pharmacokinetics (PK) parameters, using a population PK approach and bleeding and thrombotic complications. METHODS: We performed a retrospective observational single-center study, including in-patients with eGFR< 30 mL.min-1, receiving prophylactic (4500 IU.day-1) or therapeutic (175 IU.kg-1.day-1) tinzaparin. Measured anti-Xa levels were analyzed using a non-linear mixed effects modelling approach. Individual predicted tinzaparin exposure markers at steady state were calculated for each patient and dosing regimen. The PK was also evaluated through Monte-Carlo simulations, based on the final covariate model parameter estimates. RESULTS: Over a 22-month period, 802 tinzaparin treatment periods in 623 patients were analysed: two-thirds received a prophylactic dose, 66% had an eGFR<20 mL.min-1, and 25% were on renal replacement therapy. In patients for whom anti-Xa measurements were performed (n=199, 746 values), PK parameters, profiles and Cmax were comparable to those in patients without renal impairment or in healthy volunteers. In the whole population, major bleeding occurred in 2.4% and 3.5% of patients receiving prophylactic and therapeutic doses, over a median 9- and 7-days treatment period, respectively. No patients had thrombotic complication. CONCLUSION: Tinzaparin PK parameters and profiles were not affected by renal impairment. This suggests that tinzaparin, at therapeutic or prophylactic dose, could be an alternative to unfractionated heparin in hospitalized patients with severe renal impairment.

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.

Comparative Analysis of a French Prospective Series of 144 Patients with Heparin-Induced Thrombocytopenia (FRIGTIH) and the Literature.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a rare complication of heparin treatments, and only a few large patient cohorts have been reported. In this study, biological and clinical data from 144 French patients with HIT were analyzed in comparison with the literature. METHODS: The diagnosis of HIT was confirmed in all patients by an immunoassay combined with serotonin release assay. In the literature, only cohorts of at least 20 HIT patients published from 1992 were selected for a comparative analysis. RESULTS: Two-thirds of patients were hospitalized in surgery and most were treated with unfractionated heparin (83.2% vs. 16.8% with low molecular weight heparin only). Thrombotic events in 54 patients (39.7%) were mainly venous (41/54). However, arterial thrombosis was more frequent after cardiac surgery (13.2% vs. 2.4% in other surgeries, p = 0.042) with a shorter recovery time (median = 3 vs. 5 days, p < 0.001). The mortality rate was lower in our series than in the 22 selected published studies (median = 6.3% vs. 15.9%). Three genetic polymorphisms were also studied and homozygous subjects FcγRIIA RR were more frequent in patients with thrombosis (37.8 vs. 18.2% in those without thrombosis, p = 0.03). CONCLUSION: This study shows that the mortality rate due to HIT has recently decreased in France, possibly due to earlier diagnosis and improved medical care. It also confirms the strong association between polymorphism FcγRIIA H131R and thrombosis in HIT.

Potential usefulness of activated charcoal (DOAC remove®) for dRVVT testing in patients receiving Direct Oral AntiCoagulants.

INTRODUCTION: Lupus Anticoagulant testing using dilute Russell Viper Venom Time (dRVVT) is challenging in patients receiving Direct Oral AntiCoagulants (DOAC) due to potential false positive results. In a multicenter study, we evaluated the in vitro removal of DOAC by activated charcoal (DOAC remove®), allowing reliable dRVVT testing. MATERIALS AND METHODS: Patient samples were analyzed before and after treatment with DOAC remove®: 49 apixaban, 48 rivaroxaban, 24 dabigatran and 30 none. DOAC plasma concentrations were measured using anti-Xa or diluted thrombin time assays. In a subset of 28 samples, DOAC concentrations were also measured using HPLC-MS/MS following treatment with DOAC remove®. DRVVT was performed using STA-Staclot dRVVT Screen®/Confirm® (Stago) or LAC-Screening®/Confirmation® (Siemens). RESULTS: Baseline median [min-max] concentrations were 94 [<20-479] for apixaban, 107 [<20-501] for rivaroxaban and 135 ng/mL [<20-792] for dabigatran; dRVVT screen ratio/confirm ratio was positive in 47, 90 and 42% of apixaban, rivaroxaban and dabigatran samples. Treatment with DOAC remove® did not affect dRVVT results in non-DOAC patients while it resulted in DOAC concentrations <20 ng/mL in 82, 98 and 100% of samples, respectively. Concentrations were <5 ng/mL with HPLC-MS/MS in 5 out of 10, 8 out of 10 and 7 out of 8 samples, respectively. DOAC remove® corrected DOAC interference with dRVVT assays in 76, 85 and 95% of the patients, respectively. CONCLUSION: For dRVVT testing in DOAC patients, we suggest the use of DOAC remove® for every rivaroxaban sample, whereas it might only be used in positive apixaban and dabigatran samples. A residual DOAC interference cannot be ruled out in case of persisting dRVVT positive results after treatment with DOAC remove®.