The role of inflammation and antithrombin supplementation on thromboelastographic parameters during veno-venous ECMO for respiratory failure.

INTRODUCTION: Extracorporeal membrane oxygenation (ECMO) may act as a driver or propagator of systemic inflammation. In turn, cytokine release can modify thromboelastographic (TEG) tests which are commonly used for anticoagulation monitoring. In this context, antithrombin (AT) supplementation might further modify TEG. METHODS: This is a pre-specified sub-study of the "Randomized Controlled Trial of Antithrombin Supplementation During Extracorporeal Membrane Oxygenation" study (investigator-initiated, randomized, single-blind, two-arm trial) conducted in two Italian ECMO referral ICUs. Adult patients requiring vv-ECMO for respiratory failure and undergoing unfractioned heparin (UFH) administration were enrolled and randomized whether to receive AT supplementation. Plasma samples for cytokine assay (IL-8, IL-10, IL-6, IL-1ß, TNF-α and Pro-ADM) and heparinase TEG were collected from every patient before ECMO start, 24 h and 72 h after ECMO start, before ECMO removal, and 7 days after ECMO removal or upon ICU discharge whichever happened first. AT concentration, coagulation and clinical data were collected before ECMO start and at pre-fixed time points. RESULTS: Thirty-nine patients were enrolled (21 treatments, 18 controls). TEG-R had a weak-to-moderate positive correlation with IL-8, IL-6, IL-10 and TNF-α and a moderate positive correlation with Pro-ADM. TEG-ANG showed a weak negative correlation with IL-8, IL-6 and TNF-α, while TEG-MA negatively correlated with IL-8, TNF-α and Pro-ADM. AT supplementation seemed to modify the association between TEG-MA and IL-8, IL-10 and Pro-ADM; conversely, AT did not affect the relationship among TEG-R or TEG-ANG and the studied cytokines. CONCLUSIONS: High concentrations of systemic cytokines correlated with longer reaction times and decreased angle and amplitude at TEG, suggesting that an increase in inflammation is related with hypocoagulability as revealed by thromboelastography.

Effect of emicizumab-neutralizing antibodies on activated partial thromboplastin time-based clotting time test results in patients treated with emicizumab.

Background: Emicizumab is a bispecific humanized monoclonal antibody that shortens the activated partial thromboplastin time (aPTT), making aPTT-based tests unreliable. Objectives: To evaluate the efficacy of a mixture of 2 anti-idiotype monoclonal antibodies (anti-emi) in neutralizing emicizumab in samples from persons with hemophilia A treated with emicizumab. Methods: Fifty samples from persons with hemophilia A treated with emicizumab were analyzed for aPTT and factor VIII procoagulant activity; FVIII inhibitor titer was measured using Nijmegen-Bethesda assay in 50 plasma samples of additional patients (positive for FVIII inhibitor) treated with emicizumab. FVIII procoagulant activity and inhibitor titer were measured using 1-stage (Actin FS, Siemens) and chromogenic assays with bovine regents (Factor VIII Chromogenic Assay, Siemens). Emicizumab concentration was measured by modified a 1-stage assay calibrated with a drug-specific calibrator (r2 Diagnostics Inc). All the tests were performed on Sysmex CS-2400 (Sysmex) before and after the addition of anti-emi (Chugai Pharmaceutical). Results: Emicizumab concentrations measured after neutralization were <1.6 µg/mL in all samples. FVIII levels were >480 IU/dL with an aPTT of <30.8 seconds in all samples before neutralization and were <1 IU/dL with an aPTT of >70 seconds after adding anti-emi. FVIII inhibitor resulted in a false negative result in 44 of 50 samples measured with the 1-stage assay before neutralization. A good correlation (r = 0.98) was found between inhibitor titer measured using the chromogenic (insensitive to emicizumab) and 1-stage assays after neutralization. Conclusion: The anti-emi antibodies were shown to completely neutralize emicizumab, making samples pretreated with anti-emi analyzable with the 1-stage assay.

The relationship between antithrombin administration and inflammation during veno-venous ECMO.

Veno-venous Extracorporeal Membrane Oxygenation (ECMO) is used in the most severe cases of respiratory failure and further exacerbates the patients' inflammatory status. Antithrombin is supplemented during ECMO for its anticoagulant effects, but it also deploys anti-inflammatory properties. In this pre-specified ancillary study of the GATRA trial [NCT03208270] we aimed to evaluate the relationship between antithrombin and inflammation during ECMO. Forty-six patients were included in the study, 23 were randomized to receive antithrombin to maintain a level of 80-120% (study group) and 23 were randomized not to be supplemented (control group). Anticoagulation was provided in both groups with heparin infusion. Six cytokines were measured at 5 timepoints from prior to ECMO start to 7 days after ECMO removal. Cytokines decreased during the study but overall were not very different in the two groups. Testing the interaction between the study group and timepoints suggests that the administration of antithrombin led to a more rapid decrease over time of IL-6, IL-1ß, TNF-⍺ and Pro-ADM. Plasma levels of antithrombin (either endogenous or exogenous) were negatively associated with all cytokines. Inflammation decreases during ECMO but a causal effect of antithrombin administration on the reduction of inflammation (and its clinical relevance) must be confirmed by appropriately powered studies.

Analytical Performance of Different Laboratory Methods for Measuring Susoctocog-Alfa.

Recombinant porcine factor VIII (rpFVIII) is indicated for treating bleeding episodes in acquired haemophilia A, but there are few data regarding laboratory methods to adequately monitor treatment. This study involving three Italian laboratories aimed to evaluate the analytical performance of different assays for measuring rpFVIII. Five spiked rpFVIII samples (0.5-1.5 IU/mL) were analysed on three days, in triplicate, with eleven combinations of reagents (Werfen, Boston, MA, USA: SynthasIL and SynthaFax for one-stage assay, Chromogenix Coamatic FVIII for chromogenic assay), FVIII depleted plasmas (with or without von Willebrand factor-VWF) and calibrators (HemosIL human calibrator plasma, porcine calibrator diluted in FVIII deficient plasma with or without VWF). The assays were performed on ACL TOP analysers (Werfen, Boston, MA, USA). Intra- and inter-assay and inter-laboratory Coefficient of Variation (CV%) were calculated together with percentage of recovery (% recovery) on the expected value. The results showed that the reagent combinations reaching satisfactory analytical performance are: SynthasIL/human calibrator/deficient plasma+VWF (total recovery 99.4%, inter-laboratory CV 4.04%), SynthasIL/porcine calibrator/deficient plasma+VWF (total recovery 111%, inter-laboratory CV 2.75%) and Chromogenic/ porcine calibrator/deficient plasma+VWF (total recovery 96.6%, inter-laboratory CV 8.32%). This study highlights that the use of porcine standard (when available) and FVIII deficient plasma with VWF should be recommended.

Impact of a commercially available DOAC absorbent on two integrated procedures for lupus anticoagulant detection.

BACKGROUND: Lupus anticoagulant (LA)-detection in anticoagulated patients is an unmet need, which becomes even more cogent with the introduction of direct oral anticoagulants (DOAC) that may lead to false-positive results. AIMS: We aimed to investigate the effect of a commercially available DOAC absorbent on residual drug concentrations, and on integrated procedures for LA-detection. METHODS: Blood from patients treated for atrial fibrillation with either dabigatran (n = 39), rivaroxaban (n = 55), apixaban (n = 47) or edoxaban (n = 47) were collected at peak and trough, and centralized for testing with two LA integrated procedures [i.e., the silica clotting time (SCT) and dilute Russel viper venom (dRVVT)] before and after DOAC absorbent exposure. RESULTS: The commercially available DOAC absorbent investigated in this study proved effective in reducing the concentrations of all the investigated DOAC, although small residual drug was detected after absorption, especially in patients on edoxaban. Results mimicking LA were observed in patients on DOAC before absorbent exposure, especially for rivaroxaban when testing was performed with dRVVT (88% rate at peak and 20% at trough) and much less with SCT (12% at peak and 8% at trough). The correspondent rate of results mimicking LA in patients on rivaroxaban after exposure was reduced [dRVVT (peak 8%, trough 4%); SCT (peak and trough 8%)], but not abolished. CONCLUSIONS: Overall, in vitro DOAC absorbance by active charcoal compounds is a useful laboratory tool for LA-detection in patients on DOAC. Caution should however be exerted when used in daily practice.

The ADAMTS13-von Willebrand factor axis in COVID-19 patients.

BACKGROUND: Severe coronavirus disease 2019 (COVID-19) is characterized by an increased risk of thromboembolic events, with evidence of microthrombosis in the lungs of deceased patients. OBJECTIVES: To investigate the mechanism of microthrombosis in COVID-19 progression. PATIENTS/METHODS: We assessed von Willebrand factor (VWF) antigen (VWF:Ag), VWF ristocetin-cofactor (VWF:RCo), VWF multimers, VWF propeptide (VWFpp), and ADAMTS13 activity in a cross-sectional study of 50 patients stratified according to their admission to three different intensity of care units: low (requiring high-flow nasal cannula oxygenation, n = 14), intermediate (requiring continuous positive airway pressure devices, n = 17), and high (requiring mechanical ventilation, n = 19). RESULTS: Median VWF:Ag, VWF:RCo, and VWFpp levels were markedly elevated in COVID-19 patients and increased with intensity of care, with VWF:Ag being 268, 386, and 476 IU/dL; VWF:RCo 216, 334, and 388 IU/dL; and VWFpp 156, 172, and 192 IU/dL in patients at low, intermediate, and high intensity of care, respectively. Conversely, the high-to-low molecular-weight VWF multimers ratios progressively decreased with increasing intensity of care, as well as median ADAMTS13 activity levels, which ranged from 82 IU/dL for patients at low intensity of care to 62 and 55 IU/dL for those at intermediate and high intensity of care. CONCLUSIONS: We found a significant alteration of the VWF-ADAMTS13 axis in COVID-19 patients, with an elevated VWF:Ag to ADAMTS13 activity ratio that was strongly associated with disease severity. Such an imbalance enhances the hypercoagulable state of COVID-19 patients and their risk of microthrombosis.

Emicizumab, the factor VIII mimetic bi-specific monoclonal antibody and its measurement in plasma.

Objectives: Emicizumab, a monoclonal antibody mimicking the function of factor (F) VIII in the activation of FX by FIXa, is widely used for prophylaxis in hemophilia patients with or without inhibitors to FVIII. Although it is administered at fixed dose, its measurement could be occasionally required. In principle, the emicizumab procoagulant effect could be assessed by the one-stage assay (OSA) currently used to measure FVIII. However, the OSA for FVIII presents with limitations. Furthermore, owing to its potent FVIII-like activity, emicizumab interferes with the measurement of the inhibitor to FVIII, which is often needed in patients on emicizumab. Methods: We prepared test samples by spiking a FVIII-deficient plasma with graded amounts of emicizumab. We modified the OSA for FVIII and tested plasma samples for emicizumab concentrations. Furthermore the chromogenic assay (CA) for FVIII with bovine reagents was used to assess for the FVIII inhibitor in patients on emicizumab. Results: Slight modification of the OSA for FVIII (i.e., higher test plasma dilution and longer coagulometer acquisition time) made the regular OSA as a reliable laboratory tool to measure emicizumab concentration as shown by the identity of the regression (observed vs. expected) lines. Furthermore, the inhibitors to FVIII in patients on emicizumab, which were negative when measured by the regular Bethesda assay, were reliably measured by the CA assay employing bovine reagents. Conclusions: The methods currently used to measure FVIII can be easily modified to make the general clinical laboratory able to assist clinicians when dealing with patients on emicizumab.

Evaluation of an automated chromogenic assay for Factor VIII clotting activity measurement in patients affected by haemophilia A.

INTRODUCTION: The original one-stage clotting assay is still the most widely used method to measure Factor VIII clotting activity (FVIII:C) in patients with haemophilia A (HA), although the use of chromogenic assays is increasing significantly. AIM: Evaluation of the analytical performance and diagnostic accuracy of BIOPHEN™ FVIII:C (HYPHEN BioMed, Neuville-sur-Oise, France) assay on Sysmex CS-2400 (Sysmex, Kobe, Japan) analyser. METHODS: Sixty patients with haemophilia A (HA; any severity) and 120 healthy Italian subjects were included. All the assays were performed on citrate platelet-poor plasmas stored at -80°C. Chromogenic BIOPHEN™ FVIII:C was compared with the one-stage assay using Actin FS and Factor VIII deficient plasma (Siemens Healthcare Diagnostics, Marburg, Germany) on Sysmex CS-2400 and with another chromogenic automated assay (COAMATIC™ Factor VIII, CHROMOGENIX on ACL TOP analyzer; Instrumentation Laboratory, Milan, Italy). RESULTS: Intra-assay and inter-assay coefficient of variation were <6%. Linearity was good up to 1/128 dilution (r = 0.99); mean recovery was 91.7% and limit of detection was 0.2%. BIOPHEN™ FVIII:C assay showed a good correlation and diagnostic agreement with the chromogenic COAMATIC™ assay: the Spearmen's Rank correlation coefficient was 0.98 and the inter-rate agreement K Cohen coefficient was 0.61. The K coefficient was 0.91 when BIOPHEN™ FVIII:C was compared with the historical classification of the patients, demonstrating an optimal diagnostic accuracy in HA. CONCLUSIONS: BIOPHEN™ FVIII:C showed good analytical performance and diagnostic accuracy and could be considered suitable for the introduction in routine analytical panel of coagulation for the diagnosis of HA patients.

Procoagulatory state in inflammatory bowel diseases is promoted by impaired intestinal barrier function.

Inflammatory and immune mediated disorders are risk factors for arterial and venous thromboembolism. Inflammatory bowel diseases (IBD) confer an even greater risk of thromboembolic events than other inflammatory conditions. It has been shown that IBD patients display defective intestinal barrier functions. Thus, pathogen-associated molecular patterns (PAMPs) coming from the intestinal bacterial burden might reach systemic circulation and activate innate immunity receptors on endothelial cells and platelets, promoting a procoagulative state. Aim of the study was to test this hypothesis, correlating the presence of circulating PAMPs with the activation of innate immune system and the activation of the coagulatory cascade in IBD patients. Specifically, we studied lipopolysaccharide (LPS), Toll-like receptor (TLR) 2, TLR4, and markers of activated coagulation (i.e., D-Dimer and prothrombin fragment F1+2) in the serum and plasma of IBD patients. We found that LPS levels are increased in IBD and correlate with TLR4 concentrations; although a mild correlation between LPS and CRP levels was detected, clinical disease activity does not appear to influence circulating LPS. Instead, serum LPS correlates with both D-Dimer and F1+2 measurements. Taken together, our data support the role of an impairment of intestinal barrier in triggering the activation of the coagulatory cascade in IBD.

Inflammatory and prothrombotic parameters in normotensive non-diabetic obese women: effect of weight loss obtained by gastric banding.

Hypertension and diabetes are known risk factors for obesity-related thrombosis, but several studies have shown that obesity is characterised by a potentially prothrombotic inflammatory state because of activated coagulation and impaired fibrinolysis. In order to verify if obese patients-unaffected by hypertension, diabetes, dyslipidemia, cigarette smoking or inflammatory diseases-show increased prothrombotic markers and whether the weight loss induced by gastric banding normalises such parameters. Plasma levels of C reactive protein (CRP), fibrinogen, plasminogen activator inhibitor-1 (PAI-1), von Willebrand factor (VWF) and factor VII (FVII) were measured in 25 women with isolated obesity prior to, as well as 3, 6 and 12 months subsequent to gastric banding. Twenty-five healthy women served as a baseline control group. The obese women had higher CRP (p = 0.0001), fibrinogen (p = 0.014), PAI-1 (p = 0.003), VWF (p = 0.004) and FVII levels (p = 0.0001) than the normal controls, and their body mass index (BMI) positively correlated with CRP (r = 0.462, p = 0.02), fibrinogen (r = 0.426, p = 0.04) and PAI-1 (r = 0.468, p = 0.02). Twelve months after gastric banding, the median BMI had decreased from 40.0 to 34.9 (p = 0.0001); CRP from 4.18 to 1.69 µg/ml (p = 0.01); fibrinogen from 389 to 318 mg/dl (p = 0.0001); PAI-1 from 32.1 to 12.0 UI/ml (p = 0.003); VWF from 144 to 120% (p = 0.0001); and FVII from 134 to 112% (p = 0.002). Even in the absence of major cardiovascular risk factors, obese patients are characterised by a prothrombotic state. The weight loss induced by gastric banding decreases the parameters of inflammation, coagulation and impaired fibrinolysis, thus potentially reducing the thrombotic risk.