How the Hemostasis Laboratory Can Help Clinicians to Manage Patients on Oral Anticoagulants.

Oral anticoagulants are widely used to treat or prevent cardiovascular diseases in millions of patients worldwide. They are the drugs of choice for stroke prevention and systemic embolism in patients with non-valvular atrial fibrillation and prosthetic heart valves, as well as for treatment/prevention of venous thromboembolism. Oral anticoagulants include vitamin K antagonists (VKAs) and direct oral anticoagulants (DOACs). The hemostasis laboratory plays a crucial role in the management of treated patients, spanning from dose adjustment based on laboratory testing that applies to VKAs to the measurement of drug concentrations in special situations that apply to DOACs. This article aims to overview how the hemostasis laboratory can help clinicians manage patients on oral anticoagulants. Special interest is devoted to the international normalized ratio, used to manage patients on VKAs and to the measurement of DOAC concentrations, for which the role of the laboratory is still not very well defined, and most interferences of DOACs with some of the most common hemostatic parameters are not widely appreciated.

Predictive parameters for spontaneous joint bleeding during emicizumab prophylaxis.

Emicizumab is approved for prophylaxis of patients with hemophilia A (HA). Despite its efficacy in reducing bleeding, a few patients on emicizumab still experience hemarthrosis, but no tool is yet available to identify those at higher risk of spontaneous joint bleeding. To evaluate whether laboratory measurements (global coagulation assays and emicizumab concentration) and/or arthropathy scores can distinguish patients at higher risk of spontaneous joint bleeding while on emicizumab prophylaxis. Thrombin generation assay (TGA) was assessed upon the addition of tissue factor and synthetic phospholipids. Non-activated thromboelastography (NATEM) was performed in citrated whole blood. Emicizumab concentrations were measured with a modified one-stage FVIII assay. The degree of hemophilic arthropathy was assessed with the Haemophilia Joint Health Score (HJHS) and Hemophilia Early Arthropathy Detection with Ultrasound score (HEAD-US). A Cox proportional hazards model was used to evaluate the association between variables and bleeding. The predictive power of these variables was investigated by ROC analysis. Forty HA patients with and without inhibitors on emicizumab prophylaxis were enrolled in an observational cohort study. Ten of 40 developed spontaneous joint bleeding. None of the lab parameters were able to distinguish patients at higher risk of spontaneous joint bleeding. ROC analysis showed that during emicizumab prophylaxis only the presence of synovitis and a higher HEAD-US score were associated with spontaneous joint bleeding (AUC 0.84). A greater degree of arthropathy and the presence of synovitis could help to predict the risk of spontaneous joint bleeding in HA patients on emicizumab prophylaxis.

Defining a metrologically traceable and sustainable calibration hierarchy of international normalized ratio for monitoring of vitamin K antagonist treatment in accordance with International Organization for Standardization (ISO) 17511:2020 standard: communication from the International Federation of Clinical Chemistry and Laboratory Medicine-SSC/ISTH working group on prothrombin time/international normalized ratio standardization.

Calibration of prothrombin time (PT) in terms of international normalized ratio (INR) has been outlined in "Guidelines for thromboplastins and plasmas used to control oral anticoagulant therapy" (World Health Organization, 2013). The international standard ISO 17511:2020 presents requirements for manufacturers of in vitro diagnostic (IVD) medical devices (MDs) for documenting the calibration hierarchy for a measured quantity in human samples using a specified IVD MD. The objective of this article is to define an unequivocal, metrologically traceable calibration hierarchy for the INR measured in plasma as well as in whole blood samples. Calibration of PT and INR for IVD MDs according to World Health Organization guidelines is similar to that in cases where there is a reference measurement procedure that defines the measurand for value assignment as described in ISO 17511:2020. We conclude that, for PT/INR standardization, the optimal calibration hierarchy includes a primary process to prepare an international reference reagent and measurement procedure that defines the measurand by a value assignment protocol conforming to clause 5.3 of ISO 17511:2020. A panel of freshly prepared human plasma samples from healthy adult individuals and patients on vitamin K antagonists is used as a commutable secondary calibrator as described in ISO 17511:2020. A sustainable metrologically traceable calibration hierarchy for INR should be based on an international protocol for value assignment with a single primary reference thromboplastin and the harmonized manual tilt tube technique for clotting time determination. The primary international reference thromboplastin reagent should be used only for calibration of successive batches of the secondary reference thromboplastin reagent.

Characterization of Immunogenicity and Safety of COVID-19 mRNA-1273 in HIV-Positive Italian Patients with Hemophilia: A Prospective Single-Center Cohort Study.

To characterize the immunogenicity of mRNA-1273 (Moderna, Cambridge, MA, USA) vaccine in HIV-positive hemophilic patients during the third COVID-19 wave in Italy and to investigate biomarkers of coagulation and endothelial perturbation before and after complete vaccination schedule, twenty-three consecutive adult HIV-positive patients with hemophilia were included. Blood was collected before and two weeks after vaccination. We measured anti-SARS-CoV-2 spike protein antibodies to assess immunogenicity; circulating biomarkers of coagulation (protein C and D-dimer), endothelial perturbation (von Willebrand factor (VWF)) and anti-Platelet Factor 4 (PF4) antibodies were analyzed. Flow-based analysis of thrombus formation was performed in nine patients using a flow-chamber device. Two weeks after completing the vaccination schedule, all patients had anti-spike antibodies values consistent with an effective immunization. Mean (±standard deviation) basal values of protein C and VWF (106 ± 21% and 171 ± 45%, respectively) were not significantly different from data obtained two weeks after the second dose (103 ± 20%, 162 ± 43%, respectively). D-dimer median values (interquartile range) were not significantly different at baseline (442 (603-142) ng/mL) and after the second dose (477 (654-262) ng/mL). Anti-PF4 antibodies were detected in three patients with no associated clinical manifestations. No significant differences were found in flow-based analysis of thrombus formation. Our data demonstrate that in HIV-positive patients with hemophilia, SARS-CoV-2 vaccination is effective and safe, with no effects on coagulation and endothelial perturbation.

Laboratory Diagnosis of Antiphospholipid Syndrome in Anticoagulated Patients.

The laboratory diagnosis of antiphospholipid syndrome (APS) requires the measurement of solid-phase antibodies to cardiolipin or ß2-Glycoprotein-I and the search for lupus anticoagulant (LA). The diagnosis of patients whilst on anticoagulation is impaired by the difficult interpretation of results, at least for LA, owing to the fact that prolongations of clotting times induced by LA superimpose those induced by anticoagulants. This is a matter of concern as treating physicians very often need to know the APS status of their patients to make a decision on secondary antithrombotic prophylaxis. This article aims to review the effect brought about by anticoagulants on APS diagnosis and discuss the options that can be used to overcome such an effect.

Thromboplastin calibration revisited to look for possible revision of the World Health Organization recommendations.

BACKGROUND: Thromboplastin calibration is essential to determine the international sensitivity index required to calculate the international normalized ratio (INR). The procedure for calibration recommended by the World Health Organization (WHO) calls for the selection of patients on stable anticoagulation in the range of 1.5 to 4.5 INR. These patients are difficult to be recruited as the conventional therapeutic interval for warfarin is 2.0 to 3.0. A possible solution could be including patients with less intense anticoagulation in the calibration. OBJECTIVES: We sought to investigate the impact of this amended procedure on the parameters of calibration. METHODS: Eight data sets from previous calibrations of a rabbit thromboplastin that included patients on anticoagulation as required by WHO were used for this pilot study. Parameters of calibration as determined by the full data sets are identified as "full calibrations" and are considered reference. Each of the data sets were used to recalculate the calibration parameters after including patients with INRs of <4.0, <3.5, or <3.0, which were identified as "trimmed calibrations" and compared with those from the full calibrations. RESULTS: There was marginal variation of the international sensitivity index, CV, and INR that can be hardly of practical significance. CV was the most affected parameter, which increased from the full to the trimmed <3.0 calibration, but never exceeded the 3% cutoff value recommended by WHO. CONCLUSIONS: Should the results of this pilot study be confirmed for the calibration of other thromboplastins, revision of the WHO recommendations to include patients with INR from 1.5 to 4.0 is warranted.

Clinical and genetic determinants of the fatty liver-coagulation balance interplay in individuals with metabolic dysfunction.

Background & Aims: The aim of this study was to examine the determinants of the interplay between liver damage and the coagulation balance in individuals at risk of non-alcoholic fatty liver disease (NAFLD). Methods: We considered 581 healthy participants with ≥3 metabolic alterations undergoing clinical and genomic evaluation, measurement of liver stiffness (LSM) and controlled attenuation parameter (CAP) by Fibroscan, Pro-C3, coagulation balance (von Willebrand factor [vWF], factor VIII/protein C ratio [F8/PC] as the main outcome, D-dimer as marker of coagulation/fibrinolysis activation). Results: Liver fibrosis indices (both Fibrosis-4 [FIB-4] and liver stiffness measurement [LSM]), but not liver fat (CAP), were independently associated with higher F8/PC ratio (p <0.01), triggering D-dimer formation (p = 2E-21). In keeping with a causal role of liver damage in determining a procoagulant status, the main fatty liver inherited risk variant PNPLA3 p.I148M was independently associated with the F8/PC ratio (p = 0.048). Vice versa, the main determinant of the coagulation balance was ABO locus variation (p = 1E-16), through the impact on vWF (p = 8E-26). Both rs687289 ABO and factor V Leiden were independently associated with higher Pro-C3 (p <0.025), with the effect of ABO being mediated by the impact on vWF (p = 5E-10 for association with Pro-C3). Mendelian randomisation analysis was consistent with a causal association of procoagulant imbalance with heightened fibrogenesis (p = 0.001 at robust MR-Egger for Pro-C3), but not with fibrosis (for LSM; p = not significant). Conclusions: In individuals with metabolic dysfunction, liver damage severity and possibly the PNPLA3 p.I148M variant were associated with procoagulant status. Vice versa, evaluation of inherited variants in ABO and other genes influencing coagulation was consistent with a causal role of procoagulant imbalance in activation of early stages of fibrogenesis. Lay summary: In individuals with metabolic alterations at risk of metabolic fatty liver disease, there is a tendency toward heightened blood coagulation (clotting), but the cause and the impact on the progression of liver disease remain unclear. Here we show that liver damage severity and metabolic alterations, but not hepatic fat, are mainly responsible for heightened coagulation in patients with metabolic fatty liver disease. By using genetic approaches, we showed that hepatic inflammation due to lipotoxicity may favour heightened coagulation, which in turn can trigger liver fibrosis, igniting a vicious cycle that leads to progressive liver disease.

Pro-coagulant imbalance in patients with community acquired pneumonia assessed on admission and one month after hospital discharge.

OBJECTIVES: Patients hospitalized because of community-acquired-pneumonia (CAP) are at risk of cardiovascular diseases. Although plasma procoagulant imbalance play a role, mechanisms are not completely understood. We aimed to investigate whether there is a measurable state of procoagulant imbalance following inflammation determined by CAP. METHODS: We analyzed blood from 51 CAP patients at admission and 51 healthy subjects (HS) for (i) pro and anticoagulants, (ii) thrombin generation (TG) with or without thrombomodulin (TM), which is the physiologic activator of the protein C anticoagulant pathway and(iii) by assessing the ratio between von Willebrand-factor (VWF) and its protease ADAMTS13. Thirty patients were re-analyzed one month after discharge when CAP was resolved. RESULTS: Median levels of TG parameters, including the endogenous thrombin potential (ETP), the ETP-TM-ratio (with/without TM), peak-thrombin and velocity index were higher in patients at baseline than HS. In particular, the median (IQR) ETP-TM-ratio in patients vs. HS was 0.88 (0.83-0.91) vs. 0.63 (0.48-0.71), p<0.001. Factor (F)VIII, a potent procoagulant involved in TG was higher in patients at baseline than HS [195 U/dL (100-388) vs. 127(108-145)], p<0.001]. The ratio of VWF/ADAMTS13 was higher at baseline than HS. Cumulatively, the findings indicate a state of pro-coagulant imbalance, which (although reduced), remained high [i.e., ETP-TM-ratio, 0.80 (0.74-0.84); FVIII, 152 U/dL (122-190)] one month after discharge when the infection was resolved. CONCLUSIONS: Patients with CAP possess a state of pro-coagulant imbalance, which remains substantially high, even when the infection is resolved. The findings suggest CAP patients as candidates for antithrombotic prophylaxis even after the resolution of infection. Clinical trials are warranted to assess the benefit/risk ratio of prophylaxis extension.

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.

Plasma levels of extracellular vesicles and the risk of post-operative pulmonary embolism in patients with primary brain tumors: a prospective study.

Primary brain tumors are associated with an increased risk of pulmonary embolism (PE), particularly in the early post-operative period. The pathophysiological mechanisms of PE are poorly understood. This study aims to describe prospectively extracellular vesicles (EVs) levels and investigate whether or not their variations allow to identify patients at increased risk of post-operative PE. Consecutive meningioma or glioma patients candidate to tumor resection were included in the study if a pulmonary perfusion scan (Q-scan) performed before surgery ruled out PE. EVs derived from platelets (CD41+) or endothelial cells (CD144+), tissue factor-bearing EVs (CD142+) and their procoagulant subtype (annexin V+) were analyzed by flow cytometry before surgery (T0), within 24 h (T1), two (T2) and seven days (T7) after surgery. Q-scan was repeated at T2. Ninety-three patients with meningioma, 59 with glioma and 76 healthy controls were included in the study. CD142+ and annexin V+/CD142+ EVs were increased at T0 in meningioma and glioma patients compared to healthy controls. Twenty-nine meningioma (32%) and 16 glioma patients (27%) developed PE at T2. EVs levels were similar in meningioma patients with or without PE, whereas annexin V+ and annexin V+/CD142+ EVs were significantly higher at T1 and T2 in glioma patients with PE than in those without. Procoagulant EVs, particularly annexin V+/CD142+, increase after surgery and are more prevalent in glioma patients who developed PE after surgery than in those who did not.

Assessment of Platelet Thrombus Formation under Flow Conditions in Adult Patients with COVID-19: An Observational Study.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is associated with systemic inflammation, which may dysregulate platelet function. Total Thrombus-Formation Analysis System (T-TAS) is a flow-chamber device that analyses platelet-mediated thrombus formation in capillary channels through the following parameters: (1) the area under the flow-pressure curve (AUC), (2) occlusion start time (OST), time needed to reach OST, and (3) occlusion time (OT), time needed to reach the occlusion pressure. METHODS AND FINDINGS: Sixty-one COVID-19 patients admitted to intensive, subintensive, and low intensive care were prospectively enrolled according to the time of admission: group A (up to 8 days) (n = 18); group B (from 9 to 21 days) (n = 19), and group C ( > 21 days) (n = 24). T-TAS measurements were performed at enrolment and after 7 days. Median OST was similar among groups. AUC was lower in group A compared to B (p = 0.001) and C (p = 0.033). OT was longer in group A compared to B (p = 0.001) and C (p = 0.028). Platelet count (PC) was higher in group B compared to A (p = 0.024). The linear regression showed that OT and AUC were independent from PC in group A (OT: 0.149 [95% confidence interval [CI]: -0.326 to 0.624], p = 0.513 and AUC: 0.005 [95% CI: -0.008 to 0.017], p = 0,447). In contrast, in group B, PC was associated with OT (-0.019 [-0.028 to 0.008], p = 0.023) and AUC (0.749 [0.358-1.139], p = 0,015), similarly to group C. Conversely, patients with different illness severity had similar T-TAS parameters. CONCLUSION: COVID-19 patients display an impaired platelet thrombus formation in the early phase of the disease compared to later stages and controls, independently from illness severity.

Procoagulant Imbalance in Klinefelter Syndrome Assessed by Thrombin Generation Assay and Whole-Blood Thromboelastometry.

CONTEXT: Klinefelter syndrome (KS) is a condition at increased risk of thrombosis compared to 46,XY men. OBJECTIVE: This work aimed to investigate the coagulation balance of KS patients by thrombin generation assay (TGA) and thromboelastometry. METHODS: An observational, cross-sectional study was conducted at 3 tertiary endocrinological centers in Milan, Italy. Fifty-eight KS patients and 58 age-matched healthy controls were included. Anticoagulant or antiplatelet therapy and known coagulation disorders were exclusion criteria. TGA was performed in platelet-poor plasma (PPP) and platelet-rich plasma (PRP). Whole-blood thromboelastometry and activities of coagulation factors were assessed. Endogenous thrombin potential (ETP), the area under the thrombin generation curve, assessed with and without thrombomodulin (ETP-TM+ and ETP-TM-), and their ratio (ETP ratio), were considered as indexes of procoagulant imbalance. RESULTS: Patients with KS displayed higher PPP-ETP-TM+ (mean 1528 vs 0.1315 nM × min; P < .001), PPP-ETP ratio (0.78 vs 0.0.70; P < .001), factor (F)VIII (135% vs 0.107%; P = .001), fibrinogen (283 vs 0.241 mg/dL; P < .001), and FVIII/protein C ratio (1.21 vs 0.1.06; P < .05) compared to controls. Protein C was comparable in the 2 groups. Similar results were observed in PRP. The ETP ratio was positively associated with FVIII (ρ = 0.538, P < .001) in KS. Thromboelastometry parameters confirmed evidence of hypercoagulability in KS. CONCLUSION: Patients with KS display a procoagulant imbalance expressed by increased thrombin generation both in PPP and PRP, which is at least in part explained by increased FVIII levels. The procoagulant imbalance, which was confirmed by thromboelastometry, may be responsible for the thrombotic events observed in these patients. Further investigation on the benefit/risk ratio of antithrombotic prophylaxis is warranted.

No changes of parameters nor coagulation activation in healthy subjects vaccinated for SARS-Cov-2.

Background: Recent reports of thrombotic events after SARS-Cov-2 vaccination raised concern. However, modifications of the most common coagulation parameters after vaccination are unknown. Aims: We measured parameters of coagulation including (i) basic coagulation tests, (ii) procedures aimed to assess the ex-vivo potential capacity to generate thrombin and (iii) in vivo thrombin activity. We also assessed anti-platelet factor 4 (aPF4) with two methods. Design: Laboratory measurements were performed for a cohort of subjects (n = 30) before (baseline) and after (7 and 21days after first dose, and 14days after second dose) SARS-Cov-2 vaccination. Results: All subjects received the Pfizer-BioNTech vaccine, and none developed symptomatic thrombotic events during the study period. None of the parameters showed clinically relevant variations at different time-points before and after vaccination. Only platelet count showed a slight increase, and F1.2 and the thrombin generation parameters ETP and ETP-TM ratio, showed a small decline, at the last time-point after vaccination when compared to baseline. aPF4 was negative in all the subjects, except two, who were positive (one with the chemiluminescent and the other with the ELISA assay). Conclusions: The study shows no modifications of the coagulation parameters nor the presence of biochemical signs of coagulation activation following the administration of the Pfizer-BioNTech vaccine.

Thrombin Generation in Preterm Newborns With Intestinal Failure-Associated Liver Disease.

Background and Aim: Intestinal failure-associated liver disease (IFALD) affects one-fifth of neonates receiving parenteral nutrition (PN) for more than 2 weeks. We aimed to define the effect of IFALD on hemostasis of preterm infants. Methods: This is an ancillary analysis of a prospective study aimed at defining coagulation in preterm infants. We included neonates exposed to PN (at least 14 days), in full-enteral feeding. We compared thrombin generation in the presence of thrombomodulin, defined as endogenous thrombin potential-ETP, PT, aPTT between infants with IFALD vs. those without (controls), at birth, and after 30 days. IFALD was defined as conjugated bilirubin ≥1 mg/dl. Results: We enrolled 92 preterm infants (32 IFALD; 60 controls). Cholestatic patients had a lower birthweight, longer exposure to PN, and longer hospitalization. Infants with IFALD showed longer median PT (12.8-vs.-12 sec; p = 0.02) and aPTT (39.2-vs.-36.5 sec; p = 0.04) than controls, with no difference in ETP. Conclusions: Despite prolonged PTs and aPTTs infants with IFALD had similar ETP than those without.