Platelet function testing: Current practice among clinical centres in Northern Europe.

INTRODUCTION: Platelet function tests are used to screen and diagnose patients with possible inherited platelet function defects (IPFD). Some acquired platelet dysfunction may be caused by certain drugs or comorbidities, which need to be excluded before testing. AIMS: To identify current practice among centres performing platelet function tests in Northern Europe. METHODS: A total of 14 clinical centres from Sweden (six), Finland (two), Denmark (two), Norway (one), Estonia (two) and Iceland (one) completed the survey questionnaire, the population capture area of about 29.5 million. RESULTS: Six of the 14 (42.8%) centres providing platelet function assessment represent comprehensive treatment centres (EUHANET status). A Bleeding score (BS) or ISTH bleeding assessment tool (ISTH BAT score) is evaluated in 11/14 (78.6%) centres and family history in all. Five/14 centres (35.7%) use structured preanalytical patient instructions, and 10/14 (71.4%) recorded questionnaire on the preassessment of avoidance of any drugs or natural products affecting platelet functions. Preliminary investigations of screening tests of coagulation are performed in 10/14 (71.4%), while in 4/14 (28.6%), the diagnostic work-up of IPFD and von Willebrand disease (VWD) is performed simultaneously. The work-up of IPFD includes peripheral blood smear in 10/14 (71.4%), platelet aggregometry in all, flow cytometry in 10/14 (71.4%) and Platelet Function Analysis (PFA) in 3/11 (28.6%). Molecular genetic diagnosis is available in 7/14 (50%) centres. CONCLUSIONS: The considerable variability in the current practice illustrates the need for harmonization between the Northern European centres according to the international registers (i.e. EUHASS) and IPFD guidelines (ISTH, EHA).

Non-phthalate plasticizer DEHT preserves adequate blood component quality during storage in PVC blood bags.

BACKGROUND AND OBJECTIVES: Commercial blood bags are predominantly made of polyvinyl chloride (PVC) plasticized with di(2-ethylhexyl) phthalate (DEHP). DEHP is favourable for storage of red blood cells (RBC). Historically, removal of DEHP from blood bags has been linked to unacceptable haemolysis levels. Oncoming regulatory restrictions for DEHP due to toxicity concerns increase the urgency to replace DEHP without compromising RBC quality. Di(2-ethylhexyl) terephthalate (DEHT) is one suggested substitute. The aim of this study was to compare PVC-DEHT to PVC-DEHP blood bags using additive solutions saline-adenine-glucose-mannitol (SAGM) and phosphate-adenine-glucose-guanosine-saline-mannitol (PAGGSM), to determine whether DEHT can maintain acceptable component quality. MATERIALS AND METHODS: RBC concentrates (N = 64), platelet concentrates (N = 16) and fresh frozen plasma (N = 32) were produced from whole blood collected into either DEHT or DEHP plasticized systems. Using a pool-and-split study design, pairs of identical RBC content were created within each plasticizer arm and assigned either SAGM or PAGGSM. Storage effects were assessed weekly for 49 days (RBC), 7 days (platelets) and before/after freezing (plasma). RESULTS: Though haemolysis was slightly higher in DEHT, all study arms remained below half of the European limit 0·8%. K+ was lower in DEHT than in DEHP independent of additive solution. The metabolic parameters were not influenced by choice of plasticizer. Platelet activation/metabolism and plasma content were similarly preserved. CONCLUSION: Our study demonstrates that the plasticizer DEHT provides adequate blood component quality. We propose DEHT as a strong future candidate for replacement of DEHP in blood bags.

The Silence Speaks, but We Do Not Listen: Synonymous c.1824C>T Gene Variant in the Last Exon of the Prothrombin Gene as a New Prothrombotic Risk Factor.

BACKGROUND: Thrombosis is a major global disease burden with almost 60% of cases related to underlying heredity and most cases still idiopathic. Synonymous single nucleotide polymorphisms (sSNPs) are considered silent and phenotypically neutral. Our previous study revealed a novel synonymous FII c.1824C>T variant as a potential risk factor for pregnancy loss, but it has not yet been associated with thrombotic diseases. METHODS: To determine the frequency of the FII c.1824C>T variant we have sequenced patients' DNA. Prothrombin RNA expression was measured by quantitative PCR. Functional analyses included routine hemostasis tests, western blotting and ELISA to determine prothrombin levels in plasma, and global hemostasis assays for thrombin and fibrin generation in carriers of the FII c.1824C>T variant. Scanning electron microscopy was used to examine the structure of fibrin clots. RESULTS: Frequency of the FII c.1824C>T variant was significantly increased in patients with venous thromboembolism and cerebrovascular insult. Examination in vitro demonstrated increased expression of prothrombin mRNA in FII c.1824T transfected cells. Our ex vivo study of FII c.1824C>T carriers showed that the presence of this variant was associated with hyperprothrombinemia, hypofibrinolysis, and formation of densely packed fibrin clots resistant to fibrinolysis. CONCLUSION: Our data indicate that FII c.1824C>T, although a synonymous variant, leads to the development of a prothrombotic phenotype and could represent a new prothrombotic risk factor. As a silent variant, FII c.1824C>T would probably be overlooked during genetic screening, and our results show that it could not be detected in routine laboratory tests.

Substantial prevalence of subclinical cardiovascular diseases in patients with hemophilia A evaluated by advanced electrocardiography.

BACKGROUND: Patients with hemophilia A (PWHA) have reportedly lower mortality due to cardiovascular disease (CVD) compared to the general population. AIM: To evaluate signs of CVD in asymptomatic PWHA using advanced electrocardiography (A-ECG). METHODS: PWHA (n = 29, median [interquartile range] age 57 [47-70] years) and age-matched male controls (n = 29, 59 [48-68] years) were evaluated. Digital resting 12­lead ECGs were retrospectively analysed using both conventional and A-ECG techniques including derived vectorcardiography and waveform complexity. Previously validated multivariate A-ECG scores designed to detect: 1) cardiac disease in general, 2) left ventricular systolic dysfunction (LVSD), 3) coronary artery disease or coronary microvascular disease (CAD/CMVD), or 4) left ventricular hypertrophy defined as left ventricular electrical remodelling (LVH/LVER), were quantified and compared between PWHA and controls. RESULTS: Compared to controls, PWHA had a higher probability of having cardiac disease (median [interquartile range] 84.6 [32.5-99.5] vs. 0.6 [0.2-8.2]%), LVSD (4.1 [1.3-12.9] vs. 0.9 [0.5-3.2]%), CAD/CMVD (84.3 [35.6-96.6] vs. 6.7 [0.8-24.4]%), and LVH/LVER (17 [5/29] vs. 0 [0/29]%). Compared to patients with non-severe HA (n = 20), patients with severe HA (n = 9) showed a non-significant trend towards lower probability of cardiac disease, CAD/CMVD, LVSD and LVH/LVER. CONCLUSION: In PWHA, A-ECG exhibits changes more indicative of overt or subclinical CVD compared to controls, and there is a tendency for lower scores for CVD in patients with severe compared to non-severe HA. These results suggest that PWHA ≥ 40 years could be at higher risk for CVD than age-matched controls and that A-ECG could potentially be used for early detection.

Intra- and inter- individual rivaroxaban concentrations and potential bleeding risk in patients with atrial fibrillation.

BACKGROUND: Routine laboratory monitoring of rivaroxaban and dose adjustment relating to exposure is currently not recommended. However, in certain clinical situations, assessment of rivaroxaban levels is desirable. OBJECTIVES: To examine inter- and intra-subject plasma rivaroxaban variability in patients with atrial fibrillation (AF) and to correlate these results to clinical outcomes. PATIENTS/METHODS: We included 60 patients with AF treated with rivaroxaban: half on 20 mg daily (R20) and half on 15 mg daily (R15). Three trough and peak blood samples were collected with an interval of 6-8 weeks apart. Plasma rivaroxaban concentration was measured directly by liquid chromatography-tandem mass-spectrometry (LC-MS/MS) and indirectly by anti-Xa for rivaroxaban, prothrombin time (PT), and activated partial thromboplastin time (APTT). RESULTS: Patients on R15 were older (76 ± 6 vs 71 ± 6 years), had lower creatinine clearance (60 ± 26 vs 99 ± 32 mL/min), higher CHADS2 (2.5 ± 1.2 vs 1.8 ± 1.3), all p < 0.01, but had similar rivaroxaban concentrations in trough samples to patients on R20. There was no significant intra-individual variability for trough or peak rivaroxaban concentration assessed by LC-MS/MS, anti-Xa, or PT. Trough rivaroxaban levels determined by LC-MS/MS (48 ± 30 vs 34 ± 26, p = 0.02) and anti-Xa, but not with PT and APTT, were higher in patients with bleeding than in patients without it. CONCLUSIONS: There is a pronounced inter-, but not intra-individual variability in the rivaroxaban trough levels in patients with AF. Assessment of trough rivaroxaban concentration with LC-MS/MS or anti-Xa, but not with APTT or PT, may help to identify patients at increased risk of bleeding.

Clinical evaluation of laboratory methods to monitor exposure of rivaroxaban at trough and peak in patients with atrial fibrillation.

PURPOSE: The one-dose daily regime of rivaroxaban could cause a pronounced variability in concentration and effect of which a deeper knowledge is warranted. This study aimed to evaluate the typical exposure range and effect of the direct factor Xa (FXa)-inhibitor rivaroxaban in a cohort of well-characterized patients with atrial fibrillation (AF). METHODS: Seventy-one AF patients (72 ± 8 years, 55 % men) were treated with rivaroxaban 15 mg/20 mg (n = 10/61) OD. Trough (n = 71) and peak (n = 30) plasma concentrations determined by liquid chromatography-tandem mass-spectrometry (LC-MS/MS) were compared to the coagulation assays anti-FXa for rivaroxaban, prothrombin time-international normalized ratio (PT-INR) (venous samples and point-of-care assay (POC) CoaguChek XS Pro), and aPTT. RESULTS: Median rivaroxaban plasma concentrations by LC-MS/MS were 34 (range 5-84) and 233 ng/ml (range 120-375) at trough and peak, respectively. A strong correlation between LC-MS/MS and the anti-FXa assay was found (p < 0.001) for both trough (r (2) = 0.92) and peak (r (2) = 0.91) samples. PT-INR results from the POC assay, but not from the conventional PT assay, correlated significantly with LC-MS/MS in peak samples exclusively (r (2) = 0.41, p < 0.001). CONCLUSIONS: In "real-life" AF patients treated with rivaroxaban, we observed a pronounced variability in plasma concentrations at trough and to a lesser extent at peak measured by LC-MS/MS. The anti-FXa assay performed well upon rivaroxaban levels in a normal exposure range, although LC-MS/MS remains the only method that covers the whole concentration range with accuracy. Interestingly, the POC assay for PT-INR could be useful to indicate high exposure to rivaroxaban in emergency situations although further validation is required.

Effect of prothrombin Belgrade mutation, causing antithrombin resistance, on fibrin clot properties.

INTRODUCTION: Prothrombin Belgrade mutation is the result of the c.1787G>A substitution in the prothrombin gene. It is located in the antithrombin and sodium binding site and leads to impaired inactivation of thrombin by antithrombin, resulting in antithrombin resistance and thrombotic disorders. However, it negatively affects sodium binding and may have hypocoagulant effects. Considering that prothrombin Belgrade mutation mechanism is still not fully elucidated and that sodium binding is important for thrombin affinity towards fibrinogen, our aim was to determine whether this mutation affects fibrin clot formation and lysis. METHODS: Using HEK293T cell line, recombinant wild type and mutated prothrombin were generated by transient transfection. Samples that correspond to plasma of a non-carrier, heterozygous and homozygous carriers were reconstituted using prothrombin deficient plasma and recombinant proteins. Reconstituted samples were used in OHP assay (Overall Hemostasis Potential) to determine kinetic profiles of coagulation and fibrinolysis. Clot turbidity assay was performed to observe kinetics of clot formation and lysis more closely. Fibrin clots formed in reconstituted plasma samples were analyzed by confocal microscopy to determine density of fibrin network. Fibrin clots were additionally observed using electron microscopy to determine thickness of individual fibrin fibers. RESULTS: No significant difference found in OHP, OCP, OFP, and fibrin network density between wild type, heterozygous, and homozygous carrier reconstituted plasma samples. There were significant differences between samples for slope and slope time parameters in kinetic profiles and fibrin fiber thickness. CONCLUSIONS: Results indicate that prothrombin Belgrade mutation has no significant impact on fibrinolysis, however it may affect kinetics of clot formation and its architecture.

Extracellular vesicles in plasma and cerebrospinal fluid in patients with COVID-19 and neurological symptoms.

INTRODUCTION: Increased levels of extracellular vesicles (EVs) are associated with haemostatic disturbances in various clinical settings. However, their role in COVID-19 patients is still not fully clear. In the present study we investigated EVs in plasma from patients with COVID-19 and neurological symptoms in relation to the activation of coagulation. METHODS: Nineteen COVID-19 patients with neurological symptoms and twenty-three aged-matched healthy individuals were included. Global coagulation assays were performed and levels of EVs were determined by flow-cytometry in plasma and cerebrospinal fluid (CSF). RESULTS: A procoagulant state characterized by significantly increased overall coagulation- (OCP) and overall haemostatic potential (OHP), diminished overall fibrinolytic potential (OFP) together with a denser fibrin structure was found in patients with COVID-19. Flow cytometry revealed elevated levels of plasma circulating EVs derived from neutrophils (MPO+) and platelets (CD61+), as well as EVs expressing phosphatidylserine (PS+) and complement component C5b-9 (TCC+) in patients with COVID-19 compared with controls. The concentrations of PS+, CD61+ and TCC+ EVs were positively correlated with OCP and OHP in COVID-19 patients. Moreover, we identified CD61+, MPO+ and endothelial cell-derived EVs, as well as EVs exposing PS and TCC in the CSF of patients suffering from neurological symptoms during COVID-19. CONCLUSION: The unique finding in this study was the presence of EVs in the CSF of COVID-19 patients with neurologic manifestations as well as higher expression of complement protein on circulating plasma EVs. EVs may indicate blood-brain barrier damage during SARS-COV-2 infection.

Clinical Implications of Discrepancy between One-Stage Clotting and Chromogenic Factor IX Activity in Hemophilia B.

BACKGROUND: Discrepancy in factor IX activity (FIX:C) between one-stage assay (OSA) and chromogenic substrate assay (CSA) in patients with hemophilia B (PwHB) introduces challenges for clinical management. AIM: To study the differences in FIX:C using OSA and CSA in moderate and mild hemophilia B (HB), their impact on classification of severity, and correlation with genotype. METHODS: Single-center study including 21 genotyped and clinically characterized PwHB. FIX:C by OSA was measured using ActinFSL (Siemens) and CSA by Biophen (Hyphen). In addition, in vitro experiments with wild-type FIX were performed. Reproducibility of CSA was assessed between three European coagulation laboratories. RESULTS: FIX:C by CSA was consistently lower than by OSA, with 10/17 PwHB having a more severe hemophilia type by CSA. OSA displayed a more accurate description of the clinical bleeding severity, compared with CSA. A twofold difference between OSA:CSA FIX:C was present in 12/17 PwHB; all patients had genetic missense variants in the FIX serine protease domain. Discrepancy was also observed with diluted normal plasma, most significant for values below 0.10 IU/mL. Assessment of samples with low FIX:C showed excellent reproducibility of the CSA results between the laboratories. CONCLUSION: FIX:C was consistently higher by OSA compared with the CSA. Assessing FIX:C by CSA alone would have led to diagnosis of a more severe hemophilia type in a significant proportion of patients. Our study suggests using both OSA and CSA FIX:C together with genotyping to classify HB severity and provide essential information for clinical management.

Evaluation of coagulation assays versus LC-MS/MS for determinations of dabigatran concentrations in plasma.

BACKGROUND: Dabigatran is an oral direct thrombin inhibitor for which routine laboratory monitoring is currently not recommended. However, there are situations in which measurements of the drug and its effect are desirable. We therefore compared and validated different coagulation methods for assessments of dabigatran in clinical samples in relation to measurements of plasma dabigatran, without the purpose of establishing effective and safe concentrations of dabigatran in plasma. METHODS: Samples were obtained from 70 atrial fibrillation patients treated with dabigatran etexilate. Plasma concentrations were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and were compared with coagulation methods Hemoclot thrombin inhibitors (HTI) and Ecarin clotting assay (ECA), as well as with prothrombin time-international normalized ratio (PT-INR) and activated partial thromboplastin time (aPTT). RESULTS: A wide range of dabigatran concentrations was determined by LC-MS/MS (<0.5-586 ng/mL). Correlations between LC-MS/MS results and estimated concentrations were excellent for both HTI and ECA overall (r(2) = 0.97 and 0.96 respectively, p < 0.0001), but the precision and variability of these assays were not fully satisfactory in the low range of dabigatran plasma concentrations, in which ECA performed better than HTI. aPTT performed poorly, and was normal (<40 s) even with dabigatran levels of 60 ng/mL. PT-INR was normal even at supratherapeutic dabigatran concentrations. CONCLUSION: LC-MS/MS is the gold standard for measurements of dabigatran in plasma. Alternatively, either HTI or ECA assays may be used, but neither of these assays is dependable when monitoring low levels or to infer total absence of dabigatran. The aPTT assay is relatively insensitive to dabigatran, and normal aPTT results may be observed even with therapeutic dabigatran concentrations.

Thrombin generation in acute cardioembolic and non-cardioembolic ischemic stroke.

OBJECTIVE: Increased thrombin generation, as measured by the Calibrated Automated Thrombogram (CAT), has recently been reported to predict ischemic stroke, especially stroke with a cardioembolic source. However, there are few studies on thrombin generation using CAT in patients with manifest ischemic stroke, particularly in patients with cardioembolic stroke not yet on anticoagulation. MATERIALS AND METHODS: Therefore, a prospective cohort study of 205 stroke patients > 45 years of age was performed. They were recruited during their hospital stay or shortly thereafter. Inclusion criteria were ischemic stroke or TIA within two weeks and no atrial fibrillation (AF) in the history or at inclusion. Patients received a thumb ECG device in order to detect silent AF. Blood samples were collected at inclusion and after 1 month. Thrombin generation in plasma after addition of tissue factor was assessed in patients and in healthy controls. RESULTS: Mean age of patients was 72 ± 7 years and 43% were females. Peak thrombin concentrations were variable among stroke patients but overall significantly higher at both time points (p < 0.0001) compared to controls, and tended to be highest in patients in whom paroxysmal atrial fibrillation was subsequently documented. CONCLUSION: Thrombin generation in patients with acute cardioembolic and non-cardioembolic schemic stroke/TIA is variable but overall higher compared to healthy subjects. The long-term prognostic value of thrombin generation in patients with a recent ischemic stroke deserves further investigation.

Elevated plasma complement factor H related 5 protein is associated with venous thromboembolism.

Venous thromboembolism (VTE) is a common, multi-causal disease with potentially serious short- and long-term complications. In clinical practice, there is a need for improved plasma biomarker-based tools for VTE diagnosis and risk prediction. Here we show, using proteomics profiling to screen plasma from patients with suspected acute VTE, and several case-control studies for VTE, how Complement Factor H Related 5 protein (CFHR5), a regulator of the alternative pathway of complement activation, is a VTE-associated plasma biomarker. In plasma, higher CFHR5 levels are associated with increased thrombin generation potential and recombinant CFHR5 enhanced platelet activation in vitro. GWAS analysis of ~52,000 participants identifies six loci associated with CFHR5 plasma levels, but Mendelian randomization do not demonstrate causality between CFHR5 and VTE. Our results indicate an important role for the regulation of the alternative pathway of complement activation in VTE and that CFHR5 represents a potential diagnostic and/or risk predictive plasma biomarker.

Biological Variation in Rotational Thromboelastometry in Patients with Atrial Fibrillation Receiving Rivaroxaban.

Rotational thromboelastometry (ROTEM) is a viscoelastic hemostasis test used primarily in the management of bleeding after trauma or in cardiac surgery. To allow safe and valid clinical interpretation of test results, objective specifications for analytical performance are needed, which are generally based on biological variation within (CVI) and between (CVG) individuals. The aim of this study was to evaluate biological variation in ROTEM in patients receiving rivaroxaban. Sixty patients with atrial fibrillation on stable rivaroxaban therapy were included, from whom blood was collected on six occasions: three times at trough and three at peak rivaroxaban concentrations. ROTEM® Extem and LowTF were measured as well as rivaroxaban concentration, PT, APTT, and anti-Xa. Within- (CVI) and between-subject (CVG) biological estimates were calculated. Knowledge of these biological variation components will help to establish the appropriate objective analytical performance specifications for ROTEM analysis.

Using a low-molecular weight heparin-calibrated anti-factor Xa assay to assess the concentration of apixaban and rivaroxaban.

INTRODUCTION: Direct oral anticoagulant (DOAC)-inhibiting factor Xa (FXa-DOAC) are being increasingly used as prophylaxis of venous thromboembolism and for prevention of stroke in patients with atrial fibrillation. In contrast to vitamin K antagonists, DOACs do not require monitoring in general. However, it is sometimes of value in the acute setting, for instance when considering a reversal agent in uncontrolled bleeding in patients on DOAC. METHODS: We evaluated if a low-molecular weight heparin (LMWH)-calibrated anti-factor Xa assay could be used to estimate FXa-DOAC concentration in the concentration range <100 ng/mL by spiking known concentrations of FXa-DOAC and from those result calculate the FXa-DOAC concentration from the response of the LMWH assay. This procedure was then evaluated by comparing the result with a drug-calibrated chromogenic assay and liquid chromatography tandem mass spectrometry (LC-MS/MS) on clinical plasma samples from patients treated with apixaban or rivaroxaban. RESULTS: Although the measuring range was narrower for the LMWH-calibrated assay, concentrations recalculated from the LMWH assay was comparable with those measured by the drug-calibrated method when compared with LC-MS/MS. CONCLUSION: We suggest that an LMWH-calibrated anti-factor Xa assay can be used after characterization of the response of FXa-DOACs to give guidance on the concentration of apixaban and rivaroxaban. Shorter turnaround time than LC-MS/MS and the greater availability than drug-calibrated chromogenic assays could make this a valuable option in the acute setting.

[Routine screening with APTT is not indicated before surgery]. / Rutinmässig screening med APTT är inte indicerad före operation.

Activated partial thromboplastin time (APTT) is widely practiced in preoperative screening. The value of using this test to predict the risk of perioperative bleeding is not well documented in Sweden. In this article, a literature review is performed to determine whether unselected APTT testing can predict abnormal perioperative bleeding. The current literature does not support coagulation screening with APTT in routine perioperative bleeding assessment, as preoperative screening with APTT has a low sensitivity for detection of clinically significant bleeding disorder. While a comprehensive bleeding history is crucial, the APTT test should only be performed on patients with a history of increased bleeding tendency. The conclusion of this literature review is that patients with a negative bleeding history do not require routine screening with APTT prior to surgery, which, if implemented, would lead to a more cost-effective perioperative routine.

Practical viewpoints on the diagnosis and management of heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT, type II) is an immune-mediated disorder due to antibodies formed against heparin-platelet factor 4 complexes, usually appearing at days 5 to 14 after initiation of heparin. It is important to recognize HIT because heparin prophylaxis or treatment paradoxically associates with new venous and/or arterial thrombosis. Early clinical suspicion and diagnosis together with proper pharmacotherapy and close laboratory monitoring are the cornerstones for successful management. This includes monitoring of Thrombocytopenia, its Timing to heparin administration, appearance of new Thrombosis or resistance to treatment, and differential diagnosis by exclusion of o Ther causes (the 4T's). Specific attention should be paid to the absence or presence of thrombosis and to tailoring thromboprophylaxis or anticoagulant therapy with a nonheparin alternative. Even in the absence of HIT-associated thrombosis, an active policy for prolonged thromboprophylaxis is demanded. Rapid and reliable assays should be developed for diagnosis and anticoagulation monitoring to secure safe management with nonheparins. Semiquantitative testing for on-call hours should be available and later confirmed as clinically needed. Alternative therapeutic options are available, but because their use is infrequent, experienced coagulation treatment centers should provide guidance in the treatment and in laboratory monitoring. Most of the evidence in HIT is grade IC, and thus the best evidence is provided by clinical experience. New anticoagulants and platelet inhibitors may offer future alternatives in the management of HIT, but the current treatment options provide the best experience and benefit. The joint clinical and laboratory guidelines provided in this article along with two practical case scenarios were prepared by a Nordic expert panel. They will be valuable for hematologists and colleagues who do not routinely encounter HIT.

The in vitro addition of idarucizumab to plasma samples from patients increases thrombin generation.

Dabigatran interferes with many coagulation tests. To overcome this obstacle the use of idarucizumab as an in vitro antidote to dabigatran has been proposed. The aim of this study was to test the effect of idarucizumab as an in vitro antidote to dabigatran in ex vivo plasma samples from routine clinical patients examined by a thrombin generation assay (TGA). From 44 patients with atrial fibrillation five blood samples were collected. Thrombin generation was measured in all samples before and after the addition of idarucizumab. When idarucizumab was added to baseline plasma (no dabigatran), it caused a significantly shorter Lag Time and Time to Peak Thrombin, and a higher Peak Thrombin and Endogenous Thrombin Potential (ETP) of TGA. Similar results were obtained when idarucizumab was added to dabigatran-containing plasma, with TGA parameters comparable to baseline + idarucizumab plasma, but not to baseline plasma. In summary, our study showed that in vitro addition of idarucizumab to plasma samples from patients increases thrombin generation. The use of idarucizumab to neutralize dabigatran in patient plasma samples as well as the clinical relevance of in vitro increased thrombin generation induced by idarucizumab needs further investigation.

Overall hemostasis potential and aPTT-clot waveform analysis as powerful laboratory diagnostic tools for identification of hemophilia A patients with unexpected bleeding phenotype.

INTRODUCTION: Traditionally used laboratory methods do not always accurately reflect bleeding severity in hemophilia A (HA) patients. The ability of three global assays for identifying patients with unexpected bleeding phenotype was investigated. METHODS: Overall hemostasis potential (OHP), aPTT-clot waveform analysis (aPTT-CWA), endogenous thrombin potential (ETP), FVIII activities, and prothrombin fragment 1 + 2 concentrations were measured in 62 HA patients (30 severe and 32 non-severe) and 27 male controls. Bleeding phenotype was determined using our proposed scoring system including age at first joint bleed, number of target joints, and number of joint/muscle bleeds per year. Bleeding score ≤ 4 defined patients with mild bleeding phenotype (N = 27); score ≥ 5 defined severe bleeding phenotype (N = 35). RESULTS: The receiver operating characteristic analysis performed for distinguishing patients with severe and mild bleeding phenotype yielded following values of area under the curve: 0.910 (FVIII); 0.891 (aPTT-CWA parameter DELTA); 0.769 (OHP); and 0.634 (ETP). Unexpected bleeding phenotype was identified in 11/62 HA patients: 8/32 (25%) non-severe HA patients had severe, while 3/30 (10%) severe HA patients had mild bleeding phenotype, and global assays enabled the identification of all these patients. OHP and DELTA were revealed as the most reliable parameters for bleeding phenotype determination (10/11 and 9/11 unexpected results, respectively). CONCLUSION: This study emphasizes OHP and aPTT-CWA as a powerful laboratory diagnostic tool in identifying HA patients with unexpected bleeding presentations, with the best results achieved by combining both assays. Global assays should not completely replace FVIII activity measurement but should be a part of the HA diagnostic algorithm.