Testosterone Replacement Therapy in Klinefelter Syndrome-Follow-up Study Associating Hemostasis and RNA Expression.

BACKGROUND: Men with Klinefelter syndrome (KS) develop hypergonadotropic hypogonadism, are in need of testosterone replacement therapy (TRT), and present with a more than 4-fold increased risk of thrombosis. TRT in KS has the potential to modify thrombotic risk, but data are scarce. AIM: To assess effects of 18 months of TRT on hemostasis in KS and identify genes associated with the prothrombotic phenotype. METHODS: Untreated and TRT-treated men with KS were included at baseline and matched to healthy controls. TRT was initiated in untreated KS and all groups were reassessed after 18 months of follow-up. Thrombin generation was evaluated with or without thrombomodulin, and fibrin clot lysis was evaluated by turbidity measurements. RNA expression was assessed in blood, fat, and muscle tissue of patients with TRT-treated KS and controls. RESULTS: Thrombin generation with thrombomodulin was slightly increased in untreated KS, but overall KS was not associated with a hypercoagulable state. KS presented with fibrinolytic impairment associated with higher body fat and higher levels of fibrinogen. Eighteen months of TRT in KS was associated with a reduction in body fat and fibrinogen, attenuating the prothrombotic profile. The expression of ENPP4 was higher in men with KS and served as a key player among a group of genes associated with impaired fibrinolysis. CONCLUSION: KS is associated with a specific expression profile contributing to fibrinolytic impairment and increased thrombotic risk in the patients. TRT in patients with KS has the potential for alleviating the prothrombotic phenotype, in particular by reducing body fat and fibrinogen.

Myocardial injury after non-cardiac surgery and per operative fibrin metabolism in patients undergoing hip-fracture surgery: an observational study.

Myocardial injury after non-cardiac surgery (MINS) is associated with a 2-3-fold increased risk of subsequent major cardiovascular events and postoperative mortality. The pathological mechanism behind MINS is not fully uncovered. We hypothesized that patients with MINS following hip fracture surgery would have an altered haemostatic balance pre- and postoperative compared with patients without MINS. This was investigated in a prospective single-centre observational study including patients consecutively. The outcomes were changes in thrombin generation, fibrinogen/fibrin turnover, tissue plasminogen activator, plasminogen activator inhibitor-1 and fibrin structure measurements in patients developing MINS and patients who did not. Outcomes were measured preoperatively and two hours postoperatively. Seventy-two patients were included whereof 26 (36%) patients developed MINS. D-dimer delta values were significantly higher in patients developing MINS than in patients who did not (p = 0.01). After adjusting for age, sex, smoking, alcohol abuse, atrial fibrillation, anticoagulant medication preoperative CRP, preoperative creatinine and duration of surgery, the association remained significant (p = 0.04). There were no significant changes in thrombin generation, in markers of fibrinogen/fibrin turnover besides D-dimer, or in fibrin structure measurements pre- and postoperatively between patients with and without MINS. As such, a relationship between the coagulative and fibrinolytic activity and MINS cannot be ruled out in patients with MINS after hip fracture surgery. Registration: The study was an observational sub-study to a multicentre randomised clinical trial registered at ClinicalTrials.gov (NCT02344797).

Reduced fibrin clot lysis in Klinefelter syndrome associated with hypogonadism.

Objective: Klinefelter syndrome (KS) is associated with increased risk of thrombosis. Hypogonadism and accumulating body fat in KS have a potential impact on fibrinolysis. In this study, we assessed the fibrinolytic system and the association with testosterone levels in KS. Design: This study is a cross-sectional comparison of men with KS and age-matched male controls. Methods: Fibrin clot lysis was evaluated by turbidity measurements and by measuring levels of individual fibrinolytic proteins in plasma samples. Fibrin clot structure was evaluated by scanning electron microscopy. Total testosterone was measured by liquid chromatography-tandem mass spectrometry. Body fat was evaluated by dual-energy X-ray absorptiometry. Results: In this study, 45 men with KS and 45 age- and education-matched controls were included. Men with KS had a 24% reduction in fibrin clot lysis compared with controls (46.2 ± 17.1 vs 60.6 ± 18.8 %/h, P = 0.0003) and higher levels of fibrinogen, factor XIII (P ≤ 0.01), and plasminogen activator inhibitor type 1 (P = 0.04). Men with KS had lower total testosterone (P = 0.008) and higher body fat (P = 0.001). In KS, reduced fibrin clot lysability was associated with higher fibrinogen and body fat related to decreasing total testosterone and hypogonadism among men with KS. Fibrin clot structure was not different compared to KS and controls. Conclusions: Fibrin clot lysis in KS was markedly reduced, potentially contributing to a prothrombotic state and increasing thrombotic risk. Hypogonadism in KS was associated with increased fibrinogen and total body fat, predicting reduced fibrin clot lysis.

Effect of remote ischemic preconditioning on fibrin formation and metabolism in patients undergoing hip fracture surgery: a randomized clinical trial.

Remote ischemic preconditioning (RIPC) prior to surgery has recently been shown to reduce the risk of myocardial injury and myocardial infarction after hip fracture surgery. This study investigated whether RIPC initiated antithrombotic mechanisms in patients undergoing hip fracture surgery. This trial was a predefined sub-study of a multicentre randomized clinical trial. Adult patients with cardiovascular risk factors undergoing hip fracture surgery between September 2015 and September 2017 were randomized 1 : 1 to RIPC or control. RIPC was initiated before surgery with a tourniquet applied to the upper arm and it consisted of four cycles of 5 min of forearm ischemia followed by five minutes of reperfusion. The outcomes such as surgery-induced changes in thrombin generation, fibrinogen/fibrin turnover, tissue plasminogen activator, plasminogen activator inhibitor-1 and fibrin structure measurements were determined preoperatively (prior to RIPC) and 2 h postoperatively. One hundred and thirty-seven patients were randomized to RIPC (n = 65) or control (n = 72). There were no significant changes in thrombin generation, fibrinogen/fibrin turnover or fibrin structure measurements determined pre and postoperatively between patients in the RIPC and control groups. Subgroup analyses on patients not on anticoagulant therapy (n = 103), patients receiving warfarin (n = 17) and patients receiving direct oral anticoagulant therapy (n = 18) showed no significant changes between the RIPC-patients and controls. RIPC did not affect changes in thrombin generation, fibrin turnover or fibrin structure in adult patients undergoing hip fracture surgery suggesting that the cardiovascular effect of RIPC in hip fracture surgery is not related to alterations in fibrinogen/fibrin metabolism.

Reductions in plasmin inhibitor and fibrinogen predict the improved fibrin clot lysis 6 months after obesity surgery.

Prothrombotic and metabolic variables are decreased after obesity surgery, and fibrin clot lysis is increased. It is unknown how fibrinolytic variables are affected, and whether fibrinolytic and metabolic changes predict the enhanced clot lysis. Study aims were to determine fibrinolytic biomarkers before and 6 months after Roux-en-Y gastric bypass (RYGB) and to identify predictors of the RYGB-induced increase in clot lysis. Women (n = 42) and men (n = 18) with obesity underwent RYGB, and factor XIII (FXIII), thrombin activatable fibrinolysis inhibitor (TAFI), plasminogen and plasmin inhibitor (PI) were measured before and 6 months after surgery. Regression analyses identified determinants of the RYGB-induced increase in clot lysis among changes in fibrinogen and in fibrinolytic and metabolic variables. Results showed that after RYGB, FXIII, TAFI, plasminogen and PI were reduced (P < .0005). Reductions in PI (ß = -0.59) and fibrinogen (ß = -0.35), together with age (ß = -0.22) and male sex (ß = 0.22), predicted the enhanced clot lysis with the model explaining 56% (P < .0005). Predictors of the reduction in PI were reductions in cholesterol (ß = 0.37) and glucose (ß = 0.29), together with male sex (ß = -0.28), whereas reductions in fibrinogen were predicted by lowering of interleukin-6 (IL-6) (ß = 0.32). In conclusion, fibrinolytic variables were reduced 6 months after RYGB. Targeting PI and fibrinogen, by reducing metabolic variables such as glucose, cholesterol and IL-6, has a profibrinolytic effect in obesity.

Citrullination of fibrinogen by peptidylarginine deiminase 2 impairs fibrin clot structure.

BACKGROUND: Citrullination is the post-translational conversion of arginine into citrulline in proteins. The reaction is catalyzed by peptidylarginine deiminase (PAD), of which five isoforms exist. Fibrinogen is a substrate for PAD2 and PAD4, and citrullinated fibrinogen (cFBG) has been detected in patients with inflammatory diseases. In purified systems, cFBG is known to inhibit the release of fibrinopeptide A (FPA) and B (FPB) and impairs fibrin polymerization. However, the effect of cFBG on fibrin structure and fibrinolysis in a plasma environment remains unclear. We hypothesized that citrullination of fibrinogen impairs fibrin properties. METHODS: Fibrinogen was citrullinated by recombinant PAD2 and PAD4. The impact of cFBG on fibrin structure was investigated by turbidity measurements in fibrinogen-deficient plasma spiked with cFBG or native fibrinogen. RESULTS: Citrullination of fibrinogen by PAD2 dose-dependently reduced the rate of fibrin polymerization, as well as the overall hemostasis potential of fibrin, the maximum velocity of fibrin formation, the fibrin mass/length ratio, and the lysis of fibrin clots. CONCLUSION: Citrullination of fibrinogen by PAD2 affects not only fibrin polymerization but also fibrin fiber properties, indicating that the fibrin network formed in the presence of cFBG may influence hemostasis. Our results suggest that citrullination of fibrinogen alters the composition of fibrin fibers which may lead to a looser fibrin network that is more susceptible to fibrinolysis and thereby affecting the hemostatic balance.

Klinefelter syndrome and testosterone treatment: a national cohort study on thrombosis risk.

OBJECTIVES: Klinefelter syndrome (KS), 47,XXY, can be viewed as a disease model for investigating the risk of thrombosis in male hypogonadism and the subsequent risk related to testosterone treatment. We describe rates of thrombotic risk factors, thrombosis and thrombosis mortality in KS and the association with testosterone treatment. METHODS: National registry-based matched cohort study with follow-up from 1995 to 2016 set in Denmark. For the study, 1155 men with KS were each matched by year and month of birth to 100 men from the background population. First thrombotic events and thrombosis mortality was evaluated by event rates and hazard ratios (HRs) and by applying testosterone treatment as a time-dependent covariate. RESULTS: The KS cohort had higher incidence of venous thromboembolism relative to the comparison cohort (HR, 3.95; 95% CI, 2.83-5.52). Total thrombotic deaths were increased in KS (HR, 1.76; 95% CI, 1.18-2.62), and all-cause mortality was increased in KS following arterial thrombosis (HR 1.73; 95% CI 1.22-2.47). Only 48.7% of men with KS redeemed prescriptions for testosterone. Untreated men with KS were on average born 12 years before those treated, and the majority of untreated men with KS with available biochemistry were hypogonadal. Testosterone treatment in KS was associated with a non-significant decrease in venous thromboembolism and thrombotic deaths. CONCLUSION: Thrombosis and thrombotic deaths are increased in KS. Only half of the men with KS ever received testosterone treatment, despite overt hypogonadism in the non-treated. Testosterone treatment in Klinefelter syndrome was insignificantly associated with lower incidence rates of venous thrombosis and thrombotic deaths.

Testosterone treatment and association with thrombin generation and coagulation inhibition in Klinefelter syndrome: A cross-sectional study.

BACKGROUND: The background for the increased occurrence of thrombosis seen in Klinefelter syndrome (KS) is unknown. The aim was to compare thrombin generation and coagulation inhibition between men with KS and controls, and to investigate whether coagulation in KS was associated with testosterone treatment (TT), and as such, measures of androgen action. METHODS: Untreated men with KS (U-KS) or testosterone treated men with KS (T-KS) were included. KS groups were matched by age and education to groups of control males with no history of TT. Blood samples were collected after overnight fasting. Low tissue factor (1pM) thrombin generation was expressed as lag time (min), time to peak (min), peak (nmol/L), and endogenous thrombin potential (nmol/L × min, ETP). Coagulation inhibitors, sex hormones, and haematocrit were measured. Matched groups were compared by Student's t-test or Wilcoxon rank sum test. Among KS, TT status as an outcome predictor was evaluated by linear regression. RESULTS: 18 U-KS and 27 T-KS with corresponding controls participated. Thrombin generation was not different comparing U-KS and T-KS with respective control groups. Among KS, ETP was lower in T-KS compared with U-KS and inversely associated with testosterone, LH-testosterone ratio and haematocrit. CONCLUSION: Neither U-KS nor T-KS expressed a pro-coagulant state compared with controls. Thrombin generation among KS was inversely associated with androgen action and lower in T-KS compared with U-KS. Whether TT is capable of lowering thrombotic risk among men with KS needs to be assessed prospectively.

Adherence to dabigatran etexilate in atrial fibrillation patients intended to undergo electrical cardioversion.

AIMS: Effective anticoagulation in patients undergoing electrical cardioversion (ECV) for symptomatic atrial fibrillation is important to prevent adverse events. High medication adherence is a requirement. In patients with newly diagnosed atrial fibrillation (n = 169) who were intended to undergo ECV, the aim of this study was to measure self-reported short- and long-term adherence, evaluate whether dabigatran plasma concentrations reflect adherence, measure treatment satisfaction and assess whether adherence and treatment satisfaction are correlated. METHODS AND RESULTS: Plasma concentrations (liquid-chromatography tandem mass spectrometry), the 8-point Morisky Medication Adherence Scale (MMAS-8) and the Anti-Clot Treatment Scale (ACTS) were measured after 3 weeks and 7 weeks of treatment. Combined mean peak (1-3 h after intake) and trough (10-16 h after intake) plasma concentrations were 175 (SD 109) ng/mL and 75 (SD 45) ng/mL, respectively. There was no relationship between short-term adherence (last 3 days) or long-term adherence (last 3-4 weeks) and plasma concentrations, unless the last intake was more than 48 h ago. After 7 weeks high, moderate, and low adherence, according to the MMAS-8, was seen in 74%, 21%, and 5% of patients, respectively. Treatment satisfaction was high (median ACTS score 68.5, range 46-75 points). Treatment satisfaction and adherence were not correlated. CONCLUSION: The percentage of patients in the high adherence group (74%) was lower than expected, which is a matter of concern. Dabigatran plasma concentrations could not detect short- or long-term non-adherence, unless the drug was last taken more than 48 h ago. Treatment satisfaction did not correlate with adherence.

Nintedanib in Idiopathic Pulmonary Fibrosis: Practical Management Recommendations for Potential Adverse Events.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease with a dismal survival rate of only 3 years and no curative pharmacological therapy. The recent approval of 2 anti-fibrotic drugs (nintedanib and pirfenidone) that slow disease progression has provided some hope for patients. However, effectively managing anti-fibrotic treatment can be a challenge due to tolerability issues, the presence of pulmonary and extra-pulmonary comorbidities, and the need for concomitant medications in many patients. In general, making clear evidence-based decisions can be difficult for physicians because patients with comorbidities are often excluded from clinical trials. Since currently anti-fibrotic drugs are the only effective therapeutics capable of slowing disease progression, it is imperative that all treatment options are thoroughly evaluated and exhausted in each individual, irrespective of complicating factors, to permit the best outcome for the patient. In this review, we present data from clinical trials, post hoc analyses, post-marketing surveillance, and real-world studies that are relevant to the management of nintedanib treatment. In addition, we also provide practical recommendations developed by a multidisciplinary panel of experts for the management of nintedanib treatment in patients with IPF associated complications and those experiencing gastrointestinal side effects.

Anabolic Androgenic Steroid Abuse: The Effects on Thrombosis Risk, Coagulation, and Fibrinolysis.

Anabolic androgenic steroid (AAS) abuse surged during the 1980s and is seen in approximately 1 in 20 of all males today. A wide spectrum of AAS compounds and abuse regimens are applied and AAS abuse has been associated with an unfavorable cardiovascular profile. The aim of this review is to critique the collected data concerning effects of AAS abuse on thrombosis risk through presentation of condensed evidence from studies investigating AAS-induced changes in coagulation, fibrinolysis, and cardiovascular risk markers. AAS abuse inflicts a procoagulant distribution of cardiovascular risk markers including dyslipidemia and atherosclerosis proneness. AAS abuse overall stimulates synthesis of coagulation factors, inhibitors, and fibrinolytic proteins resulting in both increased global coagulation and stimulation of fibrinolysis. Overall, supported by many case reports and some epidemiological studies, AAS abuse is associated with an increased risk of thrombosis. However, to provide clear evidence for a causal relationship between AAS abuse and thrombosis risk, future studies need to address a range of potential biases, insufficient methodology, and other shortcomings of the current literature as highlighted in this review.

Procoagulant State in Current and Former Anabolic Androgenic Steroid Abusers.

BACKGROUND: Anabolic androgenic steroid (AAS) abusers are considered at increased risk of cardiovascular morbidity and mortality. We hypothesized that current and former AAS abuse would induce a procoagulant shift in the haemostatic balance. METHODS: Men 18 to 50 years of age were included as current AAS abusers, former AAS abusers or controls. Morning blood samples were collected after overnight fasting. Thrombin generation (lag time, time to peak, peak height, and endogenous thrombin potential [ETP]) and coagulation factor II (prothrombin), VII and X, antithrombin, protein C, free protein S and tissue factor pathway inhibitor (TFPI) were assessed. Groups were compared by ANOVA or Kruskal-Wallis test and probabilities were corrected for multiple comparisons. Associations were evaluated using linear regression models. RESULTS: ETP was increased around 15% in current (n = 37) and former (n = 33) AAS abusers compared with controls (n = 30; p < 0.001). Prothrombin and factor X were increased ≥10% in AAS abusers and prothrombin was a predictor of ETP (p < 0.0005). Lag time and time to peak were increased 10 to 30% in current AAS abusers (p < 0.001) and associated with higher concentrations of TFPI, antithrombin, protein C and protein S (p < 0.0005; = 0.005). Multivariate linear regression, with all coagulation inhibitors as covariates, identified TFPI to be independently associated with lag time and time to peak (p < 0.0005). CONCLUSION: Thrombin generation is augmented in current and former AAS abusers, reflecting a procoagulant state, with altered concentrations of coagulation proteins. Prospective studies are needed to clarify whether these findings translate into an increased thrombotic risk in AAS abusers potentially even after cessation.

Comprehensive characteristics of the anticoagulant activity of dabigatran in relation to its plasma concentration.

BACKGROUND: Issues with laboratory measurement of dabigatran include: 1. Do coagulation assays reflect dabigatran plasma concentrations? 2. Do samples from patients treated with dabigatran have the same coagulability as dabigatran-spiked samples from healthy volunteers? 3. What is the long-term stability of dabigatran after storage at -80 °C? This study aims to evaluate these questions. MATERIALS AND METHODS: Ecarin chromogenic assay (ECA), a laboratory-developed diluted thrombin time (LD-dTT), prothrombin time (PT) and activated partial thromboplastin time (APTT) and ROTEM® were used to measure dabigatran anticoagulant activity and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to measure dabigatran plasma concentrations. ROTEM® (EXTEM, INTEM, FIBTEM) was performed in whole blood and the other assays in platelet poor plasma (PPP), both in samples spiked with dabigatran (0, 25, 50, 100, 250, 500 and 1000 ng/mL) from healthy donors and in ex vivo samples from patients treated with dabigatran etexilate. Citrated PPP samples were frozen and stored at -80 °C, 1, 3, 6 and 12 months until analysis. RESULTS: EXTEM and FIBTEM clotting time (CT), ECA and LD-dTT correlate well with dabigatran plasma concentrations. With the exception of few ROTEM® parameters, there were no differences between spiked and patient samples. Samples were stable for at least 12 months at -80 °C. CONCLUSIONS: EXTEM and FIBTEM CT, ECA and LD-dTT are suitable for measuring the effect of dabigatran in treated patients. In general, results from spiked plasma samples are similar to those of patient samples. Storage of dabigatran plasma samples for up to 12 months does not influence measured levels.

Analytical and between-subject variation of thrombin generation measured by calibrated automated thrombography on plasma samples.

BACKGROUND: The Calibrated Automated Thrombography (CAT) is an in vitro thrombin generation (TG) assay that holds promise as a valuable tool within clinical diagnostics. However, the technique has a considerable analytical variation, and we therefore, investigated the analytical and between-subject variation of CAT systematically. Moreover, we assess the application of an internal standard for normalization to diminish variation. METHODS: 20 healthy volunteers donated one blood sample which was subsequently centrifuged, aliquoted and stored at -80 °C prior to analysis. The analytical variation was determined on eight runs, where plasma from the same seven volunteers was processed in triplicates, and for the between-subject variation, TG analysis was performed on plasma from all 20 volunteers. The trigger reagents used for the TG assays included both PPP reagent containing 5 pM tissue factor (TF) and PPPlow with 1 pM TF. Plasma, drawn from a single donor, was applied to all plates as an internal standard for each TG analysis, which subsequently was used for normalization. RESULTS: The total analytical variation for TG analysis performed with PPPlow reagent is 3-14% and 9-13% for PPP reagent. This variation can be minimally reduced by using an internal standard but mainly for ETP (endogenous thrombin potential). The between-subject variation is higher when using PPPlow than PPP and this variation is considerable higher than the analytical variation. CONCLUSION: TG has a rather high inherent analytical variation but considerable lower than the between-subject variation when using PPPlow as reagent.

Translation, Cultural Adaptation, and Psychometric Properties of the Danish Version of the Anti-Clot Treatment Scale.

Background The Anti-Clot Treatment Scale (ACTS) is a 17-item, 2-factor (Burdens and Benefits), patient-reported outcome instrument to evaluate patient satisfaction with oral anticoagulant treatment. Objectives This study aimed to translate and culturally adapt the English version of the ACTS into Danish and to subsequently validate the Danish version in a population of patients treated with dabigatran etexilate for atrial fibrillation. Methods The ACTS was translated into Danish and culturally adapted. This prospective phase 4 study included 232 respondents who completed the Danish ACTS after 1 month of treatment with dabigatran etexilate for atrial fibrillation. Psychometric properties were evaluated. For test-retest reliability, the ACTS was measured twice, 2 weeks apart, in a subgroup of 50 stable patients. Results Generally, a high level of treatment satisfaction was found. Confirmatory factor analysis showed a suboptimal fit for the two-factor model of the original version. Using modification indices of confirmatory factor analysis, a four-factor model had the best fit. Cronbach's α for internal consistency was acceptable at 0.78. There was good test-retest reliability with intraclass correlation at 0.80. Smallest detectable changes (SDCs) for individual patients were 5.89 points for the total ACTS, 5.57 for the reverse Burdens, and 3.34 for Benefits scores. Group SDCs were 0.39, 0.37, and 0.22 respectively. Substantial ceiling effects limit the ability to detect improvement at the high end of the scale. Conclusion The Danish version of the ACTS has inadequate structural validity. Reliability was acceptable. Ceiling effects challenge detection of improvement of treatment satisfaction in clinical practice in this patient population.

Measurement of dabigatran: previously demonstrated Hemoclot® Thrombin Inhibitor assay reagent instability on Sysmex CS-2100i is no longer an issue.

The Hemoclot® Thrombin Inhibitor (HTI) assay has been recommended for measurement of dabigatran concentrations in specific clinical situations. Traditionally, reagents for biochemical assays are prepared from instructions found in the package insert. For the HTI reagents the manufacturer recommends incubating the reagents much longer than indicated in the package insert. These recommendations are added to the application sheets designed for different analyzers. Many clinicians and laboratory personnel may be unaware of the discrepancy between the two instructions, resulting in incorrect handling of the reagents. The aim of this study was to investigate the effect of the two different preparation methods on reagent stability and test results. For the standard concentration range, reagent stability on Sysmex CS-2100i was only two hours instead of the eight hours indicated by the producer when following package insert instructions (incubation time: 15 min). Stability was increased to five hours when following the application sheet (incubation time: 60 min). Two years later, the study was repeated using samples of patients treated with dabigatran etexilate. This time, reagent stability was at least six hours. Since the reagent composition was unchanged, the increased stability could be due to changed logistics by the supplier, with stock and transfer closer by. Previously demonstrated HTI reagent instability is no longer an issue at our laboratory. The reliability of results of clinical studies in which the assay has been used is potentially compromised.

Cholestatic liver injury as a side-effect of dabigatran and the use of coagulation tests in dabigatran intoxication and after reversal by idarucizumab in bleeding and sepsis.

Idarucizumab, an antidote specific for dabigatran, became available recently. Dabigatran is not associated with increased risk of hepatotoxicity in comparison with warfarin, but it is seen as a rare side-effect. Cases of cholestatic liver injury due to dabigatran have not been reported previously. We present a case of severe gastro-intestinal bleeding with underlying dabigatran intoxication in a patient with renal failure and the effect of reversal of dabigatran using idaruzicumab on coagulation assays. International normalized ratio (INR) and activated partial thromboplastin time (APTT) results were elevated in a setting of sepsis, possibly due to liver failure. INR and APTT can be elevated if sepsis is complicated by disseminated intravascular coagulation (DIC) or liver failure, making it challenging to determine dabigatrans contribution to their prolongation. A rebound effect after administration of idarucizumab and slow elimination of dabigatran due to reduced kidney function could be detected using the Hemoclot® diluted thrombin time (dTT) in this situation, in contrast to with non-dilutional assays. Before admission, cholestatic liver injury started shortly after initiation of dabigatran etexilate therapy. As no other cause was found, this liver injury was likely to be drug-induced. Bleeding cessated promptly after administration of idarucizumab in dabigatran intoxication. In conclusion, the anticoagulant effect of dabigatran can be measured by Hemoclot® dTT in sepsis and cholestatic liver injury was seen as a possible rare side-effect of dabigatran treatment.

Fast form alpha-2-macroglobulin - A marker for protease activation in plasma exposed to artificial surfaces.

OBJECTIVES: Investigation of the blood compatibility requires a number of sensitive assays to quantify the activation of the blood protein cascades and cells induced by biomaterials. A global assay measuring the blood compatibility of biomaterials could be a valuable tool in such regard. In this study, we investigated whether an enzyme-linked immunosorbent assay (ELISA), that specifically measures the electrophoretic "fast form" of α2-macroglobulin (F-α2M), could be a sensitive and global marker for activation of calcium dependent and in-dependent proteases in plasma exposed to biomaterials in vitro. METHODS: A F-α2M specific monoclonal antibody was generated and applied in an ELISA setup. Using the F-α2M ELISA, we investigated activation of calcium dependent and in-dependent proteases by polyvinylchloride (n=10), polytetrafluoroethylene (n=10) and silicone (n=10) tubings as well as glass tubes (n=10). RESULTS: We found that F-α2M is a sensitive marker for activation of both calcium dependent and in-dependent proteases. A significant difference between F-α2M concentrations in the control sample and plasma exposed to the artificial surfaces was found (p>0.001). This was observed both in the presence and absence of calcium. Furthermore, the highest F-α2M concentration was in both cases found in plasma incubated with glass. CONCLUSIONS: Our findings demonstrate that F-α2M is a sensitive marker for detection of protease activation in plasma by artificial surfaces. Potentially, levels of F-α2M could be a global marker of the blood compatibility of biomaterials.

Evaluation of a reduced centrifugation time and higher centrifugal force on various general chemistry and immunochemistry analytes in plasma and serum.

Background Centrifugation of blood samples is an essential preanalytical step in the clinical biochemistry laboratory. Centrifugation settings are often altered to optimize sample flow and turnaround time. Few studies have addressed the effect of altering centrifugation settings on analytical quality, and almost all studies have been done using collection tubes with gel separator. Methods In this study, we compared a centrifugation time of 5 min at 3000 × g to a standard protocol of 10 min at 2200 × g. Nine selected general chemistry and immunochemistry analytes and interference indices were studied in lithium heparin plasma tubes and serum tubes without gel separator. Results were evaluated using mean bias, difference plots and coefficient of variation, compared with maximum allowable bias and coefficient of variation used in laboratory routine quality control. Results For all analytes except lactate dehydrogenase, the results were within the predefined acceptance criteria, indicating that the analytical quality was not compromised. Lactate dehydrogenase showed higher values after centrifugation for 5 min at 3000 × g, mean bias was 6.3 ± 2.2% and the coefficient of variation was 5%. Conclusions We found that a centrifugation protocol of 5 min at 3000 × g can be used for the general chemistry and immunochemistry analytes studied, with the possible exception of lactate dehydrogenase, which requires further assessment.

Tissue factor pathway inhibitor relates to fibrin degradation in patients with acute deep venous thrombosis.

Reduced concentration of tissue factor pathway inhibitor is a risk factor for development of deep venous thrombosis, whereas elevated concentrations of tissue factor pathway inhibitor are observed in patients with acute myocardial infarction and disseminated intravascular coagulation. Presently, we studied the association between inflammation, endothelial cell perturbation, fibrin degradation and the concentration of tissue factor pathway inhibitor in patients suspected for acute deep venous thrombosis. We determined the tissue factor pathway inhibitor -33T/C polymorphism, free and total tissue factor pathway inhibitor, C-reactive protein, von Willebrand factor and D-Dimer in 160 consecutive patients admitted to hospital with a tentative diagnosis of acute deep venous thrombosis. Deep venous thrombosis was identified in 57 patients (18 distal and 39 proximal). The distribution of the tissue factor pathway inhibitor genotypes between patients with and without deep venous thrombosis showed a trend toward significant deviation (P = 0.08). The concentrations of free and total tissue factor pathway inhibitor, C-reactive protein, von Willebrand factor and D-Dimer were significantly higher in patients with deep venous thrombosis than in patients without deep venous thrombosis (P < 0.001 for all quantities). The significant relationship between free and total tissue factor pathway inhibitor and deep venous thrombosis persisted when adjusted for the tissue factor pathway inhibitor -33T/C polymorphism, C-reactive protein, von Willebrand Factor and potentially confounding clinical conditions (P < or = 0.004), but disappeared when adjusted for D-Dimer (P > or = 0.10). We conclude that patients suffering from acute deep venous thrombosis express significantly higher concentrations of tissue factor pathway inhibitor than patients without deep venous thrombosis. The significant relationship is not associated with the -33T/C polymorphism, inflammation or endothelial cell perturbation, but is most likely related to release of tissue factor pathway inhibitor from fibrin deposits.