Karin Strandberg

Effects of the oral, direct thrombin inhibitor dabigatran on five common coagulation assays

Dabigatran is an oral, reversible thrombin inhibitor that has shown promising results in large clinical trials. Laboratory monitoring is not needed but the effects on common coagulation assays are incompletely known. Dabigatran was added to plasma from healthy subjects in the concentration range 0-1,000 μg/l and analysed using several reagents for activated thromboplastin time (APTT), prothrombin time (PT), fibrinogen, antithrombin, and activated protein C resistance. Typical trough concentrations are about 50 μg/l, peak concentrations 100-300 μg/l. At 100 μg/l all APTT-results were prolonged. The concentration required to double APTT ranged between 227 and 286 μg/l, the responses for all five reagents were similar. PT-reagents were much less affected with almost no samples above INR 1.2 at 100 μg/l. The effect was sample dilution dependent with PT Quick type more sensitive than PT Owren type methods. If a patient on dabigatran has prolonged APTT, >90 seconds, and Quick PT INR>2 or Owren PT INR>1.5 over-dosing or accumulation of dabigatran should be considered. Two of four fibrinogen reagents underestimated the fibrinogen concentration considerably at expected peak concentration. Methods based on inhibition of thrombin over-estimated the antithrombin concentration, but not Xa-based. The APC-resistance methods over-estimated the APC-ratio, which may lead to miss-classification of factor V Leiden patients as being normal. Different coagulation assays, and even different reagents within an assay group, display variable effects at therapeutic concentrations of dabigatran. Some of these assay variations are of clinical importance, thus knowledge is needed for a correct interpretation of results.

Effects of the oral, direct factor Xa inhibitor apixaban on routine coagulation assays and anti-FXa assays

Apixaban is an oral direct factor Xa inhibitor developed for the prophylaxis and treatment of thromboembolic disorders. Laboratory monitoring is not necessary, but the effects on common coagulation reagents and assays constitute clinically valuable information.

Explorative investigation of biomarkers of brain damage and coagulation system activation in clinical stroke differentiation

IntroductionA simple and accurate method of differentiating ischemic stroke and intracerebral hemorrhage (ICH) is potentially useful to facilitate acute therapeutic management. Blood measurements of biomarkers of brain damage and activation of the coagulation system may potentially serve as novel diagnostic tools for stroke subtypes.MethodsNinety-seven stroke patients were prospectively investigated in a multicenter design with blood levels of brain biomarkers S100B, neuron specific enolase (NSE), glial fibrillary acidic protein (GFAP) as well as a coagulation biomarker, activated protein C – protein C inhibitor complex (APC-PCI), within 24 hours of symptom onset.ResultsEighty-three patients (86 %) had ischemic stroke and fourteen patients (14 %) had ICH. There were no differences in S100B (p = 0.13) and NSE (p = 0.67) levels between patients with ischemic stroke or ICH. However, GFAP levels were significantly higher in ICH patients (p = 0.0057). APC-PCI levels were higher in larger ischemic strokes (p = 0.020). The combination of GFAP and APC-PCI levels, in patients with NIHSS score more than 3, had a sensitivity and negative predictive value of 100 % for ICH in our material (p = 0.0052).ConclusionThis exploratory study indicated that blood levels of biomarkers GFAP and APC-PCI, prior to neuroimaging, may rule out ICH in a mixed stroke population.

Clinical probability assessment and D-dimer determination in patients with suspected deep vein thrombosis, a prospective multicenter management study.

OBJECTIVES To investigate the reliability of a combined strategy of clinical assessment score followed by a local D-dimer test to exclude deep vein thrombosis. For comparison D-dimer was analysed post hoc and batchwise at a coagulation laboratory. DESIGN Prospective multicenter management study. SETTING Seven hospitals in southern Sweden. SUBJECTS 357 patients with a suspected first episode of deep vein thrombosis (DVT) were prospectively recruited and pre-test probability score (Wells score) was estimated by the emergency physician. If categorized as low pre-test probability, D-dimer was analysed and if negative, DVT was considered to be ruled out. The primary outcome was recurrent venous thromboembolism (VTE) during 3 months of follow up. RESULTS Prevalence of DVT was 23.5% (84/357). A low pre-test probability and a negative D-dimer result at inclusion was found in 31% (110/357) of the patients of whom one (0.9%, [95% CI 0.02-4.96]) had a VTE at follow up. Sensitivity, specificity, negative predictive value and negative likelihood ratio for our local D-dimer test in the low probability group were 85.7%, 74.5%, 98.2%, and 0,19 respectively compared to 85.6%, 67,6%, 97.9% and 0,23 using batchwise analysis at a coagulation laboratory. CONCLUSION Pre-test probability score and D-dimer safely rule out DVT in about 30% of outpatients with a suspected first episode of DVT. One out of 110 patients was diagnosed with DVT during follow up. No significant difference in diagnostic performance was seen between local D-dimer test and the post hoc batch analysis with the same reagent in the low probability group.

Comparison and evaluation of a Point-of-care device (CoaguChek XS) to Owren-type prothrombin time assay for monitoring of oral anticoagulant therapy with warfarin

BACKGROUND The standardized test used for evaluating the effect of warfarin is the prothrombin time (PT) which is measured and expressed in international normalized ratio (INR). Regular control of treatment intensity is required since inappropriate dosage increases the risk for complications. Portable point-of-care analytical instruments for measurement of capillary whole blood PT have been available for the last decades. The purpose of this study was to compare and evaluate INR values obtained by the point-of-care device CoaguChek XS, to Owren PT in a hospital setting. MATERIALS AND METHODS In 397 warfarin-treated patients, capillary whole blood was analyzed with the CoaguChek XS and the results were compared to analysis of venous plasma samples with the Owren PT assay. To study reproducibility and rule out preanalytical errors, a subgroup of 152 patients had two capillary blood samples analyzed with the CoaguChek XS. RESULTS In 397 patients, with a median age(+/-2SD) of 69.0(50-88) years, there was a positive correlation between results from the CoaguChek XS and the Owren-type PT assay (r=0.94;p<0.001) and concordance of 88.2%. The mean INR difference (S.D) was 0.02 (0.22). Comparison of the 152 double samples analyzed with the CoaguChek XS, produced a positive correlation of 0.99; p<0.001. CONCLUSIONS The CoaguChek XS presents reproducible, highly comparable results with Owren PT at therapeutic levels of INR. The CoaguChek XS seems to produce better results than the earlier CoaguChek S, probably due to a new method of PT measurement where levels of fibrinogen and haematocrit do not affect the outcome.

A new method to measure plasma levels of activated protein C in complex with protein C inhibitor in patients with acute coronary syndromes

Our newly devised immunofluorometric sandwich assay for measuring plasma concentrations of activated protein C (APC) in complex with protein C inhibitor (PCI) was compared with testing for conventional markers of myocardial damage CKMB (creatine kinase MB), TNI (troponin I) and hypercoagulability (D-dimer, TAT) in 76 patients with suspected myocardial infarction (MI). APC–PCI complex levels in samples drawn on admission did not correlate with CKMB in the simultaneously drawn sample but correlated closely with maximal CKMB, which reflects MI size (r = 0.52). The areas under the receiver operating characteristics (ROC) curves calculated for the APC–PCI complex results obtained upon admission did not differ significantly from the corresponding values for CKMB, TNI or TAT. Our results show that in patients at risk for MI, the APC–PCI concentration is a sensitive and independent marker that can identify a subgroup of MI patients with normal CKMB but an increased APC–PCI level upon admission. It remains to be determined whether these patients would benefit from early intensive anticoagulant treatment.

Monitoring Low Molecular Weight Heparins at Therapeutic Levels: Dose-Responses of, and Correlations and Differences between aPTT, Anti-Factor Xa and Thrombin Generation Assays

Background Low molecular weight heparins (LMWH’s) are used to prevent and treat thrombosis. Tests for monitoring LMWH’s include anti-factor Xa (anti-FXa), activated partial thromboplastin time (aPTT) and thrombin generation. Anti-FXa is the current gold standard despite LMWH’s varying affinities for FXa and thrombin. Aim To examine the effects of two different LMWH’s on the results of 4 different aPTT-tests, anti-FXa activity and thrombin generation and to assess the tests’ concordance. Method Enoxaparin and tinzaparin were added ex-vivo in concentrations of 0.0, 0.5, 1.0 and 1.5 anti-FXa international units (IU)/mL, to blood from 10 volunteers. aPTT was measured using two whole blood methods (Free oscillation rheometry (FOR) and Hemochron Jr (HCJ)) and an optical plasma method using two different reagents (ActinFSL and PTT-Automat). Anti-FXa activity was quantified using a chromogenic assay. Thrombin generation (Endogenous Thrombin Potential, ETP) was measured on a Ceveron Alpha instrument using the TGA RB and more tissue-factor rich TGA RC reagents. Results Methods’ mean aPTT at 1.0 IU/mL LMWH varied between 54s (SD 11) and 69s (SD 14) for enoxaparin and between 101s (SD 21) and 140s (SD 28) for tinzaparin. ActinFSL gave significantly shorter aPTT results. aPTT and anti-FXa generally correlated well. ETP as measured with the TGA RC reagent but not the TGA RB reagent showed an inverse exponential relationship to the concentration of LMWH. The HCJ-aPTT results had the weakest correlation to anti-FXa and thrombin generation (Rs0.62–0.87), whereas the other aPTT methods had similar correlation coefficients (Rs0.80–0.92). Conclusions aPTT displays a linear dose-respone to LMWH. There is variation between aPTT assays. Tinzaparin increases aPTT and decreases thrombin generation more than enoxaparin at any given level of anti-FXa activity, casting doubt on anti-FXa’s present gold standard status. Thrombin generation with tissue factor-rich activator is a promising method for monitoring LMWH’s.

Activated Protein C-Protein C Inhibitor Complex in Patients With Abdominal Aortic Aneurysms : Is It Associated With Diameter and Growth Rate?

Increased thrombin activation was documented in patients with abdominal aortic aneurysm (AAA). Activated protein C—protein C inhibitor (APC—PCI) complex, a new biological marker of thrombin generation, was measured in a population of 232 patients with AAA and a control group, and the association between aneurysm size, growth rate, and APC—PCI was studied. The patients were divided into cohorts according to AAA diameter and compared with a control group. APC—PCI was significantly higher in all AAA cohorts (n = 232; median, 0.36 µg/L; 10th to 90th percentile, 0.18-1.01) compared with the control group (n = 41; median, 0.19 µg/L; 10th to 90th percentile, 0.11-0.31; P ≤ .001). APC—PCI correlated with AAA diameter (r = .22; P = .001), body mass index (r = −.19; P = .004), and age (r = .19; P = .004). APC—PCI did not correlate with AAA growth rate (r = .11; P = .14).

Evaluation of a rapid automated assay for analysis of von Willebrand ristocetin cofactor activity.

A commercially available turbidometric assay has been evaluated for the measurement of von Willebrand factor ristocetin cofactor activity (VWF:RCo). The assay is simple, rapid, and can be cost-effectively performed on automated coagulation analyzers. This study’s aim is to illustrate the performance of the automated VWF:RCo assay and its capacity to identify patients with von Willebrand disease (VWD). By direct comparison with a conventional VWF:RCo assay, performed on an aggregometer, the concordance between the two assays was 96%. With minor modifications, the automated assay showed a detection level of 0.03 kIU/L with linearity to 2.00 kIU/L. The imprecision of the automated assay was reduced compared to the conventional assay procedure with CV of 6.8% at the 1.00 kIU/L level and 8.6% at the 0.30 kIU/L level. The automated VWF:RCo assay was also suitable as a screening test to detect VWD in patients investigated for the cause of an increased bleeding tendency. In this situation the automated VWF:RCo assay was tested simultaneously with an automated immunoassay for von Willebrand antigen. Receiver operating curves for the diagnosis of VWD showed a greater area under the curve for the automated VWF:RCo assay compared to the immunoassay, 0.98 vs. 0.94, although the difference did not reach significance. In conclusion, the modified automated VWF:RCo assay shows better precision, lower detection limit, is faster to perform with a lower cost per test compared to the conventional aggregometer based VWF:RCo activity method and is an alternative to an antigen immunoassay as a screening test for VWD.

APC-PCI complex concentration is higher in patients with previous venous thromboembolism with Factor V Leiden

Resistance to activated protein C (APC) is a major risk factor for venous thrombosis that is caused by a point-mutation in the gene of blood coagulation factor V [1,2]. This mutation (FV Leiden) renders the FVmolecule less susceptible to inactivation by APC, and is seen in 20–40% of patients with VTE [3]. It is thus the most common hereditary risk factor for VTE among Caucasians. An increased resistance to APC determined with a thrombin generation-based test in patients with the FV Leiden mutation has shown a good correlation between APC sensitivity ratios (APCsr) and the risk of venous thrombosis in carriers of FV Leiden [4,5]. D-dimer measurements have also been used for predicting recurrent VTE and measured after withdrawal of oral anticoagulant treatment D-dimer had some predictive value with regard to recurrence, but the overall discriminative power was quite poor (AUC 0.59 ± 0.03) [6–10]. The risks of DVT with the joint presence of high D-dimer and FV Leiden was increased 12.4-fold and were thus supra-additive in a prospective case–control study [11]. There is a need for more specific laboratory markers that can be used to predict an elevated risk for recurrent VTE, especially in patients without hereditary risk factors for thrombophilia. A new sensitive sandwich immunofluorimetric assay for measurements of the complex between APC and the protein C inhibitor (APC–PCI complex) has been devised [12,13]. This method employs a monoclonal antibody that is specific for the loop-inserted form of the inhibitor, i.e., PCI that is in complex withAPC.Un-complexed native PCI, which exists in a 10-fold molar excess over the APC–PCI complex has no affinity for the antibody. As a so-called reporter antibody, a monoclonal antibody against protein C is used. Because of the activation of protein C by thrombin bound to thrombomodulin, and the subsequent inactivation of APC by PCI, the concentration of the APC–PCI complex is a sensitive indicator of the degree of activation of blood coagulation. Increased levels of APC–PCI have been observed in hyper-coagulation states both affecting the venous and arterial systems such as deep vein thrombosis (DVT), pulmonary embolism, disseminated intravascular coagulation and myocardial infarction (MI) [14–16]. Measured with our method in patients with VTE and MI, ROC-analysis demonstrated an equal or better discriminative capacity than othermarkers of hypercoagulability such as F1+2, TAT, SFM andD-dimer [17–18]. The aim of the present study is to find out if there is a difference in APC–PCI concentration indicating different degrees of thrombin generation between carriers and non-carriers of the FV Leiden mutation in patients with a previous history of VTE. Venous blood was drawn after information and written consent. It was collected in 5 mL vacuum tubes (Stabilyte, Biopool, Umeå, Sweden) containing citrate with a low pH, which precludes in vitro formation of the APC–PCI complex [19]. The APC–PCI complex concentration was measured using the previously described DELFIA assay, which has a functional sensitivity in Stabilyte-plasma of 0.032 lg L. Using Stabilyte tubes, the concentration in healthy individuals was 0.07–0.26 lg L with a mean and median of 0.13 lg L [19]. The within-run coefficient of variation was 4.8% at 0.15 lg L and 3.2% at 0.40 lg L [19]. The between-run coefficient of variation was 7.1% at 0.15 lg L and 5.8% at 0.41 lg L (n 1⁄4 38). Warfarin treatment reduces the APC– PCI concentration [13]. APC–PCI measurements were made on one occasion on samples stored at )70 C. The samples were frozen and then thawed once more for measurement of D-dimer with Auto D-dimer (Biopool AB, Umeå, Sweden). Statistical analyses were performed with SPSS version 11.5 for Windows (SPSS Inc., Chicago, IL, USA). Non-normally distributed data were expressed as median and percentiles (10th and 90th). The Mann–Whitney U-test was used for bivariate analysis of continuous variables, the chi-squared test for nominal variables and Spearman’s rank correlation test for correlations. P-values <0.05 were considered significant. We compared the APC–PCI complex concentration, in 182 consecutive patients who were in the recovery phase after cessation of anticoagulant treatment for VTE, with D-dimer. At the time for blood sampling, all patients had normal protein Correspondence: K. Strandberg, Department of Clinical Chemistry, Lund University, Malmö University Hospital, S-205 02 Malmö, Sweden. Tel.: +46 40 331459; fax: +46 40 336255; e-mail: karin. strandberg@klkemi.mas.lu.se