[Show abstract][Hide abstract] ABSTRACT: Background:
Direct oral anticoagulant dabigatran was first introduced as a fixed-dose drug without routine coagulation monitoring, but current recommendations suggest that diluted thrombin time can be used to estimate plasma drug level. The aim of this study was to assess a diluted thrombin time assay based on the same thrombin reagent already used for traditional thrombin time measurements that reliably measure low to intermediate plasma dabigatran levels.
We included 44 patients with atrial fibrillation who started treatment with dabigatran 150 mg (23 patients) or 110 mg (21 patients) twice a day. Blood samples were collected at baseline (no dabigatran) and 2-4 weeks after the beginning of dabigatran therapy at trough and at peak. Plasma dabigatran levels were measured with diluted thrombin time and compared to liquid chromatography with tandem mass spectrometry as the reference method. The performance of the diluted thrombin time was compared to Hemoclot® Thrombin Inhibitor and Ecarin Chromogenic Assay.
In ex vivo plasma samples, diluted thrombin time highly correlated with the liquid chromatography with tandem mass spectrometry (Pearson's R = 0.9799). In the low to intermediate range (dabigatran concentration ≤ 100 µg/L) diluted thrombin time correlated significantly more closely to the liquid chromatography with tandem mass spectrometry (R = 0.964) than Hemoclot® Thrombin Inhibitor (R = 0.935, p = 0.05) or Ecarin Chromogenic Assay (R = 0.915, p < 0.01). It was also the only functional assay without any significant bias in the low to intermediate range. Both trough and peak diluted thrombin time values were similar to liquid chromatography with tandem mass spectrometry.
We conclude that the diluted thrombin time assay presented in this study reliably detects dabigatran and that it is superior to the Hemoclot® Thrombin Inhibitor assay in the low to intermediate range.
[Show abstract][Hide abstract] ABSTRACT: Background Laboratory diagnosis of lupus anticoagulant (LA) as a part of the diagnosis of antiphospholipid syndrome (APS) is based on prolongation of at least one coagulation assay (diluted Russel's viper venom (dRVVT) or activated partial thromboplastin time (APTT)) which normalizes after addition of phospholipids. Both assays may be influenced by anticoagulants and therefore LA should not be tested during warfarin or heparin treatment. New (direct) oral anticoagulants (N(D)OAC based on direct inhibition of thrombin (dabigatran (DAB)) or factor Xa (rivaroxaban (RIV) and apixaban (API)) are approved for the treatment of venous thromboembolism (VTE). Therefore N(D)OAC may be used in the treatment of patients with APS where LA diagnosis is critical for the decision about the VTE treatment. It has been shown (primarily in-vitro) that N(D)OAC may influence LA testing.
Objectives We have tested the effects of N(D)OAC on the assays routinely used for the diagnosis of LA in patients treated with these drugs in real life settings.
Methods Plasmas from patients with atrial fibrillation treated with DAB (n=30), RIV (n=20) and API (n=17) and known not to have LA were tested using dRVVT (LA screen and LA confirm Life Diagnostics) and APTT (PTT LA Diagnostica Stago and Actin FS Siemens Healthcare Diagnostics) assays. According to the diagnostics algorithm, the dRVVT and APTT ratio <1.2 was considered negative, >1.4 positive, while if the ratio was 1.2-1.4 LA could not be ruled out. Plasma concentrations of drugs varied between 8-172 μg/L for DAB, 8-437 μg/L for RIV and 36-178 μg/L for API.
Results Only 8 (27%) out of 30 plasmas from patients treated with DAB were negative, while 4 (13%) plasmas were positive for LA In all but 2 samples confirmation tests were also prolonged. DAB concentration correlated with both dRVVT (r=0.75 p<0.0001) and both APTT (r=0.55, p<0.005) assays, while ratio >1.2 was observed even with the lowest concentration. Negative LA was observed only in one sample from the patients treated with RIV (drug concentration 8 μg/L), while half of the samples diagnosed positive for LA. Confirmation tests were prolonged with RIV concentration above 100 μg/l. RIV concentration correlated with both dRVVT assays (r=0.86 p<0.0001) and both dRVVT and APTT ratio (r=0.56, p=0.01). LA diagnosed positive in 7 (41%) samples from patients treated with API. Eight samples (47%) were diagnosed negative for the presence of LA. Correlation between API concentration and dRVVT confirm test was observed (r=0.83, p<0.001) and it seems that a concentration >100 μg/L was associated with the prolongation of this confirmatory assay.
Conclusions Our results suggest that the risk of overestimation of LA detection is present in samples from patients treated with N(D)OAC, particularly RIV. Therefore LA testing should not be performed in patients during the treatment with N(D)OAC. Prolongation of confirmation assays may be helpful for the recognition of false positivity, especially in the case of DAB.
Disclosure of Interest None declared
No preview · Article · Jun 2015 · Annals of the Rheumatic Diseases
[Show abstract][Hide abstract] ABSTRACT: Routine laboratory monitoring is currently not recommended in patients receiving dabigatran despite its considerable variation in plasma concentration. However, in certain clinical situations, measurements of the dabigatran effect may be desirable. We aimed to assess the variability of dabigatran trough and peak concentration and explore the potential relationship between dabigatran concentration and adverse events. We included 44 patients with atrial fibrillation who started treatment with dabigatran 150 mg (D150) or 110 mg (D110) twice daily. They contributed 170 trough and peak blood samples that were collected 2-4 and 6-8 weeks after dabigatran initiation. Plasma dabigatran concentration was measured by LC-MS/MS and indirectly, by selected coagulation tests. D110 patients were older (74±7 versus 68±6 years), had lower creatinine clearance (68±21 versus 92±24 mL/min) and higher CHA2 DS2 -VASc score (3.1±1.3 versus 2.3±0.9) compared to D150 patients (all p<0.05), but both had similar dabigatran concentrations in both trough and peak samples. Dabigatran concentrations varied less in trough than in peak samples (17.0±13.6 versus 26.6±19.2 %, p=0.02). During the 12-month follow-up, 4 patients on D150 and 6 on D110 suffered minor bleeding. There was no major bleeding or thromboembolic event. Patients with bleeding had significantly higher average trough dabigatran concentrations (93±36 versus 72±62 μg/L, p=0.02) than patients without bleeding, while peak dabigatran values had no predictive value. Dabigatran dose selection according to the guidelines resulted in appropriate trough concentrations with acceptable repeatability. High trough concentrations may predispose patients to the risk of minor bleeding. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
No preview · Article · May 2015 · Basic & Clinical Pharmacology & Toxicology
[Show abstract][Hide abstract] ABSTRACT: IntroductionFrequent PT (INR) testing may represent a problem for patients on warfarin treatment, and capillary or small-volume tubes may be more appropriate for such patients. A demand for small-volume tubes also comes from pediatric wards. Yet, while various small-volume tubes are available, they have not been properly evaluated.Methods
Three small-volume tubes were tested (MiniCollect 3.8% citrate, MiniCollect 3.2% citrate and Microvette EDTA) and compared with a standard 4.5-mL 3.2% citrated tube. Samples were taken by venipuncture from the back of the hand and by capillary sampling from the tip of the finger. The measures were compared with those after standard venipuncture of the arm fold. A total of 180 samples, using different combinations of tubes and sampling sites, were collected from 30 volunteers.ResultsThere were no differences in the results obtained using citrate tubes for venous samples in comparison with those obtained by standard sampling, while the results when using EDTA tubes were not comparable to those obtained by standard sampling (P < 0.001), expressing systematically lower values (by about 10%). The results observed after capillary sampling were significantly different to those obtained after standard sampling.Conclusions
The MiniCollect 3.2% tube may be used for PT (INR) venipuncture samples when withdrawal of a small amount of blood is preferable, while EDTA tubes should not be used for PT (INR) testing.
No preview · Article · May 2015 · International journal of laboratory hematology
[Show abstract][Hide abstract] ABSTRACT: Introduction
The oral direct thrombin inhibitor dabigatran is increasingly used to prevent thromboembolic stroke in patients with atrial fibrillation (AF). Routine laboratory monitoring is currently not recommended, but measurements of dabigatran and/or its effect are desirable in certain situations. We studied dabigatran exposure and compared different tests for monitoring of dabigatran in a real-life cohort of AF patients.
Material and methods
Ninety AF patients (68 ± 9 years, 67% men, mean CHADS2 score 1.5) were treated with dabigatran 150 (n = 73) or 110 mg BID (n = 17). Trough plasma concentrations of total and free dabigatran by liquid chromatography-tandem mass-spectrometry (LC-MS/MS) were compared to indirect measurements by Hemoclot thrombin inhibitors (HTI) and Ecarin clotting assay (ECA), as well as PT-INR and aPTT.
Total plasma dabigatran varied 20-fold (12–237 ng/mL with 150 mg BID) and correlated well with free dabigatran (r2 = 0.93). There were strong correlations between LC-MS/MS and HTI or ECA (p < 0.001) but these assays were less accurate with dabigatran below 50 ng/mL. The aPTT assay was not dependable and PT-INR not useful at all. There were weak correlations between creatinine clearance (Cockcroft-Gault) and LC-MS/MS, HTI and ECA (p < 0.001 for all). A high body weight with normal kidney function was associated with low dabigatran levels.
HTI and ECA reflect the intensity of dabigatran anticoagulation, but LC-MS/MS is required to quantify low levels or infer absence of dabigatran. Most real life patients with a normal creatinine clearance had low dabigatran levels suggesting a low risk of bleeding but possibly limited protection against stroke.
No preview · Article · Oct 2014 · Thrombosis Research
[Show abstract][Hide abstract] ABSTRACT: Patients with haemophilia A have seriously impaired thrombin generation due to an inherited deficiency of factor (F)VIII, making them form unstable fibrin clots that are unable to maintain haemostasis. Data on fibrin structure in haemophilia patients remain limited. Fibrin permeability, assessed by a flow measurement technique, was investigated in plasma from 20 patients with severe haemophilia A treated on demand, before and 30 minutes after FVIII injection. The results were correlated with concentrations of fibrinogen, FVIII and thrombin-activatable fibrinolysis inhibitor (TAFI), and global haemostatic markers: endogenous thrombin potential (ETP) and overall haemostatic potential (OHP). Fibrin structure was visualized using scanning electron microscopy (SEM). The permeability coefficient Ks decreased significantly after FVIII treatment. Ks correlated significantly with FVIII levels and dosage, and with ETP, OHP and levels of TAFI. SEM images revealed irregular, porous fibrin clots composed of thick and short fibers before FVIII treatment. The clots had recovered after FVIII replacement almost to levels in control samples, revealing compact fibrin with smaller intrinsic pores. To the best of our knowledge, this is the first description of fibrin porosity and structure before and after FVIII treatment of selected haemophilia patients. It seems that thrombin generation is the main determinant of fibrin structure in haemophilic plasma.
Full-text · Article · Nov 2013 · Thrombosis and Haemostasis
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
No preview · Article · Sep 2013 · Scandinavian journal of clinical and laboratory investigation
[Show abstract][Hide abstract] ABSTRACT: Purpose: Atrial fibrillation (AF) is a major cause of thromboembolic stroke. The oral direct thrombin inhibitor dabigatran is increasingly used in patients with AF. Routine laboratory monitoring is currently not recommended for dabigatran, but there are situations (e.g. surgery, bleeding or thromboembolism during treatment, compliance) when measurements of the drug and/or its effect are desirable. We compared measurements of dabigatran plasma concentrations with four functional coagulation tests in a well-defined real-life cohort of patients with AF.
Methods: We studied 73 patients with AF, median age 68 (range 49-86) years, 66% men, mean CHADS2 score 1.6. 58 were treated with dabigatran 150 mg BID, 15 with 110 mg BID. Pre-dose samples were obtained in the morning. Plasma concentrations were measured using liquid chromatography with mass-spectrometry (LC-MS/MS).
Methods: for indirect determination of dabigatran were: Hemoclot thrombin inhibitors (HTI; Hyphen) and Ecarin clotting assay (ECA; Stago), as well as PT-INR (Owren reagent SPA+, Stago) and aPTT (Silica reagent, Automate, Stago). Creatinine clearance (CrCl) was calculated by the Cockcroft-Gault formula.
Results: The median dabigatran concentration by LC-MS/MS was 51 ng/mL with marked variation (range 9-163). There were moderate to strong correlations between LC-MS/MS and effects estimated by HTI (r2 = 0.76) and ECT (r2 = 0.81, p<0.001 for both). The lowest calibrator for HTI was 30 ng/mL. APTT correlated more weakly with dabigatran concentrations (r2=0.40; p<0.001); aPTT could be normal even at therapeutic dabigatran concentrations and could be prolonged despite low concentrations. PT-INR did not correlate with LC-MS/MS. Median CrCl was 86 mL/min (range 39-155) and there was no obvious correlation between CrCl and plasma concentrations, HTI or ECT.
Conclusions: We found a moderate to strong correlation between dabigatran concentrations in plasma and the HTI and ECA assays. These methods may thus be used to roughly estimate the intensity of dabigatran anticoagulation in a clinical setting. However, low values should be cautiously interpreted, especially for HTI. LC-MS/MS is the gold standard and the only method that can be used to monitor low levels or infer absence of dabigatran. The aPTT assay was insufficiently sensitive to dabigatran and PT-INR is not useful at all. Most real life patients with a normal CrCl had plasma concentrations of dabigatran in the low range (compared to FDA-data from the RE-LY study). Thus, the bleeding risk may not be imminent while protection against stroke could be limited in prothrombotic real life AF patients.
Full-text · Article · Aug 2013 · European Heart Journal
[Show abstract][Hide abstract] ABSTRACT: The soluble fibrin monomer (sFM) assay, like the D-dimer (DDi) assay, has the potential to be used both as an aid in the diagnosis of disseminated intravascular coagulation (DIC) and as a thrombotic marker. It differs from DDi in that it is a much earlier produced fragment produced only by thrombin action on fibrinogen, whereas DDi is a much later produced fragment formed by plasmin cleavage of cross-linked fibrin.
In our study, we compared two commercially available automated sFM assays in the routine hospital setting using samples obtained from the general hospital ward and the emergency room. The results obtained with the two automated assays (Stago LIA sFM assays and the LPIA-Iatro SF assay) were compared with each other and with the results obtained using the routine semiquantitative hemagglutination assay.
The study showed that both automated assays were comparable with each other. No patient sample previously classified as positive would be missed, but with the higher sensitivity in the automated tests, more samples are positive.
In conclusion, we suggest that both automated tests are suitable for routine laboratory use. Both assays had the advantage over the hemagglutination assay in that previously frozen samples could be used, and the assays are easier and quicker to perform. The LIA sFM Stago has slightly better sensitivity but has a tendency to lower specificity than the Iatro SF test.
No preview · Article · Jun 2013 · International journal of laboratory hematology
[Show abstract][Hide abstract] ABSTRACT: 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.
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).
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.
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.
No preview · Article · Jun 2013 · European Journal of Clinical Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Multiple electrode aggregometry (MEA) is used to measure platelet function. Pneumatic tube transport systems (PTS) for delivery of patient samples to a central laboratory are often used to reduce turnaround time for vital analyses. We evaluated the effects of PTS transport on platelet function as measured by MEA. Duplicate samples were collected from 58 individuals. One sample was sent using PTS and the other was carried by personnel to the lab. Platelet function was measured by means of a Multiplate® analyzer using the ADP test, ASPI test, COL test, RISTO test and TRAP test. Samples transported using PTS showed a reduction of AUC-values of up to a 100% of the average as compared to samples carried by personnel and a majority showed reductions of AUC-values greater than 20% of the average. Bias±95% limits of agreement for the ADP test were 26±56% of the average. Bias±95% limits of agreement for the ASPI test were 16±58% of the average. Bias±95% limits of agreement for the COL test were 20±54% of the average. Bias±95% limits of agreement for the RISTO were 14±79% of the average. Bias±95% limits of agreement for the TRAP test were 19±45% of the average. We conclude that PTS transport affect platelet activity as measured by MEA. We advise against clinical decisions regarding platelet function on the basis of samples sent by PTS in our hospital settings.
No preview · Article · May 2013 · Thrombosis Research
[Show abstract][Hide abstract] ABSTRACT: Laboratory investigations are an inevitable part of diagnosis of hemostatic disturbances. Screening analyses are usually performed in general clinical chemistry laboratories and are available 24 hours. They include platelet counts, PT(INR), aPTT, levels of fibrinogen, D-dimer, and antithrombin. More detailed investigation of coagulation abnormalities is reserved for special coagulation laboratories and includes determination of single coagulation factors (FII, FV, FVII, FVIII, FIX, FX, FXI, FXII, FXIII); anticoagulants (antibodies against coagulation factors (particularly FVIII and FIX), and lupus anticoagulant); coagulation inhibitors (PS, PS); fibrinolysis factors (plasminogen, t-PA) and inhibitors (plasmin inhibitor, PAI-1) and functional platelet analyses. Additional analyses of markers of coagulation activation and global hemostatic assays may also be performed. Genetic investigations are primarily aimed at detection of mutation in genes for FVIII, FIX and VWF, and polymorphisms associated with thrombophilia (prothrombin mutation and FV Leiden mutation). To help clinicians choose appropriate tests in the diagnosis of hemostatic abnormalities several investigation “packages” may be offered (bleeding tendency, venous thrombosis, arterial thrombosis, hereditary thrombophilia).
[Show abstract][Hide abstract] ABSTRACT: Disseminated intravascular coagulation (DIC), thrombotic thrombocytopenic purpura, and hemolytic uremic syndrome are serious medical conditions associated with hemostasis activation that require rapid diagnosis and treatment, which are discussed in this chapter. Heparin-induced thrombocytopenia (HIT) is a rare but serious condition where diagnosis is based on a clinical score and laboratory findings while alternatives to heparin (i.e. argatroban, danaparoid) should be used for the treatment.
[Show abstract][Hide abstract] ABSTRACT: Obesity is a known risk factor for venous and arterial thrombosis but the mechanisms are still unclear. In women, obesity is correlated with low-grade inflammation and recent data show that BMI is positively associated with thrombin generation. We explored the correlations between obesity, inflammation and thrombin generation in women with increased thrombotic risk by looking at a cohort of women with prior venous thrombosis. One hundred and fifty-six women age 18-65 years were enrolled at diagnosis of first venous thromboembolism (VTE). Plasma samples were obtained at least 3 weeks after cessation of anticoagulant treatment. Thrombin generation was determined with the calibrated automated thrombography (CAT) assay and the Innovance ETP assay. Thrombin generation started later but was more pronounced with higher endogenous thrombin generation potential (ETP) determined with CAT in patients with obesity. The Innovance ETP assay showed results consistent with CAT. Furthermore, patients with obesity had significantly higher levels of fibrinogen, C-reactive protein and plasminogen activator inhibitor-I (PAI-I) than patients without obesity. Increased levels of fibrinogen were the main determinant of the prolonged lag-time in patients with obesity whereas higher levels of prothrombin could account for the difference in the ETP between the groups. We found an association between BMI and ETP values using two different methods to measure thrombin generation. Obesity correlated with increased thrombin generation in women with VTE and the main determinants of this hypercoagulable state were increased levels of fibrinogen and prothrombin. This shows a possible link between obesity, low-grade inflammation and increased thrombin generation in women at increased risk for future thrombosis.
No preview · Article · Mar 2013 · Blood coagulation & fibrinolysis: an international journal in haemostasis and thrombosis
[Show abstract][Hide abstract] ABSTRACT: Background:
Microparticles (MPs) are small membrane vesicles (0.1-1 μm) released from various cells after activation and/or apoptosis. There are limited data about their role in hemophilia A.
Patients and methods:
Blood samples were taken before and 30 min after FVIII injection in 18 patients with severe hemophilia A treated on demand. Flow-cytometric determination of total MPs (TMPs) using lactadherin, platelet MPs (PMPs) (CD42a), endothelial MPs (EMPs) (CD144) and leukocyte MPs (LMPs) (CD45) was performed. The results were compared with data on endogenous thrombin potential (ETP), overall hemostatic potential (OHP), fibrin gel permeability and thrombin-activatable fibrinolysis inhibitor (TAFI).
Results and conclusions:
TMPs and PMPs decreased after treatment (to 1015 ± 221 [SEM] and 602 ± 134 × 10(6) L(-1) ) in comparison with values before treatment (2373 ± 618 and 1316 ± 331; P < 0.01). EMPs also decreased after treatment (78 ± 12 vs. 107 ± 13; P < 0.05) while LMPs were not influenced. Both TMP and PMP counts were inversely correlated, moderately but statistically significantly, with data on OHP, ETP, fibrin network permeability and TAFI/TAFIi (P < 0.05 for all). EMP counts were correlated only with ETP (P < 0.05), while LMP counts did not show any correlation. TMP and PMP counts were also inversely correlated with FVIII levels (P < 0.05). TMP, PMP and EMP counts decreased after on-demand treatment with FVIII concentrate in hemophilia A patients. The decrease in circulating MPs, which were inversely correlated with hemostatic activation, may imply that MPs are incorporated in the hemostatic plug formed after FVIII substitution at the site of injury.
No preview · Article · Dec 2012 · Journal of Thrombosis and Haemostasis