Interpreting the International Normalized Ratio (INR) in individuals receiving argatroban and warfarin

The effects of argatroban, a direct thrombin inhibitor, on the International Normalized Ratio (INR), activated partial thromboplastin time (aPTT) and functional factor X during warfarin co-administration were established to provide means to interpret INRs during argatroban/warfarin co-therapy. Twenty-four subjects receiving warfarin (7.5 mg, day 1; 3-6 mg/day, days 2-10) and argatroban (1-4 microg/kg/min over 5 h, days 1-11) were assessed daily for these coagulation parameters prior to argatroban infusion (warfarin "monotherapy") and at its conclusion ("co-therapy"). Argatroban increased aPTTs dose-dependently. Co-therapy INR increased linearly with monotherapy INR, with slope sensitive to argatroban dose and thromboplastin used. Prediction errors for monotherapy INRs were < or =+/- 0.4 for argatroban 1-2 microg/kg/min but > or = +/-1.0 for higher doses. Despite co-therapy INRs >7, no major bleeding occurred. Factor X remained > or =37% of normal. Therefore, the predictable effect of argatroban (< or =2 microg/kg/min only) [corrected] on INRs during warfarin co-therapy allows for reliable prediction of the level of oral anticoagulation.     

Monitoring direct thrombin inhibitors with a plasma diluted thrombin time

Activated partial thromboplastin time (aPTT) monitoring of direct thrombin inhibitors (DTIs) is vulnerable to interference from many sources. If the baseline aPTT is prolonged, as occurs with lupus inhibitors, alternative methods are required to monitor DTI levels. We compared the plasma diluted thrombin time (1:4 dilution of patient plasma with normal plasma) and the aPTT in patient samples spiked with argatroban, bivalirudin, or lepirudin at three concentration levels. Each drug was spiked into five samples with lupus inhibitors, five samples with deficient vitamin K-dependent factors, three samples with elevated D-dimers, and eight samples with normal baseline aPTT values. The aPTT overestimated the spiked DTI concentration in all samples with lupus inhibitors, low levels of vitamin K-dependent factors, and elevated D-dimers. In samples with normal baseline aPTTs, the aPTT failed to correctly estimate the spiked drug concentration in four of 24 samples spiked with argatroban, seven of 24 spiked with lepirudin, and three of 24 spiked with bivalirudin. The plasma diluted thrombin time was not affected by lupus inhibitors, low vitamin K-dependent factor levels or elevated D-dimer levels and correctly estimated the spiked drug level in 63 of 63 samples spiked with argatroban, 63 of 63 samples spiked with bivalirudin, and 62 of 63 samples spiked with lepirudin. In conclusion, the plasma diluted thrombin time appears to be a viable alternative to the aPTT for monitoring DTI levels, especially in patients with lupus inhibitors or low levels of vitamin K-dependent factors.     

Effects of lepirudin, argatroban and melagatran and additional influence of phenprocoumon on ecarin clotting time

INTRODUCTION: Direct thrombin inhibitors (DTI) prolong the ecarin clotting time (ECT). Oral anticoagulants (OA) decrease prothrombin levels and thus interact with actions of DTIs on the ECT method during concomitant therapy. MATERIALS AND METHODS: Actions of lepirudin, argatroban and melagatran on ECT were investigated in normal plasma (NP) and in plasma of patients (n=23 each) on stable therapy with phenprocoumon (OACP). Individual line characteristics were tested statistically. RESULTS: Control ECT in OACP was prolonged compared to NP (50.1+/-0.9 vs. 45.7+/-0.8 s; p<0.001). Lepirudin prolonged the ECT linearly. Argatroban and melagatran delivered biphasic dose-response curves. OA showed additive effects on the ECT of lepirudin but not of argatroban and melagatran. Both in NP and OACP, the first and second slopes of melagatran were steeper compared to argatroban (primary analysis; p<0.001). When using the same drug, slopes in OACP were steeper than in NP (secondary analysis; p<0.001). At similar molar concentrations, the crossing points of both slopes were significantly higher with melagatran (323.1+/-11.0 s in NP and 333.2+/-8.2 s in OACP) than with argatroban (219.6+/-14.7 and 248.4+/-15.2 s) corresponding to ratios of 7.1+/-0.2 and 6.7+/-0.2 (melagatran) vs. 4.8+/-0.3 and 4.9+/-03 with argatroban (p<0.0001). DISCUSSION: The patterns of interactions between vitamin K antagonists and DTI effects are different for bivalent (increase of slope without affecting linearity) and monovalent inhibitors (slight increase or alteration of nonlinear slopes), but there are also differences between the two monovalent inhibitors on thrombin inhibition as determined by ECT.     

Anticoagulant activity and pharmacokinetic properties of a sub-cutaneously administered mixed micellar formulation of argatroban in experimental animals

We have studied the anticoagulant properties of a novel mixed micellar formulation containing 14 mg/ml argatroban administered by the sub-cutaneous (s.c.) route to rats, rabbits, dogs and primates. Blood samples were taken at various times post-treatment for the determination of the thrombin time (TT), Ecarin clotting time (ECT) and the activated partial thromboplastin time (aPTT). Plasma levels of argatroban were determined in the dog and primate. Mixed micelles alone (0.15 M sodium glycocholate and 0.15 M egg lecithin) were without effect on the clotting parameters. The mixed micellar formulation of argatroban dose-dependently increased all three clotting parameters in the rat (1-4 mg/kg), the rabbit (1 and 2 mg/kg), the dog (1 and 2 mg/kg) and the primate (0.25 and 0.5 mg/kg). In each case the TT was the most sensitive parameter, followed by the ECT and the aPTT. The duration of action of argatroban in each species was dose dependent and varied from 3 h in the rat to 6 h in the dog. In the latter, the mixed micelle formulation had a significantly increased plasma half-life and mean residence time without affecting the overall area under the curve. The increases in the clotting time were strongly correlated with the plasma levels of argatroban and were linear across the range of concentrations obtained in the dog and the primate, although the aPTT plasma concentration response curve was very flat. Species differences were noted between the increase in clotting time for a given plasma concentration, with the primate being more sensitive than the dog (e.g. 4.7 times more so in terms of the ECT). Thus, a mixed micellar formulation of argatroban, which markedly enhances its solubility, could be useful as a potential anticoagulant for sub-cutaneous administration.     

Differences in the clinically effective molar concentrations of four direct thrombin inhibitors explain their variable prothrombin time prolongation

Four direct thrombin inhibitors (DTIs), lepirudin, bivalirudin, argatroban, and melagatran, differ in their ability to prolong the prothrombin time (PT). Paradoxically, the DTI in clinical use with the lowest affinity for thrombin (argatroban) causes the greatest PT prolongation. We compared the effects of these DTIs on various clotting assays and on inhibition of human and bovine factor Xa (FXa). On a mole-for-mole basis, lepirudin was most able to prolong the PT, activated partial thromboplastin time (APTT), and thrombin clotting time (TCT), whereas argatroban had the least effect. At concentrations that doubled the APTT (argatroban, 1 micromol/l; melagatran, 0.5 micromol/l; bivalirudin, 0.25 micromol/l; lepirudin, 0.06 micromol/l), the rank order for PT prolongation was: argatroban > melagatran > bivalirudin > lepirudin. Although the Ki's associated with inhibition of human FXa by melagatran (1.4 micromol/l) and argatroban (3.2 micromol/l) approach their therapeutic concentrations, inhibition of FXa did not appear to be a major contributor to PT prolongation, since argatroban also prolonged the PT of bovine plasma (despite a Ki for bovine FXa of 2,600 micromol/l). Only melagatran inhibited prothrombinase-bound FXa. We conclude that the differing effects of the DTIs on PT prolongation are primarily driven by their respective molar plasma concentrations required for clinical effect. DTIs with a relatively low affinity for thrombin require high plasma concentrations to double the APTT; these higher plasma concentrations, in turn, quench more of the thrombin generated in the PT, thereby more greatly prolonging the PT.     

Effect of prothrombin time outlier exclusion rules on ISI calibration in multicentre studies

International Sensitivity Index (ISI) calibration is based on prothrombin time (PT) determinations in fresh plasma samples of healthy (normal) individuals and patients treated with vitamin K-antagonists (VKA). The ISI is calculated from the slope of the orthogonal regression line of the log(PT) results. The ISI calibration model is based on the assumption that the mean logarithms of the PT's of the normals are found on the orthogonal regression line derived using patients’ samples. According to World Health Organization (WHO) guidelines, patients’ samples with International Normalized Ratio (INR) beyond the 1.5 to 4.5 interval shall be excluded for ISI calibration. According to the WHO guidelines, outlier samples are defined as those at a perpendicular distance from the orthogonal regression line greater than 3 residual standard deviations, and shall be excluded as well. The purpose of the present study was to assess the effect of sample exclusion on ISI calibration, using the data base of three historic multicenter studies performed in 1990, 1995 and 2005, respectively. Various rules for sample exclusion were tried. In comparison to calibration without any exclusion, between-laboratory variation of the ISI was slightly reduced by sample exclusion using the WHO rule. Furthermore, the adequacy of the ISI calibration model was improved. The effect of the WHO sample exclusion rule on the mean value of the ISI of the current International Standards for thromboplastins was not greater than approximately 1%. It is concluded that the WHO rule for sample exclusion is appropriate for reliable ISI calibration.     

Additive effects of anticoagulants: recombinant human activated protein C and heparin or melagatran, in tissue factor-activated umbilical-cord plasma

Severe sepsis in children or adults may cause a life-threatening coagulopathy, with widespread consumption of activated protein C (APC); recombinant human APC (rhAPC) is a promising candidate anticoagulant treatment. We investigated the effects of rhAPC and other anticoagulants on coagulation triggered by adding small quantities of lipidated tissue factor to human umbilical-cord plasma in vitro. rhAPC, unfractionated heparin (UH), and melagatran (a direct thrombin inhibitor) were studied individually, and in combinations of rhAPC with either UH or melagatran. rhAPC alone dose-dependently prolonged the activated partial-thromboplastin time (aPTT) but not the prothrombin time (PT), and dose-dependently suppressed two indices of thrombin generation, namely prothrombin fragment F 1.2 (F 1.2) generation and thrombin-antithrombin (TAT) complex formation. UH alone dose-dependently prolonged the aPTT but not the PT, while melagatran alone dose-dependently prolonged both the aPTT and the PT. Adding either UH or melagatran dose-dependently augmented the capacity of rhAPC to suppress F 1.2 generation (with addition of UH showing a greater effect) and TAT formation (with addition of melagatran showing a greater effect). Both the capacity of UH to prolong the aPTT and the capacity of melagatran to prolong the aPTT and the PT were augmented by adding rhAPC. In our in-vitro study, adding either UH or melagatran augmented the capacity of rhAPC to suppress thrombin generation in human umbilical-cord plasma, with the anticoagulant effect of melagatran being more predictable than that of UH. Hence, combining rhAPC with melagatran might be a valuable therapeutic option in patients with severe sepsis.     

Effect of melagatran on prothrombin time assays depends on the sensitivity of the thromboplastin and the final dilution of the plasma sample

Prothrombin time (PT) assays are clotting methods that measure the activity of vitamin K-dependent coagulation factors (F) II, VII, and X. There are three main types of PT assays in general usage, namely the Quick assay, Owren's assay and PT dry chemistry test cards. PT assays were initially developed to monitor dose-adjustments of vitamin K antagonists such as warfarin. The aim of the present study was to investigate whether commercially available PT assays are suitable for evaluating the anticoagulant activity of direct thrombin inhibitors. Melagatran, a reversible direct thrombin inhibitor, was added to human plasma at concentrations ranging from 0.1 to 2.0 micromol/l. Seventeen different commercially available PT kits were used, including thirteen Quick reagents, two Owren reagents and two PT test cards. The sensitivity of the different reagents, expressed as the concentration of melagatran that doubled the prothrombin time (IC50) varied widely, with Thromboplastin S and Thromboplastin HS being the most sensitive (IC50 = 0.9 micromol/l). The reagents with apparently the lowest sensitivity were the two Owren reagents Nycotest PT and SPA 50 with an IC50 of 2.2 and 2.9 micromol/L, respectively. This is most likely due to a higher dilution of melagatran in these assays compared to the dilution in the Quick assays. The results were also dependent on the International Sensitivity Index (ISI) of each reagent. The concentration of melagatran that produced an International Normalized Ratio (INR) of 2 was calculated from dose-response curves for each assay, and these results revealed that reagents with a high ISI value gave an INR of 2 at much lower concentrations of melagatran (0.5-0.7 micromol/L) than those with an ISI-values around one (0.9-1.2 micromol/L). It was found that INR depends not only on the plasma concentration of melagatran, but also on the sensitivity of the PT reagent and on the final dilution of the plasma sample in the prothrombin time assay. Thus, since the same melagatran concentration can be associated with widely varying PT/INR results depending on the specific assay used it is concluded that PT assays and INR can not be used to monitor melagatran activity.     

Comparing the prothrombin time INR versus the APTT to evaluate the coagulopathy of acute trauma

INTRODUCTION: In trauma patients, PT/INR or aPTT cutoffs of > or =1.5x normal are often used as triggers for the transfusion of plasma. MATERIAL AND METHODS: To evaluate the ability of the PT/INR or aPTT to predict low coagulation factor levels, these tests were compared to coagulation factor levels in samples with artificially prepared single and multiple factor deficiencies, 9 heparin-contaminated samples, 10 lupus inhibitor-containing samples, 21 samples with elevated factor VIII levels, and 35 samples from acute trauma patients. RESULTS AND CONCLUSIONS: The PT/INR and aPTT showed comparable sensitivity for single or multiple factor deficiencies in artificially deficient plasmas, but the PT/INR was more sensitive than the aPTT to low coagulation factor levels in actual trauma patients (sensitivity 84% versus 50%). The aPTT can show false positives with lupus anticoagulants and heparin contamination and false negatives in samples with elevated factor VIII. Thus, in the acute trauma setting, the PT/INR cutoff is a more reliable indicator of reduced coagulation factor levels.     

Bivalirudin

Bivalirudin is a direct thrombin inhibitor (DTI) frequently used for anticoagulation in the setting of invasive cardiology, particularly percutaneous coronary intervention (PCI). Bivalirudin has a unique pharmacologic profile: unlike other marketed DTIs, it undergoes predominant non-organ elimination (proteolysis), and has the shortest half-life (approximately 25 min). Its affinity for thrombin is intermediate between that of lepirudin (highest) and argatroban (lowest)--this helps explain why it interferes with functional clotting assays to an extent intermediate between that achieved by these two other DTIs. This effect is best known for the PT (INR)--higher affinity for thrombin corresponds to lower molar DTI requirements to prolong the APTT; in turn, lower concentrations required for APTT prolongation (and, presumably, in-vivo effect) result in reduced PT (INR) prolongation. Bivalirudin is primarily used for its first FDA-approved indication, namely anticoagulation during percutaneous transluminal coronary angioplasty ("balloon angioplasty"), the most frequent type of PCI. Bivalirudin is also indicated for PCI with provisional use of glycoprotein IIb/IIIa antagonist therapy, and for patients with, or at risk of, heparin-induced thrombocytopenia (HIT), or HIT with thrombosis syndrome (HITTS), undergoing PCI. The bivalirudin development program has used a "quadruple" endpoint comprising a "triple" efficacy endpoint plus major bleeding - this approach anticipated the subsequent emphasis on strategies to improve clinical outcomes through bleeding reduction. Besides summarizing the key trials evaluating bivalirudin use for acute coronary syndrome (especially employing PCI), we review also the studies of bivalirudin as anticoagulant for "on-" and "off-pump" cardiac surgery, including both HIT and non-HIT situations.     

Critical role of factors II and X during coumarin anticoagulation and their combined measurement with a new Fiix-prothrombin time

Vitamin K antagonists (VKA) are monitored with prothrombin time (PT) based assays that are equally sensitive to reductions in factors II, VII or X. We compared the effect of vitamin K dependent (VKD) coagulation factors on PT and also on rotational thromboelastometric (ROTEM) parameters. The PT was equally sensitive to reductions in factors II, VII or X but ROTEM parameters correlated poorly with the PT in anticoagulated patients´ plasmas. ROTEM parameters were more affected by mild and moderate reductions in FII or FX than by FVII or FIX which had little influence except at very low coagulant activity. We developed a modified PT that was sensitive only to reductions in factors II and X. The Fiix-PT (Fiix-INR) correlated well with PT (INR) but the Fiix-INR fluctuated less than the INR in an anticoagulated patient reflecting its insensitivity to FVII. The ROTEM results suggest that mild to moderate reductions in factors II or X are more important during clot formation than factors VII or IX. Reductions in FII and X may better reflect anticoagulation with VKA than FVII or IX. The new Fiix-PT may more accurately reflect the degree of therapeutic anticoagulation in patients treated with VKA than the current PT which is subject to a confounding variation caused by FVII.     

Impact of dabigatran on a large panel of routine or specific coagulation assays. Laboratory recommendations for monitoring of dabigatran etexilate

Due to low bioavailability and high inter-individual variability, monitoring of dabigatran may be required in specific situations to prevent the risk of bleedings or thrombosis. The aim of the study was to determine which coagulation assay(s) could be used to assess the impact of dabigatran on secondary haemostasis. Dabigatran was spiked at concentrations ranging from 4.7 ng/ml to 943.0 ng/ml in pooled citrated human platelet-poor plasma. The following clotting assays were performed: prothrombin time (PT); activated partial thromboplastin time (aPTT); thrombin time (TT); ecarin clotting time (ECT); ecarin chromogenic assay (ECA); prothrombinase-induced clotting time (PiCT); activated clotting time (ACT); Hemoclot Thrombin Inhibitor (HTI) and thrombin generation assay (TGA). A concentration-dependent prolongation of PT, dPT, and aPTT was observed with aPTT being the more sensitive test. The results varied mostly due to the clotting reagent. HTI, ECT and TGA were the most sensitive tests but are not available 24 hours a day. In addition, HTI showed a linear correlation with a good reproducibility. Dabigatran induced a concentration-dependent delay and inhibition of tissue factor-induced TGA. Cut-offs related with higher risk of bleedings or thrombosis were defined for each reagent of aPTT and HTI. In conclusion, aPTT could be used for the monitoring of dabigatran and as screening test for the risk of overdose. However, because of its higher sensitivity, good reproducibility, excellent linear correlation at all doses, its simplicity of use, and possibilities of automation, HTI should be considered as the gold-standard.     

European Concerted Action on Anticoagulation--comparison of fresh plasma and whole blood multicentre ISI calibrations of CoaguChek Mini and TAS PT-NC whole blood prothrombin time point-of-care monitors

A procedure for using citrated fresh plasmas for International Sensitivity Index (ISI) calibration of two types of whole blood point-of-care test (POCT) prothrombin time (PT) monitor systems has been assessed in a multicentre study. The CoaguChek Mini and TAS PT-NC systems gave higher ISI with whole blood samples than with fresh plasma calibrations. However. there was good agreement between whole blood and fresh plasma monitor system International Normalised Ratio (INR) and the reference INR of target samples. Reliable INR can therefore be obtained with both whole blood and plasma samples on these two POCT systems based on their respective ISI. With the CoaguChek Mini system, the plasma calibration ISI can also be used to derive reliable INR with whole blood PT results. This was not possible with the TAS PT-NC system.     

A comparative study of conventional versus new,magnesium-poor Vacutainer® Sodium Citrate blood collection tubes for determination of prothrombin time and INR

Introduction

Conventional Vacutainer® Sodium Citrate blood collection tubes contain a relatively high concentration of contaminating magnesium ions, which may result in shortening of the prothrombin time (PT) and the International Normalized Ratio (INR). Recently the manufacturer of Vacutainer® Sodium Citrate tubes introduced new tubes with a magnesium-poor stopper. The magnesium concentration in the new low-Mg tubes is significantly lower than that in the conventional plastic tubes. The purpose of the present study was to compare PT and INR determined in specimens drawn with the new tubes to those drawn with the conventional tubes.

Materials and Methods

Venous blood specimens were collected from 22 healthy persons and 65 patients treated with vitamin K-antagonists using conventional Vacutainer® Sodium Citrate tubes and new, low-Mg Vacutainer® tubes. PT and INR were determined with four thromboplastin reagents, i.e., three brands of recombinant human tissue factor and one brand of combined rabbit brain reagent. Magnesium concentrations were determined in the citrate plasmas with a colorimetric method.

Results

The differences in PT, INR and International Sensitivity Index (ISI) between the two tubes were significant when using three recombinant human thromboplastin reagents, but were not significant when using the rabbit thromboplastin. The PT and INR differences between the tubes correlated with the magnesium concentration differences (P < 0.001). The INR bias between the four reagents was greater for specimens drawn with conventional tubes than the INR bias for specimens drawn with the new tubes.

Conclusion

Agreement of INR between reagents is improved by using magnesium-poor tubes.     

Multicentre ISI assignment and calibration of the INR measuring range of a new point-of-care system designed for home monitoring of oral anticoagulation therapy

The new CoaguChek XS system is designed for use in patient selftesting. It is the successor of the current CoaguChek S system. The detection principle is based on the amperometric measurement of the thrombin activity initiated by starting the coagulation cascade using a human recombinant thromboplastin. This study was performed to assign the International SEnsitivity Index (ISI) to the new test according to the WHO guidelines for thromboplastins and plasmas used to control anticoagulant therapy, and to establish the measuring range of the new system. At four study sites a total of 90 samples of normal donors and 291 samples of warfarin-, phenprocoumon- or acenocoumarol-treated patients were included in the study. The ISI value of the new test was assigned against the human recombinant reference thromboplastin rTF/95 at each site using the samples from stabilized patients in the International Normalized Ratio (INR) range between 1.5 and 4.5 only. The new point-of-care system's measuring range between 0.8 and 8 INR was calibrated against the mean INR of rTF/95 and AD149 using polynomial regression. ISIs were (CV of the slope): Site 1: ISI 0.99 (1.1%); Site 2: ISI 1.02 (2.0%); Site 3: ISI 1.03 (1.1%); Site 4: ISI 1.00 (1.4%). All regression lines calculated from patient-only data pass through the normal donor data points. All CVs of the slopes of the orthogonal regression lines are well below 3%, thus fulfilling the requirements of the WHO guidelines. The mean ISI for the new CoaguChek XS PT Test is 1.01.     

In-vitro profile and ex-vivo anticoagulant activity of the direct thrombin inhibitor dabigatran and its orally active prodrug, dabigatran etexilate

Dabigatran is a reversible and selective, direct thrombin inhibitor (DTI) undergoing advanced clinical development as its orally active prodrug, dabigatran etexilate. This study set out to determine the molecular potency and anticoagulant efficacy of dabigatran and its prodrug dabigatran etexilate. This was achieved through enzyme inhibition and selectivity analyses, surface plasmon resonance studies, platelet aggregation, thrombin generation and clotting assays in vitro and ex vivo. These studies demonstrated that dabigatran selectively and reversibly inhibited human thrombin (Ki: 4.5 nM) as well as thrombin-induced platelet aggregation (IC(50): 10 nM), while showing no inhibitory effect on other platelet-stimulating agents. Thrombin generation in platelet-poor plasma (PPP), measured as the endogenous thrombin potential (ETP) was inhibited concentration-dependently (IC(50): 0.56 microM). Dabigatran demonstrated concentration-dependent anticoagulant effects in various species in vitro, doubling the activated partial thromboplastin time (aPTT), prothrombin time (PT) and ecarin clotting time (ECT) in human PPP at concentrations of 0.23, 0.83 and 0.18 microM, respectively. In vivo, dabigatran prolonged the aPTT dose-dependently after intravenous administration in rats (0.3, 1 and 3 mg/kg) and rhesus monkeys (0.15, 0.3 and 0.6 mg/kg). Dose- and time-dependent anticoagulant effects were observed with dabigatran etexilate administered orally to conscious rats (10, 20 and 50 mg/kg) or rhesus monkeys (1, 2.5 or 5 mg/kg), with maximum effects observed between 30 and 120 min after administration, respectively. These data suggest that dabigatran is a potent, selective thrombin inhibitor and an orally active anticoagulant as the prodrug, dabigatran etexilate.     

Calibration of local systems with lyophilized calibrant plasmas improves the interlaboratory variability of the INR in the Italian external quality assessment scheme

Calibration with lyophilized calibrant plasmas certified in terms of PT with International Reference Preparations for thromboplastin has been proposed to minimize the effect of coagulometers on the INR. Aim of this study was to test the ability of local calibration with lyophilized calibrant plasmas, combined with a modified statistical approach, to improve the interlaboratory variability of the INR measured on two test plasmas (one coumarin and one artificially-depleted) by participants in the External Quality Assessment Scheme (EQAS). Sets of lyophilized calibrant and test plasmas were sent to the participants in the EQAS, who were asked to determine PT with their own reagent/ instrument combination (local system). Results were returned as PT together with information on the type of local system, the stated International Sensitivity Index (ISI) and the geometric mean of PTs determined by testing with the local system fresh plasmas from 20 healthy subjects. Ninety-two participants using 9 and 11 brands of reagents and instruments returned results. The CV of the INR determined with the stated ISI for the coumarin (Mean INR = 4.39) and artificially-depleted (Mean INR = 4.23) test plasmas were 11.2% and 10.3% and were reduced on the average by 34% and 54%, respectively, when the INR was calculated with the local ISI. In conclusions, results from this field study involving laboratories and testing systems representative of the real situation in oral anticoagulant monitoring in our country, indicate that local calibration by artificially-depleted plasmas, combined with the proposed statistical approach, is suitable to improve the interlaboratory agreement on the INR.     

Prothrombin time, activated partial thromboplastin time and dilute Russell's Viper Venom times are not shorter in patients with the prothrombin G20210A mutation, and dilute Russell's Viper Venom time may be longer

Introduction

Prothrombin G20210A (PT20210) carriers have increased prothrombin levels and increased risk for venous thrombosis. We hypothesized PT20210 carriers would have decreased PT, aPTT, and dRVVT clotting times.

Methods

We reviewed 1186 thrombotic risk panels that included PT, aPTT, dRVVT, and PT20210 genotype with potential confounding variables, excluding samples consistent with anticoagulant therapy or lupus anticoagulant presence. We examined relationships of PT20210 with PT, aPTT, and dRVVT correcting for covariates using multivariate regression. We confirmed associations in 1876 separate panel results and a group of homozygotes for PT20210 and used general linear models to determine if associated tests predict PT20210 status.

Results

Neither PT, aPTT, nor dRVVT was shorter in PT20210 carriers. Contrary to our hypothesis, PT20210 was significantly associated with higher dRVVT (p = 0.001), but not PT or aPTT. dRVVT differences were significant in a replicate sample p = 0.035 and an additional sample of PT20210 homozygotes (p = 0.02). Of all variables available, only dRVVT predicted PT20210 carrier status (p = 0.0008, AUC = 0.64).

Conclusions

We observed an association between longer dRVVT and the prothrombin G20210A mutation in a retrospective observational study. These findings merit further study in large well-characterized clinical cohorts and laboratory research experiments.     

European Concerted Action on Anticoagulation (ECAA). Minimum number of centres for reliable International Sensitivity Index calibration of CoaguChek and TAS point-of-care whole blood monitors

INTRODUCTION: Prothrombin time (PT) test systems require multicentre calibration for reliable International Sensitivity Index (ISI). Multicentre calibration of CoaguChek Mini and TAS PT-NC point-of-care test (POCT) systems is less precise than conventional PT testing. The aim of the present study was to determine the number of centres required to give reliable ISI and International Normalised Ratio (INR) with these two POCT whole blood PT monitors. MATERIALS AND METHODS: A simulation study, based on results of a 10-centre calibration exercise, was performed to assess reliability of ISI and INR when the number of centres was reduced from 10 to 2. RESULTS AND CONCLUSIONS: With both systems, the range of ISI and INR deviation increased as the number of centres was reduced. For the CoaguChek Mini, at least five centres were needed for satisfactory INR deviation in 95% of calibrations. With the TAS PT-NC, three centres gave satisfactory INR at this level. The number of centres required for multicentre calibration of these two POCT PT systems is greater than the two proposed by World Health Organisation (WHO) Guidelines for conventional PT testing.     

In vitro study of the anticoagulant effects of edoxaban and its effect on thrombin generation in comparison to fondaparinux

Introduction

Edoxaban, an oral direct factor Xa (FXa) inhibitor, is in Phase III development for prevention and treatment of thromboembolic disorders. Fondaparinux is an approved indirect FXa inhibitor. This study compared the effects of edoxaban and fondaparinux on thrombin generation (TG) using the calibrated automated thrombogram (CAT). Secondary objectives included evaluation of edoxaban and inhibition of coagulation parameters (prothrombin time [PT], activated partial thromboplastin time [aPTT]), anti-FXa activity and clotting times.

Materials and Methods

Pooled citrated platelet-poor plasma from healthy subjects was spiked with edoxaban (0.02-3.65 μM) or fondaparinux (0.15-1.18 μM). Parameters of TG were calculated using Thrombinoscope™ software. PT ratios and aPTT were measured in the presence of different thromboplastin reagents. Exogenous anti-FXa was measured using Rotachrom HBPM (Stago) and a specific assay developed for direct FXa inhibitors (Hyphen BioMed).

Results

Edoxaban exhibited a 3-fold greater concentration-dependent effect than fondaparinux across TG parameters (except endogenous thrombin potential). Edoxaban also produced a concentration-dependent prolongation of PT ratio and aPTT. The magnitude of concentration-dependent increase was related to thromboplastin reagent. In contrast to edoxaban, fondaparinux was inactive on these clotting tests. Linear correlations were observed between plasma concentration of edoxaban and anti-FXa activity and results of clotting time assays.

Conclusions

TG evaluation by the CAT method, coagulation tests, and anti-FXa and clotting assays demonstrated concentration-dependent effects of edoxaban. The PT and aPTT prolongation are reagent dependent; correction of PT ratio by international normalized ratio does not reduce variability in response. The greater effect of edoxaban vs. fondaparinux may be related to the broader activity of direct FXa inhibitors compared with indirect FXa inhibitors.