Testing for dabigatran and rivaroxaban by clinical laboratories

Rivaroxaban and dabigatran are among the newest anticoagulants, and measuring their concentration in patients is a new challenge for clinical laboratories. We analyzed data from the ECAT proficiency program to determine how well the assays are performing in clinical laboratories internationally. Most laboratories received a passing grade (Z score <3) for the results of their dabigatran and rivaroxaban testing. Failing Z scores were not associated with any particular method. With dabigatran, some homemade calibrators gave higher results than the commercially available calibrators. There were no significant differences among the instruments or the 5 reagents in use, but results showed inter‐laboratory variability that could have clinical significance. The 3 reagents with the lowest number of users had poor inter‐laboratory precision. Ten different anti‐Xa reagents were in use for rivaroxaban testing. One reagent gave lower results than other reagents at 100 ng/mL but not at 300 ng/mL. There were no significant differences among the different rivaroxaban calibrators or instruments. In conclusion, inter‐laboratory precision could be improved for both dabigatran and rivaroxaban assays. Homemade dabigatran calibrators differed from commercially available calibrators, and there was a statistically significant difference between some of the rivaroxaban reagents. About 10% of results received failing Z scores or passed but fell in a range that require the laboratory to investigate for bias or other inaccuracy in their method. Am. J. Hematol. 91:E464–E467, 2016. © 2016 Wiley Periodicals, Inc.     

Measurement of dabigatran and rivaroxaban in primary prevention of venous thromboembolism in 106 patients, who have undergone major orthopedic surgery: an observational study

No routine coagulation laboratory test is recommended during rivaroxaban or dabigatran treatment. However measuring drug concentration and/or anticoagulant activity can be desirable in some special clinical settings, such as bleeding, thrombosis recurrence or emergency surgery. The effects of dabigatran etexilate and rivaroxaban on various coagulation assays have been previously studied in normal plasma spiked with increasing concentrations of the drug. In contrast, few data are available in routinely treated patients. In order to perform and to interpret the results of these tests, it is necessary to determine the usual responses of patient’s plasma. We have used several coagulation tests in a prospective study including 106 patients receiving thromboprophylactic treatment with dabigatran 150 or 220 mg od and rivaroxaban 10 mg od for major orthopaedic surgery. The most common tests—prothrombin time (PT) and activated partial thromboplastin time (aPTT)—give results, which vary according to the reagent used. To overcome this limitation, we advocate the use of plasma calibrators, which decreases the inter-laboratory heterogeneity of results. Anti-Xa measurement and Hemoclot, a thrombin diluted clotting assay, are specific assays which have been proposed for rivaroxaban and dabigatran respectively. These tests, conventional PT, aPTT and thrombin generation (TG) have been performed. We demonstrated that measurements of both drugs can determine reliably the drug concentration in patients’ plasmas. PT is more prolonged with rivaroxaban than with dabigatran. Interestingly, the pattern of TG was clearly different in relation to the difference in the mechanism of action of the two new anticoagulants. A significant inter-individual variability of response is detected. Rivaroxaban—mean Cmax 140 ng/mL (extremes 0–412) induces a greater increase of PT than dabigatran. aPTT is sensitive to dabigatran. Rivaroxaban concentrations were in good agreement with two other studies while unexplained lower than expected concentrations were found in dabigatran patients receiving 220 mg once a day [mean Cmax 60 ng/mL (extremes 0–320)]. An interference by pantoprazole, a drug which reduces dabigatran absorption, could explain the observed lower than expected results.     

Harmonization of fibrinogen assay results: study within the framework of the Dutch project 'Calibration 2000'

In a Dutch project for harmonization of fibrinogen assays, the commutability of potential calibrators for fibrinogen was assessed by means of a twin-study design, which is, in essence, a multicentre, split-patient sample, between-field-methods protocol. The study consisted of simultaneous analysis of fresh-frozen patient plasmas and three potential calibrators for fibrinogen by 48 Dutch laboratories forming 24 couples. The state-of-the-art intralaboratory standard deviation was used to assess the commutability of the potential calibrators. The potential calibrators were commutable for the Clauss, but not for the prothrombin time (PT)-derived assays. One potential calibrator was used in an attempt to harmonize fibrinogen assay results in a Dutch field study. The interlaboratory coefficient of variation (CV) of three out of four test samples could be reduced significantly using the common calibrator. The average overall CV for the four test samples was 10.3% using the routine measurements and 7.8% using the common calibrator. Despite the reduction in the overall CV, the bias between Clauss and PT-derived assay results in two coumarin test samples could not be eliminated.     

Preparation of a purified fibrinogen calibration material for Clauss method and turbidimetric immunoassay possessing biological activity and antigenicity

Fibrinogen plays a major role in basic coagulation tests such as prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT). These show high interlaboratory variation because of inaccurate instrumental calibration. The same is true of the fibrinogen assay, despite its being a quantitative assay. Most medical laboratories use automated coagulometers and commercially available calibration materials (calibrators) to obtain an accurate fibrinogen value, but, when checked, calibrators have been found to deviate from the assigned value. The Japan Society of Laboratory Medicine (JSLM) has identified the need for a reliable plasma fibrinogen standard. To enhance the reliability of calibrator fibrinogen values and thereby remedy the poor precision and accuracy of plasma fibrinogen testing, we undertook the preparation of a standard calibration material applicable to both the Clauss method and turbidimetric immunoassay (TIA). The calibrator was prepared from fresh human plasma by glycine precipitation and virus inactivation followed by affinity chromatography to remove contaminated plasminogen. In the resulting product, clottable fibrinogen accounted for 95% of total protein and within-run precision showed a CV of less than 1.8%. We believe the preparation will become a candidate material for laboratory and manufacturer use in Japan.     

Effect on Plasma Protein S Activity in Patients Receiving the Factor Xa Inhibitors

Aims: Measurement of protein S (PS) activity in patients taking direct oral anticoagulants (DOACs) using reagents based on a clotting assay results in falsely high PS activity, thus masking inherited PS deficiency, which is most frequently seen in the Japanese population. In this study, we investigated the effect of factor Xa (FXa) inhibitors on PS activity using the reagent on the basis of the chromogenic assay, which was recently developed in Japan. Methods: The study enrolled 152 patients (82 males and 70 females; the average age: 68.5±14.0 years) receiving three FXa inhibitors (rivaroxaban, edoxaban, and apixaban). PS activity was measured using the reagents on the basis of the clotting and chromogenic assays. Results: PS activity measured by the clotting assay reagents exhibited falsely high values depending on the plasma concentrations of FXa inhibitors in patients taking either rivaroxaban or edoxaban. However, none of the three FXa inhibitors affected PS activity when measured using the chromogenic assay. Conclusion: In patients taking rivaroxaban or edoxaban, inherited PS deficiency is likely missed because the levels of PS activity measured using the reagents based on the clotting assay are falsely high. However, we report that three FXa inhibitors do not affect PS activity measured by the chromogenic assay. When measuring the levels of PS activity in patients undergoing DOACs, the principles of each reagent should be understood. Furthermore, plasma samples must be collected at the time when plasma concentrations of DOACs are lowest or the DOAC-Stop reagent should be used.     

Determination of an international sensitivity index of thromboplastin reagents using a WHO thromboplastin as calibrator for plasma spiked with rivaroxaban

Rivaroxaban and other direct factor Xa inhibitors are used at fixed doses without drug monitoring and dose adjustment. Patients may require determination of the anticoagulant effect during treatment. The aim of this study was to develop a method to reduce the differences between thromboplastin reagents and coagulation analysers for determination of the anticoagulant effect of rivaroxaban in human plasma. Purity of rivaroxaban extracted from commercially available drug was confirmed by mass spectrometry, elemental analysis and 1H-NMR spectroscopy. Coagulation times of pooled human plasma spiked with 50-900 ng/ml rivaroxaban were analysed. Thromboplastin reagents, WHO RBT/90, Innovin, RecombiPlasTin 2G, STA Neoplastin Plus, Technoclot PT Plus and Thromborel S, the manual Kolle-Hook method and the KC10 analyser were used. An international sensitivity index (ISI) was determined for each reagent and coagulation method using the RBT/90 thromboplastin reagent as reference. The orthogonal, used for the determination of the ISI of coumarin plasmas, and ordinary regression analyses were compared. The results showed than increasing concentrations of rivaroxaban prolonged coagulation values of all thromboplastin assays linearly (r?(2)=?0.96 and r(2)?=?0.99, respectively). The coefficient of variation between the slopes of the dilution curves and the ratios of the thromboplastin reagents were reduced using the international normalized ratio (INR) and ISI calculated for rivaroxaban. The ISIs of the thromboplastin reagents ranged from 0.73 to 1.67 as compared with the WHO reagent using the manual technique. The coefficient of variations between the thromboplastin reagents comparing the orthogonal and the ordinary regression analysis were 6.8 versus 3.7% (Kolle-Hook method, P?=?0.0011) and 8.5 versus 4.8% (KC10 method, P?相似文献   

Evaluation of automatic and semiautomatic coagulation assay instruments

The precision of five instruments (Coag-A-Mate, Auto-Fi, Coagulyzer, Electra 600 and Fibrometer) in performing activated partial thromboplastin time (PTT) assays were compared using three different coagulation reagent systems (Ortho, Dade, and General Diagnostics) utilizing normal and abnormal control reagents. There were wide variations in mean PTT values and co-efficient of variation (CV) among all instruments and for each instrument utilizing different reagents. The smallest CV was noted for the Coagulyzer using Dade reagents, the Coag-A-Mate using Dade and General Diagnostic reagents and the Auto-Fi using General Diagnostic reagents. Auto-Fi values were always longer than those determined with the Fibrometer whereas the optical detection instruments usually gave shorter values. The range of normal values for the prothrombin time (PT) and PTT assay was recorded simultaneously on blood samples of 52 healthy donors utilizing the Coagulyzer with Ortho reagents, the Coag-A-Mate with General Diagnostic reagents and the Auto-Fi with Dade reagents. No differences were noted between male and female donors. There was a poor correlation among PT results recorded by all instruments. For the PTT assay, results obtained with the Coag-A-Mate and the Auto-Fi had a correlation of 0.84. The normal mean plus 2 or 3 standard deviations determined by the Auto-Fi and the Coag-A-Mate were used to classify blood samples from 263 patients as normal or abnormal when assayed by each instrument. For the PTT assay, coincidence was attained in 91.2% of the samples and discrepancies (a blood classified abnormal by one instrument and normal by the other) were evenly distributed for both instruments. For the PT assay, coincidence occurred in 85.1% of the cases and there was a statistically significant trend (P<o.05) for the Auto-Fi in classifying abnormal samples categorized normal by the Coag-A-Mate.     

Use of EDTA samples for prothrombin time measurement in patients receiving oral anticoagulants

BACKGROUND AND OBJECTIVES: Oral anticoagulant therapy is commonly called for in health care. Hitherto sampling for prothrombin time (PT) has been measured on blood collected into a coagulation tube and diluted in citrate solution. Blood samples anticoagulated with EDTA are used for hematologic tests and the same sample could also be available for PT. DESIGN AND METHODS: We studied 107 patients on oral anticoagulant therapy. Samples were taken by from both coagulation tubes (citrate) and EDTA tubes. The PT from both samples was measured with an ACL 7000 analyzer and reported in seconds and as an international normalized ratio (INR). The regression equation between citrate and EDTA samples was calculated in both units. We studied the clinical significance of INR results from both sample types and compared the effect of different combined thromboplastin reagents on the correlation equation between citrate and EDTA samples. RESULTS: The regression equation for PT by Owren's PT reagent from citrate (y) and EDTA (x) plasma was y = 1.11 x -0.24 INR, R(2)= 0.99. We observed no clinically significant difference between INR results from citrate and EDTA samples using the regression equation for INR calculation from EDTA samples. ISI depends on sample type (dilution, anticoagulant) and the difference is 0.117, 10%. We calculated ISI for EDTA samples and no clinically significant difference was seen between citrate and EDTA INR results. INTERPRETATION AND CONCLUSIONS: A good correlation was observed between INR results with citrate and EDTA samples from patients receiving oral anticoagulants using Owren's PT reagent with the same citrate calibration. Using the regression equation (INR or sec) for analysis of INR results from EDTA samples is clinically acceptable and offers the possibility of using EDTA samples for PT measurement with citrate calibrators. Reagent international sensitivity index (ISI) values for citrate and EDTA samples differ from each other. ISI determination for EDTA samples requires mathematical calculation of EDTA ISI as in the present study or EDTA-based ISI calibrators. The regression equation for INR from citrate and EDTA samples depended on the reagent used, not only on sample dilution or anticoagulant.     

Laboratory measurement of the non-vitamin K antagonist oral anticoagulants: selecting the optimal assay based on drug,assay availability,and clinical indication

Although the non-vitamin K antagonist oral anticoagulants (NOACs) do not require routine monitoring, there are special circumstances in which laboratory measurement may be warranted. The objectives of this review are to summarize evidence on the influence of the NOACs on coagulation tests and provide practical guidance to clinicians on measurement and interpretation of coagulation assays in NOAC-treated patients. Selection of an appropriate assay for NOAC measurement depends on the drug, clinical objective, and assay availability. Separate suggestions for assay selection are provided depending on whether specialized assays are available or whether choice is limited to conventional coagulation assays such as the prothrombin time (PT) and activated partial thromboplastin time (APTT). The dilute thrombin time (TT) and ecarin-based assays are able to quantify dabigatran across a broad range of concentrations, but are not widely available. A normal TT excludes clinically relevant levels. A normal APTT probably excludes excess levels of dabigatran, but does not rule out typical on-therapy drug concentrations. The PT is insufficiently sensitive to dabigatran to be useful in most situations. Factor Xa inhibitors may be quantified with an anti-Xa assay calibrated with drug-specific standards. A normal PT probably excludes excess levels of rivaroxaban and edoxaban, but not typical on-therapy levels of these agents. The PT is less sensitive to apixaban. Depending on the sensitivity of the thromboplastin reagent, a normal PT may not exclude excess levels of apixaban. The APTT has inadequate sensitivity to factor Xa inhibitors and is not recommended for their measurement.     

ISI/INR system in Japan: experience from simultaneous measurement of the same plasma at four different laboratories

In 1984, the Scientific and Standardization Committee (formerly ICTH) recommended the use of the International Sensitivity Index and International Normalized Ratio (ISI/INR) System for the monitoring of oral anticoagulant therapy. This system was introduced because the sensitivity of thromboplastin reagents used for the measurement of prothrombin time (PT) was widely different and comparison among hospitals employing different reagents was virtually impossible. In this study, we simultaneously measured the plasma from 7 patients with warfarin therapy at 4 different institutions for PT seconds, PT-INR, thrombotest (TT) seconds and TT-INR. The comparison between these laboratories revealed clinically important variances between the 4 laboratories even when PT was converted to PT-INR. Laboratory 1 and laboratory 3 were using the same thromboplastin reagents for the measurement of PT. The PT (seconds) in both laboratories showed similar numbers, but when they converted into INR, the variances were significant (maximum coefficient of variance 10.44). We investigated the reason why these differences occurred and found that the PT seconds (11.40) for normal control at laboratory 3 were somewhat larger than those of other laboratories. If we assume that PT-INR is identical to TT-INR, the estimated PT (second) for normal control at laboratory 3 can be calculated from TT-INR, and was found to be 10.56 +/- 0.10 seconds. This was nearly the same as the one that was used at laboratory 1. In conclusion, there still exist some difficulties that must be overcome before the ISI/INR system can be used reliably, and we suggest attention be given to the PT seconds used as normal control plasma.     

Measurement of factor VIII activity using one‐stage clotting assay: a calibration curve has not to be systematically included in each run

Summary. Coagulation factor VIII (FVIII) is usually evaluated using activated partial thromboplastin time‐based one‐stage clotting assays. Guidelines for clotting factor assays indicate that a calibration curve should be included each time the assay is performed. Therefore, FVIII measurement is expensive, reagent‐ and time‐consuming. The aim of this study was to compare FVIII activities obtained using the same fully automated assay that was calibrated once (stored calibration curve) or each time the assay was performed. Unique lots of reagents were used throughout the study. We analysed 255 frozen plasma samples from patients who were prescribed FVIII measurement including treated and untreated haemophilia A patients. Twenty‐six runs were performed on a 28‐week period, each including four lyophilized control and at most 10 patient plasma samples. In control samples, FVIII activities were not significantly different when the assay was performed using the stored calibration curve or was daily calibrated. The same applied to FVIII activities in patient plasma samples that were not significantly different throughout the measuring range of activities [68.3% (<1–179) vs. 67.6% (<1–177), P = 0.48] and no relevant bias could be demonstrated when data were compared according to Bland and Altman. These results suggest that in the studied technical conditions, performing the FVIII assay using a stored calibration curve is reliable, for at least 6 months. Therefore, as far as the same lots of reagents are used, it is not mandatory to include a calibration curve each time the FVIII assay was performed. However, this strategy has to be validated if the assay is performed in different technical conditions.     

Factor VIII activity of BAY 94‐9027 is accurately measured with most commonly used assays: Results from an international laboratory study

Introduction

Discrepancies in the measurement of modified factor VIII (FVIII) products have been recognized, highlighting the need for adjustments in clinical laboratory practices to ensure effective monitoring of patients treated with these products, particularly using the one‐stage (activated partial thromboplastin time [aPTT]) assay.

Aim

To assess the ability of clinical laboratories to measure the activity of BAY 94‐9027, a PEGylated extended half‐life FVIII product, using routine (predominantly one‐stage) assays in clinical laboratories

Methods

Blinded samples of FVIII‐deficient plasma spiked with defined levels of BAY 94‐9027 and a recombinant FVIII product comparator were provided to 52 clinical laboratories that routinely conduct FVIII testing. Samples were provided at 3 concentrations (low, medium and high), and laboratories analysed the samples using routine in‐house one‐stage and, when available, chromogenic assays. Acceptable spiked recovery (accuracy) of the local laboratory methods to measure BAY 94‐9027 was the primary endpoint of the study.

Results

Accurate FVIII measurements were obtained at all concentrations for both products using the chromogenic assay and most of the commonly used one‐stage reagents, both ellagic acid and silica based. Two specific silica‐based reagents, APTT‐SP and PTT‐A, underestimated BAY 94‐9027 levels at all concentrations, consistent with previous findings.

Conclusions

FVIII activity of BAY 94‐9027 was accurately measured with most commonly used one‐stage assays used in routine clinical practice. The chromogenic assay was also accurate. It is recommended that clinical laboratories identify and avoid specific inappropriate reagents, such as the APTT‐SP and PTT‐A, in their one‐stage assays for FVIII monitoring.     

Laboratory Measurement of the Anticoagulant Activity of the Non–Vitamin K Oral Anticoagulants

Background

Non–vitamin K oral anticoagulants (NOACs) do not require routine laboratory monitoring. However, laboratory measurement may be desirable in special situations and populations.

Objectives

This study’s objective was to systematically review and summarize current evidence regarding laboratory measurement of the anticoagulant activity of dabigatran, rivaroxaban, and apixaban.

Methods

We searched PubMed and Web of Science for studies that reported a relationship between drug levels of dabigatran, rivaroxaban, and apixaban and coagulation assay results. Study quality was evaluated using QUADAS-2 (Quality Assessment of Diagnostic Accuracy Studies 2).

Results

We identified 17 eligible studies for dabigatran, 15 for rivaroxaban, and 4 for apixaban. For dabigatran, a normal thrombin time excludes clinically relevant drug concentrations. The activated partial thromboplastin time (APTT) and prothrombin time (PT) are less sensitive and may be normal at trough drug levels. The dilute thrombin time (R² = 0.92 to 0.99) and ecarin-based assays (R² = 0.92 to 1.00) show excellent linearity across on-therapy drug concentrations and may be used for drug quantification. For rivaroxaban and apixaban, anti-Xa activity is linear (R² = 0.89 to 1.00) over a wide range of drug levels and may be used for drug quantification. Undetectable anti-Xa activity likely excludes clinically relevant drug concentrations. The PT is less sensitive (especially for apixaban); a normal PT may not exclude clinically relevant levels. The APTT demonstrates insufficient sensitivity and linearity for quantification.

Conclusions

Dabigatran, rivaroxaban, and apixaban exhibit variable effects on coagulation assays. Understanding these effects facilitates interpretation of test results in NOAC-treated patients. More information on the relationship between drug levels and clinical outcomes is needed.     

Determination of rivaroxaban in human plasma samples

Rivaroxaban is one of the novel oral direct factor Xa inhibitors, which is effective in preventing thromboembolic complications at fixed doses (i.e., once daily), without the need for dose adjustment according to laboratory monitoring. Nearly 60% of rivaroxaban is cleared from circulation by glomerular filtration, 30% of which is excreted as active drug. Therefore, as renal elimination plays a pivotal role in the metabolism of this drug, impairment of renal function may be important during anticoagulation with rivaroxaban over long periods of time. The assessment of the anticoagulant effect/concentration of rivaroxaban may thus be useful in special patient populations such as in the elderly and eldest, during acute diseases with concurrent dehydration, before surgery, during bleeding or thrombotic episodes, or to verify adherence to therapy. Rivaroxaban prolongs prothrombin time in a dose-dependent, linear fashion. Activated partial thromboplastin time (APTT) is also prolonged, but in an exponential manner. Substantial differences in test results might be generated by different thromboplastin and APTT reagents. One-step prothrombin-induced clotting time assay is sensitive to low concentrations of rivaroxaban. Chromogenic substrate assays specific for factor Xa are also sensitive to rivaroxaban. Several initiatives are currently ongoing to standardize the various methods to determine rivaroxaban in human plasma samples, some of which will be summarized in this article along with the dose-dependent effects of rivaroxaban on relevant coagulation parameters. Therefore, although rivaroxaban prolongs all coagulation assays used to assess the anticoagulant effects of most anticoagulants, the most specific assay cannot be identified at present. Moreover, clinical trials are needed to determine the relationship of assay results with bleeding or thrombotic complications.     

Comparison of modes of prothrombin time reporting in patients with advanced liver disease associated with viral hepatitis

Background Prothrombin time is widely utilized for evaluation of liver disease severity. However, its standardization of modes of reporting is not established for universal purpose. Variability in thromboplastin reagents leads to large intralaboratory and interlaboratory differences in PT results. Objective The aim of this study was to establish standardization of modes of PT reporting by the interchangeability analysis of prothrombin time in patients with advanced liver disease associated with viral hepatitis measured with different thromboplastin reagents by the use of various methods of expression, i.e. prothrombin time (PTs), prothrombin activity percentage (PTp), prothrombin time ratio (PTr) and International Normalized Ratio (INR). Methods we prospectively collected blood samples from 61 patients with advanced liver disease associated with viral hepatitis, control patients were on warfarin (n = 20). PT was measured on a STA-R with six thromboplastin reagents. PT was expressed in PTs, PTr, PTp, and INR. Neoplastin was selected as reference reagent for comparison of methods of reporting. Results The study revealed the closest agreement of the results in study population between Neoplastin and the other five reagents, and the regression lines of these reagents were close to each other, when the results were expressed in PTp while INR, PTs and PTr is not valid for comparison of patients with liver disease. In patients on oral anticoagulant therapy, only INR standardize PT results. Conclusion we conclude that, in patients with liver disease, only activity percentage expression may provide a common international scale of PT reporting.     

Japanese collaborative study for fibrinogen assay: variability of the fibrinogen assay between different laboratories does not improve when a common calibrator is used

Several national and local external quality assurance schemes have been developed to improve the plasma fibrinogen assay in Japan over the past 30 years. Now most commercial calibrant plasma may be calibrated against an International Standard preparation, in order to achieve agreement of results obtained by different laboratories. However, we have never achieved satisfactory results, according to an external quality control survey regarding the fibrinogen assay. Therefore, we distributed two kinds of fibrinogen standards to be used as common calibrators, along with three plasma samples, among 183 general laboratories in Japan. The results of this collaborative study showed that the assigned value for the commercially available calibrators remained problematic. Furthermore, it was concluded that the between-laboratory variability could not be improved beyond a certain degree of standardization, even if a common calibrator was used for the Clauss-derived assay carried out by an automatic coagulometer.     

International external quality assessment for measurements of direct oral anticoagulants: results and recommendations

There is limited information regarding the performance of tests for direct oral anticoagulants (DOACs). To generate more knowledge, the accuracy of DOAC tests were evaluated using external quality assessment data from multiple years. This data demonstrated a good correlation for the tests with a small overall interlaboratory variability (10% for dabigatran, rivaroxaban and apixaban and 12% for edoxaban). The greatest differences between the various reagents were observed for rivaroxaban, especially for concentrations below 100 ng/ml. In conclusion, the results show overall reliable DOAC levels with some differences between reagent groups. Important finding: clinical decision criteria could be affected by the choice of reagent.     

Comparison of five specific assays for determination of dabigatran plasma concentrations in patients enrolled in the START‐Laboratory Register

Introduction

Several specific assays are commercially available to determine dabigatran anticoagulant activity. Aims of this multicenter and multiplatform study were to compare five methods for dabigatran measurement and investigate their performances in the low concentration range.

Methods

Dabigatran levels were analyzed in 295 plasma samples from patients enrolled in the START‐Laboratory Register by the following methods using dedicated calibrators and controls: STA‐ECA II (Diagnostica Stago), standard and low range Hemoclot Thrombin Inhibitors (Hyphen BioMed), Direct Thrombin Inhibitor Assay (Instrumentation Laboratory), Direct Thrombin Inhibitor Assay (Siemens), Technoclot DTI (Technoclone).

Results

Methods showed variable agreement with the Hemoclot Thrombin Inhibitors assay used as reference test, with modest under‐ or overestimations (Bland‐Altman bias from ?17.3 to 4.0 ng/mL). Limits of detection and quantification varied depending on the assay (4‐52 and 7‐82 ng/mL, respectively). Between‐run precision and accuracy were good for all methods for both quality control levels. Assay's repeatability assessed at very low dabigatran concentrations (from 10 to 60 ng/mL) was also acceptable, variability generally increased at lower drug levels.

Conclusion

The five dabigatran‐specific assays evaluated in this study provided reliable assessment of dabigatran plasma levels, although showing different performances.

     

Revisiting the anticardiolipin test and its standardization

Although the importance of the anticardiolipin test in diagnosis of antiphospholipid syndrome (APS) is widely accepted, there remains much misunderstanding about the strengths and weaknesses of this assay. Several disorders result in formation of low levels of the antibody, hence the anticardiolipin test is not specific when results are low positive. In general, the higher the anticardiolipin level the greater the likelihood of a diagnosis of APS. Hence there have been numerous efforts to enable reproducible measurement of anticardiolipin levels. Standard calibrators were introduced to construct calibration curves from which levels of unknown samples can be derived. Those standard calibrators were made by mixing varying quantities of high positive with normal sera. More recently, calibrators derived from monoclonal anticardiolipin antibodies have been introduced. There are advantages and disadvantages with both types of calibrators. Determination of a precise and reproducible anticardiolipin level is difficult, whatever the calibrators used, because the assay is dependent on several variable components, any of which may fail on any given day. Utilization of a semi-quantitative measure (low, medium, high) may suffice in most clinical settings and would be less subject to error. Validated ELISA kits may offer greater reproducibility, since there is less variability than bench assays set up in very different laboratories. Whether using a kit or a bench assay, meticulous attention to detail offers the best opportunity for precision and reproducibility.     

Quality control of ISI/INR system in oral anticoagulant therapy

Ten controlled plasmas and 4 thromboplastin reagents were distributed to 75 laboratories for measurement of prothrombin time (PT). The results were converted to prothrombin activity (PA). prothrombin ratio (PR) and international normalized ratio (INR). Among the 4 different ways of expression, the discrepancy between the results obtained by the different reagents was the smallest when the results were expressed by INR. However, the discrepancy of the results was still remained because of the different assay methods used routinely at these laboratories, even with the use of ISI/NR system (Instrumentation effects). In this study, reference curve of prothrombin time: INR was created using INR reference plasmas, which were selected from commercial control plasma using a standard thromboplastin reagent. INR's of 108 patients' plasmas were calculated by ISI/INR system on one hand, and by the use of the reference curve of the prothrombin: INR on the other hand. It was found that the discrepancy of the results obtained by different assay methods was significantly improved by the latter method, compared to those obtained by the conventional ISI/INR system.