Determination of dabigatran, rivaroxaban and apixaban by ultra-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) and coagulation assays for therapy monitoring of novel direct oral anticoagulants.

BACKGROUND: Three novel direct oral anticoagulants (DOACs) have recently been registered by the Food and Drug Administration and European Medicines Agency Commission: dabigatran, rivaroxaban, and apixaban. To quantify DOACs in plasma, various dedicated coagulation assays have been developed. OBJECTIVE: To develop and validate a reference ultra-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) method and to evaluate the analytical performance of several coagulation assays for quantification of dabigatran, rivaroxaban, and apixaban. METHODS: The developed UPLC-MS/MS method was validated by determination of precision, accuracy, specificity, matrix effects, lower limits of detection, carry-over, recovery, stability, and robustness. The following coagulation assays were evaluated for accuracy and precision: laboratory-developed (LD) diluted thrombin time (dTT), Hemoclot dTT, Pefakit PiCT, ECA, Liquid anti-Xa, Biophen Heparin (LRT), and Biophen DiXal anti-Xa. Agreement between the various coagulation assays and UPLC-MS/MS was determined with random samples from patients using dabigatran or rivaroxaban. RESULTS: The UPLC-MS/MS method was shown to be accurate, precise, sensitive, stable, and robust. The dabigatran coagulation assay showing the best precision, accuracy and agreement with the UPLC-MS/MS method was the LD dTT test. For rivaroxaban, the anti-factor Xa assays were superior to the PiCT-Xa assay with regard to precision, accuracy, and agreement with the reference method. For apixaban, the Liquid anti-Xa assay was superior to the PiCT-Xa assay. CONCLUSIONS: Statistically significant differences were observed between the various coagulation assays as compared with the UPLC-MS/MS reference method. It is currently unknown whether these differences are clinically relevant. When DOACs are quantified with coagulation assays, comparison with a reference method as part of proficiency testing is therefore pivotal.

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.

Cerebral activation related to implicit sequence learning in a Double Serial Reaction Time task.

Using functional magnetic resonance imaging (fMRI), we examined the distribution of cerebral activations related to implicitly learning a series of fixed stimulus-response combinations. In a novel - bimanual - variant of the Serial Reaction Time task (SRT), simultaneous finger movements of the two hands were made in response to pairs of visual stimuli that were presented in a fixed order (Double SRT). Paired stimulus presentation prevented explicit sequence knowledge occurring during task practice, which implied that a dual task paradigm could be avoided. Extensive prescanning training on randomly ordered stimulus pairs allowed us to focus on the acquisition of implicit sequence knowledge. Activation specifically related to the acquisition of fixed sequence knowledge was highly significant in the right ventrolateral prefrontal cortex. The medial prefrontal and right ventral premotor cortex were more indirectly related with such procedural learning. We conclude that this set of activations reflects a stage of implicit sequence learning constituted by components of (i) spatial working memory (right ventral prefrontal cortex), (ii) response monitoring and selection (medial prefrontal cortex), and (iii) facilitated linkage of visuospatial cues to compatible responses (right ventral premotor). Comparing the random-order stimulus-response actions with fixed sequences showed activations in dorsal premotor and posterior parietal cortices, consistent with a dorsal pathway dominance in real-time visuomotor control. The relative long time during which performance improves in the DoSRT provides an opportunity for future study of various stages in both general skill and fixed sequence learning.

Cerebral activation related to skills practice in a double serial reaction time task: striatal involvement in random-order sequence learning.

We used functional Magnetic Resonance Imaging (fMRI) to examine the distribution of cerebral activation related to prolonged skill practice. In a bimanual variant of the Serial Reaction Time Task (SRT), simultaneous finger movements of the two hands were made in response to randomly ordered pairs of visual stimuli (Double SRT, DoSRT). Extended practice by a week of daily performance resulted in gradual decrease of reaction times, associated with an increased involvement of the ventral putamen and globus pallidus, reaching statistical significance only on the left side (Statistical Parametric Mapping, SPM99). This increase was complementary to a decrease of cortical activations. The striatal activation after training on random order stimuli indicates that the striatum is not exclusively involved in sequence learning. This extended function implies a role in the acquisition of basic visuomotor skills that includes the specific selection of the appropriate muscles in response to independent stimuli.

Exclusion and diagnosis of deep vein thrombosis in outpatients by sequential noninvasive tools.

Phlebography is the reference gold standard for the diagnosis of deep vein thrombosis (DVT), but due to its invasive nature and associated side effects it has been replaced by compression ultrasonography (CUS). Patients suspected of DVT are subjected to leg vein CUS that actually confirms DVT in only 16 to 28% of outpatients in large prospective management studies. CUS has a high positive predictive value of more than 98% for proximal DVT but usually misses calf vein thrombosis. Its negative predictive value for proximal DVT is about 97-98%, on the basis of which repeated scanning at day 7 after a negative first CUS (serial CUS) in outpatients with a first suspicion of DVT is advocated. Serial ultrasonography is costly and can be simplified and improved by the addition of clinical score and D-dimer testing. The safe exclusion of DVT by a rapid sensitive D-dimer test in combination with clinical score and/or CUS requires a negative predictive value of >99%. The negative predictive value for DVT is determined by the sensitivity of the rapid ELISA D-dimer test and the prevalence of DVT in subgroups of outpatients suspected of the condition. The prevalence of DVT in outpatients with a low, moderate and high clinical score varies widely from 3-10%, 15-30% and >70%, respectively. The combination of a low clinical score (prevalence DVT 3-5%) and a negative rapid ELISA D-dimer alone test will have a very high negative predictive value of >99.9% to exclude DVT without the need of CUS testing. The combination of a negative CUS and a negative rapid ELISA D-dimer test safely excludes DVT in patients with suspected DVT irrespective of the clinical score. The combination of a negative CUS, a low clinical score and a positive ELISA D-dimer but <1000 ng/ml excludes DVT with a negative predictive value of >99% without the need to repeat CUS. Patients with a negative CUS, scan but a positive ELISA D-dimer, and a moderate or high clinical score are still at risk with a probability of DVT of 3-5% and 20-30%, respectively and are thus candidates for repeated ultrasound scanning. The rapid ELISA D-dimer first followed by risk-based no, single or repeated CUS will be the most cost-effective strategy.

Brain activation related to the representations of external space and body scheme in visuomotor control.

Regional cerebral blood flow was assessed during reaching movements with either target or finger selection. Measurements were performed with positron emission tomography in normal subjects. We thus identified two patterns of cerebral activation representing parietal command functions based on either external space or body scheme information. Directing the right-hand index finger toward one target dot in an array of five was related to activations distributed over dorsal extrastriate visual cortex (putative area V3A), along the parieto-occipital sulcus (putative V6/V6A) and the posterior intraparietal sulcus (IPS). Right-hemisphere dominance was present at the occipital extension of posterior IPS. Positioning one right-hand finger of five on the middle target dot was related with anterior IPS activation, extending over the marginal gyrus of the left inferior parietal lobe. The latter indicated a parietal role in prehension, independent of the shape of the target reached for. In both conditions of the reaching task, instructions for movement were auditorily given by random numbers 1 to 5, thus excluding visual cueing. The observed lateralization of movement-related parietal functions helps to explain neurological symptoms such as ideomotor apraxia and spatial hemineglect.

Implicit sub-unit learning in a serial reaction time task in humans.

In the present serial reaction time task experiment (SRT), a fixed 12-item sequence was practiced in order to evaluate the effect on response times to 3-item sub sequences (triplets) in a subsequent random sequence. Subjects were visually cued to press one out of four keys with a corresponding right-hand finger. The occurrence of implicit sequence knowledge was evidenced by the increase in mean response time when the transition was made from the final 12-item sequence block to the subsequent random block. In the stimulus-set applied, a total of 36 triplets could be constructed, of which 24 triplets were encountered only during the random blocks (random-only triplet set) (RO-set), whereas 12 triplets were also part of the sequence used in the sequence blocks (sequence-also triplet set) (SA-set). Approximately 35% of the triplets that comprised the two random blocks were also presented in the sequence blocks. There was no difference in mean response times between the triplet sets in the random block that preceded the sequence blocks. In the final random block, however, the SA-set induced significantly faster responses as compared with the RO-set. We argue that stimulus response associations within the SA-set are responsible for the difference in response times between the two triplet sets in the final random block.

Exclusion of deep venous thrombosis with D-dimer testing--comparison of 13 D-dimer methods in 99 outpatients suspected of deep venous thrombosis using venography as reference standard.

In a direct assay comparison we evaluated the diagnostic performance of 10 novel D-Dimer assays for the exclusion of deep venous thrombosis (DVT). In addition, 3 conventional ELISA D-Dimer assays were included as reference tests. The study was performed in 99 consecutive outpatients referred to the emergency department for clinical suspicion of DVT. Venography was used as reference standard and demonstrated the presence of DVT in 50 patients (6 patients with isolated distal DVT and 44 patients with proximal DVT). The qualitative D-Dimer assays Minutex and SimpliRED and the quantitative BC DD showed overall sensitivities (for proximal and distal DVT) of only 80-83% with specificities that ranged from 87 to 94%. Overall sensitivity was 94% for the qualitative INSTANT I.A. and 98% for the quantitative Turbiquant at a cut-off level equal to the detection limit. Using different cut-off levels a sensitivity of 100% for proximal DVT and for proximal as well as distal DVT could be obtained for NycoCard, IL DD, Liatest, Tinaquant and VIDAS D-Dimer assays with specificities that ranged from 31% (NycoCard) to 71% (VIDAS) for proximal DVT and from 12% (NycoCard) to 47% (IL DD) for overall DVT. At a cut-off level equal to the upper limit of the reference range only Tinaquant and VIDAS showed a sensitivity of 100% for proximal as well as for distal DVT with a specificity of 39% and 41% respectively. The results of this study suggest that the VIDAS and Tinaquant D-Dimer assays have the highest sensitivity for the exclusion of DVT in outpatients. In outpatients that have a low or moderate pretest probability for DVT, these tests may be used in management studies where anticoagulation is withheld on the basis of D-Dimer testing alone.

Strategies for the safe and effective exclusion and diagnosis of deep vein thrombosis by the sequential use of clinical score, D-dimer testing, and compression ultrasonography.

Patients with suspected deep vein thrombosis (DVT) are subjected to leg vein compression ultrasonography (CUS) that confirms DVT in only 20 to 30% of patients. A positive CUS is consistent with DVT irrespective of clinical score. The sequential use of a simple clinical score assessment, a rapid sensitive enzyme-linked immunosorbent assay (ELISA) D-dimer test and CUS to safely exclude DVT is promising. The clinical score is a validated clinical model of complaints, signs, and symptoms, on the basis of which a pretest clinical probability for DVT can be estimated as low, moderate, and high. The safe exclusion of DVT by a rapid sensitive D-dimer test in combination with clinical score or CUS necessitates a negative predictive value of more than 99%. The negative predictive value for DVT is determined by the sensitivity of the rapid ELISA D-dimer test and the prevalence of DVT in subgroups of outpatients with suspected DVT. The prevalence of DVT in outpatients with a low, moderate, and high clinical score varies widely from 3 to 10%, 15 to 30% and more than 70%, respectively. A negative rapid ELISA D-dimer and a low clinical score (prevalence DVT 3 to 5%) will have a very high negative predictive value of more than 99.5% to exclude DVT without the need of CUS testing. A negative ELISA D-dimer test and a first-negative CUS safely exclude DVT in patients with a moderate clinical score with a negative predictive value of more than 99.5%, therefore obviating the need to repeat CUS. The use of a rapid ELISA D-dimer testing in patients with a high clinical score is not recommended. A negative CUS, a low clinical score, and a positive ELISA D-dimer, even less than 1000 ng/mL exclude DVT with a nega tive predictive value of more than 99%. Patients with a negative CUS, but a positive ELISA D-dimer, and a moderate or high clinical score have a probability of DVT of 3 to 5% and 20 to 30%, respectively, and are thus candidates for repeated CUS testing. The proposed sequential use of the clinical score assessment, a rapid ELISA D-dimer test, and CUS will be the most cost-effective diagnostic strategy for DVT because of a significant reduction of CUS examinations and gain of time for the patient and physician in charge.

Synaptic transmission in the rat dentate gyrus after adrenalectomy.

Granule cells in the rat hippocampal dentate gyrus contain intracellular receptors for the adrenal hormone corticosterone. Activation of these receptors seems essential for granule cell viability, since removal of the adrenal gland (adrenalectomy) results within three days in apoptotic-like degeneration of granule cells. In the present study we used extracellular in vitro recording methods to study the synaptic transmission in the dentate gyrus of adrenalectomized animals, in sham-operated controls and adrenalectomized rats treated with a low dose of corticosterone. We found that particularly three days after adrenalectomy orthodromic field responses in the dentate gyrus were reduced in amplitude. Corticosterone-treated rats did not show this impairment of synaptic transmission. Antidromically-evoked field responses were also reduced after adrenalectomy, which indicates that postsynaptic cell properties rather than signal transduction in the synapses are under steroid control. Responses to paired pulse stimulation were only marginally affected, suggesting that interneuronal networks may be less affected by the hormones than the principal cells. These electrophysiological data indicate that adrenalectomy induced apoptotic-like degeneration in the hippocampal dentate gyrus is clearly associated with impaired processing of incoming information.

Four agglutination assays evaluated for measurement of von Willebrand factor (ristocetin cofactor activity)

The concentration of von Willebrand factor (vWf) in patients' plasma can be determined by measuring the ristocetin cofactor activity (vWf R:Co). However, this vWf R:Co assay is time consuming, which limits its routine use. Several commercial vWf R:Co tests, based on agglutination of lyophilized fixed platelets, are available. We evaluated the slide tests and aggregometer assays from Behring and Organon Teknika and compared them with the classic vWf R:Co aggregometer method. The within-run and between-run precisions of the two slide tests were better than those of the aggregometer methods. The correlation studies between the four commercial assays and the classic aggregation method were based on 23 plasma samples (range: 15-450% vWf R:Co). The correlation coefficients, which ranged from 0.923 to 0.950, did not differ significantly (P > 0.1). All four commercial assays gave significantly lower vWf R:Co values than the classic aggregation method (P < 0.01). We conclude that commercially available fixed platelets can be used for the rapid measurement of vWf R:Co with a slide test. The use of the aggregometer is time consuming and may result in a lower precision.

One-step chromogenic equivalent of activated partial thromboplastin time evaluated for clinical application.

We evaluated the clinical usefulness of a recently developed semi-automated one-step chromogenic equivalent of activated partial thromboplastin time (APTT; Behring). This simple test is easily adaptable for automation. Generally, the results with this chromogenic one-step APTT were at least as precise as those obtained with comparative coagulometric methods. The chromogenic one-step APTT showed, both in vitro and in vivo, adequate sensitivity to congenital intrinsic factor deficiency but no sensitivity to Factor VII deficiency. Unlike a two-step coagulometric APTT (Dade), the one-step chromogenic APTT seemed sensitive to activation products of the contact system, which are present in immunoadsorbed factor-deficient plasma. The in vitro sensitivity of the chromogenic APTT to heparin was comparable with that of a coagulometric APTT, but the sensitivity to heparin in patients' samples differed slightly. The chromogenic APTT is relatively insensitive to anomalies in the fibrinogen-fibrin conversion. Finally, we observed discrepancies between the chromogenic and coagulometric APTT results for plasma of patients with disseminated intravascular coagulation. We conclude that this one-step chromogenic APTT warrants further evaluation for possible use as a routine test for the clinical laboratory.

[Macro-creatine kinase: not all increased CK-MB activity signifies a heart infarct]. / Macro-creatine-kinase: niet iedere verhoogde CK-MB-activiteit duidt op een hartinfarct.

Assay of creatine kinase MB isoenzyme plays an important role in the diagnosis of acute myocardial infarction. An increase in CK-MB is frequently interpreted by the clinician as objective evidence of myocardial cell damage. However, increases of CK-MB may be found in several circumstances in which patients have not sustained an acute myocardial infarction. An important cause of elevated CK-MB values unrelated to acute MI is the presence of macro-creatine kinases in the patient's plasma. With immuno-inhibition procedures macro-CK is often measured as CK-MB, leading to falsely elevated CK-MB. In this paper macro-CKs, their clinical importance and their interference with CK-MB determination are discussed.

Interaction of human plasma kallikrein and its light chain with alpha 2-macroglobulin.

Human plasma kallikrein participates in the contact activation system of plasma. The light chain of kallikrein contains the enzymatic active site; the heavy chain is required for binding to high molecular weight kininogen and for surface-dependent activation of coagulation. This study has examined the functional contributions of the heavy chain of kallikrein and of high molecular weight kininogen in the inactivation of kallikrein and of its isolated light chain by alpha 2-macroglobulin (alpha 2M). Irreversible inhibition was observed for both kallikrein and its light chain, with the initial formation of a reversible enzyme-inhibitor complex. The second-order rate constants for these reactions were 3.5 X 10(5) and 4.8 X 10(5) M-1 min-1 for kallikrein and its light chain, respectively. When present in excess, high molecular weight kininogen decreased the rate of kallikrein inactivation by alpha 2M, whereas the rate of inactivation of the light chain was unaffected by high molecular weight kininogen. Although at a drastically reduced rate, high molecular weight kininogen was cleaved by alpha 2M-bound kallikrein. Sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis was used to study complex formation between alpha 2M and kallikrein or its light chain. Under reducing conditions, four kallikrein-alpha 2M complexes were observed. Three of these complexes consisted of alpha 2M and the light chain of kallikrein (Mr 123 000, 235 000, and 330 000). Two alpha 2M-kallikrein light chain complexes incorporated [3H]diisopropyl fluorophosphate ( [3H]DFP) whereas the Mr 330 000 complex did not react with [3H]DFP.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of human plasma kallikrein and its light chain with C1 inhibitor.

The light chain of human plasma kallikrein contains the enzymatic active site. The inactivation of kallikrein and of its isolated light chain by C1 inhibitor was investigated to assess the functional contributions of the heavy-chain region of kallikrein and of high molecular weight kininogen to this reaction. The second-order rate constants for the inactivation of kallikrein or its light chain were respectively 2.7 X 10(6) and 4.0 X 10(6) M -1 min -1. High molecular weight kininogen did not influence the rate of kallikrein inactivation. The nature of the complexes formed between kallikrein or its light chain and C1 inhibitor was studied by using sodium dodecyl sulfate (SDS) gradient polyacrylamide slab gel electrophoresis. Kallikrein as well as its light chain combined with C1 inhibitor to form stable stoichiometric complexes that were not dissociated by SDS and that exhibited apparent molecular weights (Mr's) of 185 000 and 135 000, respectively, on nonreduced SDS gels. Reduction of the kallikrein-C1 inhibitor complex gave a band at Mr 135 000 that comigrated with the complex seen for the light chain-C1 inhibitor complex. During the inactivation of both kallikrein and its light chain, a Mr 94 000 fragment of C1 inhibitor was formed which was unable to inactivate or bind kallikrein or its light chain. Kallikrein inactivated by diisopropyl phosphofluoridate did not form SDS-stable complexes with C1 inhibitor. These results demonstrate that the functional binding site for C1 inhibitor is localized in the light chain of kallikrein.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and functional characterization of the active light chain of activated human blood coagulation factor XI.

Human blood coagulation Factor XIa was reduced and alkylated under mild conditions. The mixture containing alkylated heavy and light chains was subjected to affinity chromatography on high Mr kininogen-Sepharose. Alkylation experiments using [14C]iodoacetamide showed that a single disulfide bridge between the light and heavy chains was broken to release the light chain. The alkylated light chain (Mr = 35,000) did not bind to high Mr kininogen-Sepharose while the heavy chain (Mr = 48,000), like Factors XI and XIa, bound with high affinity. The isolated light chain retained the specific amidolytic activity of native Factor XIa against the oligopeptide substrate, pyroGlu-Pro-Arg-p-nitroanilide. Km and kcat values for this substrate were 0.56 mM and 350 s-1 for both Factor XIa and its light chain, and the amidolytic assay was not affected by CaCl2. However, in clotting assays using Factor XI-deficient plasma in the presence of kaolin, the light chain was only 1% as active as native Factor XIa. Human coagulation Factor IX was purified and labeled with sodium [3H]borohydride on its carbohydrate moieties. When this radiolabeled Factor IX was mixed with Factor XIa, an excellent correlation was observed between the appearance of Factor IXa clotting activity and tritiated activation peptide that was soluble in cold trichloroacetic acid. Factor XIa in the presence of 5 mM CaCl2 activated 3H-Factor IX 600 times faster than Factor XIa in the presence of EDTA. In the absence of calcium, Factor XIa and its light chain were equally active in activating 3H-Factor IX. In contrast to Factor XIa, the light chain in this reaction was inhibited by calcium ions such that, in the presence of 5 mM CaCl2, Factor XIa was 2000 times more effective than its light chain. Neither phospholipid nor high Mr kininogen and kaolin affected the activity of Factor XIa or its light chain in the activation of 3H-Factor IX. These observations show that the light chain region of Factor XIa contains the entire enzymatic active site. The heavy chain region contains the high affinity binding site for high Mr kininogen. Furthermore the heavy chain region of Factor XIa plays a major role in the calcium-dependent mechanisms that contribute to the activation of Factor IX.