The danger of relying on the APTT and PT in patients on DOAC therapy, a potential patient safety issue.

Prolongation of the activated partial thromboplastin time (APTT) and prothrombin time/international normalized ratio (PT/INR) correlates poorly with plasma concentrations of direct oral anticoagulant agents (DOACS) including direct thrombin and direct Xa inhibitors. It has been repeatedly demonstrated that patients can have normal APTT and PT/INR with a therapeutic plasma concentration of a DOAC. Clinicians can no longer rely on a normal APTT and PT to determine that an anticoagulated patient is safe to undergo an invasive procedure. Laboratory scientists need to play a key and active role in educating clinicians about the limitations of the APTT and PT in patients on DOAC prophylaxis or therapy.

Comparison of the effect of the anti-Xa direct oral anticoagulants apixaban, edoxaban, and rivaroxaban on coagulation assays.

INTRODUCTION: The purpose of this study is to compare the effect of increasing concentrations of direct anti-Xa oral anticoagulants (DOAC) apixaban-, edoxaban-, and rivaroxaban-enriched plasma samples on various prothrombin time (PT), activated partial thromboplastin time (APTT), heparin calibrated anti-Xa methods, and other coagulation assays. METHODS: Apixaban, edoxaban, or rivaroxaban was dissolved in dimethylsulfoxide and added to pooled normal plasma to obtain concentrations ranging from 0 ng/mL to approximately 600 ng/mL. The samples were tested using Innovin(®) , Neoplastine(®) CI Plus, Recombiplastin 2G, and Thromborel(®) S for PT testing and Actin, Actin(®) FS, Actin(®) FSL, APTT-Automate, and SynthaSIL for APTT. Samples were also tested using four different anti-Xa methods calibrated for unfractionated heparin or low molecular weight heparin. Special coagulation assays included antithrombin activity, lupus anticoagulant assays, and others. For special coagulation assays, the concentration of DOAC that resulted in a >15% change from baseline value was determined. DOAC quantification was performed using liquid chromatography-tandem mass spectrometry. RESULTS: All PT and APTT reagents demonstrated a higher sensitivity for edoxaban and rivaroxaban than for apixaban. Anti-Xa methods were able to detect low concentrations of DOAC. DOACs effected special coagulation assays to differing degrees, with lupus anticoagulant testing using dilute viper venom, the most sensitive test to the presence of anti-Xa DOAC. CONCLUSION: No PT or APTT reagent system effectively detected apixaban. All anti-Xa methods demonstrated sensitivity to low concentrations of DOAC. Dilute viper venom methods are exquisitely sensitive to anti-Xa DOAC, suggesting potential use of this assay for screening or measuring these drugs.

A single test to assay warfarin, dabigatran, rivaroxaban, apixaban, unfractionated heparin, and enoxaparin in plasma.

UNLABELLED: Essentials Simple and fast assaying of different anticoagulants (ACs) is useful in emergent situations. We used highly diluted prothrombin time (dPT) or highly diluted Fiix-PT (dFiix-PT) to assay ACs. Both tests could quantify target specific anticoagulants and warfarin anticoagulation. Improved results were consistently observed with the dFiix-PT compared with the dPT. SUMMARY: Background Assaying anticoagulants is useful in emergency situations or before surgery. Different specific assays are currently needed depending on the anticoagulant. Objectives We hypothesized that levels of warfarin, dabigatran, rivaroxaban, apixaban, and heparins could be measured with use of the diluted prothrombin time (dPT) and diluted Fiix-PT (dFiix-PT), using highly diluted thromboplastin (TP). The latter test is affected only by reduced levels of active factors II and X but corrects test plasma for other deficiencies Methods Increasing TP dilutions were used to identify suitable dilutions to measure dabigatran, rivaroxaban, apixaban, unfractionated heparin (UFH), and enoxaparin. Calibrators containing known amounts of direct oral anticoagulants (DOACs) were used to make standard curves. Citrated plasma samples were obtained from patients taking warfarin or DOACs with known drug concentrations as determined by specific assays. Results The dFiix-PT at a TP dilution of 1:1156 could be used to measure all of the drugs tested at therapeutic concentrations except for fondaparinux. The dPT achieved the same but required two TP dilutions (1:750 and 1:300). The warfarin effect could be assessed by using dFiix-PT at 1:1156 with a PT ratio identical to the international normalized ratio. Six different TPs yielded similar results, but two were less sensitive. Dabigatran, rivaroxaban, and apixaban could be accurately measured in patient samples using both dilute PT assays, but a better correlation was consistently observed between the dFiix-PT and specific assays than with the dPT. Conclusion The dFiix-PT using a single dilution of TP may be suitable to assess the anticoagulant effects of warfarin, dabigatran, rivaroxaban, apixaban, heparin, and enoxaparin.

Verifying the performance characteristics of the TEG5000 thromboelastogram in the clinical laboratory.

OBJECTIVE: To verify the manufacturer performance claims of the TEG5000 with traditional laboratory methods. MATERIALS AND METHODS: Samples were concurrently measured using the TEG5000 analyzer and either PT, APTT, fibrinogen, factor activities, platelet count, or platelet function testing using whole blood or platelet-rich plasma methods. RESULTS: Within-run imprecision yielded coefficient of variation (CV) of <5%. There was no correlation of PT or APTT with R time. Only Factor VIII and factor XII activity significantly correlated with R time. There was significant correlation between k and angle with FBG, PLT count, and factor levels. There was weak inverse correlation between angle results and measures of platelet function. All laboratory methods were significantly correlated with MA. There were significant differences between citrated whole blood and fresh citrated plasma for angle and MA, and between fresh and frozen plasma for R time and MA. We demonstrated a high % inhibition noted with normal, drug naïve donors, especially with ADP PLT mapping (50% inhibition), but less so with AA PLT mapping (20% inhibition). For TEG platelet mapping, 19/22 (86.3%) and 17/22 (77.3%) results were concordant with traditional aggregation results. CONCLUSION: We demonstrated both the lack of, and strong correlation between laboratory tests and the TEG parameters.

The laboratory's 2015 perspective on direct oral anticoagulant testing.

The introduction of direct oral anticoagulant (DOAC) therapy into clinical use in the past 5 years has had significant impact on the clinical laboratory. Clinicians' desire to determine plasma drug presence or measure drug concentration, and more recent observations regarding the limitations and utility of coagulation testing in the setting of DOAC treatment, suggest that early published recommendations regarding laboratory testing should be reassessed. These initial recommendations, furthermore, were often based on drug-spiked plasma studies, rather than samples from patients receiving DOAC therapy. We have demonstrated that reagent sensitivity varies significantly whether drug-spiked samples or samples from DOAC-treated patients are tested. Data from drug-enriched samples must therefore be interpreted with caution or be used as a guide only. We present laboratory assays that can be used to determine drug presence and to measure drug concentration, and provide recommended testing algorithms. As DOAC therapy may significantly impact on specialty coagulation assays, we review those tests with the potential to give false-positive and false-negative results.

Direct Oral Anticoagulants (DOACs) in the Laboratory: 2015 Review.

Direct oral anticoagulant therapies, including direct anti-Xa and thrombin inhibitors have recently been introduced and may have advantages over vitamin K antagonists such as warfarin. This review describes briefly the clinical utility and mechanism of action of these agents. Detailed information is provided on effect of these agents on routine assays including the APTT and PT as well as their impact on specialty laboratory assays. Also included are the use of drug specific assays and a discussion of alternative methods to determine relative drug concentration, such as evaluating drug calibrators in APTT and PT assays and using heparin calibrated anti-Xa assays to measure direct Xa inhibitors.

Assessing nonvitamin K antagonist oral anticoagulants (NOACs) in the laboratory.

Nonvitamin K antagonist oral anticoagulants (NOACs) are being used with increasing frequency due to their safety profile, ease of use, and given that therapeutic monitoring is not required. As these agents have only recently been FDA approved, their effect on routine and specialty coagulation assays is not well appreciated. This article discusses NOAC effect on routine coagulation assays and whether these assays can be used to estimate drug concentration as well as which assays are suitable to quantitate drug concentration in plasma. Also reviewed is the use of manufactured drug calibrators to determine reagent responsiveness and the effect of these agents on various special coagulation assays.

Effects of storage and thawing conditions on coagulation testing.

INTRODUCTION: Current recommendations for coagulation testing storage and thawing are based on historical studies that were performed using unbuffered 3.8% sodium citrate. We sought to measure the effects of freezing and thawing conditions 3.2% buffered sodium citrate plasma samples that have been stored in vials with either snap or sealed screw tops, frozen in -70 °C freezer or dry ice and thawed either capped or uncapped. METHODS: Shed blood samples were pooled and then aliquoted into four snap top and four screw tops vials. Half the vials were stored in a -70 °C freezer, and half on dry ice for at least 16 h. Afterwards, half the frozen samples were thawed in 37 °C waterbath capped, and other half were thawed capped. After thawing cycles, samples were tested for PT, activated partial thromboplastin time (APTT), fibrinogen, D-dimer, factor assays, von Willebrand factor activity, plasminogen, antithrombin, protein C and lupus anticoagulant. RESULTS: Prothrombin time, APTT, factor X, and lupus anticoagulant testing were affected by all vials, freezing and thawing conditions, whereas fibrinogen, D-dimer, von Willebrand activity or protein C were not affected by any vial, freezing or storage condition. CONCLUSIONS: Storage vials, freezing and thawing condition affect coagulation testing, although these differences may not be clinically significant.

Lymphangioleiomyomatosis Presenting with Perirenal Hemorrhage.

Lymphangioleiomyomatosis (LAM) is a rare debilitating disease of unknown etiology, classically described as almost exclusively affecting women of childbearing age. The disease most commonly involves the lungs and is characterized by hamartomatous smooth muscle cell proliferations along blood vessels, airways and lymphatics. Most patients present with pulmonary symptoms, including shortness of breath, recurrent pneumothorax and pleural effusions. Extrapulmonary manifestations of LAM as the initial presentation of the disease are highly unusual. We present the case of a patient in whom LAM was incidentally discovered when the patient presented with retroperitoneal hemorrhage from a ruptured renal angiomyolipoma.

Evaluating the use of commercial drug-specific calibrators for determining PT and APTT reagent sensitivity to dabigatran and rivaroxaban.

Suitable laboratory methodologies for quantifying the non-vitamin K oral anticoagulants (NOAC) include liquid chromatography-tandem mass spectrometry (LC-MS/MS) or drug-calibrated assays such as the dilute thrombin time for dabigatran or anti-Xa measurements for rivaroxaban. In situations when these tests are unavailable, it has been suggested that using commercial drug calibrators on APTT and PT assays would theoretically provide reagent sensitivity to these drugs. The purpose of this study was to determine whether commercial drug calibrators deliver similar reagent sensitivity information as samples from patients receiving dabigatran or rivaroxaban as part of their routine care. Two laboratory sites tested commercial calibrator material for dabigatran and rivaroxaban (Hyphen Biomedical) using PT and APTT reagents and data was compared to samples collected from patients taking NOACs that were quantified by LC-MS/MS. Correlation statistics and calculating the amount of drug required to double the clotting time of normal plasma were performed. All drug calibrator material correlated more strongly (R²> 0.95) for any reagent/drug combination than patient samples (R² ranged from 0.29-0.86). Dabigatran calibrator results and patient data were equivalent for SynthASil and PTT-A APTT reagents. The dabigatran and rivaroxaban calibrator material over-estimated drug sensitivity for all PT reagents when compared to sensitivity data calculated based on drug levels obtained by LC-MS/MS from patient samples. In conclusion, drug-specific calibrators overestimated reagent sensitivity which may underestimate in vivo drug concentration in a given patient. Further studies are required to assess whether this method of determining relative sensitivity of NOAC on routine coagulation assays should be recommended.

Serial haemostatic monitoring of dogs with multicentric lymphoma.

Lymphoma is the most common haematopoietic malignancy in dogs and it has been associated with hypercoagulability and subsequent thromboembolism. The objectives of this study were to serially characterize the haemostatic status of dogs with multicentric lymphoma. Thromboelastography, thrombin-antithrombin complex concentration and routine haematology and coagulation panels were measured. Twenty-seven dogs were included in the study and 15 completed the study in remission. At presentation, 81% (22/27) of dogs with multicentric lymphoma had altered haemostatic profiles consistent with hypercoagulability. Laboratory evidence of hypercoagulability did not resolve during treatment or for up to 1 month following attainment of clinical remission. Accelerated rate of clot formation at the time of chemotherapeutic protocol completion was associated with decreased survival time. We concluded that dogs with multicentric lymphoma were frequently hypercoagulable from presentation through 4 weeks after the completion of chemotherapy. Increased angle and shortened K in dogs that have successfully completed their chemotherapeutic protocol may be associated with shorter survival times.

A rare case of diffuse alveolar hemorrhage following oral amphetamine intake.

Diffuse alveolar hemorrhage (DAH) is a clinical syndrome, which refers to injury to the capillaries, arterioles and venules, leading to red blood cell accumulation in the distal air spaces. It is defined by the clinical triad of hemoptysis, anemia and progressive hypoxemia. Chest radiographs reveal non-specific patchy or diffuse bilateral pulmonary consolidation. Multiple conditions are associated with DAH, of which Wegener's granulomatosis is the most frequent, and underlying disease determines the prognosis and treatment. This case describes DAH as a result of oral amphetamine abuse in a young patient of which the diagnosis was established by laboratory, clinical and radiologic findings. The patient experienced a rapid recovery without significant sequelae.

Differential activation of the prefrontal cortex and amygdala following psychological stress and colorectal distension in the maternally separated rat.

Visceral hypersensitivity is a hallmark of many clinical conditions and remains an ongoing medical challenge. Although the central neural mechanisms that regulate visceral hypersensitivity are incompletely understood, it has been suggested that stress and anxiety often act as initiating or exacerbating factors. Dysfunctional corticolimbic structures have been implicated in disorders of visceral hypersensitivity such as irritable bowel syndrome (IBS). Moreover, the pattern of altered physiological responses to psychological and visceral stressors reported in IBS patients is also observed in the maternally separated (MS) rat model of IBS. However, the relative contribution of various divisions within the cortex to the altered stress responsivity of MS rats remains unknown. The aim of this study was to analyze the cellular activation pattern of the prefrontal cortex and amygdala in response to an acute psychological stressor (open field) and colorectal distension (CRD) using c-fos immunohistochemistry. Several corticoamygdalar structures were analyzed for the presence of c-fos-positive immunoreactivity including the prelimbic cortex, infralimbic cortex, the anterior cingulate cortex (both rostral and caudal) and the amygdala. Our data demonstrate distinct activation patterns within these corticoamygdalar regions including differential activation in basolateral versus central amygdala following exposure to CRD but not the open field stress. The identification of this neuronal activation pattern may provide further insight into the neurochemical pathways through which therapeutic strategies for IBS could be derived.

Characterization of thrombelastography over time in dogs with hyperadrenocorticism.

Canine hyperadrenocorticism (HAC) is a common endocrinopathy often associated with hypercoagulability, thrombosis and thromboembolism and it can contribute to increased morbidity and mortality. The condition results in increased, unregulated secretion of glucocorticoids (GCs). While prospective identification of hypercoagulability is challenging, thrombelastography (TEG) is a diagnostic tool that enables the detection of hypercoagulability in a clinical setting. The objective of this prospective cohort study was to serially assess coagulation in dogs with HAC using TEG to test the hypothesis that dogs with HAC have increased TEG maximal amplitude (MA) and that the MA would normalize once clinical control was achieved. Twenty-three dogs with naturally occurring HAC were enrolled and hemostatic (including TEG, platelet function, thrombin-antithrombin complexes and coagulation panel) and hematological variables were measured at presentation. TEG was serially monitored until clinical resolution of HAC was attained. At presentation, most dogs with HAC had increased MA values, increased thrombin-antithrombin complexes and many were hyperfibrinogenemic. Platelet function analyzer-100 (PFA-100) closure times were significantly prolonged. TEG tracings did not normalize in either medically- or surgically-managed dogs, but fibrinogen concentrations decreased. It seems that dogs with HAC have a complex coagulopathy in which hypercoagulability and platelet hyporeactivity or dysfunction might occur simultaneously. As TEG tracings did not normalize in well-controlled dogs, it is unlikely that increased blood GCs are solely responsible for TEG alterations seen in dogs with HAC.

Performance of coagulation tests in patients on therapeutic doses of dabigatran: a cross-sectional pharmacodynamic study based on peak and trough plasma levels.

BACKGROUND: Knowledge of anticoagulation status during dabigatran therapy may be desirable in certain clinical situations. OBJECTIVE: To determine the coagulation tests that are most useful for assessing dabigatran's anticoagulant effect. METHODS: Peak and trough blood samples from 35 patients taking dabigatran 150 mg twice daily, and one sample each from 30 non-anticoagulated individuals, were collected. Mass spectrometry and various coagulation assays were performed. 'Therapeutic range' was defined as the range of plasma dabigatran concentrations determined by mass spectrometry between the 2.5th and 97.5th percentiles of all values. RESULTS: The therapeutic range was 27-411 ng mL(-1) . The prothrombin time (PT) and activated partial thromboplastin time (APTT), determined with multiple reagents, and activated clotting time (ACT) were insensitive to therapeutic dabigatran: 29%, 18% and 40% of samples had a normal PT, APTT, and ACT, respectively. However, normal PT, ACT and APTT ruled out dabigatran levels above the 75th percentile. The thrombin clotting time (TCT) correlated well and linearly with dabigatran levels below the 50th percentile, but was unmeasurable above it. The dilute thrombin time, ecarin clotting time and ecarin chromogenic assay showed linear correlations with dabigatran levels over a broad range, and identified therapeutic and supratherapeutic levels. CONCLUSIONS: The prothrombin time, APTT and ACT are often normal in spite of therapeutic dabigatran plasma levels. The TCT is useful for detecting minimal dabigatran levels. The dilute thrombin time and chromogenic and clotting ecarin assays accurately identify therapeutic and supratherapeutic dabigatran levels. This trial is registered at www.clinicaltrials.gov (#NCT01588327).

Neuronal expression of the ubiquitin ligase Nedd4-2 in rat dorsal root ganglia: modulation in the spared nerve injury model of neuropathic pain.

Neuronal hyperexcitability following peripheral nerve lesions may stem from altered activity of voltage-gated sodium channels (VGSCs), which gives rise to allodynia or hyperalgesia. In vitro, the ubiquitin ligase Nedd4-2 is a negative regulator of VGSC α-subunits (Na(v)), in particular Na(v)1.7, a key actor in nociceptor excitability. We therefore studied Nedd4-2 in rat nociceptors, its co-expression with Na(v)1.7 and Na(v)1.8, and its regulation in pathology. Adult rats were submitted to the spared nerve injury (SNI) model of neuropathic pain or injected with complete Freund's adjuvant (CFA), a model of inflammatory pain. L4 dorsal root ganglia (DRG) were analyzed in sham-operated animals, seven days after SNI and 48 h after CFA with immunofluorescence and Western blot. We observed Nedd4-2 expression in almost 50% of DRG neurons, mostly small and medium-sized. A preponderant localization is found in the non-peptidergic sub-population. Additionally, 55.7 ± 2.7% and 55.0 ± 3.6% of Nedd4-2-positive cells are co-labeled with Na(v)1.7 and Na(v)1.8 respectively. SNI significantly decreases the proportion of Nedd4-2-positive neurons from 45.9 ± 1.9% to 33.5 ± 0.7% (p<0.01) and the total Nedd4-2 protein to 44% ± 0.13% of its basal level (p<0.01, n=4 animals in each group, mean ± SEM). In contrast, no change in Nedd4-2 was found after peripheral inflammation induced by CFA. These results indicate that Nedd4-2 is present in nociceptive neurons, is downregulated after peripheral nerve injury, and might therefore contribute to the dysregulation of Na(v)s involved in the hyperexcitability associated with peripheral nerve injuries.