Performance of direct oral anticoagulant (DOAC) testing by hemostasis laboratories: The Australasian/Asia-Pacific experience.

INTRODUCTION: Direct oral anticoagulants (DOACs) reflect anticoagulation agents given to treat or prevent thrombosis, having largely replaced vitamin K antagonists (VKAs) such as warfarin. DOACs are given in fixed daily doses and generally do not need monitoring. However, there may be a variety of reasons that justify measurement of plasma DOAC levels in individual patients. METHODS: We report updated findings for DOAC testing in our geographic region, using recent data from the RCPAQAP, an international external quality assessment (EQA) program, currently with some 40-60 participants in each of the different DOAC (rivaroxaban, apixaban, dabigatran) modules, to assess laboratory performance in this area. Data has been assessed for the past 5 years (2019-2023 inclusive), with 20 samples each per DOAC. RESULTS: Data shows a limited repertoire of assays in use, and mostly consistency in reported numerical values when assessing proficiency samples. Available assays mostly comprised reagents from four manufacturing suppliers. There was good consistency across what participants identified as 'DOAC detected', but some variability when participants attempted to grade DOAC levels as low vs moderate vs high. Inter-laboratory/method coefficient of variation (CVs) were generally <15% for each DOAC, when present at >100 ng/mL. CONCLUSION: We hope our findings, reflecting on mostly consistent reporting of DOAC levels and interpretation provides reassurance for clinicians requesting these measurements, and helps support their implementation in regions where there is a paucity of test availability.

Testing for the lupus anticoagulant: the good, the bad, and the ugly.

Lupus anticoagulant (LA) represents 1 of the laboratory criteria for classification of patients as having definite antiphospholipid syndrome (APS). The other 2 laboratory criteria are anticardiolipin antibodies and anti-beta2-glycoprotein I antibodies. At least 1 of these antiphospholipid antibody (aPL) tests need to be positive, with evidence of persistence, together with evidence of at least 1 clinical criterion for APS, before a patient can be classified as having definite APS. LA and other aPL assays are also important for diagnosis or exclusion of APS, as well as for risk stratification, with triple-positive patients carrying the greatest risk. Whereas LA is identified through "uncalibrated" clot-based assays, the other aPL assays (anticardiolipin and anti-beta2-glycoprotein I antibodies) represent immunological assays, identified using calibrated solid-phase methods. Because LA is identified using clot-based assays, it is subject to considerable preanalytical and analytical issues that challenge accurate detection or exclusion of LA. In this narrative review, we take a look at the good, the bad, and the ugly of LA testing, primarily focusing on the last 10 years. Although harmonization of LA testing as a result of International Society on Thrombosis and Haemostasis guidance documents and other international activities has led to improvements in LA detection, many challenges remain. In particular, several anticoagulants, especially direct oral anticoagulants and also vitamin K antagonists, given as therapy to treat the pathophysiological consequences of aPL, especially thrombosis, interfere with LA assays and can generate false-positive or false-negative LA findings. Overcoming these diagnostic errors will require a multifaceted approach with clinicians and laboratories working together.

Artificial intelligence in the pre-analytical phase: State-of-the art and future perspectives.

The use of artificial intelligence (AI) has become widespread in many areas of science and medicine, including laboratory medicine. Although it seems obvious that the analytical and post-analytical phases could be the most important fields of application in laboratory medicine, a kaleidoscope of new opportunities has emerged to extend the benefits of AI to many manual labor-intensive activities belonging to the pre-analytical phase, which are inherently characterized by enhanced vulnerability and higher risk of errors. These potential applications involve increasing the appropriateness of test prescription (with computerized physician order entry or demand management tools), improved specimen collection (using active patient recognition, automated specimen labeling, vein recognition and blood collection assistance, along with automated blood drawing), more efficient sample transportation (facilitated by the use of pneumatic transport systems or drones, and monitored with smart blood tubes or data loggers), systematic evaluation of sample quality (by measuring serum indices, fill volume or for detecting sample clotting), as well as error detection and analysis. Therefore, this opinion paper aims to discuss the state-of-the-art and some future possibilities of AI in the preanalytical phase.

Pearls and Pitfalls in the Measurement of Direct Oral Anticoagulants.

Due to their widespread use, testing for direct oral anticoagulants (DOACs) has become urgent in certain clinical situations. Screening based on widely available, rapid, and simple hemostasis assays such as prothrombin time, activated partial thromboplastin time, or even diluted Russel Viper venom time may provide sufficient evidence of "over-coagulation" and could be used "in small/peripheral/spoke laboratories" as an emergency strategy, but is not thought to be reliable for driving clinical decision making. Given their good correlation with plasma concentration, urine dipsticks may be considered a valuable alternative for emergency screening, although their performance is dependent on renal function, may vary depending on the time since the last urination, and there may be problems of interfacing with the laboratory/hospital information system. Separation methods based on liquid chromatography and mass spectrometry may be clinically questionable, since they measure the concentration rather than the actual inhibitory effect of DOACs, are relatively expensive, cumbersome and time consuming, and therefore seem unsuitable for most conditions requiring urgent clinical decision making. A proposed approach therefore involves establishing a network of routine clinical laboratories, designating a reference center where DOAC tests could be available 24/7, establishing a clear diagnostic care pathway for ordering the tests from the laboratory and standard operating procedures for performing them, the use of the diluted thrombin time for dabigatran and anti-FXa assays (drug-calibrated) for rivaroxaban, apixaban, and edoxaban, as well as providing expert advice throughout the testing process, from ordering to interpretation of results.

D-dimer diagnostics: can I use any D-dimer assay? Bridging the knowledge-to-action gap.

A State of the Art lecture titled "D-dimer Diagnostics: Can I use any D-dimer assay? Bridging the Knowledge-to-Action gap" was presented at the International Society on Thrombosis and Haemostasis Congress in 2023, included in the session on the clinical impact of variability in commonly used coagulation assays. Here, we review the role of D-dimer, primarily in the outpatient diagnosis of patients with venous thromboembolism (VTE) when combined with clinical decision rules. We focus on the recent large management trials that have studied adjustments of VTE exclusion thresholds for D-dimer based on either prior clinical probability of VTE or patient age, and the resultant benefit of reduced imaging for VTE and improved diagnostic efficiency. In this context, we report on the significant variability between D-dimer results and the multiple D-dimer assays in use worldwide using data from international external quality assurance programs. This variability is particularly high at typical VTE exclusion thresholds. We discuss the potential clinical impact of D-dimer assay substitution on accuracy of diagnosis and risk stratification of patients with VTE. Finally, we summarize relevant new data on this topic presented during the 2023 International Society on Thrombosis and Haemostasis Congress and outline future priorities urgently needed to harmonize D-dimer results and reporting that will require international collaboration among multiple stakeholders with an overall goal to close this knowledge-to-action gap.

Variable Performance of D-dimer Testing by Hemostasis Laboratories: The Australasian/Asia-Pacific Experience.

D-dimers represent the breakdown products of fibrin. Thus, elevated plasma D-dimers will arise following a thrombotic event, such as a deep vein thrombosis or a pulmonary embolism, and therefore, a nonelevated D-dimer is used to effectively exclude such events. D-dimers are also elevated in a range of other conditions, for example, during disseminated intravascular coagulation. D-dimer levels may also be associated with prognostic value. For example, highly raised D-dimer levels can be associated with worsening clinical features in coronavirus disease 2019. Thus, D-dimer testing represents a commonly requested hemostasis test, often performed in 24/7 laboratories. Unfortunately, D-dimer testing is neither standardized nor harmonized across manufacturers or laboratories. Indeed, considering reporting units and the magnitude of units, up to 28 different combinations may be reported by laboratories. We provide updated findings for D-dimer testing in our geographic region, using recent data from the Royal College of Pathologists of Australasia Quality Assurance Programs, an international external quality assessment program, currently with over 450 participants in the D-dimer module. Data show a wide variety of assays in use and variable outcomes in reported numerical values when assessing proficiency samples. D-dimer testing mostly comprised reagents from three main manufacturing suppliers, with a small number of users of reagents from other manufacturers. Reported results showed important differences in numerical values for the same homogeneous tested samples when normalized to a single reporting unit (e.g., mg/L). Nevertheless, despite using different test reagents and reporting, most participants uniformly identified D-dimer values as below or above a "detection" cut-off for samples that were constructed to be below or above most cut-off values. As expected, mixed findings were reported for samples containing levels around expected cut-off values. We hope that our findings, reflecting on the heterogeneity of test reagents and test data, help improve diagnostic testing for D-dimer testing and facilitate harmonization and standardization, in the future.

Simplified Method for Removing Direct Oral Anticoagulant Interference in Mechanical Coagulation Test Systems-A Proof of Concept.

BACKGROUND: Direct oral anticoagulants (DOACs) cause unwanted interference in various hemostasis assays, including lupus anticoagulant (LA) testing, where false positive and false negative identification may occur. DOAC Stop (DS) is an activated charcoal (AC) product used to specifically and effectively adsorb DOACs from test plasma. This process normally requires plasma treatment, centrifugation and plasma separation prior to tests, but inexperienced operators may also inadvertently transfer residual AC particles, thereby potentially adversely affecting clot detection. METHODS: We hypothesized that residual DS might not be problematic for mechanical clot detection. We therefore investigated the potential impact of DS and a new DS liquid (DS-L) product on clotting tests using a mechanical clot detection system. Varying concentrations of DS were added to normal and abnormal plasmas with and without DOAC presence. Clotting tests including PT, APTT and dRVVT were performed directly in the analyzer without plasma/DS centrifugation. RESULTS: DS up to double the recommended treatment level had only minor effects on all test results, despite completely obscuring visibility in the plasma/reagent mix. This confirms that the centrifugation step may be able to be omitted when using mechanical detection systems. CONCLUSIONS: Should DS carryover into treated plasmas occur, this should not cause issues with testing performed on mechanical clot-sensing devices. Moreover, we hypothesize that DS can be used directly in these systems, without the need for centrifugation, thereby simplifying its many potential applications.

Unfractionated heparin: optimizing laboratory monitoring and reducing unwanted interference in everyday hemostasis test practice.

Unfractionated heparin (UFH) serves as a commonly used anticoagulant. It is widely utilized for a variety of reasons, including to 1) anticoagulate patients and help treat and / or prevent thrombosis, 2) maintain patency in artificial blood flow circuits, and 3) anticoagulate blood samples collected for laboratory testing (typically for biochemical assays or blood gas analysis). As such, the presence of UFH is nearly ubiquitous in a hospital setting. Therefore, in laboratory practice, UFH may be present in samples intended for monitoring patients on UFH therapy or intended for biochemical tests, or it may interfere with other (hemostasis) laboratory tests. The aim of this manuscript is to review the role of UFH from the perspective of optimizing laboratory testing to monitor UFH therapy and to avoid or overcome unwanted interference with other laboratory tests.

Algorithm for Rapid Exclusion of Clinically Relevant Plasma Levels of Direct Oral Anticoagulants in Patients Using the DOAC Dipstick: An Expert Consensus Paper.

BACKGROUND: With the widespread use of direct oral anticoagulants (DOACs), there is an urgent need for a rapid assay to exclude clinically relevant plasma levels. Accurate and rapid determination of DOAC levels would guide medical decision-making to (1) determine the potential contribution of the DOAC to spontaneous or trauma-induced hemorrhage; (2) identify appropriate candidates for reversal, or (3) optimize the timing of urgent surgery or intervention. METHODS AND RESULTS: The DOAC Dipstick test uses a disposable strip to identify factor Xa- or thrombin inhibitors in a urine sample. Based on the results of a systematic literature search followed by an analysis of a simple pooling of five retrieved clinical studies, the test strip has a high sensitivity and an acceptably high negative predictive value when compared with levels measured with liquid chromatography tandem mass spectrometry or calibrated chromogenic assays to reliably exclude plasma DOAC concentrations ≥30 ng/mL. CONCLUSION: Based on these data, a simple algorithm is proposed to enhance medical decision-making in acute care indications useful primarily in hospitals not having readily available quantitative tests and 24/7. This algorithm not only determines DOAC exposure but also differentiates between factor Xa and thrombin inhibitors to better guide clinical management.

Diagnostic value of D-dimer in differentiating Multisystem Inflammatory Syndrome in Children (MIS-C) from Kawasaki disease: systematic literature review and meta-analysis.

Coronavirus disease 2019 (COVID-19) is frequently associated with thrombo inflammation, which can predispose to developing of life-threatening conditions in children such as the multisystem inflammatory syndrome (MIS-C) and Kawasaki disease. Because of the consistent overlap in pathogenesis and symptoms, identifying laboratory tests that may aid in the differential diagnosis of these pathologies becomes crucial. We performed an electronic search in PubMed, Web of Science and Scopus, without date or language restrictions, to identify all possible studies reporting D-dimer values in separate cohorts of children with MIS-C or Kawasaki disease. Three multicenter cohort studies were included in our analysis, totaling 487 patients (270 with MIS-C and 217 with Kawasaki disease). In this meta-analysis, significantly higher D-dimer values were found in MIS-C compared to Kawasaki disease in all three studies, yielding an SMD of 1.5 (95 % CI, 1.3-1.7) mg/L. Thus, very high D-dimer values early in the course of disease should raise the clinical suspicion of MIS-C rather than Kawasaki disease. Further studies should be planned to identify harmonized D-dimer diagnostic thresholds that may help discriminate these conditions.

Venous Thrombosis in Airborne Viral Infections: Is Coronavirus Disease 2019 now Any Different from Influenza?

One of the hallmarks of coronavirus disease 2019 (COVID-19), particularly in complicated cases (i.e., requiring hospitalization or intensive care support), is persistent hemostasis activation, which may be associated with a vast array of thrombotic episodes involving both the arterial and venous systems. The renewed emphasis on the relationship between viral infections and venous thrombosis paves the way for determining whether a more common and often underestimated infection disease, such as influenza, may also be associated with a significant burden of venous thrombotic episodes, and how this eventual thrombotic risk compares to that seen in COVID-19, both in the past and with newer variants. Our review of studies comparing the burden of venous thromboembolism (VTE) in patients with COVID-19 or influenza revealed that the thrombotic risk appears to be significantly higher in patients with COVID-19 but remains certainly not meaningless in those with influenza, particularly in subjects infected by highly virulent strains (i.e., H1N1), in those who develop pneumonia and require intensive care support. In these specific clinical settings, the adoption of tailored thromboprophylaxis may be indicated though more studies are compellingly needed on this matter. As COVID-19 variants emerge, there is a possibility that the VTE burden of COVID-19 will decrease, and progress to that of other respiratory viruses.

Evolution of Hemostasis Testing: A Personal Reflection Covering over 40 Years of History.

There is no certainty in change, other than change is certain. As Seminars in Thrombosis and Hemostasis celebrates 50 years of publication, I felt it appropriate to reflect on my own 40-year plus scientific career. My career in the thrombosis and hemostasis field did not start until 1987, but the subsequent 35 years reflected a period of significant change in associated disease diagnostics. I started in the Westmead Hospital "coagulation laboratory" when staff were still performing manual clotting tests, using stopwatches, pipettes, test tubes, and a water bath, which we transported to the hospital outpatient department to run our weekly warfarin clinic. Several hemostasis instruments have come and gone, including the Coag-A-Mate X2, the ACL-300R, the MDA-180, the BCS XP, and several StaR Evolution analyzers. Some instruments remain, including the PFA-100, PFA-200, the AggRAM, the CS-5100, an AcuStar, a Hydrasys gel system, and two ACL-TOP 750s. We still have a water bath, but this is primarily used to defrost frozen samples, and manual clotting tests are only used to teach visiting medical students. We have migrated across several methodologies in the 45-year history of the local laboratory. Laurel gel rockets, used for several assays in the 1980s, were replaced with enzyme-linked immunosorbent assay assays and most assays were eventually placed on automated instruments. Radio-isotopic assays, used in the 1980s, were replaced by an alternate safer method or else abandoned. Test numbers have increased markedly over time. The approximately 31,000 hemostasis assays performed at the Westmead-based laboratory in 1983 had become approximately 200,000 in 2022, a sixfold increase. Some 90,000 prothrombin times and activated partial thromboplastic times are now performed at this laboratory per year. Thrombophilia assays were added to the test repertoires over time, as were the tests to measure several anticoagulant drugs, most recently the direct oral anticoagulants. I hope my personal history, reflecting on the changes in hemostasis testing over my career to date in the field, is found to be of interest to the readership, and I hope they forgive any inaccuracies I have introduced in this reflection of the past.

The Role of the von Willebrand Factor Collagen-Binding Assay (VWF:CB) in the Diagnosis and Treatment of von Willebrand Disease (VWD) and Way Beyond: A Comprehensive 36-Year History.

The von Willebrand factor (VWF) collagen binding (VWF:CB) assay was first reported for use in von Willebrand diagnostics in 1986, by Brown and Bosak. Since then, the VWF:CB has continued to be used to help diagnose von Willebrand disease (VWD) (correctly) and also to help assign the correct subtype, as well as to assist in the monitoring of VWD therapy, especially desmopressin (DDAVP). However, it is important to recognize that the specific value of any VWF:CB is predicated on the use of an optimized VWF:CB, and that not all VWF:CB assays are so optimized. There are some good commercial assays available, but there are also some "not-so-good" commercial assays available, and these may continue to give the VWF:CB "a bad reputation." In addition to VWD diagnosis and management, the VWF:CB found purpose in a variety of other applications, from assessing ADAMTS13 activity, to investigation into acquired von Willebrand syndrome (especially as associated with use of mechanical circulatory support or cardiac assist devices), to assessment of VWF activity in disease states in where an excess of high-molecular-weight VWF may accumulate, and lead to increased (micro)thrombosis risk (e.g., coronavirus disease 2019, thrombotic thrombocytopenic purpura). The VWF:CB turns 37 in 2023. This review is a celebration of the utility of the VWF:CB over this nearly 40-year history.

Epidemiology and Predisposing Factors of Post-COVID Venous Thrombosis: A Concise Review.

Long-coronavirus disease 2019 (COVID-19) represents a heterogeneous clinical syndrome characterized by a pathologic continuum of signs, symptoms, and also laboratory/radiologic abnormalities that may persist for a long time after recovering from an acute severe acute respiratory syndrome-coronavirus disease 2 infection. Among the various components of this postviral condition, the risk of venous thromboembolism in patients hospitalized for COVID-19 remains considerably higher after discharge, especially in older individuals, in men, in patients with longer hospital stays and more aggressive treatment (e.g., mechanical ventilation and/or intensive care), when thromboprophylaxis is not used, and in those with a persistent prothrombotic state. Patients who have these predisposing factors should be monitored more closely to intercept any thrombosis that may occur in a post-COVID time-related manner but may also benefit from extended thromboprophylaxis and/or antiplatelet therapy.

Effects of Recombinant SARS-CoV-2 Spike Protein Variants on Platelet Morphology and Activation.

Platelets are central elements of hemostasis and also play a pivotal role in the pathogenesis of thrombosis in coronavirus disease 2019. This study was planned to investigate the effects of different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recombinant spike protein variants on platelet morphology and activation. Citrated whole blood collected from ostensibly healthy subjects was challenged with saline (control sample) and with 2 and 20 ng/mL final concentration of SARS-CoV-2 recombinant spike protein of Ancestral, Alpha, Delta, and Omicron variants. Platelet count was found to be decreased with all SARS-CoV-2 recombinant spike protein variants and concentrations tested, achieving the lowest values with 20 ng/mL Delta recombinant spike protein. The mean platelet volume increased in all samples irrespective of SARS-CoV-2 recombinant spike protein variants and concentrations tested, but especially using Delta and Alpha recombinant spike proteins. The values of both platelet function analyzer-200 collagen-adenosine diphosphate and collagen-epinephrine increased in all samples irrespective of SARS-CoV-2 recombinant spike protein variants and concentrations tested, and thus reflecting platelet exhaustion, and displaying again higher increases with Delta and Alpha recombinant spike proteins. Most samples where SARS-CoV-2 recombinant spike proteins were added were flagged as containing platelet clumps. Morphological analysis revealed the presence of a considerable number of activated platelets, platelet clumps, platelet-monocyte, and platelet-neutrophils aggregates, especially in samples spiked with Alpha and Delta recombinant spike proteins at 20 ng/mL. These results provide support to the evidence that SARS-CoV-2 is capable of activating platelets through its spike protein, though such effect varies depending on different spike protein variants.