Assessing Direct Oral Anticoagulants in the Clinical Laboratory.

Direct oral anticoagulants (DOACs) have significant advantages over vitamin K antagonists including lack of need for routine laboratory monitoring. However, assessment of DOAC effect and concentration may be important to guide clinical management including need for DOAC reversal, particularly in acute or emergent situations. In this manuscript, the authors describe tests to screen for DOAC presence and tests that have demonstrated equivalence to gold standard testing for quantifying DOAC exposure. They also discuss the effect of DOACs on other coagulation assays and strategies for monitoring unfractionated heparin in patients with concomitant DOAC exposure.

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.

International Council for Standardization in Haematology Guidance for New Lot Verification of Coagulation Reagents, Calibrators, and Controls.

The clinical laboratory uses commercial products with limited shelf life or certain expiry dates requiring frequent lot changes. Prior to implementation for clinical use, laboratories should determine the performance of the new reagent lot to ensure that there is no significant shift in reagent performance or reporting of patient data. This guideline has been written on behalf of the International Council for Standardization in Haematology (ICSH) to provide the framework and provisional guidance for clinical laboratories for evaluating and verifying the performance of new lot reagents used for coagulation testing. These ICSH Working Party consensus recommendations are based on good laboratory practice, regulatory recommendations, evidence emerged from scientific publications, and expert opinion and are meant to supplement regional standards, regulations, or requirements.

International Council for Standardization in Haematology Field Study Evaluating Optimal Interpretation Methods for Activated Partial Thromboplastin Time and Prothrombin Time Mixing Studies.

CONTEXT.­: The prothrombin time (PT) and activated partial thromboplastin time (APTT) are screening tests used to detect congenital or acquired bleeding disorders. An unexpected PT and/or APTT prolongation is often evaluated using a mixing test with normal plasma. Failure to correct ("noncorrection") prolongation upon mixing is attributed to an inhibitor, whereas "correction" points to factor deficiency(ies). OBJECTIVE.­: To define an optimal method for determining correction or noncorrection of plasma mixing tests through an international, multisite study that used multiple PT and APTT reagents and well-characterized plasma samples. DESIGN.­: Each testing site was provided 22 abnormal and 25 normal donor plasma samples, and mixing studies were performed using local PT and APTT reagents. Mixing study results were evaluated using 11 different calculation methods to assess the optimal method based on the expected interpretation for factor deficiencies (correction) and noncorrection (inhibitor effect). Misprediction, which represents the failure of a mixing study interpretation method, was assessed. RESULTS.­: Percentage correction was the most suitable calculation method for interpreting PT mixing test results for nearly all reagents evaluated. Incubated PT mixing tests should not be performed. For APTT mixing tests, percentage correction should be performed, and if the result indicates a factor deficiency, this should be confirmed with the subtraction III calculation where the normal pooled plasma result (run concurrently) is subtracted from the mixing test result with correction indicated by a result of 0 or less. In general, other calculation methods evaluated that performed well in the identification of factor deficiency tended to have high misprediction rates for inhibitors and vice versa. CONCLUSIONS.­: No single method of mixing test result calculation was consistently successful in accurately distinguishing factor deficiencies from inhibitors, with between-reagent and between-site variability also identified.

Preanalytical Variables in Hemostasis Testing.

Hemostasis testing performed in clinical laboratories are critical for assessing hemorrhagic and thrombotic disorders. The assays performed can be used to provide the information required for diagnosis, risk assessment, efficacy of therapy, and therapeutic monitoring. As such, hemostasis tests should be performed to the highest level of quality, including the standardization, implementation, and monitoring of all phases of the testing, which include the preanalytical, analytical, and post-analytical phases. It is well established that the preanalytical phase is the most critical component of the testing process, being the hands-on activities, including patient preparation for blood collection, as well as the actual blood collection, including sample identification and the post-collection handling to include sample transportation, processing, and storage of samples when testing is not performed immediately. The purpose of this article is to provide an update to the previous edition of coagulation testing-related preanalytical variables (PAV) and, when properly addressed and performed, can reduce the most common causes of errors in the hemostasis laboratory.

Ultracentrifugation for Coagulation Testing.

Lipemia is known to potentially affect coagulation testing. It may be detected with newer coagulation analyzers that are validated to assess hemolysis, icterus, and lipemia (HIL) in a plasma sample. In samples with lipemia where accuracy of the test result is compromised, strategies for mitigating the lipemia interferences would be required. The tests affected by lipemia are those using chronometric, chromogenic, immunologic, or other light scattering/reading principles. Ultracentrifugation is one process that has been effectively demonstrated to remove lipemia from blood samples to allow for more accurate measurements. In this chapter, a description of one ultracentrifugation method is provided.

Hemostasis and Thrombosis: An Overview Focusing on Associated Laboratory Testing to Diagnose and Help Manage Related Disorders.

Hemostasis is a complex but balanced process that permit normal blood flow, without adverse events. Disruption of the balance may lead to bleeding or thrombotic events, and clinical interventions may be required. Hemostasis laboratories typically offer an array of tests, including routine coagulation and specialized hemostasis assays used to guide clinicians for diagnosing and managing patients. Routine assays may be used to screen patients for hemostasis-related disturbances but may also be used for drug monitoring, measuring efficacy of replacement or adjunctive therapy, and other indications, which may then be used to guide further patient management. Similarly, "specialized" assays are used for diagnostic purposes or may be used to monitor or measure efficacy of a given therapy. This chapter provides an overview of hemostasis and thrombosis, with a focus on laboratory testing that may be used to diagnose and help manage patients suspected of hemostasis- and thrombosis-related disorders.

Protocols for D-Dimer Measurements for Aid to Diagnosis or Exclusion of Venous Thromboembolism.

Measuring D-dimer is commonly used as a surrogate to indicate a clot-forming process, with subsequent lysis. This test has two primary intended uses: (1) as aid to diagnosis of various conditions and (2) venous thromboembolism (VTE) exclusion. If the manufacturer cites a VTE exclusion claim, the D-dimer test must only be used in evaluating patients with a non-high or unlikely pretest probability for pulmonary embolism and deep vein thrombosis. D-dimer kits with only aid to diagnosis claim should not be used for VTE exclusion. The intended use of the D-dimer may vary by region, and readership should consult manufacturer instructions for use to assure proper use of the assay. In this chapter, several methods for measuring D-dimer will be described.

An Acute Need Inspiration: Autoneutralization of Lupus Anticoagulants in Affected Prothrombin Times (PT) and Activated Partial Thromboplastin Times (APTT).

Lupus anticoagulants are antibodies directed to phospholipids (PL) and in particular represent an in vitro phenomenon where these antibodies bind to PL in coagulation reagents creating an artificial prolongation of the activated partial thromboplastin time (APTT) and sometimes also prothrombin time (PT) clotting times. Prolongation of LA-induced clotting times is typically not associated with bleeding risk. However, the degree of prolongation may cause some trepidation for clinicians that will be performing delicate surgeries or those with high bleeding risks, so a mechanism to alleviate their anxiety may be prudent. As such, an autoneutralizing method to mitigate or eliminate the LA effect on the PT and APTT may be beneficial. In this document, the detailing of an autoneutralizing procedure to diminish the LA effect on the PT and APTT will be provided.

Ecarin-Based Methods for Measuring Thrombin Inhibitors.

Ecarin is a venom from the saw-scaled viper, Echis carinatus, which catalyzes prothrombin into meizothrombin. This venom is used in several hemostasis laboratory assays, including ecarin clotting time (ECT) and ecarin chromogenic assays (ECA). The use of these ecarin-based assays was first implemented as a tool for monitoring the infusion of a direct thrombin inhibitor, hirudin. Subsequently, this method has been more recently employed for measuring either the pharmacodynamic or pharmacokinetic properties of the oral direct thrombin inhibitor, dabigatran. In this chapter, the procedure for performing manual ECT and automated and manual ECA for measuring thrombin inhibitors is described.

Preparing Surrogate Abnormal Quality Control Samples for Platelet Function Studies and Viscoelastic Testing.

In the United States, published options for clinical laboratories to perform quality control (QC) procedures less stringent than the regulatory requirements (Clinical and Laboratory Improvement Act, CLIA) based on risk assessment, although the laboratory must perform to manufacturer's minimum requirements. The US requirements for internal quality control requires at least two levels of control material every 24 h of patient testing. For some coagulation testing, the recommended quality control may be a normal sample or commercial controls that do not address all reporting components of the test. Additional circumstances and difficulties in achieving this minimum QC requirement can be due to either (1) nature of the sample type (i.e., whole blood sample requirements), (2) lack of commercial or suitable control material, or (3) unusual or rare samples. The purpose of this chapter is to provide provisional guidance for laboratory sites to prepared samples to verify the performance of reagents and testing performance of platelet function studies and viscoelastic measurements.

Post-analytical Issues in Hemostasis and Thrombosis Testing: An Update.

There are typically three phases identified as contributing to the total testing process. The preanalytical phase starts with the clinician and the patient, when laboratory testing is being considered. This phase also includes decisions about which tests to order (or not), patient identification, blood collection, blood transport, sample processing, and storage to name a few. There are many potential failures that may occur in this preanalytical phase, and these are the topic of another chapter in this book. The second phase, the analytical phase, represents the performance of the test, which is essentially covered in various protocols in this book and the previous edition. The third is the post-analytical phase, which is what occurs after sample testing, and is the topic of the current chapter. Post-analytical issues are generally related to reporting and interpretation of test results. This chapter provides a brief description of these events, as well as guidance for preventing or minimizing post-analytical issues. In particular, there are several strategies for improved post-analytical reporting of hemostasis assays, with this providing the final opportunity to prevent serious clinical errors in patient diagnosis or management.

The Future of Laboratory-Developed Tests in Hemostasis Laboratories.

Laboratory-developed tests (LDTs) are widely used in clinical hemostasis laboratories. The extent to which LDTs are regulated varies greatly around the world, and proposed changes to regulations have raised concerns about the future of LDTs in clinical laboratories. It is increasingly difficult to justify the use of an LDT where a commercially available method with regulatory approval is available. Conversely, where there is no suitable test with regulatory approval and there is a compelling clinical need, using an LDT outweighs the risk associated with not performing the test. We argue that LDTs are still required in specialist clinical laboratories to fulfill unmet clinical needs, and in lower middle-income countries where they are a vital resource.

Factor VIII and Factor IX Activity Measurements for Hemophilia Diagnosis and Related Treatments.

Accurate measurement of clotting factors VIII (FVIII) or IX (FIX) is vital for comprehensive diagnosis and management of patients with hemophilia A or B. The one-stage activated partial thromboplastin time (aPTT)-based clotting assay is the most commonly used method worldwide for testing FVIII or FIX activities. Alternatively, FVIII and FIX chromogenic substrate assays, which assess the activation of factor X, are available in some specialized laboratories. The choice of reagent or methodology can strongly influence the resulting activity. Variation between one-stage FVIII or FIX activities has been reported in the measurement of some standard and extended half-life factor replacement therapies and gene therapy for hemophilia B using different aPTT reagents. Discrepancy between one-stage and chromogenic reagents has been demonstrated in some patients with mild hemophilia A or B, the measurement of some standard and extended half-life factor replacement therapies, and the transgene expression of hemophilia A and B patients who have received gene therapy. Finally, the measurement of bispecific antibody therapy in patients with hemophilia A has highlighted differences between chromogenic assays. It is imperative that hemostasis laboratories evaluate how suitable their routine assays are for the accurate measurement of the various hemophilia treatment therapies.

Activated Partial Thromboplastin Time and Prothrombin Time Mixing Studies: Current State of the Art.

Mixing studies have long been in the clinical laboratory armamentarium for investigating unexpected, prolonged activated partial thromboplastin time (aPTT) or prothrombin time (PT). The purpose of the mixing study is to identify whether the aPTT/PT prolongation is secondary to a factor deficiency versus an inhibitor, which would present as a "corrected" and "noncorrected" mixing study, respectively. The differentiation between a factor deficiency and inhibitor may likely further direct clinical decisions, including additional diagnostic testing or factor replacement therapy. While aPTT/PT mixing studies are simple tests to perform, there is a lack of standardization for both the testing protocol and the interpretation of what is considered to be a corrected or noncorrected mixing study result. This review will describe the common indications for the mixing test, preanalytic variables that may affect mixing study performance, and describe several methods for interpreting the results of aPTT and PT mixing tests.

From Ink Pens to Computers: A Personal Look Back at Landmark Changes during 5 Decades as a Clinical Laboratory Scientist in U.S. Hemostasis Laboratories.

In 2023, Seminars in Thrombosis and Hemostasis will be celebrating its 50th anniversary, and similarly this will also mark my 5th decade of working in, or association with, laboratories that perform hemostasis testing. My career started at a large military medical center, but I also worked at several other facilities, including military dispensaries, community hospitals, and a large academic institution. The difference between each type of hemostasis laboratory was as expected, with larger facilities having better instrumentation and more prolific test menus. However, whether one worked in a large academic center, or a small rural hospital, regulatory changes affected every clinical laboratory to the same degree. Advances in technology also eventually affected every hemostasis laboratory, but these salient changes were more likely to occur earlier at the larger institutions. As Seminars in Thrombosis and Hemostasis celebrates its 50th anniversary, that milestone triggered recollection about those salient events that occurred during my own career in hemostasis testing. As such, I describe (my impression) the top ten landmark changes that altered laboratory practice at the facilities where I worked during the past 5 decades.

The myths behind DOAC measurement: Analyses of prescribing information from different regulatory bodies and a call for harmonization.

For more than a decade, US laboratories have failed to implement solutions to help their clinicians in managing complex situations or patients on direct oral anticoagulants (DOACs). The problem may find different origins, among which is the position of the Food and Drug Administration, which categorized these drugs as monitoring- and measurement-free, whereas other regulatory bodies like the European Medicines Agency or the Therapeutic Goods Administration in Australia were more conservative on the principle that the absence of proof (of monitoring/measurement benefits) is not proof of an absence (of monitoring/measurement needs). Pivotal clinical studies that led to the approval of DOACs were presented as devoid of such testing, although some companies considered monitoring as a solution to improve their benefit/risk ratio. In this JTH In Clinics issue, we report more than a decade of development that has permitted the activation of smart laboratory solutions to qualify or quantify DOACs and discuss myths and misconceptions around technical and regulatory requirements that support the current reluctance of implementing these technologies in most US laboratories. Use of DOACs is ever expanding, with DOAC prescriptions now exceeding those of other anticoagulants, including vitamin K antagonists, in some geographies. As this use increases, the likely need to measure DOAC exposure will also increase. Measurement of DOACs does not represent any technical difficulty. That these laboratory tests are not available in some locations suggests disparities in patient care, and we suggest it is time to address such inequalities.