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.

Immature platelets and platelet reactivity in patients with acute ST-segment Elevation Myocardial Infarction using whole blood flow cytometry with SYTO-13 staining.

BACKGROUND: Reduced effect of antiplatelet therapy has been reported in patients with ST-segment elevation myocardial infarction (STEMI). Multiple factors may concur to explain this, including increased amount of highly reactive immature platelets. OBJECTIVES: To investigate the association between immature platelets and reactivity determined with multicolour flow cytometry using the SYTO-13 dye in STEMI patients. METHODS: We conducted an observational study of 59 patients with acute STEMI. Blood samples were obtained within 24 h after admission and after loading doses of dual antiplatelet therapy. For comparison, samples were obtained from 50 healthy individuals. Immature platelets and platelet reactivity were investigated using multicolour flow cytometry including the SYTO-13 dye that binds to platelet RNA and thus provides a method for subdividing platelets into immature and mature platelets. Additionally, we assessed platelet aggregation, serum-thromboxane B2 levels and standard immature platelet markers. RESULTS: Immature platelets were more reactive than mature platelets in both STEMI patients and healthy individuals (p-values < 0.05). STEMI patients had lower platelet aggregation and thromboxane B2 levels than healthy individuals. We found a positive association between automatically determined immature platelet markers and CD63 expression on activated platelets (Spearman's rho: 0.27 to 0.58, p-values < 0.05). CONCLUSIONS: Our study shows that immature platelets identified with a multicolour flow cytometric method using the SYTO-13 dye are more reactive than mature platelets in patients with acute STEMI and in healthy individuals. The presence of immature platelets may be important for the overall platelet reactivity, which may have implications for the effect of antiplatelet therapy.

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.

Platelet Function and Maturity and Related microRNA Expression in Whole Blood in Patients with ST-Segment Elevation Myocardial Infarction.

BACKGROUND: Reduced effect of antiplatelet therapy has been reported in patients with ST-segment elevation myocardial infarction (STEMI). MicroRNAs (miRs) may influence platelet function and maturity, and subsequently the effect of antiplatelet therapy. OBJECTIVES: We aimed to explore the association between miR expression and platelet function and maturity in patients with acute STEMI and healthy individuals. METHODS: We performed an observational study of STEMI patients admitted directly to primary percutaneous coronary intervention. Patients were treated with antiplatelet therapy according to guidelines. Within 24 hours after admission, blood samples were obtained to measure: the expression of 10 candidate miRs, platelet function markers using advanced flow cytometry, platelet aggregation, serum thromboxane B2, and platelet maturity markers. Furthermore, blood samples from healthy individuals were obtained to determine the normal variation. RESULTS: In total, 61 STEMI patients and 50 healthy individuals were included. STEMI patients had higher expression of miR-21-5p, miR-26b-5p, and miR-223-3p and lower expression of miR-150-5p, miR423-5p, and miR-1180-3p than healthy individuals. In STEMI patients, the expression of miR-26b-5p showed the most consistent association with platelet function (all p-values <0.05, Spearman's rho ranging from 0.27 to 0.41), while the expression of miR-150-5p and miR-223-3p showed negative associations with platelet function. No association between miR expression and platelet maturity markers was observed. CONCLUSION: In patients with STEMI, the expression of six miRs was significantly different from healthy individuals. The expression of miR-26b-5p may affect platelet function in acute STEMI patients and potentially influence the effect of antiplatelet therapy.

Laboratory diagnosis of von Willebrand disease in the age of the new guidelines: considerations based on geography and resources.

von Willebrand disease (VWD) is considered the most common bleeding disorder and arises from deficiency and/or defect in the adhesive plasma protein von Willebrand factor (VWF). Diagnosis of VWD requires clinical assessment and is facilitated by laboratory testing. Several guidelines for VWD diagnosis exist, with the latest American Society of Hematology, International Society on Thrombosis and Haemostasis, National Hemophilia Foundation, and World Federation of Hemophilia 2021 guidelines presenting 11 recommendations, some of which have drawn controversy. In the current narrative review, we provide additional context around difficulties in laboratory diagnosis/exclusion/typing of VWD, with a focus on developing countries/resource-poor settings. In particular, there are many variations in assay methodology, and some methods express high assay variability and poor low-level VWF sensitivity that compromises their utility. Although we favor an initial 4-test assay panel, comprising factor (F) VIII coagulant activity, VWF antigen, VWF glycoprotein Ib binding (VWF:GPIbR or VWF:GPIbM favored over VWF Ristocetin cofactor) and VWF collagen binding, we also provide strategies for laboratories only able to incorporate an initial 3-test assay panel, as favored by the latest guidelines, to improve diagnostic accuracy.

Focal Anticoagulation by Somatic Gene Transfer: Towards Preventing Cardioembolic Stroke.

Cardioembolic stroke (CS) has emerged as a leading cause of ischaemic stroke (IS); distinguished by thrombi embolising to the brain from cardiac origins; most often from the left atrial appendage (LAA). Contemporary therapeutic options are largely dependent on systemic anticoagulation as a blanket preventative strategy, yet this does not represent a nuanced or personalised solution. Contraindications to systemic anticoagulation create significant unmedicated and high-risk cohorts, leaving these patients at risk of significant morbidity and mortality. Atrial appendage occlusion devices are increasingly used to mitigate stroke risk from thrombi emerging from the LAA in patients ineligible for oral anticoagulants (OACs). Their use, however, is not without risk or significant cost, and does not address the underlying aetiology of thrombosis and CS. Viral vector-based gene therapy has emerged as a novel strategy to target a spectrum of haemostatic disorders, achieving success through the adeno-associated virus (AAV) based therapy of haemophilia. Yet, thrombotic disorders, such as CS, have had limited exploration within the realm of AAV gene therapy approaches-presenting a gap in the literature and an opportunity for further research. Gene therapy has the potential to directly address the cause of CS by localised targeting of the molecular remodelling that serves to promote thrombosis.

Harmonization of Hemostasis Testing Across a Large Laboratory Network: An Example from Australia.

Harmonization and standardization of laboratory tests and procedures carry a variety of benefits. For example, within a laboratory network, harmonization/standardization provides a common platform for test procedures and documentation across different laboratories. This enables staff to be deployed across several laboratories, if required, without additional training, since test procedures and documentation are the "same" in the different laboratories. Streamlined accreditation of laboratories is also facilitated, as accreditation in one laboratory using a particular procedure/documentation should simplify the accreditation of another laboratory in that network to the same accreditation standard. In the current chapter, we detail our experience regarding the harmonization and standardization of laboratory tests and procedures related to hemostasis testing in our laboratory network, NSW Health Pathology, representing the largest public pathology provider in Australia, with over 60 separate laboratories.

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.

Auto-validation of Routine Coagulation/Hemostasis Assays with Reflex Testing of Abnormal Test Results.

Hemostasis laboratories play a crucial role in the diagnosis and treatment of individuals with bleeding or thrombotic disorders. Routine coagulation assays, including the prothrombin time (PT)/international normalized ratio (INR), and activated partial thromboplastin time (APTT), are used for various purposes. These include as a screen of hemostasis function/dysfunction (e.g., possible factor deficiency) and for monitoring of anticoagulant therapy, such as vitamin K antagonists (PT/INR) and unfractionated heparin (APTT). Clinical laboratories are also under increasing pressure to improve services, especially response (test turnaround) times. There is also a need for laboratories to try to reduce error rates and for laboratory networks to standardize/harmonize processes and policies. Accordingly, we describe our experience with the development and implementation of automated processes for reflex testing and validation of routine coagulation test results. This has been implemented in a large pathology network compromising 27 laboratories and is under consideration for expansion to our larger network (of 60 laboratories). These rules have been custom-built within our laboratory information system (LIS), perform reflex testing of abnormal results, and fully automate the process of routine test validation for appropriate results. These rules also permit adherence to standardized pre-analytical (sample integrity) checks, automate reflex decisions, automate verification, and provide an overall alignment of network practices in a large network of 27 laboratories. In addition, the rules enable clinically significant results to be quickly referred to hematopathologists for review. We also documented an improvement in test turnaround times, with savings in operator time and thus operating costs. Finally, the process was generally well received and determined to be beneficial for most laboratories in our network, in part identified by improved test turnaround times.

Laboratory Testing for Activated Protein C Resistance (APCR): An Update.

Activated protein C resistance (APCR) reflects a hemostatic state defined by a reduced ability of activated protein C (APC) to affect an anticoagulant response. This state of hemostatic imbalance is characterized by a heightened risk of venous thromboembolism. Protein C is an endogenous anticoagulant that is produced by the hepatocytes and undergoes proteolysis-mediated activation to APC. APC in turn degrades activated Factors V and VIII. APCR describes a state of resistance by activated Factors V and VIII to APC-mediated cleavage of these factors, thereby promoting amplified thrombin production and a potentially procoagulant state. This resistance of APC may be inherited or acquired. Mutations in Factor V are responsible for the most frequent form hereditary APCR. The predominant mutation, a G1691A missense mutation at Arginine 506, the so-called Factor V Leiden [FVL], causes a deletion of an APC-targeted cleavage site in Factor Va, thereby rendering it resistant to inactivation by APC. There are a variety of laboratory assays for APCR, but this chapter focuses on a procedure using a commercially available clotting assay that utilizes a snake venom and ACL TOP analyzers.

An Overview of Laboratory Testing for Antiphospholipid Antibodies.

Antiphospholipid antibodies (aPL) represent a group of autoantibodies directed against phospholipids. These antibodies may arise in a number of autoimmune conditions, of which the antiphospholipid (antibody) syndrome (APS) is best recognized. aPL can be detected by various laboratory assays, essentially comprising both solid-phase (immunological) assays and "liquid-phase" clotting assays identifying so-called lupus anticoagulants (LA). aPL are associated with various adverse pathologies, including thrombosis and placental/fetal morbidity and mortality. The type of aPL present, as well as the pattern of reactivity, is variously associated with the severity of the pathology. Thus, laboratory testing for aPL is indicated to help assess the future risk of such events, as well as representing certain "classification" criteria for APS, also used as surrogates for diagnostic criteria. The current chapter overviews the laboratory tests available to measure aPL and their potential clinical utility.

Antiphospholipid Antibody Testing for Anti-cardiolipin and Anti-ß2 Glycoprotein I Antibodies Using Chemiluminescence-Based Panels.

Antiphospholipid (antibody) syndrome (APS) is a prothrombotic condition with increased risk for thrombosis and pregnancy-related morbidity. In addition to clinical criteria related to these risks, APS is characterized by the persistent presence of antiphospholipid antibodies (aPL), as detected in the laboratory using a potentially wide variety of assays. The three APS criteria-related assays are lupus anticoagulant (LA), as detected using clot-based assays, and the solid-phase assays of anti-cardiolipin antibodies (aCL) and anti-ß2 glycoprotein I antibodies (aß2GPI), with immunoglobulin subclasses of IgG and/or IgM. These tests may also be used for the diagnosis of systemic lupus erythematosus (SLE). In particular, APS diagnosis/exclusion remains challenging for clinicians and laboratories because of the heterogeneity of clinical presentations in those being evaluated and the technical application and variety of the associated tests used in laboratories. Although LA testing is affected by a wide variety of anticoagulants, which are often given to APS patients to prevent any associated clinical morbidity, detection of solid-phase aPL is not influenced by these anticoagulants, and this thus represents a potential advantage to their application. On the other hand, various technical issues challenge accurate laboratory detection or exclusion of aPL. This report describes protocols for the assessment of solid-phase aPL, specifically aCL and aß2GPI of IgG and IgM class by means of a chemiluminescence-based assay panel. These protocols reflect tests able to be performed on the AcuStar instrument (Werfen/Instrumentation Laboratory). Certain regional approvals may also allow this testing to be performed on a BIO-FLASH instrument (Werfen/Instrumentation Laboratory).

Heparin-Induced Thrombotic Thrombocytopenia (HITT) and Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT): Similar but Different.

Heparin-induced thrombocytopenia (HIT) represents an autoimmune process whereby antibodies are formed against heparin in complex with platelet factor 4 (PF4) after heparin administration. These antibodies can be detected by a variety of immunological assays, including ELISA (enzyme-linked immunosorbent assay) and by chemiluminescence on the AcuStar instrument. However, pathological HIT antibodies are those that activate platelets in a platelet activation assay and cause thrombosis in vivo. We would tend to call this condition heparin-induced thrombotic thrombocytopenia (HITT), although some workers instead use the truncated abbreviation HIT. Vaccine-induced (immune) thrombotic thrombocytopenia (VITT) instead reflects an autoimmune process whereby antibodies are formed against PF4 after administration of a vaccine, most notably adenovirus-based vaccines directed against COVID-19 (coronavirus disease 2019). Although both VITT and HITT reflect similar pathological processes, they have different origins and are detected in different ways. Most notable is that anti-PF4 antibodies in VITT can only be detected immunologically by ELISA assays, tending to be negative in rapid assays such as that using the AcuStar. Moreover, functional platelet activation assays otherwise used for HITT may need to be modified to detect platelet activation in VITT.

An Overview of Laboratory Testing for ADAMTS13.

ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) is also called von Willebrand factor (VWF) cleaving protease (VWFCP). ADAMTS13 acts to cleave VWF multimers and thus reduce plasma VWF activity. In the absence of ADAMTS13 (i.e., in thrombotic thrombocytopenia purpura, TTP), plasma VWF can accumulate, in particular as "ultra-large" VWF multimers, and this can lead to thrombosis. Relative deficiencies in ADAMTS13 can also occur in a variety of other conditions, including secondary thrombotic microangiopathies (TMA). Of contemporary interest, COVID-19 (coronavirus disease 2019) may also be associated with relative reduction of ADAMTS13 and also pathological accumulation of VWF, with this likely contributing to the thrombosis risk seen in affected patients. Laboratory testing for ADAMTS13 can assist in the diagnosis of these disorders (i.e., TTP, TMA), as well as in their management, and can be achieved using a variety of assays. This chapter therefore provides an overview of laboratory testing for ADAMTS13 and the value of such testing to assist the diagnosis and management of associated disorders.

Automated and Rapid ADAMTS13 Testing Using Chemiluminescence: Utility for Identification or Exclusion of TTP and Beyond.

Thrombotic thrombocytopenic purpura (TTP) is a prothrombotic condition caused by a significant deficiency of the enzyme, ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). In the absence of adequate levels of ADAMTS13 (i.e., in TTP), plasma VWF accumulates, in particular as "ultra-large" VWF multimers, and this leads to pathological platelet aggregation and thrombosis. In addition to TTP, ADAMTS13 may be mildly to moderately reduced in a range of other conditions, including secondary thrombotic microangiopathies (TMA) such as those caused by infections (e.g., hemolytic uremic syndrome (HUS)), liver disease, disseminated intravascular coagulation (DIC), and sepsis, during acute/chronic inflammatory conditions, and sometimes also in COVID-19 (coronavirus disease 2019)). ADAMTS13 can be detected by a variety of techniques, including ELISA (enzyme-linked immunosorbent assay), FRET (fluorescence resonance energy transfer) and by chemiluminescence immunoassay (CLIA). The current report describes a protocol for assessment of ADAMTS13 by CLIA. This protocol reflects a rapid test able to be performed within 35 min on the AcuStar instrument (Werfen/Instrumentation Laboratory), although certain regional approvals may also permit this testing to be performed on a BioFlash instrument from the same manufacturer.

Identification of ADAMTS13 Inhibitors in Acquired TTP.

Thrombotic thrombocytopenic purpura (TTP) is a prothrombotic condition caused by a deficiency of ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). In turn, ADAMTS13 (also called von Willebrand factor (VWF) cleaving protease (VWFCP)) acts to cleave VWF multimers and thus reduce plasma VWF activity. In the absence of ADAMTS13 (i.e., in TTP), plasma VWF accumulates, in particular as "ultra-large" VWF multimers, and this leads to thrombosis. In most patients with confirmed TTP, ADAMTS13 deficiency is an acquired disorder due to the development of antibodies against ADAMTS13, which either promote clearance of ADAMTS13 from circulation or cause inhibition of ADAMTS13 activity. The current report describes a protocol for assessment of ADAMTS13 inhibitors, being antibodies that inhibit ADAMTS13 activity. The protocol reflects the technical steps that help identify inhibitors to ADAMTS13, whereby mixtures of patient plasma and normal plasma are then tested for residual ADAMTS13 activity in a Bethesda-like assay. The residual ADAMTS13 activity can be assessed by a variety of assays, with a rapid test able to be performed within 35 minutes on the AcuStar instrument (Werfen/Instrumentation Laboratory) used as an example in this protocol.

Laboratory Testing for von Willebrand Disease Using a Composite Rapid 3-Test Chemiluminescence-Based von Willebrand Factor Assay Panel.

von Willebrand disease (VWD) is the most commonly reported inherited bleeding disorder and may alternatively occur as an acquired von Willebrand syndrome (AVWS). VWD/AVWS develops from defects and/or deficiency in the adhesive plasma protein von Willebrand factor (VWF). VWD/AVWS diagnosis/exclusion remains challenging because of the heterogeneity of VWF defects and the technical limitations of many VWF tests, as well as the VWF test panels (number and type of tests) chosen by many laboratories. Laboratory testing for these disorders utilizes evaluation of VWF level and activity, with activity assessment needing several tests due to the many functions performed by VWF in order to help counteract bleeding. This report explains procedures for evaluating VWF level (antigen; VWF:Ag) and activity by means of a chemiluminescence-based panel. Activity assays comprise collagen binding (VWF:CB) and a ristocetin-based recombinant glycoprotein Ib-binding (VWF:GPIbR) assay that reflects a contemporary alternative to classical ristocetin cofactor (VWF:RCo). This 3-test VWF panel (Ag, CB, GPIbR [RCo]) reflects the only such composite panel available on a single platform and is performed on an AcuStar instrument (Werfen/Instrumentation Laboratory). Certain regional approvals may also allow this 3-test VWF panel to be performed on the BioFlash instrument (Werfen/Instrumentation Laboratory).