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.

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.

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.

Platelet-activating functional assay resolution in vaccine-induced immune thrombotic thrombocytopenia: differential alignment to PF4 ELISA platforms.

Background: Anti-platelet factor 4 (PF4) antibodies in vaccine-induced immune thrombotic thrombocytopenia (VITT) appear to be transient, with discrepant persistence depending on the platform used for detection. Objectives: We aimed to report a longitudinal study of antibody persistence using 2 ELISA platforms and 2 platelet-activating functional assays in a clinical cohort of patients with VITT referred for follow-up testing. Methods: In total, 32 Australian patients with VITT or pre-VITT, confirmed by expert adjudication, with samples referred for clinical follow-up were included. Clinical follow-up assays, including Stago and Hyphen ELISAs, procoagulant platelet flow cytometry, and modified PF4-serotonin-release assay, were performed according to the pattern of reactivity for that patient at diagnosis. Results: The median follow-up was 24 weeks after diagnosis. A general decline in anti-PF4 antibody levels and platelet-activating capacity over time was observed with a more rapid median time to resolution of 16 weeks by functional assay vs 24 weeks by Stago ELISA. Decline in platelet-activating antibody levels detected by functional assays mirrored Stago ELISA titer but not Hyphen. However, 87% of patients received a documented second vaccination and 74% received an mRNA booster with no reported adverse events. Conclusion: Anti-PF4 antibodies persist longer than functional platelet-activating antibodies in VITT but do not warrant avoidance of subsequent vaccinations. Persistence detection is assay-dependent. Stago ELISA may be a surrogate where functional assays are unavailable for follow-up testing of confirmed patients with VITT.

Autoimmune Diseases Affecting Hemostasis: A Narrative Review.

Hemostasis reflects a homeostatic mechanism that aims to balance out pro-coagulant and anti-coagulant forces to maintain blood flow within the circulation. Simplistically, a relative excess of procoagulant forces can lead to thrombosis, and a relative excess of anticoagulant forces can lead to bleeding. There are a wide variety of congenital disorders associated with bleeding or thrombosis. In addition, there exist a vast array of autoimmune diseases that can also lead to either bleeding or thrombosis. For example, autoantibodies generated against clotting factors can lead to bleeding, of which acquired hemophilia A is the most common. As another example, autoimmune-mediated antibodies against phospholipids can generate a prothrombotic milieu in a condition known as antiphospholipid (antibody) syndrome (APS). Moreover, there exist various autoimmunity promoting environments that can lead to a variety of antibodies that affect hemostasis. Coronavirus disease 2019 (COVID-19) represents perhaps the contemporary example of such a state, with potential development of a kaleidoscope of such antibodies that primarily drive thrombosis, but may also lead to bleeding on rarer occasions. We provide here a narrative review to discuss the interaction between various autoimmune diseases and hemostasis.

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.

D-dimer: old dogmas, new (COVID-19) tricks.

D-dimer is a fibrin degradation product encompassing multiple cross-linked D domains and/or E domains present in the original fibrinogen molecule, whose generation is only theoretically possible when hemostasis and fibrinolysis pathways are concomitantly activated. D-dimer measurement has now become a pillar in the diagnosis/exclusion and prognostication of venous thromboembolism (VTE) and disseminated intravascular coagulation (DIC), when incorporated into validated clinical algorithms and especially using age-adjusted diagnostic thresholds. Although emerging evidence is also supporting its use for predicting the duration of anticoagulant therapy in certain categories of patients, the spectrum of clinical applications is constantly expanding beyond traditional thrombotic pathologies to the diagnosis of acute aortic dissection, acute intestinal ischemia and cerebral venous thrombosis among others, embracing also clinical management of coronavirus disease 2019 (COVID-19). Recent findings attest that D-dimer elevations are commonplace in patients with severe acute respiratory syndrome (SARS-CoV-2) infection (especially in those with thrombosis), its value predicts the clinical severity (up to death) of COVID-19 and remains more frequently increased in COVID-19 patients with post-discharge clinical sequelae. Further, D-dimer-based anticoagulant escalation may be associated with a lower risk of death in patients with severe SARS-CoV-2 infection and, finally, D-dimer elevation post-COVID-19 vaccination mirrors an increased risk of developing vaccine-induced thrombocytopenia and thrombosis (VITT).

What We Know (and Do not Know) Regarding the Pathogenesis of Pulmonary Thrombosis in COVID-19.

The clinical course of coronavirus disease 2019 (COVID-19) is often complicated by the onset of venous thrombosis and thromboembolism (VTE), encompassing also pulmonary thrombosis. Recent statistics attests that the cumulative frequency of VTE can be as high as 30% in COVID-19 hospitalized patients, increasing to nearly 40 to 70% (depending on systematic screening) in those with severe illness, mechanical ventilation, or intensive care unit admission. The risk of venous thrombosis seems mostly limited to the active phase of disease, and is directly associated with some genetic (i.e., inherited prothrombotic predisposition) and demographical factors (male sex, overweight/obesity), disease severity (risk increasing progressively from hospitalization to development of severe illness, being the highest in patients needing mechanical ventilation and/or intensive care), presence and extent of pulmonary disease, coexistence of multiple risk factors (immobilization, mechanical ventilation, co- or superinfections), along with increased values of inflammatory and thrombotic biomarkers. At least three different phenotypes of pulmonary thrombosis may develop in COVID-19 patients, one caused by typical embolization from peripheral venous thrombosis (e.g., deep vein thrombosis), a second type triggered by local inflammation of nearby pulmonary tissue, and a third one mostly attributable to the prothrombotic state consequent to the pronounced systemic inflammatory response (i.e., the so-called cytokine storm) that is frequently observed in COVID-19. Although the pathogenesis of these three conditions has different features, their discrimination is essential for diagnostic and therapeutic purposes. The prognosis of COVID-19 patients who develop pulmonary thrombosis is also considerably worse than those who do not, thus probably needing frequent monitoring and more aggressive therapeutic management.

Mechanisms of Thrombosis in Heparin-Induced Thrombocytopenia and Vaccine-Induced Immune Thrombotic Thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombotic thrombocytopenia (VITT) are rare, iatrogenic immune-mediated conditions with high rates of thrombosis-related morbidity and mortality. HIT is a long-recognized reaction to the administration of the common parenterally administered anticoagulant heparin (or its derivatives), while VITT is a new, distinct syndrome occurring in response to adenovirus-based vaccines against coronavirus disease 2019 and potentially other types of vaccines. A feature of both HIT and VITT is paradoxical thrombosis despite a characteristic low platelet count, mediated by the presence of platelet-activating antibodies to platelet factor 4. Several additional factors have also been suggested to contribute to clot formation in HIT and/or VITT, including monocytes, tissue factor, microparticles, endothelium, the formation of neutrophil extracellular traps, complement, procoagulant platelets, and vaccine components. In this review, we discuss the literature to date regarding mechanisms contributing to thrombosis in both HIT and VITT and explore the pathophysiological similarities and differences between the two conditions.

D-dimers-"Normal" Levels versus Elevated Levels Due to a Range of Conditions, Including "D-dimeritis," Inflammation, Thromboembolism, Disseminated Intravascular Coagulation, and COVID-19.

D-dimers reflect a breakdown product of fibrin. The current narrative review outlines how D-dimers can arise in normal individuals, as well as in patients suffering from a wide range of disease states. D-dimers in normal individuals without evident thrombosis can arise from background fibrinolytic activity in various tissues, including kidney, mammary and salivary glands, which ensures smooth flow of arising fluids where any blood contamination could be immediately lysed. In addition, healthy individuals can also regularly sustain minor injuries, often unbeknown to them, and wound healing follows clot formation in these situations. D-dimers can also arise in anxiety and following exercise, and are also markers of inflammation. Lung inflammation (triggered by microbes or foreign particles) is perhaps also particularly relevant, since the hemostasis system and fibrinolysis help to trap and remove such debris. Lung inflammation in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may contribute to D-dimer levels additive to thrombosis in patients with COVID-19 (coronavirus disease 2019). Indeed, severe COVID-19 can lead to multiple activation events, including inflammation, primary and secondary hemostasis, and fibrinolysis, all of which may contribute to cumulative D-dimer development. Finally, D-dimer testing has also found a role in the diagnosis and triaging of the so-called (COVID-19) vaccine-induced thrombotic thrombocytopenia.

Heparin: The Journey from Parenteral Agent to Nasal Delivery.

Although the worldwide usage of direct oral anticoagulants has continuously increased over the past decade, heparin remains an important weapon in the current arsenal of anticoagulant drugs. Parenteral heparin administration (i.e., either intravenously or subcutaneously) has represented for decades the only possible route for generating a significant anticoagulant effect, although being notoriously associated with some important drawbacks such as discomfort and risk of low compliance, thus paving the way to searching for more amenable means of administration. We provide here an updated analysis of animal and human studies that have explored the feasibility, suitability, and efficiency of heparin administration through the unconventional nasal route, as a possible alternative to the more traditional parenteral injection. The major hurdles that contribute to impair intranasal absorption and systemic delivery of heparin are represented by its relatively high molecular weight and negative charge. Therefore, although pure drug administration would not be associated with efficient nasal adsorption, or by systemic biological activity (i.e., anticoagulant effect), the combination of low molecular weight heparins and absorption enhancers such as surfactants, mucoadhesive, cyclodextrins, polyethylenimines and encapsulation into (nano)carriers seems effective to at least partially improve drug transport through the nasal route and allow systemic delivery in animals. Besides generating anticoagulant effects, intranasal heparin administration can also produce local pleiotropic effects, mostly related to anti-inflammatory properties, such as attenuating airway allergic inflammation or inhibiting the binding of the spike protein of some coronaviruses (including severe acute respiratory syndrome coronavirus 2) to their host cell receptors. This preliminary evidence represents a valuable premise for planning future studies in humans aimed at establishing the pharmacokinetics and biological activity of locally and systemically delivered intranasal heparin formulations.

Complement Levels at Admission Reflecting Progression to Severe Acute Kidney Injury (AKI) in Coronavirus Disease 2019 (COVID-19): A Multicenter Prospective Cohort Study.

Background: Dysregulation of complement system is thought to be a major player in development of multi-organ damage and adverse outcomes in patients with coronavirus disease 2019 (COVID-19). This study aimed to examine associations between complement system activity and development of severe acute kidney injury (AKI) among hospitalized COVID-19 patients. Materials and Methods: In this multicenter, international study, complement as well as inflammatory and thrombotic parameters were analyzed in COVID-19 patients requiring hospitalization at one US and two Hungarian centers. The primary endpoint was development of severe AKI defined by KDIGO stage 2+3 criteria, while the secondary endpoint was need for renal replacement therapy (RRT). Complement markers with significant associations with endpoints were then correlated with a panel of inflammatory and thrombotic biomarkers and assessed for independent association with outcome measures using logistic regression. Results: A total of 131 hospitalized COVID-19 patients (median age 66 [IQR, 54-75] years; 54.2% males) were enrolled, 33 from the US, and 98 from Hungary. There was a greater prevalence of complement over-activation and consumption in those who developed severe AKI and need for RRT during hospitalization. C3a/C3 ratio was increased in groups developing severe AKI (3.29 vs. 1.71; p < 0.001) and requiring RRT (3.42 vs. 1.79; p < 0.001) in each cohort. Decrease in alternative and classical pathway activity, and consumption of C4 below reference range, as well as elevation of complement activation marker C3a above the normal was more common in patients progressing to severe AKI. In the Hungarian cohort, each standard deviation increase in C3a (SD = 210.1) was independently associated with 89.7% increased odds of developing severe AKI (95% CI, 7.6-234.5%). Complement was extensively correlated with an array of inflammatory biomarkers and a prothrombotic state. Conclusion: Consumption and dysregulation of complement system is associated with development of severe AKI in COVID-19 patients and could represent a promising therapeutic target for reducing thrombotic microangiopathy in SARS-CoV-2 infection.