To diagnose and manage thrombotic microangiopathies (TMA) correctly, it is essential to accurately determine the activity of ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif, member 13). This characteristic specifically facilitates the differentiation between thrombotic thrombocytopenic purpura (TTP) and other forms of thrombotic microangiopathy (TMAs), ensuring that the right treatment is administered for the identified disorder. Commercial quantitative assays of ADAMTS13 activity, encompassing both manual and automated methods, exist; some furnish results within the hour, but availability is confined to specialized diagnostic centers requiring specialized equipment and personnel. Biodegradation characteristics Technoscreen ADAMTS13 Activity, a commercially available, rapid, semi-quantitative screening test, is based on flow-through technology coupled with an ELISA activity assay. No specialized equipment or personnel are needed for this simple screening tool. The colored end point is measured against a reference color chart, featuring four levels of color intensity corresponding to ADAMTS13 activity levels (0, 0.1, 0.4, and 0.8 IU/mL). The screening test's indication of reduced levels demands further quantification. The assay's design facilitates its implementation in nonspecialized labs, distant sites, and immediate-care settings.
A consequence of low levels of ADAMTS13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13, is the prothrombotic disorder, thrombotic thrombocytopenic purpura (TTP). ADAMTS13, also termed von Willebrand factor (VWF) cleaving protease (VWFCP), carries out the task of cleaving VWF multimers, thereby reducing plasma VWF's functional capacity. Without ADAMTS13, typically observed in thrombotic thrombocytopenic purpura (TTP), plasma von Willebrand factor (VWF) builds up, specifically as extremely large multimeric forms, ultimately causing a thrombotic event. Among patients with definitively confirmed thrombotic thrombocytopenic purpura (TTP), ADAMTS13 deficiency often originates as an acquired condition. This is due to the generation of antibodies that either promote the elimination of ADAMTS13 from the blood or inhibit the crucial functions of this enzyme. XMU-MP-1 inhibitor The current report outlines a procedure for assessing ADAMTS13 inhibitors, substances that are antibodies obstructing ADAMTS13 activity. The technical steps of the protocol identify ADAMTS13 inhibitors by testing mixtures of patient and normal plasma for residual ADAMTS13 activity using a Bethesda-like assay. Using various assays, the residual ADAMTS13 activity can be quantified, with the AcuStar instrument (Werfen/Instrumentation Laboratory) providing a rapid 35-minute test, as shown in this protocol.
A critical lack of the ADAMTS13 enzyme, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13, leads to the prothrombotic disorder known as thrombotic thrombocytopenic purpura (TTP). Thrombotic thrombocytopenic purpura (TTP) is characterized by a deficiency of ADAMTS13, which results in excessive accumulation of ultra-large von Willebrand factor (VWF) multimers in the plasma. This, in turn, leads to problematic platelet aggregation and the formation of blood clots. Apart from its presence in TTP, ADAMTS13 levels might be subtly to moderately lowered in a diverse range of conditions, encompassing secondary thrombotic microangiopathies (TMA), such as those resulting from infections (e.g., hemolytic uremic syndrome (HUS)), liver disease, disseminated intravascular coagulation (DIC), sepsis, acute/chronic inflammatory conditions, and sometimes COVID-19 (coronavirus disease 2019). Various techniques, including ELISA (enzyme-linked immunosorbent assay), FRET (fluorescence resonance energy transfer), and chemiluminescence immunoassay (CLIA), allow for the detection of ADAMTS13. This CLIA-validated report describes a protocol for measuring ADAMTS13 activity. The protocol describes a rapid test, complete within 35 minutes, that can be done on the AcuStar instrument (Werfen/Instrumentation Laboratory). In certain regions, approval might be given for the use of the BioFlash instrument for this same procedure.
ADAMTS13, a member of the disintegrin and metalloproteinase family with a thrombospondin type 1 motif, is also identified as the von Willebrand factor cleaving protease, VWFCP. The action of ADAMTS13 is to cleave VWF multimers, consequently reducing plasma VWF activity levels. Plasma von Willebrand factor (VWF), particularly in the form of large multimers, accumulates in the absence of ADAMTS13, a scenario characteristic of thrombotic thrombocytopenic purpura (TTP), and this accumulation can trigger thrombosis. ADAMTS13's relative insufficiencies extend to a number of other circumstances, including secondary thrombotic microangiopathies (TMA). Of current clinical significance, the coronavirus disease 2019 (COVID-19) infection may be linked to both a decline in ADAMTS13 activity and a pathological buildup of von Willebrand factor (VWF), a factor likely involved in the observed thrombotic predisposition of patients. The identification and treatment of thrombotic thrombocytopenic purpura (TTP) and thrombotic microangiopathies (TMAs) can benefit from ADAMTS13 laboratory testing, which can be performed using various assays. This chapter, consequently, presents an overview of the laboratory testing process for ADAMTS13 and its importance in assisting with the diagnosis and treatment of connected diseases.
As the gold standard for detecting heparin-dependent platelet-activating antibodies, the serotonin release assay (SRA) is essential to the diagnosis of heparin-induced thrombotic thrombocytopenia (HIT). Following the 2021 adenoviral vector COVID-19 vaccination, a case of thrombotic thrombocytopenic syndrome was documented. The vaccine-induced thrombotic thrombocytopenic syndrome (VITT) was a severe immune response causing platelet activation, presenting with unusual blood clots, low platelet count, very elevated D-dimer levels in the blood, and a high death rate despite intensive treatment, including anticoagulation and plasma exchange. While the antibodies in both heparin-induced thrombocytopenia (HIT) and vaccine-induced thrombotic thrombocytopenia (VITT) are directed at platelet factor 4 (PF4), important clinical distinctions in their actions are evident. Modifications to the SRA became essential to better identify functional VITT antibodies. Functional platelet activation assays are irreplaceable in the diagnostic procedure for identifying heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombocytopenia (VITT). We explore the implementation of SRA for the quantification of HIT and VITT antibodies.
A well-documented, iatrogenic complication of heparin anticoagulation, heparin-induced thrombocytopenia (HIT), has substantial health consequences. A significantly different consequence of adenoviral vaccines, including ChAdOx1 nCoV-19 (Vaxzevria, AstraZeneca) and Ad26.COV2.S (Janssen, Johnson & Johnson) against COVID-19, is vaccine-induced immune thrombotic thrombocytopenia (VITT), a newly recognized severe prothrombotic complication. The diagnostic process for HIT and VITT encompasses laboratory testing of antiplatelet antibodies via immunoassays, followed by a confirmation step using functional assays to identify platelet-activating antibodies. The detection of pathological antibodies requires functional assays due to the inconsistent sensitivity and specificity of immunoassays. A method using whole blood flow cytometry to detect procoagulant platelets in the blood of healthy donors, as a response to plasma from patients possibly affected by HIT or VITT, is presented in this chapter. A technique for identifying healthy individuals qualified for HIT and VITT testing is elaborated.
Adverse reactions associated with the adenoviral vector COVID-19 vaccines, including AstraZeneca's ChAdOx1 nCoV-19 (AZD1222) and Johnson & Johnson's Ad26.COV2.S vaccine, led to the recognition of vaccine-induced immune thrombotic thrombocytopenia (VITT) in 2021. VITT, a severe immune-mediated platelet activation syndrome, is diagnosed in an estimated 1-2 patients per 100,000 vaccinations. VITT, a condition characterized by thrombocytopenia and thrombosis, can develop within 4 to 42 days following the initial vaccine dose. Platelet factor 4 (PF4) is the target of platelet-activating antibodies produced by individuals affected by this condition. An antigen-binding assay (enzyme-linked immunosorbent assay, ELISA) and a functional platelet activation assay are both recommended by the International Society on Thrombosis and Haemostasis for the diagnostic evaluation of VITT. This functional assay for VITT, namely multiple electrode aggregometry (Multiplate), is detailed herein.
Heparin-dependent IgG antibodies, a key component in immune-mediated heparin-induced thrombocytopenia (HIT), bind to heparin/platelet factor 4 (H/PF4) complexes, leading to platelet activation. A wide spectrum of assays can be employed to scrutinize heparin-induced thrombocytopenia (HIT), differentiated into two fundamental groups. Antigen-based immunoassays initially detect all antibodies targeted against H/PF4, acting as a preliminary diagnostic approach. Crucial for final confirmation, functional assays identify only those antibodies possessing the capacity to activate platelets, thus establishing a diagnosis of pathological HIT. While the serotonin-release assay (SRA) has served as the gold standard for decades, easier alternatives have become increasingly common over the past ten years. This chapter will address whole blood multiple electrode aggregometry, a validated approach for the functional diagnosis of heparin-induced thrombocytopenia (HIT).
The immune system's response to heparin involves the formation of antibodies that target the heparin-platelet factor 4 (PF4) complex, leading to heparin-induced thrombocytopenia (HIT) after heparin administration. Gender medicine These antibodies can be identified through diverse immunological procedures, including ELISA (enzyme-linked immunosorbent assay) and chemiluminescence using the AcuStar device.