The reduction in turbidity, a consequence of bead agglutination, demonstrates a linear dependence on VWFGPIbR activity. The VWFGPIbR assay, employing a VWFGPIbR/VWFAg ratio, exhibits excellent sensitivity and specificity in differentiating type 1 VWD from type 2. A detailed protocol for the VWFGPIbR assay is detailed in the subsequent chapter.

Often identified as the most commonly reported inherited bleeding disorder, von Willebrand disease (VWD) is sometimes found in a different form, acquired von Willebrand syndrome (AVWS). VWD/AVWS arises from flaws or insufficiencies within the adhesive plasma protein, von Willebrand factor (VWF). The task of diagnosing or ruling out VWD/AVWS is complicated by the heterogeneity of VWF defects, the technical limitations of many VWF tests, and the varying VWF test panels (the number and types of tests) chosen by different laboratories. Assessment of VWF levels and activity through laboratory testing is crucial for diagnosing these disorders, with activity measurements requiring multiple tests given VWF's multifaceted role in mitigating bleeding. This report lays out the procedures to evaluate VWF level (antigen, VWFAg) and activity, relying on a chemiluminescence-based testing platform. Biological life support Within activity assays, there are two key components: collagen binding (VWFCB) and a ristocetin-based recombinant glycoprotein Ib-binding (VWFGPIbR) assay, a modern alternative to the traditional ristocetin cofactor (VWFRCo). The VWF panel (Ag, CB, GPIbR [RCo]), comprising three tests, is the only composite panel available on a single platform and is conducted using an AcuStar instrument (Werfen/Instrumentation Laboratory). Sirtuin activator Regional approvals are required for the use of the BioFlash instrument (Werfen/Instrumentation Laboratory) to execute the 3-test VWF panel.

In the US, clinical laboratory quality control procedures, under risk-assessment protocols, can deviate from the Clinical Laboratory Improvement Amendments (CLIA) standards; however, the manufacturer's minimum requirements remain binding. Every 24 hours of patient testing necessitates at least two levels of control material, as per US internal quality control requirements. Some coagulation tests' quality control guidelines might recommend a normal specimen or commercial controls, but these may not fully capture all the reported results of the test. Obstacles preventing compliance with the minimum QC requirements could be rooted in (1) the characteristics of the sample type (like complete blood samples), (2) the lack of sufficient or suitable commercial control materials, or (3) the occurrence of rare or unusual sample compositions. Laboratory sites are offered preliminary guidance in this chapter on sample preparation techniques for confirming reagent efficacy and assessing the performance of platelet function studies and viscoelastic measurements.

The diagnosis of bleeding disorders and the ongoing monitoring of antiplatelet therapy necessitate platelet function testing. The gold standard assay, light transmission aggregometry (LTA), has been employed globally for sixty years, continuing to be widely used. The process, while demanding access to expensive equipment and time investment, also needs an experienced investigator to evaluate the results. Laboratories experience fluctuating results due to the lack of standardized protocols. The 96-well plate-based Optimul aggregometry method, analogous to LTA principles, endeavors to standardize agonist concentrations. The key to this lies in pre-coating 96-well plates with seven levels of each lyophilized agonist (arachidonic acid, adenosine diphosphate, collagen, epinephrine, TRAP-6 amide, and U46619). These plates are suitable for storage at ambient room temperature (20-25°C) for a maximum of 12 weeks. To assess platelet function, 40 liters of platelet-rich plasma are introduced into each well, the plate is then secured on a plate shaker, and light absorbance is subsequently monitored to evaluate platelet aggregation. This method minimizes the necessary blood volume, enabling thorough platelet function analysis without the requirement for specialized training or the purchase of costly, dedicated equipment.

Historically, light transmission aggregometry (LTA) has served as the gold standard for platelet function testing, a procedure often performed in dedicated hemostasis labs because of its hands-on and time-consuming methodology. However, advanced automated testing systems facilitate standardization and the execution of tests within the routine procedures of laboratories. Platelet aggregation measurement procedures on the CS-Series (Sysmex Corporation, Kobe, Japan) and CN-Series (Sysmex Corporation, Kobe, Japan) platforms for routine hematological analysis are described. Further descriptions are provided regarding the disparate approaches used by the analyzers. The CS-5100 analyzer's protocol requires the preparation of final diluted agonist concentrations via the manual pipetting of reconstituted agonist solutions. Prepared agonist dilutions, eight times more concentrated than the intended working level, are precisely diluted in the analyzer to acquire the desired level before testing. Within the CN-6000 analyzer, the auto-dilution feature ensures the automatic preparation of agonist dilutions and the resultant final working concentrations.

A method for quantifying endogenous and infused Factor VIII (FVIII) in patients undergoing emicizumab therapy (Hemlibra, Genetec, Inc.) will be detailed in this chapter. Emicizumab, a bispecific monoclonal antibody, is utilized in the treatment of hemophilia A, including cases with inhibitors. In its novel mechanism of action, emicizumab emulates FVIII's in-vivo role by binding FIXa and FX together. Exosome Isolation The laboratory must accurately assess the effect of this drug on coagulation tests and employ a chromogenic assay unaffected by emicizumab to reliably measure FVIII coagulant activity and inhibitors.

Prophylactic administration of emicizumab, a bispecific antibody, in several countries, has proven effective in preventing bleeding episodes in severe hemophilia A, and is occasionally used for moderate hemophilia A patients. Hemophilia A sufferers, with and without factor VIII inhibitors, can employ this medication, as it is not a target for these inhibitors. In most instances, emicizumab's fixed weight-based dosing obviates the need for laboratory monitoring; however, a laboratory test may be necessary in the event of unforeseen bleeding episodes, particularly for a patient with hemophilia A who has undergone prior treatment. This chapter examines the performance metrics of a one-stage clotting assay, specifically regarding its use in measuring emicizumab.

Through the application of various coagulation factor assay methods, clinical trials have evaluated the treatment effects of extended half-life recombinant Factor VIII (rFVIII) and recombinant Factor IX (rFIX). Despite the standardization of reagent combinations for routine usage, diagnostic laboratories may use different combinations during field trials of EHL products. This review's core theme is evaluating the choice of one-stage clotting and chromogenic Factor VIII and Factor IX assays, examining the influence of assay principle and components on measured results, specifically considering the effects of various activated partial thromboplastin time reagents and factor-deficient plasma types. A tabulated presentation of findings, categorized by method and reagent group, is intended to aid laboratories in assessing how their reagent combinations perform against others, for the diverse options of EHLs available.

Thrombotic microangiopathies can be distinguished, in part, from thrombotic thrombocytopenic purpura (TTP) by an ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif, member 13) activity level found to be less than 10% of its normal range. Inherited or developed TTP exists, with acquired immune-mediated TTP frequently observed. This type stems from autoantibodies that interfere with ADAMTS13 activity or promote its removal. Basic 1 + 1 mixing studies, designed to identify inhibitory antibodies, are supplemented by Bethesda-type assays. These assays quantify the loss of function observed in a series of mixtures created from test plasma and normal plasma. Not all patients display inhibitory antibodies; in these scenarios, ADAMTS13 deficiency may be a direct consequence of clearing antibodies, antibodies that remain undetectable through functional assays. Recombinant ADAMTS13, a component of common ELISA assays, is used to detect clearing antibodies. The preferred assay, although it cannot distinguish between inhibitory and clearing antibodies, is based on its ability to detect inhibitory antibodies. The principles, performance characteristics, and practical considerations for employing a commercial ADAMTS13 antibody ELISA and a generic approach to Bethesda-type assays for detecting inhibitory ADAMTS13 antibodies are presented in this chapter.

An accurate evaluation of ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif, member 13) activity is crucial in distinguishing thrombotic thrombocytopenic purpura (TTP) from other thrombotic microangiopathies during a diagnostic procedure. The initial assays' excessive demands for time and effort in execution made them unsuitable for managing acute scenarios. This frequently led to treatment protocols reliant solely on clinical findings, with necessary laboratory validation tests coming days or weeks later. Fast results, generated by rapid assays, can now influence immediate diagnostic and treatment protocols. Fluorescence resonance energy transfer (FRET) or chemiluminescence assays can offer results in less than an hour, notwithstanding the requisite for specific analytical platforms. The time to generate results from enzyme-linked immunosorbent assays (ELISAs) is about four hours, though the assays themselves do not require equipment beyond commonly used ELISA plate readers that are present in many laboratories. The following chapter explores the principles, operational performance, and practical aspects of using ELISA and FRET assays to determine ADAMTS13 activity levels in plasma samples.