A linear correlation is evident between VWFGPIbR activity and the decrease in turbidity, attributable to bead agglutination. 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.
Von Willebrand disease (VWD), frequently reported as the most common inherited bleeding disorder, may sometimes be manifested as the acquired form of the syndrome, von Willebrand syndrome (AVWS). VWD/AVWS results from imperfections or insufficiencies in the adhesive plasma protein known as von Willebrand factor (VWF). VWD/AVWS diagnosis or exclusion is complex due to the variety of VWF defects, the technical shortcomings of numerous VWF tests, and the differences in VWF test panels (in the number and type of tests) employed by various labs. Diagnosing these disorders involves laboratory testing for VWF levels and activity, the assessment of which necessitates multiple tests because of the wide range of VWF's functions in combating bleeding. A chemiluminescence-based panel serves as the basis for this report's explanation of procedures for evaluating VWF levels (antigen; VWFAg) and its activity. Selleckchem KHK-6 Collagen-binding (VWFCB) and ristocetin-based recombinant glycoprotein Ib-binding (VWFGPIbR) assays, which are contemporary alternatives to the classical ristocetin cofactor (VWFRCo), are included in activity assays. This 3-test VWF panel (Ag, CB, GPIbR [RCo]) stands alone as the sole composite panel available on a single AcuStar instrument (Werfen/Instrumentation Laboratory). Antioxidant and immune response Regional approvals are required for the use of the BioFlash instrument (Werfen/Instrumentation Laboratory) to execute the 3-test VWF panel.
Clinical laboratories in the United States may, based on risk assessment, employ quality control protocols that fall short of regulatory requirements, such as those established under the Clinical Laboratory Improvement Amendments (CLIA), but must meet the manufacturer's minimum specifications. Patient testing, in accordance with US internal quality control regulations, necessitates at least two levels of control material for every 24-hour period. A normal sample or commercial controls could be used for quality control in certain coagulation tests, however, these may not include all of the test components that are part of the reporting results. Difficulties in meeting the requisite QC threshold may arise from (1) the kind of sample (e.g., whole blood), (2) the scarcity of appropriate commercial control substances, or (3) the peculiarity or rarity of the samples examined. This chapter gives preliminary guidance to laboratory sites on how to prepare samples for verifying the accuracy and performance of reagents, platelet function tests, and viscoelastic measurements.
The diagnosis of bleeding disorders and the ongoing monitoring of antiplatelet therapy necessitate platelet function testing. Light transmission aggregometry (LTA), the gold standard assay, has persisted as a globally recognized method for sixty years, maintaining its widespread use. Despite requiring expensive equipment and being a time-consuming procedure, the interpretation of the results must be carried out by a well-versed investigator. Laboratories experience fluctuating results due to the lack of standardized protocols. Optimul aggregometry, a 96-well plate-based method, leverages the foundational principles of LTA, aiming for standardized agonist concentrations. This is achieved through pre-coated 96-well plates, housing seven concentrations of lyophilized agonists (arachidonic acid, adenosine diphosphate, collagen, epinephrine, TRAP-6 amide, and U46619). These plates are stored at ambient room temperature (20-25°C) for a maximum duration of twelve weeks. Platelet function testing involves the addition of 40 liters of platelet-rich plasma to each well, followed by placement on a plate shaker, and subsequent determination of platelet aggregation through light absorbance changes. This technique allows for a complete platelet function analysis, with reduced blood volume requirements, without the need for specialized training or the acquisition of costly, dedicated tools.
The longstanding gold standard of platelet function testing, light transmission aggregometry (LTA), is typically conducted in specialized hemostasis laboratories due to its demanding, manual procedure. Yet, modern automated testing procedures establish a framework for standardization and enable testing routines in typical laboratory environments. We present the methods for measuring platelet aggregation on both the CS-Series (Sysmex Corporation, Kobe, Japan) and CN-Series (Sysmex Corporation, Kobe, Japan) blood coagulation analysis systems. A more detailed explanation of the differing methodologies employed by both analyzers follows. Reconstituted agonist solutions are manually pipetted to generate the final diluted concentrations required by the CS-5100 analyzer. The dilutions of agonists, initially eight times more concentrated than the final working level, are correctly further diluted within the analyzer before being used for testing. The CN-6000 analyzer's auto-dilution feature automatically generates the agonist dilutions and the final operational concentrations.
This chapter's focus is on describing a method for measuring both endogenous and infused Factor VIII (FVIII) in patients undergoing emicizumab therapy (Hemlibra, Genetec, Inc.). Emicizumab, a bispecific monoclonal antibody, is utilized in the treatment of hemophilia A, including cases with inhibitors. The action of emicizumab is distinct, embodying FVIII's in-vivo function of linking FIXa and FX through a binding mechanism. Microscopes and Cell Imaging Systems The laboratory's comprehension of this drug's impact on coagulation tests is critical, necessitating the utilization of a suitable chromogenic assay unaffected by emicizumab to ascertain FVIII coagulant activity and inhibitors.
As a prophylactic against bleeding, emicizumab, a bispecific antibody, has gained widespread adoption in various countries for individuals with severe hemophilia A, and occasionally in those with moderate hemophilia A. 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 comprehensively describes how a one-stage clotting assay performs in the context of emicizumab quantification.
Clinical trials have used diverse approaches in coagulation factor assays to evaluate the efficacy of therapies employing extended half-life recombinant Factor VIII (rFVIII) and recombinant Factor IX (rFIX). Different reagent combinations might be employed by diagnostic laboratories for everyday testing or for evaluating EHL products in the field. A key subject of this review is the selection criteria for one-stage clotting and chromogenic Factor VIII and Factor IX methodologies, analyzing the impact of assay principle and component variations on results, particularly considering the effects of different activated partial thromboplastin time reagents and factor-deficient plasma. For practical laboratory guidance, we tabulate the results for each method and reagent group, contrasting local reagent combinations with others, for all available EHLs.
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. TTP is a condition that can be present from birth or developed later in life. The most common manifestation is acquired immune-mediated TTP, which is characterized by autoantibodies that inhibit or increase clearance of ADAMTS13. Mixing tests, fundamental to detecting inhibitory antibodies, involve combining basic samples of 1+1, and Bethesda-type assays, precisely quantifying the loss of functionality in blended samples of test plasma and normal plasma, are well-suited for this purpose. Not every patient exhibits inhibitory antibodies, potentially leading to ADAMTS13 deficiency solely due to the presence of undetectable clearing antibodies in functional assessments. Clearing antibodies are detected via capture with recombinant ADAMTS13 in ELISA assays. The preferred assay, although it cannot distinguish between inhibitory and clearing antibodies, is based on its ability to detect inhibitory antibodies. The present chapter examines a commercial ADAMTS13 antibody ELISA, focusing on its principles, performance, and practical applications, as well as a general method for Bethesda-type assays for the detection of inhibitory ADAMTS13 antibodies.
Determining the precise activity level of ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif, member 13) is essential for distinguishing thrombotic thrombocytopenic purpura (TTP) from other thrombotic microangiopathies in a diagnostic context. Given their cumbersome nature and lengthy duration, the original assays were unsuitable for immediate application in the acute phase, making treatment dependent primarily on clinical evaluations, with supporting laboratory assays performed considerably later, after days or even weeks. Newly available rapid assays provide results with the speed necessary to impact immediate diagnostic and therapeutic decisions. Fluorescence resonance energy transfer (FRET) or chemiluminescence assays can offer results in less than an hour, notwithstanding the requisite for specific analytical platforms. Enzyme-linked immunosorbent assays (ELISAs) can generate outcomes in approximately four hours; however, these assays do not require equipment beyond the commonplace ELISA plate readers that are routinely present in many laboratories. Regarding ADAMTS13 activity quantification in plasma, this chapter presents the principles, performance evaluations, and practical implications of both ELISA and FRET assays.