Conversely, a multitude of technical obstacles impede the precise laboratory identification or dismissal of aPL. A chemiluminescence assay panel is employed in this report to describe protocols for the evaluation of solid-phase antiphospholipid antibodies (aPL), specifically anti-cardiolipin (aCL) and anti-β2-glycoprotein I (a2GPI) antibodies of IgG and IgM isotypes. These protocols specify tests which can be performed using the AcuStar instrument, a product of Werfen/Instrumentation Laboratory. Regional approval is a necessary condition for performing this testing on a BIO-FLASH instrument manufactured by Werfen/Instrumentation Laboratory.
Antibodies known as lupus anticoagulants specifically target phospholipids (PL). This creates an in vitro situation where these antibodies bind to PL in coagulation reagents, resulting in an artificially extended activated partial thromboplastin time (APTT) and occasionally, the prothrombin time (PT). The lengthening of clotting times, induced by LA, is generally not connected with an increased likelihood of bleeding. Nevertheless, the extended procedure duration could provoke concern among surgeons conducting intricate surgical procedures, or those anticipating high bleeding risks. Therefore, a strategy to mitigate their anxiety is potentially beneficial. In summary, a method of autoneutralization designed to curtail or eliminate the LA effect on the PT and APTT could be helpful. The document contains a detailed explanation of an autoneutralizing technique designed to lessen the effects of LA on PT and APTT.
The impact of lupus anticoagulants (LA) on routine prothrombin time (PT) assays is often limited by the high phospholipid content present in thromboplastin reagents, effectively neutralizing the antibodies' action. The dilution of thromboplastin in the creation of a dilute prothrombin time (dPT) screening test is instrumental in enhancing the assay's sensitivity to lupus anticoagulants (LA). Substitution of tissue-derived reagents with recombinant thromboplastins leads to demonstrably enhanced technical and diagnostic capabilities. An elevated screening test for LA does not definitively indicate the presence of an LA, as other coagulation abnormalities can also lengthen clotting times. The characteristically reduced clotting time observed in confirmatory testing, utilizing undiluted or less-dilute thromboplastin, underscores the platelet-dependent nature of lupus anticoagulants (LA), in comparison to the screening test results. When coagulation factor deficiencies are present, known or suspected, mixing studies are useful in correcting the deficiencies and revealing the inhibitory properties of lupus anticoagulants. This increases the diagnostic specificity. LA testing, while typically confined to Russell's viper venom time and activated partial thromboplastin time measurements, often overlooks deficiencies detected by dPT. Routinely including dPT in testing improves the identification of clinically significant antibodies.
Given the potential for misleading results, including both false positives and false negatives, testing for lupus anticoagulants (LA) in the context of therapeutic anticoagulation is generally contraindicated, although the detection of LA in these situations can still be medically relevant. Employing strategies such as combining test methods with anticoagulant neutralization techniques can prove beneficial, but are not without drawbacks. An extra analytical path is supplied by prothrombin activators in the venom of Coastal Taipans and Indian saw-scaled vipers; these activators are unaffected by vitamin K antagonists, thereby avoiding the consequences of direct factor Xa inhibitors. Coastal taipan venom's Oscutarin C, a phospholipid- and Ca2+-dependent compound, is utilized in a dilute phospholipid formulation for an LA screening assay, the Taipan Snake Venom Time (TSVT). Cofactor-independent, the ecarin fraction extracted from Indian saw-scaled viper venom, effectively serves as a confirmatory test for prothrombin activation, the ecarin time, because the absence of phospholipids prevents interference by lupus anticoagulants. By focusing solely on prothrombin and fibrinogen in coagulation factor assays, enhanced specificity is achieved compared to other LA assays. Similarly, the thrombotic stress vessel test (TSVT), used as a preliminary screening test, demonstrates strong sensitivity for LAs discovered in other assays and sometimes reveals antibodies undetectable by other methods.
Phospholipids are a focus of antiphospholipid antibodies, a type of autoantibody (aPL). A spectrum of autoimmune conditions might lead to the development of these antibodies, with antiphospholipid (antibody) syndrome (APS) being a significant one. Various laboratory assays can detect aPL, encompassing both solid-phase (immunological) tests and liquid-phase clotting assays for the identification of lupus anticoagulants (LA). Thrombosis, placental and fetal complications, and mortality are all adverse outcomes that can be connected to the presence of aPL. MI-503 chemical structure The severity of the pathology is frequently linked to the particular aPL type present, as well as the manner in which it reacts. Accordingly, the laboratory examination of aPL is indicated for evaluating the potential future threat posed by such occurrences, along with its role in defining criteria for the classification of APS, functioning as a substitute for diagnostic criteria. Biofouling layer This chapter provides an overview of the laboratory tests used to measure aPL and their applicability in clinical practice.
Laboratory testing for Factor V Leiden and Prothrombin G20210A genetic variations aids in establishing the amplified susceptibility to venous thromboembolism in a select patient cohort. Among the various methods used for laboratory DNA testing of these variants, fluorescence-based quantitative real-time PCR (qPCR) is prominent. This method stands out for its speed, simplicity, reliability, and robustness in determining genotypes of interest. This chapter's method is based on polymerase chain reaction (PCR) to amplify the patient's DNA region of interest, followed by the use of allele-specific discrimination techniques for genotyping on a quantitative real-time PCR (qPCR) platform.
The liver is the site of synthesis for Protein C, a vitamin K-dependent zymogen which is integral to the regulation of the coagulation pathway. Exposure of protein C (PC) to the thrombin-thrombomodulin complex leads to its activation and formation of activated protein C (APC). medication-overuse headache APC and protein S, in a coordinated effort, regulate thrombin production by targeting and inactivating factors Va and VIIIa. Protein C's (PC) crucial regulatory function in the coagulation cascade is evident in deficiency states. Heterozygous PC deficiency increases susceptibility to venous thromboembolism (VTE), whereas homozygous deficiency poses a significant threat to the fetus, potentially resulting in life-threatening conditions like purpura fulminans and disseminated intravascular coagulation (DIC). A screening for venous thromboembolism (VTE) frequently includes protein C, alongside protein S and antithrombin. The protocol described in this chapter, a chromogenic PC assay, determines the amount of functional plasma PC by employing a PC activator. The intensity of the color change precisely mirrors the sample's PC concentration. Functional clotting-based assays and antigenic assays are alternative methods; nonetheless, this chapter omits their associated protocols.
Venous thromboembolism (VTE) risk is elevated by the presence of activated protein C (APC) resistance (APCR). The understanding of this phenotypic characteristic was initially enabled by a factor V mutation. This mutation, involving a change from guanine to adenine at nucleotide 1691 in the factor V gene, produced the replacement of arginine, at position 506, with glutamine. This mutated FV resists the proteolytic attack launched by the complex of activated protein C and protein S. Apart from these factors, various other elements also contribute to APCR, such as differing F5 mutations (for example, FV Hong Kong and FV Cambridge), protein S deficiency, elevated levels of factor VIII, the use of exogenous hormones, pregnancy, and the post-partum period. These conditions, collectively, result in the observable expression of APCR and a concomitant increase in VTE risk. The need to accurately detect this phenotype among the large affected population poses a significant public health challenge. Currently, two types of assays are employed: clotting time-based assays, with multiple variations, and thrombin generation-based assays, including the ETP-based APCR assay. Due to the perceived singular connection between APCR and the FV Leiden mutation, assays measuring clotting time were specifically crafted to identify this inherited clotting disorder. Nevertheless, additional occurrences of abnormal protein C resistance have been reported, but they were not included in these clotting evaluations. The APCR assay, based on ETP technology, has been proposed as a universal coagulation test apt to assess these various APCR conditions. This comprehensive data set positions it as a potential screening method for coagulopathic conditions before any therapeutic procedures are carried out. The current method for the ETP-based APC resistance assay's execution is presented in this chapter.
The reduced anticoagulant action of activated protein C (APC) characterizes a hemostatic state known as activated protein C resistance (APCR). The presence of hemostatic imbalance is directly correlated with an elevated risk of venous thromboembolism. The proteolysis-mediated transformation of hepatocyte-produced protein C, an endogenous anticoagulant, yields activated protein C (APC). Following activation, APC leads to the degradation of Factors V and VIII. The APCR state is defined by activated Factors V and VIII's resistance to APC-mediated cleavage, resulting in an amplification of thrombin production and a procoagulant tendency. Antigen-presenting cells (APCs) may exhibit resistance that is either innate or acquired. Factor V gene mutations are directly associated with the most frequent form of hereditary APCR. The most common mutation, a G1691A missense mutation at Arginine 506, also called Factor V Leiden [FVL], removes an APC-targeted cleavage site from Factor Va, thereby preventing its inactivation by the APC protein.