This assay can be used to determine antibody activity in patients with disorders such as heparin-induced thrombocytopenia (HIT) and vaccine-induced thrombotic thrombocytopenia (VITT)

This assay can be used to determine antibody activity in patients with disorders such as heparin-induced thrombocytopenia (HIT) and vaccine-induced thrombotic thrombocytopenia (VITT). This causes a reduction in opacity allowing more light to pass through PRP. Light transmission through the cuvette is proportional to the degree of platelet aggregation and is measured by the photocell over time. The advantage of this protocol is that it is a simple, reliable assay that can be applied to assess antibody activity in thrombotic conditions. Light transmission aggregometry does not require the use of radioactive reagents and is technically less demanding compared with 14C-serotonin release assay, another common assay for detecting antibody activity. Key features ? This protocol can be used to assess platelet function and to detect platelet activating antibodies in diseases such as HIT and VITT. ? Does not require radioactive reagents, requires an aggregometer; based on the light transmission aggregometry protocol, adapted for detection of VITT and other platelet-activating antibodies. ? Two positive controls are required for reliable detection of antibodies in diseases such as HIT/VITT, namely a weak HIT/VITT antibody and a physiological agonist. ? For detection of HIT/VITT antibodies, it is essential to use donors known to have platelets reactive to these antibodies to avoid false negative results. Keywords: Platelet Urocanic acid activation, Antibody, Aggregation, Immune thrombosis, Heparin-induced thrombocytopenia, Vaccine-induced thrombotic thrombocytopenia Background Platelets play an essential role in hemostasis, bleeding, and thrombosis. Upon activation, platelets undergo shape changes and aggregate, leading to platelet clumping. The rate and extent of aggregation can be measured by light transmission via a platelet aggregometer. Platelet-rich plasma (PRP), which is turbid, is stirred in a test cuvette. When the agonist is added, the platelets will form increasingly larger aggregates and the PRP will begin to clear, allowing more light to pass through. This increase in light transmittance is directly proportional to the amount of aggregation. First described by Born in the 1960s (Born, 1962), light transmission aggregometry is considered a assay for platelet function, often assigned as the first step for analyzing platelet dysfunction in hemorrhagic patients (Kottke-Marchant and Corcoran, 2002; Hayward, 2008; Gadisseur et al., 2009; p150 Hayward et al., 2009; Podda et al., 2012). To detect the presence and activity of pathogenic antibodies, patient sera or purified patient IgG can be used. Purified IgG confirms that the IgG is the component that induces platelet activation, thus excluding other potential platelet activating agents in patient sera. Standardization of the use of light transmission aggregometry to assess platelet function has Urocanic acid been published by the International Society of Thrombosis and Haemostasis (Cattaneo et al., 2013). Another method of analyzing platelet activation is whole-blood aggregometry (Park et al., 2012; Mencarini et al., 2021). This method measures the change in electrical impedance between two electrodes to indicate platelet aggregation. Although this process accounts for the effect of all blood cells on platelet function, it cannot determine with accuracy the direct or indirect contributions of other blood cells to the platelet activation observed. To avoid confounding factors in whole blood, light transmission aggregometry using PRP is an ideal, relatively simple, and reliable method to specifically determine platelet activity. The 14C-serotonin release assay (SRA) and the heparin-induced platelet activation (HIPA) test are also common assays used to test antibody activity (Arepally et al., 1995; Greinacher et al., 2012 and 2022). They are based on the principle that incubation of patient serum (containing the pathogenic antibody) will activate donor platelets resulting in release of intraplatelet serotonin (SRA) and platelet aggregation (HIPA). SRA is a Urocanic acid sensitive method used to detect pathogenic antibody in heparin-induced thrombocytopenia (HIT) (Warkentin et al., 2015) and more recently, vaccine-induced thrombotic thrombocytopenia (VITT) (Leung et al., 2022). For SRA, washed platelets are incubated with radiolabeled serotonin. Upon activation, platelets release radiolabeled serotonin, and this is measured by a beta counter. Despite the high sensitivity of SRA, this method is labor intensive and technically demanding, requiring the user to have.