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 gold standard 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; 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 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 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 radiation training and certification, access to radiation laboratory, and waste disposal streams for the radioactive waste. Due to this, platelet aggregometry is a simpler, more convenient method of assessing platelet activation.

The HIPA test, developed in 1991, is based on the visual assessment of platelet aggregates (Greinacher et al., 1991). It is performed in microtiter plates, allowing multiple samples and platelet donors to be tested simultaneously, uses washed platelets, and is generally considered more sensitive than platelet aggregation tests (Greinacher et al., 1994; Tardy et al., 2020). Experienced personnel are required to conduct this assay, as platelet washing can over-activate platelets, giving false positive results, and estimation of platelet aggregation by eye can be imprecise. In comparison, platelet aggregometry is technically less demanding, does not rely on personnel to be experienced in identifying visual changes in platelet morphology, and the real-time trace clearly shows when platelet activation has taken place. By using platelets from donors pre-determined to be reactive to HIT antibodies, the sensitivity of the assay should approach that of HIPA (Chong et al., 1993). Platelet aggregometry has certain limitations as it cannot be used if donors have low platelet counts and, unlike whole-blood aggregometry, some sample preparation is required.

Here, we provide details on an updated version of Born’s method for determining antibody activity by platelet aggregation (Figure 1). This protocol is improved by standardization, with added appropriate controls and use of reactive donor platelets to increase accuracy, and adapted to test IgG, along with serum and plasma. This protocol can be used for research purposes, as well as in clinical laboratories for testing of HIT/VITT antibodies.

Figure 1. Schematic diagram of the platelet aggregometry assay. Centrifuged tubes show the different layers of blood cells and plasma. PPP: platelet-poor plasma; PRP: platelet-rich plasma.