Chronic spontaneous urticaria (CSU) is a disorder where urticaria (hives) occurs without an identifiable provoking factor and persists for more than six weeks (Zuberbier et al., 2022; Kolkhir et al., 2022). CSU is thought to result from the pathogenic activation of mast cells and basophils causing them to release histamine and other proinflammatory mediators. Based on experimental and clinical evidence, it has been recognized that a significant proportion of CSU cases have autoimmune etiology (Konstantinou et al., 2013; Kolkhir et al., 2017; Bracken et al., 2019).The autoimmune nature of CSU is further supported by the autologous serum skin test (ASST), an in vivo assay of mast cell activation induced by intradermal injection of a patient's serum into themselves (Sahiner et al., 2011; Hide et al., 1993; Sabroe et al., 1999; Konstantinou et al., 2009). Nearly 50% of patients with CSU will develop a wheal-and-flare response at the point of injection when inoculated with their own serum. Unfortunately, positive ASST results are not unique to patients with CSU and have been noted in a substantial proportion of patients with allergic or non-allergic rhinitis, multiple drug allergy syndrome, and even in healthy control subjects (Taskapan et al., 2008). The utility of the ASST is further limited by the fact that it cannot be used in patients treated with antihistamines, a status that is quite common for patients with CSU in clinical practice (D'Auria et al., 2019).

Historically, laboratorians have exploited the tendency of donor basophils to release histamine on exposure to CSU patient serum in the basophil histamine release assay (BHRA) for the in vitro assessment of CSU (Konstantinou et al., 2013; Schoepke et al., 2019; Hoffmann et al., 2015). More recently, flow cytometry has been employed to measure the expression of activation-linked cell surface antigens including CD63 in a more readily automatable indirect basophil activation test (BAT) (Konstantinou et al., 2013; D'Auria et al., 2019; Schoepke et al., 2019; Irinyi et al., 2013; Frezzolini et al., 2006; Szegedi et al., 2006; De Swerdt et al., 2005). Numerous studies have shown that positive indirect BAT results are more frequent in CSU patients than in non-CSU controls (Moñino-Romero et al., 2023; Marcelino et al., 2021; HOssein Zadeh Attar et al., 2019; Irinyi et al., 2013; Netchiporouk et al., 2016; De Swerdt et al., 2005). Furthermore, it has been suggested that positive results for BAT tests might not only be linked to high disease activity in both adults and children but may also serve as predictors for how rapidly the patient will respond to treatment (Netchiporouk et al., 2016; Gericke et al., 2017; Curto-Barredo et al., 2016).

One of the challenges in performing indirect BAT is the intra-individual variability in the response of donor basophils in the assays. While some donor basophils are highly reactive to pathologic serum samples, others are not. Conversely, some donor basophils are non-specifically activated by healthy control serum. In common laboratory practice, this variability in donor responsiveness is accounted for by testing patient samples with multiple donors in separate experiments (Gentinetta et al., 2011). Arbitrary rules are established to assess the results when activation for individual donors varies (Marcelino et al., 2021; D'Auria et al., 2019). We have developed a procedure for prescreening individual donors and creating a pool of selected blood donors to optimize the pooled donor basophil activation test (PD-BAT) assay performance and ensure similar results for samples tested from run to run.