Introduction

Sensitisation to inhalant allergens such as house dust mites, cats and dogs, is commonly associated with asthma, but is neither necessary nor sufficient for disease expression. Similarly, a positive skin prick test (SPT) or IgE test to a food does not equate to clinical food allergy, and false positive results are common. Challenge testing (either oral food challenge or inhaled allergen challenge) can be offered to patients, but is time consuming, carries the risk of severe reaction and is not suitable for all patients. Therefore, tests with superior diagnostic accuracy than IgE that are safe to conduct in all patients would be of value in clinical practice, especially amongst patients sensitised to many allergens.
Basophils and mast cells (MC) are the two primary effector cells in allergic responses (1). Cellular degranulation triggers the release of preformed and newly synthesized mediators inducing a potent biological response in a sensitized person following allergen exposure (2). While basophils are found in the circulation, MC are localized in peripheral tissues. The two cell types may have different roles in an allergic response but this is currently poorly understood (2, 3).
The more accessible circulating basophils have been used as cell models for studying allergy (4). Basophils account for <1% of blood leukocytes however, making purification a challenge. To obviate the need for purification, the basophil activation test (BAT) was developed using immediately analysed fresh whole blood (4, 5). Following stimulation of whole blood with allergen (or control), the responsiveness of the basophils can be quantified using fluorochrome-coupled antibody markers of basophil activation (e.g. CD63 and CD203c) by flow cytometry. The advantage of the BAT is that it takes account of many factors which influence basophil responsiveness to an allergen such as IgG4/IgE ratio (6, 7), heterogeneity of sIgE to allergen components (8), medication (9) and innate responsiveness of the cells (10). The disadvantages are that blood needs to be analysed immediately after being drawn (5), requiring the allergy clinic to have instant access to a staffed flow cytometry facility. In addition, 10-20% of people carry “non-releaser” basophils, which are non-responsive in the BAT, despite having clinical allergy (11). Consequently, this test is not generally available for clinical diagnostics, but used only in specialist laboratories for hymenoptera venom and drug allergy testing.
The passive BAT, which uses basophils from a donor that are passively sensitized with the serum from the patient, was developed as an alternative method that circumvents some of these problems (12). Stored serum samples from subjects can be analysed in batches, providing greater flexibility and allowing humoral factors to be investigated separately from cellular factors (7). However, the donors’ basophils must be stripped of endogenous IgE with a mild acid treatment before the cells can be passively sensitized with patient serum samples, which can damage the donor basophils and lead to auto-basophil activation (13) and reduced sensitivity (14). Due to these limitations, passive BAT has only been used in a few studies. Although the passively sensitized approach has also been used on basophilic cell lines such as RBL-2H3, there are a number of disadvantages, including the gradual loss of cell responsiveness within weeks of cultures (15).
Recently, Bahri et al developed a robust and reproducible effector cell assay based on human progenitor cell derived MC, the mast cell activation test (MAT). This assay appeared to confer superior diagnostic accuracy in distinguishing peanut allergic from peanut tolerant subjects compared with existing diagnostics such as sIgE (to whole peanut or Ara h 2), SPT and BAT (16). In this study we use a similar approach to generate functional progenitor cell-derived basophils (PCB) and provide detailed characterization of basophil differentiation, and demonstrate the functionality and reproducibility of this technique. We then explore the potential clinical application of progenitor cell basophil activation test (PCBAT) by passively sensitizing the cells with sera/plasma from five groups of patients with allergic asthma and food allergy and testing degranulation to two allergens (cat and peanut).