3.4 Allergen tetramers bind the total basophil population in sensitized individuals irrespective of activation
Conventional understanding of basophils capturing polyclonal IgE via their FcεRI suggests that all basophils of an allergic patient have the potential to bind IgE with specificity to an allergen molecule. However, typically only a fraction of the basophils will degranulate and express CD63 following stimulation with that allergen. To examine whether the CD63+ basophils differed from CD63basophils in terms of allergen binding specificity and capability, the staining intensities of the allergen tetramers were re-examined following stratification of basophils based on CD63 expression afterin vitro stimulation (Figure 2B, 3B ). Both the CD63 and the CD63+ basophils from all 41 BV-allergic individuals showed strong staining with [Api m 1]4-APC as compared to streptavidin-APC. Importantly, the CD63 and CD63+ fractions bound similar amounts of Api m 1 (Suppl. Figure 4A ). Similarly, [Lol p 1]4-APC showed high degrees of binding to both CD63+ and CD63 basophils from the 50 RGP-allergic individuals, albeit with slightly but significantly lower levels on the CD63 subset (Suppl. Figure 4B ). Thus, in sensitized individuals, all basophils may have surface bound allergen-specific IgE, but not all will degranulate upon binding to allergen.
3.5 Greater diagnostic ability of basophil staining than activation with recombinant allergen tetramers
The BV-allergic and RGP-allergic individuals in this study were diagnosed based on their clinical history, SPT and/or allergen-specific serum IgE, as per clinical standards. To compare the predictive values of the BAT and CytoBas for allergy, ROC of their outcomes were determined. For each individual, the highest CD63+frequency was selected from the serial dilutions of allergen tetramer (Figure 2B; Figure 3B ). CD63 positivity following stimulation with [Api m 1]4 was <10% for all control subjects and >10% in 40 of 41 BV-allergic individuals (Figure 4A ). Incubation with [Lol p 1]4resulted in <5% of basophils becoming CD63+in all controls and >5% in 42/50 RGP allergic patients (Figure 4B ).
Staining intensities with the allergen tetramers were determined by calculating the ratio of MFI from staining with 1 µg/ml tetramer-APC over 1 µg/ml streptavidin-APC. This MFI ratio was <2 for all control subjects stained with [Api m 1]4-APC, whereas all 41 patients with BV allergy had a ratio >2 (Figure 4C ). Furthermore, the MFI ratios for all patients were higher than for any of the controls, resulting in an area under the curve (AUC) in the ROC curve of 10,000. This was slightly higher than the AUC for the ROC curve of the [Api m 1]4 BAT (9792). Similarly, all control subjects showed a ratio <2 for [Lol p 1]4-APC staining, whereas 48/50 RGP-allergic patients had a ratio >2 (Figure 4D ). As a result, the AUC for the ROC curve (9980) was substantially higher than that of the ROC curve for the BAT of [Lol p 1]4 (9500). In conclusion, the CytoBas approach with single allergens showed high specificity and sensitivity for BV (Api m 1) and RGP (Lol p 1) allergies with improved performance over BAT.