Basophil Activation and Histamine Release Tests in Relation to Omalizumab Response in Peanut‐Allergic Children

Abbreviations

BAT

basophil activation test

BHRA

basophil histamine release assay

DBPCFC

double‐blind, placebo‐controlled food challenge

EC50

basophil sensitivity

To the Editor,

1.

The growing prevalence of food allergy (FA) worldwide [1] poses an increasing challenge for public health and a significant burden on affected individuals and their families. Among children, food allergy remains the leading cause of anaphylaxis, with peanut allergy as the primary cause of severe food‐related allergic reactions [2]. Standard care includes allergen avoidance and access to self‐injectable epinephrine for emergency situations. However, these measures are reactive rather than preventive, leaving a need for effective long‐term therapeutic options.

Recent advancements in treatment options, such as oral immunotherapy (OIT) and biological agents, show promise in altering the natural course of FA. Among biologics, Omalizumab (Xolair), an anti‐IgE humanised monoclonal antibody, offers potential as either monotherapy or in combination with OIT in severe FA cases [3]. By binding to free circulating IgE, Omalizumab inhibits IgE‐mediated responses, specifically preventing the binding of IgE to FcεRI receptors on effector cells like basophils and mast cells. This inhibition reduces cell degranulation and, consequently, the release of pro‐inflammatory mediators, thereby targeting FA at a fundamental immunological level.

To explore the performance of BAT and BHRA results in predicting Omalizumab treatment and the correlation to clinical response, a single‐centre, double‐blind, placebo‐controlled study was conducted at the Allergy Centre, Odense University Hospital, Denmark (TOFAC: Treatment with Omalizumab in food‐allergic children; clinicaltrials.gov: ID NCTO4037176) [4]. The study included peanut‐allergic children aged 6–17 years with a cumulative threshold dose at or below 443 mg peanut protein determined by double‐blind, placebo‐controlled food challenges [DBPCFC]. Participants were randomised to receive Omalizumab or a placebo (3:1) and were re‐evaluated after three and 6 months by DBPCFC to assess treatment efficacy. Patients' characteristics are given in reference [4]. Our focus was to evaluate the performance of the Basophil Activation Test (BAT) and the Basophil Histamine Release Assay (BHRA) in predicting and correlating with clinical outcomes following Omalizumab therapy.

BAT and BHRA are two in vitro tests that measure basophil degranulation in response to allergens, with distinct methodologies and endpoints [5]. BAT assesses basophil degranulation by measuring the cell surface expression of exposed granular membrane markers, specifically CD63, on activated basophils, while BHRA quantifies histamine release directly from activated basophils. Both tests have been instrumental in identifying patients responsive to Omalizumab in conditions such as asthma and chronic spontaneous urticaria, and are increasingly used to assess treatment efficacy [6, 7].

In our study, BAT and BHRA were conducted as previously described [5], with minor modifications. Results from both BAT and BHRA were compared at multiple time points—before, during, and after Omalizumab treatment. This evaluation allowed us to observe whether BAT and BHRA could effectively reflect changes in basophil reactivity in response to Omalizumab and if these changes aligned with clinical outcomes during DBPCFC. Specifically, we sought to determine the predictive potential of BAT and BHRA for distinguishing between the Omalizumab group and the placebo group and identifying children likely to exhibit higher allergen tolerance post‐treatment.

In untreated or placebo‐treated peanut allergic children, peanut extract induced a dose‐dependent upregulation of CD63 expression (in BAT) and histamine release (in BHRA), with consistent results observed across sample points (Table 1; Figure 1a). Omalizumab‐treated children, however, showed a marked reduction in maximal basophil reactivity and histamine release, with some cases exhibiting complete suppression of basophil activation even at high allergen doses (Figure 1a).

TABLE 1.

Summary of results.

Test type	Data type	Untreated/Placebo‐treated group Mean ± SEM (Samples: N = 45)	Omalizumab‐treated group Mean ± SEM (Samples: N = 26)	p	Optimal cut‐off	Sensitivity (%)	Specificity (%)	ROC AUC (95% CI)	p	Tjur's r 2
BAT	Anti‐FcƐR1‐reactive basophils (%)	70.5 ± 3.1	44.0 ± 5.5	< 0.0002	62.4	73	76	0.76 (0.77–0.98)	< 0.0001	0.23
	Maximal basophils reactivity (%)	83.1 ± 2.3	34.0 ± 6.4	< 0.0001	67.5	81	87	0.88 (0.77–0.98)	< 0.0001	0.52
	Sensitivity (EC50) ng/mL	55.4 ± 15.7	36,306 ± 8925	< 0.0001	245	85	98	0.93 (0.86–1.0)	< 0.0001	0.70
	AUC (AU)	7,186,867 ± 328,696	3,018,207 ± 625,293	< 0.0001	6,411,701	81	80	0.84 (0.74–0.95)	< 0.0001	0.39
BHRA	Anti‐IgE‐induced histamine release (ng/mL)	21.9 ± 2.4	16.1 ± 2.1	0.061	16.7	66	67	0.63 (0.50–0.77)	0.061	0.04
	Maximum histamin release (ng/mL)	52.0 ± 3.7	23.0 ± 3.8	< 0.0001	36	77	73	0.84 (0.74–0.94)	< 0.0001	0.35
	Sensitivity (EC50) ng/ml	1321 ± 351	44,084 ± 8461	< 0.0001	3263	81	91	0.93 (0.87–0.99)	< 0.0001	0.58
	AUC (AU)	4,531,947 ± 362,386	1,548,956 ± 314,748	< 0.0001	2,802,856	77	71	0.86 (0.77–0.95)	< 0.0001	0.39

Note: Goodness of logistic regression model fit was measured by Tjur's r ². All data, except BHRA anti‐IgE induced histamine release, fit the logistic regression model with high significance (p < 0.0001). All sample results prior to treatment and following treatment cessation (at Day 0 and 9 month), were included in the untreated/placebo group patients, together with results from patients receiving placebo at 3 and/or 6 months, totaling 45 sample points. All results from patients receiving Omalizumab after 3 and/or 6 months of treatment were categorised under the Omalizumab group (26 sample points in total).

FIGURE 1.

Peanut‐extract (filled circles) and anti‐FcεRI induced CD63 expression (%) (BAT) and anti‐E induced histamine release (ng/mL) (BHRA) (open circles) in blood from the same patient at baseline (point 0, prior to treatment) and at 3 months of Omalizumab treatment (sample point 3). The blue line represents the non‐linear fit of dose vs. response generated using by GraphPad Prism 8.3.1 (a). Linear regression analysis of associations between BAT and BHRA EC50 results and last tolerated challenge step. Filled circles represent patients receiving Omalizumab, while open circles represent patients receiving untreated/placebo patients (b). The results of the Basophil Activation Test (BAT) and Basophil Histamine Release Assay (BHRA) are depicted as a function of the last tolerated challenge step for patients receiving Omalizumab at the 3‐month point, represented by filled circles. Open circles denote the untreated condition at the study's initiation (time point 0) (c). Receiver Operating Characteristic (ROC) Curve Analysis for Predicting Omalizumab Treatment in Peanut Allergic Children. Each curve evaluates the diagnostic ability of these tests to predict Omalizumab treatment in children with peanut allergies. The x‐axis represents the false positive rate (1‐specificity), and the y‐axis represents the true positive rate (sensitivity). The diagonal dashed lines indicate a no‐discrimination threshold, where the test performance is equivalent to chance (d).

Importantly, both BAT and BHRA reflected significant increases in EC50 values—a measure of basophil sensitivity—following Omalizumab treatment, indicating an increase in the allergen dose required to trigger a response (Table 1; Figure 1a). Additionally, parameters like the area under the curve (AUC) and maximum histamine release in BHRA demonstrated significant reductions, while the last tolerated peanut challenge step in DBPCFC increased in correlation with these changes (Figure 1b). Such findings underscore Omalizumab's impact on reducing basophil sensitivity to allergens, an effect that appears to correspond with improved clinical tolerance levels.

Furthermore, our study highlighted the ability of pre‐treatment EC50 values from both BAT and BHRA to predict post‐treatment allergen tolerance, with higher baseline EC50 values associated with better treatment outcomes (Figure 1c). By using receiver‐operating characteristic (ROC) curve analysis, we identified specific cutoff values for BAT and BHRA parameters that achieved high sensitivity and specificity in distinguishing Omalizumab‐treated children from those in the placebo group. BAT EC50, in particular, demonstrated strong predictive performance, suggesting it could serve as a robust marker of Omalizumab treatment response (Figure 1d).

In clinical practice, these findings could support the use of BAT and BHRA as complementary tools in assessing and predicting the efficacy of Omalizumab in children with peanut allergy. While these tests currently function as investigational markers, their potential to provide insights into patient‐specific treatment responses and allergen tolerance thresholds could guide personalised FA management strategies.

Nonetheless, certain limitations should be noted. The sample size of our study was modest, and additional research involving larger, more diverse cohorts would be beneficial to further validate the observed associations. Moreover, although BAT and BHRA proved useful in this context, they are not yet standardised for routine clinical use in predicting FA treatment outcomes. Further, the BHRA typically requires about 10‐fold higher concentrations of stimuli to achieve a comparable level of reactivity in basophils. These differences may arise from several factors, such as the distinct mechanisms of activation and variations in the laboratory setup where BAT uses whole blood and BHRA replaces plasma with PIPES buffer [5]. Additionally, BAT was conducted on the same day as blood collection, whereas BHRA was performed the following day.

Despite these differences, BAT and BHRA were equally effective in predicting Omalizumab treatment and clinical response in peanut allergic children. This comparison highlights their complementary roles in evaluating treatment efficacy, suggesting both tests can be valuable in guiding personalised therapeutic strategies for managing peanut allergy and improving patient safety.

Author Contributions

C.G.M. and C.B.J. conceived the study design. C.N. and P.S.S. performed the basophil activation test and the histamine release test. All authors were involved in data interpretation. C.N. performed the data analysis of the basophil activation test and the histamine release test data and wrote the first manuscript draft. All authors reviewed and approved the final manuscript.

Conflicts of Interest

P.S.S. is head of R & D at Reflab. Other authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.