The Basophil Activation Test in the Diagnosis of Cow’s Milk Allergy in Children: A Narrrative Review

1. Introduction

Cow’s milk allergy (CMA) is one of the most common food allergies in infants and young children, typically manifesting as an adverse immune response to proteins present in cow’s milk [1]. In high-income countries, the prevalence of CMA is estimated to be approximately 2-3% [1]. CMA presents often within the first few months of life, usually within two months after the introduction of cow’s milk based infant formula [1]. Symptoms range in severity.

The initial evaluation of suspected food allergy typically involves obtaining a detailed clinical history and perform a complete physical examination, eventually supplemented by skin prick testing (SPT) and measurement of allergen-specific immunoglobin E (sIgE) levels [2]. While these diagnostic tools demonstrate high sensitivity in identifying sensitized individuals, their specificity is limited [2,3]. As a result, these tests cannot definitively confirm or exclude a diagnosis of food allergy. The Cow’s Milk related Symptom Score (CoMiSS TM) was developed to increase awareness among health care professionals for CMA [4].

Currently, the oral food challenge (OFC) remains the gold standard for diagnosing CMA [5]. However, the OFC is associated with several disadvantages: it is time-consuming, resource-intensive and will cause symptoms if positive, carrying a risk of severe allergic reactions including anaphylaxis [5]. These limitations underscore the need for safer, more specific diagnostic tools that can reduce reliance on OFCs.

The basophil activation test (BAT) is a laboratory test that measures how strongly basophils react to certain allergens such as cow’s milk proteins [6]. The aim of this article is to provide an overview of the current knowledge on the use of the BAT in the diagnosis of CMA in children.

2. Materials and Methods

This narrative review was conducted by searching PubMed/MEDLINE for articles published up to March, 2026. Search terms included combinations of: “cow’s milk allergy,” “basophil activation test, “BAT”, “food allergy,” “oral food challenge,” “IgE-mediated,” “children,” and “pediatric.” Additional references were identified through manual screening of reference lists of relevant articles and review papers. No formal inclusion or exclusion criteria were applied; studies were selected based on relevance to the diagnostic role of the BAT in pediatric cow’s milk allergy.

3. Results

3.1. Cow’s Milk Allergy: Definition and Classification

CMA is caused by an adverse immune response to proteins present in cow’s milk, predominantly caseins (α1-, α2-, β-, and κ-casein) and whey proteins (α-lactalbumin and β-lactoglobulin) [7]. CMA can manifest through different immunological mechanisms, primarily classified into IgE-mediated, non-IgE-mediated, or a combination of both [7].

The incidence of CMA varies by region and diagnostic criteria. Data from the EuroPrevall birth cohort provide one of the most reliable population-based estimates, confirming CMA in only 0.54% of children with double-blind, placebo-controlled [8]. Interestingly, nearly one-quarter of the confirmed cases had negative cow’s milk–specific IgE [sIgE), suggesting a non-IgE-mediated mechanism [8]. These findings highlight both the relatively low prevalence of confirmed CMA and the substantial proportion of children who outgrow the condition during early childhood.

Understanding the different types and clinical presentations of CMA is crucial for effective management and improving quality of life for affected children and their families.

3.2. Cow’s Milk Allergy: Pathophysiology

In IgE mediated allergy, the immune system aberrantly produces during the “sensitization” phase IgE antibodies against cow’s milk proteins, which bind to the surface of mast cells and basophils [9]. Upon subsequent exposure, the “activation” phase occurs: IgE on mast cells recognizes allergenic epitopes, triggering rapid degranulation and release of inflammatory mediators responsible for acute allergic reactions [9].

In contrast, non-IgE-mediated reactions are thought to result from different mechanisms, including Th1-driven immune activity and interactions between T cells, mast cells, and neurons that can alter intestinal smooth muscle function and motility [10,11]. These reactions typically develop more slowly, often hours to days after exposure, and predominantly affect the gastrointestinal tract [10,11]. The immune response involves local inflammation, epithelial damage, and recruitment of immune cells, leading to chronic or delayed gastrointestinal symptoms [10,11].

Understanding these divergent mechanisms is crucial for the interpretation of diagnostic tools such as the BAT, which is primarily informative in IgE-mediated reactions but may provide additional insights in complex or mixed presentations.

3.3. Cow’s Milk Allergy: Clinical Presentation

The clinical presentation of CMA encompasses a wide spectrum of symptoms affecting multiple organ systems [12]. Gastrointestinal manifestations include diarrhea and constipation, vomiting, and colic; respiratory symptoms may present as wheezing or coughing; and cutaneous signs commonly include eczema and urticaria [7,12]. The symptom profile varies with age: infants more frequently exhibit gastrointestinal involvement, whereas older children are more likely to present with respiratory or dermatologic manifestations [11,13,14]. The heterogeneity of CMA presentations, involving various tissues and organ systems, complicates clinical identification and may contribute to diagnostic uncertainty or delayed diagnosis [11].

3.4. Cow’s Milk Allergy: Diagnosis

The diagnosis of CM is primarily based on a detailed assessment of clinical symptoms in conjunction with allergy testing [11]. The 2024 ESPGHAN Position Paper discusses various tools for the diagnosis of CMA. A diagnostic elimination diet for 2 to 4 weeks, followed by a controlled oral food challenge, is recommended as the cornerstone of diagnostic confirmation, as re-exposure under supervised conditions remains the gold standard for establishing cow’s milk allergy [11].

SPT and sIgE indicate sensitization rather than clinical allergy and results should always be interpreted alongside the clinical history and in most cases confirmed by an elimination diet followed by elimination diet followed by a supervised oral food challenge [13]. Component resolved diagnostics (CRD) offers a further refinement by measuring specific IgE against cow’s milk protein components [15,16]. It may help identify sensitization, predict the risk of severe reactions. However, evidence that CRD offers superior diagnostic accuracy over conventional allergy tests remains inconclusive and it cannot replace OFC [17]. While DBPCFC remains the gold standard, an open OFC is generally sufficient for confirming CMA in routine clinical practice, especially during the first year of life [13]. Despite its diagnostic value, the OFC is resource-intensive, time-consuming and carries an inherent risk of allergic reactions, including anaphylaxis [25]. These limitations underscore the need for non-invasive biomarker that could reduce the number of OFCs required and support decision-making [25]. More recently, the BAT has emerged as possibly promising diagnostic approach, offering additional insight into IgE-mediated mechanisms.

3.5. Basophil Activation Test

Basophils are round cells measuring approximately 5–10 μm in diameter and containing multilobed nuclei with densely packed chromatin [18].

Their characteristic appearance helps for differentiation from other circulating leukocyte populations [19]. Histamine, present at about 1–2 pg per cell, represents the major constituent of these granules and plays a central role in the basophil’s contribution to allergic inflammation [20]. Upon activation, basophils release granule mediators into the surrounding environment through exocytosis involving multiple pores formed in the plasma membrane [20].

Classically, basophil activation and subsequent degranulation occur when high-affinity IgE receptors (FcεRI) are cross-linked by IgE antibodies bound to their specific antigen and upregulate surface activation markers including CD45, CD11b, CD11c, CD62L, CD203c, and CD63[21]. However, basophils may also be stimulated to release mediators through IgE-independent pathways [22].

The BAT is a flow-cytometry–based assay that identifies allergen reactivity by assessing whether a suspected allergen can cross-link surface-bound IgE on peripheral blood basophils and trigger degranulation [23,24]. Introduced in 1991 as a diagnostic tool, the BAT gained momentum following the discovery of CD63 as a reliable activation marker [23,24]. CD63, normally associated with granule membranes, becomes expressed on the cell surface during degranulation and histamine release. BAT protocols employing CD63 detection typically using dual staining with anti-CD63 and anti-IgE monoclonal antibodies have demonstrated strong diagnostic accuracy for IgE-mediated allergic conditions [23,24].

Commercially available systems include Flow-CAST (Bühlmann Laboratories), BASOTEST (Becton Dickinson) and the Allergenicity test (Beckman Coulter) [25,26]. Each differing in their choice of basophil identification and activation markers [25,26]. Flow-CAST , the most widely used of these systems, has undergone analytical validation studies aimed at achieving compliance with EU in Vitro Diagnostic Regulation requirements, representing an important step towards broader regulatory recognition [25,26].

BAT is widely used as a diagnostic tool in various food allergies. Several studies have compared the performance of the BAT to other diagnostic methods to evaluate its diagnostic accuracy and reliability.

In the study by Ciepiela et al. the BAT was compared to sIgE for diagnosing CMA in children [27]. Flow cytometry was used to analyze allergen-induced CD203c upregulation [27]. The sensitivity of this method was found to be comparable to that of traditional diagnostic techniques, while its specificity exceeded that of serum IgE testing [27]. Consequently, the BAT represents a highly effective tool for confirming CMA in pediatric patients [27].

The diagnostic performance of BAT in CMA was also compared to traditional methods, using the OFC as the reference standard in a study by Bartha et al., which focused on the ability of BAT to predict tolerance to baked milk and fresh milk in children in a cohort of 150 children [28]. BAT demonstrated the highest diagnostic accuracy for both baked milk and fresh milk allergy [28]. Using optimal cut-off values, BAT minimized the need for OFCs compared with the other tests while maintaining very high diagnostic precision; in children under 2 years of age, BAT drastically reduced the number of required OFCs and achieved 100% diagnostic accuracy for BM [28]. Additionally, further analyses indicated that BAT was the only biomarker capable of distinguishing both the severity of allergic reactions and the reaction threshold during OFCs, suggesting that BAT may have potential for enhanced risk stratification in patients with cow’s milk allergy [28].

Santos et al. reported a sensitivity of 98% and a specificity of 96% for the BAT in diagnosing peanut allergy in peanut-sensitized children [29]. Similarly, Berghea et al. demonstrated a sensitivity of 85.0% and a specificity of 77.0% when applying the test to wheat allergy [30]. These results highlight broader clinical application of the BAT.

The BAT is not only a diagnostic tool but can also be used to assess the severity of allergic reactions [29,31,32]. According to the study by Rubio et al., a strong correlation was observed between the severity of clinical allergic reactions and the extent of basophil activation, as reflected by each patient’s individual basophil reactivity [32]. Another study, performed by S. Ford et al. also suggest that the BAT provides insights into the severity of clinical reactivity and the degree of allergen tolerance [31].

Several studies have also highlighted the added value of BAT in guiding OFCs [29,33]. In the study by Ruinemans-Koerts et al., 80% of children showed complete concordance between the results of the double-blind placebo-controlled food challenge (DBPCFC) and the BAT [33]. The BAT provides a safe in vitro alternative to the DBPCFC that can accurately identify IgE-mediated CMA without exposing children to an oral allergen challenge [33]. By achieving perfect diagnostic accuracy in IgE-sensitized patients, BAT can substantially reduce the need for DBPCFC, reserving oral challenges only for selected cases where immunological mechanisms remain unclear [29,33].Given these findings, the BAT appears not only valuable for determining the optimal timing of OFC but also potentially useful for monitoring the progression of desensitization and the development of tolerance in children undergoing therapeutic interventions [28,29,33].

Taken together, these studies consistently demonstrate that the BAT offers diagnostic accuracy at least comparable to and in several populations exceeding, that of SPT and sIgE, while simultaneously providing functional information on reaction severity and threshold that conventional test cannot deliver.

The diagnostic value of the BAT, however, is not uniform across all clinical presentations of CMA and its interpretation differs depending on whether baked or fresh milk allergy is being assessed. This distinction has both immunological and clinical dimensions. Ford et al. demonstrated across 132 children that basophil reactivity, casein-specific IgE, and milk SPT wheal size all differed significantly across five clinical tolerance groups, and were markedly higher in baked milk-reactive compared to baked milk-tolerant children [31]. In the BAT2 study, BAT was the only biomarker able to predict both the severity and the reaction threshold for baked and fresh milk challenges, but the performance profiles diverged: for baked milk, BAT achieved 71% sensitivity and 100% specificity in identifying severe reactors, reflecting the narrower, more extreme phenotype of children who react to heat-denatured protein [28]. For fresh milk, the picture was different. BAT reached 96% sensitivity for identifying low-threshold reactors, but specificity fell to only 41%, because the much broader range of sensitization among fresh milk-reactive children makes it harder to discriminate [28]. This performance asymmetry has a practical implication: a positive BAT result carries a different predictive weight depending on which allergen form is being challenged. In the context of baked milk, a positive BAT result identifies a small, high-risk subgroup in whom reaction during OFC is both likely and likely to be severe [28]. For fresh milk, BAT’s high sensitivity makes it a strong rule-out tool, but its modest specificity means a positive result alone does not reliably define who will react clinically [28]. These differences underscore that BAT cut-offs and clinical interpretation cannot be applied uniformly across challenge types, and that future standardization efforts should define type-specific thresholds for baked and unheated milk challenges separately.

3.6. Limitations of the BAT

The BAT is not yet universally used for diagnosing food allergies, despite its high diagnostic potential, due to several limitations and challenges. There is a lack of validation and standardization, which prevents the universal application of the test across different laboratories and protocols [34]. A detailed protocol is needed to determine which allergens should be used and how relevant they are for different patient groups. It is also essential to identify which activation markers are most effective in measuring responses across various patient populations [34]. According to a study, 5–10% of patients have non-responsive basophils, which do not upregulate CD203c or CD63 when stimulated by IgE-mediated allergens, further limiting the implementation of BATs [35]. Furthermore, basophil responses can be influenced by the patient’s medication use and clinical condition, while variability in allergen extracts may reduce the reproducibility of the results [35].

The choice of stimulant, whole allergen extract versus isolated molecular components (recombinant or purified) has important consequences for BAT performance, particularly in CMA. Whole-protein extracts contain the full complement of allergen molecules and their native conformational structures, and as such tend to elicit stronger basophil responses [36].

For cow’s milk specifically, extract-based stimulation was confirmed as superior: Chhing et al. showed that even in concordant cases, component-stimulated basophils displayed on average 6% lower activation rates than their extract-stimulated counterparts and that discordance was more pronounced in patients with lower total IgE [36].

This has implications for standardization: laboratories using component-based protocols for cow’s milk BAT should be aware of the elevated risk of false-negative results, particularly in patients with lower total IgE or undergoing immunotherapy.

Basophils can be stimulated with single allergen concentration or with a serial log dilution series [37,38,39]. The single concentration approach is operationally simpler, faster, and less costly: one concentration of allergen is applied and the percentage of CD63- or CD203c-positive basophils is read as the primary outcome [39]. This reflects how strongly basophils respond at a given dose. Its main limitation is that it is sensitive to the chosen concentration: too low and even truly allergic patients may not show activation; too high and the dose-response plateau may obscure differences between patients with varying degrees of sensitization [37,38,39]. A serial log titration approach addresses this by generation full dose response curve, that reflects both reactivity and sensitivity and may improve discrimination between tolerant and reactive patients, as well as between mild and severe reactors [37,38,39]. Increased cost, complexity and processing time limit feasibility in routine clinical settings [37,39].

A further practical limitation relates to the stability if basophils ex vivo. Traditionally, BAT required analysis within 4-6 hours of blood collection, which restricts the test to centers with on-site flow cytometry capacity [40,41,42]. With recent developments, CD203c upregulation remains stable in EDTA or heparin-anticoagulated blood stored at 4 °C for up to 24 hours [40,41,42]. More recently studies showed that processed samples can be stored at room temperature or 2-8 °C for up to 7 days before analysis, and cryopreservation approaches using glycerol-assisted protocols at -20 °C or glycerol-free storage at -80 °C have shown feasibility for long-term storage of up to 6-12 months [40,41,42]. These advances are relevant for multi-center studies.

According to the BAT User’s Manual, the test requires specialized equipment and trained personnel, which limits its accessibility [39].

Reimbursement and formal regulatory recognition of the BAT remain limited in many healthcare systems [43]. In several countries, the test does not yet meet the criteria required for inclusion under standard health insurance coverage, reflecting the broader need for prospective clinical validation and harmonized protocols. The cost of the test varies depending on the center and the type of allergy [43].

3.7. The Basophil Activation Test and Oral Immune Therapy

While elimination diets remain the cornerstone of management, they do not actively alter the natural course of the allergy. This has led to growing interest in approaches that aim to accelerate tolerance acquisition, most notably oral immunotherapy (OIT) [44,45].

OIT may be considered for children with confirmed IgE-mediated CMA, particularly when dietary avoidance proves insufficient, negatively affects daily functioning, or significantly diminishes quality of life [44,46]. According to the EAACI Guidelines, OIT can be offered to children from approximately 4 to 5 years of age with persistent CMA, with the intention of raising the reaction threshold and reducing the likelihood of accidental reactions [46]. This recommended age range reflects the observation that most children who naturally outgrow CMA typically achieve tolerance before entering school [46].

As mentioned earlier, the BAT is considered the most promising test for confirming food allergies without the need for OFC [28,29,33]. Typically, OFC is used to evaluate eligibility for initiating immunotherapy in food allergy patients [44]. Several studies have shown that basophil activation changes during allergen-specific immunotherapy, with a reduction in basophil activation observed in patients undergoing OIT for food allergies such as cow’s milk, peanut, and egg [47]. This allows for monitoring clinical response to OIT and reducing the number of OFC required [47]. his section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.

4. Discussion

The available literature indicates that the BAT demonstrates particularly high specificity and may therefore more effectively discriminate between mere sensitization and clinically relevant CMA compared with conventional IgE-based diagnostics [27,29]. This distinction is of considerable clinical importance, especially in patients presenting with discordant findings between clinical history and sIgE levels. In such cases, BAT has been proposed as a valuable adjunctive diagnostic tool, particularly when the indication for an OFC is uncertain and the risk–benefit balance requires careful consideration .

Several studies have reported an association between increased basophil activation and greater clinical reaction severity [28,29]. However, these findings have not been consistently replicated. The heterogeneity across studies likely contributes to this inconsistency, including variability in the definition and grading of reaction severity, differences in the activation markers employed (e.g., CD63 versus CD203c), and methodological variation in allergen preparations and concentrations [35]. As a result, the predictive value of BAT for reaction severity remains inconclusive. Further methodological standardization and well-designed prospective studies are required to clarify its prognostic potential [34].

In the context of the OIT, dynamic changes in basophil reactivity suggest that the BAT may serve as a biomarker for immunological modulation and treatment response [47]. Nonetheless, current evidence remains limited and is derived from relatively small and heterogeneous study populations [34]. Robust longitudinal data are necessary before the BAT can be reliably implemented as a monitoring tool in clinical practice [34].

Despite its promising diagnostic performance, widespread clinical implementation of the BAT is currently hindered by the lack of international standardization [34,39]. Consensus on allergen sources and preparation, activation markers, assay protocols, and diagnostic cut-off values is essential to ensure reproducibility and inter-laboratory comparability [34,39]. In addition, economic evaluations are warranted to determine whether the implementation of the BAT is cost-effective compared with the current reliance on oral food challenges, taking into account not only direct healthcare costs but also patient burden and resource utilization [39,43]. Moreover, large-scale prospective multicenter trials are needed to definitively define the position of the BAT within the diagnostic algorithm of CMA [39].

Future research addressing diagnostic accuracy, prognostic value, therapeutic monitoring, and health-economic impact may further support the integration of the BAT into personalized allergy management strategies.

5. Conclusions

In conclusion, the BAT represents a promising and potentially practice-changing tool in the diagnosis of CMA. Its high specificity may enable more accurate discrimination between sensitization and clinically relevant allergy, thereby reducing unnecessary OFCs and improving risk stratification.

However, despite its strong diagnostic potential, clinical implementation remains constrained by limited standardization and insufficient large-scale validation. Well-designed prospective multicenter studies are essential to firmly establish the position of BAT within the diagnostic algorithm of CMPA and to support its integration into routine clinical practice.

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