Evaluation of Eosinophil Indices in Pediatric Patients with Cow’s Milk Allergy

Ayşegül Kırankaya Çatal; Levent Deniz; Meltem Erol

doi:10.4274/BMB.galenos.2025.2024-01-013

Abstract

Objective

This study aimed to evaluate eosinophil indices and other inflammatory parameters in pediatric patients with cow’s milk allergy (CMA), which are easily accessible and may be useful in predicting CMA.

Method

This retrospective study included 39 people in the patient group and 123 people in the control group. The records of the patients diagnosed with CMA by specific immunoglobulin E (IgE) tests were compared with patients without CMA. The study examined demographic data of the patients, including age at presentation and gender, as well as laboratory findings. The study included children under 24 months of age who underwent a specific IgE test for CMA and had simultaneous hemogram results.

Results

Neutrophil-to-eosinophil ratio (NER), derived neutrophil-to-lymphocyte ratio (dNLR), and leukocyte-to-eosinophil ratio (LER) were significantly lower in the CMA than control groups. On the other hand, eosinophil, eosinophil-to-monocyte ratio (EMR), and eosinophil-to-lymphocyte ratio (ELR) were significantly higher than control groups. Specific IgE levels were observed to have a negative correlation with LER, NER, and dNLR, and a positive correlation with eosinophil, EMR, and ELR. For a LER ≤22.2 cut-off value, sensitivity was 64.1%, and specificity was 73.2%.

Conclusion

In our study, LER and EMR seem to be useful parameters to predict CMA in children. This study’s findings may indicate that leukocytes and eosinophils, could be crucial in the pathogenesis of CMA cases. Eosinophil counts and eosinophil indices, readily obtainable through a complete blood count, can serve as parameters for distinguishing CMA.

Keywords:

Child, cow’s milk allergy, eosinophil, eosinophil-to-monocyte ratio, leukocyte-to-eosinophil ratio

Introduction

Cow’s milk allergy (CMA) is an immunologic reaction to one or more milk proteins (1). CMA stands out as a prevalent form of food allergy among children aged below 24 months in developed nations (2); the prevalence in this age group was calculated to be 2-7.5% (3). Adverse reactions after cow’s milk ingestion can occur at any age after birth, even in breastfed infants. The immune response to cow’s milk proteins can be mediated by immunoglobulin E (IgE) or be independent of IgE (4). In most children, CMA develops with IgE-mediated reaction (5), and Th2 lymphocytes and eosinophils also play a significant role in the response of inflammation resulting from allergen binding to specific IgE (6). Platelets and neutrophils are indicators that play crucial roles in inflammation. Today, easy accessibility to these blood parameters allows them to be used to diagnose and monitor many diseases. According to studies, the platelet-lymphocyte ratio (PLR), neutrophil-lymphocyte ratio (NLR), and inflammation indices obtained from hemogram parameters are effective in diagnosing and monitoring cardiovascular diseases, malignancies, chronic inflammatory diseases, and allergic diseases like: allergic rhinitis, atopic dermatitis, and asthma (7-10).

It is crucial to avoid unnecessary initiation of elimination diets in children with CMA. The diagnosis of food allergy is still based on the principle that the causative allergen should be removed from the diet, and the symptoms should recur when added. Currently, there is no single commonly accepted diagnostic laboratory test to demonstrate an adverse immune response to cow’s milk proteins (2). Although eosinophil indices have been studied in patients with allergic rhinitis, asthma and nasal polyps (10), there is an insufficient number of studies on these indices in children with CMA in the literature.

We aimed to evaluate eosinophil indices and other inflammatory parameters in pediatric patients with CMA, which are easily accessible and may be helpful in predicting CMA.

Materials and Methods

This retrospective study analyzed patient data from a University of Health Sciences Turkey, İstanbul Bağcılar Training and Research Hospital between September 2022 and September 2023. The records of the patients diagnosed with CMA by specific IgE tests were compared with children without CMA. The Ethical Committee of the University of Health Sciences Turkey, İstanbul Bağcılar Training and Research Hospital, gave its approval for the study (date: 27/10/2023, number: 2023/10/05/061). The research was carried out in accordance with the guidelines established by the Helsinki Declaration.

This research included 162 patients. The study included 39 people in the patient group and 123 age-and-gender-matched people in the control group. The study examined demographic data of the patients, including age at presentation and gender, as well as laboratory findings. The study included children under 24 months of age who underwent a specific IgE test for CMA and had simultaneous hemogram results. The study excluded parasitic diseases, malignancy, hematological diseases, and known infectious and systemic inflammatory diseases, as these conditions may affect eosinophilia levels. Systemic inflammation index (SII) was defined as (neutrophil×platelet)/lymphocyte; SIRI was defined as (neutrophil×monocyte)/lymphocyte; dNLR was defined as neutrophil count/(leukocyte count-neutrophil count); aggregate index of systemic inflammation (AISI) was defined as (neutrophil×platelet×monocyte)/lymphocyte. Eosinophil indices, including leukocyte-to-eosinophil ratio (LER), neutrophil-to-eosinophil ratio (NER), eosinophil-to-monocyte ratio (EMR), and eosinophil-to-lymphocyte ratio (ELR) were calculated based on hemogram parameters. In whole blood samples, hemogram parameters were measured by a Mindray BC-6800 Plus device (Shenzhen Mindray Bio-Medical Electronics Co), and specific IgE was measured using the chemiluminescent immunoassay method with the Immulite 2000 (Siemens Healthcare Diagnostics).

Statistical Analysis

To evaluate the normal distribution of continuous data, the Shapiro-Wilk test was employed. Continuous data were displayed as either mean with standard deviation or median with the 25^thand 75^th percentiles. The Mann-Whitney U test or Student’s t-test was employed to compare continuous variables. The diagnostic performance of the laboratory parameters was evaluated through the receiver operating characteristic curve. The relationships between parameters were evaluated using Spearman correlation analyses. Statistical analyses were conducted using IBM SPSS v. 26.0 (IBM Corp., Armonk, NY, US) and GraphPad Prism 8.0 (GraphPad Software, San Diego, California, US). A significance level (p-value) of less than 0.05 was considered significant.

Results

The demographic data of our study are given in Table 1. There was no statistical difference in age, gender, leukocyte, platelet, monocyte, neutrophil, lymphocyte values, NLR, PLR, MLR, systemic inflammation response index, SII, and AISI between the patients with CMA and the control group (p>0.05). NER, dNLR and LER were significantly lower in the patients with CMA, whereas eosinophil, ELR and EMR were significantly higher than controls (p<0.001), as shown in Table 1 and Figure 1.

LER achieved the highest area under the curve (AUC) value of 0.716 [95% confidence interval (CI) =0.640-0.784] at a cut-off value of 22.2. EMR had an AUC value of 0.711 (95% CI=0.634-0.779) at a cut-off value of 0.30. ELR achieved an AUC value of 0.703 (95% CI =0.626-0.772) at a cut-off value of 0.06. NER achieved an AUC value of 0.703 (95% CI=0.626-0.772) at a cut-off value of 8.75 (Table 2).

In Spearman correlation, serum specific IgE levels were observed to have a negative correlation with LER (r=-0.340, p<0.001), NER (r=-0.319, p<0.001), and dNLR (r=-0.162, p=0.040), and positively correlated with eosinophil (r=0.337, p<0.001), EMR (r=0.330, p<0.001), and ELR (r=0.318, p<0.001) (Table 3).

Discussion

The study participants’ age and gender were similar in all groups, ensuring that variations in inflammation marker levels can be attributed to specific factors rather than demographic differences. NER, dNLR, and LER were significantly lower in the patients with CMA. On the other hand, eosinophil, ELR, and EMR were significantly higher in the patients with CMA than in the controls. In determining the CMA group, the highest AUC was observed in LER. Moreover, LER had a higher correlation with specific IgE levels.

CMA is a prevalent food allergy among infants (11). Early recognition and appropriate management of CMA are crucial for the well-being of affected infants. The first step in the immune system’s response to cow’s milk protein allergy (CMPA) is a T-cell-dependent reaction (12). As a result, proinflammatory cytokines [interleukin (IL)-5, IL-13, and IL-14] are secreted by Th2 cells (13). This activates B-cells, leading to the secretion of IgE. When the same food allergen is ingested again, IgE binds to eosinophils, basophil, and mast cell surfaces and activates these cells, causing the release of mediators such as histamine that produce typical symptoms including anaphylaxis, laryngospasm, bronchospasm, angioedema, rhinitis, and urticaria within minutes to two hours (14). Allergic reactions to cow milk can be categorized into two main types: Immediate, which are typically IgE-mediated, and late-onset, encompassing both non-IgE-mediated and mixed IgE and cell-mediated reactions (15). IgE-mediated food allergy occurs with the development of food allergen-specific IgE, which develops after first contact with an allergen. Symptoms may be mild or may progress to anaphylaxis, which can be life-threatening. The oral food challenge has been regarded as the gold standard in diagnosing CMPA (16). Nevertheless, food intolerance and severe eosinophilia may cause symptoms to recur. Currently, no widely accepted diagnostic laboratory test will detect an undesirable immune system response to cow’s milk proteins.

Eosinophils play a crucial role in immuno-inflammatory reactions in CMA (17). Because current indicators do not accurately represent inflammatory processes, their usefulness in this disease is limited. Thus, it is crucial to search for new biomarkers capable of detecting and monitoring the dynamics of inflammation. Neutrophilic lipocalin associated with gelatinase (NGAL) and chemerin, markers associated with neutrophilic inflammation, was shown to be at higher levels in the CMA patient group than in the control group. Statistically significant correlations have been shown between IL-10, TNF-α, calprotectin, NGAL and WBC levels in children with CMA (18). Furthermore, the leukocyte adherence inhibition test has been proposed to discriminate antigen-specific immunoreactivity in non-IgE-mediated CMA (19). According to research, eosinophil-related indicators like eosinophil cationic protein and eosinophil protein X, were linked to intestinal inflammation in infants with atopic eczema and food allergies (20). Numerous studies have suggested that NLR and ELR could function as effective inflammatory indicators for differentiating between intermittent and persistent allergic rhinitis. Analyses have revealed that T-helper 2 lymphocytes, neutrophils, and eosinophils are all actively involved in the late-phase immune response that follows allergen exposure (21).

Study Limitations

The limitations of this study were its retrospective and single-center design. Total IgE, and clinical characteristics could not be obtained from all patient data.

Conclusion

In our study, LER and EMR seem to be useful parameters to predict CMA in children. These findings may indicate that leukocytes and eosinophils could play an important role in the pathogenesis of CMA. Eosinophil counts and eosinophil indices, readily obtainable through a complete blood count, can serve as parameters for distinguishing CMA. Understanding the immune response, particularly the role of eosinophils and leukocytes, could provide valuable insights into the pathogenesis, diagnosis, and potential predictors of tolerance in CMA. Studies on this subject are limited in the literature. We believe it will contribute to the literature. Moreover, more comprehensive studies are needed.

Ethics

Ethics Committee Approval: The Ethical Committee of the University of Health Sciences Turkey, İstanbul Bağcılar Training and Research Hospital, gave its approval for the study (date: 27/10/2023, number: 2023/10/05/061).

Informed Consent: Retrospective study.

Authorship Contributions

Concept: A.K.Ç., L.D., M.E., Design: A.K.Ç., L.D., M.E., Data Collection or Processing: A.K.Ç., L.D., M.E., Analysis or Interpretation: A.K.Ç., L.D., M.E., Literature Search: A.K.Ç., L.D., M.E., Writing: A.K.Ç., L.D., M.E.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support.

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