**2. Materials and Methods**

The prospective study was conducted for 7 years (2014–2020). During the first 5 years (2014–2018), chronic milk-FPIES was diagnosed in 57 children. They lived in the region of West Pomerania and were patients of the Paediatric Gastroenterology and Rheumatology Clinic, Gastroenterology Outpatient Clinic or Allergy Outpatient Clinics in Szczecin.

In the last 2 years of study (2019–2020) we continued the observation of the study group and we did not include new patients.

The patients were selected from children with symptoms indicating CMA. The suspicion of CMA was the premise for including a milk-free diet for 2–12 weeks, depending on the symptoms (Figure 1). After the symptoms had resolved or decreased, the milk oral food challenge (OFC) was performed. A negative OFC outcome ruled out CMA. A positive OFC was the basis for CMA recognition. FPIES was diagnosed in these children, with a delayed response during OFC (symptoms occurred above 2 h after milk ingestion), in whom occurred vomiting, pallor, and diarrhea, and often also severe dehydration.

The criteria for including a child in the study were: chronic milk-FPIES, age (up to 4 months), absence of coexisting chronic diseases, and a parental/legal guardian's written consent for the child to participate in the controlled study. Consent also included the storage and publication of the collected data. Only 55 children (33 boys, 60.0%) at the age of 1.6–2.6 months (median 2.2 months) were included in the study. After 19 months of treatment, three patients discontinued their participation in the project (they did not report for control milk provocations). Ultimately, 52 children (94.5%) completed the study.

diseases.

**Figure 1.** Gastrointestinal disorders of non-IgE mediated CMA diagnosis scheme \* CMA—cow's milk allergy; \*\* OFC—Oral Food Challenge; \*\*\* CMA diseases according to symptoms: FPIES food protein-induced enterocolitis syndrome, FPIAP—food protein-induced proctocolitis, FPIE food protein-induced enteropathy syndrome, EGID—syndromes of eosinophilic gastrointestinal **Figure 1.** Gastrointestinal disorders of non-IgE mediated CMA diagnosis scheme \* CMA—cow's milk allergy; \*\* OFC—Oral Food Challenge; \*\*\* CMA diseases according to symptoms: FPIES—food protein-induced enterocolitis syndrome, FPIAP—food protein-induced proctocolitis, FPIE—food protein-induced enteropathy syndrome, EGID—syndromes of eosinophilic gastrointestinal diseases.

The criteria for including a child in the study were: chronic milk-FPIES, age (up to 4 months), absence of coexisting chronic diseases, and a parental/legal guardian's written consent for the child to participate in the controlled study. Consent also included the storage and publication of the collected data. Only 55 children (33 boys, 60.0%) at the age of 1.6–2.6 months (median 2.2 months) were included in the study. After 19 months of Each patient had a medical and allergic history (recorded recurrent adverse reactions) and underwent physical examination. Every 6 months medical examination was carried out and a follow-up milk OFC was performed. If adverse symptoms occurred after milk ingestion, the provocation was stopped. A positive OFC outcome affirmed the persistence of CMA and was a premise for continuing a milk-free diet. A negative OFC indicated that the child had developed tolerance to milk.

treatment, three patients discontinued their participation in the project (they did not report for control milk provocations). Ultimately, 52 children (94.5%) completed the study. Each patient had a medical and allergic history (recorded recurrent adverse reactions) and underwent physical examination. Every 6 months medical examination was At the time of chronic milk-FPIES diagnosis and during follow-up visits (usually once per year, during the OFC procedure) blood morphology and cow's milk-specific IgE (sIgE) concentration in serum were tested. In addition, skin prick tests (SPTs) with food allergens were performed (Figure 2).

carried out and a follow-up milk OFC was performed. If adverse symptoms occurred after milk ingestion, the provocation was stopped. A positive OFC outcome affirmed the persistence of CMA and was a premise for continuing a milk-free diet. A negative OFC indicated that the child had developed tolerance to milk. During the diagnostic elimination diet and in the treatment of chronic milk-FPIES, a milk-free diet was administered: either the milk of mothers remaining on a milk-free diet or extensively hydrolyzed infant formulae (eHF; lactose-free casein hydrolysate). Children diagnosed with severe milk allergy received free amino acids formulae (AAF).

At the time of chronic milk-FPIES diagnosis and during follow-up visits (usually once per year, during the OFC procedure) blood morphology and cow's milk-specific IgE (sIgE) concentration in serum were tested. In addition, skin prick tests (SPTs) with food allergens were performed (Figure 2). The age of chronic milk-FPIES diagnosis was also the age of introduction of a diagnostic milk-free diet. A severe form of CMA was diagnosed according to WAO and ESPGHAN recommendations [4,14]. In all children the diagnosis of FPIES followed the 2017 criteria [7]. Previously, we used the Sicherer et al. criteria [8].

The open OFC procedures were always commenced in hospital conditions, under the control of a nurse and a doctor, with access to anti-shock drugs [15]. After a negative lip test (a drop of milk), gradually higher doses of milk were administered every 15 min (1, 2, 5, 10, 20, 50, and 100 mL). Infants younger than 6 months received at least 100 mL of milk. Patients remained under observation for at least 4–8 h following the end of OFC [15–18]. The provocation was continued at home for the next 6 days. Every day, parents administered the milk mixture corresponding in volume to one meal (older children—up to 250 mL), the

information about possible adverse reactions was recorded in the observation card. After 6 days (or earlier, if side effects had occurred), the doctor examined the OFC outcome. In total, the results of 139 milk OFCs were analyzed.

**Figure 2.** Diagnostic and treatment scheme in the study group of children with chronic FPIES dependent on cow's milk protein intake. OFC \*—Oral Food Challenge.

During the first OFC all the children had an intravenous entry, while during the next OFC, only those with elevated milk sIgE levels.

Cow's milk sIgE concentration was determined by FEIA method in CAP system with automatic UniCAP apparatus from Phadia. The determination parameters were the range between 0.35–100 kU/L; accuracy between 2–9.1%; sensitivity < 2 kU/L; repeatability at 98%; specificity at 100%. Recommended range: healthy patients < 0.35 kU/L, atopics > 0.35 kU/L.

Skin prick tests (SPTs) with food allergens were performed with a modified method on the skin on the patient's back. The positive control was histamine solution (1 mg/1 mL), while the negative one—a diluent. Commercial Allergopharma and ALK food solutions were used. The wheal and erythema size were measured after 20 min. SPT was regarded positive when the sum of the half of the longest diameter and the midpoint orthogonal diameter of the wheal with allergen was at least equal in diameter to the histamine wheal and at least 3 mm higher than the negative control.

Finally, all data were collected in electronic form in MS Excel spreadsheet and were subject to statistical analysis. Continuous variables were described by median, minimum,

1

and maximum values. Discrete variables were described by their abundance and frequency of occurrence.

The research was approved by the PUM Bioethics Committee No KB-0012/80/14. The research was financed by the statutory activities (WNoZ-319-01/s/12/2013–2020) and by the NCN grant No 2016/21/N/NZ7/03409. The presented results are part of the ongoing project.
