Update Diagnostic Tests of Cow Milk Allergy

Update Diagnostic Tests of Cow Milk Allergy

Widodo Judarwanto, Allergy Online Clinic Jakarta Indonesia

Cow’s milk allergy (CMA) is thought to affect 2–3% of infants. The signs and symptoms are nonspecific and may be difficult to objectify, and as the diagnosis requires cow’s milk elimination followed by challenge, often, children are considered cow’s milk allergic without proven diagnosis. Because of the consequences, a correct diagnosis of CMA is pivotal. Open challenges tend to overestimate the number of children with CMA. The only reliable way to diagnose CMA is by double-blind, placebo-controlled challenge (DBPCFC).

The prevalence of cow’s milk allergy (CMA) is estimated to be between 2% and 3% in infants and marginally lower in older children. The percentage of parents that believe their child has CMA (or any other food allergy), however, amounts to between 5% and 20% . Signs and symptoms of CMA are nonspecific and often difficult to objectify. Due to diagnostic burdens, the number of children treated for CMA is probably two to three times higher than justified. A wrong diagnosis of CMA may not only result in somatisation but also in insufficient topical treatment of eczema, fear for or problems with the introduction of solids, and dietary deficiencies. Moreover, the long-term elimination of cow’s milk protein (CMP) in a sensitised child without CMA may elicit severe adverse reactions when cow’s milk is reintroduced. Careful diagnosis of CMA, therefore, is of utmost importance.

Adverse reactions to CMP can be present from birth, even in exclusively breast-fed infants. Not all reactions are of allergic nature. In 2001, the EAACI published a report on the terminology of adverse reactions. The umbrella phrase, food hypersensitivity, covers non-allergic food hypersensitivity (traditionally named ‘food intolerance’) and allergic food hypersensitivity (food allergy). The latter requires an underlying immune mechanism. Most children with CMA have immunoglobulin E (IgE)-mediated allergy as a manifestation of their atopic constitution, with or without atopic eczema, asthma, or allergic rhinitis. A small group have cell-mediated allergy with gastro-intestinal symptoms.

A comprehensive history (including a family history of atopy) and careful physical examination form the foundation of both algorithms. The risk of atopy increases if a parent or sibling has atopic disease (20–40% and 25–35%, respectively), and is higher still if both parents are atopic (40–60%).In comparison to cow’s milk formula‐fed infants, exclusive breast feeding during the first 4–6 months of life reduces the risk for CMPA and most severe allergic manifestations during early infancy. The distinction between breast‐fed and formula‐fed infants reflects the importance of ensuring an adequate duration of breast feeding. Management principles also differ. The management of breast‐fed infants depends on reducing the maternal allergen load and strict avoidance of CMP in supplementary feeding. It is recommended that exclusive or partial breast feeding is continued, unless alarm symptoms require a different management. The earlier CMPA develops, the greater the risk of growth retardation

Cow’s milk challenge

After the elimination of CMP from the child’s or the mother’s diet, signs and symptoms should disappear within a few days. Atopic eczema, when caused by CMA, may take 4 weeks to improve sufficiently. Upon challenge, renewed confrontation with CMP results in recurrence of the presenting signs and symptoms.

Challenges may be performed either open or double-blindly. With open challenges, both the staff performing the test and the parents know that the child is given CMP and in what amount. Double-blind placebo-controlled food challenges (DBPCFC) are designed to withhold this information both to the parents and the staff until afterwards. They are performed with placebo and verum in random order. CMP is concealed in a way that both test feedings look and taste similarly. DBPCFCs are superior to open challenges, but they are difficult to perform, require extensive preparation and are relatively expensive.

Open challenge

Open challenges can be used as the first diagnostic step. Challenges should follow an approved protocol, suited for the circumstances. In 1995, a national protocol for CMA diagnosis and treatment was introduced in Dutch well-baby clinics, including a simple open challenge, which is still in use. After 2 weeks of elimination diet, the child ingests 10 ml of original formula while being supervised for 1 h; on the three following days, the formula is given in increasing amounts. While originally, the protocol was developed to avoid inappropriate CMA diagnoses, presently, it is thought that it contrarily may induce falsely positive results and should be followed by DBPCFC. Preset conviction, subjective symptom appreciation and random symptom fluctuation increase the risk of falsely positive results. Between 27% and 70% of open-challenge CMA diagnoses are rejected after DBPCFC.

Apart from this, a generally accepted open challenge procedure has only been published in 2007, when a European working group formulated a protocol for CMA diagnosis in general practice meant for children with relatively mild signs and symptoms. The child’s own formula is given in increasing amounts over 3 h. In our opinion, however, open challenges should only be used to reject CMA.

CMP administration schedules during challenge

Step Open challenge; child’s own formula (Ref 34) DBPCFC; hypoallergenic formula, with 5 g of Protifar® (Ref 28)
T (min) Dose (ml) CMP (mg)a T (min) Dose (ml) CMP (mg)
1 0 Drop on lips 0 0.1 2
2 15 0.5 8 15 1 18
3 45 1.0 15 30 10 176
4 75 3.0 45 45 20 352
5 105 10 150 60 30 528
6 135 30 450 80 40 704
7 165 50 750 110 60 1,056
8 195 100 1,500 150 90 1,584
Total 195 2,925 250 4,400

aBased on a mean protein content of 1.5 g/100 ml; depending on brand, the protein content of infant formula is between 1.3 and 1.6 g/100 ml; of follow-on formula, 1.7 and 1.9 g/100 ml


Double-blind challenges are the gold standard. In 2007 as well, the Health Council of the Netherlands issued a report asking for the general introduction of DBPCFCs. Dutch paediatric allergy centres and many hospitals already practice DBPCFCs . They can be performed in well-baby clinics and general practices as well, as long as basic precautions have been met, including thorough knowledge of the procedure, careful patient selection and the equipment to block adverse reactions. The importance of DBPCFCs is underscored by the fact that between 13% and 30% of placebo tests are eliciting adverse reactions . There is no generally accepted protocol for DBPCFC. The protocol presented here is used in the Wilhelmina Children’s Hospital.

  • Preparation The diet should be CMP free for at least 2 weeks. The patient’s condition should be stable, especially concerning skin symptoms. Topical corticosteroids may be continued, but antihistamines should be discontinued at least one week in advance. Preparation includes a thorough history for previous adverse reactions.
  • Safety Tests should be performed in a day-care setting or during admission. Depending on the severity of previous signs and symptoms, a monitoring device and intravenous access may be needed. Clemastine and epinephrin for parenteral use must be at hand. The personnel should be well trained, also regarding the management of (rare) severe acute reactions.
  • Test material The child is given his/her own hypoallergenic formula or expressed breast milk. The research kitchen prepares coded bottles with (verum) or without (placebo) 5 g Protifar® powder (Nutricia/SHS), containing 4.4 g CMP, per 250 ml formula.
  • Procedure Placebo and verum are administered on separate days, preferably 1 week apart. The test formula is given in increasing dosages at fixed intervals. Adverse reactions are recorded. After a negative test, the child remains under supervision for 2 h, after a positive test for 4 h. The parents are asked to report late reactions. After the second test, a follow-up period of at least 48 h is observed before the seal is broken.
  • Evaluation The test is discontinued when the child experiences objective adverse reactions, subjective reactions that persist for 30 min or longer or repeated short-lived subjective reactions. Allergic and non-allergic reactions are assessed separately and in the light of the child’s history. The DBPCFC is considered negative when verum did not elicit adverse reactions or when reactions following verum are not worse than following placebo. Even DBPCFCs, however, may not provide unequivocal results

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Interpretation of DBPCFCs

Verum challenge Placebo challenge Test outcome
Positive Negative Positive
Strongly positive Positive Positive
Positive Positive Negative
Negative Negative Negative
Positive Strongly positive Negative
Adapted from Vlieg-Boerstra BJ, Bijleveld CMA, van der Heide S et al (2004) Development and validation of challenge materials for double blind, placebo-controlled food challenges in children. J Allergy Clin Immunol 113:341–346.

Risk assessment

Although it is impossible to predict reaction severity during food challenges, some rules apply. Severe adverse reactions are more likely with previous severe reactions, with previous reactions on very low CMP doses, in older children, in children suffering from asthma and after prolonged exclusion of cow’s milk.

Severe reactions with CMP challenges, however, are rare. During over 12 years of open challenges in Dutch well-baby clinics , no severe adverse events have been reported to the supervising committee (K.I. van Drongelen, Netherlands Nutrition Centre, personal communication). With the DBPCFC protocol presented here, over 500 challenges have been performed without any severe adverse events. Hence, CMP challenges can be safely performed in general practices, provided basic safety precautions are met. High-risk challenges should be performed in the hospital.

Cow’s milk reintroduction

When CMA is refuted, standard formula and dairy products can safely be reintroduced in the diet of the child or the breast-feeding mother. Sometimes the child’s illness has put so much strain on the parents that the help of a dietician is required to complete the transition to a normal diet. When, nevertheless, adverse reactions develop after reintroduction, this may be due to the natural course of the underlying condition (eczema), but often expresses the preset conviction of the parents that notwithstanding the test outcome, their child is suffering from CMA. These signs or symptoms are likely to disappear when the introduction is continued.

Laboratory investigation

The role of laboratory tests in CMA diagnosis is debatable. The tests used in clinical practice only reveal sensitisation to CMP, which is not necessarily followed by clinically relevant allergy. Over 50% of sensitised children do not have food allergy . In our experience, positive skin prick tests and allergen-specific IgE tests tend to be falsely interpreted as proof of CMA. This is not at all innocuous.

Although there is a strong positive correlation between the level of allergen-specific IgE and the chance of having CMA, unequivocally high specific IgE titres are rare and may occur in non-allergic children. In general practice, therefore, laboratory tests are seldom helpful. The only way to prove CMA is through elimination and challenge.

  • IgE-mediated CMA. Skin-prick testing (SPT) with fresh milk or commercial reagents and ImmunoCAP-RAST (for determining specific IgE against cow’s milk proteins as caseins, lactoglobulin, and alpha-lactalbumin), are the currently available tests. The performance characteristics of these tests have been described in different settings. In children older than two years, an SPT reaction with a wheal diameter ≥8mm or milk specific IgE level ≥15,0 ku/L, the likelihood is 95% that the child will have a positive milk challenge. The corresponding wheal size in children younger than two years is 6mm diameter and milk-specific IgE ≥5,0ku/L respectively. Studies on specific caseloads are limited by several factors inherent to the characteristics of the studied group, such as clinical conditions (AD, asthma, GI), their severity, the percentage of polyallergic patients, and age and geographical differences. Thus, the use of sensitization tests is dependent on the clinical setting and on the pre-test probability of disease. For this reason, the whole matter has been subjected to systematic review and metanalysis in the preparation of the Diagnosis and Rationale Against Cow’s Milk Allergy (DRACMA) guidelines. Including studies published up to September 2009, the DRACMA panel reviewed the evidence summaries and the draft guidelines, and made recommendations on diagnosis.

According to these recommendations,

  • A formal challenge with cow’s milk remains the best diagnostic test.
  • It should be performed with physician supervision regardless of food-specific IgE value.
  • If challenge is positive, out of a research setting sensitization tests may not be necessary.
  • In settings where oral food challenge is not considered a requirement for making a diagnosis of IgE-mediated cow’s milk allergy, a positive SPT and/or ImmunoCAP (cut-off: 0.35 kUI/L) can be used and diagnostic tests in case of high pre-test probability.
  • In such settings, a negative SPT and/or ImmunoCAP with whole milk (cut-off: 0.35 kUI/L) can be used as rule-out tests in case of low pre-test probability.
  • In any case of high uncertainty, challenges remain necessary.

The prognostic and diagnostic utility of using specific proteins at ImmunoCAP or microarrayed platforms remains to be established.

  • Non-IgE-mediated CMA: There are no reliable tests for the diagnosis of non-IgE mediated CMA. Initial diagnosis is based on a suggestive history and absence of positive SPT or ImmunoCAP-RAST. In these patients, the diagnosis primarily relies on a successful milk avoidance diet with clinical relapses after re-exposure to cow’s milk proteins. In patients with atopic dermatitis and eosinophil esophagitis in whom non-IgE mediated cow’s milk allergy is suspected, Atopy Patch testing (APT) may be a helpful diagnostic tool.
  • Elimination-challenge testing: Food challenges remain the definitive procedure for the diagnosis of CMA. If the symptoms substantially improve or disappear after 2-4 weeks on an elimination diet, an open challenge with a formula based on whole cow’s milk protein should be performed. Clinicians should be aware that the severity of a past reaction might not predict the severity of a challenge reaction, particularly after a period of dietary exclusion. Previous mild reactions may be followed by anaphylactic reactions in some infants with CMA. For this reason, open challenges should ideally be performed in a setting where resuscitation facilities are available. In a case of cow’s milk-induced anaphylaxis, a challenge is contraindicated unless SPTs and/or specific IgE measurements show improvement. In these cases, the challenge should always be performed in a hospital setting.
  • Positive challenge: CMA confirmed If symptoms of CMA re-appear, the suspected diagnosis of CMA is confirmed and the infant should be maintained on an elimination diet using a milk substitute (discussed below) for at least 6 months. The challenge is then repeated. If it is possible to follow the infant with IgE-mediated allergy with SPTs and/ or specific IgE determination, improvement of these tests would help in choosing the time point of challenge. Supplementary feeding should be introduced carefully to avoid accidental intake of cow’s milk protein. Protocols of milk challenges have been published in different guidelines. Of importance, all guidelines emphasize the rules for milk challenge in immediate CMA, while the interpretation of delayed reactions occurring up to 7, 9, or 14 days is more controversial. The diagnosis of delayed reaction may be difficult because when the child returns home, multiple environmental factors (infections, dietary factors, emotional, casual contacts, sports-related physical activity) may impinge diagnostic interpretation. Frequently, immediate and delayed symptoms are present concomitantly in the same child.
  • Negative challenge: No CMA Children who do not develop symptoms on the cow’s milk formula during challenge and up to one week after follow-up can resume their normal diet, although they should still be carefully monitored. Clinicians should advise parents to be attentive for delayed reactions, which may evolve over several days following the challenge.


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