TESTS FOR HYPERSENSITIVITY REACTIONS

VI. TESTS FOR HYPERSENSITIVITY REACTIONS

Because of the large variety of hypersensitivity and other untoward reactions to foods and food additives there is no simple all-embracing in vivo or in vitro test method to detect the causes of such reactions. In immediate (type I) allergic reactions, the allergen is usually

a protein inducing IgE production, which can be shown with both skin and challenge tests as well as with in vitro tests in a laboratory. Patch tests and other skin tests are the tests of choice for delayed allergies. In experimental work, several in vitro tests are also available.

The grade of intolerance to food additives may vary from time to time. The symp- toms, especially those caused by nonprotein food additives, are so ambiguous that our comprehension of the exact number and severity of hypersensitivity reactions to nonpro- tein food additives is incomplete.

A. Skin Tests

Prick testing is the simplest way to test immediate allergies in vivo. Drops of allergen Prick testing is the simplest way to test immediate allergies in vivo. Drops of allergen

Scratch testing is a less standardized method than prick testing for immediate al- lergy. It is, however, still in use because many nonstandardized test substances are often used and the assessment of a scratch test result may be easier than that of a prick test result. Scratches approximately 5 mm long are made with a lancet or with a puncture needle on the forearm, upper arm, or back skin. Bleeding is avoided. Allergen solutions for prick/scratch tests are applied to the scratches, and the results are read 15–20 min later. Freeze-dried and other powdered allergens can also be used. They are moistened with 0.1 N aqueous sodium hydroxide solution. Histamine hydrochloride, 10 mg/mL, is the positive control, and only the longest diameter perpendicular to the scratch is measured. Reactions of at least the size elicited by histamine are usually clinically significant.

Intracutaneous (intradermal) tests are needed only for special purposes. A dispos- able tuberculin syringe with a small (26-gauge) needle is filled with approximately 0.1 mL of allergen solution for intradermal testing. Usually 0.02–0.05 mL of test solution is injected intradermally, preferably in the forearm skin, but the back skin can also be used.

A bleb 3–5 mm in diameter is formed. Histamine hydrochloride, 0.1 mg/mL, is used as the positive reference, and the test vehicle (usually saline or phosphate-buffered saline with phenol or parabens as preservative) as the negative control. The results are read at

Figure 3 Skin-prick testing. Allergen drops are pierced with prick test lancets, each drop with a new one.

Figure 4 Skin-prick testing. Results are read 15 min later, and the reactions are compared with that produced by histamine (H in the picture) and with that produced by the diluent for the allergens

15 min, and reactions larger than that induced by histamine are regarded as positive. Higher or lower concentrations of allergens are used if the threshold of positivity is needed. Allergens for prick, scratch, and intradermal tests . Among food additives, spices are the only group in which there are protein allergens capable of producing type I allergic reactions. There are only a few commercially available allergen solutions, and that is why many test solutions must be prepared by the investigator (Niinima¨ki and Hannuksela, 1981).

Scratch-chamber tests were introduced for testing foodstuffs as such when there are no commercially available allergens and the allergenicity of the test substance is easily lost (Hannuksela and Lahti, 1977). The test is performed like an ordinary scratch test, but the test material is covered with a small aluminum chamber (Finn Chamber, Epitest Ltd, Hyryla¨, Finland) for 15 min. The result is read according to the criteria for scratch tests. Scratch chamber tests are used especially for testing fresh material such as potato, apple, and carrot, but they can also be used for testing spices.

Patch tests are needed for detecting contact allergies. In the modern test technique, test substances are usually incorporated in white soft paraffin (WSP) and applied to the skin in small aluminum chanbers (Finn Chamber). Fluids, usually alcoholic and aqueous solutions of allergens, are adsorbed in filter paper disks placed on the bottom of the test chamber. The test chambers, filled to half volume, are fixed on the back skin of the patient

for 48 h (Figures 5 and 6 ). The results are read 20–30 min after the removal of the cham- bers and once again at 72–96 h (D2 and D3–D4). Both irritant and allergic reactions may develop. Primary irritant reactions are at their maximum on D2 and allergic reactions on

D2–D4 (Bandmann and Fregert, 1982) ( Figures 7 and 8 ).

Figure 5 Patch testing. Allergens, most of which are incorporated in petrolatum, are applied to

Figure 6 Patch testing. Test chambers are fixed on the back skin with a porous tape for 48 h.

Figure 7 Patch test result at 48 h. An allergic reaction comprises ‘‘mini-eczema’’ (redness and infiltration with tiny vesicles), which tends to spread to a larger area than the chamber size.

Parabens, sorbic acid, propylene glycol, and spices are the most common contact allergens among food additives. Parabens are tested as a 15% mixture of methyl-, ethyl-, and propyl-p-hydroxybenzoates, sorbic acid at 2.5% in WSP, propylene glycol at 2–30% in water, and spices as ground powders.

Open application tests and rub tests are used in searching for the causes of contact urticarial and other immediate reactions and sometimes also in detecting delayed contact allergies. In open application tests a small amount (approximately 0.1 mL) of test sub- stance is applied to apparently healthy skin on a 5 ⫻ 5 cm area of the extensor side of the upper extremity or on the back skin (Hannuksela, 1986a, b). Allergic (ICU) reactions are seen in 15–20 min, nonimmunologic reactions in an hour ( Figure 9 ). It is also possible that in spite of a patient history suggestive of contact urticaria, normal-looking skin is nonreactive but a positive reaction can be seen in diseased skin. Therefore open application tests are often done on the back of the hand.

The rub test is a modification of the open application test. The test substance is gently rubbed into a skin area (usually the hands) where the reaction previously occurred.

A positive open application test result comprises a wheal and flare reaction, but in

Figure 8 Patch test result at 48 h. An irritant reaction appears as redness and possible infiltration without vesicles. There is no spreading of the reaction.

test, small vesicles may be the only response, appearing within 15–30 min and disap- pearing within a couple of hours (Hjorth and Roed-Petersen, 1976; Ko¨pman and Hannuk- sela, 1983).

The repeated open application test (ROAT) is done in questionable cases of delayed contact allergies in whom the significance of the reaction seen is not clear (Hannuksela and Salo, 1986). Approximately 0.1 mL of test substance is applied twice daily to a 5 ⫻

Figure 9 Nonimmunologic contact urticaria reaction from 5% benzoic acid in petrolatum 40 min

5 cm area on the volar aspect of the forearm or on the back skin. In positive cases, eczema- tous dermatitis appears on the test area, usually on D2–D5. If no sign of dermatitis is seen in a week, the test is discontinued and the result is considered to be negative.

In the use test, the product suspected to be responsible for the reaction is used as usual and the skin response is followed for up to 1 month. Both weak allergens and primary irritant substances may produce reactions in the use test.

The Prausnitz–Ku¨stner test (the passive transfer test) provides a method for demon- stration of IgE-mediated reactions in nonsensitized individuals. Aliquots of 0.1 mL of serum derived from a patient are injected intradermally into a healthy person or a monkey. Prick, scratch, or intradermal tests are performed on the infection sites 24–72 h later. If the patient’s serum contains specific IgE, the test result in the healthy person or monkey is positive. Previously, the Prausnitz–Ku¨stner test was often used, but now radioimmuno- assay tests have replaced it in most cases. It is noteworthy that in the Prausnitz–Ku¨stner test, infective viruses may be transfered from the patient to another person. Hence, tests for the presence of HBAg and HTLV III antibodies must be carried out before the patient’s serum is injected in the test subject.

B. Oral Challenge Tests

Allergic reactions to spices appear most often as local reactions in the mouth and adjacent areas (see Section III). The simplest way to assess the role of spices and other substances suspected of producing such reactions is to perform an oral mucosal test, spreading the substances on the oral mucosa (Niinima¨ki and Hannuksela, 1981). Within 15–20 min, swelling of the oral mucosa, lips, or throat; an attack of allergic rhinitis; and/or conjunctivi- tis may appear. In rare cases, a systemic reaction (urticaria) may take place.

In the peroral challenge test the substance to be tested is given to the patient in nontransparent gelatin capsules, preferably in a double-blind manner. The dose should be about the average daily dose used by the population (Table 11). In some cases, individual doses are needed.

Table 11 An Example of Test Battery for Peroral Challenge Tests with Food Additives

Substance

Amount

Placebo (wheat starch)

100 ⫹ 200 mg

Benzoic acid

200 ⫹ 400 mg

BHT ⫹ BHA, 1:1

Sorbic acid

200 ⫹ 400 mg

β-Apocarotenal ⫹ β-carotene

200 ⫹ 400 mg

Sodium nitrite

50 ⫹ 100 mg

Sodium metabisulfite

10 ⫹ 20 mg

Sodium glutamate

100 ⫹ 200 mg

Azo dyes a 5 ⫹ 10 mg

Note : Dose given to patients in nontransparent gelatin capsules at 1-h intervals.

a Tartrazine, new coccine, and Sunset Yellow (or paraorange). Only in countries in which azo dyes are allowed to be added

In cases of urticaria, possible reactions are followed up to 24 hr, although the reac- tion is usually seen within 1–4 h (Hannuksela and Lahti, 1986). In patients with purpura and in those with suspected internal exacerbation of contact dermatitis, a longer exposition and follow-up time (up to several days) may be needed.

Subjective sensations, such as nausea, vertigo, and itching, are frequent after peroral challenge tests. However, they are always regarded as negative challenge test results. Only clearcut reactions are to be regarded as positive results. In chronic and recurrent urticaria,

a marked worsening of urticaria with or without angioneurotic edema is suggestive of real hypersensitivity reaction. In cases of purpura, a widespread attack of purpural patches, and in cases of internal exacerbation of contact dermatitis, eczema and/or pompholyx reactions, are significant. The assessment of respiratory symptoms must also be based on objective criteria. In rhinitis, the number of sneezes may be counted, the mucus flow from the nose measured, and the number of handkerchiefs used counted. The nasal obstruction is measured usually by a peak flow meter (nasal peak expiratory flow) or a rhinomano-

meter. The asthmatic response is expressed as PEF (peak expiratory flow) or FEV 1 (forced expiratory volume in 1 s). Usually, a drop of 20% is regarded as significant. The respiratory reaction may be delayed up to 8 h. All equivocal and positive challenge tests should be repeated.

The importance of the double-blind challenge test technique was clearly demon- strated in a study by Hannuksela and Lahti (1986). Peroral challenge tests with common food additives were made in patients with chronic urticaria, in patients with atopic derma- titis, and in patients with eczematous dermatitis as a control group. Positive and equivocal reactions were seen in all groups, and there were no differences between the various addi- tives and the placebo in their capacity to produce these reactions ( Table 12 ). This result indicates that the double-blind technique ought to be the only way to make peroral chal- lenge tests with these substances, reactions to which are poorly established and difficult to assess.

Whether or not to avoid antihistamines, peroral steroids, and antiasthmatic drugs during peroral challenges is a very important question. When urticaria or asthma is in a steady state and the patient is nearly or totally free of symptoms without medication, the drugs mentioned before should be avoided during challenge tests. Patients with severe asthma need their medication, even if the drugs may have some influence on the results of the challenge tests.

A restriction diet free from important food additives is recommended for some days to some weeks preceding the peroral challenge test (Juhlin, 1981). The need for the diet is, however, a matter of controversy. We now know that aspirin produces a refractory period of a couple of days during which further intake of aspirin does not produce symp- toms. Whether or not this is also true with food additives remains to be investigated.

C. In Vitro Tests

1. Determination of Specific IgE Demonstration of specific circulating antibodies of the IgE class provides evidence of an

immunologic sensitization. However, it has been shown that low levels of IgE antibodies to inhalant or food allergens are often present without clinical manifestation (Haahtela and Jaakonma¨ki, 1981).

The most used technique to measure IgE antibodies is the Radio Allergo Sorbent

Table 12 Results of Double-Blind, Placebo-Controlled Peroral Challenge Tests with Food Additives

Disease group

Contact Substance(s) result

Amount

Atopy dermatitis Wheat starch (placebo)

per capsule

Urticaria

100 mg

equivocal 1 a — — positive

1 b — 1 c Sodium metabisulfite

9 mg

equivocal 1 a 1 a — positive

— — Benzoic acid

200 mg

equivocal 1 a — — positive

BHT ⫹ BHA d 50 mg

equivocal 2 a 2 a — positive

— — β-Apocarotenal ⫹ β-Carotene 1:1

200 mg

equivocal

1 a — positive

1 c — Note : Carried out in 44 patients with chronic urticaria, in 91 patients with atopic dermatitis, and in 123 patients

with contact dermatitis as a comparison group. The dose was 1 ⫹ 2 capsules given at 1-h intervals. The reactions were followed up for 24 h.

a Retest 4 days later negative. b Retest 4 days later positive. c Retest not done due to severe symptoms in the first challenge. d BHT ⫽ butylhydroxytoluene, BHA ⫽ butylhydroxyanisole.

Source : Hannuksela and Lahti, 1986.

evidence that, with the exception of spices (Toorenenberger and Dieges, 1985), adverse reactions to food additives are mediated by IgE. Therefore there are as yet no commercial RAST tests for food additives available.

2. Basophil Degranulation Test and Histamine Release The basophil degranulation test using either whole blood or washed leukocytes has been

advocated to diagnose immediate hypersensitivity to various allergens (Benveniste, 1981). The results correlate well with histamine release from leukocytes, particularly from baso- phils, and with the RAST. However, virtually nothing is known about the possibilities of using these tests in the diagnosis of hypersensitivity reactions to food additives.

3. Lymphocyte Transformation Test The lymphocyte transformation test (LTT) is widely used as an in vitro test for delayed

allergies. It is also suitable for examination of several contact allergies such as those toward cobalt (Veien and Svejgard, 1978), chromium (Jung, 1969), gold (Denman and Denman, 1968), and nickel (MacLeod et al., 1970; Kim and Scho¨pf, 1976; Silvennoinen- Kassinen, 1980). In cases of drug reactions, several antibiotics, chemotherapeutics, and many other drugs have been found to induce blast formation from lymphocytes in LTT

It is possible that LTTs could be used for the investigation of food additives sus- pected of producing delayed-type reactions. For complex mixtures of many chemicals, such as essential oils and spices, the LTT might not be successful, but it is worth trying in selected cases for simpler chemicals such as sorbic acid and parabens.

4. Migration Inhibition Test The leukocyte migration inhibition test has been used, for example, in nickel allergy

(Mirza et al., 1975). This test could obviously also be used for studies on contact allergies from food additives.

5. Cytotoxic Test It has been claimed that the white blood cells of food allergy patients die and disintegrate

in the presence of the food to which the patient is sensitive (Black, 1956). In the cytotoxic test, circulating white blood cells are exposed in vitro to food extracts, and the number of dead cells is counted under a microscope. The test has been found to be difficult to reproduce and unreliable (Sethi et al., 1987), and the interindividual results may vary grossly from time to time (Ruokonen, 1982).

Summarizing, at the moment a controlled peroral challenge is the only reliable way to test systemic reactions to food additives. Skin tests are irreplaceable in the diagnosis of many skin reactions.