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Skin Testing: Concepts and Realities
Skin Testing: Concepts and Realities MARIE BRITT RHYNE, M.D.I*
The skin envelops the entire surface of the child's body and is easily accessible to the examiner's eye. It is, tberefore, well suited for use as a test organ to determine response to foreign substances. Although methods for applying test substances to the skin were reported by investigators in the latter half of the nineteenth century, it was not until von Pirquet described his cutaneous tuberculin test in 1911 that this procedure became a valuable diagnostic aid. The introduction of skin testing in a scientific manner by this astute clinical investigator provided a means of obtaining fundamental information on immunologic phenomena in children and adults. 25 An understanding of the nature of allergic responses and the relationship of the responses to common clinically manifested symptom complexes, the developmental processes of the child's capacity to respond, the methods for eliciting the desired response, the character and concentration of materials used for testing, and the significance of the observed reaction is necessary for the physician to utilize skin tests as an immunologic diagnostic procedure. TYPES OF ALLERGIC RESPONSES
Four types of allergic responses have been described. 6 The key to understanding their relationship to clinical disorders is a knowledge of the mechanisms involved in antibody and cell induced immune damage. Antibody activity is carried by globulins found in tbe serum, external secretions, and tissue fluids. These globulins, known as immunoglobulins (Ig), have been classified by their physicochemical properties. Five classes of human immunoglobulins are now recognized: IgG, IgA, IgM, IgD, and IgE. Distinct subclasses are also recognized for several From the Allergy Foundation of America Laboratory, Department of Pediatrics, The Johns Hopkins University School of Medicine and Hospital, Baltimore, Maryland. Supported by Projects 212 and H-188 (R) from the Children's Bureau, Department of Health, Education and Welfare . ... Presently Special Research Fellow, National Center for Health Services, Research and Development, and Graduate Student, Department of Chronic Diseases, The Johns Hopkins University School of Hygiene and Public Health. Pediatric Clinics of North America-Vol. 16, No.1, February, 1969 227
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MARIE BRITT RHYNE
immunoglobulins. Each class and subclass has been chemically identified and purified, and for most some of the biologic capabilities are known. The child is capable of responding to an allergen with a great many different immunoglobulins, each of which seems to have a specialized function. Antibodies capable of combining with antigen and a tissuefixing site activate enzyme-mediated processes through which cells release vasoactive substances (histamine, serotonin, bradykinin, and slow reacting substance of anaphylaxis).2 Antibodies capable of "fixing complement" activate biochemical pathways through which cells are destroyed by the components of complement. 15 Nine proteins are required for the cytotoxic activity of complement. They participate sequentially in a series of reactions which eventually lead to the enzymatic formation of a hole in the surface of the cell coated with antibody. Such cells lose their ability to maintain osmotic equilibrium; they swell and burst. The sequence of reactions need not go to completion to produce biologic effects. At various stages in the reaction sequence chemotactic substances are released which attract polymorphonuclear leukocytes into the area of immune injury. At other stages the smooth muscle contractors, anaphylatoxins, may be released. 22 Anaphylactic Response Anaphylactic responses are enzyme-mediated processes triggered by allergen-antibody interactions involving an antibody class which has both an allergen-combining site and a tissue-fixing site. Tissue-bound antibody is activated on combination with allergen to trigger the enzyme pathways leading to a release of vasoactive substances, without the occurrence of cell death. 16 Urticaria and Angioedema. Extensive urticaria and angioedema may be an expression of a cutaneous allergic response of the anaphylactic type. Vasoactive substances released from cells by this reaction may be multiple. Inhibition of symptoms and signs· of the reaction is often not accomplished by use of antihistaminics alone. Atopic Disorders. Many atopic persons who develop clinical symptoms of atopic dermatitis (infantile eczema), seasonal hay fever, nonseasonal rhinitis, sinusitis and eustachitis, or bronchial asthma readily produce a specialized class of antibodies, presumably IgE, after ordinary exposure to otherwise innocuous substances. This antibody, called skinsensitizing or reaginic antibody, has "par excellence" the property of binding to tissue cells and interacting with its allergen to cause a response typical of the anaphylactic reaction. Cytolytic or Cytotoxic Response Some IgG and IgM antibodies are chemically capable of combining with an antigenic' component of a tissue cell or an antigen which has
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become intimately associated with tissue cells. They usually produce their effects locally by activating the biochemical pathway through which cells are destroyed by the components of complement. 21 Thrombocytopenic Purpura. Generalized purpura due to sensitivity is a clear example of a cytolytic reaction. Such reactions are initiated by antibody reacting with an allergen which has become intimately associated with platelets. 23 Toxic Complex Syndrome or Response Antibodies have multiple combining sites, and many are capable of cross linking with polyvalent allergens to form sticky complexes. Circulating antigen-antibody complexes large enough to be entrapped by a vascular basement membrane can localize in such organs as the skin, the kidney and the spleen, where they fix complement and cause local inflammation by the release of chemotactic substances which attract polymorphonuclear leukocytes into the area of immune injury.4 Serum Sickness. The clinical manifestations of serum sickness result from two types of allergic responses. Symptoms and signs of joint pains, leukopenia, lymphadenopathy, and hematuria are ascribed to a toxic complex response. Symptoms of fever, urticaria, periorbital edema, and asthma are evoked by the anaphylactic response. Delayed Sensitivity Response In this immune response the appearance of small lymphocytes, presumably activated by antigen which recognizes an antibody-like antigen site on the surface of the cell, is followed by in-migration of monocytes. These cellular elements plug small vessels near the site of antigen localization and can produce tissue necrosis through ischemia or through release of chemical mediators th~t produce cell death directly.2o Allergic Contact Dermatitis. Allergic contact dermatitides characterized by redness, edema, papules, vesiculation, weeping, crusting, and itching reactions in the child's skin are delayed sensitivity responses. Usually, once allergic sensitization is established in even a small area of the skin, the entire surface of the skin is capable of reacting.
THE CHILD'S CAPACITY TO RESPOND
Demographic and Environmental Factors
Age and Maturation. The child's physiological capacity to respond to an allergen is subject to control by various demographic, genetic, and environmental factors. In an epidemiologic study designed to measure the prevalence of asthma and atopic dermatitis in randomly selected populations of l-year-old children and elementary school children in the state of Maryland, those defined as cases by interview were matched
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for sex, race, age, and residence with noncases and scratch skin-tested with extracts of several common inhalant and food allergens standardized at 0.1 mg. protein nitrogen (10,000 PNU) per milliliter. In the one-year-old population, significant differences (P < .05) between cases and control reactivity rates were noted for ragweed, wheat, and eggwhite, but only one allergen, eggwhite, gave a rate greater than 2 per cent in any group. The positive percentage for that allergen was 4.48 for cases and 1.49 for controls. In the elementary school group significant differences in the allergen reactivity rates of asthmatic and nonasthmatic children were noted for ragweed, orchard grass, house dust, and feathers. Four allergens, ragweed, orchard grass, house dust, and alternaria, gave a rate greater than 10 per cent in the case group. The highest was for ragweed; the positive percentage for that allergen was 13.7 for cases and 3.6 for controls. Asthmatic children with current symptoms had a significantly higher rate of reaction to house dust and to feathers than did asthmatic children without current symptoms. The positive percentage for house dust was 19.1 for current and 8.0 for noncurrent cases; for feathers, 12.7 for current and 4.3 for noncurrent cases. These data suggest that children demonstrate an increasing capacity to develop reaginic antibody (presumably IgE) to ordinary environmental substances with increasing age and exposure, and that the potential for response is greater for the asthmatic child with current symptoms than for either the asthmatic child without current symptoms or the nonasthmatic child. Some of the observed differences in response with age are probably related to structural differences of the skin itself. About 35 years ago Carey and Gay 3 reported that reactions of newborn infants to intracutaneous tests of a 1: 10,000 dilution of ergamine acid phosphate were consistently much smaller than those observed in adults, erythema and wheal being about half the diameter of that noted in the adult reactions. The capacity to accept passive sensitization to reaginic antibody was present even in the skin of a 16-hour-old infant, but stronger concentrations of test solutions were required to produce reactions comparable to those observed in adults. The synthetic mechanism for IgG develops and matures during the first 2 years of the child's life, usually beginning at 4 to 6 weeks of age and reaching the lower level of that attained by the normal adult during the second year of life. 27 In the fetus small quantities of the high-molecular weight immunoglobulin, IgM, are often present. It seems plausible that this originates in the fetus, as synthesis starts very rapidly postnatally and continues at such a rate that adult levels are often attained by age 9 months. The development and maturation of the synthetic mechanisms for delayed sensitivity remain to be studied. Studies with Rhus allergens
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under controlled circumstances have demonstrated that infants below the age of 1 year have a depressed ability to react to these allergens; children between 1 and 3 years of age assume an intermediate position, while children between 3 and 8 years are readily sensitized and show an intensity of reaction comparable to that seen in adults. 8 It seems likely that the fault lies not so much in the skin itself as in the ability to make delayed type "antibodies," as irritants readily induce inflammation in the skin of older infants and young children. However, the immature structure of the skin may be an important factor for young infants, as they usually react negatively or only slightly to injections of turpentine, iodoform, and other irritants into the skin. Positive reactions to such irritants are more common in infants over 2 months of age. 1 Sex. In the Maryland studies the skin reactivity rate for the allergen mixed feathers was significantly greater in school-age female asthmatics (13.4 per cent) than in school-age male asthmatics (4.7 per cent), but this was believed to be due to increased exposure rather than to differences in capacity to respond, as no significant differentiation in reactivity rates was noted for other allergens. Sex differences for other antibody classes have not been evaluated. Several of the congenital varieties of hypogammaglobulinemia are male sex-linked, but the acquired varieties occur in both males and females. Race. In the Maryland studies race does not appear to be an important demographic variable for reaginic antibody. No significant differentiation in reactivity rates was noted for asthmatic white and Negro children. Residence and Social Class. Detectable differences in reactivity rates between asthmatic children living in urban, suburban, and rural areas of Maryland were observed. This is especially true for ragweed, for which the highest reactivity rates were found in the suburban asthmatic children (21.7 per cent), the lowest in the rural asthmatic children (8.3 per cent). Significantly higher reactivity rates were also detectable for three allergens, ragweed, grass, and house dust, in asthmatic children of families of upper class strata. While the factors responsible for the observed differences in skin reactivity have not been elucidated, it is likely that they are related to differences in the concepts of hygienic living habits and the environmental exposures encompassed in the family living patterns of children of different social strata and different residential areas.
THE DIAGNOSTIC PROCEDURE
The anaphylactic_ response, the toxic complex response, and the delayed sensitivity response are immunological phenomena demonstrable by skin tests.
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Anaphylactic Response Skin tests for this type of response are most frequently employed in the evaluation of children with respiratory or dermatologic manifestations of atopy. They are used to evaluate either atopic or nonatopic children with symptoms of local anaphylactic reaction, such as urticaria and angioedema, recurrent gastroenteritis and vernal conjunctivitis, or a history of symptoms of a generalized anaphylactic reaction with shock. Selection of Allergens for Testing. Allergens which induce the anaphylactic reaction are frequently ordinary contact ants of daily living. Testing extracts of such allergens are standardized in terms of either total nitrogen content or protein nitrogen concentration per milliliter, or by the weight of the dried allergen material in a given volume of extracting fluid. Such extracts are notoriously variable in potency, making precise comparisons of the results in patients tested in different clinics or physician offices with different preparations unsatisfactory. As all "purified" allergens to date have been shown to be of a proteinous nature, standardization on a protein nitrogen concentration basis is the preferred method. Food Allergens. Foods capable of acting as allergens tend to be proteins of either animal or plant origin. Milk, meat, poultry, fish, egg, dried legumes, nuts, chocolate, and seeds are the most frequent producers of anaphylactic reactions in children. The frequency of ingestion of foods with great allergenic potentiality and the amounts consumed help determine their importance as allergens. The amounts and types of foods ingested are frequently a function of the cultural habits of the child's family. For instance, cow's milk allergy follows and parallels the development of successful artificial feeding of infants with cow's milk formulas in this country.7 Preferential food habits noted in various ethnic groups, such as Italians, Orientals, and Mexicans, and in families living in different regions of this country, probably account for some 'of the observed differences in the frequencies and patterns of food allergy in children reported by physicians practicing in widely different geographic areas. Food allergens used for testing should reflect the food habits of the child under study. Inhalant Allergens. Inhalant allergens which induce anaphylactic reactions appear to be proteins with a molecular weight of about 30,000 to 40,000. 12 • 24 The allergenic environment of the child is important, and differences in the availability of and degree of exposure to various allergens help determine their significance as allergens at different ages in the child's life. Allergens that contaminate the house air may sensitize the child earlier in life than seasonal external environmental allergens, such as pollen and mold spores, which are present in the air only for limited periods of the year. These usually require several years of exposure to produce clinical signs of sensitivity.
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Indoor airborne allergens originate from home furnishings, from animals, and from material in the building attacked by molds. House dust is a mixture of many materials. Current studies suggest that the mite is probably one of its major allergens. 14 The major biologic fibers used in manufacturing stuffed chairs and sofas, pillows, mattresses, box springs, and stuffed toys in this country are cotton and kapok. In other cultures different fibers are commonly used. The animal or epidermal allergens found in the house air are emitted fromfeathers found in furnishings and from house pets such as parakeets, rabbits, cats, dogs, gerbilles, and guinea pigs. The rural child may also be exposed in barns or sheds to the indoor airborne allergens exnitted from farm animals such as horses, cows, and chickens. Microbes in the indoor air originate within the house from such areas as defective timber, wallpaper paste, stored books, clothes, and toys. In spite of ventilation, Peincillium is the mold spore dominating the indoor air of most homes, while Cladosporium (Hormodendrum) is usually the dominant spore in the indoor air.ll Knowledge of the common inhalant allergens in the child's environment, together with a detailed assessment of the nature, place, and tixning of the allergic responses noted in the child, is necessary to determine what substances to employ as test allergens. Outdoor airborne allergens appear to originate from vegetation above the ground. Pollens from wind-pollinated plants are the principal outdoor airborne allergens in most parts of the world. They appear in the air only when the plant is in flower. Only a very few plants possess pollens that contain antigens with properties suitable to sensitize the child and promote anaphylactic responses. Plants known to be of allergenic significance in the continental United States are listed in Table l. This outline was prepared by the author from material drawn from the book Hay Fever Plants by Wodehouse2 6 and from data obtained from correspondence with 15 pediatric allergists practicing in various parts of this country. Only two plants, ragweed and Bermuda grass, were noted to be real troublemakers for children in the United States. Most fungus spores from outdoor vegetation have a marked seasonal incidence and a clear diurnal periodicity. Cladosporium is the most prevalent spore type in Europe and North America, and while often present through the year usually has a marked rise from June to September. Each spore type varies in quantity from year to year, depending on the weather, the presence of diseases in crops, and the general condition of the vegetation in the neighborhood. Debris from insects may be inhaled in sufficient quantities to produce anaphylactic responses if the insects occur in large number. In the Great Lakes region of the United States the caddis fly swarms in xnidsummer and sheds a loose scaly epithelium from its wings which is capable of acting as an allergen. 18 Insect Allergens. Serious anaphylactic reactions to insect stings are
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MARIE BRITT RHYNE
Table 1. Plants oj Allergenic Importance in the Continental Unites States GEOGRAPHIC AREA
Northeastern: New
Eng~
land, New York, New Jersey, Pennsylvania Middle Atlantic: Delaware, Maryland, District of Columbia Virginias and Carolinas:
Virginia, West Virginia, N orth Caro~ lina, South Carolina North Central: Ohio, Kentucky, Indiana, Michigan, Illinois, Iowa, Eastern Missouri, Wisconsin, Southern Ontario Southan: Florida, Tennessee, Georgia, Alabama, Mississippi, Louisiana, Arkansas, Southern Missouri, Oklahoma, Eastern Texas Southlvtst: Arizona, New Mexico, Western Texas
TREES
Elm, maples, poplars, birches, oaks
GRASSES
Sweet vernal, June, orchard, timothy, annual blue grass Orchard, timothy
Maples, oaks, eastern sycamore, hickory, birches, paper mulberry Maples, elm, red ce- June, annual blue dar, junipers, hickgrass, orchard, ories (pecan), oaks sweet vernal, redtop, Bermuda (in southern areas) Ash, cottonwood, oak, June, orchard, timothy, redtop maple, elm
WEEDS
MAJOR POLLENS
Short and giant ragweeds, plantain Short and giant ragweeds, plantain
Ragweeds, grasses
Short ragweed, sorrel dock
Short ragweed, Bermuda grass, hickories (pecan)
Short ragweed
Short ragweed
Ragweeds, orchard grass
Mountain cedar, red cedar) poplar (cottonwood), birch, hickories (pecan), oak, live oak, privets, scrub elm, paper mulberry Mulberry, olive, mountain cedar, Arizona cottonwood
Timothy, orchard, Bermuda
Amaranths, spiny carelessweeds, western water.. hemp, giant and short ragweed, Russian thistle
Bermuda grass, hickories (pecan), ragweeds
Bermuda, Johnson
Rabbit bush, Rus... ian thistle, amaranths (Chenopodium)
Southern California
Olive, Chinese elm, coast, live oak, hickories (walnut)
Bermuda, saltgrass
Bermuda grass, mountain cedar, chenopods, amaranths Bermuda, saltgrass
North Pacific: Northern California, Nevada, Oregon, Washington
Acacia, cottonwoods, boxelder (maple), alder, birch, oak, hickories (walnut)
Timothy, orchard, redtop, blue grass, fescue, velvet, Western rye
Rocky Mountain: Idaho,
Cottonwoods, boxelder (maple), birch, Rocky Mountain cedar Elm, oak
Timothy, redtop, orchard, June, fescues, wheat groups
Curly dock, pigweed, scale family, lambs quarter, Russian this.. tle, sages Saltbushes, Russian Weeds (none of thistle, sagebrush, major importance) maypigweed, weed, curly dock, sheep sorrel Russian thistle, Russian thistle, sagebrushes sagebrushes, ragweeds
Montana, Wyoming, Utah, Colorado
Plains and Prairies: North Dakota, South Dakota, Nebraska, Kansas, Minnesota
Orchard, bluegrass, timothy, red top, Bermuda, Johnson (in southern areas)
Russian thistle; taU, short, and western ragweods
Ragweeds, Russian thistle
limited to the Hymenoptera, which include bee, hornet, wasp, and yellow jacke~. These reactions are due to allergens usually present in both the venom and the bodies of the insects. Rarely can children identify these insects accurately, but multiple cross sensitivities to family members are the rule, and exact identification is not necessary, because testing to the allergens of all members should be conducted on every study child. Bites from household insects such as fleas and bedbugs may produce an allergic response known as papular urticaria, but this is a separate entity from the urticaria and angioneurotic edema of the anaphylactic response.
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Incomplete Allergens (Haptens). Most allergens naturally encountered in the child's environment are complete allergens and are capable of inducing anaphylactic responses by themselves. However, there are many low molecular weight substances which can act as allergens if they can become conjugated with a carrier substance, usually a protein, in the body. Of the many chemicals capable of acting as incomplete allergens, those of greatest concern are drugs, such as penicillin, which are administered as therapeutic agents. Sensitization may occur to the chemical itself or to one of its metabolic products. Suitable conjugated allergens for testing of simple chemicals or metabolic products of such chemicals are not available for general use, although study of the number and chemical characteristics of the allergens produced during the metabolism of penicillin has been the focus of several investigators' work, 13. 17 There are wide differences of opinion as to whether all allergens known to occur in the child's environment should be tested or only the limited number known to be associated with the production of clinical symptoms. The author prefers to test a limited number of the commoner potent allergens known to be present in the environment with the understanding that further and repeated tests will be performed during subsequent evaluations if the course of the symptoms warrants additional studies. Potent allergens capable of producing anaphylactic type responses and common to the environment of children living in the Maryland area are presented in Table 2. Methods of Testing. Cutaneous and intracutaneous tests are used to elicit anaphylactic responses. An aqueous solution of the allergen is prepared by extracting the appropriate allergenic material with slightly alkaline buffered saline. The solvent containing the allergen is separated from the residual material by clarification or preliminary filtration. Usually it is dialysed against buffered saline to dispose of irritating substances and concentrated to the desired standard volume. The extract is sterilized by bacterial filtration and tested for both aerobic and anaerobic organisms. It can then be standardized on the basis of protein nitrogen content and applied to the skin by one of three methods, prick, scratch, or intradermal tests. There are differences of opinion regarding the relative value of these methods. All have distinct advantages and disadvantages. Scratch tests are performed by making a superficial cut into the skin with a vaccinostyle needle or scalpel without drawing blood. The extract of the allergen is then applied to the cut. Although tests are usually read in 20 minutes, sensitive patients may develop large whealing reactions before that time. Such test sites can be wiped free of allergen with an alcohol sponge as soon as large reactions appear to prevent continuous absorption of allergen and the development of a systemic anaphylactic reaction. Prick tests are performed by placing a drop of an aqueous
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MARIE BRI'IT RHYNE
Table 2.
Diagnostic Skin Tests Jar Common Allergens in the Maryland Area HOUSEHOLD
POLLENS
INHALANTS
Weeds Ragweed, giant Ragweed, short Plaintain, Eng\.
Feather mix Dust Kapok Pyrethrum
Grasses Timothy Orchard Trees Birch Elm Hickory Maple Oak Sycamore Paper mulberry FOODS
Grains Wheat flour Oat Cornmeal Rice Dairy products Milk (cow) Eggwhite Meats Beef Chicken Fish Vegetables String beans Lima beans Peas Onions Potato, white Tomato
EPIDERMALS
Cat hair Dog hair Horse dander MOLD SPORES
Mixed 1 to 4 (Indoor group) 1. Aspergillus fumigatus 2. Penicillium mix 3. Mucor racemosus 4. Fusarium Mixed 5 to 8 (Outdoor group) 5. Alternaria tenuis 6. Helminthosporium 7. Hormodendrum hordei 8. Botrytis cinerea INSECTS
Mixed Hymenoptera 1. Honeybee 2. Hornet 3. Wasp 4. YeIIow jacket Household insects Mosquito
Fruits Orange Banana Beverages Coffee Chocolate Other Peanut Black pepper Mustard
solution of allergen on the child's skin and pricking the skin through the drop. They are read in the same manner as scratch tests. Diluent control tests are always performed. If dermatographism is marked, anti-
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histaminic medication should be given about 30 minutes before subsequent testing. The criteria the author uses for interpretation of cutaneous tests are: Negative
= no reaction or no different from control
1+ 2+ 3+
= erythema less than 10 mm. in diameter = erythema greater than 10 mm. in diameter with no wheal = wheal with surrounding erythema = wheal less than 10 mm. in diameter with pseudopods,
4+
= wheal
±
disregarding erythema greater than 10 mm. in diameter with pseudopods, disregarding erythema
Criteria are arbitrary and differ from clinic to clinic, but they do permit a more quantitative estimation of skin reactivity than does a simple recording of tests as negative, positive, or doubtful. The intensity of the reaction is a measure of the degree of sensitivity, but it does not necessarily correspond to the clinical importance of the allergen to the child. Cutaneous testing is commendable in view of its simplicity. Syringes and needles are not required and the risk of systemic reactions is minimal. In addition few children find scratch testing really unpleasant, so that reluctance to cooperate is minimized. Its chief drawback is its relative insensitivity. Intracutaneous tests are about a hundred times more sensitive than cutaneous tests. 9 They are performed by injecting a small amount, 0.02 ml. is usually sufficient, of aqueous solution of allergen into the epidermis. The precaution of starting with an intracutaneous injection of a 1: 100 or 1: 1000 dilution of the allergen will usually suffice to prevent systemic reactions, but serious drawbacks to the exclusive use of the intracutaneous tests are the large number of syringes needed to make a complete examination and the great reluctance of young children to accept such multiple "shots." The author uses intracutaneous testing to supplement scratch testing when the latter gives a negative or weakly positive reaction to a suspected allergen. Scratch testing is usually performed with allergen extracts standardized at 0.01 mg. protein nitrogen (1000 PNU) per milliliter. A negative scratch test is followed by intracutaneous testing with allergen extracts standardized at 0.001 mg. protein nitrogen per milliliter. Intracutaneous tests with extracts of a concentration greater than 0.01 mg. protein nitrogen per milliliter are rarely performed, as such materials may be chemically irritating to the child's skin and produce false positive reactions. Significance of Reactions. The variability of potency of allergenic extracts must be kept in mind in evaluating negative reactions. This is particularly true for the household inhalant group and for certain foods, but it can be a problem with any allergen. The effects of certain drugs, especially antihistaminics, on the size of the skin reaction have been
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MARIE BRITT RHYNE
studied with conflicting results. Further evaluations with chemically standardized allergens are needed to determine the significant effects, if any, of these agents upon the intensity of the anaphylactic response in skin tests. Subcutaneous injections of adrenalin do have an inhibitory effect on the reactivity of the skin which persists for about 60 minutes after administered. Ii False positive reactions occur when the allergen extracts contain nonspecific chemical irritants or when poor testing technique results in mechanical irritation of the skin. When the test materials and the testing technique are satisfactory, positive reactions are immunologically specific. However, they mayor may not be related to the clinical syndrome for which the child is being evaluated. A thorough knowledge of the child's clinical history and physical findings, and continued observations for several months or even years, are usually necessary to properly interpret the relationship of the skin test results to the child's clinical symptoms. Use of Passive T1'ansfer Test. Prausnitz and Kiistner19 described the passive transfer test for anaphylactic reactions, and theirs are eponymous names for the test. In this test the serum of the child under study is injected into the skin of a normal recipient. If reaginic antibodies are present in the serum, passive sensitization of the injected site of the recipient's skin will result within 24 hours. Injection of the allergen at the same skin site can produce a local anaphylactic response. This test is direct evidence of the presence of reaginic antibodies in the study child's serum. For clinical diagnostic purposes the usefulness of this test is limited by the danger of homologous serum jaundice. Its usefulness in scientific investigations of the interaction of allergens and reaginic antibody in vivo is undisputed. Toxic Complex Syndrome Responses Local toxic complex reactions or Arthus reactions are produced by antibodies capable of interacting with allergen to forin' microprecipitates. Frequently the child will produce anaphylactic antibodies or delayed sensitivity "antibodies" as well as toxic complex forming antibodies to the same allergen, making it difficult for the evaluator to distinguish one skin test from the other. The anaphylactic response may modify or mask the Arthus reaction, and either or both may modify the delayed reactions. Test Alle1'gens. Allergens eliciting serum sickness or the toxic complex syndrome tend to be substances administered to the child by injection. In the past sera derived from animals were the frequent causative agents. Today drugs, particularly penicillin, are the more common offenders. Some fungi, especially Aspergillus fumigatus, and a wide variety of vegetable dusts from hay, straw, bagasse, cotton, hemp, paprika, malt,
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and nuts are regarded as possible causes of toxic complex syndromes involving the lung and should be employed as test allergens in the study of children, particularly those living in rural areas, with recurrent or chronic bronchopulmonary disease. Methods of Testing. Intracutaneous tests are used to elicit toxic complex reactions. The speed and intensity of the reaction is a function of the degree of sensitivity. It usually appears in 3 to 6 hours as a nodular, infiltrated, erythematous reaction and starts to resolve at 24 hours. Corticosteroid treatment can inhibit the reaction. Delayed Sensitivity Response Skin tests for delayed responses are best seen in tests for contact sensitivity and for the allergy of infection. After the intradermal injection of specific allergens into sensitized children, no gross inflammation or reaction is visible for 5 to 24 hours. After this interval, induration and erythema appear and reach peak intensity at 16 to 72 hours. If the reaction is marked, there may be edema, vesicles, and bullae with residual pigmentation. Erythema is variable, but firm induration is the hallmark of a positive reaction. Test Allergens. Allergic contact dermatitis due to poison ivy and the related poison sumac and poison oak oleoresins, to topical medications such as the antihistaminics, mercury, and benzocaine, and to certain ingredients in clothing, particularly rubber, occurs during childhood and infancy. Sensitizers such as nickel and the dichromates, which are important allergens in the production of allergic contact dermatitis in adults, rarely cause such dermatitis in children. 10 Skin tests with bacterial and viral antigens give delayed reactions which are of diagnostic value. Tuberculin antigens are most frequently used. Tuberculin preparations now in general use are old tuberculin (OT), prepared by concentration of the autoclaved culture filtrate of Mycobacterium tuberculosis and standardized to contain 100,000 tuberculin units per milliliter, and purified protein derivatives (PPD) of tuberculin, prepared by ammonium sulfate or trichloracetic acid precipitation of culture filtrates. They cannot be standardized in terms of one another because of qualitative factors which produce differences in reactions in children with varying degrees of sensitivity. Other bacterial antigen extracts frequently used in the study of infections in children are histoplasmin, coccidioidin, and blastomycin. Methods of Testing. Von Pirquet used the cutaneous (scratch) method in introducing the tuberculin test. Many other methods have been used, including patch tests, but the most satisfactory is the intracutaneous or Mantoux test. It is the most reliable for quantitative studies, because the dose can be graded, a known amount injected and the resulting reaction measured. Patch test reactions in children must be interpreted with care, since
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MARIE BRITT RHYNE
concentrations of chemicals that are "standard" for testing adults may produce nonspecific irritant reactions in early life. Patches should be placed on grossly normal, nonhairy skin. Suspected contactants are placed on a 2-inch square patch, preferably made of Dermicel (Johnson and Johnson) which contains an "acrylic mass" adhesive. Usually the patch is kept in place for 48 hours and then removed. A reading is made 20 to 60 minutes later, after the effect of pressure has worn off. Significant reactions present the changes in the skin characteristic of eczema and can be read as: Negative
1+
2+
3+
= no reaction = erythema and papules = erythema and papules
= erythema
and vesicles
= marked edema and vesicles Patch tests properly applied in the young are as reliable as those performed on older children and adults.
4+
SUMMARY
The developmental processes of the child's capacity to respond have an important inHuence upon his reactions to various allergens and upon the evolution of an allergic state. The physiological capacity to respond, particularly manifested by the maturing of the immune system, is subject to control by various genetic and environmental factors. Social expectations are manifestations of the sense of values and framework of knowledge of the cultural or ethnic group to which the child's family belongs and influence the child's pattern of exposure to most allergens. Allergen skin testing can be performed as a diagnostic aid at any age and for many types of allergic responses. It can be limited or extensive, but it should be based on clinical observations of reactions to exposure so that treatment is planned for the child, not the results of the tests. ACKNOWLEDGMENT The helpful comments on an outline and draft of this article by Dr. Shirley P. Borkowf and Miss Annette Ellinghaus are gratefully acknowledged.
REFERENCES 1. Adelsberger, L.: Das Verhalten der IdndlichenHaut gegeniiber verschiedenen Reizen. Zeitschr. fur Kinderh., 43:373, 1927. 2. Austen, K. F., and Humphrey, J. H.: In vitro studies of the mechanism of anaphylaxis. Adv. Immunol., 3:1, 1963.
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3. Carey, T. N., and Gay, L. N.: Skin reactions in infants. J. Allergy, 5:488, 1934. 4. Cochrane, C. G.: Mediators of the Arthus and related reactions. Progr. Allergy, 11:1,1967. 5. Cooke, R. A.: Allergy in Theory and Practice. Philadelphia, W. B. Saunders Co., 1947. 6. Coombs, R. R. A., and Gell, P. G. H.: The classification of allergic reactions underlying disease. In Gell, P. G. H., and Coombs, R. R. A., eds.: Clinical Aspects of Immunology. Philadelphia, F. A. Davis Co., 1963. 7. Dees, S. C.: Allergy to cow's milk. PEDIAT. CLIN. N. AMER., 6:881, 1959. 8. Epstein, W. L.: Contact-type delayed hypersensitivity in infants and children: Induction of RHUS sensitivity. Pediatrics, 27:51, 1961. 9. Fineman, A. H.: Studies of hypersensitiveness. XXIII. A comparative study of the intradermal, scratch and conjunctival tests in determining the degree of pollen sensitivity. J. Immunol., 11:465, 1926. 10. Fisher, A. A.: Contact Dermatitis. Philadelphia, Lea and Febiger, 1967. 11. Gregory, P. H.: The Microbiology of the Atmosphere. London, Leonard Hill (Books), Ltd., 1961. 12. King, T. P., Norman, P. S., and Lichtenstein, L. M.: Studies on ragweed pollen allergens. V. Ann. Allergy, 25:541, 1967. 13. Levine, B. B.: Immunochemical mechanisms of drug allergy. Ann. Rev. Med., 17:23, 1966. 14. Miyamoto, T., Oshima, S., Ishizaki, T., and Sato, S.: Allergenic identity between the common floor mite (Dermatophagoides farinae Hughes 1961) and house dust as a causative antigen in bronchial asthma. J. Allergy, 42:14, 1968. 15. Muller-Eberhard, H. J.: Chemistry and reaction mechanisms of complement. Adv. Immunol., 8:1, 1968. 16. Osler, A. G., Lichenstein, L. M., and Levy, D. A.: In vitro studies of human reaginic allergy. Adv. Immunol., 8:183, 1968. 17. Parker, C. W.: Drug reactions. In Samter, M., ed.: Immunological Diseases. Boston, Little, Brown & Co., 1965. 18. Parlato, S. J.: The sandfly (caddis fly) as an exciting cause of allergic coryza and asthma. II. Its relative frequency. J. Allergy, 1:307, 1930. 19. Prausnitz, C., and Kustner, H.: Studien uber der Dberempfindlichkeit. Zentralblat. Bakteriol., 86:160, 1921. Translated by Prausnitz, C., in Gell, P. G. H., and Coombs, R. R. A., eds.: Clinical Aspects of Immunology. Philadelphia, F. A. Davis Co., 1963. 20. Raffel, S.: Delayed (cellular) hypersensitivity. In Samter, M., ed.: Immunological Diseases. Boston, Little, Brown & Co., 1965. 21. Rapp, H. J., and Borsos, T.: Complement research. Fundamental and applied. J.A.M.A., 198:1347, 1966. 22. Rhyne, M. B., and Frank, M. M.: The nature of allergy. J. Pediat., 73:459, 1968. 23. Shulman, N. R.: Immunoreactions involving platelets. I. A steric and kinetic model for formation of a complex from a human antibody, quinidine as a haptene, and platelets; and for fixation of complement by the complex. J. Exper. Med., 107:665, 1958. 24. Stanworth, D. R.: Fundamental approach to the mechanisms of asthma. Tr. World Asthma Conference, Eastbourne, 1965. 25. Von Pirquet, C. E.: Allergy. Arch. Int. Med., 7:259, 383, 1911. 26. Wodehouse, R. P.: Hay Fever Plants. Walthaus, Chronica Botanica Co., 1945. 27. Zak, S. J., and Good, R. A.: ImmunocheInical studies of human serum gammaglobulins. J. CUn. Invest., 38:579, 1959. Department of Chronic Diseases School of Hygiene and Public Health The Johns Hopkins University Baltimore, Maryland 21205
The skin envelops the entire surface of the child's body and is easily accessible to the examiner's eye. It is, tberefore, well suited for use as a test organ to determine response to foreign substances. Although methods for applying test substances to the skin were reported by investigators in the latter half of the nineteenth century, it was not until von Pirquet described his cutaneous tuberculin test in 1911 that this procedure became a valuable diagnostic aid. The introduction of skin testing in a scientific manner by this astute clinical investigator provided a means of obtaining fundamental information on immunologic phenomena in children and adults. 25 An understanding of the nature of allergic responses and the relationship of the responses to common clinically manifested symptom complexes, the developmental processes of the child's capacity to respond, the methods for eliciting the desired response, the character and concentration of materials used for testing, and the significance of the observed reaction is necessary for the physician to utilize skin tests as an immunologic diagnostic procedure. TYPES OF ALLERGIC RESPONSES
Four types of allergic responses have been described. 6 The key to understanding their relationship to clinical disorders is a knowledge of the mechanisms involved in antibody and cell induced immune damage. Antibody activity is carried by globulins found in tbe serum, external secretions, and tissue fluids. These globulins, known as immunoglobulins (Ig), have been classified by their physicochemical properties. Five classes of human immunoglobulins are now recognized: IgG, IgA, IgM, IgD, and IgE. Distinct subclasses are also recognized for several From the Allergy Foundation of America Laboratory, Department of Pediatrics, The Johns Hopkins University School of Medicine and Hospital, Baltimore, Maryland. Supported by Projects 212 and H-188 (R) from the Children's Bureau, Department of Health, Education and Welfare . ... Presently Special Research Fellow, National Center for Health Services, Research and Development, and Graduate Student, Department of Chronic Diseases, The Johns Hopkins University School of Hygiene and Public Health. Pediatric Clinics of North America-Vol. 16, No.1, February, 1969 227
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immunoglobulins. Each class and subclass has been chemically identified and purified, and for most some of the biologic capabilities are known. The child is capable of responding to an allergen with a great many different immunoglobulins, each of which seems to have a specialized function. Antibodies capable of combining with antigen and a tissuefixing site activate enzyme-mediated processes through which cells release vasoactive substances (histamine, serotonin, bradykinin, and slow reacting substance of anaphylaxis).2 Antibodies capable of "fixing complement" activate biochemical pathways through which cells are destroyed by the components of complement. 15 Nine proteins are required for the cytotoxic activity of complement. They participate sequentially in a series of reactions which eventually lead to the enzymatic formation of a hole in the surface of the cell coated with antibody. Such cells lose their ability to maintain osmotic equilibrium; they swell and burst. The sequence of reactions need not go to completion to produce biologic effects. At various stages in the reaction sequence chemotactic substances are released which attract polymorphonuclear leukocytes into the area of immune injury. At other stages the smooth muscle contractors, anaphylatoxins, may be released. 22 Anaphylactic Response Anaphylactic responses are enzyme-mediated processes triggered by allergen-antibody interactions involving an antibody class which has both an allergen-combining site and a tissue-fixing site. Tissue-bound antibody is activated on combination with allergen to trigger the enzyme pathways leading to a release of vasoactive substances, without the occurrence of cell death. 16 Urticaria and Angioedema. Extensive urticaria and angioedema may be an expression of a cutaneous allergic response of the anaphylactic type. Vasoactive substances released from cells by this reaction may be multiple. Inhibition of symptoms and signs· of the reaction is often not accomplished by use of antihistaminics alone. Atopic Disorders. Many atopic persons who develop clinical symptoms of atopic dermatitis (infantile eczema), seasonal hay fever, nonseasonal rhinitis, sinusitis and eustachitis, or bronchial asthma readily produce a specialized class of antibodies, presumably IgE, after ordinary exposure to otherwise innocuous substances. This antibody, called skinsensitizing or reaginic antibody, has "par excellence" the property of binding to tissue cells and interacting with its allergen to cause a response typical of the anaphylactic reaction. Cytolytic or Cytotoxic Response Some IgG and IgM antibodies are chemically capable of combining with an antigenic' component of a tissue cell or an antigen which has
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become intimately associated with tissue cells. They usually produce their effects locally by activating the biochemical pathway through which cells are destroyed by the components of complement. 21 Thrombocytopenic Purpura. Generalized purpura due to sensitivity is a clear example of a cytolytic reaction. Such reactions are initiated by antibody reacting with an allergen which has become intimately associated with platelets. 23 Toxic Complex Syndrome or Response Antibodies have multiple combining sites, and many are capable of cross linking with polyvalent allergens to form sticky complexes. Circulating antigen-antibody complexes large enough to be entrapped by a vascular basement membrane can localize in such organs as the skin, the kidney and the spleen, where they fix complement and cause local inflammation by the release of chemotactic substances which attract polymorphonuclear leukocytes into the area of immune injury.4 Serum Sickness. The clinical manifestations of serum sickness result from two types of allergic responses. Symptoms and signs of joint pains, leukopenia, lymphadenopathy, and hematuria are ascribed to a toxic complex response. Symptoms of fever, urticaria, periorbital edema, and asthma are evoked by the anaphylactic response. Delayed Sensitivity Response In this immune response the appearance of small lymphocytes, presumably activated by antigen which recognizes an antibody-like antigen site on the surface of the cell, is followed by in-migration of monocytes. These cellular elements plug small vessels near the site of antigen localization and can produce tissue necrosis through ischemia or through release of chemical mediators th~t produce cell death directly.2o Allergic Contact Dermatitis. Allergic contact dermatitides characterized by redness, edema, papules, vesiculation, weeping, crusting, and itching reactions in the child's skin are delayed sensitivity responses. Usually, once allergic sensitization is established in even a small area of the skin, the entire surface of the skin is capable of reacting.
THE CHILD'S CAPACITY TO RESPOND
Demographic and Environmental Factors
Age and Maturation. The child's physiological capacity to respond to an allergen is subject to control by various demographic, genetic, and environmental factors. In an epidemiologic study designed to measure the prevalence of asthma and atopic dermatitis in randomly selected populations of l-year-old children and elementary school children in the state of Maryland, those defined as cases by interview were matched
I
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MARIE BRITT RHYNE
for sex, race, age, and residence with noncases and scratch skin-tested with extracts of several common inhalant and food allergens standardized at 0.1 mg. protein nitrogen (10,000 PNU) per milliliter. In the one-year-old population, significant differences (P < .05) between cases and control reactivity rates were noted for ragweed, wheat, and eggwhite, but only one allergen, eggwhite, gave a rate greater than 2 per cent in any group. The positive percentage for that allergen was 4.48 for cases and 1.49 for controls. In the elementary school group significant differences in the allergen reactivity rates of asthmatic and nonasthmatic children were noted for ragweed, orchard grass, house dust, and feathers. Four allergens, ragweed, orchard grass, house dust, and alternaria, gave a rate greater than 10 per cent in the case group. The highest was for ragweed; the positive percentage for that allergen was 13.7 for cases and 3.6 for controls. Asthmatic children with current symptoms had a significantly higher rate of reaction to house dust and to feathers than did asthmatic children without current symptoms. The positive percentage for house dust was 19.1 for current and 8.0 for noncurrent cases; for feathers, 12.7 for current and 4.3 for noncurrent cases. These data suggest that children demonstrate an increasing capacity to develop reaginic antibody (presumably IgE) to ordinary environmental substances with increasing age and exposure, and that the potential for response is greater for the asthmatic child with current symptoms than for either the asthmatic child without current symptoms or the nonasthmatic child. Some of the observed differences in response with age are probably related to structural differences of the skin itself. About 35 years ago Carey and Gay 3 reported that reactions of newborn infants to intracutaneous tests of a 1: 10,000 dilution of ergamine acid phosphate were consistently much smaller than those observed in adults, erythema and wheal being about half the diameter of that noted in the adult reactions. The capacity to accept passive sensitization to reaginic antibody was present even in the skin of a 16-hour-old infant, but stronger concentrations of test solutions were required to produce reactions comparable to those observed in adults. The synthetic mechanism for IgG develops and matures during the first 2 years of the child's life, usually beginning at 4 to 6 weeks of age and reaching the lower level of that attained by the normal adult during the second year of life. 27 In the fetus small quantities of the high-molecular weight immunoglobulin, IgM, are often present. It seems plausible that this originates in the fetus, as synthesis starts very rapidly postnatally and continues at such a rate that adult levels are often attained by age 9 months. The development and maturation of the synthetic mechanisms for delayed sensitivity remain to be studied. Studies with Rhus allergens
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under controlled circumstances have demonstrated that infants below the age of 1 year have a depressed ability to react to these allergens; children between 1 and 3 years of age assume an intermediate position, while children between 3 and 8 years are readily sensitized and show an intensity of reaction comparable to that seen in adults. 8 It seems likely that the fault lies not so much in the skin itself as in the ability to make delayed type "antibodies," as irritants readily induce inflammation in the skin of older infants and young children. However, the immature structure of the skin may be an important factor for young infants, as they usually react negatively or only slightly to injections of turpentine, iodoform, and other irritants into the skin. Positive reactions to such irritants are more common in infants over 2 months of age. 1 Sex. In the Maryland studies the skin reactivity rate for the allergen mixed feathers was significantly greater in school-age female asthmatics (13.4 per cent) than in school-age male asthmatics (4.7 per cent), but this was believed to be due to increased exposure rather than to differences in capacity to respond, as no significant differentiation in reactivity rates was noted for other allergens. Sex differences for other antibody classes have not been evaluated. Several of the congenital varieties of hypogammaglobulinemia are male sex-linked, but the acquired varieties occur in both males and females. Race. In the Maryland studies race does not appear to be an important demographic variable for reaginic antibody. No significant differentiation in reactivity rates was noted for asthmatic white and Negro children. Residence and Social Class. Detectable differences in reactivity rates between asthmatic children living in urban, suburban, and rural areas of Maryland were observed. This is especially true for ragweed, for which the highest reactivity rates were found in the suburban asthmatic children (21.7 per cent), the lowest in the rural asthmatic children (8.3 per cent). Significantly higher reactivity rates were also detectable for three allergens, ragweed, grass, and house dust, in asthmatic children of families of upper class strata. While the factors responsible for the observed differences in skin reactivity have not been elucidated, it is likely that they are related to differences in the concepts of hygienic living habits and the environmental exposures encompassed in the family living patterns of children of different social strata and different residential areas.
THE DIAGNOSTIC PROCEDURE
The anaphylactic_ response, the toxic complex response, and the delayed sensitivity response are immunological phenomena demonstrable by skin tests.
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Anaphylactic Response Skin tests for this type of response are most frequently employed in the evaluation of children with respiratory or dermatologic manifestations of atopy. They are used to evaluate either atopic or nonatopic children with symptoms of local anaphylactic reaction, such as urticaria and angioedema, recurrent gastroenteritis and vernal conjunctivitis, or a history of symptoms of a generalized anaphylactic reaction with shock. Selection of Allergens for Testing. Allergens which induce the anaphylactic reaction are frequently ordinary contact ants of daily living. Testing extracts of such allergens are standardized in terms of either total nitrogen content or protein nitrogen concentration per milliliter, or by the weight of the dried allergen material in a given volume of extracting fluid. Such extracts are notoriously variable in potency, making precise comparisons of the results in patients tested in different clinics or physician offices with different preparations unsatisfactory. As all "purified" allergens to date have been shown to be of a proteinous nature, standardization on a protein nitrogen concentration basis is the preferred method. Food Allergens. Foods capable of acting as allergens tend to be proteins of either animal or plant origin. Milk, meat, poultry, fish, egg, dried legumes, nuts, chocolate, and seeds are the most frequent producers of anaphylactic reactions in children. The frequency of ingestion of foods with great allergenic potentiality and the amounts consumed help determine their importance as allergens. The amounts and types of foods ingested are frequently a function of the cultural habits of the child's family. For instance, cow's milk allergy follows and parallels the development of successful artificial feeding of infants with cow's milk formulas in this country.7 Preferential food habits noted in various ethnic groups, such as Italians, Orientals, and Mexicans, and in families living in different regions of this country, probably account for some 'of the observed differences in the frequencies and patterns of food allergy in children reported by physicians practicing in widely different geographic areas. Food allergens used for testing should reflect the food habits of the child under study. Inhalant Allergens. Inhalant allergens which induce anaphylactic reactions appear to be proteins with a molecular weight of about 30,000 to 40,000. 12 • 24 The allergenic environment of the child is important, and differences in the availability of and degree of exposure to various allergens help determine their significance as allergens at different ages in the child's life. Allergens that contaminate the house air may sensitize the child earlier in life than seasonal external environmental allergens, such as pollen and mold spores, which are present in the air only for limited periods of the year. These usually require several years of exposure to produce clinical signs of sensitivity.
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Indoor airborne allergens originate from home furnishings, from animals, and from material in the building attacked by molds. House dust is a mixture of many materials. Current studies suggest that the mite is probably one of its major allergens. 14 The major biologic fibers used in manufacturing stuffed chairs and sofas, pillows, mattresses, box springs, and stuffed toys in this country are cotton and kapok. In other cultures different fibers are commonly used. The animal or epidermal allergens found in the house air are emitted fromfeathers found in furnishings and from house pets such as parakeets, rabbits, cats, dogs, gerbilles, and guinea pigs. The rural child may also be exposed in barns or sheds to the indoor airborne allergens exnitted from farm animals such as horses, cows, and chickens. Microbes in the indoor air originate within the house from such areas as defective timber, wallpaper paste, stored books, clothes, and toys. In spite of ventilation, Peincillium is the mold spore dominating the indoor air of most homes, while Cladosporium (Hormodendrum) is usually the dominant spore in the indoor air.ll Knowledge of the common inhalant allergens in the child's environment, together with a detailed assessment of the nature, place, and tixning of the allergic responses noted in the child, is necessary to determine what substances to employ as test allergens. Outdoor airborne allergens appear to originate from vegetation above the ground. Pollens from wind-pollinated plants are the principal outdoor airborne allergens in most parts of the world. They appear in the air only when the plant is in flower. Only a very few plants possess pollens that contain antigens with properties suitable to sensitize the child and promote anaphylactic responses. Plants known to be of allergenic significance in the continental United States are listed in Table l. This outline was prepared by the author from material drawn from the book Hay Fever Plants by Wodehouse2 6 and from data obtained from correspondence with 15 pediatric allergists practicing in various parts of this country. Only two plants, ragweed and Bermuda grass, were noted to be real troublemakers for children in the United States. Most fungus spores from outdoor vegetation have a marked seasonal incidence and a clear diurnal periodicity. Cladosporium is the most prevalent spore type in Europe and North America, and while often present through the year usually has a marked rise from June to September. Each spore type varies in quantity from year to year, depending on the weather, the presence of diseases in crops, and the general condition of the vegetation in the neighborhood. Debris from insects may be inhaled in sufficient quantities to produce anaphylactic responses if the insects occur in large number. In the Great Lakes region of the United States the caddis fly swarms in xnidsummer and sheds a loose scaly epithelium from its wings which is capable of acting as an allergen. 18 Insect Allergens. Serious anaphylactic reactions to insect stings are
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Table 1. Plants oj Allergenic Importance in the Continental Unites States GEOGRAPHIC AREA
Northeastern: New
Eng~
land, New York, New Jersey, Pennsylvania Middle Atlantic: Delaware, Maryland, District of Columbia Virginias and Carolinas:
Virginia, West Virginia, N orth Caro~ lina, South Carolina North Central: Ohio, Kentucky, Indiana, Michigan, Illinois, Iowa, Eastern Missouri, Wisconsin, Southern Ontario Southan: Florida, Tennessee, Georgia, Alabama, Mississippi, Louisiana, Arkansas, Southern Missouri, Oklahoma, Eastern Texas Southlvtst: Arizona, New Mexico, Western Texas
TREES
Elm, maples, poplars, birches, oaks
GRASSES
Sweet vernal, June, orchard, timothy, annual blue grass Orchard, timothy
Maples, oaks, eastern sycamore, hickory, birches, paper mulberry Maples, elm, red ce- June, annual blue dar, junipers, hickgrass, orchard, ories (pecan), oaks sweet vernal, redtop, Bermuda (in southern areas) Ash, cottonwood, oak, June, orchard, timothy, redtop maple, elm
WEEDS
MAJOR POLLENS
Short and giant ragweeds, plantain Short and giant ragweeds, plantain
Ragweeds, grasses
Short ragweed, sorrel dock
Short ragweed, Bermuda grass, hickories (pecan)
Short ragweed
Short ragweed
Ragweeds, orchard grass
Mountain cedar, red cedar) poplar (cottonwood), birch, hickories (pecan), oak, live oak, privets, scrub elm, paper mulberry Mulberry, olive, mountain cedar, Arizona cottonwood
Timothy, orchard, Bermuda
Amaranths, spiny carelessweeds, western water.. hemp, giant and short ragweed, Russian thistle
Bermuda grass, hickories (pecan), ragweeds
Bermuda, Johnson
Rabbit bush, Rus... ian thistle, amaranths (Chenopodium)
Southern California
Olive, Chinese elm, coast, live oak, hickories (walnut)
Bermuda, saltgrass
Bermuda grass, mountain cedar, chenopods, amaranths Bermuda, saltgrass
North Pacific: Northern California, Nevada, Oregon, Washington
Acacia, cottonwoods, boxelder (maple), alder, birch, oak, hickories (walnut)
Timothy, orchard, redtop, blue grass, fescue, velvet, Western rye
Rocky Mountain: Idaho,
Cottonwoods, boxelder (maple), birch, Rocky Mountain cedar Elm, oak
Timothy, redtop, orchard, June, fescues, wheat groups
Curly dock, pigweed, scale family, lambs quarter, Russian this.. tle, sages Saltbushes, Russian Weeds (none of thistle, sagebrush, major importance) maypigweed, weed, curly dock, sheep sorrel Russian thistle, Russian thistle, sagebrushes sagebrushes, ragweeds
Montana, Wyoming, Utah, Colorado
Plains and Prairies: North Dakota, South Dakota, Nebraska, Kansas, Minnesota
Orchard, bluegrass, timothy, red top, Bermuda, Johnson (in southern areas)
Russian thistle; taU, short, and western ragweods
Ragweeds, Russian thistle
limited to the Hymenoptera, which include bee, hornet, wasp, and yellow jacke~. These reactions are due to allergens usually present in both the venom and the bodies of the insects. Rarely can children identify these insects accurately, but multiple cross sensitivities to family members are the rule, and exact identification is not necessary, because testing to the allergens of all members should be conducted on every study child. Bites from household insects such as fleas and bedbugs may produce an allergic response known as papular urticaria, but this is a separate entity from the urticaria and angioneurotic edema of the anaphylactic response.
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Incomplete Allergens (Haptens). Most allergens naturally encountered in the child's environment are complete allergens and are capable of inducing anaphylactic responses by themselves. However, there are many low molecular weight substances which can act as allergens if they can become conjugated with a carrier substance, usually a protein, in the body. Of the many chemicals capable of acting as incomplete allergens, those of greatest concern are drugs, such as penicillin, which are administered as therapeutic agents. Sensitization may occur to the chemical itself or to one of its metabolic products. Suitable conjugated allergens for testing of simple chemicals or metabolic products of such chemicals are not available for general use, although study of the number and chemical characteristics of the allergens produced during the metabolism of penicillin has been the focus of several investigators' work, 13. 17 There are wide differences of opinion as to whether all allergens known to occur in the child's environment should be tested or only the limited number known to be associated with the production of clinical symptoms. The author prefers to test a limited number of the commoner potent allergens known to be present in the environment with the understanding that further and repeated tests will be performed during subsequent evaluations if the course of the symptoms warrants additional studies. Potent allergens capable of producing anaphylactic type responses and common to the environment of children living in the Maryland area are presented in Table 2. Methods of Testing. Cutaneous and intracutaneous tests are used to elicit anaphylactic responses. An aqueous solution of the allergen is prepared by extracting the appropriate allergenic material with slightly alkaline buffered saline. The solvent containing the allergen is separated from the residual material by clarification or preliminary filtration. Usually it is dialysed against buffered saline to dispose of irritating substances and concentrated to the desired standard volume. The extract is sterilized by bacterial filtration and tested for both aerobic and anaerobic organisms. It can then be standardized on the basis of protein nitrogen content and applied to the skin by one of three methods, prick, scratch, or intradermal tests. There are differences of opinion regarding the relative value of these methods. All have distinct advantages and disadvantages. Scratch tests are performed by making a superficial cut into the skin with a vaccinostyle needle or scalpel without drawing blood. The extract of the allergen is then applied to the cut. Although tests are usually read in 20 minutes, sensitive patients may develop large whealing reactions before that time. Such test sites can be wiped free of allergen with an alcohol sponge as soon as large reactions appear to prevent continuous absorption of allergen and the development of a systemic anaphylactic reaction. Prick tests are performed by placing a drop of an aqueous
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MARIE BRI'IT RHYNE
Table 2.
Diagnostic Skin Tests Jar Common Allergens in the Maryland Area HOUSEHOLD
POLLENS
INHALANTS
Weeds Ragweed, giant Ragweed, short Plaintain, Eng\.
Feather mix Dust Kapok Pyrethrum
Grasses Timothy Orchard Trees Birch Elm Hickory Maple Oak Sycamore Paper mulberry FOODS
Grains Wheat flour Oat Cornmeal Rice Dairy products Milk (cow) Eggwhite Meats Beef Chicken Fish Vegetables String beans Lima beans Peas Onions Potato, white Tomato
EPIDERMALS
Cat hair Dog hair Horse dander MOLD SPORES
Mixed 1 to 4 (Indoor group) 1. Aspergillus fumigatus 2. Penicillium mix 3. Mucor racemosus 4. Fusarium Mixed 5 to 8 (Outdoor group) 5. Alternaria tenuis 6. Helminthosporium 7. Hormodendrum hordei 8. Botrytis cinerea INSECTS
Mixed Hymenoptera 1. Honeybee 2. Hornet 3. Wasp 4. YeIIow jacket Household insects Mosquito
Fruits Orange Banana Beverages Coffee Chocolate Other Peanut Black pepper Mustard
solution of allergen on the child's skin and pricking the skin through the drop. They are read in the same manner as scratch tests. Diluent control tests are always performed. If dermatographism is marked, anti-
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237
histaminic medication should be given about 30 minutes before subsequent testing. The criteria the author uses for interpretation of cutaneous tests are: Negative
= no reaction or no different from control
1+ 2+ 3+
= erythema less than 10 mm. in diameter = erythema greater than 10 mm. in diameter with no wheal = wheal with surrounding erythema = wheal less than 10 mm. in diameter with pseudopods,
4+
= wheal
±
disregarding erythema greater than 10 mm. in diameter with pseudopods, disregarding erythema
Criteria are arbitrary and differ from clinic to clinic, but they do permit a more quantitative estimation of skin reactivity than does a simple recording of tests as negative, positive, or doubtful. The intensity of the reaction is a measure of the degree of sensitivity, but it does not necessarily correspond to the clinical importance of the allergen to the child. Cutaneous testing is commendable in view of its simplicity. Syringes and needles are not required and the risk of systemic reactions is minimal. In addition few children find scratch testing really unpleasant, so that reluctance to cooperate is minimized. Its chief drawback is its relative insensitivity. Intracutaneous tests are about a hundred times more sensitive than cutaneous tests. 9 They are performed by injecting a small amount, 0.02 ml. is usually sufficient, of aqueous solution of allergen into the epidermis. The precaution of starting with an intracutaneous injection of a 1: 100 or 1: 1000 dilution of the allergen will usually suffice to prevent systemic reactions, but serious drawbacks to the exclusive use of the intracutaneous tests are the large number of syringes needed to make a complete examination and the great reluctance of young children to accept such multiple "shots." The author uses intracutaneous testing to supplement scratch testing when the latter gives a negative or weakly positive reaction to a suspected allergen. Scratch testing is usually performed with allergen extracts standardized at 0.01 mg. protein nitrogen (1000 PNU) per milliliter. A negative scratch test is followed by intracutaneous testing with allergen extracts standardized at 0.001 mg. protein nitrogen per milliliter. Intracutaneous tests with extracts of a concentration greater than 0.01 mg. protein nitrogen per milliliter are rarely performed, as such materials may be chemically irritating to the child's skin and produce false positive reactions. Significance of Reactions. The variability of potency of allergenic extracts must be kept in mind in evaluating negative reactions. This is particularly true for the household inhalant group and for certain foods, but it can be a problem with any allergen. The effects of certain drugs, especially antihistaminics, on the size of the skin reaction have been
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MARIE BRITT RHYNE
studied with conflicting results. Further evaluations with chemically standardized allergens are needed to determine the significant effects, if any, of these agents upon the intensity of the anaphylactic response in skin tests. Subcutaneous injections of adrenalin do have an inhibitory effect on the reactivity of the skin which persists for about 60 minutes after administered. Ii False positive reactions occur when the allergen extracts contain nonspecific chemical irritants or when poor testing technique results in mechanical irritation of the skin. When the test materials and the testing technique are satisfactory, positive reactions are immunologically specific. However, they mayor may not be related to the clinical syndrome for which the child is being evaluated. A thorough knowledge of the child's clinical history and physical findings, and continued observations for several months or even years, are usually necessary to properly interpret the relationship of the skin test results to the child's clinical symptoms. Use of Passive T1'ansfer Test. Prausnitz and Kiistner19 described the passive transfer test for anaphylactic reactions, and theirs are eponymous names for the test. In this test the serum of the child under study is injected into the skin of a normal recipient. If reaginic antibodies are present in the serum, passive sensitization of the injected site of the recipient's skin will result within 24 hours. Injection of the allergen at the same skin site can produce a local anaphylactic response. This test is direct evidence of the presence of reaginic antibodies in the study child's serum. For clinical diagnostic purposes the usefulness of this test is limited by the danger of homologous serum jaundice. Its usefulness in scientific investigations of the interaction of allergens and reaginic antibody in vivo is undisputed. Toxic Complex Syndrome Responses Local toxic complex reactions or Arthus reactions are produced by antibodies capable of interacting with allergen to forin' microprecipitates. Frequently the child will produce anaphylactic antibodies or delayed sensitivity "antibodies" as well as toxic complex forming antibodies to the same allergen, making it difficult for the evaluator to distinguish one skin test from the other. The anaphylactic response may modify or mask the Arthus reaction, and either or both may modify the delayed reactions. Test Alle1'gens. Allergens eliciting serum sickness or the toxic complex syndrome tend to be substances administered to the child by injection. In the past sera derived from animals were the frequent causative agents. Today drugs, particularly penicillin, are the more common offenders. Some fungi, especially Aspergillus fumigatus, and a wide variety of vegetable dusts from hay, straw, bagasse, cotton, hemp, paprika, malt,
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and nuts are regarded as possible causes of toxic complex syndromes involving the lung and should be employed as test allergens in the study of children, particularly those living in rural areas, with recurrent or chronic bronchopulmonary disease. Methods of Testing. Intracutaneous tests are used to elicit toxic complex reactions. The speed and intensity of the reaction is a function of the degree of sensitivity. It usually appears in 3 to 6 hours as a nodular, infiltrated, erythematous reaction and starts to resolve at 24 hours. Corticosteroid treatment can inhibit the reaction. Delayed Sensitivity Response Skin tests for delayed responses are best seen in tests for contact sensitivity and for the allergy of infection. After the intradermal injection of specific allergens into sensitized children, no gross inflammation or reaction is visible for 5 to 24 hours. After this interval, induration and erythema appear and reach peak intensity at 16 to 72 hours. If the reaction is marked, there may be edema, vesicles, and bullae with residual pigmentation. Erythema is variable, but firm induration is the hallmark of a positive reaction. Test Allergens. Allergic contact dermatitis due to poison ivy and the related poison sumac and poison oak oleoresins, to topical medications such as the antihistaminics, mercury, and benzocaine, and to certain ingredients in clothing, particularly rubber, occurs during childhood and infancy. Sensitizers such as nickel and the dichromates, which are important allergens in the production of allergic contact dermatitis in adults, rarely cause such dermatitis in children. 10 Skin tests with bacterial and viral antigens give delayed reactions which are of diagnostic value. Tuberculin antigens are most frequently used. Tuberculin preparations now in general use are old tuberculin (OT), prepared by concentration of the autoclaved culture filtrate of Mycobacterium tuberculosis and standardized to contain 100,000 tuberculin units per milliliter, and purified protein derivatives (PPD) of tuberculin, prepared by ammonium sulfate or trichloracetic acid precipitation of culture filtrates. They cannot be standardized in terms of one another because of qualitative factors which produce differences in reactions in children with varying degrees of sensitivity. Other bacterial antigen extracts frequently used in the study of infections in children are histoplasmin, coccidioidin, and blastomycin. Methods of Testing. Von Pirquet used the cutaneous (scratch) method in introducing the tuberculin test. Many other methods have been used, including patch tests, but the most satisfactory is the intracutaneous or Mantoux test. It is the most reliable for quantitative studies, because the dose can be graded, a known amount injected and the resulting reaction measured. Patch test reactions in children must be interpreted with care, since
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concentrations of chemicals that are "standard" for testing adults may produce nonspecific irritant reactions in early life. Patches should be placed on grossly normal, nonhairy skin. Suspected contactants are placed on a 2-inch square patch, preferably made of Dermicel (Johnson and Johnson) which contains an "acrylic mass" adhesive. Usually the patch is kept in place for 48 hours and then removed. A reading is made 20 to 60 minutes later, after the effect of pressure has worn off. Significant reactions present the changes in the skin characteristic of eczema and can be read as: Negative
1+
2+
3+
= no reaction = erythema and papules = erythema and papules
= erythema
and vesicles
= marked edema and vesicles Patch tests properly applied in the young are as reliable as those performed on older children and adults.
4+
SUMMARY
The developmental processes of the child's capacity to respond have an important inHuence upon his reactions to various allergens and upon the evolution of an allergic state. The physiological capacity to respond, particularly manifested by the maturing of the immune system, is subject to control by various genetic and environmental factors. Social expectations are manifestations of the sense of values and framework of knowledge of the cultural or ethnic group to which the child's family belongs and influence the child's pattern of exposure to most allergens. Allergen skin testing can be performed as a diagnostic aid at any age and for many types of allergic responses. It can be limited or extensive, but it should be based on clinical observations of reactions to exposure so that treatment is planned for the child, not the results of the tests. ACKNOWLEDGMENT The helpful comments on an outline and draft of this article by Dr. Shirley P. Borkowf and Miss Annette Ellinghaus are gratefully acknowledged.
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