Proceedings of the Task Force on guidelines for standardizing old and new technologies used for the diagnosis and treatment of allergic diseases

THE JOURNAL

OF

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Proceedings of the Task Force on Guidelines for Standardizing Old and Mew Technologies Used for the Diagnosis and Treatment of Allergic Diseases I. Leonard

Bernstein,

MD General Chairman and Guest Editor

CHAPTER 1. INTRODUCTION The development and standardization of tests to aid in the diagnosis and treatment of patients with immediate and delayed hypersensitivity disorders are important public health considerations because of the widespread prevalence of these diseases and the necessity to rely on these tests for proper diagnosis and management. The majority of primary care practitioners do not receive proper “hands-on” instruction in these methods, and even qualified specialists in allergic diseases have differing views on which tests to use and how they should be interpreted. In many instances performance and interpretation of these tests depend on the personal opinion of the physician simply because there is currently no consensus on guidelines for performing and evaluating these tests. The objective of this Task Force was to develop a reference document for standardization of relevant in vivo and in vitro diagnostic techniques in allergic diseases* and interpretation of test results. Although considerable progress is currently being made in elucidating the diagnostic use of specific organ challenge tests for allergic diagnosis, these techniques were excluded by the organizing committee because preliminary attempts to establish uniform methods and stan*The classification of human allergic diseases as proposed by Cell and Coombs will be used interchangeably throughout this document: type I, IgE-dependent diseases (immediate hypersensitivity); type II, cytotoxic antibody-mediated diseases; type III, immune complex-mediated diseases; and type IV, cellular immune diseases (delayed hypersensitivity).

dards have just begun to appear in the medical literature . Dr. Sheldon G. Cohen, Director, Allergy Branch, National Institute of Allergy and Infectious Diseases-Institute of Allergy and Infectious Diseases, National Institutes of Health, initially recruited an advisory steering committee consisting of Drs. I. Leonard Bernstein, Howard I. Maibach, Burton Zweiman, Edward A. Emmett, and Ross E. Rocklin. This committee organized the Task Force into two major sections: (1) evaluation of type I skin test and relevant laboratory procedures and (2) evaluation of type IV skin test and relative procedures. The Workshop convened in Washington, DC, under the sponsorship of the National Institute of Allergy and Infectious Diseases. Participants in workshop I included: Drs. N. Franklin Adkinson, Jr., Paul C. Atkins, Harold Baer, I. Leonard Bernstein, William A. Hook, Michael Kaliner, Robert F. Lemanske, Jr., Richard F. Lackey, Reuben Siraganian, Paul C. Turkeltaub, and Burton Zweiman. Participants in workshop II consisted of Drs. C. Edward Buckley, III, Edward A. Emmett, Howard I. Maibach, Robert L. Rietschel, Ross E. Rocklin, and Paul C. ‘Axkeltaub. Preliminary discussion of subject outlines was presented to the entire group on June 18, 1987. These summaries were followed by open discussion and critiques of all presentations. Small breakout sessions of individual workshop committees were then held for the purpose of preparing consensus statements and recommendations. These workshop drafts were presented to the plenary session on June 19, 1987, after which there 487

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was a final round of discussion devoted to critique, emendments, and recommendations from all participants. The distillation of these deliberations is the subject of this report. The chairman and participants of the Task Force extend special recognition and thanks for the diligent

Jo ALLERGY Clln! IMMUNOI.. SF’-I-EMBER ‘988

efforts of Drs. Sheldon G. Cohen and Dorothy D. Sogn in organizing and planning the meetmg at the National Institutes of Health, Bethesda, Md. The spccial efforts of program facilitator, Carol Sh;ipiro, are also acknowledged.

LEVARlT l&l WV0 TESTS S NWlEDtATE H A. Percutaneous and intracutaneous skin tests 1. Diagnostic techniques 1.1. Prick tests 1.11. Background 1.111. Historical perspective Although Blackley first documented the diagnostic potential of skin testing, the introduction of the cutaneous test for tuberculosis by von Pirquet provided the chief impetus for the development of allergy skin testing.’ The first of these tests was a scratch or epicutaneous test made by rubbing the allergen into abraded skin of the forearm. The work of Schloss permanently established the von Pirquet technique as a method for the diagnosis of allergic diseases. A few years later, Schick and Cooke independently introduced the intracutaneous test as a diagnostic method in allergic diseases. Sir Thomas Lewis’ first suggested the puncture technique as an alternative skin test. Variability in the instruments, application, and interpretation of both scratch and intracutaneous tests was recognized soon after their introduction. Perhaps as a way to avoid these problems, references to puncture tests began to appear in the early 195Os, especially among European allergologists. Squire3 first called attention to the quantitative aspects of the prick technique as a method of detecting various protein sensitizations. Although he estimated that only a small amount (3 x 10m6 ml) of the test solution was introduced through the puncture site, in effect this degree of absorption made the test a microintradermal test. Before 1970, the prick-puncture test had few adherents in the United States. Scratch tests were popular, especially among pediatricians. Intracutaneous tests were used almost exclusively in several Eastern allergy centers and by allergists trained on the Eastern seaboard. other clinicians advocated that the intracutaneous tests be used only when scratch tests were negative. Presumably, as a result of increased international exchange of information, the attitude towitrd prickpuncture tests in the United States began to change in the early 1970s. Since then, there has been a dramatic increase in the acceptance of the prick test as a prime diagnostic skin procedure in the United States. 1.112. Present applications Prick tests are widely used for confirmation of clinical immediate hypersensitivity induced by a wide variety of naturally occurring inhalant and food allergens. Under certain carefully defined circumstances, these tests are also useful in the diagnosis of drug and chemical hypersensitivity reactions. They are frequently used as reference standards for evaluating the specificity and sensitivity of specific in vitro tests that measure circulating reagins (specific IgE, specific IgGJ. They also are currently used to determine bioequivalent potencies of allergenic extracts. 1.12. Current methods 1.12 1. Instruments Since it is impossible to quantify the exact amount of injected material by prick tests, the allergic skin response is dependent on the reliability of the device, its dimensions, the depth of the puncture needle and the force, duration, and angle of application of the device. The actual devices vary from disposable 25 to 27-gauge hypodermic needles, lancets (standard, Morrow-Brown, Allergy Pricker [Dome/Hollister-Stier, West Haven, Conn.), multitest devices (eight plastic head and eight sterile disposable needle instruments), and a bifurcated scarifier.4-8 1.122. Standardization of techniques 1.122 1. Performance factors

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The reliability of the prick test is heavily dependent on the skill of the individual tester, the reliability of the test instrument, the status of skin reactivity on the day of the test, circadian rhythms, potency and stability of test extracts, especially optimum concentrations, and experimental differences between duplicate prick tests.‘, lo 1.1222. Interpretation of test results Wide variation exists in measuring, scoring, and interpreting prick skin test results. Some clinicians advocate immediate blotting of the allergen after the prick test to reduce the risk of an adverse reaction, whereas others leave the allergen in place for 20 minutes.” The method of recording the dimensions of flare, wheal, or both also varies among physicians and investigators. The size of the reaction may be recorded as a mean wheal diameter (D + d* /2), planimetry, minimal diameter of a significant wheal(>2, 3, 4, or 5 mm), comparison with a histamine equivalent prick (HEP) caused by 1 or 10 mg/ml of histamine dihydrochloride (1 HEP), European HEP scores (3 + = wheal the same size as the histamine reference, 2 + = a wheal50% of the histamine reference, and <2 -t- = a negative response), or a score related to a codeine phosphate control (a wheal of 75% or more of that elicited by a control codeine phosphate solution [25 mg/ml of the salt]).‘, “, I3 Both erythema and wheal should be measured and compared with positive (i.e., histamine or codeine) and negative controls (i.e., buffered diluent or 50% glycerol in the case of glycerinated extracts). 1.1223. Biologic equivalency tests A 1 mg/ml (5.43 mmol/L) concentration of histamine dihydrochloride (i.e., salt concentration) was established as the standard reference for skin prick testing by the Northern Society for Allergology and has since been adopted by many European investigators.’ At this concentration of histamine dihydrochloride, a positive prick test giving rise to wheal diameters ranging between 6 and 8 mm in most patients was arbitrarily defined as a unit of 1 HEP unit or reaction. According to European investigators, the inclusion of a histamine reference enables an interpolative adjustment of wheal reactions comparable with the histamine reference when the mean wheal diameter falls within the 6 to 8 mm range. This approach permits individual technicians to self-correct technical errors such as the use of too heavy or too light pricks. Prick tests with allergen extracts could then be related to the HEP by means of a threshold index (index = diameter of wheal produced by allergen divided by diameter of wheal produced by histamine). Alternatively, the relative potency of an allergen extract could be compared directly with the histamine standard solution, which is assigned a potency of 1 equivalent to 1 HEP. Results interpreted by this system vary according to differing sensitivities of individual subjects to histamine and the test allergen and assume that technical factors are constant within patients at all test sites. In addition, the different time courses or responses to histamine after 10 minutes and the allergen after 15 to 20 minutes complicate the practicality and direct comparability of this assay system. Use of the reference HEP system does not account for individual differences in histamine-releasing activity in different skin test sites, i.e., proximal vs distal areas of the arm, or in skins of different persons. Histamine-releasing substances, such as codeine sulfate, could be used as alternative reference systems, but these modifications have not been generally adopted. Moreover, other studies indicate that the reference concentration of the histamine standard might be more accurate and reproducible at a level of 10 mg / ml. I4 For these reasons, the HEP system is not yet considered an acceptable biologic method of standardizing allergen extracts in the United States. Histamine dihydrochloride is not available in the United States as an approved skin test reagent. Histamine diphosphate (0.1, 1.O, and 1.8 mg /ml of the base) is available as an approved skin test solution. There are substantial puncture skin test data in U.S. indigenous populations with the use of a 1 .O mg/ml histamine base reagent and a 25-gauge prick needle. Ninety percent of the test population responded with wheal and erythema diameters that ranged from 2 to 7 mm and from 4.5 to 32.5 mm, respectively.15 a. HEP in vivo assays One approach to assignment of biologic unitage is based on application of the 1 mg/ml I-IEP system.16 By selecting at least 20 representative allergic patients for each allergen on the basis of case history, skin prick tests, radioallergosorbent test (RAST), and relevant provocation tests, the concentration of extract giving the same wheal reaction as 1 mg/ml of histamine dihydrochloride is determined. This concentration is termed “1 HE,,” and new batches of extracts are standardized to *D

= largest

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of the wheal

and d = largest

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this concentration by comparative skin testing on at least 10 known allergic patients. When &I~~x~w skin tests were done with several dilutions of allergen extract in a much larger number of patients, the reactions did not differ significantly from the model, and the observed differences in concentratron of allergen extract were proportionate to the absolute differences in the mean wheal diameters. These studies confirmed the potential use of the HEP system of standardization. Other investigators liavc assessed the potency of 43 commercial allergen preparations in HEP units and observed an improvement in the prediction of biologic activities over those expressed in traditional units (protein nitrogen units [PNU] per milliliter or weight/volume).‘* However, the accuracy of HEP analysis is markedly affected by the number of subjects used for the assay and the use of suitable allergen standard reference preparations. Thus the 95% confidence interval ranged from one fifth to five times the true HEP value when the number of subjects in the assay was 30 to 50 persons. The high ranges within the 95% confidence limit can be narrowed by either increasing the number of subjects or decre;ling intersubject variability through careful patient selection. Accordingly, 20 carefully selected sensitive test subjects are considered the minimum required for statistically significant HEP assays. b. Allergen activity units Biologic activity may also be expressed in activity units for allergen extracts without reference to HEP.” One proposed biologic activity unit is based on the assumption that an extract previously shown to be free of irritants or nonspecific toxicity contains 10 activity units/ml when prick tests with this extract in 30 clinically sensitive persons elicit a geometric mean wheal area of 7.5 mm‘ in this population. With this system, analysis of several allergen extracts revealed a straight-line relationship between mean wheal area (millimeters squared) and extract dilutions when plotted on loglog paper. In practice, the slope of this line is flat, and relatively large increases in dose are required to effect a significant change in wheal size. Notwithstanding this limitation, the results showed that geometric wheal sizes between 47 and 120 mm’ were suitable ranges for assessing biologic activity of extracts used for skin prick tests. Another recent investigation optimized allergen assay for several standardized extracts by tirst transforming logarithmically both wheal diameter and allergen dose responses and then determining the linear portion of the dose-response curve by calculation.18 This assay was stable with regard to change in the composition of panels of atopic subjects and the time intervals between challenge and response measurement. The coefficient of variation of the assay was 18%. Estimates of potency units obtained by skin prick tests for several international reference extract preparations demonstrated significant correlation with international units of allergen potency. 1.1224. Comparison of tests performed by different instruments Many comparative trials have been reported. 7. lo. ‘X ” Routine blood lancets show the greatest variability. Although wheal size areas are significantly larger with the 27-gauge prick test needle than the Morrow-Brown needle, the variance of the former method is also greater. However, the coefficients of variation of both techniques are comparable and equally reproducible. Recent experience with rhe plastic Morrow-Brown needle suggests that it may not detect a significant number of positive reactions, In general, the multitest techniques do not compare well with either the 27-gauge needle prick method or the Morrow-Brown needle. Although there is no overall consensus, most recent investigations that compared mean wheal size data concluded that the commercial device known as the Allergy Pricker (DomeiHollister Stier) may be preferable because it is less dependent on the experience of’ the investigator. The Wyeth bifurcated smallpox scarifier has only been compared with other metht?ds in a single study.“’ 1.123. Reproducibility Reproducibility of tests performed with the same device is related to many factors including sex, time of year, circadian rhythms, light or heavy prick technique, and biologic stability of test 1xtracts.‘, I3 Reproducibility with the same device is better over time when standardized or partially purified extracts are used.14. I* 1.124. Sensitivity and specificity It is generally accepted that prick tests are less sensitive than intracutaneous tests. This is partially because larger amounts of test solutions are administered by the intracutaneous method. To compensate for this, positive prick tests require that test extracts be at least 10,000 times more concentrated than intracutaneous test solutions. This lack of sensitivity to prick tests can be partially compensated by

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avoidance of glycerinated extracts or by adding small amounts of Tween 80 (0.0005%).” The increased sensitivity at a fixed concentration of the intracutaneous test (i.e., 1 to 1000 W/V) may be responsible for a small but reproducible number of false-positive reactions. Prick tests do not appear to elicit false-positive reactions by any of the currently available methods. However, the optimal criteria for positive prick and intradermal tests are not known. One opinion is that when percutaneous test results are negative in patients with inhalant allergy, positive intracutaneous tests are not likely to indicate the presence of clinical allergy. On the other hand, in two well-established IgE-mediated anaphylactic syndromes (penicillin and venom hypersensitivity), the diagnostic predictability of intracutaneous testing is superior to that of prick tests. It is possible that a positive intracutaneous test and a negative prick test could denote clinical allergy in a less sensitive patient or the presence of an IgG reagin that cannot be detected by prick tests. When using specific purified allergens, a clear distinction between primary irritant reactions and true sensitization can be more easily established by prick than by intradermal tests.” Prick tests are generally considered more specific than the usual test strength of intracutaneous tests (1 to 1000 W/V) when both are compared with inhalation or ingestion challenges. Depending on the test allergen and criteria for a positive test, the correlation between prick tests and inhalation challenge may vary anywhere from 60% to 90%) whereas lower correlation indexes (30% to 40%) are generally observed between intracutaneous tests and inhalation challenge tests. To an extent, the sensitivity and specificity of prick and intradermal skin tests will vary according to the allergen under study, the degree of its biologic standardization, its dose, and criteria for positivity. For example, in one recent study, the sensitivity of a prick test compared with nasal provocation tests for birch allergen was 97%, whereas the corresponding specificity was 94%.19 In contrast, the sensitivity of prick tests in the timothy system was still excellent at 97%, but the specificity in this system had fallen to 70%. The specificity of prick tests in the diagnosis of food allergy is subject to other constraints. Although positive skin prick tests to cereal grains are common, they often occur in the absence of clinical symptoms. Furthermore, one must be aware of nonspecific cross-reactions among foods in the same family group. 1.125. Significance Prick tests are generally considered the most convenient and least expensive screening method for detecting allergic reactions in patients who have appropriate exposure histories. Although the degree of correlation of these tests with in vivo challenge tests and specific IgE in vitro tests varies among populations and allergens, prick tests are used most frequently to evaluate individual cases as well as populations of allergic patients. The important role of prick tests in the detection of clinical allergy necessitates standardization of their performance, measurement, and interpretation. However, until the diagnostic efficacy of prick tests is fully established with standardized allergens and methods, negative prick test results should be confirmed by more sensitive intracutaneous techniques. 1.2. Intracutaneous tests 1.2 1. Present applications Intracutaneous tests are generally used when increased test reliability, sensitivity, and reproducibility are the main goals of testing. ** They permit identification of a larger population of reactive patients, especially those with lower clinical sensitivity, i.e., patients with a negative puncture test result. In addition, low-potency allergenic extracts may best be evaluated by this method. 1.22. Current methods 1.22 1. Instruments A single unitized 0.5 ml hubless syringe with an attached hypodermic needle is preferred. The gauge of the attached hypodermic needle may vary from 26 to 30.23 1.222. Technique 1.222 1. Performance factors There may be a leakage of allergen at the injection site because of improper technique. Leakage between the syringe and needle is prevented by the use of unitized syringes and needles. Other performance factors listed under prick tests apply equally well to intracutaneous tests. 1.2222. Interpretation of skin results Various indexes such as sum of diameters, longest diameter, products of diameters, and the weighing of skin response outlines as traced on paper have been used to interpret intracutaneous results.Xp z

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Both erythema and wheal diameters should be measured. Erythema can be measured as reliably as wheal reaction. 1.223. Comparison with negative and positive controls Concurrent tests with saline solution or phosphate-buffered diluents should always be performed. In addition, a positive histamine control (0.01 mg/ml of the base) should be included to assess skin reactivity. An optional positive control of codeine phosphate (1 mg/ ml of’ the salt) may also be t.wd for this purpose. 1.224. Reproducibility The reproducibility of intracutaneous tests is affected by the same variables as those described for prick tests .24*26These include the age of the skin, the area of the body where the tests were applied, circadian rhythms, skin pigmentation, interference by concurrent medication (e.g., antihistamines), potency of the allergen test extract, and biologic stability of the test extract. 1.225. Sensitivity and specificity Since intracutaneous tests are more sensitive than prick tests, special care must be given to the preparation of test dilutions. This is further complicated by the fact that concentrated unitage may vary from allergen to allergen. As a general rule, the starting dose of intracutaneous extract solutions in patients with a negative prick test should range between IOO- and lOOO-fold dilutions of the concentrated extract solution. Thus in the case of potent allergens (100,000 allergy units [AU*]) the range of starting intracutaneous test solution, in patients with a negative puncture test is between 100 and 1000 AU. For less potent allergens of 10,000 AU concentration, the range of the proper starting intracutaneous test solutions for patients with a negative puncture test is between 10 and 100 AU. At this allergen potency, tests may be performed with solutions of 1000 AU, provided the concentrate is not preserved with 50% glycerol, or if it is, by using appropriate glycerol controls, for example, 5% glycerol in 1000 AU prepared from a 10,000 AU extract in 50% glycerol. In all cases the maximum recommended test concentration for intracutaneous tests is 1000 AU. The volumes of intracutaneous test solutions may vary from 0.01 to 0.05 ml, depending on the purpose of the test. Delivery of small volumes (~0.03 ml) is subject to wider variability. Systemic anaphylactic reactions are more likely to occur with intracutaneous tests.” In most cases such reactions can be prevented by prescreening with epicutaneous or percutaneous tests. Concurrent medications such as P-blocking agents and monoaminooxidase inhibitors, which may increase the risk of allergic adverse reactions, should be considered in assessing the risk/benefit ratio of skin testing. P-Blockers may enhance the severity of anaphylaxis and interfere with the therapeutic action of epinephrine. The therapeutic use of epinephrine is contraindicated in patients receiving monoaminooxidase inhibitors. 1.226. Significance Because intracutaneous tests are more sensitive than prick tests, false-positive reactions are more likely to occur. Therefore these tests must be interpreted in the context of the clinical history. However, intracutaneous tests may be more advantageous than prick tests in detecting patients with lower levels of clinical sensitivity and evaluating allergenic extracts of low potency. Equivalent degrees of reactivity between positive prick and positive intracutaneous skin responses may be established by precise titration of intracutaneous doses. 2. Standardization techniques 2.1. Introduction Standardization of skin testing is essential to determine patient sensitivity, as well as to standardize allergenic extracts.** In both applications the skin test procedure must be well defined and statistically valid to provide the physician with a test result that is accurate, precise, and meaningful. Accuracy in this context refers to the ability of the test to discriminate between patients with differences in skin sensitivity or between extracts of different potency. Generally, differences in patient sensitivity are not known a priori, whereas extract potency is known. As a first approximation, it can be reasonably assumed that skin test methods that accurately and precisely discriminate among extracts with known differences in potency are likely to discriminate between patients whose sensitivity differs. Because biologic assays are functional assays based on dose-response data, relative potency and compositional differences between extracts can be deduced from analyses of the induced sigmoid *See 2.11”

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dose-response curves.**,29Sinceextractdosesthat produceidenticalresponses can be considered bioequivalent,biologic assayscan be usedto establishbioequivalentdosesof extractsthat arc independent of the allergen.This strategycanalsobe usedto assignbioequivalentunits to extracts from diversesources.Similarly, differencesin patientsensitivitycanalso be accuratelyestimated from observed sigmoid dose-response curves. Thus within the limits of the variability of the skin test, patients in whom identical exracts produce identical responses at identical doses can be considered similar with respect to sensitivity to that extract. 2.11. Present applications Because of the lack of standardized methods for determining relative potency, compositional differences, and bioequivalent doses of allergenic extracts, as well as methods for establishing patient sensitivity,the Food and Drug Administration (FDA) Laboratory of Allergenic Products has developed defined methods and procedures for calculating these parameters. 3oThese methods have been described and are used by manufacturers, academic investigators, and physicians in clinical research studies. Each method has defined accuracy and precision with statistical limits to determine whether the relative potency and composition of a test and reference extract are identical or whether patients or patient populations differ in skin sensitivity to extracts from the same or diverse sources. In addition. a standardized proficiency test method has also been developed so that any skin test practitioner can determine whether the accuracy and precision of skin testing skills are within acceptable ranges. These methods are described in detail along with their statistical variability in The A4unuul of Methods of the Laboratory of Allergenic Products. The following methods for skin test bioassays have been distributed and are in use: 1. Determination of relative potency and composition of extracts a. Screening method b. Confirmatory method 2. Determination of stability of potency of allergenic products 3. Determination of patient sensitivity to allergenic extracts 4. Determination and assignment of bioequivalent units to references from diverse sources by a method that defines an allergy unit as the intradermal dilution producing a 50 mm sum of erythema diameters(IDjo EAL). This method is based on determining which mean threefold dilution of an allergy extract elicits a sum of erythema diameter of 50 mm (DSo) in a patient known to be puncture test positive (by virtue of a sum of erythema reading of 340 mm). Potent extracts usually have a D.50 of 14 (range, 13 to 15), which is arbitrarily assigned 100,000 AU. Less potent extracts based on D,, are assigned a lower AU; for example, D,, 3 11 but <13 is deesignafed as 10,000 AU/ml, and DSo2 9 but
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Laboratory of Allergenic Products selects patients known to be highly skin reactive to the allergen of interest. These highly sensitive patients (prick test sum of erythema ~-40 mm) are selected because they are at higher risk of adverse allergic reactions during immunotherapy, as well as more likely to have severe symptoms related to the allergen of interest than patients with lower skin sensitivity. 2.2 Standardization prerequisites Each method defines: 2.21. Reagents and equipment required for performance of the test: a. Bifurcated puncture needle (Allergy Laboratories of Ohio, Columbus, Ohio) b. A 0.5 ml unitized syringe with a built-in 27-gauge needle c. Transparent tape to make a permanent record of skin reaction outline d. Ruler to measure orthogonal diameters of erythema and wheal 2.22. A specified volume of injection (0.05 ml) and method for preparation of an accurate dilution series 2.23. The method of quantitation of wheal and erythema sum of longest and mid-point orthogonal diameters 2.24. Spacing (-6.0 cm) of injections to prevent overlapping of response 2.25. Clinical and statistical criteria for acceptable dose-response lines 2.26. The method of statistical analyses of the data 2.27. Statistical limits for the test result so that the investigator can interpret whether the test results fall within or outside the limits 2.28. Quality control for submission of proficiency test data to establish that the investigator can perform the method with acceptable accuracy and precision; for example, each method specifies sizes of erythema at specified histamine doses to permit other investigators to determine whether they have carried out the procedure reproducibly 2.29. The use of a positive control, for example, histamine base, 1.O or 1.8 mg/ml; the largest data base pertinent to skin reactivity to histamine in the U.S. population is based on 1 .O mg histamine base administered by the Pepys technique by means of 25-gauge test needles.15 3. Recommendations 3.1. There are proper roles for both prick and intracutaneous tests in clinical allergy. Children as young as 1 month of age may be tested if clinical indications are present. 3.2. Prick instrumentation includes 25- to 27-gauge hypodermic needles, various types of lancets, multitest devices, and a bifurcated prick instrument. The preferred intracutaneous instrument is a 0.5 ml unitized syringe with an attached 26- to 30-gauge needle. Appropriate proficiency test methods for evaluating accuracy and precision of skin testing are encouraged for both prick and intracutaneous methods. The hazards of blood contamination with the use of all instruments must be given appropriate attention. All technicians must be carefully trained in appropriate preventive techniques. 3.3. The following medications are to be avoided before any type of skin testing: a. Antihistamines, both long and short acting; a long-acting antihistamine, astemizole, may interfere for as long as 6 weeks b. Tricyclic antidepressants such as doxepin c . Chlorpromazine for at least 5 days before testing d. Hydroxyzine 1 e. Prior skin application of anesthetic-embedded patch tests because of interference with the antidromic erythema response f. Long-term use of high potency topical steroids to the skin test sites 3.4. Skin test sites on the arms or upper back are suitable for tests. Acceptable areas

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includetheventralsurfacesof both lower andupperarmsanddorsiiateralcites of upperarms.Skin adjacentto wristsandantecubitalupperfossaeshouldbe avoided.Areas of active skin inflammation should not be used. 3.5. In the case of intracutaneous skin tests, single disposable syringes are to Ix used for each test. Use of one syringe for multiple skin tests of different patients is contraindicated because of the danger of acquired immune deficiency syndrome and hepatitis B viral contamination. Individual lancets should be used for each patient. However, prick instruments may be wiped clean with benzyl isopropyl alcohol between different allergen tests in the same person. This technique is only satisfactory if a solid pricking instrument is used. However. information about the validity of this technique is anecdotal and requires confirmation by further controlled studies. Interallergen wiping is contraindicated if hollow needles are used. 3.6. There should be sufficient space between both prick and intracutaneous tests to prevent overlapping of erythema and wheals. 3.7. The appropriate dilution of FDA-approved allergenic test extracts for prick tests is the most concentrated available one. In the case of intracutaneous tests. there is a range of starting dilutions dependent on whether the patient is puncture test negative or positive. For puncture test-negative patients tested with allergens containing 100,000 AU/ml, the range of starting intracutaneous test solution doses is 100 to 1000 AU/ml. In the case of allergen extracts of 10,000 AI1 /ml potency, the range of starting intracutaneous tests should be 10 to 100 AU/ ml. Fine tuning of these dose ranges requires further study. For puncture test-positive patients, the starting doses should range between lo-“- and 10e8-fold dilutions of the concentrate, for example, 0.01 to 1 AU/ml. The maximum recommended dose for any intracutaneous test is 1000 AU/ml. In patients with a negative puncture test result tested with unstandardized extracts, extract. In patients with a positive puncture test, starting dilutions of unstandardized extracts should be 10e5- to IO-‘-fold dilutions of the concentrate. Special care should be given to preparation of intracutaneous test solutions. Separate syringes and needles should be used for each serial dilution, and there should be no reaspiration of solution once the aliquot is delivered. 3.8. The volume of prick tests is indeterminate. The volume of intracutaneous tests may vary from 0.01 to 0.05 ml, depending on the purpose of the test and the accuracy of the syringe. Delivered volumes should always be specified in recording test results. 3.9. All extracts should be stored under cold conditions to ensure stability of test extracts. In addition, all concentrated extracts should be glycerinated. Glycerinated solutions > 1% for intracutaneous tests may induce nonspecific skin reactions. Therefore appropriate glycerinated control solutions should be performed with each set of allergy tests. Wherever possible, biologically standardized extracts should be used. 3.10. Reproducibility of prick and intracutaneous tests depends on sex, age, time of year, circadian rhythms, light or heavy pricking techniques in the case of prick tests, and color of the skin. 3.11. Histamine should be used as a positive control. The optimal dose for this control is 10 mg/ml histamine dihydrochloride, which is equivalent to 6.14 mg histamine base, in the case of prick tests. If this histamine preparation is not available, doses of histamine phosphate containing either 1.0 or 1.8 mg/ml expressed as the base are adequate. Histamine controls for intracutaneous tests should also be performed at doses of 0.01 mg/ml base concentration.‘” 3.12. Both prick and intracutaneous tests should be read 15 to 20 minutes after

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FIG. 1. Dose-response effects of antigen-induced LPCR. Area of skin intradermal injection of three doses (0.1, 1.0, and 10 PNU) of pollen resents mean ( f SE) of seven subjects injected with either ragweed or with permission from Atkins PC, Martin GL, Yost R, Zweiman 9. Ann

3.13.

3.14.

3.15.

3.16.

induration observed after antigens. Each point repgrass pollen. (Reproduced Allergy 1988;60:27-30.)

application. Histamine control tests should be read 10 minutes after application. Many methods of measurement have been proposed. One technique is to measure both wheal and erythema in millimeters with a clear plastic ruler. If preservation of skin test area data is desired, measurements may be transferred to transparent tape and stored in the patient’s record. The sum of the largest diameter and its orthogonal diameter are acceptable measurement data for most clinical purposes. Comparison of specificity and sensitivity between prick and intracutaneous tests Although prick tests are generally considered more specific than intracutaneous tests, such assessments can be made only by proper attention to the biologic quality of specific allergen with well-characterized extracts and well-defined criteria for a positive test. For example, specificity of a prick test may in fact be equivalent to an intracutaneous test if the latter test is performed at a weaker concentration. It is difficult to draw conclusions about comparative data when these tests are compared at single doses. Therefore it is recommended that the entire issue of specificity of prick and intracutaneous tests requires further evaluation. Regarding analytic sensitivity, intracutaneous tests are more sensitive than prick tests. With respect to both specificity and sensitivity within a population, more research is needed to determine the predictive abilities of each test. Moreover, the dose(s) and criteria for positive tests used in such studies should be clearly defined. Wherever possible, it is preferable to conduct such studies in volunteers with proved disease entities to define the standard of comparison. If such populations are unavailable, controlled organ challenge tests may be used as alternative standards. Safety In general, intracutaneous tests should only be performed if the prick (or epicutaneous) test result is negative. If scratch or prick tests are not performed routinely, some form of threshold response of intracutaneous tests beginning at very high dilutions (i.e., lo-’ to lo-‘) is suggested. The magnitude of loca! tissue damage induced by percutaneous and intracutaneous tests may be a concern, especially if the number of positive reactions is excessive. At relatively high concentrations of intracutaneous tests, residual (late response) inflammatory effects are likely to occur. Rescreening with scratch or prick tests is a practical way to avert an untoward number of adverse local responses in the routine skin testing of patients. The risk/benefit ratio should be weighed carefully before tests are attempted in patients receiving B-blockers and monoaminooxidase inhibitors. Data regarding adverse drug interactions require more widespread dissemination to clinical practitioners. Skin tests are essential for the biologic standardization of allergenic extracts.

Task Force G&k$ines

VOLUME 82 NUMBER 3, PART 2

TABLE --

I. Time

course

of antigen-induced

LPR

-1.-

Mean diameter of edema (mm) Clinical study Solley *Mean

Hr

et al.”

of four subjects

injected

497

..._ ..._-.-._- ---_

---.“-

--_--1___

0

2

4

6

8

10

12

14

18

18-24

6

22

41

52

5s

59

60

63

fir i

S-56

with grass (100 PNU/ml),

ragweed,

altemaria

(SO0 PNU/

ML),

and gurnea

pig dandrr

Although some investigators have attempted to accomplish this with prick tests compared with known histamine equivalence data, further research is required to determine if this procedure is valid. Most of these efforts have been expressed as prick wheal area units, but further investigations of prick erythema area units should also be explored. Current intracutaneous biologic methods of standardizing allergy extracts have proved to be statistically valid. A modified technique with four threefold serial dilutions of allergy extract has simplified this method. However, computerized software mathematic models required for the statistical analysis of these assays, that is, parallel line assays or biovariate analysis of responsiveness and sensitivity, should be developed and made available as soon as possible.‘“, 29All data obtained by prick and intracutaneous tests should be recorded on permanent patient record sheets. B. Late phase cutaneous reactions 1. Introduction Although the late phase cutaneous response (LPCR) was first described in 1973, there has been no systematic effort to standardize the description or measurement of this reaction.” It was the objective of this task force to attempt to establish a definition, standardization of measurement, a general description of current pathogenic mechanisms, relationship of LPCR to late phase responses in other target organs, and clinical relevance of the LPCR. 2. Definition and description LPCRs are characterized by erythema, induration/edema, and dyesthesia that develop progressively at sites of immediate wheal and flare reactions.32”8 They become apparent between 1 and 2 hours, peak between 6 and 12 hours, and usually disappear after 24 to 48 hours. Histopathogically, they are characterized by the presence of edema, mixed cellular infiltrates, and sometimes fibrin deposition scattered throughout the dermis without the deposition of complement, IgG, IgA, or IgM or vascular damage. 3. Inciting stimuli LPCRs occur after both immune and nonimmune mast cell activation. Agents inciting immunologic activation of the mast cell that have induced LPCR include anti-IgE antibodies and the following antigens: aeroallergens (molds, pollens, danders, mites, and enzymes), insulin, and possibly some foods .39.4a The secretagogue, compound 48/80, has induced prominent LPCR but codeine has not despite the fact that it elicits a prominent immediate wheal and flare response.“‘, 32.4’.43 The propensity to develop LPCR may be dependent on the type of antigen, host sensitivity, and the concentration of injected antigen.43 4. Measurement After challenge with diverse stimuli, the intensity of the LPCR increases rapidly (doubling or tripling in size) during the first 2 hours, as noted in Fig. 1 and Tables I to III.3’-3s. .” The response plateaus between the sixth and twelfth hours, is present at 24 hours, and disappears by 48 hours after challenge. Accordingly, these reactions should be quantified between the sixth and twelfth hour (most commonly at the sixth or eighth hour) by measurements of mean diameter or area of induration/edema. Although the minimum size of the induration necessary to qualify a delayed-in-time cutaneous response as a “significant” LPCR has not been defined, no observable indurated response can be measured at 6 to 8 hours after administration of diluent or histamine.“2. 13.4’ Histologic studies of LPCR may be helpful in distinguishing these reactions from either Arthus or delayed-type hypersensitivity reactions.

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Task Force Guidelines

II. Time

course

of anti-IgE-induced

LPR Mean

Clinical

study

Hr

0.33

50 -

Dorsch et al.* Gronneberg et al.?

diameter

of edema

(mm)

1

2

4

6

8

10

24

12

35 20

32 24

45 33

48 -

36

25

-, Not done. *Eight subjects, 200 IU of rabbit antihuman IgE. (J ALLERGY CLIN IMMUNOL 1981;62:117.) ?Twenty-eight subjects, 1: 333 W/V dilution of rabbit antihuman IgE antibody.

TABLE

III.

Time

Clinical study

deShazo et al.“‘* Dor et al.? Solley et al.‘*f

course

of 48:80-induced Hr

0.26-0.33

18 18 10

LPR 0.5

19 -

1

2

3

4

5

6

8

10

12

24

24 25 27

35

25 24 38

34 38

31 36

20 19 33

26 35

17 45

-

-

-, Not done. *Three nonatopic subjects, injection, 50 kg, inner diameter. t Forty subjects, nonatopic, 50 pg, inner diameter. *Five subjects, atopic and nonatopic, 5 mg/ml, inner diameter.

5. Pathogenetic mechanisms Mast cell activation plays an important role in the pathophysiology of LPCR, as indicated by at least two lines of evidence. First, immunologic challenge with anti-IgE and antigens and nonimmune challenge with 48/80 will induce LPCR. Second, isolated mast cell granules or granule factors will elicit a LPR-like response in rodents.44 The observations that LPCRs contain numbers of diverse inflammatory cells and that intradermal injection of mediators such as platelet-activating factor, kallikrein, and leukotriene B, can induce LPR-like reactions indicate that factors other than mast cell products may also contribute to the observed changes.39,45-47 In addition, pharmacologic studies have demonstrated that the pathophysiology of the LPCR is different from the immediate cutaneous response. The LPCR is inhibited by systemic glucocorticosteroids in conventional doses (1 to 2 mg/ kg/day or less) but not by H, antihistamines, whereas the immediate response is not inhibited by systemic steroids in conventional doses but is inhibited by H, antihistamines.4s, 49 Although histamine, which is responsible for the immediate response, does not induce the LPCR, a single study has demonstrated that a combination of H, and H2 antihistamines can inhibit the LPCR.32. 4’, 5o 6. Relationship of LPCR to late phase responses in other organ systems The relationships among cutaneous, nasal, and pulmonary late phase response (LPR) have been evaluated by a number of investigators, that is, does the development of LPCR after cutaneous challenge portend a similar response to the same antigen in the nose and lung? Although some investigators have found a significant correlation, others have not.33. 5’-54Factors that may be important in these observed differences include subject selection, methods of evaluating LPR, types of antigens used, and possible differences in pathogenetic mechanisms in various target organs. 7. Clinical relevance The clinical relevance of LPCR is uncertain because its description has relied on experimental settings in which large doses of antigens were used for the skin testing of very sensitive subjects. However, the magnitude and chronicity of these reactions induced after diverse methods of mast cell activation or injection of a variety of mediators of infiammation suggest that LPCR may be a useful model in the investigation of the pathophysiologic mechanisms of disorders such as chronic u&aria and atopic dermatitis in which the precise pathophysiologic mechanisms are as yet unknown. There may be additional clinical relevance if LPCR mirrors LPR in the lower airways. The relationship between LPR after inhalation challenge and airways hyperreactivity suggests the possibility that a

9

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Task Force Grideiines

reduction of LPR may be of clinical benefit. Along these same lines, the capacity of glucocofticosteroids to reduce LPR in asthma may be an important action of these drugs. Thus it seems likely that LPR may be an important therapeutic target. 8. Recommendations 8.1. Further research is required to define the conditions of the test. These studies should be performed with standardized extracts. Dose-response assessment of individual extracts should be conducted in well-defined clinical populations (e.g., highly sensitive, moderately sensitive, and slightly sensitive subsets!. Multicenter studies are encouraged to enable the assessment of geographic variability, circadian rhythms, race, and other variables known to affect the interpretation of immediate type skin responses. 8.2. Further studies are required to evaluate optimal methods of measuring LPCR. These investigations should be done concomitantly with the dose-response techniques in a larger number of clinically sensitive or nonsensitive subjects. The development of objective techniques of measuring skin inflammation (e.g.. the laser-Doppler technique) should be encouraged. 8.3. Research activities concerning the clinical relevance of LPCR to LPR of lungs and nose should receive high priority. If these studies are done systemically in larger population groups, the results should be rewarding inasmuch as cells, mediators, and lymphokines can be adequately sampled either by bronchoalveolar lavage in the case of lungs or by nasal washes in the case of upper airways hypersensitivity. C. In vitro diagnostic tests 1. In vitro measurements of IgE antibody 1.1. Historical background Until 1966 when Ishizaka et al.55 identified reaginic activity with a novel isotypic immun~lobuiin class, IgE could only be quantitated by bioassays. The discovery of an IgE myeloma by Bennich” made it possible to raise antisera against IgE and thereby permitted immunoassay of total IgE protein and specific IgE in serum. Since then, solid phase immunoradiometric assays, first designated the RAST by Wide et al.“’ who reported the first prototype in 1967, have been widely apphed to the detection of allergen-specific IgE in serum. These tests are used as a replacement for, or an adjunct to, the traditional skin bioassay for IgE, in which allergenic extract is introduced through the skin to IgE-laden mast cells, resulting in their degranulation. As serologic methods for IgE detection become more widely used in allergy diagnosis, it becomes important to examine critically methodologic pitfalls and quality control. 1.2. Current status of in vitro testing methodology 1.2 I. Technologic approaches The most widely used version of the RAST uses allergen insolubilized on a cellulose paper disk (allergosorbent), which binds specific IgE (and other antibody classes) from serum during a first incubation. Maximum sensitivity is achieved by incubating 0.1 ml of undfuted serum with each disk overnight to allow antibodies of all classes (primarily IgE and IgG) to bind. The immu~~iobulin solid phase is then washed and iodine 125 isotope-labeled anti-IgE (Fc,) or enzyme-labeled anti-IgF (Fc,) is incubated with the washed disks in a second overnight incubation. After further washing. the radioactivity bound to the disk is counted, or in the case of enzyme-labeled antibodies, a substrate is incubated to produce a colored or fluorescent product. The radioactivity bound to the disk, or the quantity of product generated by enzyme activity is related to disk-bound IgE by a reference serum curve, by means of which unknown (test) specimens are commonly scored. During the past several years modifications to the original RAST technique have been introduced. As shown in Table IV, these involve differences in the solid phase used to fix the allergens, the type of tracer attached to the IgE detection protein, and the instruments required to read out the final signal.‘8 Difficulties in obtaining comparable results from various immunoassays for IgE antibody can be expl&md in part by differences among the allergen extracts used in each assay, the variable SUC:ceSS with which each technique insolubilizes all of the relevant allergenic molecules in a mixture, the recommended incubation times, and the threshold criteria recommended by each manufacturer. The

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Task Force Guidelines

TABLE IV. Commercially

available

RAST and alternative

solid-phase

methodologies

for in vitro

IgE quantitation Allergosorbent

Detection (label

system type)

Paper disk CNBr Diazo

Iodine 125 (v-rays) Enzyme (visible product)

Plastic surface Plastic surface

Enzyme (visible product) ELISA Enzyme (fluorescent product) FIA

Cellulose threads

Iodine 125 (y-rays),

luminescence

System name

RAST EAST VAST IP system FAST MAST

Purveyors

Pharmacia Kallestad Ventrex DRT Inc. 3M Diagnostics (formerly Allergenetics) MAST Immuno-Systems

EAST, Enzyme allergosorbent test; VAST, ventrex allergosorbent test; IP-system, immunoperoxidase system; FAST, fluorescent allergosorbent test; MAST, multiple-thread allergosorbent test; ELISA, enzyme-linked immunosorbent test. Reprinted with permission from Hamilton RG, Adkinson NF Jr. Clin Immunol Newsletter 1986;7:10-4. Copyright 1986 by Elsevier Science Publishing Co., Inc. relative performance of these newer systems is still largely unevaluated, especially for less common allergens. 1.22. Schemes for reporting results In 1983, 33 laboratories participated in a blinded evaluation of strongly positive test sera and a verifiably negative serum pool in a proficiency test program conducted by the Centers for Disease Control.59 The best concordance was obtained when each laboratory was asked to specify whether it considered the sample result positive or negative using its own criteria. The laboratories correctly identified strongly positive sera in 674 of 691 (97.5%) individual tests. Samples were correctly identified as negative in 301 of 334 (90.1%) samples. However, the use of class grading systems for reporting results resulted in wide variations among laboratories. About half of the laboratories used the modified RAST scoring method, which normalizes all test results to a single positive standard obtained by incubating serum containing 25 IU/ml of total IgE with insolubilized anti-IgE (Fc,). The other half of the laboratories used the original Phadebas class system in which RAST class is assigned on the basis of the relationship of the test serum to four or five reference serum dilutions. In the survey positive samples commonly differed by two or three class ranks. Negative samples generally spanned two classes; however, for wheat and milk there was a spread of three classes (0 to 2). The overall high rate of qualitative concordance in these laboratories (95.1%) is because many laboratories that use the modified RAST reporting scheme do not consider a class 1 result positive. The variability among reported results strongly suggests that not only are more standardized threshold criteria and reporting methods needed, but also that interlaboratory results should be compared among patients with proved clinical disease entities. Currently, in vitro IgE tests from different commercial sources are virtually impossible to compare, a state of affairs almost unprecedented in modem laboratory medicine. 1.23. Threshold criteria For most commercial IgE antibody assays, a single negative reference point is used to establish threshold criteria for a wide variety of allergen test systems. This was done for economy, since a large battery of tests is commonly performed. The difficulty lies in the fact that each allergosorbent differs somewhat in its nonspecific binding properties and the degree to which it is influenced by elevated total serum IgE.@’The manufacturer or test laboratory may choose a threshold level sufficiently high to preclude false-positive results, with problem allergosorbents demonstrating high nonspecific binding. This was the approach taken by Pharmacia in its original Phadebas scoring scheme. This conservative approach with regard to false positives led to an unacceptable level of false-negative results. This prompted Pharmacia to introduce a lower threshold value (reference point E), which defines a new positive class (class O/ 1).61 This lower threshold is still two to four times higher than a birch pollen blank, but is much closer to nonspecific binding levels in problemsome food and mold test systems. The threshold for the modified RAST method of 750 normalized counts is less than

VOLUME 82 NUMBER 3. PART 2

Task

Force

Gurdelines

two times the nonspecific binding in clean pollen systems, but may be considerably below nonspecific binding levels with more difficult allergen systems. As theory would predict, this much lower threshold increases the rate of false-positive results, which ranged from 22% to 45% in the 1383 Centers for Disease Control survey. Since the designations “positive” or “negative” often have important clinical imphcations for diagnosis and immunotherapy considerations, a consensus needs to be developed about the most acceptable compromise in defining appropriate threshold criteria for in vitro IgE testing. This tradeoff between sensitivity and specificity may need to be balanced differently depending on clinical context. For example, the diagnosis of anaphylactic sensitivity to drugs or bee stings demands maximum assay sensitivity (low threshold), since the clinical cost of missing a sensitized patient is potentially great. In contrast, for aeroallergen disease or food allergy in which low levels of IgE may not correlate with clinical disease, overdiagnosis is of greater concern so a higher threshold value may be appropriate. 1.24. Quality control The manufacturer of an in vitro IgE test bears the ethical and regulatory burdens for quality control of its commercial product. Regulatory oversight has increased recently, leading to a decline in the number of patently invalid tests offered in either kit form or commercial laboratories.hZ Most commercial laboratories are obliged to participate in quality control exercises and proficiency testing programs. However, these have only recently become available for IgE antibody testing. There is a need for ongoing quality control program in physicians’ offices where in vitro tests are often performed by less experienced personnel sometimes with minimal oversight. Even in the best of environments, high-quality results from any laboratory test require ongoing quality control programs. For m vitro IgE tests, the problem is complicated by the fact that the RAST is not a single test as is commonly spoken of, but a battery of up to 50 or more tests, each of which is subject to malfunction. Quality control data of one or two “representative” allergen disks (or other substrates) are not necessarily applicable to other allergens in the battery of tests. The National Committee for Clinical Laboratory Standards is now drafting recommendations for office-based testing, including quality control procedures. Such consensus documents are clearly overdue. Their recommendations, when available, may be mandated by reimbursement policies of third-party payers. However, the guidelines under development are general in nature and will not address the specific problems of in vitro allergy testing. 1.25. Clinical interpretation of results The clinical interpretation of in vitro IgE antibody tests is subject to the same caveats and pitfalls as the interpretation of traditional allergy skin testing.“3 There is considerable disagreement in the available literature about the actual concordance among various in vivo and in vitro tests for IgE antibody and even less agreement about the relationship between the quantity of IgE antibody and clinical risk of IgE-mediated diseases. Little comparable data are available about the correlation between in vitro tests and quantitative threshold skin tests. However, most authorities agree that in vitro IgE assays are intrinsically less sensitive than bioassays such as skin testing. However, it has not been demonstrated that the lower sensitivity of RAST and RAST analogs is clinically significant, with the possible exception of the special cases of anaphylactic sensitivities to drugs and Hymenoptera venom. In these two instances, the greater sensitivity of skin testing is probably clinically relevant. Otherwise, there is little information about the diagnostic accuracy of individual IgE antibody tests, whether performed in vivo or in vitro, in predicting clinical sensitivity in well-defined allergic diseases. 2. Standardization of in vitro measurements of IgE antibody 2.1. Current methods To use in vitro tests for the diagnosis of allergy, the actual tests must be studied to obtain information on statistical variability. At present, no information is available on the following: a. Characteristics of extracts coupled to the solid support b. The specificity and animal source of the anti-IgE reagent; since all anti-IgE reagents are derived by immunizing animals with myeloma IgE, information should be made available regarding the animal source, tests for anti-idiotypic antibody, whether the reagent is an antibody to Fc, fragment or IgE heavy chain, whether it contains antibody activity against constant region domains,

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.I. ALLERGY CLIN. IMMUNOL. SEPTEMBER 1999

C.1, C,2, or C,3, or if the reagent is a monoclonal anti-IgE antibody.64 In addition, its specificity characteristics should be clearly defined. Differences in results with various test systems could be the result of differences in this reagent. c. Whether the solid supports are maximally sensitized with each type of extract d. Lot-to-lot consistency in the manufacture and testing of supports e. Stability of reagents and coupled solid supports at varying times and temperatures. Such data could be used to provide an Arrhenius plot. This would have to be done with a wide selection of allergy supports. Review of initial stability data would help determine the total number of different allergy supports to be studied. f. Most, if not all, of the commercially available tests use a reference system to compare or interpret the radioactive counts per minute or optical density units resulting from tests with a variety of extracts. The need for a reference system has never been adequately justified. Furthermore, extrapolation of respective units of multiple test extracts in any serum from the dose-response line of the reference system implies that all extracts would yield dose-response lines whose slopes are statistically not different from that of the reference system; almost no data on this are available. g. When a system has been adequately evaluated, it should be entered into a double-blind, placebo-controlled study to determine its ability to predict the presence of allergy and intensity of symptoms in well-defined, clinical allergic entities. This is done best with a symptom-medication score procedure. Clinical evaluation should include quantitative skin testing to determine if there is a correlation between in vitro unitage and size of skin reaction. 3. Recommendations 3.1. Provision of information on source materials No in vitro test for IgE can be better than the source material used to make it. Currently, little, if any, information is available from manufacturers regarding the source and characterization of allergenic extracts used to prepare allergosorbents or the anti-IgE, that is, polyspecific or monoclonal, used in the immunoassay.62 To allow better comparison among available tests, information on source materials should be routinely provided by the manufacturer or diagnostic laboratory, either the package insert, or in readily available documents. 3.2. Movement toward quantitative reporting methods Rather than current arbitrary reference systems, it should be possible at reasonable cost to report IgE antibody test results in units that are proportional to antibody content. This is feasible because the serum dilution curves in most optimized solid-phase immunoassays are parallel, even among allergosorbents. Such a movement toward quantitative reporting would facilitate application of the usual statistical criteria for threshold selection, determination of precision and reproducibility, and ultimately quality control of performance. Second, physicians and their patients are entitled to quantitative results in allergy testing just as they are routinely supplied for almost all other immunoassays in medical use. Finally, quantitative reporting is a necessary prerequisite to efforts (recommended below) aimed at defining more precisely the diagnostic accuracy of in vitro IgE tests and the relationship between various amounts of IgE antibody and symptoms or risk of disease. 3.3. Uniform definitions of assay performance parameters To provide physician-users with estimates of assay performance that are derived from uniform standards, it is estimated that a set of operational definitions for performance parameters such as threshold, precision, parallelism, and reproducibility be developed. This will permit test manufacturers to provide performance specifications on their product in a format that will allow direct comparisons among test systems. Such guidelines are currently under development by the American Academy of Allergy and Immunology’s Committee on In Vitro Tests. 3.4. Quality control of test systems

VOLUME 82 NUMBER 3. PART 2

Task Farce GtGdelines

It is recommended that manufacturers, in consultation with relevant professional organrzations. develop recommended quality control procedures for use of their test systems. This should include data on maximum sensitization of solid supports, lot-to-lot consistency, and stability at varying times and temperatures. Such programs should take into consideration the variability among allergowrhents within a given test method. 3.5. Importance of proficiency testing program Widely subscribed proficiency testing programs provide important benefits for quality control of individual laboratory results as well as ferreting out the strengths and weaknesses of various test methodologies. An IgE antibody testing panel has now been offered by the College of American Pathologists. It is recommended that participation in this program be endorsed by appropriate professional organizations and that manufacturers encourage their clients to participate by providing incentive programs. 3.6. Additional research on clinical correlation The clinical use of most specific IgE measurements in vitro is not well established. This is because the test measures IgE antibody, but in many clinical situations it is difficult to translate the quantity of IgE antibody to the predictive value of IgE-dependent diseases. Therefore we strongly recommend investment by the industry and research agencies into additional clinical research aimed at more precisely defining the clinical predictive value of IgE antibody assays, both in vitro and in viva, for various IgE-dependent disorders. Such clinical correlation studies are essential for a needed refinement in the use of IgE tests in the diagnosis and management of allergic diseases. 4. In vitro methods of allergen standardization 4.1. Background All medicines must have a label that describes quantity and potency. Traditionally, allergenic extracts have been labeled in weight to volume-an operational value and hence not subject to verification-or protein nitrogen units (PNU), the quantity of phosphotungstic acid preciptible nitrogen. Since allergens are known to be a small percentage of the total protein, source material can be manipulated to maximize the content of proteins that contribute to the PNU value without regard to the allergenically active proteins. Consequently, these procedures yield extracts whose labeling cannot be relied on to express the allergenic activity of the contents. In 1970 a program was initiated in the FDA Laboratory of Allergenic Products to develop procedures that would permit a description of the allergenic activity of extracts as determined by comparison of laboratory and skin test reactivities. 4.2. Current methods The first extracts in which an attempt was made to provide lot-to-lot consistency were the insect venoms. These products are labeled in arbitrary units of hyaluronidase enzyme per 100 pg of protein. The next extract was that of short ragweed pollen. These are labeled in units of antigen E per mililiter (a unit of antigen E is approximately 1 p,g). Antigen E is measured by a radial immunodiffusion test. If this type of program was continued, there would be as many types of methods and label designations as there are extracts. This would result in considerable confusion in the use of these products. Therefore a new unit was developed, AU, which is based on skin bioreactivity. However. each lot is evaluated by one or more laboratory tests. All quantitative laboratory tests currently used have been found to correlate with quantitative skin test reactivity. Thus in the future, all standardized extracts used in the United States will be labeled according to a single system (AU), and the labeled value can be verified by carrying out the prescribed tests. 4.2 1. Procedures Standardization of allergenic extracts involves a series of tests. All the tests are described in detail along with statistical methods and variability in the FDA’s Manual of Methods offhe Lahorato~ of Allergenic Products.

These methods include: a. Evaluation of the source material used for extract production b. Determination of a satisfactory procedure for preparing an extract c. Tests of the extract that include total protein, radial immunodif‘fusion

503

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test for a single allergen (e.g., short ragweed antigen E and cat allergen l), enzyme assay (hyaluronidase in venoms), RAST inhibition (pollen, mite, and mold extracts). Where applicable, isoelectric focusing and the passive immunoblotting from isoelectric focusing technique are carried out to evaluate the allergenic identity of extracts and the number of IgE-binding proteins. Using an extract standardized by these methods, serum pools collected from sensitive patients can be evaluated to determine the validity and reproducibility of RAST tests. d. Test variability 1. RID: the correlation coefficient of the reference dose-response line should be at least 0.9. A single concentration for a test extract is reproducible to -+25% when estimated from the calculated regression line. 2. RAST inhibition: this procedure provides relative potency and consequently requires a reference preparation. The data are analyzed by means of parallel line statistics. Therefore the frequently used method of comparing extracts at 50% inhibition values is without special meaning when this procedure is used for standardization of extracts. All validity assays must be included in the data statistical analysis described in detail in the FDA’s Manual of Methods. Prospective evaluation of the allergen standardization procedure was carried , out in the FDA laboratory and other laboratories by assessing more than 45 sets of data from three individual workers. The variability is proportional to the number of test methods, all of which should be performed at least in duplicate. For three tests, the calculated variability was 47% to 213%, and for five tests it was 56% to 180%. 4.3. Recommendations Standardization of extracts must continue until at least the most commonly used extracts are entered into the program. If any studies of diagnostic or therapeutic efficacy are planned, the extracts used should be subjected to an appropriate battery of tests, as described in 4.2, to assure that they are suitable for extensive studies. A reference preparation should be available so that the lot under study and any future lots can be compared. Thus data obtained from any laboratory or clinic can be more readily compared with all other data. Excellent examples of this are the rapid progress made in understanding insect sting allergy and diagnostic and efficacy studies that use short ragweed pollen extracts based on their antigen E content. Reference preparations are available from the FDA and the World Health Organization. 5. Histamine release from basophils 5.1. Background 5.11. Introduction More than 75 years ago Dale et aL6’ demonstrated the presence and physiologic action of histamine in different tissues. Later work established that histamine in tissue is present in granules of cells and that in human tissue these granules are present only in basophils and mast cells. Over the years it was demonstrated that histamine or histamine-like material was released into the blood of experimental animals during anaphylactic reactions. This approach led to the demonstration that the addition of antigen or allergen to the blood or washed leukocytes of either experimentally sensitized animals or allergic persons results in the release of histamine from the cells. Basophils are the only cells in human peripheral blood that contain histamine. The interaction of specific antigen with the IgE antibody fixed to the basophil membrane initiates a secretory reaction from these cells with the release of histamine and other mediators of immediate hypersensitivity. The release of histamine from the cell is a secretory event that is modulated by the addition of a number of pharmacologic agents. The addition of plasma or serum factors is not essential for the release reaction, although normal serum will enhance the release reaction when conditions are suboptimal.

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The serum of allergic persons also contains blocking IgG antibodies that also react with the allergen. However, when histamine release measurements are done with washed leukocytes, these antibodies probably do not influence the results. This overview will discuss only the use of histamine release from peripheral blood leukocytes as a tool for investigations of immediate hypersensitivity.66 The use of histamine assays to measure the level of histamine in various body fluids (e.g., serum, urine, etc.) is more problem& and requires a number of modifications of assay procedures. Moreover, these techniques are difficult to standardize and have wide variability in different laboratories. 5.12. Present application Histamine release from human basophils is a valuable tool for in vitro investigations of allergy. In most studies of histamine release, allergen or antigen is added to washed leukocytes from venous blood. This can be simplified by eliminating the leukocyte preparation step and adding the allergen to whole heparinized blood.67 The histamine released into the supematant can then be determined directly. Histamine release from basophils (leukocytes) has been used in several types of investigations: 5.12 1. Evaluation of the allergic status of patients In ragweed-allergic persons there is a good correlation between the severity of the clinical symptoms and the extent of in vitro histamine release.68 The histamine release also correlates with the skin test and the level of serum IgE specific for ragweed antigen E. Both the antigen concentration at which 30% or 50% histamine release occurs and the maximum percentage of histamine release correlate with the clinical severity of the allergic rhinitis and the skin test. Patients with high levels of serum ragweed-specific IgE release histamine with low concentrations of antigen. Similarly, in Hymenopterasensitive patients there is good correlation between positive histamine release in vitro and skin tests with venom antigens. 5.122. Allergen studies The purification of an allergen can be followed efficiently by determining the capacity of fractions to release histamine from the cells of allergic persons. Similarly, the system is useful to study allergic cross-reactivity and the effect of allergen modification on its activity. As a result of donor variability. histamine release is not very useful for allergen standardization. 5.123. Other studies In the past passive sensitization of normal basophils followed by antigen-induced histamine release has been used to measure IgE antibodies. Obviously, this is only of historical importance and has been superseded by the RAST. Histamine release can also be used for studies of blocking antibody levels, although radioimmunoassays are presently the preferred method for such determinations. 5.2. Current methods of histamine assay The discovery of histamine and the demonstration of its biologic importance were accomplished through the use of biologic assay systems that depended on the contraction of smooth muscle after the addition of this biologically active amine. However, these early techniques have been superseded by chemical methods that are more sensitive and specific for the determination of histamine concentrations . 5.21. The chemical method for histamine determination A method for the chemical determination of histamine was first described by Shore et al. in 1959.“’ Since then this method has been modified to increase both its specificity and sensitivity. It is based on the coupling of histamine with o-phthalaldehyde at an alkaline pH to form a fluorescent product. The fluorescence of the histamine-o-phthalaldehyde complex is more intense and more stable at an acid pH, unlike the complex formed by some other amines. To remove interfering compounds, me histamine is extracted before the condensation step. Protein is removed from the sample to be analyzed by perchloric acid precipitation; the histamine is extracted into n-butanol from the alkalinized S& saturated solution. The histamine is recovered in an aqueous solution in dilute hydrochloride acid by adding heptane. This dilute hydrochloride acid solution is then used for the condensation of histamine with o-phthalaldehyde. The extraction procedure with organic solvents is essential to remove histidine and other interfering compounds before the eondensation step. A new glass microfilter-based method of binding histamine has been reported.‘” This technique is based on the finding that glass microfilters bind histamine with high affinity and selectivity. Small

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samples of blood are added to the microfilter incorporated into a microtiter plate. After incubation with antigen, the released histamine is bound to the glass microfilter. Subsequently, histamine is eluted by a mixture of perchloric acid/o-phthalaldehyde and taken through the last steps of the chemical analysis protocol. This method requires only small amounts of blood (50 pUsample) and can be completed in 2% hours. Further studies are required to validate this assay. A completely automated fluorometric technique is capable of analyzing 30 samples/hr with a precision of 1% to 2%. The sensitivity of the method is such that 0.1 to 10 rig/ml of histamine can be accurately determined. This method is convenient in handling large numbers of samples with excellent precision. The methodology for both the manual and automated histamine analysis methods has been described in detail.% 5.22. The enzyme-isotopic assay. This method involves the transfer of either carbon 14- or tritiated-labeled methyl groups from S-adenosylmethionine to unlabeled histamine by the enzyme histamine, N-methyltransferase. The labeled methyl-histamine formed is separated from histamine by extraction into organic solvent. This method has been improved by the use of the more active rat kidney histamine-N-methyltransferase enzyme, and the introduction of thin layer chromatography purification steps. Several modifications have been introduced that increase the sensitivity and specificity of this assay. In the double isotopic procedure, a trace amount of tritiated histamine is added to each sample to monitor the internal recovery. In contrast, a single-step procedure can be used for the assay of samples that contain larger amounts of histamine or with the use of special steps for very sensitive assays of histamine. In this type of assay, the extraction is done with different organic solvents that do not have to distinguish between histamine and methyl histamine. The enzymatic assays have better specificity than the chemical methods and have been used to determine histamine levels in plasma or other body fluids. However, this is a subject of controversy and is beyond the scope of this review. The sensitivity of these enzymatic methods is excellent; for example, it is possible to determine values as low as 3.5 pg/ 10 ~1. The details of this methodology have been extensively reviewed.7’ 5.23. A radioimmunoassay for histamine This method depends on the use of a proprietary antihistamine antibody for the measurement of histamine. ‘* Further work is required to validate this assay technique and compare it thoroughly with the previously discussed two established methods. 5.3. Interpretation Experimental histamine release results are expressed as a percentage of total cellular histamine. Control measurements include the histamine released in the absence of added antigen, and this value is used to calculate the specific release. In most experiments the nonspecific “blank” release should be less than 10% of the total cellular histamine. High spontaneous release of histamine from washed leukocytes has been reported in a small percentage of patients who are highly atopic or sensitive to food. The significance of that finding is not clear. Appropriate controls should also include the testing of the allergen with the cells of normal nonatopic donors to demonstrate that the allergen does not contain any cytotoxic materials. Similarly, allergens or pharmacologic agents should be tested to see whether they influence the histamine assay procedure nonspecifically and contribute to erroneous results. Histamine release results can be conveniently expressed by two parameters: (1) cell sensitivity: this is the concentration of antigen or allergen concentration expressed in units (preferably micrograms per milliliter) required to release either 30% or 50% of the cellular histamine and (2) cell reactivity: this is the maximal amount (percentage) of histamine release obtained with any amount of the antigen. A positive control in histamine release experiments should be the addition of different dilutions of an anti-IgE antiserum to the cells. In general, the cells of most persons release >lO% histamine after challenge with anti-IgE. The number of false-positive reactions to allergens determined by histamine release is low. These are defined as an allergy history and patients with a negative skin test whose leukocytes release histamine. Limited information is available about this type of reaction, the occurrence of which is rare. The incidence of false negatives is a more critical factor in the interpretation of histamine release tests. These might occur when the clinical history and skin tests indicate the presence of sensitivity, but histamine release results are negative. Even under the best of circumstances, for example, skin

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test studies with pure venom antigens, there is a significant number of people who have positive skin tests and appear to be allergic by a convincing history but fail to release histamine after challenge with appropriate allergens. The percentage of these persons may be as high as 10% to 15% This raises the issue of how to interpret a negative test result. Some patients have little histamine release at any concentration of allergen but nevertheless are very sensitive by skin tests. Cells from patients with chronic urticaria frequently do not release histamine. Similarly, leukocytes from patients receiving certain medications will fail to release histamine. Desensitization of patients may also result in changes in the degree of histamine release from leukocytes. Changes attributable to immunotherapy are variable and inconsistent. 5.4. Recommendations Histamine release from leukocytes of allergic persons is an excellent in vitro correlate of alergy. However, the skin test with endpoint titration is the simplest method of assessing the presence or absence of allergy. In vitro histamine release results can be useful adjuncts to the clinical evaluation of patients and can supply quantitative data on the degree of sensitivity to a specific allergen. As such they should be compared with the RAST assay. Both assays suffer from the occurrence of falsenegative results, that is, patients who are clinically sensitive but negative findings on these tests..” Regarding percentage of true positive or true negative tests compared with clinical sensitivity, the results appear to be similar.73 The RAST has the following advantages: (1) It requires a small amount of serum, (2) samples can be stored and processed at a central laboratory, and (3) the techniques for the radioimmunoassay are fairly simple. In contrast, histamine release requires a larger blood sample, must be performed within a relatively short time after the sample of blood is obtained, and the techniques are more complicated. The advantages of histamine assays are that they require smaller amounts of allergens, do not involve injection of allergen into the subject, and are not dependent on coupling of the allergens to immobilized support systems with the inherent problems of imtigcn modification or availability of binding sites. Furthermore, with washed leukocyte experiments, there is no competition between IgG and IgE for antigenic-binding sites and therefore IgG cannot interfere in the assays as is the case in the RAST procedure. CHAPTER 3. WORKSHOP II. RELEVANT IN VIVO AND BN VITRO f##m@~T~ OF CELL-MEDIATED IMMUNE REACTlONS (DELAYED HYPERSENSITIVITY)

TESTS

A. Patch tests 1. Introduction Allergic contact dermatitis is a chemically induced, immunologically mediated event that is typically an eczematous (histologically spongiotic) dermatitis. The inflammatory exudate is largely a cellmediated (type IV) immunologic reaction but may contain an IgE-mediated (type I) component. The onset of dermatitis typically occurs 2 to 5 days after contact with a sensitizer. Patch testing is used to verify the responsible substance. The use of patch testing to identify allergic reactions other than on mucocutaneous surfaces has not been determined. 2. Indications Patch testing is usually used to determine the causative agent in eczematous dermatitis. Dermatitis of the hands, feet, lips, anogenital region and multiple areas of the body is a clinical situation in which patch testing is useful. Additional indications include chronic occupational dermatitis, especially when a change of jobs is considered. Contact dermatitis may be superimposed on atopic dermatitis not responding to appropriate therapy and may be from medicaments and other topical agents that can be identified with patch testing. When clinical evaluation suggests that a specific allergen may be implicated in a clinical setting, patch testing can be used to confirm the diagnosis. Patch testing can also be used when allergic contact dermatitis is suspected but unproved and the allergen is unknown. Patch testing may be needed to demonstrate to a patient that there is no sensitivity to a specific substance; this is particularly useful when neuropsychiatric symptoms predominate over physical findings. In a similar manner, patch testing is useful for medicolegal purposes to include or exclude the diagnosis of allergic contact dermatitis. Finally, patch testing is useful from a public health perspective to identify exposure to known and newly introduced allergens before the medical community and industry are informed about the potential health hazard. 3. The patch test

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The patch test was introduced by Jadassohn74 in the last decade of the nineteenth century as a defined method for verifying the presence of contact dermatitis. A small area of skin was covered with a semiocclusive bandage containing the reputed causative agent. A positive test result was declared when the clinical disease state was reproduced. Procedures for performing this deceptively simple test have evolved to provide an adequate, nonirritating, controlled exposure to a defined amount of substance in a nonsensitizing and nonsensitizer-containing patch test system. Two systems are in common use; both take advantage of the low allergenicity of aiuminum.75 The “Al” test uses larger strips of foil with heat-affixed Webril pads for allergen presentation. The “Finn” chamber uses small 8 mm diameter aluminum chambers that are occlusive and permit more accurate quantification of the dose of allergen per unit area. Both the Al test and Finn chamber systems are applied to the skin and held in place by tape. The individual allergens are applied at the time of testing with both systems. A new system for allergen presentation, known as the True Test,* uses allergens that have been previously incorporated into a gel delivery system. Although many common contact allergens are currently available in the True Test system, many are not, thus limiting its current applicability to the routine series of most common antigens. 4. The allergens Many contact allergens have been identified, but allergy is common to relatively few substances. Fewer than 40 allergens produce most cases of contact dermatitis. Very little information is available concerning most of the 2800 antigens that have been putatively identified. In the past little attention has been focused on the precise identification of the actual antigen in a complex product. Contaminating chemicals and minor ingredients have at times been the actual allergen, whereas the parent compound or major component has been erroneously labeled a sensitizer. The precise chemical antigen has yet to be determined in certain complex diagnostic allergen mixtures such as balsam of Peru, which is useful to help detect perfume allergy. In other cases the hapten may be a metabolically altered product of the substance applied to the skin. Approximately 20 to 30 antigens in the usual routine series of patch tests (screening panel) can be estimated to identify 50% to 70% of the clinically relevant causes of allergic contact dermatitis.76 With the exception of medicaments that vary according to local medical custom and practice, most of this routine screening series is applicable worldwide. Selected panels of allergens based on the patient’s history can be used to supplement the screening panel of allergens to cover as completely as possible the range of exposures of the patient.” Specialized kits for specific occupations and exposures (e.g., hairdressers, machinists, shoes, plants, photoallergens, etc.) permit identification of many other significant allergens. New antigens constantly appear in the human environment and require identification and validation for appropriate patch testing. 5. The vehicle Petrolatum is the most widely used vehicle for dispersion of allergens. Although it offers good stability and simplicity for many antigens, some substances do not disperse well in this medium. Substantial variation in the applied test dose as a result of poor dispersion has been shown for at least one common antigen from some manufacturers. The quality of dispersion can be assessed by light microscopy of a test substance in petrolatum. Added substances that enhance antigen dispersion in petrolatum introduce an additional variable into patch testing and require an additional patch test control of the dispersion substance in petrolatum without antigen. Solvents such as dimethyl sulfoxide have attractive properties for promoting antigen penetration but often induce vasoactive responses, thereby making interpretation difficult. 6. Duration of patch test application Traditionally, patch tests have remained on the upper back for 48 hours.78 When 24-hour time periods are used, concordant results are common but not universa1.78.7gWhen the patch test concentration is lowered or reactivity is diminished, the 48-hour patch test will detect a somewhat greater number of sensitized persons. Other reading schedules have also been investigated. *O Although all time variables have not been explored in depth, two collaborative group studies (the International Contact Dermatitis Research Group and the North American Contact Dermatitis Group) documented that approximately 30% of relevant allergens, which are negative at a 48-hour reading, become positive at a !&how *Not

available

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reading.“~ 82Conversely,somepositive reactions that are present at 48 hours disappear by 9h hours. Theseare believed to represent irritant rather than allergic responses. Readings at 96 hours are conducted 48 hours after removal of the original 48-hour occlusive patch. 7. Reading scale Close to 2 decades of discussion in the International Contact Dermatitis Research group led to the development and refinement of the currently used nonlinear descriptive scale; this scale has been almost universally accepted7? NT, not tested; ?, doubtful reaction; + , weak (nonvesicular) but palpable reaction; + + , strong (edematous or vesicular) reaction; + + + , extreme reaction (e,g., markedly bullous or ulcerative); IR, irritant reaction; and - , negative reaction. With some experience in grading, most observers can replicate the scores of more experienced graders. This descriptive scale defines the morphologic findings only and therefore has limitations in separating irritant from allergic reactions. Although novel bioengineered techniques (laser Doppler or reflectance measurements) provide objective readings and offer the advantage of metric measurements, they have not reached the stage of replacing the descriptive scale for routine clinical observations.‘” 8. False positives and false negatives The ideal patch test provides a dose that elicits a sensitization reaction but does not induce active sensitization or an irritant response. Furthermore, the elicitation reaction should not be so extreme as to produce unnecessary discomfort. There currently exists a significant data bank of relevant variables that affect interpretative reliability for allergens in the routine battery of patch tests and for some hundreds of less frequently tested antigens.76 There is a considerable body of descriptive information outlining the sources of false-positive and false-negative reactions. The major causes of spurious outcomes are inadequate technique, which is presumably amenable to instructions and training, and the difficulty of identifying a uniform test procedure that reliably separates irritant from allergic responses. The latter problem is especially prevalent in persons with the angry back or excited skin syndrome. R4Quantitative information about the prevalence of false-positive and false-negative reactions is more difficult to obtain. Data about irritancy and the threshold required to elicit a sensitization reaction are lacking for the many thousands of less frequently tested chemicals in the human environment. 9. Reproducibility The precision of a patch test result can be considered as encompassing both repeatabifity, that is, obtaining the same result on tests performed in a similar manner at the same time, and reproducibility, obtaining of the same result over time. Furthermore, two additional components of patch test results should be evaluated. These are the similarity of the reaction on the skin and the reading and interpretation of the test. The interpretation of a single test result is susceptible to both interobserver and intraobserver variation, Several large studies have compared the results of simultaneous applications of several allergens by different application methods and interpreted by the same observers.?‘. ESThese studies revealed >70% concordance of the positive or doubtful results of each study. Sources of lack of reproducibility include varying doses of antigen on the patch test and lack of consistency of antigen dispersed within the vehicle (usually petrolatum) carrier. Nonspecific hyperreactivity caused by multiple positive results in routine panel testing, that is, the excited skin syndrome or angry back, may produce as high a rate of false positives (defined as nonreproducible reactions on a single retest) as 40%Tcu4

10. Clinical relevance Once a test is positive, an attempt must be made to determine the environmental source of exposure to the reputed allergen. This requires knowledge of the likely sources of the substance in a person’s environment and the subject’s hobbies, habits, occupation, and idiosyncrasies. Certain allergens in a routine patch test battery such as nickel and some rubber chemicals have high clinical relevance (approximately 75%), whereas others appear to have low clinical relevance (tars and colaphony). The frequency with which a linkage can be made between a positive patch test and a likely environmental source of exposure for a specific substance by means of standard screening patch test batteries varies from 17% to 85%, depending on the substance. Lack of adequately standardized allergens may be partially responsible for the lack of relevance (i.e., false-positive irritant reactions). Inadequate knowledge of the chemical environment may also be important. Despite these inherent problems among specific contactant allergens, standard screening panels of allergens have a high

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level of relevance to clinical allergy.82 Detailed information about individual chemicals in the routine patch test series and sources of exposure is available in a booklet entitled, “Exposure List,” published by the American Academy of Dermatology. 11. Miscellaneous Additional subjects of clinical importance deferred for future review include techniques for identifying photoallergic contact dermatitis, namely, photo patch testing. This involves the exposure of patch test material to ultraviolet light energy for detection of sensitization. Also deferred are the contact urticarial syndrome and proper use of the provocation use tests (repeated open application test) .86 12. Recommendtions 12.1 Patch tests The Finn chamber is the best-studied and currently the best-accepted standard test system. It is the system used exclusively by members of the North American Contact Dermatitis Group. A dose of 15 pl per chamber is recommended for allergens. The antigen concentration should be appropriate for the patch test system used. Among the common screening allergens, formaldehyde is the most sensitive to changes in patch test systems. With the Al test system, formaldehyde is tested at a 2% (aqueous) concentration, but this concentration under a Finn chamber will produce irritant reactions because of the superior occlusion afforded. Thus the formaldehyde aqueous concentration used in the Finn chambers should not exceed 1%. Ideally, the in vitro release of allergen from its test system and the penetration of allergen into skin assure that an adequate reproducible and known test dose was delivered. The ideal surface size for patch tests is not known; it would be preferable to use the smallest area that reliably detects allergy to minimize the degree of patient discomfort. Ideally, none of the patch test material should react with the testing device. Certain metal salts, for example, mercury, will etch aluminum chambers. Although polypropylene coating can prevent this phenomenon, the introduction of a possible irritant to the Finn chamber to prevent such rare occurrences is not practical for standard testing. 12.2. The allergens Precise identification of allergens should be obtained with analytic quantification when possible. The molar test concentration should be stated, along with a description of the concentration in gram units. If percentage by weight is used, the concentration should be expressed as active principle, for example, nickel ion rather than nickel sulfate. The stability of allergens should be known and the level of acceptable degradation over time stated. The recommended storage temperature for stability and the effect of light on the materials should be known. Complex mixtures of allergens may have to be studied initially to identify the offending antigen(s). Allergens in plants present a special problem. If the precise allergen is known and available, it should be used. Failing that, identification of the botanical source is mandatory, and the preparation of crude plant extracts should be standardized. For convenience, some antigens are mixed together for patch test purposes. Whenever this is performed, it is essential to verify that the mixture will still be sensitive and specific for the diagnosis of persons allergic to each of the single components. For public health purposes, it may be important to test with mixtures when the precise antigen is unknown. The development of new panels of relevant antigens is specifically encouraged to identify sources of dermatitis. 12.3 The vehicle Alternative vehicles that allow better solubilization and more even dispersion of antigens are needed to ensure optimum antigen delivery, that is, predictable bioavailability. 12.4. Duration of patch test application With present delivery systems, a 48-hour application interval is recommended. However, to identify 30% of the reactions that may develop beyond 48 hours, an additional reading at approximately 96 or 48 hours after removal of the occlusion patch is strongly encouraged. Further studies are required to ascertain whether those reactions that disappear between 48 and 96 hours are in fact irritant. Investigations of the responses between 48 hours and 1 week should determine if supplementary readings within this period may be more valid than a single 96-hour reading interval. 12.5. Reading scale Despite the universal use of the descriptive reading scale, more objective criteria for routine clinical use should be sought. The complexity of the reaction (i.e., erythema, edema, and vesicles) should

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be further studied to ascertain if dissection of the components of the response might

Gwiaiines

add clinical insight. Development of simplified quantitative instrumentation suitable for clinical use should be encouraged. 12.6. False positives and false negatives Further quantitative data in large populations are required to establish a “gold’ standard, rhat is, a concentration that is sufficient to elicit sensitization but not irr&ion.87-89 The nonirritant threshold dose response should be available for most chemicals to which there is significant human exposure. Elucidation of these objectives requires more investigation into the appropriate number of negative and positive controls for a given chemical class. Clearly, more efficient methods of separating irritants and allergens are required. More and better training in currently available methodology should allow greater numbers of skilled physicians who properly separate irritant from allergic reactions 12.7. Reproducibility Simultaneous application of allergens should help determine the error of the measurement or observation of the response. Reproducibility, that is, identical results over different time spans, is much more difficult to interpret, but suitable protocols should be devised. Interpretation of such studies will be improved by comparisons of inter- and intraobserver variability. 12.8. Clinical relevance Quality assurance of the appropriateness of concentration, vehicle, and stability is essential for future attempts to define clinical relevance. More efficient product labeling, index, and computer data banks will enhance the development of clinically useful allergen exposure tests. A more rigorous test of relevance is the application of the suspected allergenic substance to normal skin in a manner that mimics the exaggeration of normal use. This is accomplished by a provocative use test (repeated open application test), which has demonstrated that as many as 80% of standard screening allergen reactions are clinically relevant. 79Thus standard screening of the most common contactant allergens has a high level of relevance to clinical allergy and should be encouraged in clinical practice. The FDA has just approved the release of patch test kits that are prepackaged with defined concentrations of allergens. These kits may be obtained from Dermatology Services, Inc., P. 0. Box 3 166, Evanston, IL 60204. B. Tuberculin-like and recall intracutaneous tests 1. Tuberculin hypersensitivity reactions The purified protein derivative (PPD) of tuberculin is the prototype of recall antigens.gO PPDtuberculin is an ammonium sulfate precipitate of the heated aqueous uhrafiltrate of a broth culture of Mycobacferium hominis. The tuberculin skin test is elicited by the intracutaneous injection of 0.1 ml of stabilized and standardized antigen solution. The reaction begins within hours and reaches maximum size in 48 hours. The involved skin feels firm or indurated to the touch. Erythema and edema are not necessary components of the tuberculin reaction. The reaction can persist for a week or longer. With tuberculin the vesiculation and blistering response of exquisite delayed cutaneous hypersensitivity is rare. The tuberculin reaction is used to identify exposed persons at risk of infection with the tubercle bacillus. Exposure to nonpathogenic soil mycobacteria can also provoke modest degrees of cross-reacting cutaneous tuberculin hypersensitivity. Comparisons between populations that have been exposed to cross-reacting soil mycobacteria and unexposed populations such as Alaskan Eskimos indicate that a 10 mm delayed reaction diameter identifies 90% of healthy persons who have been sensitized to the human tubercle bacillus.9’ When tuberculin is used as a recall antigen to evaluate cellular immunity, the size of the skin test reaction is not important. The histologic changes provoked by tuberculin begin with changes in the distribution of specific cells and local edema of the arterioles, capillaries, venules, and lymphatics.92. 93Within 2 to 4 hours, a few basophilic leukocytes accumulate within the lumen and about small blood vessels.94Hypertrophy of the blood vessels, pericyte and endothelial cell hyperplasia, endothelial cell necrosis, and thickening of the vascular basement membrane can be seen. By 4 to 6 hours, perivascular cuffs composed of large and small lymphocytes can be detected, and the ratio of T helper to T suppressor lymphocytes in the infiltrate exceeds the ratio in the peripheral blood.95 Approximately 75% to 90% of the mononuclear cells in the perivascular infiltrate bear the cell surface phenotypes of T cells or macrophages. The perivascular changes reach a maximum in 48 hours and can appear as nodular pseudogranulomata. In the epidermis, the numbers of T helper and suppressor phenotypes are equivalent.

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In the dermal interstitium, the T-lymphocytic and monocytic infiltrate is dominated by T helper cells by 12 to 24 hours. The reaction can be accompanied by a variable but often prominent neutrophil infiltrate. Heavy fibrin deposition occurs and is responsible for the induration at the reaction site.W The perivascular changes are followed by a diffuse, less intense intervascular infiltrate composed of lymphocytes, monocytes, and a few basophils. Evidence of cell activation, as manifested by the appearance of transferrin and interleukin-2 receptors, is not detected until approximately 48 hours after antigen challenge. 2. Current applications A detailed description of the traditional methods for performing Mantoux-type tests with tuberculin and other recall antigens is available. % Recall antigens can be used to evaluate delayed-type hypersensitivity and ex vivo correlates of cellular immunity in patients with suspected immune disorders, aging, and altered nutrition. Recall antigen skin tests can be used to predict survival and detect disease-related changes in immunity or the outcome of therapy. The detection of intact delayed cutaneous hypersensitivity can be used to avoid more costly ex vivo tests of cellular immunity. Despite the usefulness of recall antigen skin tests, a major problem limits their widespread use in patients. Mantoux skin tests done by one physician cannot be readily compared with tests done by others.” This means that critical comparisons of skin test observations are often limited to those obtained by a single investigator. The clinical application of in vivo recall antigen testing is limited by the lack of standardized reliable test methods, antigens, and a better understanding of altered hypersensitivity in disease. 3. Optimal test methods Despite much past experience, the reliability of the delayed skin test is not clearly known. Assessments of the sensitivity and specificity of recall antigen skin tests depend on the choice of test methods, test antigens, antigen dose, time of measurement of the reaction, and prior exposure of the population at risk. Other sources of variation include the choice of epicutaneous, intracutaneous, or subcutaneous deposition of antigen, the choice of skin test site (proximal vs distal arm sites; right vs left arm differences, etc.), and the effect of medications.9* Extraneous sources of variation can also contribute to the test variation. In addition to the time of day and year that the test is done, “booster” and suppressive effects have been reported after repeated antigen stimulation.W-lO’ Experimental studies focused on one or another source of variation in recall antigen reactions are often not truly informative unless these sources of extraneous variation are carefully controlled. The physician who uses the test antigen needs to know how the magnitude of these extraneous sources of variation relate to between-subject variation in the test population in which the antigen is used. 4. Test antigen availability Recall antigen testing is performed with antigens developed for other purposes. These include PPD-tuberculin, histoplasmin, coccidioidin, mumps skin test antigen, trichophytin, Candida (a&icans) antigen, tetanus toxoid, and streptokinase-streptodomase. The suggestion has been made that antigens developed specifically for recall antigen testing should be categorized as orphan drugs. The “Multitest” tine panel may represent an exception to the lack of adequately characterized recall skin test antigens. lo1 In contrast to prior reports of a high prevalence of false-negative reactions with the tuberculin tine test, the “Multitest” (Merieux, Miami, Fla.) antigen tine panel yields an apparent prevalence of reactions at 48 hours that is comparable with that obtained with the Mantow-type test. ‘O**lo3 However, this interpretation of the prevalence of reactions elicited with single or multiple tine tests may be at risk because of evidence that the cellular infiltrate in the epicutaneous tine reaction does not become comparable with the classic Mantoux 48-hour intracutaneous skin test until 96 hours9” This disparity suggests that a portion of the high prevalence of Multitest reactions at 48 hours could be from immediate hypersensitivity. Until this interesting issue is resolved, tests with tine-type devices at 48 hours cannot be considered a comparable measure of delayed-type hypersensitivity or cell-mediated immunity. 5. Time of reaction measurement Large differences exist in average reaction diameters and percentage of reactors to the same antigen at 24, 48, and 72 hours. This means that experimental comparisons of skin test reactions measured at 24 hours in one subject sample are not comparable with reactions measured at 48 hours in a second subject sample.96*g7Although circumstantial evidence supports the use of the 48-hour skin test reaction,

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the optimaltime for measurement of delayed cutaneous hypersensitivity is not known ‘(a Traditional choices of the time of appearance of immediate and delayed cutaneous reactions are ha-sed on experimental studies of adoptive sensitization, which differ from the way recall antigens are used in patients. The descriptions of immediate and delayed cutaneous changes used by physicians have rarely been directly evaluated in naturally sensitized patients. One such study of several recall antigens by Buckley et al. confirms that both immediate and delayed components of cutaneous hypersensitivity are elicited by Mantoux-type skin tests and that the 48- and 72-hour reactions are least compromised by immediate hypersensitivity.%” 6. Methods The traditional standard method for performing recall antigen skin tests is the Mantoux type of intracutaneous injection of 0.1 ml of antigen solution. Other skin test methods may be appropriate but must be reconciled with the time course and histopathologic examination of the antigen-mduced changes obtained with the Mantoux-type skin test. The skin test should be initiated with the bevel of a No. 27, 0.5-inch needle directed upward and the needle held at a 15- to 20-degree angle to the skin. The needle should be inserted into the skin and channeled several millimeters through the dermis. When done correctly, the skin will dimple with slight pressure or movement at the tip of the needle. The injection of the 0.1 ml volume should produce a transient mild burning discomfort and a 5 to 10 mm wheal in the skin. In addition to standardized PPD-tuberculin, histoplasmin, coccidioidin, and the mumps skin test antigen, recall antigen skin tests may be done with noninfectious and nontoxic antigenic products of any pathogenic or nonpathogenic organism. Appropriate antigens include those from the streptococcus and staphylococcus, blastomycin, diphtheria, and tetanus toxoids, trichophyton, and antigens from common fungi such as C. albicans. When possible, relevant antigens used for prophylactic imrnunization are preferred. The size of the delayed skin test reaction should be measured 48 hours after antigen challenge. The diameter of the palpable firm area of the reaction (induration) should be estimated as the average of orthogonal diameters measured to the nearest millimeter. When clinically indicated and in mvestigative studies of recall antigens, the diameters of the wheal reaction at 0.25 hour, the erythema and edema at 6 and 24 hours, and induration at 24 and 72 hours should also be measured and averaged to describe the time of the changes in the reaction and the occurrence of adverse (240 mm,! skin test reactions. 7. Interpretation Generally applicable specific guidelines for the interpretation of recall antigen skin tests must await the availability of standardized antigens. At the least, the reaction should be identified as negative or positive, and the size of positive reactions should be measured and recorded. When tine tests with multiple antigens are collectively interpreted, the identification of a ~2 mm reaction diameter can be used as a reliable criterion for a positive test. When a single intracutaneous antigen is used, a 35 mm reaction may provide a more conservative interpretative criterion for a positive test, but nevertheless should be accompanied by reports of any 2 to 4 mm reactions. With standardized skin test antigen panels, it may be possible to use the number and size of reactions to identify relative grades of immune function or immune deficiency. Presently grades of relative immune deficiency must be defended within the reference range of the particular test antigen(s) in an appropriate control population. A clinical diagnosis of delayed curuneous anergy can be established with recall antigen skin tests when a <5% probability of negative reactions exists in comparably exposed healthy control subjects. A clinical diagnosis of delayed cutaneous anergy cannot be established with a single test antigen because the implied >95% reaction rate to the antigen does not clearly distinguish between a toxic and hypersensitivity reaction. 8. Recommendations 8.1. Development and assay of recall skin test antigens Diagnostic recall skin test antigens should be developed for the purpose of reliably and safely testing the integrity and specificity of cellular immunity and providing positive controls for skin tests with antigens from pathogenic organisms. Specific future needs include characterization of the accuracy and reliability of measured skin test reactions, the optimal time of assessment of delayed

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cutaneous hypersensitivity, and the development of stable, safe, and standardized recall antigens that elicit a significant prevalence of reactions in control populations. These issues are amenable to research focused on the development of skin test methods and antigens. Appropriate methods and antigens will facilitate the acquisition of objective clinical experience needed to understand hypersensitivity mechanisms in disease. 8.2. Uniformity of unitage The physical attributes of diagnostic antigens should be defined in centimeters/grams/seconds units such as grams per liter. When possible, antigen concentrations should also be stated in millimoles of solute. Scientific descriptions of the physical and chemical properties of candidate antigens should be sufficient to permit reproducible prediction of their biologic properties. Serial clinical studies of the efficacy of diagnostic skin test antigens are required to assure that the clinical use and potency of the antigens remain unchanged in contemporary patient populations over time. 8.3. Research and development Several specifically focused technical efforts and open-ended research could help meet these overall goals. These opportunities include: 1. Studies of potentially useful candidate test antigens, including an assessment of the reaction size elicited by different antigen doses, the storage stability of the antigen, the optimal time for measurement of skin test reactions, the prevalence and size of reactions, and the occurrence of adverse skin test reactions in subjects from the test survey population, are needed. The change in reaction size elicited by candidate antigens over time should not compromise the usefulness of the antigen during the period in which it will be used. Reasonable goals include the following: a. A 50% to 70% prevalence of reactions X5 mm b. A 2% or less prevalence of adverse reactions (i.e., 340 mm) c. A 20% or less decline in potency per year. 2. Research is needed to identify and eliminate the effect of heat labile and proteolytic components in diagnostic recall antigens to better control their storage stability and potency. 3. Skin test reactions to standardized diagnostic skin test antigens should be evaluated in selected population surveys of healthy subjects over time. These studies should at least include the following: a. Assessments of geographic, ethnic, and racial differences in reactivity to ubiquitous recall antigens and antigens that have been prepared from selected pathogens appropriate to the region or population group. b. Assessments of population immunity after known exposure to potential immunotoxins . c. Assessments of immunity in selected, independently identified subjects who are at risk of diseases in which immune function is important, 4. The reactions of standardized diagnostic skin test antigens should be evaluated over time in specific patient populations to provide continued assurance of their efficacy or identify the need for alternative diagnostic strategies. 5. Biopsies of skin test reaction sites should be done under several circumstances likely to provide substantive information. These include the folloiwng: a. Studies of the time course and meaning of reactions elicited with novel diagnostic skin test antigens should be done, including a quantitative description of time-dependent changes in the number and type of cells in hypersensitivity lesions (morphometry) and an appraisal of functional relationships between these cells. b. Studies of the meaning of intermediate or mixed-type reactions should be done in selected diseases. The use of biopsy specimens of intermediate or mixed-type reactions in unusual patients should be encouraged in the hope of relating the mechanisms responsible for the hypersensitivity reaction to the specific manifestations of disease. C. Standardization of type IV skin tests 1. Clinical performance vs analytic performance The usefulness of type IV testing to detect chemical sensitivity or cell-mediated immum>comRetence is based on eliciting a clinical response that in the former case discriminates among persons whose

VOLUME 82 NUMBER 3, PART 2

Task

Force

Gurdelines

syndrome is caused by the chemical of interest from those diseased patients whose syndrome is nor related to the chemical of interest and in the latter case between immunocompetent and anergic

patients.Theabilityto discriminate theseclinicalgroupsdefinesthe“clinicalperfor~~~a~& of the test and requires complex and expensive multicenter clinical trials to establish the diagnostic efficacy of the test. Evaluation of the analytic performance of type IV tests can be accomplished more simply, efficiently, economically, and with lower risk to patients compared with evaluation of the clinical performance of a test. Therefore before investing the time and expense required to determine the clinical performance of a test, the “analytic performance” of the test should be established. Whereas the clinical performance of a test is based on its ability to discriminate among disease states, the analytic performance of a test is based on its ability to discriminate among doses ot allergen. Thus tests that detect small differences between doses accurately and precisely with the fewest replicates would be preferable to tests whose analytic performance demonstrates lower sensitivity, precision, accuracy, and replicability. Test methods that reveal greater accuracy and precision can subsequently be selected for evaluation of the clinical performance, with a greater likelihood that their diagnostic efficacy will be superior to tests whose analytic performance demonstrates lower sensitivity, accuracy, and precision. 2. Accuracy and precision of tests of analytic performance Because analytic performance of an in vivo test depends on the skin test response’s ability to discriminate among doses of allergen rather than diseased members of the population, analytic test performance is based on dose-response data. The use of dose-response data permits application of suitable mathematic models (e.g., parallel line assay) to assessaccuracy and precision of the analytic performance of the test on the basis of the test’s ability to discriminate between two known doses of extract, that is, relative potency.28 The development of dose-response data for specified allergens or positive control reagents also facilitates the development of proficiency protocols in which investigators can determine whether they can carry out the test accurately, precisely, and with results similar to a reference group of investigators. 3. Requirements To obtain the most sensitive, accurate, and precise test requires that the dose of antigen be accurately measured and administered, the response be quantitated, and the dose-response relationship be defined so that the test with the steepest slope can be selected. To ensure that the test can be performed appropriately by multiple investigators requires the standardization of both the test procedure and the allergens. a. Allergen standardization Ideally, reference preparations of allergens should be provided to investigators so that all investigators can use a common defined allergen to generate dose-response date. Test preparations that are to be compared with the reference can include the actual reference or known dilutions of the reference. In this manner, the relative potency of the reference and test sample is ascertained a priori so that accuracy of the test method can be determined. Model standardized allergens for type IV testing should be made available in forms suitable for epicutaneous, percutaneous, and intracutaneous testing. b. Measurement of allergen dose The simplest approach to measurement of allergen dose is to consider that the test preparation behaves as a dilution of the reference. The bioavailability of the dose administered is dependent on the route of administration (epicutaneous, percutaneous, and intracutaneous). Therefore where feasible, each technique of administration must be compared to determine which yields more accurate and reproducible test results. Allergens should be available in different doses or be suitable for dilution to avoid severe overdose reactions and permit determination of the dose-response relationship. c. Measurement of response The most important impediment to determining the analytic performance of a type IV test is faihrre to quantify the response based on measurement. This is not true of tuberculin testing in which measurements are routinely performed. Measurement is the least ambiguous method of quantification and readily lends itself to routine statistical analysis. Erythema should be measured along with other inflammatory responses, for example, induration, vesiculation, to evaluate which parameter can be

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516 Task Force Guidelines

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evaluated most precisely and which produces the steepest dose-response lines. Intracutaneous testing may facilitate quantification of the allergic response and should also be compared with patch tests in special instances of contact sensitivity.‘” d. Dose-response lines Sufficient dose-response data without exposure of patients to high doses of allergen while still obtaining at least four points (including the endpoint) should be achievable with serial threefold dilutions. Linear dose-response lines have been obtained with induration, and therefore erythema should also yield linear dose-response lines. Once dose-response data are obtained, they can be standardized, that is, by comparing correlation coefficients, slopes, and range of response intensities, to determine qualitative consistency of the data base, relative potency of test and reference preparations, and evaluation of accuracy and precision of the test. Once accuracy and precision have been determined for each method of application or dosage form, they can be compared to establish which method of application provides the most accurate and precise analytic test results. 4. Recommendations 4.1. Standardized allergens should be made available in forms permitting dilution or in multiple dose forms so that dose-response relationships can be established. 4.2. Standardized allergens should be made available in dose forms suitable for each method of administration (epicutaneous, percutaneous, and intracutaneous) when feasible to facilitate comparison of the accuracy and precision of each method of administration. 4.3. Analytic performance of each test method must be established based on quantitative dose-response data before evaluation of clinical performance is carried out. 4.4. Analytic test methods that produce the most accurate and precise determinations of relative potency can then be recommended to investigators who wish to establish replicability and proficiency of testing. 4.5. Proficiency test methods for each method of application need to be developed to ensure that investigators have carried out the procedure with appropriate accuracy and precision. 4.6. Once the accuracy, precision, replicability, sensitivity, and proficiency with the analytic test method are established with a model allergen, its clinical performance can be evaluated. D. In vitro evaluation of cell-mediated immunity 1. Background The cell types that contribute to the cellular hypersensitivity reaction include lymphocytes, macrophages, and granulocytes. The techniques to be described are used to assessin vitro cellular function in patients who may have certain types of recurrent infections (e.g., fungal, mycobacterial, and protozoal), depressed cellular immunity (e.g., acquired immune deficiency syndrome, sarcoidosis, and cancer), or autoimmune disease (e.g., glomerulonephritis and thyroiditis). The best in vivo screening procedure that the clinician can use to evaluate cellular hypersensitivity is the 24- to 48-hour skin test. The development of one or more positive cutaneous responses to environmental antigens such as purified protein derivative of tuberculin, monilia, streptokinasestretodomase, or mumps, or the ability to be sensitized to a contact allergen such as dinitrochlorobenzene, usually indicates intact cellular immunity. A positive delayed skin test response results from intact lymphocyte-macrophage interaction as well as certain components of the inflammatory response. Therefore in vitro testing in such patients will not usually provide more information. However, polymorphonuclear leukocyte function is not evaluated by delayed hypersensitivity skin testing, and performance of this test would not detect defects in this system. Tests that quantify lymphocyte function detect the ability of lymphocytes to proliferate, produce mediators, mount cytotoxic responses, and regulate immune responses. Lymphocyte proliferative responses can be evaluated by the use of nonspecific mitogenic stimulants such as phytohemagglutinin, concanavalin A, or pokeweed mitogen and by specific stimuli such as soluble and cell-bound antigens. The nonspecific activation of lymphocytes measures both T and B cell function, although the kinetics

VOLUME 82 NUMBER 3. PART 2

TABLE V. Lymphokines

Task Force and their target cells Lymphokine

Migration inhibitor factor Macrophage-activating factors, including interferon-y and granulocytemonocyte colony-stimulating factor Macrophage chemotactic factor

Guidehes

517

--~---Target cell

Macrophages or monocytes

Interleukin-2 B cell-stimulating factor 1 B cell-stimulating factor 2 B cell growth factor II Interleukin-4

Lymphocytes

Neutrophil, basophil, and eosinophil chemotactic factors Leukocyte inhibitory factor Eosinophil stimulation promotor Histamine-releasing factor Interleukin-5

Polymorphonuclear leukocytes

Interleukin-3 (multicolony-stimulating factor) Interferon-y Collagen-producing factor Procoagulant (tissue factor)

Other cells

of these responses differ. In contrast, specific antigenic challenge appears to measure only T cell function. In addition, by using autologous as well as homologous serum in the cultures, one can also determine whether the patient’s serum contains factors that may interfere with the prolif&ative response. The elaboration of cytokines by lymphocytes and monocytes indicates that these ceElsarecapable of producing factors that are involved in the afferent and efferent limbs of the cellular hj!persensitivity response (Table V). Factors such as interleukin-1 and interleukin-2 are involved in the. a&vat&n and growth of T cells, whereas inflammatory mediators such as macrophage and neutrophil ehem#actic factors (CF), macrophage migration inhibitory factor (MIF), and leukocyte inhibitory factor (LIF) are involved in the expression of cellular hypersensitivity. The production of CF, MlF> and LIF has been shown to correlate with in vivo delayed-type skin reactivity, although they do not necessarily measure the function of a particular cell type (T or B cells). The ability of lymphocytes to act as cytotoxic “killer” cells in response to either al target or malignant cells has clinical relevance in patients undergoing a transplant proce&?re ‘and patients with cancer. Moreover, T cell regulation of immunoglobulin synthesis or ~~,~y production, as well as lymphocyte proliferation, also has clinical application. Excessive or diminished regulation of these immune responses can result in disorders in humoral or cell-mediated immunity or both.

2. History In 1960, Nowell’” described that phytohemagglutinin, a lectin extracted from ki nonspecifically transformed small lymphocytes into proliferating lymphoblasts in vitro. Sub in addition to plant lectins that activate all normal T cells, it was shown that a variety of a&&%6 could also induce proliferation. lo7 However, this occurred only in those persona who had pssitive delayed skin tests to these antigens. Thus in vitro proliferation to solubie antigens but not to mit@gens was shown to be a good correlate of in-vivo.delayed-type hypersensitivity. Even though proliferation correlates with in vivo delayed-type hypersensitivity, its role in the expression of the skin reaction is unclear. On the basis of what is known about the biolo@c ae&&ies of cytokines, these factors appear to be better candidates to serve this function. In fact, when these in vitro tests are correlated with skin testing in normal subjects and in patients with diseases associated with defects in delayed-type hypersensitivity, the measurement of lymphokines more often CbSefY

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Task Force Guidelines

TABLE VI. Inhibition lymphocyte function

of cell migration: in patients with

correlation depressed

between skin test reactivity cellular immunity

Patient diagnosis

Group I (61 patients) Hodgkin’s disease, sarcoid collagen disease Group II (70 patients) Hodgkin’s disease, sarcoid, collagen disease,chronic candidiasis, DiGeorge’s syndrome (22 patients) Chronic candid&is, sarcoid, collagen disease(34 patients)

Hodgkin’s disease, collagen disease(14 patients)

and in vitro Tritiated thymidine

Skin

MIF

+

+

-

-

-

-

+

-

+

+

+

parallels the results of the skin test than does proliferation (Table VI).‘O’ For this reason, this review will focus on the production and measurement of two effector lymphokines (MIF/LIF), which have been shown to be useful in in vitro correlates in a wide range of clinical disease settings. The antigen-induced inhibition of cell migration has been used as an in vitro correlate of delayedtype hypersensitivity since its original description in 1932. The development of the capillary tube method has greatly facilitated a dissection into its mechanism and the discovery of MIF.lw The latter was first described by David”’ and Bloom and Bennett”‘; it is a protein produced by sensitized lymphocytes on activation by specific antigens or mitogens. Human MIF exhibits heterogeneity in its molecular weight, isoelectric point, and glycosylation.“’ That LIF is a mediator distinct from MIF was initially established in studies that used molecular sieve chromatography. ’ I3 As shown in Fig. 2, supernatants from antigen-stimulated lymphocytes contained MIF activity that eluted with molecules having an approximate molecular weight of 23,000, and this material had no inhibitory effect on the migration of neutrophils. LIF activity was found to elute with molecules having a molecular weight of 58,000, and this fraction had no inhibitory effect on macrophage migration. Furthermore, like MIF, its production was antigen specific by the lymphocytes of the sensitized donor. 3. Application The results of migration inhibition assays correlate well with delayed cutaneous hypersensitivity reactions. One can assessthe production of LIF or MIF in response to stimulation with a variety of antigens such as environmental antigens, tissue antigens, contact allergens, or drugs, either in conjunction with in vivo testing or when in vivo testing is impractical (such as with a patient receiving steroids and anticoagulants). Lack of reactivity provides a useful screening test for immunodeficiency, since diminished lymphokine production can result from abnormal lymphocyte function, a defect in macrophage accessory cell activity, blocking factors, or a lack of antigen-specific lymphocytes. These assays can also be used to monitor patient immunocompetence during therapy or detect reactivity to drugs or antigens that may produce CM1 inflammatory diseases. 4. Current methods 4.1. Direct and indirect migration techniques Two assay methods can be used to measure cell migration, an indirect method (two-step) and a direct method (one-step). With the indirect method, lymphocytes are cultured with antigen to produce the mediator, which is then assayed on indicator cells at a different time (Table VII). With the direct method, the lymphocytes, antigen, and indicator cells are mixed together, mediator is produced locally, and its effects are measured at the same time. In the indirect method, blood lymphocytes are isolated and stimulated in vi&o by antigen. The cell-free supematants are collected and assayed for inhibitory activity on guinea pig peritoneal cells or human monocytes in capillary tubes or agar. Inhibition of migration is read at 18 to 24 hours and correlates qualitatively (but not quantitatively) with cutaneous delayed hypersensitivity reactions in the host. For example, lymphocytes from agammaglobulinemic children with intact delayed hypersensitivity produce MIF, whereas cells from patients with DiGeorge’s syndrome, which is characterized by normal antibody and a lack of delayed hypersensitivity, do not produce MI.F.‘14 The advantage of the two-step method is that it allows a dissection of the cellular immune response; one can separately assess the ability of patients’ lymphocytes to elaborate MIF if normal indicator cells are provided and also to examine whether patients’ monocytes respond to autologous or homologous MIF preparations. The disadvantages are that it

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POSITIVE

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Guidelines

NEGATIVE

GlJhKA PIG MACROPh!AGES

0

ItIl

Ill

I!h

L!lb

ItlI

m

Ish In

FiG. 2. Supernatant preparations were obtained from monilia or PPD-positive lymphocytedonors (/B#, as well as from monilia or PPD-negative donors (f&bf) and filtered on cotumna. The fractions were assayed for migration inhibitory activity on gluinaa and leukocytes from normal subjects lacking sensitivity to these ant&ens. &tz indicete both the antigen used to produce the supernatant and the inhibition of migration of the resulting fractions.

requires large amounts of blood from patients and is time-consuming. In addition, because of the 10% to 20% incidence of false-negative results, the responses should be repeated in an individual patient to confirm the findings. However, false-positive results generally do not occur if the proper antigen controls are carried out. The direct method involves the use of peritoneal exudate cells from sensitized animals or peripheral buffy coat cells from sensitive donors in capillary tubes or agar. The cells are e specific antigen, and MIF or LIF produced locally by lymphocytes results in migration. The advantage of this assay over the indirect method is that it requires less time to requires fewer cells (if the microassay is used), and can give an answer in 18 disadvantage with this technique is that a negative result may be difficult to interpret; that is, no inhibition of migration may not only result from a lymphocyte defect (no MIF or LIF) or lwk of response to the antigen, but it may also represent a failure of the indicator cell to respoml to the mediator. This distinction may be quite important in evaluafing patients with cutaneous anorgy who may have more than one defect. Thus the direct leukocyte migration test may be a useful in vitro screening test to assess delayed-type hypersensitivity in patients. One or more positive responses to antigens may be taken to mean intact lymphocyte-monocyte interaction. A negative response may require the use of the indirect assay technique to further delineate the defect. 4.2. Generation of LIF and MIF Lymphocytes isolated from a number of sources may be used to generate LIF and MIF, including blood, lymph nodes, and spleen.“5 These lymphokines have been produced in resporrse to a variety of stimulants, including specific antigens (tuberculin, monilia, and s~toki~~-~~~~), mitogens (coneanavalin A, phytohemagglutinin), mixed lymphocyte reactions, cornple and IgG contaming immune complexes. The antigen preparations are usually the same as for skin testing, and it is important to use dose responses of each stimulant to detect differences in the sensitivity of patients’ cells. MIF and LIF are produced by both T and B cells, null cells, and large granular lymphocytes. 4.3. Preparation of neutrophils

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520 Task Force Guidelines TABLE

VII. Cell migration

CLIN. IMMUNOL. SEPTEMBER 1988

assay systems

Direct (one step)

Lymph node or spleen explants (animal) Peritoneal exudate cells (many animal species) Capillary tube Agarose drop Human lymphocytes mixed with exudate cells Capillary tube Electrophoretic field Human buffy coat leukocytes Capillary tube Agar plate

Indirect (two

steps)

1. Culture of human lymphocytes separately to produce mediators (MIF or LIF) 2. Assay of culture supematants with indicator cells a. Peritoneal exudate cells b. Human buffy coat leukocytes c. Human monocytes d. Human PMN leukocytes Capillary tube Agar plate

Whole blood is allowed to sediment through dextran and is then subjected to Ficoll-Hypaque centrifugation. ‘I33II6 After removal of the mononuclear cell layer and the underlying Ficoll-Hypaque, the remaining cell pellet consists of fieutrophils and some erythrocyte contamination. 4.4. Preparation of monocytes Monocytes are isolated from the mononuclear cell fraction of the Ficoll-Hypaque centrifugation by further centrifugation on a Percoll continuous gradient.“’ 4.5. Capillary vs agar technique The migration inhibition technique initially used extensively in humans was a direct assay of the inhibition of migration of human peripheral blood buffy coat cells or an indirect assay that used guinea pig peritoneal macrophages. Indicator cells were allowed to migrate from capillary tubes placed in chambers containing medium and antigen or lymphokine-containing supematant. ‘I33I’* The capillary tube assay is very labor intensive and at times requires two people working half a day. Large numbers of indicator cells and lymphokine samples are required to perform this assay, and there is difficulty in quantitating the results. With the agar technique, purified leukocytes and antigen are placed in wells made in semisolid agarose in Petri dishes.“’ Leukocytes are allowed to migrate under the agarose, and the areas of migration are measured. With indirect techniques the purified indicator cells are placed in the wells and inhibited by coculture with appropriate lymphokine-containing supematants (Fig. 3). This remains a frequently performed technique for the detection of LIF but is generally not applicable to the measurement of MIF activity, since macrophages do not migrate well under the agar. Like the capillary tube assay, this method requires large volumes of whole blood and large quantities of antigen. The number of leukocytes that can be isolated limits the size of the experiment that can be performed in a day. The agarose microdroplet assay was originally described by Harrington and Stastny”’ with guinea pigs and tias adapted for human studies by McCoy et al. ‘*’ Small volumes of agarose and indicator cells (polymorphonuclear leukocytes or macrophages / monocytes) are mixed and placed in the bottom of a 96-well microtiter plate. The agarose is allowed to harden, and areas of migration of leukocytes out of the agarose droplet in each well are measured. The agarose microdroplet assay has several advantages over the capillary tube assay, including the ability to test for MIF or LIF activity, the use of several different antigens over a range of concentrations, technical simplicity, rapid completion, requirements for smaller numbers of cells (50% to 75% less), and a smaller lymphokine sample (80% or less). 4.6. Measurement of lymphokine activity MIF and LIF containing supematants are added to monocyte/neutrophil indicator cells placed in either capillary tubes or agarose plates. The areas of migration are measured at 24 hours at low magnification by using a microscope with a grid attachment.“* The percentage of migration inhibition is calculated by the formula:

VOLUklE 82 NUMBER 3, PART 2

Task Force

FIG. 3. Migration plicate

in alternate

of polymorphonuclear wells of control

% Migration

(large

inhibition

leukocytes under agarose. circles) and LIF-containing

Samples supernatants

~~ideiines

tested in quadru(sma//circ/es).

mean migration diameter of’ cells in test supematant = 1 x loo mean migration diameter of cells in control supematant

This migration assay has standard deviations ranging from 9% to 15% (mean, 6.9%). Greater than 20% migration inhibition represents significant MIF or LIF activity as determined by the MannWhitney rank sum test (p < 0.05). 4.7. Reproducibility and interpretation In assessing LIF or MIF production by sensitized lymphocytes, false-positive results may occur, since there may be nonlymphokine factors that directly inhibit random migration. These factors include antigen-antibody complexes and antigen itself. This may be more of a problem when interpreting the results of a direct migration inhibition assay. There is a 10% incidence of false-negative results in which the indicator cells do not respond to LIF or MIF because of individual variation. In such cases the assay should be repeated with another set of indicator cells to confirm deficient lymphocyte function. In general, population studies are recommended for the investigation of a disease rather than dependence on individual results. A positive result for LIF or MIF activity in response to antigen stimulation may in’dicate lymphocyte sensitization but may also result from nonspecific mitogenic activity of the test antigen. Furthermore, lymphocyte sensitization does not necessarily imply a cause-and-effect relationship between antigen reactivity and the pathogenesis of the disease. 5. Recommendations Whenever possible, more than one in vitro test of lymphocyte function should be measured, since each assay may detect a distinct subpopulation of cells. Furthermore, the present evidence indicates that lymphocytes are compartmentalized; that is, cells in the blood may be functionally different from those in the lymph nodes, spleen, or other organs. Therefore sampling of blood lymphocytes alone may not yield representative results. An evaluation should include quantitation of the numbers of T cells (and their subsets) and B cells, determination of proliferative responses to mitogens and specific antigens, measurement of at least one lymphokine, and determination of a cytotoxic response,

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If any of these functions are abnormal, assessment of suppressor cell activity would then be indicated. It should be pointed out that the precise role of suppressor cells in disease is presently an area of active investigation, and in many instances it is difficult to determine whether a defect is primary or secondary to the disease process. Because of their complexity, not all of these in vitro assays are carried out in most clinical laboratories; they are presently restricted to research centers that specialize in basic and applied investigation. Moreover, because of the nature of these assays, that is, they are biologic phenomena and subject to considerable variation, the results should be interpreted with caution. Therefore in an individual patient, a negative result should be repeated to confirm abnormal cellular function. For this reason, these tests are more useful when applied to the study of groups or populations of patients with certain diseases rather than to individual patients. Although they are not usually diagnostic, these tests are of clinical value for purposes of identifying certain pathogenetic factors, monitoring the results of therapy, and the clinical course of patients with depressed cellular function. With the advent of recombinant deoxyribonucleic acid technology, a number of lymphokines and cytokines have already been cloned and are mass produced. In the future, this should lead to the development of radioimmunoassays that are more sensitive and specific than the existing bioassays. Thus the availability of radioimmunoassay kits will allow more widespread application of in vitro cell-mediated tests to office and clinic settings. This should also permit the systematic investigation of their correlation with a variety of disease states. CHAPTER 4. CONCLUSIONS The participants of this workshop conclude that the National Institute of Allergy and Infectious Diseases (NIAID) should consider implementation of the following suggestions extracted from the workshop summaries of the Task Force on Guidelines for Standardizing Old and New Technologies Used for the Diagnosis and Treatment of Allergic Diseases. A. NIAID intramural evaluation of Task Force recommendations 1. Diagnostic type I skin tests a. Information about the precision and accuracy of current devices should be disseminated. Commercial developers of new instruments should be required to evaluate results compared with reference instruments. b. The use of bioequivalent extracts, expressed in AU or other agreed on equivalency units such as 1 HEP, should be encouraged for both research and clinical practice. c. The importance of extract stability should be stresed both to physicians and commercial manufacturers of extracts used for skin tests and relevant IgE in vitro tests. d. A consensus should be developed on standardized criteria for interpretation of type I skin tests. e. Variability of skin tests should be minimized by defining the conditions of the test as described in these proceedings. f. Proficiency test methods to evaluate the accuracy and precision of puncture and intracutaneous test methods should be developed and disseminated to the allergy community. 2. The LPCR a. Furtber research about concentration and dose-response effects in larger populations of clinically sensitive persons is encouraged. b. Cooperative studies will be required to standardize details about the time of reading and measurement of these tests. Development of more refined techniques of measuring skin inflammation should be vigorously pursued. c. Individual research products and/or multicenter cooperative programs concerning the relevance of late phase cutaneous response to late phase responses in the upper and lower airways are strongly encouraged. Such studies show promise of detecting persons who might have increased susceptibility to the effects of allergic inflammation. 3. Diagnostic type I in vitro tests a. Because of the proliferation of commercial in vitro diagnostic techniques, there is an urgent need to develop a consensus on recommendations for use of well-characterized reagents in these tests.

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b. Development of quantitative reporting systems of these tests should be explored as rapidly as possible. c. Federal and state agencies should encourage improved methods of quality control by commercial reference laboratories and individual physicians who perform these tests in their offices. d. The concept of national proficiency programs of these tests should be endorsed by both private and public sectors. e. The use of standardized extracts as reagents in these tests should become a prerequisite to licensing of these products. f. To protect the public against overmarketing of poorly characterized in vitro tests, the NIAID should consider the publication of lay literature on this subject in accordance with the principles elaborated on in these proceedings. g. It was generally agreed that basophilic histamine release is an excellent in vitro correlate of IgE-dependent reactions. The current technology of histamine measurements by chemical fluorometry and enzyme isotopic assays is well established. Further research should be encouraged to establish the validity of new histamine radioimmunoassays based on the availability of antihistamine monoclonal antibodies. 4. Diagnostic type IV skin tests a. There should be continuing research on establishing methods to improve patch test procedures, with particular emphasis on the size of the patch test, dispersion of the test agent within the vehicle of the patch, and the validity of various reading times. b. There should be increased emphasis on the importance of dose-response assays to determine appropriate concentrations of relevant allergens in clinical practice. In this regard, more efforts are required to develop linear reading scales by objective techniques. c. There should be continuing surveillance concerning the relevance of new allergenic contactants. These should be incorporated into routine patch test panels as soon as their reliability has been confirmed. d. Efforts should be channeled and supported to differentiate irritant from allergic reactions. e. Multicenter cooperative studies should be designed to standardize recall intracutaneous allergens, with special emphasis on the stability of these substances and rep&ability of the test conditions. Such studies should include standardization by quantitative dose-response tests. f. Cooperative studies are also urged to assess reliability of recall antigens in normal populations and immune deficient subsets over time. Built into the design of these experiments should be consideration of variables such as geographic locality, ethnic and racial factors, circadian or supracadian rhythms, and age. g. Demonstration programs should be encouraged for the purpose of establishing proficiency criteria in the performance of both patch and recall antigen tests. 5. Diagnostic type IV in vitro tests Although tests of lymphocyte function often have unpredictable variability and may not be useful in the diagnosis of individual cases, they are useful objective immunologic indexes in larger-scale population studies. Meaningful correlative studies in population surveys of patch and recall antigens should be encouraged. B. Consultation with NIAID Allergy Advisory Panel The Allergy Advisory Panel of NIAID should review these recommendations, with the goal of encouraging NIAID to offer specific contracts or request for proposals grant programs directed to implementation of key recommendations in this document. Multicentered, cooperative studies would be particularly amenable to these approaches. C. Permanent Diagnostic Guidelines Task Force Committee It is recommended that participants and/or representatives of the current Task Force be nominated to a permanent ad hoc advisory committee that would serve as a consultative resource for assigning priorities to request for proposals grant programs, for ongoing surveillance, and for future modification of recommendations pertaining to diagnostic modalities.

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