------------------------------------------------------------------------------ TITLE: EVALUATION OF THE ALLERGIC PATIENT Skin Endpoint Titration Versus Radioallergosorbent Testing SOURCE: Dept. of Otolaryngology, UTMB, Grand Rounds DATE: October 24, 1990 RESIDENT PHYSICIAN: Mark L. Nichols, M.D. FACULTY: Karen H. Calhoun, M.D. DISCUSSANT: J. R. B. Hutchinson, M.D., Atlanta, GA DATABASE ADMINISTRATOR: Melinda McCracken, M.S. ------------------------------------------------------------------------------- "This material was prepared by resident physicians in partial fulfillment of educational requirements established for the Postgraduate Training Program of the UTMB Department of Otolaryngology/Head and Neck Surgery and was not intended for clinical use in its present form. It was prepared for the purpose of stimulating group discussion in a conference setting. No warranties, either express or implied, are made with respect to its accuracy, completeness, or timeliness. The material does not necessarily reflect the current or past opinions of members of the UTMB faculty and should not be used for purposes of diagnosis or treatment without consulting appropriate literature sources and informed professional opinion." I. Basic Immunology A. Immunologic mechanisms of hypersensitivity are based on Gell and Coombs Classification, there are 6 types. 1. Type I: Immediate Hypersensitivity a. Antigen binds to 2 molecules of IgE or IgG4 attached to the receptor site (Fc) on the surface of a Basophil or a Mast Cell. b. This is followed by a release of chemical mediators: histamine, serotonin, bradykinins, leukotrienes, eosinophil chemotactic factor; which ultimately result in vasodilatation and increased vascular permeability as well as bronchoconstriction. c. Examples: allergic rhinitis, bronchial asthma, anaphylactic shock 2. Type II: Cytotoxic Antibody (IgG, IgM) a. IgG or IgM binds to the cell surface bound antigen activating complement which leads to lysis or agglutination of the target cell. b. Examples: hemolytic anemia, hemolytic disease of the newborn 3. Type III: Immune Complex a. Antigen - antibody complex combines with complement to form immune complexes, these deposit in the tissue. b. Vasoactive amines are liberated and inflammation of the involved tissue occurs c. Examples: autoimmune disease, serum sickness, some types of nephritis 4. Type IV: Delayed Hypersensitivity a. Sensitized T-cells (lymphocytes) cause target cell injury or death by direct cell attachment or release of mediators. b. Examples: Contact dermatitis, graft and tumor rejection. 5. Type V: Stimulating Antibody a. IgG antibodies reacting with tissue receptors b. Example: Graves disease 6. Type VI: Antibody Dependant Cell Mediated Cytotoxicity (ADCC) K-cell, Null cell (non T or B) a. K cell exert a cytotoxic effect on target cells that are coated with antibody, without being previously sensitized. b. This cytotoxic interaction is complement independent, brought about by a K-cell which has Fc receptors binding to the Fc portion of the antibody molecule on the cell surface. B. Basic Cell Types Involved in Immunity 1. Phagocytic cells: Tissue macrophages and circulating monocytes. a. Do not specifically interact with antigen. b. Capable of concentrating antigen on their surface for presentation to T and B lymphocytes. c. Macrophages release biologically active soluble substances: prostaglandins and interleukins which regulate lymphocyte activity. 2. Immunologically Reactive Cells: lymphocytes and plasma cells. a. Lymphocytes (2) types: (1) B-cells: release specific immunoglobulins (IgG,M,A,D,E) or antibodies responsible for humoral immunity following exposure to antigens. (a) primary and secondary humoral immune responses. (b) when antibody binds to antigen on the surface of the B-cell, it can clonally differentiate into many plasma cells which release immunoglobulin specific for that antigen. (2) T-cells: thymus dependant, heterogeneous group of cells responsible for cellular immunity (a) Can function as effector cells in transplant rejection, delayed type hypersensitivity, have specificity for antigens of infecting viruses and foreign cells but also self: major histocompatibility complex antigens. (b) Helper T-cells: interact with B- cells to allow them to produce antibody, interact with other T- cells to produce cell mediated responses. (c) Suppressor T-cells inhibit humoral or cell mediated responses. (d) Null cells: not identifiable as T or B cells, Participate in ADCC (K- cell), Natural Killer cell capable of eliminating cells which have become spontaneously malignant, there is increased NK cell activity when exposed to interferon. 3. Mechanism of Type I Immediate Hypersensitivity Reactions a. IgE is the primary immunoglobulin involved with the immediate hypersensitivity response. (1) Initial exposure to the antigen produces clonal differentiation of memory cells (B-cells, plasma cells) capable of producing antigen specific IgE. (2) Secondary exposure - much greater production of IgE (3) IgE is present in thee skin, respiratory secretions and serum, as well as the GI tract. (4) IgE binds via Fc receptor portion of molecule to the surface of tissue mast cells and circulating basophils. (5) When two IgE molecules are bridged on a subsequent exposure to antigen, the mast cell degranulates releasing vasoactive mediators which are responsible for the physiologic changes seen in allergy, most are secondary to histamine. (a) increased vascular permeability (b) smooth muscle contraction in the respiratory and GI tract (c) local increase in eosinophils (d) symptoms: sneezing, rhinorrhea, irritation, lacrimation, increased bronchial secretions b. The basic defect in allergy is probably cell- mediated (1) Evidence indicates that the overproduction of IgE is not the primary defect, it is probably a defect in the suppressor T-cells controlling the production of IgE. 4. Inhalant Antigens: there are 4 major classes of organic proteins ranging from 2-60 microns in size. a. Pollens: seasonal flux, trees in spring, grasses in summer, weeds in the fall. (1) peak in early AM, attenuated by central A/C. b. Molds: (1) Alternaria is the most prominent allergen (2) Flourish in humid climates and low altitude. (3) May be found in dry climates wherever moisture collects (plumbing etc.) (4) Symptoms usually perennial. c. Dusts: (1) Housedust is comprised of 28 known or suspected allergens, most important is the house dust mite. (2) Symptoms usually perennial secondary to central A/C. d. Epidermals: (1) Most virulent of antigens. (2) Includes hair/dander of domesticated animals (dogs,cats,horses, cattle). II. Skin Endpoint Titration: Theory and Practice A. Background: 1. Developed by Hansel in 1935, used serial dilution testing to establish guidelines for coseasonal treatment of hay fever. 2. Rinkel studied Hansel's techniques and began using SET routinely in his practice. a. Skin tested patients with 5 fold dilutions (1:5), using biologic skin sensitivity to determine initial therapeutic allergen potency, treatment vial contains varying allergen concentrations. B. Traditional Allergy Testing: many disadvantages 1. Screening is performed with prick testing, the reaction is read as a measurement of the amount of wheal and erythema, scale 0 to 4+, it is assumed that a 3+ or 4+ reaction correlates with a high probability of a clinically relevant atopic sensitization. 2. If prick testing is negative or equivocal, a single dilution test is done intradermally, rarely with a concentration of greater than 1:5000 w/v 3. Treatment is begun usually at a concentration of 1:5,000 with each antigen at the same strength, treatment escalates to a maximum tolerated dose. 4. Much less specific than SET testing, the final dose of single dilution - based immunotherapy is limited by the most reactive antigen. 5. This may result in clinically important but less reactive antigens failing to achieve the necessary dilution strength required to relieve symptoms. 6. General Allergists rarely test with dilutions stronger than a 1:1000 or 1:5000 w/v because many believe that above these concentrations that there are too many false positive reactions. C. Advantages of SET: compared to single dilutional techniques the advantages are both qualitative and quantitative. 1. Qualitative advantages: a. In comparison to the scratch and prick techniques, SET offers safety, readability and standardization (1) tests are begun at dilute doses, unlikely to give adverse reactions (2) standardized doses are administered via standard techniques (3) positive responses are well defined and are not equivocal b. SET is more sensitive c. A range of allergens is available for testing and antigen vials can be prepared from almost any agent that is suspected from the patients history 2. Quantitative advantages: a. SET allows for therapy institution at higher initial antigen doses. (1) this permits more rapid achievement of the maximally tolerated dose or symptom relieving dose (2) this ultimately results in fewer injections, fewer office visits, greater convenience for the patient and reduced expense compared with conventional techniques b. The potency of each allergen included in a multiantigen vial can be varied independently according to the degree of sensitivity exhibited. (1) dosages for each allergen can then be advanced equally (2) this is unlike the conventional single dilution technique as practiced by general allergists. 3. SET allows for reassessment of quantitative sensitivity during peak season (coseasonal) or anytime symptom relief is inadequate so that antigen doses may be appropriately adjusted. 4. Studies have demonstrated that SET based immunotherapy results in much higher antigen doses than single dilutional technique based therapy (Lee, Knicker and Campos 1982) SET patients demonstrated a greater reduction of total and specific IgE than traditional groups. D. Disadvantages of SET 1. Not tolerated well by some children. 2. Cannot be used in patients with severe dermatologic conditions, may lead to false positives in patients with dermatographism. 3. False positive results not uncommon. 4. No clinically significant correlation between the degree of sensitivity exhibited by skin testing and the severity of the patients symptoms. 5. Does not indicate the final antigenic dose required for therapeutic relief of symptoms. 6. Skin test responses do not decrease consistently with clinical improvement. E. Techniques 1. May be used with any dilution progression, the method proposed by Rinkel uses 1:5 dilutions serially, others have been tried which vary from 1:2.5 to 1:20 concentration and were found to be less sensitive and specific. 2. Sterile vials are obtained containing 4cc sterile normal saline diluent with 0.4% phenol as preservative. 3. Most allergen extracts are delivered as 1:20 w/v concentration. a. The basic testing board should include representative allergens from the four groups of aeroallergens. (1) 2 or 3 each of the appropriate tree, grass, and weed pollens as indicated by area survey and what other allergists in the locale have found to be useful. (2) 2 or 3 appropriate molds including alternaria. (3) Epidermal antigens suspicious from the patients history should be included. (4) House dust mite should always be included, it typically is supplied as 1:20 w/v extract. 4. Most extracts use glycerin-saline as the solvent for allergen extract usually a 50% concentration. a. To standardize the amount of glycerin in all #1 dilutions, all #1 dilutions should be prepared equal to 1:100 w/v except house dust mite which will be 1:500 w/v concentration. b. If too high a concentration of glycerin is injected subcutaneously or intradermally, the injection is painful, irritating, and often results in a false positive wheal and flare. 5. The #1 dilution is prepared by adding 1cc of 1:20 antigen concentrate to 4cc of diluent and mixed thoroughly to make a 1:100 dilution. a. 1cc of the #1 dilution is then added to 4cc of saline diluent to make a #2 dilution or 1:500 concentration. b. These steps are repeated making consecutive 1:5 dilutions through vial #10 for each antigen. 6. Once prepared the test boards should be kept refrigerated at 2 degrees C when not in use and remade every 6 weeks if left unrefrigerated and every 3 months if refrigerated. 7. SET is based on intracutaneous wheals. a. Requires practice and skill to get reproducible results b. Volume required to produce a wheal 4 mm in diameter that is blanched with sharp margins is 0.01cc. c. To prevent allergenic and infectious contamination, disposable sterile 26 gauge syringes with 3/8 inch needles with prefixed hubs are used. d. The needle is inserted in the most superficial layer of the skin, bevel down, until the bevel is immersed in the skin, injecting 0.01cc of the antigen. e. Screening wheals of different antigens of the same dilution are applied vertically in succession, one over the other on the lateral surface of the upper arm, at least 3cm apart. SET wheals of the same antigen are placed horizontally across the lateral surface of the arm with the weaker dilutions applied medially. 8. SET Starting Points a. The skin may respond positively to a dilution which is weaker than that to which the patient is sensitive, but not vice-versa unless immunodeficient. b. The modified RAST test has demonstrated that no one has a true sensitivity of greater than a #6 dilutional endpoint (1:312,000), thus one may start safely with a #6 dilution without risk to the patient if the patient gives a history of severe allergic problems or angioneurotic edema or asthma. c. Typically can start with a #4 dilution if the history is negative, if this test is negative then one may progress to the #2 dilution for testing, reading the wheals after a 10-15 minute waiting period. d. The endpoint is the dilution that provides the first positive wheal, i.e. a wheal that has enlarged by 2mm greater than the immediately preceding negative wheal and it also initiates progressively larger positive wheals 2mm or greater on application of consecutively stronger 1:5 dilutions. The endpoint also indicates the dilution at which immunotherapy may be started safely either preseasonally or coseasonally. e. Controls are important for several reasons. (1) A negative control is required to identify a positive reaction. Usually dilutions are made from a 50% concentration of phenolated saline diluent, made in successive dilutions. A #2 dilution of glycerin without the antigen serves as negative control. Some may react to the glycerin alone if in too high a concentration. (2) Positive control - if all the tests are negative will need to check immunocompetence. A #3 dilution of histamine phosphate is used as a control. f. Variations in the whealing response occur in as many as 30% of tests applied, and may indicate the concurrent presence of food allergies. (1) Flash response: a hypersensitivity response occurring to a dilution weaker than the true endpoint, i.e. a 25mm response to a #4 or #6 dilution. (a) Not usually reproducible, may identify true endpoint by starting at a weaker dilution and advancing to endpoint. (b) Often must wait 24 hours to repeat the test. (c) Usually indicates an outdated antigen (d) Don't base immunotherapy on this dilution. (2) Hourglass Response (a) Progressively larger dilutions produce less positive skin responses until at some point the classic pattern of progressively larger wheals appear. (b) Endpoint is that dilution that initiates progressively increased responses of 2mm or more. (3) Plateau Response (a) Identical size positive whealing response occurs to each progressively stronger dilution until the classic pattern emerges with positive wheal growth. (b) The positive responding wheal that initiated the progressive response is the endpoint. III. Radioallergosorbent Test or RAST A. Background information 1. RAST is a radioimmunoassay test developed in the late 60's for the detection of specific serum IgE antibodies. Initial studies demonstrated a 96% efficiency, sensitivity and specificity. 2. The modified RAST is the form now used, introduced by Fadal and Nalebuff in 1977 with the advantages of increased test sensitivity without a loss in specificity. B. Method - Principles of RAST 1. Soluble allergens bound to solid phase support (paper disc) to create a stable immunosorbent media. 2. The paper disc is incubated with the test sera, specific IgE antibody will bind to the solid phase allergen. 3. The paper disc is then washed to remove all unbound sera and IgE. 4. The disc is then exposed to rabbit anti-human IgE antibodies which are radiolabeled. It interacts with the Fc determinant portion on human IgE bound to the solid phase allergen. 5. The unbound anti-IgE is washed off the disc and the disc is then quantified by a scintillation counter. C. RAST Utility in Allergy Diagnosis 1. It reliably detects allergen specific IgE antibodies in the serum and accurately assays its serum concentration, thus the degree of sensitization. 2. It has been successfully employed to determine IgE levels for specific antigens such as animal danders, pollens, stinging insect venoms, viruses and other substances shown to produce elicit specific IgE antibody. 3. RAST has been demonstrated to be dependable in the detection of IgE mediated food reaction. D. Correlation of RAST with Sensitivity 1. Modified RAST scoring system Class Counts Interpretation 0 250-500 negative 1/0 501-750 equivocal 1 751-1600 usually positive 2 1601-3600 usually positive with 3 3601-8000 increasing level of specific 4 8001-18,000 IgE 5 18,001-40,000 2. MRT Class I a low positive score, IgE is detectable but may not be clinically relevant. Class I usually associated with a positive skin test. a. Only 50% of the patients respond when suitably challenged. b. Studies have demonstrated that MRT scores of less than class 2 have skin reactivity not significantly different from a 2% glycerin negative control. 3. Class 2 MRT and above (>1601 counts) represent positive scores with increasing level of detectable specific IgE antibody and degrees of sensitivity. a. More than 95% of the patients with these scores respond to properly performed nasal or conjunctival challenge tests. E. RAST Allergen Screening 1. Concept useful as a method of controlling costs in allergy diagnosis and therapy. 2. Mini RAST screens available with about 5 antigens which are representative of their specific allergen group. 3. Useful for the detection of truly atopic patients and to avoid performing multiple tests on those not allergic. 4. Less than 1% of atopic patients should be negative to a panel of 5 selected allergens. 5. The screen has been demonstrated to be 97% sensitive in comparison to large RAST test screens. 6. Positive responders should have additional testing performed. F. Advantages of RAST over SET 1. Convenient for the patient, single blood sample required. 2. No risk of allergic reaction to injected antigens 3. Results are quantitative 4. Results are not influenced by antihistamines or other medications. 5. Useful in apprehensive patients and children 6. Useful in patients with dermatologic conditions in whom skin testing is contraindicated. 7. Of use in patients unable to stop medications which could influence test results. 8. Less time consuming for patient than skin testing 9. RAST is the most reproducible procedure for identifying allergies, reagents can be kept for years without losing potency. G. Disadvantages of RAST 1. More expensive on a per test basis than SET 2. Takes longer to get results - 3 days. H. Inappropriate Uses of RAST 1. As a screening test without examination by a physician. 2. In a patient with a positive history of sensitivity in whom non-specific therapy is effective in alleviating symptoms. 3. In rhinitis patients already doing well on allergen immunotherapy. 4. In symptomatic patients who have had properly performed skin tests at adequate concentration with potent extracts. 5. In patients with total IgE levels less than 10 units/ml. IV. Treatment A. Avoidance B. Pharmacologic 1. Antihistamines 2. Antihistamine-decongestant combinations 3. Cromolyn sodium 4. Steroids - topical, systemic C. Immunotherapy 1. Rationale a. Block mediator release, relieve allergic symptoms. b. T-suppressor cells and IgG4 antibody play a role. c. When allergen is injected IgG4 antibody specific for the allergen is produced. d. Elevated IgE levels decline during immunotherapy secondary to IgG4 blocking the synthesis of IgE production by neutralizing the antigen. e. IgG4 antibodies are then preferentially produced to IgE antibodies by a separate set of B and T cells. f. Antigen specific T suppressor cells may suppress plasma cell production of IgE. 2. Indications: a. Symptoms correspond with specific allergen exposure. b. Positive tests confirm the presence of IgE antibody. c. The patient is unresponsive to environmental controls d. The allergen cannot be avoided e. Inadequate response to pharmacologic management f. When the severity of symptoms outweighs the hassles and cost of immunotherapy. g. When the patient is likely to comply with the rigid schedule of injections for 2-3 years. 3. SET Based Immunotherapy a. Therapy for each antigen is begun at a strength of 5 times the endpoint dilution b. Each succeeding dose is increased by 5 times the endpoint dilution. c. Several antigens are mixed in the same treatment vial. d. Injections are given 1-2 times weekly until the maximum tolerated dose is reached, then the frequency of injections is decreased to the minimum required to maintain the therapeutic effect. e. The maximum tolerated dose has been reached when: (1) An erythematous, hot, injection site occurs. (2) Clinical symptoms occur shortly after injection. (3) A systemic reaction occurs after injection. f. 2-3 years of therapy are usually adequate to maintain tolerance, if symptoms recur then another course of therapy is instituted. 4. RAST Based Immunotherapy a. Can start immunotherapy based on RAST class scores with confidence, using a well documented schedule. b. Must place a small dose skin test challenge before institution of immunotherapy. c. The RAST based schedule allows delivery of higher doses of antigen earlier than SET based therapy. d. Higher levels of blocking IgG4 antibody are produced earlier in RAST based therapy versus SET based therapy. 5. Immunotherapy Failures: Causes a. Incorrect diagnosis: wrong antigen treated b. Inadequate environmental control measures c. Maintenance doses inadequate d. Maintenance doses too high e. Presence of concomitant food or chemical sensitivity. f. Underlying medical problems not addressed such as nasal polyps or chronic sinusitis. g. Patient uses medications that cause nasal congestion. i.e. reserpine, oral contraceptives etc. V. SET versus RAST A. Both have similar advantages, they provide a quantitative and qualitative diagnosis, they indicate the type and degree of allergy. B. Comparison between SET and RAST: Summary 1. SET is more sensitive than RAST, however there is a propensity for intracutaneous tests to have false positive results, thus requiring negative and positive controls. 2. RAST is more specific than SET, it disallows unnecessary allergen or patient treatment that may result after skin testing. 3. RAST allows for a stronger starting immunotherapy dose than SET based therapy. 4. Studies have indicated that RAST therapy based groups elicited a greater amount of IgG4 blocking antibody than SET based methods, both RAST and SET elicited a much greater amount of blocking antibody than the classical single dilution based methods. 5. RAST is more convenient for both the patient and physician. 6. RAST testing is more expensive than SET on a per test basis, taking longer to get results than SET, but is more reproducible than SET. 7. SET being less expensive is also more time and labor intensive requiring experienced personnel to perform, however the results are immediate. 8. Neither test has been shown to be an effective guide to the final therapeutic dose. 9. RAST and SET may be useful as a guide to a safe initial immunotherapy dose and for quantifying patient sensitivity. 10. A comparison of skin testing and RAST in situations where the prevalence of disease is low, i.e. 20%, the predictive value of a positive skin test is less than 50%, however the corresponding value of a positive RAST test is 96%. 11. When test specificity i.e. for RAST is high - 95% , there is a 50% chance that one test in a panel of 15 tests will labelled as falsely positive. VI. Practice Trends Otolaryngologists and Allergists: A. A survey by the California Medical Association Scientific Panel on Allergy was published in August 1985. 1. Survey consisted of responses from 103 Allergists and 186 Otolaryngologists. 2. Of the Otolaryngologists, only 18% surveyed treated allergies in their practices. 3. Skin testing was performed by a majority of both the Allergists and Otolaryngologists, with most of the allergists using the 2 step prick, intracutaneous test. A majority of the Otolaryngologists used the SET method. 4. RAST was used rarely or not at all by 90% of the allergists and 80% of the Otolaryngologists. 5. For the diagnosis of food or inhalant allergies, intracutaneous methods were used mostly by both groups, but immunotherapy was rarely used to treat food allergies. ---------------------------------------------------------------------------- DISCUSSANT'S REMARKS: ALLERGY TESTING J. R. B. Hutchinson, M.D., Atlanta GA April 27, 1995 1. When allergy testing has been performed by in vitro methods e.g. Rast, it is imperative that a test wheal be applied prior to beginning immunoRx since the patient has never been exposed to the antigen during testing, as in the case of skin testing. The discussant stated that this " should " be done. 2. Cat control is extremely difficult. Cats go and do as they please and in my experience it is the exception rather than the rule that one can control this. Since the patient would prefer to remove the allergist from their environment rather than lose the cat, I have just advised them of the limited benefit and increased risks involved and moved on from there. Only in the case of an asthmatic have I found it necessary to insist on removal of the pet. This was done to protect the physician as well as impress upon the parent the importance of this matter. 3. Astemizole blocks the reactivity of the skin and precludes skin testing and thus removes an important monitor for patients on immunoRx. This effect has been reported to last up to seven weeks and I have seen it last for four in some of my patients. It would also preclude performance of a test wheal prior to starting immunoRx and thus I do not use this medication if immunotherapy is a possibility. 4. While it is unfortunate that there is a division amongst allergy physicians treating allergy patients it may be best to refer to the " General " Allergist by their long formal name as agreed to many years ago. This is an allergist certified by the conjoint board of Internal Medicine and Pediatric Allergy. 5. The role of immunoRx appears to be of diminished importance with the advent of non sedating antihistamines and steroid nasal sprays. These give significant relief and in the patient that has a seasonal problem only, are usually safe. Life style changes in our society make compliance more difficult than in past times. Socioeconomic issues have changed the outlook on therapy as well. The percentage of patients requiring immunoRx has thus decreased over the past decade. While immunoRx is effective in the majority of patients; the length of therapy, expense, time consumed and other factors need be considered when offering this therapy. ------------------------------------------------------------------------ Bibliography 1. Becker, G: Introduction to Otolaryngic Allergy; AAO-HNS, 1986. 2. King, WP: Efficacy of a screening radioallergosorbent test. Arch. Otolaryngol. 108:81, 1982. 3. Krause, HF Ed.: Otolaryngic Allergy and Immunology; Saunders, 1989. 4. Lee, KJ Ed.: Immunology and Allergy; Otolaryngol Clin North Am., 1985. Nov. 18(4). 5. Lee, LK; Knicker, WT; Campos, T: Aggressive coseasonal immunotherapy in mountain cedar allergy., Arch. Otolaryngol. 108:787,1982. 6. Mayer, DB; Nelson, HS: Use of modified radioallergosorbent testing in determining initial immunotherapy doses., Otolaryngol. Head Neck Surg. 93:335, 1985. 7. Mc Millan, T.: Nasal Allergy: Grand Rounds Presentation, Jan 17, 1990. 8. Nalebuff, DJ; Fadal, RG; Ali, M: Determination of initial dose for ragweed hypersensitivity with modified RAST, Otolaryngol. Head Neck Surg., 89:271, 1981. 9. Osguthorpe, JD;Ed.: Current developments in Otolaryngic allergy; Ear Nose Throat J. 1990 Jan. 69:1, 6-70. 10. Radford, ER; Berkowitz, EC: The use of intradermal tests and relevance of negative controls in patients with negative or equivocal modified RAST test scores to inhalant allergens; Laryngoscope 97:675, 1987. 11. Terr, AI; Weiskopf, A; Sporacino, R: Allergy practice in California - diagnostic and therapeutic methods used by Allergists and Otolaryngologists; West J. Med. 1985 Aug; 143:276-279. 12. Trevino, RJ: IgG levels after treatment with antigen vials based on scratch testing, intradermal testing and modified RAST testing; Otolaryngol. Head Neck Surg. 1986, 95:307. ------------------------------END---------------------------------------------