Radioallergosorbent test (RAST) VII Ö1 1 reliable tool or poor substitute? Edward W. Hein, M.D. An in vitro method, the radioallergosorbent test (RAST) has been developed for the detection of allergen-specific antibodies of the IgE class. Review of the literature shows that in comparison to skin testing, the RAST has a high degree of correlation (60% to 90% depending on the antigen); however, this method is not as sensitive as other tests (50% false-negative). The RAST is affected by blocking antibodies (IgG), resulting in false-negative values and high levels of IgE that bind on the allergen discs, giving false- positive findings. Because of these problems, RAST is somewhat limited for use in the clinical setting. Index terms: Allergy and immunology • Radioallergosor- bent test (RAST) Cleve Clin Q 50:361-366, Fall 1983 Skin testing has been the traditional method for diagnosis of IgE-mediated allergic disorders. This bioassay is highly sensitive, cost-effective, and safe when used by experienced personnel. In 1967, Wide et al,1 in Sweden, reported a new technique capable of detecting the minute quantities of allergen-specific IgE antibodies that circulate in the serum of allergic patients. This laboratory procedure, called RAST (radioallergosorbent test), utilized a solid- phase radioimmunoassay method. During the last decade this in vitro test has been refined and is now a commercially 1 Departments of Pediatric and Adolescent Medi- available laboratory test for clinical laboratories and, in kit cine, and Allergy and Immunology, The Cleveland form, for physicians' offices. Proponents of this new Clinic Foundation. (E.W.H., Head, Section Pediatric method claim that its results are more objective, safer for Allergy and Clinical Immunology.) Submitted for pub- lication May 1983; accepted June 1983. the patient, and not affected by symptoms or medication. 361 Downloaded from www.ccjm.org on September 24, 2021. For personal use only. All other uses require permission. '362 Cleveland Clinic Quarterly Vol. 50, No. 2 Mechanism of the radioallergosorbent test reference B had binding ability similar to 10 units The radioallergosorbent test is used to identify of IgE in the PRIST system (Fig. 1). the presence of IgE antibodies against specific In the Phadebas RAST scoring system, serum allergens. Allergens are coupled to solid-phase with bound radioactivity less than that obtained polysaccharides (Sephadex) or linked to filter pa- with standard D (which has been tested against per discs by cyanogen bromide. These solid-phase its homologous birch disc) is considered to have allergens are then exposed to serum containing an undetectable level of allergen-specific IgE. To antibodies to the allergens; during incubation, correlate these values with results of allergy skin antibodies to the allergen, including IgE antibod- testing, the reference curve is divided into classes. ies, bind to the allergen. After being washed to Values < 0.35 PRU/ml, class 0; values 0.35-0.7 remove unbound antibodies, the solid-phase al- PRU/ml, class I; 0.7-3.5 PRU/ml, class II; 3.5- lergen-antibody complex is exposed to radioiod- 17.5 PRU/ml, class III; > 17.5 PRU/ml, class inated, purified anti-IgE antibody. This complex IV. The higher the score, the more specific IgE is washed again, and the bound radioactivity is is present. measured. The radioactivity bound to the disc is The principal criticism of the Phadebas RAST an indirect measurement of the amount of spe- method is that sensitivity is set too high, thereby cific IgE present in the patient's serum. missing a significant amount of antibody, which 4 One of the greatest difficulties with RAST has may be clinically important. Muittari found pos- been understanding the confusing scoring sys- itive nasal and bronchial provocation responses in 50% of the patients with negative RAST test tems for determining test results. The present 5 Phadebas RAST (PRU) scoring system uses four results. Deuschal and Johansson also described reaginic reference standards: standard A, which 18 patients with allergic rhinitis (positive by prov- is pooled serum from patients highly sensitive to ocation, history, and skin tests) who failed to have birch pollen allergen; standard B, which is a detectable levels of allergen-specific IgE by the fivefold dilution of standard A; standard C, which Phadebas RAST. By diluting reference D five- is a fivefold dilution of standard B; and standard fold, (the cutoff point was lowered to 0.2 PRU/ D, which is a twofold dilution of standard C.2 To ml), they improved the sensitivity of the tests. Their test data showed significant skin reactivity determine the RAST units (PRU/ml), references 6 A, B, C, and D are assigned values of 17.5, 3.5, in all these patients. Nalebuff observed that a 0.7, and 0.35 PRU/ml respectively. In accord tenfold dilution of reference standard D with with this assignment, the counts for each refer- human cord serum (to 0.1 PRU/ml) still gave a ence serum versus assigned PRU/ml are plotted binding value greater than twice that obtained logarithmically. The PRU of each unknown sam- from negative controls. As a result of these ob- ple can be determined by noting where its count servations and in response to many criticisms falls on this reference curve. The PRU is only a leveled at the test for its allegedly low sensitivity, a new modified RAST system was developed by relative term and does not quantitate specific IgE; 2 however, Lundkvist3 observed that the Phadebas Fadal and Nalebuff. This system extends the initial incubation period from three to 18 hours. It increases the test volume from 50 to 100 yul. Class IV The discs are removed from the original tubes and placed in fresh tubes before counting. To Class III keep counts constant from day to day despite B variation in temperature and isotope decay, a Class II time control is incorporated using 25 units of C IgE, which reacts with the RAST isotope, and Class 1 the time required to reach 25,000 counts is de- : Class 0^ termined. In this assay the lower limit of detect- able levels of allergen-specific IgE, or the cutoff level, is 1.5 times the binding of negative control. Therefore, the cutoff point is 750 counts. Serum 0.35 0.7 3.5 17.5 between 250 and 750 counts is recorded as neg- PRU ative, indicating allergen-specific IgE antibody Fig. 1. Serial dilutions of reference standard A (PRU) are below detectable levels. The lower cutoff point plotted against the radioactivity counts for each dilution. Downloaded from www.ccjm.org on September 24, 2021. For personal use only. All other uses require permission. Fall 1985 Radioallergosorbent test (RAST) 363 of this system is 0.02 units of IgE (Fig. 2). Because the bound radioactivity varies in direct propor- tion to the square root of the change in serum concentration, a reference curve is established by using 5X multiples of the cutoff point. This ref- erence curve is then divided into classes to sim- plify reporting. The negative control has a count of 750. It is felt that samples falling into class I or 0 contain insignificant amounts of specific IgE. The higher the class, the more specific IgE is present. Proponents of the modified RAST scor- ing system allege that the diagnostic sensitivity of the test has increased, thus permitting identifi- cation of patients with lower, but clinically signif- icant IgE antibody levels. Clearly, a physician (25) 0.022 0.10 0.50 2.50 12.50 62.50 faces a dilemma in determining the significance IgE (U/ml) of the laboratory test in relationship to the clinical Fig. 2. The reference standard is a known serum sample con- condition of the patient and in choosing the taining 25 International units IgE per ml, which has been deter- appropriate test. mined to have a radioactivity count of 25,000 cps. The lower cutoff point is set at 500 counts, which is a fivefold dilution of the reference standard. Serum samples with counts below 500 cps contain insig- Comparison between radioallergosorbent and nificant amounts of specific IgE. Test results are reported as classes, other diagnostic tests for atopy depending on the amount of specific IgE present. Initial studies of RAST have compared its abil- and herein lies the controversy. Proponents of ity to identify patients with allergy to danders, skin testing maintain that RAST is not sensitive pollens, or foods with that of provocation tests enough and will miss a significant number of and skin tests. Agreement between these proce- allergies. Proponents of RAST maintain that skin dures has ranged from 59% for allergies to house testing is too sensitive and that the additional dust to 93% for fish allergies. Wide et al1 showed allergies identified by skin testing may not be a 68% agreement between results of 140 intra- clinically significant. dermal tests and RAST using 14 different anti- Recently an independent evaluation of these gens. Stenius and Wide7 found 80% agreement RAST systems was completed at the Mayo Clinic between RAST and prick skin tests with extracts by Santrach et al8 (Table). Forty patients with of house dust and house dust mite in patients allergic rhinitis to at least one of nine different with histories suggesting house dust allergy. In pollens (ragweed, English plantain, orchard, tim- the Stenius study, patients with discordant prick othy, alder, elm, oak, birch, and maple) were test and RAST results usually had positive skin studied by provocation testing, skin testing (prick tests and negative RAST; in most cases the neg- and intradermal), Phadebas RAST, Fadal/ ative RAST results correlated with negative Nalebuff modified RAST, and an expanded provocation results.