The Importance of Glycerin-Containing Negative Control Tests in Allergy Research Studies that Use Intradermal Skin Tests

OBJECTIVE: We sought to assess skin whealing with glycerin-containing control injections for intradermal skin tests. DESIGN: Observational. METHODS: Wheal sizes were measured at 0, 10, and 15 minutes after intradermal injection of 0.01 and 0.02 mL of phenolated normal saline and 0.5% and 5% concentrations of glycerin in the same quantity of phenolated saline. RESULTS: Intradermal injection of 0.01 mL of phenolated saline produced an average 4.9-mm wheal, which expanded to 5.2 mm at 10 minutes and to 6.0 mm at 15 minutes. Intradermal injection of 0.02 mL of phenolated saline produced a 6.4-mm wheal, which expanded to 7.0 mm at 10 minutes and 8.0 mm at 15 minutes. The addition of glycerin produced proportionally larger wheals. CONCLUSIONS: Because glycerin increases whealing beyond that with phenolated saline, skin tests containing glycerin must be compared with glycerin-containing negative controls. Intradermal skin tests that fail to compare findings in this manner contain an inherent methodologic flaw and are uninterpretable. A major issue in allergy testing is deciding whether the observed skin response is truly indicative of the patient having a clinically relevant, IgE-mediated reaction. 1 Skin test results are influenced by many variables, including patient skin response, specific technique, and tester consistency. Wheal measurement, comparisons with positive and negative control solutions, and interpretation are of equal importance. The development of in vitro methods for allergy diagnosis has helped to independently verify the accuracy of skin tests. In some cases, poor standardization of antigen sources and testing techniques has been shown to lead to discrepancies between skin tests and in vitro IgE antibody results of more than 100-fold. 2 It is also possible for skin tests to be falsely negative, as has been shown by comparing IgE blood tests with both skin tests and challenge tests. 3 Conversely, skin tests may be falsely positive because of nonspecific irritants, such as glycerin, present in allergen solutions. 4,5 Recommendations for immunotherapy must be based on clinical appropriateness as related to valid testing of proposed therapeutic agents. Recent reports by Nelson et al 6 and Wood et al 7 have suggested that skin prick tests (SPTs), even when negative, are sufficiently sensitive to diagnose clinical atopy without the need for further intradermal skin tests (IDTs). Both authors describe performance of a single IDT with injection of 0.02 mL of antigen solution. The basic tenant of their methodology is that all wheals resulting from an IDT measuring 6 mm or greater, accompanied by erythema, are to be recorded as positive. Nelson et al took measurements at 15 minutes, and Wood et al took measurements at an unspecified time. We were concerned that their methodology for IDTs created many false-positive results. This led to the condemnation of IDTs by these authors, stating that “a positive intradermal skin test response to Timothy grass in the presence of a negative skin prick test response to Timothy grass did not indicate the presence of clinically significant sensitivity to Timothy grass.” 6 We found the conclusion based on their particular IDT method to be suspect for 2 reasons: (1) it categorically assumes that an injection of 0.02 would produce a wheal of less than 6 mm and (2) it ignores the effects of small concentrations of the preservative glycerin, used in most all allergy test solutions, on whealing. Either assumption would lead to frequent false-positive skin test interpretations and discredit 60 years of intradermal testing. We therefore sought to evaluate control tests appropriate for use with the methodologies published in the general allergy literature to determine whether a 6-mm wheal with erythema should appropriately be interpreted as a positive or as a negative test.