CHAPTER 10
Blending Skin Testing and In Vitro Testing in Clinical Practice
It is a basic axiom of the field that the diagnosis of allergy is made on clinical grounds. Clinical evaluation of the patient, however, does not identify the offenders. The most direct method of confirmation of inhalant offenders is by mucosal challenge, which remains a useful research tool but has not been popular with patients since Leonard Noon first used it in 1911. Skin testing, in several forms, remains the benchmark against which all other allergy tests are measured. However, in vitro testing has become increasingly popular since the characterization of immunoglobulin E (IgE) in 1967. To this day, disagreement continues between proponents of skin testing and those of in vitro testing about the validity and clinical usefulness of each of these methods. The modern practitioner of otolaryngic allergy should understand both, and may actually employ both in the practice. With a firm grasp of the principles of each method, it is possible to move back and forth between them, utilizing whichever is most appropriate for the circumstances at hand.
PRINCIPLES COMMON TO SKIN TESTING AND IN VITRO METHODS
Many types of skin test are available. Undoubtedly the most accurate and reproducible, however, is the method of intradermal testing in which progressively stronger concentrations of antigen are used to determine the end point of reactivity. This method, initially known as skin end-point titration (SET), is the classic form of the broader class now referred to as intradermal dilutional testing (IDT). IDT presents the advantage of bearing a reproducible relationship with results obtained from in vitro radioallergosorbent testing (RAST). Although variants of RAST have now been introduced, some using radioactive markers and others using enzymatic or fluorometric methods (enzyme-linked immunosorbent assay, ELISA), only the Fadal-Nalebuff modification of the RAST scoring system (F/N mRAST) parallels IDT. In this text, RAST refers to F/N mRAST, and to other tests that are truly equivalent to F/N mRAST Whether the system employed by an individual practitioner also bears this relationship has to be determined. Likewise, SET refers to the comprehensive approach first espoused by Rinkel, whereas IDT describes the more recently accepted techniques that allow for increased efficiency of testing.
For testing: |
Initial screening is performed with a limited number of antigens |
Testing is performed with individual antigens rather than mixes |
For treatment: |
Treatment is based on clinical judgment, not just test results |
End point indicates safe starting dose |
Testing and treatment may safely begin at 1:312,500 w/v |
Initial treatment is performed with separate vials for high and low reactors |
Adapted with permission from Mabry RL. Blending skin endpoint titration and in vitro methods of clinical practice. Otolaryngol Clin North Am 1992;25:61-70.
Several principles of allergy testing and immunotherapy, some initially derived from experience with IDT and others based on principles learned using RAST, are equally applicable to both methodologies (Table 10-1). These principles are discussed in the appropriate chapters elsewhere in this text, but they bear repetition here to put in context the relationship between IDT and RAST.
PRINCIPLES OF SCREENING (FOR IN VITRO AND IN VIVO TESTING)
It has been clearly shown, using RAST testing, that an antigen screening panel comprising a significant grass, weed, and tree for the area, plus two molds, house dust mite, and animal danders chosen on the basis of exposure, is effective for inhalant allergy.1 The effectiveness of this “miniscreen” of six antigens (plus animal danders as necessary) may be enhanced by increasing the number of antigens to provide a “midiscreen” of two grasses, one weed, two trees, three molds, and a dust mite (plus animal danders).2 If the results of such a screen are negative, the chances of the patient being significantly allergic to other inhalants are extremely small. On the other hand, if positive reactors are noted, further testing is probably needed for additional antigens in these classes. Although this concept was initially developed from RAST as a means of demonstrating a cost-effective in vitro approach, the entire screening concept is equally valid for skin testing using IDT.
For many years, skin testing was performed and immunotherapy administered using a series of mixed antigens from the same general family. Thus, it was not uncommon for patients to be tested and treated using “weed mix,” “tree mix,” or “grass mix.” RAST technology and a technique called RAST inhibition have shown that it is more effective to test and treat for individual antigens. Patients may not be sensitive to all the antigens contained in the mix, and although a sensitivity to one or more allergens gives a positive response on skin testing, patients will ultimately receive injections containing antigenic material to which they are not allergic but to which they are likely to become allergic through repeated exposure in this fashion. Another factor to be considered is that because of the presence of multiple antigens in the mixture, each one effectively dilutes the concentration of all the other antigens. For this reason, testing and subsequent immunotherapy are recommended with individual antigens, not mixes.
One exception to the previously mentioned concept is the use of mixtures for screening in patients not strongly suspected of allergy. In the case of skin testing, this involves testing with an antigen mix (e.g., grass mix, weed mix, etc.). A similar RAST screen is exemplified by the microscreen, utilizing two RAST disks. The first (seasonal) disk contains antigens for two grasses, two weeds, and two trees. The second (perennial) disk contains antigens for a dust mite and a common mold (Alternaria). A negative screening test makes it unlikely that the patient will demonstrate positive reactions when tested with individual grasses, weeds, trees, dust mites, or molds. It should be noted that a negative screening test of this type is indicative of the absence of allergy, but does not totally rule it out, especially in the face of a strongly suggestive history. More importantly, if the screening test result is positive, retesting with specific antigens is necessary to make a more definitive diagnosis and prepare for immunotherapy. Thus, many people prefer not to use this screening approach, but rather utilize the miniscreen of individual components described earlier.
Regarding treatment, whether testing is by skin test or RAST, the decision to institute immunotherapy is based on the clinician’s evaluation of the patient rather than simply on an abnormal laboratory test result. This situation rarely arises in patients undergoing skin testing. However, the temptation is very real for some physicians who depend entirely on RAST for diagnosis to let the laboratory do their thinking for them. The process of blindly ordering immunotherapy for every patient with one or more positive results on inhalant RAST involves no clinical judgment and may subject patients to unnecessary treatment. This dependence on in vitro results instead of clinical judgment has given rise to the pejorative term in vitro allergist. Both the American Academy of Otolaryngic Allergy and the American Academy of Allergy, Asthma, and Immunology have adopted position statements against this “remote practice of allergy”
TREATMENT VIALS: IN VIVO AND IN VITRO
A concept that is very important when considering the mechanics of mixing treatment vials is that, whether determined by IDT or F/N mRAST, the end point defines a safe antigen starting dose. More precisely, because of the slight sensitivity differences in the two techniques, immunotherapy based on in vitro results is generally started one dilution weaker than the F/N mRAST class, that is, at the RAST minus one level. This means that an F/N mRAST class III would be equated with an IDT end point of #4 (1:12,500 w/v). Although this relationship may vary from antigen to antigen, it is sufficiently constant to be a basis for moving freely between the two methodologies.3 This is discussed more completely in Chapter 8, but it should be evident that it is possible to incorporate into treatment vials antigens whose end points have been determined from both IDT and RAST. This relationship is depicted in Table 10-2.
Before RAST was commonly used, in an attempt to start at an anticipated nonreacting concentration, IDT testing was sometimes begun using extremely dilute antigens. This gave rise to whealing patterns such as the “hourglass” (see Unusual Whealing Reactions in Chapter 5). This pattern caused significant confusion among novice practitioners. After experience with RAST, it was found that it was very rarely necessary to begin skin testing with antigen concentrations weaker than the #6 IDT dilution (1:312,500 w/v). Likewise, treatment could almost always be started at this strength, even if a skin test wheal at this concentration produced a wheal larger than usual.
IDT end point | Antigen concentration** | RAST-1*** |
|---|---|---|
#1 | 1:100 | 1:500 |
#2 | 1:500 | 1:2500 |
#3 | 1:2,500 | 1:12,500 |
#4 | 1:12,500 | 1:62,500 |
#5 | 1:62,500 | 1:312,500 |
#6 | 1:312,500 | 1:312,500**** |
* F/N mRAST; may be true of other RAST and ELISA systems.
** Weight/volume, if “concentrate” is 1:20.
*** Usual regimen, in which “end point” is considered one dilution weaker than RAST score (RAST-1).
**** It is rarely necessary to begin treatment at strengths weaker than #6 dilution.
Adapted with permission from Mabry RL. Blending skin endpoint titration and in vitro methods of clinical practice. Otolaryngol Clin North Am 1992;25:61–70.
Whether testing is done by IDT or RAST, the quantitation of results provided by these methods allows treatment to be begun at the highest safe dose. RAST-based immunotherapy confirmed the clinical impression gained from IDT-based treatment that patients with very high allergen-specific IgE levels (F/N mRAST levels IV or V) were very labile in their reactions to immunotherapy, requiring low initial antigen doses and cautious advancement.4 Doses of antigens to which hypersensitivity was lower, on the other hand, could be advanced more rapidly and with less risk for reaction. This led to the practice of initially splitting treatment vials, whether based on skin tests or in vitro methods, to include highly reacting antigens (RAST classes IV and V; IDT end point #6, #5, or #4) in one vial, to be advanced cautiously, and antigens to which the patient is less sensitive in another vial, to be advanced more quickly.
CHARACTERISTICS OF SKIN TESTS
Skin testing (in this context the term applies to all forms of IDT) has both advantages and disadvantages (Tables 10-3 and 10-4). A skin-testing session may take about an hour, but at the end of that time the results are available, with no further delay. Because of the effect on skin reactivity of antihistamines, tricyclic antidepressants, and some tranquilizers, patients must omit these for several days before skin testing. Patients whose skin is hyperreactive (dermatographia) may exhibit false-positive results on skin testing. For this reason, positive and negative controls must be a part of each skin-testing session. The financial investment required to purchase equipment and supplies for skin testing is modest. However, persons performing the test must be trained, and supervision must be provided until they are experienced. Although single-dilution prick testing or single-dilution intradermal tests give only a rough approximation of the degree of sensitivity of the patient, IDT is quantitative and reproducible. After skin testing, one knows exactly how the patient will react to the antigen that will be used in the treatment vial, as the patient has undergone a bioassay with that exact substance (from the same stock vial).
Accuracy: |
May be quantified (intradermal titration) |
Safety: |
Testing antigen identical to treating antigen |
Convenience: |
Rapidly performed |
Little equipment required |
Results quickly available |
Adapted with permission from Mabry RL. Blending skin endpoint titration and in vitro methods of clinical practice. Otolaryngol Clin North Am 1992;25:61-70.
Accuracy: |
Placement of accurate wheals depends on skill and experience |
Reading and interpretation of results are subject to variability |
Safety: |
There is risk for anaphylaxis or systemic reaction |
Convenience: |
Procedure is time-consuming |
Multiple sticks are uncomfortable |
Patient must omit medications (antihistamines, others) |
Adapted with permission from Mabry RL. Blending skin endpoint titration and in vitro methods of clinical practice. Otolaryngol Clin North Am 1992;25:61-70.
