The Mechanics and Interpretation of Multiple-Prick Testing

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The Mechanics and Interpretation of Multiple-Prick Testing

John H. Krouse

Prick testing, also known as epicutaneous testing, is among the earliest forms of skin testing employed for the diagnosis of inhalant allergy. Prick testing has been used for over 100 years, and has been the primary testing methodology, both in the United States and internationally, for the diagnosis of allergic sensitivity. In prick testing, a small amount of antigen is introduced into the superficial skin through a small puncture. The reactivity of the skin is then noted, and the response is judged to be positive or negative. There are several approaches to prick testing, and a variety of devices have been developed to aid in the placement of tests.

Devices for skin testing can be broadly divided into two categories: single-prick devices and multiple-prick devices. With single-prick devices, antigen is placed onto a small puncture wound created in the skin with a needle or lancet, or it is delivered with a multiple-tined device that introduces the antigen into the skin at the same time the puncture is made. Common devices used for single-prick testing include the Morrow-Brown needle (Antigen Laboratories, Liberty, Missouri) and the DuoTip device (Lincoln Diagnostics, Inc., Decatur, Illinois).

Multiple-prick devices utilize the same basic principle, introducing a small amount of antigen directly into the superficial skin, but these devices contain a set of testing arms linked together into a single unit. Through the use of these multiple-prick devices, several tests can be placed into the skin simultaneously. In addition, research suggests that these multiple-prick devices provide more reliable, accurate, and replicable testing results, even when used by less experienced examiners.1,2 Multiple-prick devices available for testing include the Multi-Test II (Lincoln Diagnostics, Inc.) and the Quintest (Hollister-Stier, LLC, Spokane, Washington). Because these multiple-prick devices appear to be superior in their testing properties to single-prick methods, only multiple-prick testing are discussed in this chapter.

♦ The Mechanics of Multiple-Prick Testing

With all forms of skin testing, patient safety is a primary concern. The patient is first questioned about relevant medical history, including prior testing for allergy, asthma, and history of anaphylaxis. In addition, the patient is asked about medications that may interfere with skin testing (e.g., antihistamines, tricyclic antidepressants) and medications that may increase the risk of testing (e.g., beta-blockers). Once the complete history is obtained, and the testing is described to the patient, the testing procedure may begin. Testing is usually done on the volar forearms, so the forearms are cleaned with alcohol or another acceptable antimicrobial agent. If testing is done on the back or anterior thighs, these areas are cleaned in a similar manner.

In the practice of skin testing, regardless of the type of testing performed, it is essential to assess the ability of the skin to react to an allergen challenge. A positive response to an antigen depends on the release of histamine from activated mast cells and the interaction of that histamine with receptors in the skin that stimulate edema and induration. Medications such as antihistamines can interfere with receptor binding and can therefore prevent response even though the patient is truly allergic to the antigen. Skin testing, therefore, requires the placement of a histamine challenge to act as a positive control, assuring that the skin will develop whealing in response to the presence of histamine. Some patients may be dermatographic, responding with histamine release to minor skin trauma or even light pressure, so that the placement of the skin testing device alone could create nonspecific whealing. To assess for this effect, it is important to utilize a negative, saline control in testing. The introduction of normal saline should not create any whealing with a prick test.

In using multiple-prick test devices, antigens are often preloaded into a tray that may contain up to 24 different antigens in individual wells, and may accommodate up to three individual testing devices. As already noted, it is important to use two of the wells for control solutions of histamine and saline, delivering these controls simultaneously with delivery of the specific antigens. Although consideration can be given to using one of the wells for a glycerin control, glycerin reactivity in prick testing is very rare.

Prick tests may be placed on various skin surfaces, although the volar forearms provide an accessible and reliable location for testing. It is important to realize that not all skin sites are equal in reactivity. The middle and upper back are the most responsive to skin testing, although the forearm does provide a useful and less sensitive location. It is practical to place up to 16 individual tests on each forearm, so that by using both arms, up to 32 prick tests may be placed in one individual testing session. In the rare case in which more antigens will be tested, the anterior thighs or back also provide excellent areas for placement of prick tests. In addition, the anterior thigh is often a preferred site in young children, because the amount of skin surface at the volar forearm is limited in these small patients.

The skin is first marked with a washable pen to indicate the location of each of the testing panels. Each device is then individually applied to the skin at the outlined location with moderate pressure, rocking it from side to side and from back to front to provide adequate delivery of the test substances. Small droplets will remain at the testing sites once the device is removed, and should not be wiped away for at least 5 minutes. In addition, patients should be advised to avoid moving their arms for at least 5 minutes, to avoid cross-contamination of puncture sites. The puncture sites will begin to develop whealing with positive antigens within a few minutes. Complete development of whealing and erythema occurs by 20 minutes, at which time the tests are measured.

Although various methods for evaluating test results are utilized, the most accurate method for assessing positivity involves the measurement of wheal sizes. While noting that the erythema surrounding the wheal is useful (the so-called flare), it is the whealing response that is specific for histamine release. The wheal size for each individual antigen test site is measured and recorded. Most guidelines suggest that a positive wheal is defined as a wheal that is at least 3 mm in diameter and at least 3 mm greater in diameter than a saline (negative) control wheal. The size of the wheal is recorded rather than simply recording the response as positive versus negative because the size of whealing can be used to roughly estimate the strength of response.

♦ Interpretation of Results with Multiple-Prick Testing

Prick testing can provide a useful measurement of reactivity to antigens. It is a qualitative test, meaning that results primarily reflect whether or not an individual is allergic to a specific antigen. Although wheal sizes can be used to estimate the degree of sensitivity within very broad parameters, in this case they must be viewed as semiquantitative at best. Using the information gained from multiple-prick testing, immunotherapy can be prepared and safely delivered, but approaches to immunotherapy must be different from those based on intradermal dilutional testing (IDT).

The first priority in the delivery of immunotherapy is patient safety. Because the administration of antigen to sensitized patients can have life-threatening consequences, it is vital to treat the patient with concentrations of antigens to which they will have a very low risk of an adverse reaction. In nonquantitative testing, in contrast to quantitative testing such as IDT and modified quantitative testing (MQT), no attempt is made to quantify the degree of responsiveness. For that reason, the only safe manner in which to initiate immunotherapy is to prepare initial treatment vials with very dilute concentrations of antigens. If therapy is based on prick testing alone, many allergists choose the safest approach, which is to prepare treatment vials in which all antigens to be treated are initiated at 1 : 1,000,000 weight per volume (w/v) concentrations to significantly decrease the likelihood of a significant systemic reaction. In quantitative testing, in contrast, endpoints have been derived that have been assayed through testing to be safe on the skin. For that reason, with quantitative testing, starting concentrations of antigen can be much higher, even as high as 1 : 500 w/v.

In another method of utilizing multiple-prick testing results to guide immunotherapy, prick test responses can be divided into two or more categories on the basis of wheal size, for example, high responders and low responders.3 One such method that can be used is to dichotomize prick testing results in a semiquantitative fashion into two discrete categories: low level and high level. This classification assumes that the strength of the reaction on the skin is an accurate representation of the underlying allergic sensitivity. By utilizing this two-level classification system, vial preparation can include two distinct levels of antigen concentration. For practical purposes, discrimination of sensitivity based on wheal diameters alone is inaccurate, and it is therefore not recommended that the physician divide treatment vials into more than two antigen strengths based on prick testing alone.

One specific two-level system that can be used easily and appears to have good effectiveness employs two ranges of wheal diameters to define two-allergen sensitivity; these ranges are then assigned specific intradermal dilutional titration endpoint values for vial preparation. Testing results can be categorized by placing wheal diameters of 3 to 8 mm in the low-level sensitivity group, and wheal sizes of 9 mm or greater in the high-level sensitivity group. The low-level group would be arbitrarily assigned an intradermal dilutional titration endpoint 4, because it has a lesser degree of skin reactivity, and the high-level group would be assigned an intradermal dilutional titration endpoint 6, based on its greater degree of skin response. Again, it must be stressed that this approach is only semiquantitative because estimates of skin sensitivity are made only on the basis of response to a single prick test. Without the precision and refinement of either IDT or blended techniques such as MQT, semiquantitative approaches such as the one described here should be used carefully, and only by experienced physicians.

In those cases in which semiquantitative approaches are used to prepare immunotherapy vials, the use of an intradermal vial test from each new vial is essential in confirming the safety of that vial.4 For the vial test, a 4-mm intradermal wheal is placed in the upper arm using the mixture from the prepared vial to be tested. A period of 10 minutes is allowed to elapse, and the size of the wheal is measured. If the wheal that is generated by this vial test is greater than 13 mm in diameter, the vial is considered to be overly potent, and injections are not given from that vial. It is diluted fivefold and a vial test from this diluted vial is applied. If the wheal diameter is less than 13 mm, immunotherapy injections may proceed at that time. If the wheal is precisely 13 mmin diameter, the vial is acceptable, although the initiation of therapy is delayed until the next visit, at least 72 hours from the time of the vial test.

♦ Implications of Multiple-Prick Testing

When used appropriately, multiple-prick testing can be an effective way of establishing qualitatively whether an individual is allergic to a specific antigen or antigens. It can also be used as a basis for the preparation of treatment vials for immunotherapy. It is clear that the ability to prepare precise treatment vials quantitatively is limited by utilizing this method alone. In cases in which patients have difficulty tolerating injections based on prick testing, or if it becomes difficult to advance patients beyond the level that they would be expected to achieve, it may be necessary to retest the patient using a quantitative technique such as IDT or MQT. These approaches would be expected to more accurately define the precise antigen sensitivities and allow preparation of a more specific treatment set for the patient.

♦ Conclusion

Multiple-prick testing techniques can be a significant addition to the testing options available to the practicing allergist. When used properly, in conjunction with a careful history and general evaluation, they can accurately assess the presence or absence of allergic sensitivity in patients. They can also be used to guide the preparation of treatment vials, on either a qualitative or a semiquantitative basis. When employed with care, they are safe and effective methods for assisting in diagnosing and treating allergy.