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Skin Testing Techniques
SKIN TESTING
0889-8561/01 $15.00 i.OO
SKIN TESTING TECHNIQUES William K. Dolen, MD
As a method for detection of allergen specific immunoglobulin E (IgE), skin testing has been in routine clinical use for nearly 100 years. During this time, various methods and devices have been used, and no one method or device has emerged as a standard. Devices for skin testing were discussed in the article by Oppenheimer elsewhere in this issue. This article reviews techniques appropriate for use in clinical practice, focusing on advantages and limitations. Techniques for research applications are described in detail in the article by Dreborg. For the purpose of study, skin test methods can be described as epicutaneous (or percutaneous) or intradermal (or intracutaneous). EPICUTANEOUS SKIN TESTING
The epicutaneous methods currently in general use are prick testing, puncture testing, and scarification. The term prick/puncture, often used in the literature, is a misnomer because the prick and puncture methods differ significantly. Because scratch testing, in which a linear abrasion is made with a knife or needle before application of extract, is uncomfortable and traumatic, it is used less widely. Pepys described the modified prick test (Fig. 1) that is in general use worldwide? In this method, a drop of extract is placed on the skin. A sharp needle is introduced laterally into the skin at an angle, and the most superficial layers of the skin are lifted up with no downward pressure on the skin, introducing a minute amount of extract.6Although of limited use for precision research purposes (see article by Dreborg),
From the Allergy and Immunology Section, Departments of Pediatrics and Medicine, Medical College of Georgia, Augusta, Georgia IMMUNOLOGY AND ALLERGY CLINICS OF NORTH AMERICA VOLUME 21 NUMBER 2 * MAY 2001
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Figure 1. The modified prick test. A drop of extract is placed on a cleansed skin testing site. The device is introduced though the drop into the superficial layers of the epidermis, exerting no downward pressure on the skin. The device is then lifted away from the skin.
this method is well suited for general use and is the technique of choice for dermatographic patients. As reviewed in Oppenheimer’s article, a variety of testing devices is available. In puncture testing, a drop of extract is placed on the skin and the testing device, often a modified blood lancet, is placed perpendicular to the skin. When a lancet is used (Fig. 2), it is placed gently on the skin and tapped gently with a finger, or held on the skin with gentle pressure for one second. This method, recommended by the European Academy of Allergy and Clinical Immunology, is considered highly reproducible.2 A bifurcated needle, originally designed for smallpox vaccinations, also may be used (Fig. 3). This device is placed on the skin with enough downward pressure to form a small indentation in the skin and rocked forward and backward and from side to side. This method has been adopted by the Office of Biologics of the US Food and Drug Administration (FDA). The other devices for puncture testing described in the article by Oppenheimer are placed on the skin with enough downward pressure to create an indentation in the skin. Because puncture methods to some extent involve downward pressure on the skin, they are less appropriate for dermatographic individuals than modified prick testing, although they are easier to learn and use. Some devices can be dipped in the extract before skin application. Classic scarification, in which a knife blade abrades an area of skin before the extract is applied, is no longer commonly used because of difficulties discussed in the article by Oppenheimer. Some modern devices in common use, however, are designed to be dipped in extract,
SKIN TESTING TECHNIQUES
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Figure 2. Puncture testing with a lancet. A drop of extract is placed at the chosen skin testing site. The lancet is hbld between the thumb and middle finger. The tip of the index finger is used to tap the lancet tip into the skin, and a small amount of pressure is maintained for one second before the device is withdrawn.
Figure 3. Puncture testing with a bifurcated needle. A drop of extract is placed at the skin testing site. The device is held between the thumb and index finger and pressed gently into the skin. It is rocked forward and back and from side to side.
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applied to the skin with downward pressure, and twisted, producing a small abrasion. This technique is termed rotation. In scratch testing, a knife blade or needle abrades an area of skin in a linear fashion, producing a superficial scratch. The extract is then applied. INTRACUTANEOUS SKIN TESTING Intracutaneous, or intradermal, skin testing was popularized by Robert Cooke in the early 20th century (see article by Cohen and King). Until recently, it was the primary method used by allergistimmunologists trained in many of the Eastern centers. Intradermal testing remains the method of choice for testing with low potency extracts and substances such as venoms and drugs. As described in the article by Nadarajah, et al, intradermal tests are used in the skin endpoint titration (SET) method preferred by many otolaryngologists. Testing is performed with a disposable tuberculin syringe (0.5-1.0 mL) and small (26-30 gauge) needle. A small amount (about 0.02 mL) of dilute extract is injected into the superficial layers of skin, making wheals approximately the same size: Intradermal testing is more difficult and time consuming than the epicutaneous methods and is more uncomfortable for the patient. Irritant reactions occur commonly and can be difficult to distinguish from truly positive reactions, particularly when any reaction greater than that of the saline control is considered positive. EPICUTANEOUSVERSUS INTRACUTANEOUS SKIN TESTING Many of the allergy-immunology clinicians who employ intraderma1 tests for inhalant allergy testing use a 50- to 100-fold dilution (1:500 weight/volume [w/v] or 1:lOOO w/v) of the stock extract (1:lO w/v or 1:20 w / v ) ~when an epicutaneous test with the stock extract is negative or equivocal, but clinical suspicion of allergic sensitization is high.' Although this approach does increase the clinical sensitivity of skin testing, it does so with a loss of clinical specificity. In the case where an epicutaneous test with a potent extract was negative, the clinical relevance of a positive intradermal test for inhalants (e.g., pollens, fungi, animal danders) has not been clearly established. This subject is reviewed in the article by Nelson. In testing for drug or venom hypersensitivity, most clinicians perform a preliminary epicutaneous test followed by a single intradermal test or titrated intradermal tests, as these materials are less potent compared with pollen and most other inhalant extracts. There is a general consensus among allergist-immunologists that intradermal testing for food hypersensitivity is rarely, if ever, indicated.' Most allergist-immunologists also agree that the use of titrated intradermal skin tests by the SET method, as proposed by Rinkel and
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277
modernized by Willoughby7and others, to determine an immunotherapy starting dose is not necessary. This method, lucidly described in the article by Nadarajah, et al, employs extract concentrations as great as 1:lOO w/v if lower concentrations do not yield positive results. LOCATION FOR SKIN TESTING For reasons discussed in the article by Nelson, the areas most favored for epicutaneous testing are the back and volar surface of the forearms. Because of the potentially increased risk for a systemic reaction, the forearms or upper arms are the sites of choice for testing with drugs and venoms. Intradermal testing usually is performed on the lateral upper arms or the volar surface of the forearms. PREPARATION FOR SKIN TESTING Systemic anaphylaxis from skin testing, particularly the epicutaneous methods, is an exceptionally rare event. Nonetheless, a physician should be present when skin testing is performed, and medications for treatment of systemic reactions should be available. Before testing, the technician should verify that the patient has not been taking medications that might interfere with testing. The selected test site is cleansed with alcohol and allowed to dry. Test sites are marked. In general, tests should be spaced as far apart as practical. Many clinicians prefer 5-cm spacing, but 2-cm spacing is acceptable? When tests are spaced too closely together, a large test result may make it difficult to interpret results of other tests in its vicinity. After tests are applied, the sites may be blotted gently4but the site should not otherwise be disturbed or traumatized. Reactions should be measured or graded beginning 15 to 20 minutes after the first test is applied. Shining a light on a test site from the side may be helpful if the patient’s skin texture or color makes test grading difficult. In measuring a reaction, it is important not to disturb the test site itself. In particular, touching or rubbing a test site is unwise because small amounts of extract (or histamine) may be transferred from site to site by the technician’s finger. If an undisturbed test site becomes larger within 30 minutes of the initial reading, the larger reaction should be recorded. After tests are graded, a nonmedicated cream, ointment base, or lotion may be applied to testing sites to help relieve itching. The physician may order a rapid-onset antihistamine if itching is unusually severe. Patients should be observed for at least 30 minutes after skin testing is finished. SKIN TEST FORM A skin test form is the permanent record of skin test results. As discussed in the article by Esch, allergen extracts chosen for inclusion
278
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should reflect the physician's knowledge of local botany, aerobiology, allergenic foods, other substances commonly and rarely implicated in allergic diseases, and relevant cross-reactivity. Guidelines for designing a skin testing form have been published in the Allergy Diagnostic Testing Practice Parameter.' An ideal skin testing form should list the name, address, telephone number of the physician, patient's name, and date of testing. For quality control purposes, it should contain the name or initials of the person actually doing the testing. The exact method or methods used for testing (e.g.,prick, puncture, rotation, intradermal), the device, and location of testing should be noted. When a grading system is used, the criteria for recording results should be specified. The skin testing sheet also should report extract concentrations used for testing (e.g., 1:20 w/v or 50,000 AU/mL) and list an unambiguous name for all extracts tested. The term mite is inexact; Dermutophugoidesfurinue would be preferable. When possible, the English common name should be used in addition to abbreviated binomial Latin nomenclature. If mixes are used, there should be a listing of components. It i: desirable to arrange extracts in groups according to their botanic classification. Records of extract source, lot number, and expiration date may be kept separately. The sheet also should note the concentration of histamine or other positive control substances used, and the composition of the substance used for the negative control. Results of positive and negative control tests should be recorded.
RECORDING RESULTS Epicutaneous Methods
Methods for recording of results in research applications are discussed extensively in the article by Dreborg. Most methods are not practical for use in a busy clinical practice. One of several semiquantitative grading systems in clinical use is presented in the article by Tripathi and Peterson. A wheal size of 3 mm generally is considered positive; however, this is only appropriate if the glycerosaline negative control site is negative. Some techniques and devices can produce wheals larger than 3 mm in some patients, particularly individuals with dermatographism. Wheals less than 3 mm in diameter may be considered equivocally positive by some clinicians when the negative control site is negative. For reporting quantitative results, some clinicians report diameter of wheal and diameter of flare; a wheal 4 mm in diameter with a 23mm-diameter flare would be recorded as 4/23. Simply reporting positive or negative results is not recommended.' lntradermal Testing
The procedures for recording intradermal test results are the same as those used for epicutaneous testing. When testing is performed as
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described previously (i.e., following a negative or equivocal epicutaneous test) and with a single concentration (such as 1:lOOO w/v), the literature does not provide an evidence-based definition of a positive result. Thus, authorities state “any reaction larger than the negative control may indicate the presence of specific IgE antibody.”’ For this reason, a semiquantitative grading system would not be advisable for reporting results of intradermal testing. At a minimum, diameter of wheal and flare should be recorded. Because glycerin is a skin irritant, the negative control should contain glycerin in the same concentration as the substances tested. SUMMARY
Skin testing is a simple, safe method for determination of immediate hypersensitivity. It is reliable in skilled hands, and results are reproducible when standardized extracts are employed. Epicutaneous methods have greater specificity than intradermal testing and have acceptable sensitivity in most clinical situations when potent extracts are employed. Although puncture testing has greater reproducibility than the modified prick method (see article by Dreborg), the latter is the method of choice in dermatographic patients. References 1. Bemstein IL, Storms WW: Practice parameters for allergy diagnostic testing. Ann Allergy 75553-625, 1995 2. Dreborg S, Frew A: Allergen standardization and skin tests [position paper]. Allergy 48(suppl):49-82, 1993 3. Nelson HS: Effect of distance between sites and region of the body on results of skin prick tests. J Allergy Clin Immunol 197596401,1996 4. Ownby DR, Anderson JA An improved prick skin test procedure for young children. J Allergy Clin Immunol69:533-535,1982 5. Pepys J: Skin testing. Br J Hosp Med 14412417, 1975 6. Vanselow NA Skin testing and other diagnostic procedures. In Sheldon JM, Lovell RG, Mathews KP (ed): A Manual of Clinical Allergy. Philadelphia, WB Saunders, 1967, p 55-77 New concepts in hunotherapy. Otolaryngol Clin North Am 25:717. Willoughby JW: 100,1992
Address reprint requests to William K. Dolen, MD Allergy and Immunology Section Medical College of Georgia Augusta, GA 30912
0889-8561/01 $15.00 i.OO
SKIN TESTING TECHNIQUES William K. Dolen, MD
As a method for detection of allergen specific immunoglobulin E (IgE), skin testing has been in routine clinical use for nearly 100 years. During this time, various methods and devices have been used, and no one method or device has emerged as a standard. Devices for skin testing were discussed in the article by Oppenheimer elsewhere in this issue. This article reviews techniques appropriate for use in clinical practice, focusing on advantages and limitations. Techniques for research applications are described in detail in the article by Dreborg. For the purpose of study, skin test methods can be described as epicutaneous (or percutaneous) or intradermal (or intracutaneous). EPICUTANEOUS SKIN TESTING
The epicutaneous methods currently in general use are prick testing, puncture testing, and scarification. The term prick/puncture, often used in the literature, is a misnomer because the prick and puncture methods differ significantly. Because scratch testing, in which a linear abrasion is made with a knife or needle before application of extract, is uncomfortable and traumatic, it is used less widely. Pepys described the modified prick test (Fig. 1) that is in general use worldwide? In this method, a drop of extract is placed on the skin. A sharp needle is introduced laterally into the skin at an angle, and the most superficial layers of the skin are lifted up with no downward pressure on the skin, introducing a minute amount of extract.6Although of limited use for precision research purposes (see article by Dreborg),
From the Allergy and Immunology Section, Departments of Pediatrics and Medicine, Medical College of Georgia, Augusta, Georgia IMMUNOLOGY AND ALLERGY CLINICS OF NORTH AMERICA VOLUME 21 NUMBER 2 * MAY 2001
273
274
DOLEN
Figure 1. The modified prick test. A drop of extract is placed on a cleansed skin testing site. The device is introduced though the drop into the superficial layers of the epidermis, exerting no downward pressure on the skin. The device is then lifted away from the skin.
this method is well suited for general use and is the technique of choice for dermatographic patients. As reviewed in Oppenheimer’s article, a variety of testing devices is available. In puncture testing, a drop of extract is placed on the skin and the testing device, often a modified blood lancet, is placed perpendicular to the skin. When a lancet is used (Fig. 2), it is placed gently on the skin and tapped gently with a finger, or held on the skin with gentle pressure for one second. This method, recommended by the European Academy of Allergy and Clinical Immunology, is considered highly reproducible.2 A bifurcated needle, originally designed for smallpox vaccinations, also may be used (Fig. 3). This device is placed on the skin with enough downward pressure to form a small indentation in the skin and rocked forward and backward and from side to side. This method has been adopted by the Office of Biologics of the US Food and Drug Administration (FDA). The other devices for puncture testing described in the article by Oppenheimer are placed on the skin with enough downward pressure to create an indentation in the skin. Because puncture methods to some extent involve downward pressure on the skin, they are less appropriate for dermatographic individuals than modified prick testing, although they are easier to learn and use. Some devices can be dipped in the extract before skin application. Classic scarification, in which a knife blade abrades an area of skin before the extract is applied, is no longer commonly used because of difficulties discussed in the article by Oppenheimer. Some modern devices in common use, however, are designed to be dipped in extract,
SKIN TESTING TECHNIQUES
275
Figure 2. Puncture testing with a lancet. A drop of extract is placed at the chosen skin testing site. The lancet is hbld between the thumb and middle finger. The tip of the index finger is used to tap the lancet tip into the skin, and a small amount of pressure is maintained for one second before the device is withdrawn.
Figure 3. Puncture testing with a bifurcated needle. A drop of extract is placed at the skin testing site. The device is held between the thumb and index finger and pressed gently into the skin. It is rocked forward and back and from side to side.
276
DOLEN
applied to the skin with downward pressure, and twisted, producing a small abrasion. This technique is termed rotation. In scratch testing, a knife blade or needle abrades an area of skin in a linear fashion, producing a superficial scratch. The extract is then applied. INTRACUTANEOUS SKIN TESTING Intracutaneous, or intradermal, skin testing was popularized by Robert Cooke in the early 20th century (see article by Cohen and King). Until recently, it was the primary method used by allergistimmunologists trained in many of the Eastern centers. Intradermal testing remains the method of choice for testing with low potency extracts and substances such as venoms and drugs. As described in the article by Nadarajah, et al, intradermal tests are used in the skin endpoint titration (SET) method preferred by many otolaryngologists. Testing is performed with a disposable tuberculin syringe (0.5-1.0 mL) and small (26-30 gauge) needle. A small amount (about 0.02 mL) of dilute extract is injected into the superficial layers of skin, making wheals approximately the same size: Intradermal testing is more difficult and time consuming than the epicutaneous methods and is more uncomfortable for the patient. Irritant reactions occur commonly and can be difficult to distinguish from truly positive reactions, particularly when any reaction greater than that of the saline control is considered positive. EPICUTANEOUSVERSUS INTRACUTANEOUS SKIN TESTING Many of the allergy-immunology clinicians who employ intraderma1 tests for inhalant allergy testing use a 50- to 100-fold dilution (1:500 weight/volume [w/v] or 1:lOOO w/v) of the stock extract (1:lO w/v or 1:20 w / v ) ~when an epicutaneous test with the stock extract is negative or equivocal, but clinical suspicion of allergic sensitization is high.' Although this approach does increase the clinical sensitivity of skin testing, it does so with a loss of clinical specificity. In the case where an epicutaneous test with a potent extract was negative, the clinical relevance of a positive intradermal test for inhalants (e.g., pollens, fungi, animal danders) has not been clearly established. This subject is reviewed in the article by Nelson. In testing for drug or venom hypersensitivity, most clinicians perform a preliminary epicutaneous test followed by a single intradermal test or titrated intradermal tests, as these materials are less potent compared with pollen and most other inhalant extracts. There is a general consensus among allergist-immunologists that intradermal testing for food hypersensitivity is rarely, if ever, indicated.' Most allergist-immunologists also agree that the use of titrated intradermal skin tests by the SET method, as proposed by Rinkel and
SKIN TESTING TECHNIQUES
277
modernized by Willoughby7and others, to determine an immunotherapy starting dose is not necessary. This method, lucidly described in the article by Nadarajah, et al, employs extract concentrations as great as 1:lOO w/v if lower concentrations do not yield positive results. LOCATION FOR SKIN TESTING For reasons discussed in the article by Nelson, the areas most favored for epicutaneous testing are the back and volar surface of the forearms. Because of the potentially increased risk for a systemic reaction, the forearms or upper arms are the sites of choice for testing with drugs and venoms. Intradermal testing usually is performed on the lateral upper arms or the volar surface of the forearms. PREPARATION FOR SKIN TESTING Systemic anaphylaxis from skin testing, particularly the epicutaneous methods, is an exceptionally rare event. Nonetheless, a physician should be present when skin testing is performed, and medications for treatment of systemic reactions should be available. Before testing, the technician should verify that the patient has not been taking medications that might interfere with testing. The selected test site is cleansed with alcohol and allowed to dry. Test sites are marked. In general, tests should be spaced as far apart as practical. Many clinicians prefer 5-cm spacing, but 2-cm spacing is acceptable? When tests are spaced too closely together, a large test result may make it difficult to interpret results of other tests in its vicinity. After tests are applied, the sites may be blotted gently4but the site should not otherwise be disturbed or traumatized. Reactions should be measured or graded beginning 15 to 20 minutes after the first test is applied. Shining a light on a test site from the side may be helpful if the patient’s skin texture or color makes test grading difficult. In measuring a reaction, it is important not to disturb the test site itself. In particular, touching or rubbing a test site is unwise because small amounts of extract (or histamine) may be transferred from site to site by the technician’s finger. If an undisturbed test site becomes larger within 30 minutes of the initial reading, the larger reaction should be recorded. After tests are graded, a nonmedicated cream, ointment base, or lotion may be applied to testing sites to help relieve itching. The physician may order a rapid-onset antihistamine if itching is unusually severe. Patients should be observed for at least 30 minutes after skin testing is finished. SKIN TEST FORM A skin test form is the permanent record of skin test results. As discussed in the article by Esch, allergen extracts chosen for inclusion
278
WLEN
should reflect the physician's knowledge of local botany, aerobiology, allergenic foods, other substances commonly and rarely implicated in allergic diseases, and relevant cross-reactivity. Guidelines for designing a skin testing form have been published in the Allergy Diagnostic Testing Practice Parameter.' An ideal skin testing form should list the name, address, telephone number of the physician, patient's name, and date of testing. For quality control purposes, it should contain the name or initials of the person actually doing the testing. The exact method or methods used for testing (e.g.,prick, puncture, rotation, intradermal), the device, and location of testing should be noted. When a grading system is used, the criteria for recording results should be specified. The skin testing sheet also should report extract concentrations used for testing (e.g., 1:20 w/v or 50,000 AU/mL) and list an unambiguous name for all extracts tested. The term mite is inexact; Dermutophugoidesfurinue would be preferable. When possible, the English common name should be used in addition to abbreviated binomial Latin nomenclature. If mixes are used, there should be a listing of components. It i: desirable to arrange extracts in groups according to their botanic classification. Records of extract source, lot number, and expiration date may be kept separately. The sheet also should note the concentration of histamine or other positive control substances used, and the composition of the substance used for the negative control. Results of positive and negative control tests should be recorded.
RECORDING RESULTS Epicutaneous Methods
Methods for recording of results in research applications are discussed extensively in the article by Dreborg. Most methods are not practical for use in a busy clinical practice. One of several semiquantitative grading systems in clinical use is presented in the article by Tripathi and Peterson. A wheal size of 3 mm generally is considered positive; however, this is only appropriate if the glycerosaline negative control site is negative. Some techniques and devices can produce wheals larger than 3 mm in some patients, particularly individuals with dermatographism. Wheals less than 3 mm in diameter may be considered equivocally positive by some clinicians when the negative control site is negative. For reporting quantitative results, some clinicians report diameter of wheal and diameter of flare; a wheal 4 mm in diameter with a 23mm-diameter flare would be recorded as 4/23. Simply reporting positive or negative results is not recommended.' lntradermal Testing
The procedures for recording intradermal test results are the same as those used for epicutaneous testing. When testing is performed as
SKIN TJ3TING TECHNIQUES
279
described previously (i.e., following a negative or equivocal epicutaneous test) and with a single concentration (such as 1:lOOO w/v), the literature does not provide an evidence-based definition of a positive result. Thus, authorities state “any reaction larger than the negative control may indicate the presence of specific IgE antibody.”’ For this reason, a semiquantitative grading system would not be advisable for reporting results of intradermal testing. At a minimum, diameter of wheal and flare should be recorded. Because glycerin is a skin irritant, the negative control should contain glycerin in the same concentration as the substances tested. SUMMARY
Skin testing is a simple, safe method for determination of immediate hypersensitivity. It is reliable in skilled hands, and results are reproducible when standardized extracts are employed. Epicutaneous methods have greater specificity than intradermal testing and have acceptable sensitivity in most clinical situations when potent extracts are employed. Although puncture testing has greater reproducibility than the modified prick method (see article by Dreborg), the latter is the method of choice in dermatographic patients. References 1. Bemstein IL, Storms WW: Practice parameters for allergy diagnostic testing. Ann Allergy 75553-625, 1995 2. Dreborg S, Frew A: Allergen standardization and skin tests [position paper]. Allergy 48(suppl):49-82, 1993 3. Nelson HS: Effect of distance between sites and region of the body on results of skin prick tests. J Allergy Clin Immunol 197596401,1996 4. Ownby DR, Anderson JA An improved prick skin test procedure for young children. J Allergy Clin Immunol69:533-535,1982 5. Pepys J: Skin testing. Br J Hosp Med 14412417, 1975 6. Vanselow NA Skin testing and other diagnostic procedures. In Sheldon JM, Lovell RG, Mathews KP (ed): A Manual of Clinical Allergy. Philadelphia, WB Saunders, 1967, p 55-77 New concepts in hunotherapy. Otolaryngol Clin North Am 25:717. Willoughby JW: 100,1992
Address reprint requests to William K. Dolen, MD Allergy and Immunology Section Medical College of Georgia Augusta, GA 30912