Multitest (Skin Test Antigens for Cell-Mediated Immunity)

MULTITEST® CMI

Connaught

Skin Test Antigens for Cell-Mediated Immunity

Diagnostic Aid

Action And Clinical Pharmacology: The delayed cutaneous responses associated with the ubiquitous antigens in the Multitest battery appear to be typical cellular hypersensitivity reactions. A relatively small amount of soluble antigen is introduced into the epidermia and superficial dermal tissue by puncture. Circulating T-cells (lymphocytes), sensitized to the antigen from prior contact, react with the antigens in the skin and induce a specific immune response which includes mitosis (blastogenesis) and the release of numerous soluble mediators (lymphokines). Specific lymphokines initiate inflammation (vasculitis and edema) that is manifest after several hours.

The intensity of the overall dermal inflammation reaches its peak 24 to 72 hours after antigen application and is resolved within days or weeks.

Delayed hypersensitivity skin testing may be useful in evaluating individuals suspected of having primary or acquired immune deficiency disorders in which cell-mediated immunity is decreased or absent.

An assessment of cellular hypersensitivity reactions has been shown to be useful in many conditions with other products. Protein calorie malnutrition often complicated by the increasing frequency and severity of infection. Malnutrition of moderate or severe degree (weight for height less than 70% of standard) is almost invariably associated with impaired immune response. Delayed hypersensitivity is one of the immunocompetence response criteria in moderate to severe protein calorie malnutrition. Even marginal malnutrition may be associated with alterations in immunocompetence.

Delayed hypersensitivity tests again have been shown to be a comparative immunocompetence parameter. Marginal malnutrition has been shown to have a predictive value for morbidity and mortality. The presence of deficiencies of minerals, other trace elements and certain vitamin such as ascorbic acid has been associated with decreased delayed hypersensitivity response. Other factors such as diabetes mellitus, uremia, and certain acquired immune deficiency disorders can decrease delayed hypersensitivity. Skin testing at periodic intervals may be useful to determine if a state of immunodepression persists or if delayed hypersensitivity skin test responsiveness returns to normal. Delayed hypersensitivity testing has been useful to assess nutritional and immunocompetence criteria in pre- and postsurgical evaluations to detect high risk groups and adopt improved nutritional support and therapeutic programs.

Indications And Clinical Uses: For detection of anergy (nonresponsiveness to antigens) by means of delayed hypersensitivity skin testing.

Cutaneous anergy may indicate functional impairment of, or abnormalities in, the cellular immune system. Delayed cellular hypersensitivity is a valuable measure of immune response because it involves a complex series of immunologic, cellular, mediator-associated, and vascular effects.

Numerous investigators have shown a positive correlation between defective cell-mediated immunity, as indicated by anergy to multiple skin test antigens, and disseminated cancer. The occurrence of anergy may be correlated to some extent with advanced stage disease. The demonstration of anergy can be a negative prognostic factor in certain malignant diseases since diminished cutaneous reactivity has been associated with poor prognosis.

In general, patients capable of displaying normal delayed hypersensitivity skin test reactions to standard skin test antigens may have a better prognosis, whereas those who remain anergic or who exhibit significantly impaired reactivity, tend to experience a poor response to therapy, an increased incidence of recurrences and a shortened survival.

Table I summarizes data obtained from studies with the Multitest CMI antigens in France, in a normal population and in cancer patients. Table I illustrates differences in skin test responses between the sexes and between healthy persons compared to those with cancer. Clinical studies conducted in Canada and the U.S. using similar products manufactured by Institut Merieux show comparable results to those obtained in France.

Delayed hypersensitivity skin testing is not an absolute determinant of immune system dysfunction, and any such interpretation must be avoided. Delayed cutaneous hypersensitivity may be diminished or absent when there is in vitro evidence that T-lymphocyte function remains intact, and when antibody associated immunity and phagocytic function appear normal.

Until data are available for infants and children (to age 16), skin testing with Multitest CMI is recommended only for subjects 17 years of age or greater.

Contra-Indications: Do not apply at sites involving acneiform, infected or inflamed skin. Although severe systemic reactions to diphtheria and tetanus antigens are rare, persons known to have a history of such reactions should be tested with Multitest CMI only after the test heads containing these antigens have been removed.

Manufacturers’ Warnings In Clinical States: Epinephrine should be available in case of severe reactions.

Precautions: Discard the applicator after use. Do not reuse. The sterility is guaranteed only when the seals on the individual test heads are intact. Do not attempt to resterilize or reuse the applicator.

If periodic testing is done more frequently than every 2 months, then the test sites should be rotated so that retesting is not conducted at the same site sooner than 2 months.

Reactivity to delayed hypersensitivity skin test antigens may decrease or disappear temporarily as a result of: febrile illness; measles and other viral infections; live virus vaccination including measles, mumps, rubella, and poliomyelitis vaccines.

Individuals may acquire skin testing sensitivity resulting from either immunization or infection.

Epinephrine should be available in case of severe reactions.

Information for the Patient: Patients should be informed of the types of test site reactions that may be expected.

Drug Interactions: It is possible to observe loss of reactivity in patients undergoing treatment with drugs or procedures that suppress immunity, such as: corticosteroids, chemotherapeutic agents, antilymphocyte globulin and irradiation.

Drug Laboratory Test Interactions : The effect of repeated skin testing on specific antibody levels is not yet known, consequently those doing in vitro testing must be cognizant of the fact that repeated skin testing may alter antibody levels.

Pregnancy: Animal reproduction studies have not been conducted with Multitest CMI. It is also not known whether Multitest CMI can cause fetal harm when administered to a pregnant woman or can affect reproduction capacity. The skin test should be given to a pregnant woman only if clearly needed. Pregnancy may result in a decreased level of sensitivity to the test antigens.

Children: The safety and effectiveness in children below the age of 17 have not been established.

Adverse Reactions: Vesiculation, ulceration or necrosis may occur in highly sensitive subjects at the test site. Pain or pruritus at the test site may be relieved by topical glucocorticoids or ice pack.

Systemic reactions may occur in those persons sensitive to allergenic media components.

Dosage And Administration: Carefully follow each step of instructions below. Preparation of the Site and Multitest CMI: 1. Remove the skin test from refrigeration approximately 1 hour before use.

2. Select only test sites that permit sufficient surface area and s.c. tissue to allow adequate penetration of all points on all 8 test heads. Preferred sites are the volar surfaces of the arms and the back. Skin of the posterior thighs may be used if necessary. If several tests are planned, alternating forearms is desirable. Avoid hairy areas when possible because interpretation of reactions will be more difficult.

3. Cleanse test site with alcohol and allow to dry completely before testing. Ether or acetone may also be used.

4. Tear off the foil strip covering the test heads.

5. Tap the device on a hard surface, foil side up, to release antigen from top of cap.

6. Remove the protective plastic cap on each preloaded test head by twisting cap clockwise and counterclockwise. Carefully lift cap away from points.

Application of Multitest CMI: 1. Point “T-bar” end toward a constant reference point such as the elbow or head of the subject being tested to eliminate later identification problems with antigens or the control. The antigens and control are numbered clockwise, beginning top right through 8 on the round plastic platform supporting each test head.

2. Keep the skin at test site taut.

3. Press loaded unit into the skin with sufficient pressure to puncture the skin and allow adequate penetration of all points. Maintain firm contact for at least 5 seconds. During application the device should be “rocked” back and forth and side to side without removing any of the test heads from the skin sites. Bleeding rarely occurs with proper pressure.

4. If adequate pressure is applied it will be possible to observe: a. The puncture marks of the 9 tines on each of the 8 test heads. b. An imprint of the circular platform surrounding each test head. c. Residual antigen and glycerin at each of the 8 sites. If any of the above 3 criteria are not fully followed, the test results may not be reliable.

5. Identify the area of test sites by drawing one line above test sites No. 8 and No. 1, and another below test sites No. 5 and No. 4. The lines should be drawn about 1/4 inch from the puncture patterns and made with an indelible marker so that they will remain clearly visible on the skin for at least 48 hours.

6. Allow residual antigens and glycerin to remain on the skin surface for at least 3 minutes then gently dab with a gauze pad so as not to cross-contaminate test sites with antigen.

7. Discard applicator after use. Do not reuse.

Reading and Recording Results: 1. Reading should be done in good light. Read the test sites at both 24 and 48 hours, if possible. The largest reaction recorded from the 2 readings at each test site should be used. If 2 readings are not possible, a single 48 hour reading is recommended. The time for maximal reactivity to the various antigens may vary in different people. This may depend on the presence of antibodies.

2. A positive reaction from any of the 7 delayed hypersensitivity skin test antigens is induration of 2 mm or greater providing there is no induration at the negative control site. Fewer than 2% of healthy volunteers tested during clinical studies exhibited detectable induration from the glycerin negative control solution. Should induration occur at the negative control site, then indurated reactions from individual antigens must exceed the size of induration from the negative control solution by 2 mm or greater to be considered positive. Erythema without induration is of no significance. The size of the induration reactions with Multitest CMI may be smaller than those obtained with other intradermal procedures.

3. Determine the size of an indurated area at any test site by inspection, palpation with gentle finger stroking, and measurements across 2 diameters at right angles. R

Scoring: Periodic testing with Multitest CMI can be conducted to determine if a state of anergy persists, or if skin test reactivity returns. A 2 part scoring system has been developed for the test battery that consists of the number of positive antigens and the total induration resulting from all positive antigen test sites. Whenever positive skin test reactions occur, the scoring system can be used to assist in determining the degree of skin test reactivity and whether there is a trend over a period of time toward greater or lesser skin test responsiveness. The scoring system can be illustrated by this hypothetical example: Three of the 7 delayed hypersensitivity skin test antigens administered simultaneously produce indurated reactions measuring 3 mm and 5 mm respectively. The resulting score in this hypothetical case is 3/10 mm. The figure 3 represents the number of antigens positive, while 10 mm represents the sum total of induration from all three positive test sites. If a patient is anergic, the score is 0/0 mm, signifying no reaction to any antigen and no resulting induration.

Occasionally, a small and diffused reaction (2 to 3 mm) may be present at one time of testing and not at another. The disappearance or recurrence of such a reaction is probably not significant, providing positive reaction sizes to other antigens remain relatively constant. However, the disappearance or substantial reduction in size of a larger, well defined reaction may be significant, even though the total score is not appreciably affected.

Availability And Storage: Multitest CMI is a disposable, plastic applicator consisting of 8 sterile test heads preloaded with the following 7 delayed hypersensitivity skin test antigens and glycerin negative control for percutaneous administration: Tetanus Toxoid Antigen, Diphtheria Toxoid Antigen, Streptococcus Antigen, Tuberculin Old, Candida Antigen, Trichophyton Antigen, and Proteus Antigen. The test provides a quick, convenient and uniform procedure for delayed cutaneous hypersensitivity testing.

The 8 test heads of the Multitest CMI applicator are numbered 1 through 8 and are preloaded with the following test substances: (see manufacturer’s package insert for illustration).

Test Head No. 1 – Tetanus Toxoid Antigen: Tetanus Toxoid Antigen is a sterile, glycerinated solution containing tetanus toxoid prepared from the culture filtrate of C. tetani, inactivated and detoxified with formaldehyde. Residual formaldehyde does not exceed 0.02%. The potency of each lot is determined by a comparison of responses obtained by the intradermal injection of the lot and a reference Tetanus Toxoid into sensitized guinea pigs. Each mL of Tetanus Toxoid Antigen in 70% W/V glycerin is biologically equivalent to 550 000 Merieux Tetanus Units.

Test Head No. 2 – Diphtheria Toxoid Antigen: Diphtheria Toxoid Antigen is a sterile glycerinated solution containing diphtheria toxoid prepared from the culture filtrate of C. diptheriae, inactivated and detoxified with formaldehyde. Residual formaldehyde does not exceed 0.02%. The potency of each lot is determined by a comparison of responses obtained by the intradermal injection of the lot and a reference Diphtheria Toxoid into sensitized guinea pigs. Each mL of Diphtheria Toxoid Antigen in 70% W/V glycerin is biologically equivalent to 1 100 000 Merieux Diphtheria Units.

Test Head No. 3 – Streptococcus Antigen: Streptococcus Antigen is a sterile, glycerinated solution containing culture filtrate of Streptococcus (Group C) inactived with phenol. Residual phenol does not exceed 0.5%. The potency of each lot is determined by a comparison of responses obtained by the intradermal injection of the lot and a reference Streptococcus preparation into sensitized guinea pigs. Each mL of Streptococcus Antigen in 70% W/V glycerin is biologically equivalent to 2 000 Merieux Streptococcus Units.

Test Head No. 4 – Tuberculin, Old: Tuberculin, Old is a sterile glycerinated solution containing standardized culture filtrates of M. tuberculosis (C, D and PN) and M. bovis (Vallee). The potency of each lot is standardized by a comparison of responses obtained by the intradermal injection of the lot and the U.S. Standard Tuberculin, Old into sensitized guinea pigs. Each mL of Tuberculin, Old in 70% W/V glycerin is biologically equivalent to 300 000 U.S. Tuberculin Units (T.U.).

Test Head No. 5 – Glycerin Negative Control: Glycerin Negative Control is a 70% W/V sterile glycerin solution identical to the glycerin solution that serves as a vehicle for the skin test antigens.

Test Head No. 6 – Candida Antigen: Candida Antigen is a sterile glycerinated solution containing culture filtrate of C. albicans inactivated with phenol. Residual phenol does not exceed 0.5%. The potency of each lot is determined by a comparison of responses obtained by the intradermal injection of the lot and a reference Candida preparation into sensitized guinea pigs. Each mL of Candida Antigen in 70% W/V glycerin is biologically equivalent to 2 000 Merieux Candida Units.

Test Head No. 7 – Trichophyton Antigen: Trichophyton Antigen is a sterile glycerinated solution containing culture filtrate of Trichophyton mentagrophytes inactivated by the addition of phenol. Residual phenol does not exceed 0.5%. The potency of each lot is determined by a comparison of responses obtained by the intradermal injection of the lot and a reference Trychophyton preparation into sensitized guinea pigs. Each mL of Trichophyton Antigen in 70% W/V glycerin is biologically equivalent to 150 Merieux Trichophyton Units.

Test Head No. 8 – Proteus Antigen: Proteus Antigen is a sterile glycerinated solution containing culture filtrate of P. mirabilis inactivated by the addition of phenol. Residual phenol does not exceed 0.5%. The potency of each lot is determined by a comparison of responses obtained by the intradermal injection of the lot and a reference Proteus preparation into sensitized guinea pigs. Each mL of Proteus Antigen in 70% W/V glycerin is biologically equivalent to 150 Merieux Proteus Units.

Each individual carton contains 1 preloaded Skin Test Antigens for Cell-Mediated Immunity. Boxes of 12. Store at room temperature not exceeding 25°C. Avoid the proximity of any heat sources.

MULTITEST® CMI Connaught Skin Test Antigens for Cell-Mediated Immunity Diagnostic Aid

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