Skin Tests And Their Applications Dr. Bhavesh Patel Principal V.P. and R.P.T.P. Science College Vallabh Vidyanagar Email – bhavesh1968@rediffmail.com

Skin Tests Types Skin tests involving toxin - antitoxin neutralization Schick test Dick test Delayed hypersensitivity skin tests Tuberculin test Lepromin test Allergic skin tests Prick test Patch test Skin end point titration PK procedure

Skin tests involving toxin - antitoxin neutralization In a number of infections the lethal toxin produced by the pathogen and the antitoxin in the blood neutralizes the effect of toxin and does not produce any reaction. In contrast to this, if antitoxin is not present then the injected toxin produces immunological reactions on the skin like erythema and swelling at the site of injection. This visible immunological reaction can be used in determining the susceptibility of persons against particular diseases. Schick test Dick test

Schick test It is used to determine the susceptibility of a person to diphtheria (Corynebacterium diphtheriae). Named after its inventor, Béla Schick (1877-1967) The Schick test is no longer in use. The availability of safe and effective toxoid preparation has made susceptibity tests unnecessary.

Schick test Procedure A small amount (0.1 ml) of diluted (1/50 MLD) diphtheria toxin is injected intradermally into the forearm of the person. Minimum lethal dose (MLD, also LDmin) is the least amount of drug that can produce death in a given animal species under controlled conditions . Similar dose of inactivated toxin (heated at 70°C, 30 min.) is injected intradermally into the other arm as a control. Readings are taken after 1, 4 and 7 days.

Schick test Results Results can be interpreted as: Positive reaction: when the test results in a red necrotic area of 5-10 mm diameter within 24 hrs, reaching a maximum within 4-7 days. Positive test indicates susceptibility to diphtheria and thus requires immunization. Negative reaction: No erythema on any arm indicating that the toxin has been neutralized by the antitoxin & the person is immune to diphtheria Pseudo-positive: when there is only a red colored inflammation within 6-24 hrs and it disappears rapidly (within 4 days). This reaction is same on both arms and indicates immunity

Dick Test The test is used for diagnosis and determination of susceptibility for scarlet fever. Scarlet fever is a type of acute pharyngitis with erythematous rash, caused by Streptococcus pyogenes strains producing erythrogenic toxin or Dick toxin. The test of only of historical importance as scarlet fever is no longer a serious disease.

Dick Test Dick test is a neutralization test. 0.2 ml of standard toxin of Streptococcus pyogenes is injected intradermally in one forearm and the heat inactivated toxin is injected intradermally in another forearm , as control. Readings are taken after 24 hrs. Positive result- erythema of 1 cm diameter which indicates susceptibility towards scarlet fever and requires immediate immunization.

Delayed Type Hypersensitivity Skin tests Delayed type hypersensitivity (Type IV hypersensitivity) is checked for the diagnosis/prognosis of diseases like tuberculosis, leprosy. DTH involves activation of T cells by Ags that are typically cell bound (CMI). This cell bound Ag is recognized by specific receptors located on the membrane of the T lymphocyte. DTH reactions evolve slow slowly and consist of a mixed cellular reaction involving lymphocytes and macrophages. The reaction is not induced by circulating Abs but by but by sensitized T cells which on contact with specific Ag release lymphokines that cause biological effects on macrophages, leucocytes and tissue cells. Tuberculin test Lepromin test

Delayed Type Hypersensitivity

Delayed Type Hypersensitivity Skin tests 1. Tuberculin test Tuberculin is a glycerol extract of the tubercle bacillus. Purified protein derivative (PPD) tuberculin is a precipitate of non-species-specific molecules obtained from filtrates of sterilized, concentrated cultures. It was first described by Robert Koch in 1890. The test is named after Charles Mantoux, a French physician who developed on the work of Koch and Clemens von Pirquet to create his test in 1907.

Tuberculin test Procedure A standard dose of 5 Tuberculin units (0.1 mL) is injected intradermally (between the layers of dermis) and read 48 to 72 hours later. This intradermal injection is termed the Mantoux technique. A person who has been exposed to the bacteria is expected to mount an immune response in the skin containing the bacterial proteins. In persons not sensitized to tuberculin, the injection leads to a negative reaction as there is no immune response.

Tuberculin test Results The reaction is read by measuring the diameter of induration (palpable raised hardened area) across the forearm (perpendicular to the long axis) in millimeters. If there is no induration, the result should be recorded as "0 mm". Erythema (redness) should not be measured.

Classification of tuberculin reaction The results of this test must be interpreted carefully. The person's medical risk factors determine at which increment (5 mm, 10 mm, or 15 mm) of induration the result is considered positive  A positive result indicates exposure to tubercule bacillus. Negative reaction indicates susceptibility of person to disease and requires immediate immunization

Classification of tuberculin reaction False positive result Due to the test's low specificity, most positive reactions in low-risk individuals are false-positives. A false positive result may be caused by nontuberculous mycobacteria or previous administration of BCG vaccine. Prior vaccination with BCG may result in a false-positive result for many years afterwards.  False negative result Those that are immunologically compromised, especially those with HIV and low CD4 T cell counts, frequently show negative results from the PPD test. This is because the immune system needs to be functional to mount a response to the protein derivative injected under the skin.

Tuberculin test Heaf test The Heaf test is a tuberculin skin test formerly used in the United Kingdom, but discontinued in 2005. The equivalent Mantoux test positive levels done with 10 TU (0.1 mL 100 TU/mL, 1:1000) are <5 mm induration (Heaf 0-1) 5–15 mm induration (Heaf 2) >15 mm induration (Heaf 3-4)

Tuberculin test Negative & Positive Mantoux test

Applications of tuberculin test To diagnose active infection Measure the prevalence of infection in a community To select susceptibles for BCG vaccination or as an indication of successful vaccination. Tuberculin test of cattle is useful to control bovine tuberculosis.

Delayed Type Hypersensitivity Skin tests 2. Lepromin Test Lepromin test is based on delayed type of hypersensitivity (DTH) Kensuke Mitsuda published the first paper leading to the lepromin reaction (1919).  It involves the injection of a standardized extract of the inactivated "leprosy bacillus",(Mycobacterium leprae or "Hansen's Bacillus") under the skin. It is not recommended as a primary mode of diagnosis but is used to determine what type of leprosy a person has.

Lepromin Test Procedure An extract sample of inactivated Hansen's Bacillus is injected just under the skin, usually on the forearm, so that a small lump pushes the skin upward. The lump indicates that the antigen has been injected at the correct depth. The injection site is labeled and examined 3 days and 28 days later to see if there is a reaction.

Lepromin Test The response to the intradermal injection of lepromin is diphasic, consisting of two separate events. The first is the early reaction of Fernandez, which consists of erythema and induration developing in 24-48 hrs and remains for 3-5 days. The second phase is more meaningful and is called the late reaction of Mitsuda. It starts in four weeks 1-2 weeks reaching a peak in and gradually in the next few weeks.

Lepromin Test The reaction consists of an indurated skin nodule which may ulcerate. Histologically, there is infiteration of lymphocytes and epitheloid cells. The Mitsuda late reaction is a measure of CMI induced by the injected lepromin itself. It thus distinguishes between persons who can mount a CMI response against the lepra bacillus Ags and those who cannot.

Applications of lepromin test To classify lesions of leprosy patients The lepromin test is positive for tuberculoid type of leprosy, negative in lepromatous type and variable in dimorphous and intermminate type. To assess the prognosis and response to treatment A positive reaction indicates good prognosis and a negative a bad prognosis To assess resistance of individuals to leprosy Only lepromin positive persons in leprosaria as lepromin negative persons are more prone to develop the disease.

Skin Testing for Allergies Allergy is a hypersensitive disorder of the immune system An antigenic substance that can trigger the allergic state are called allergens. It can be a protein or a chemically complex low molecular substance. Most allergens are poorly immunogenic and most people don’t respond to them adversely. Strictly, allergy is one of four forms of hypersensitivity and Type I hypersensitivity or /immediate hypersensitivity. It is characterized by excessive activation of mast cells and basophils by IgE leaing to the release of pharmocologically active chemicals. Common allergic reactions include eczema, hives, urticaria, hay fever, asthma attacks, food allergies and reactions to the venom, stinging insects such as wasps and bees

Skin Testing for Allergies Type I hypersensitivity reactions occur when Ag combines with cell fixed Abs (Ig E). It is characterized by excessive activation of mast cells and basophiles by IgE leading to the release of pharmacologically active chemicals (vasoactive amines). Clinical syndrome include anaphylaxis and atopy

Skin Testing for Allergies- Methods Skin allergy testing  is a method for medical diagnosis of allergies. Skin testing for allergies is used to identify the substances that are causing allergy symptoms. The classical skin test in atopy is Type I wheal and flare reaction in which allergen introduced into the skin leads to the release of preformed mediators causing increased vascular permeability, local edema. Skin allergy tests can be performed by Prick test Patch test Skin end point titration PK procedure

Skin Testing for Allergies 1.Prick test In the prick (scratch) test, a few drops of the purified allergen are gently pricked on to the skin surface, usually the forearm. This test is usually done in order to identify allergies to pet dander, dust, pollen, foods or dust mites. Intradermal injections are done by injecting a small amount of allergen just beneath the skin surface. The test is done to assess allergies to drugs like penicillin or bee venom.

Skin Testing for Allergies 2. Patch test The patch test simply uses a large patch which has different allergens on it. The patch is applied onto the skin, usually on the back. The allergens on the patch include latex, medications, preservatives, hair dyes, fragrances, resins and various metals. When a patch is applied the subject should avoid bathing or exercise for at least 48 hours.

Skin Testing for Allergies 3. Skin end point titration Skin end point titration (SET) uses intradermal injection of allergens at increasing concentrations to measure allergic response. To prevent a severe allergic reaction, the test is started with a very dilute solution. After 10 minutes, the injection site is measured to look for growth of wheal. 2 mm of growth in 10 minutes is considered positive. If 2 mm of growth is noted, then a second injection at a higher concentration is given to confirm the response. The end point is the concentration of antigen that causes an increase in the size of the wheal followed by confirmatory whealing. If the wheal grows larger than 13 mm, no further injection are given since this is considered a major reaction.

Skin Testing for Allergies 4. Prausnitz- Kustner (PK) Test PK test is used to test people for life threatening allergens such as bee venom  The test has been replaced by the safer skin prick test. The PK test involves transferring serum from the test subject to another healthy person. This is a pathway for transmission of blood-borne diseases like Creutzfeldt–Jakob disease, AIDS, and others, which is why the test is no longer recommended.

Prausnitz- Kustner (PK) Test Procedure Serum, suspected to contain IgE antibodies, is drawn from allergic patient. This serum is injected intradermally into a non-allergic person. The suspected antigens are then intradermally injected into the non-allergic person 24-48 hours later. If the person being tested for an allergy has made antibodies for the antigen, this will cause a local reaction in the non-allergic person when the antibodies mix with the antigen. A positive PK test usually appears as a wheal and flare. This demonstrates a type I hypersensitivity reaction, and the allergic reaction to the injected antigen is confirmed for the patient being tested. Individuals who are sensitive to an allergen can undergo this indirect mode o of testing to determine if their serum contains specific Ig E to the allergen, without having to come into direct contact with the allergen.

Skin Testing for Allergies Results If an immune response is seen in the form of a rash, urticaria , or anaphylaxis (bronchoconstrictive and vasodilatory effects, symptoms such as asthma or rhinitis) it is concluded that the patient has a hypersensitivity (or allergy) to that allergen. In all cases where the test is positive, the skin will become raised, red and appear itchy. The results are recorded- larger wheals indicating that the subject is more sensitive to that particular allergen. A negative test does not mean that the subject is not allergic; s imply that either the right concentration was not used or the body failed to elicit a response. SET can be useful in these cases.

Demonstration of Immune Complexes

Immune complex disease,Type III hypersensitivity Type III hypersensitivity is due to damage by Ag-Ab complexes. These complexes precipitate in and around blood vessels causing local inflammation and massive complement activation Clinical syndromes include Arthus reaction –local deposition Serum sickness – generalized deposition

Demonstration of Immune Complex Methods for detection of circulating immune complexes Polyetylene glycol assay Immunofluorescence Platelet aggregation test C1q binding test

Demonstration of Immune Complexes 1. Polyetylene glycol assay Polyethylene glycol (PEG) can precipitate high molecular weight complexes The test serum is added to a buffer containing PEG Ag-Ab immune complexes will be precipitated by PEG and monitored by laser nephelometery

Demonstration of Immune Complexes 2. IF Tissue samples examined by immunofluorescence for presence of IgG and complement

Demonstration of Immune Complexes 3. Platelet aggregation test Ag-Ab complexes have the ability to aggregate blood platelets Clumps can be detected visually with a microscope or electronically by changes in turbidity measured in a platelet aggregometer

Demonstration of Immune Complexes 4. C1q binding test C1q is bound to an inert solid phase support (e.g. sides of a test tube) Serum containing immune complex is added, binding of immune complex and C takes place After incubation, serum is removed and a second incubation is performed with radio labeled anti IgG Abs The amount of radioactivity remaining after this second incubation correlates with the concentration of immune complexes in the patient’s serum.