Laboratory Procedures And Tests

Revised by Kenneth R. WatsonD.O.* SkinTests

FungusExaminations

Biopsies

Surgical „Excision

PunchBiopsy

ScissorsBiOPsy sh.aye„„B.iopsy Biopsy, Handling

Cytodiaqnosis Additional studies Acknow|ed.qment Bibliography

In addition to the usual laboratory procedures used in the workup of medical patients, certain special tests are of importance in the field of dermatology. These include skin tests, fungus examinations, biopsies, and immunologic diagnosis. For special problems, additional testing methods are suggested in the sections on the various diseases. skin tests

There are three types of skin tests:

• Intracutaneous

The intracutaneous tests and the scratch tests can have two types of reactions: an immediate wheal reaction or a delayed reaction. The immediate wheal reaction develops to a maximum in 5 to 20 minutes. This type of reaction is elicited in testing for the cause of urticaria, atopic dermatitis, and inhalant allergies. This immediate wheal reaction test is seldom used for determining the cause of skin diseases.

The delayed reaction to intracutaneous skin testing is exemplified best by the tuberculin skin test. Tuberculin is available in two forms: as purified protein derivative and as a tuberculin tine test.

The purified protein derivative test is performed by using tablets that come in two strengths and by injecting a solution of either one intracutaneously. If there is no reaction after the test with the first strength, then the second strength may be employed.

The tuberculin tine test (Mantoux) is a simple and rapid procedure using OTK. Nine prongs, or tines, covered with OTK are pressed into the skin. If at the end of 48 or 72 hours there is more than 2 mm of induration at the site of any prong insertion, the test is positive.

Patch tests are used commonly in dermatology and offer a simple and accurate method of determining whether a patient is allergic to any of the testing agents. There are two different reactions to this type of test: a primary irritant reaction and an allergic reaction. The primary irritant reaction occurs in most of the population if they are exposed to agents (in appropriate concentrations) that have skin-destroying properties. Examples of these agents include soaps, cleaning fluids, bleaches, "corn" removers, and counterirritants. The allergic reaction indicates that the patient is more sensitive than normal to the agent being tested. This test reaction is idiosyncratic and not necessarily related to concentration or dose. It also shows that the patient has had a previous exposure to that agent or a cross-sensitizing agent.

The technique of the patch test is simple, but the interpretation of the test is not. For example, consider a patient presenting with dermatitis on top of the feet. It is possible that shoe leather or some chemical used in the manufacture of the leather is causing the reaction. The procedure for a patch test is to cut out a 1/2-inch-square piece of the material from the inside of the shoe, moisten the material with distilled water, place it on the skin surface, and cover it with an adhesive band or some patch-test dressing. The patch test is left on for 48 hours. When the patch test is removed, the patient is considered to have a positive patch test if there is any redness, papules, or vesiculation under the site of the testing agent. Delayed reactions to allergens can occur, and, ideally, a final reading should be made after 96 hours (4 days), or, in other words, 2 days after the patch is removed.

The patch test can be used to make or confirm a diagnosis of poison ivy dermatitis, ragweed dermatitis, or contact dermatitis caused by medications, cosmetics, or industrial chemicals. Fisher (1995) and Adams (1990) compiled lists of chemicals, concentrations, and vehicles to be used for eliciting the allergic type of patch test reaction. Most tests can be performed very simply, however, as in the case of the shoe-leather dermatitis. One precaution is that the test must not be allowed to become wet in the 48-hour period.

A patch test kit, T.R.U.E. Test (Glaxo), includes ready-to-apply self-adhesive allergen tapes.

A method of testing for food allergy is to use the Rowe elimination diet. The procedure is to limit the diet to the following basic foods, which are known to be hypoallergenic: lamb, lemon, grapefruit, pears, lettuce, spinach, carrots, sweet potato, tapioca, rice and rice bread, corn sugar, maple syrup, sesame oil, gelatin, and salt. The patient is to remain on this basic diet for 5 to 7 days. At the end of that time, one new food can be added every 2 days. The following foods can be added early: beef, white potatoes, green beans, milk (along with butter and American cheese), and white bread with puffed wheat. If there is a flare-up of the dermatitis, which should occur within 2 to 8 hours after ingestion of an offending food, the new food should be discontinued for the present. More new foods are added until the normal diet, minus the allergenic foods, is regained.

Keeping a "diet diary" of all foods, medicines, oral hygiene items, or anything injected or inhaled can sometimes be a retrospective way of identifying an allergen. The skin reaction usually occurs less than 8 hours after ingestion.

fungus examinations

The KOH preparation is a simple office laboratory procedure for the detection of fungal organisms present in skin and nails. It is accomplished by scraping the diseased skin and examining the material with the microscope. The skin scrapings are obtained by abrading a scaly diseased area with a scalpel. If a blister is present, the underside of the blister is examined. The material is deposited on a glass slide and then covered with 20% aqueous potassium hydroxide solution and a coverslip. The preparation can be gently heated or allowed to stand at room temperature for 15 to 60 minutes. The addition of dimethyl sulfoxide to the KOH preparation eliminates the need to heat the specimen. A diagnostically helpful pale violet stain can be imparted to the fungi if the 20% KOH solution is mixed with an equal amount of Parker's Super Quink permanent blue-black ink. The slide is then examined microscopically for fungal organisms ( Fig 2-1).

Figure 2-1. Fungi from a skin scraping as seen with microscope in a KOH preparation. (Top) Low-power lens (100*) view. (Bottom) High-power lens (450*) view of area outlined above. ( Dr. D. Gibson)

For culture preparation, a portion of the material from the scraping can be implanted on several different types of agar including: mycobiotic agar, IMA inhibitory mold agar, BHI with blood, chloramphenicol, gentamicin agar, and Sabouraud's glucose agar. A white or variously colored growth is noted in approximately 1 to 3 weeks (Fig.2-2). The species of fungus can be identified by morphology on the culture plate, biochemical characteristics, and microscopic morphology with a lacto-phenol cotton blue stain of a smear from the fungal colony.

biopsies

The biopsy and microscopic examination of a questionable skin lesion may be invaluable. The histologic appearance of many skin conditions may be diagnostic, particularly if correlated with the clinic findings. It is imperative that there be good communication between the physician performing the biopsy and the histopathologist. The instruments and materials needed to perform a skin biopsy are discussed in Chapter^.

There are four principal techniques for performing skin biopsies: (a) surgical excision with suturing, (b) punch biopsy, (c) excision with scissors, and (d) shave biopsy. The decision in favor of one method depends on such factors as location of the biopsy, cosmetic result desired, depth of the disease being looked for, type of lesion to be removed (flat or elevated), and simplicity of technique. For example, vesicles should be completely excised in an attempt to keep the roof intact, and scalp biopsy specimens should extend into the subcutis to include the bulbs of terminal follicles.

sauer note

The skin biopsy specimen must include adequate tissue for proper interpretation by the pathologist.

Surgical Excision

The technique of performing surgical excision biopsies with suturing of the skin is well known. In general, this type of biopsy is performed if a good cosmetic result is desired and if the entire lesion is to be removed. The disadvantage is that this procedure is the most time-consuming of the three techniques, and it is necessary for the patient to return for removal of the sutures. It is important that a sharp scalpel be used to reduce compression artifact, and that care is taken not to crush the specimen with the forceps.

Punch Biopsy

Punch biopsies can be done rather rapidly, with or without suturing of the wound. A punch-biopsy instrument of appropriate size is needed. Disposable biopsy punches are available. A local anesthetic is usually injected at the site. The operator rotates the instrument until it penetrates to the subcutaneous level. The circle of tissue is then removed. Bleeding can be stopped with pressure or by the use of one or two sutures. An elliptical wound instead of a circular wound can be produced by stretching the skin perpendicular to the desired suture line before the punch is rotated. The resultant scar, after suturing, is neater. Punch biopsies may be inadequate for evaluation of vesiculobullous diseases and must be deep enough to include subcutaneous fat if used for diagnosis of panniculitis or tumors in a subcutaneous location. In most instances, pigmented lesions should not be punched unless they can be completely excised.

Scissors Biopsy

The third way to remove skin tissue for a biopsy specimen is to excise the piece with sharp pointed scissors and stop the bleeding with light electrosurgery, Monsel solution, or aluminum chloride solution. This latter procedure is useful for certain types of elevated lesions and in areas in which the cosmetic result is not too important. The greatest advantage of this procedure is the speed and the simplicity with which it can be done.

Shave Biopsy

A scalpel or razor blade can be used to slice off a lesion. This can be performed superficially or deeply. Hemostasis can be accomplished by pressure, light electrosurgery, Monsel solution, or aluminum chloride solution.

Biopsy Handling

The biopsy specimen must be placed in an appropriate fixture, usually 10% formalin. If the specimen tends to curl, it can be stretched out on a piece of paper or cardboard before fixing. Mailing specimens in formalin during winter may result in freezing artifact. This may be avoided by the addition of 95% ethyl alcohol, 10% by volume.

cytodiagnosis

The Tzanck test is useful in identifying bullous diseases such as pemphigus and vesicular virus eruptions (herpes simplex and herpes zoster). The technique and choice of lesions are important. For best results, select an early lesion. In the case of a blister, remove the top with a scalpel or sharp scissors. Blot the excess fluid with a gauze pad. Then gently scrape the floor of the blister with a scalpel blade. Try not to cause bleeding. Make a thin smear of the cells on a clean glass slide. If you are dealing with a solid lesion, squeeze the material between two slides. The slide may be air dried, but it can also be fixed by placing it in 95% ethanol for 15 seconds. Stain the slide with Wright-Giemsa stain, or hematoxylin and eosin.

In addition to skin testing, fungus examination, biopsies, and cytodiagnosis, there are certain tests for specific skin conditions that are discussed in connection with the respective diseases.

additional studies

The deposition of immunoglobulin and complement may be detected by direct immunofluorescence. This is an extremely valuable technique for the diagnosis of lupus erythematosus and autoimmune bullous diseases. It is performed on a frozen section, and therefore the biopsy specimen must be received fresh or in Michel's solution.

Immunohistology is particularly helpful in the accurate diagnosis and classification of neoplasms. It is possible to identify specific antigens in a routinely processed tissue section by attaching a labeled antibody. For example, malignant melanoma may be identified using antibodies directed against S-100 protein and melanoma-specific antigen (HMB-45). Epithelial tumors are immunoreactive for cytokeratins, and malignant lymphoma for leukocyte common antigen.

DNA technology may be very useful. In situ hybridization allows recognition of specific DNA or RNA sequences using a gene probe in frozen or paraffin tissue sections. For example, a variety of different viruses, including herpes simplex, cytomegalovirus, and a human papillomavirus, can be identified using this technique.

The polymerase chain reaction is a technique that amplifies defined DNA sequences. It may be used to identify microorganisms and genetic diseases. It is so sensitive that false positive tests can occur from contamination.

acknowledgment

I would like to acknowledge the assistance of Dr. Cindy Essmeyer and members of her staff, Marcella Godinez, M.T., Katrin Boese, M.T., and Tammy Thorne, M.T., in preparation of the section on fungus examination. I would also like to acknowledge Clint Gillespie, of Photographic Services, for the kodachromes.

*Department of Pathology, St. Luke's Hospital, Kansas City, Missouri

BIBLIOGRAPHY

Skin Tests

Adams RM. Occupational skin disease. Orlando, FL, Grune & Stratton, 1990. Fisher AA. Contact dermatitis, ed 4. Philadelphia, Lea & Febiger, 1995.

Fungus Examinations

Koneman EW, Roberts GD. Practical laboratory mycology, ed 3. Baltimore, Williams & Wilkins, 1985. Isenberg HD, ed. Essential procedures for clinical microbiology. ASM Press, 1998. (See Ch.ap:.19 for additional references.)

Biopsies

Ackerman AB. Histopathologic diagnosis of inflammatory skin diseases. Philadelphia, Lea & Febiger, 1978:149.

Epstein E, Epstein E Jr. Skin surgery, ed 6. Philadelphia, WB Saunders, 1987.

Lever WF, Schaumburg-Lever G. Histopathology of the skin, ed 7. Philadelphia, JB Lippincott, 1990.

Immunodiagnosis

Vassileva S. Immunofluorescence in dermatology. Int J Dermatol 1990;332:153. General References

Beare JM, Bingham EA. The influence of the results of laboratory and ancillary investigations in the management of skin disease. Int J Dermatol 1981;20:653. The authors conclude that the number of ancillary investigations required by an experienced clinician is quite extraordinarily small, including only biopsy, patch testing, sedimentation rate, and Wood's light.

Hurwitz RM, Hood AF. Pathology of the skin: Atlas of clinical-pathological correlation. Stamford, CT, Appleton & Lange, 1998.

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