ApPENDIX ONE Useful Techniques In the body of this book, many investigations have been recommended that are of help in confirming the various diagnoses. Many of these (routine histology, bacteriology) are well known to most readers, but we suspect that others are less widely used. This appendix contains details of a number of procedures that may be helpful to the nonspecialist. Cellophane Tape Test Transparent self-adhesive plastic tape (Cellotape, Scotch tape) is extremely useful in confirming the diagnosis of pityriasis versicolor. Three to four centi­ meters of the tape are stuck over an active area and firmly rubbed with the back of a fingernail. When removed, the tape will show an exact replica of the scaly lesion that it has covered. This in itself is diagnostic of pityriasis versi­ color, but further confirmation can be obtained if the tape is dipped for one minute into a drop of 1 percent gentian violet, blotted, and mounted on a microscope slide. Examination will show the spores and germ tubes of the Pityrosporum. The cellophane tape test is negative in early cases of tinea imbricata, which may sometimes be confused with pityriasis versicolor. Diascopy It is often helpful to know whether or not a dermal papule has a vascular ele­ ment. This can best be recognized by determining whether it blanches on pres­ sure, but as the pressing finger will entirely cover a small lesion, it is more 244 Appendix One Useful Techniques helpful if the pressure is exerted with some hard, flat material that is trans­ parent. This permits the nodule to be seen while it is under pressure. Such instruments may be a plastic tongue depressor or spatula, a magnifying lens, or some other reasonably thick piece of glass. An ordinary microscope slide can be used, but they have been known to break under pressure and lacerate the patient. Diascopy is of particular value in examination of nodules in a patient sus­ pected of having lupus vulgaris. The apple-jelly nodule is often rather erythe­ matous, and only under diascopy will the typical greenish-yellow color be clearly revealed. The Matchstick Test An even older technique for demonstrating an apple-jelly nodule, located in the upper part of the dermis, and covered by a taut, stretched epidermis, involves taking an ordinary wooden match that has been sharpened to a point and plac­ ing it vertical to the papule. Light pressure with a finger will cause the match to penetrate the epidermis and stand upright without support. If the lesion is deep in the dermis and the epidermis is not thinned, the point of the match will break under pressure. Both these tests are positive in lupus vulgaris and lupoid leishmaniasis and negative in tuberculoid granulomata. Dutz Technique For the many bacterial or parasitic lesions that show crusting or ulceration, cotton swabs are usually used to obtain suitable material for examination or culture. This method is often ineffective in ulcerated lesions and of no use at all if a sample is needed from the depths of a dermal infection. It is better to use a dental broach. These instruments, used by dentists to abrade dentine, are steel needles, the distal part being surrounded by metallic barbs of different sizes. The finer broaches may easily be pushed into the core of an infected area, producing a very small puncture wound. If they are gently rotated before removal, they will become coated with tissue, which can be used for a stab culture, to make a direct smear for bacteriologic examination, or to inoculate a bacterial plate. If the lesion is crusted, it is usually possible to insert the broach from the side into the main body of the swelling and so completely avoid any surface contamination that may be present. The technique has been found useful for leishmaniasis, leprosy, anthrax, and yaws. (Reference: Dutz, W, Kohout, E: Dermatologic diagnosis by using the hemocytometer and the dental broach. Int J Dermatol1982; 21:410.) Leprosy Skin Smear 245 Lepromin Test If lepromin is available and kept at a suitable temperature, it can be used as a diagnostic aid. It seems to have become traditional to use the left forearm some two inches distal to the elbow fold to inject 0.1 ml intradermally. Although the WHO suggests that readings should be taken after 28 days, those taken after three weeks are acceptable. The diameter of the resultant papule is measured in millimeters and recorded. 0-3 mm negative 3-6mm + 6-9mm ++ Over 10 mm +++ Unfortunately, both false negative and false positive reactions are possible, the former perhaps because the injection has been too deep and the latter because an unclean needle has caused a small intradermal infection. The combination of clinical, bacteriologic, and histopathologic findings usu­ ally makes the lepromin an unnecessary luxury. Leprosy Skin Smear It may be impossible to detect any M. /eprae in a lesion of tuberculoid leprosy, but in all other types the mycobacterium is present in varying numbers. The demonstration of its presence is an essential part of the clinical investigation of borderline and lepromatous disease. How to Do the Skin-Slit The following equipment is necessary: 1. a supply of clean, preferably unused microscope slides; 2. an alcohol lamp; 3. numerous gauze or cotton swabs; 4. 1 percent cetrimide or other nonflammable skin cleanser; 5. a scalpel handle fitted with a No. 15 Bard Parker blade. The purpose of this investigation is to detect the presence of leprosy organ­ isms. It is not a blood test. The lesion should be squeezed between the forefinger and thumb of one hand until it is suitably blanched. The skin is then cleaned, an incision 3 to 4 mm long is made, and, the blade being turned at right angles, one of the walls of the cut is firmly scraped to remove a drop of tissue fluid. This is smeared onto a clean slide over an area approximately I cm in diameter 246 Appendix One Useful Techniques (two or three smears can be made on each slide). If the lesion has been firmly held, the smear will not be bloodstained. Sometimes the incision will bleed after the smear has been taken-a small wisp of cotton will ensure rapid coagulation. The scalpel blade is wiped with cetrimide, flamed on the alcohol lamp, and cleaned again before the next smear is taken. Which sites are studied should be recorded so that further investigations in three or six months' time will be from the same lesions and so be truly comparable. Where to Do the Skin-Slit Patients in the borderline group (BT, BB, or BL) will not have a generalized eruption, and there is no point in taking smears from skin that is not clinically and visibly involved. Six different sites should be tested to obtain an overall picture of the activity and severity of the disease. For lepromatous patients, the following routine is recommended. The shirtless patient sits on a stool with his back to the operator and smears are taken from the following sites: 1. left ear lobe; 2. right ear lobe; 3. lesion from the back of the left arm; 4. lesion somewhere on the back; 5. lesion from the right arm; 6. the patient turning toward the operator, lesion on the knee or thigh. It is helpful to take smears from both ear lobes, because the unexpected pres­ ence of bacilli can be taken as evidence that the patient's disease is becoming completely lepromatous. This technique is not particularly scientific, as an unmeasured quantity of fluid is smeared over an unmeasured area on the slide; different operators will produce smears that differ widely in thickness. It is urged that a patient's smears always be taken by the same operator, thus minimizing the possibility of extensive technical variation. How to Count the Skin-Slit All smears should be fixed by gentle heating and then stained. Four solutions are needed: Solution A 10 percent carbol fuchsin in 90 percent absolute alcohol Solution B 5 percent phenol in 95 percent distilled water Solution C 0.5 percent hydrochloric acid in 70 percent alcohol Solution D 1 percent methylene blue Leprosy Skin Smear 247 Step 1 Combine 1 ml of solution A with 9 ml of solution B and cover the smear for 20 minutes. It may be gently heated, but not dried out, for a minute at the start of staining. After this, the slide should be washed with running water. Step 2 Decolorize the smear by pouring on a small amount of solution C for a few seconds and wash again with running water. If the smear is still red, decolorize further, until there is nothing left but a pale pink tinge. Step 3 Counterstain with solution D for two minutes, wash and leave to dry. The stain is now ready to be counted. Under an oil-immersion lens, the mycobacteria should appear as red-stained rods. Other tissues are slightly blue. Ridley's logarithmic index is used to count the organisms. One hundred oil­ immersion fields are examined on each smear, and the total number of bacilli are recorded as follows: 1-9 organisms in 100 fields 1 + 10-99 organisms in 100 fields 2 + 1-9 organisms in every field 3 + 10-99 organisms in every field 4 + More than 100 organisms in every field 5 + It is possible but very unusual to find more than 1,000 bacilli in each field. This must be counted as 6 + . The bacterial index (BI) is then recorded as the average from all the six sites examined. What to Count Unfortunately, even with successful antileprosy treatment the BI takes a very long time to diminish significantly, as dead M.