Investigations (Outline of common investigations in Dermatology) Col. (Dr.) S. Radhakrishnan, MD, DNB, Professor & HOD Dr. Hema Sadar, MBBS, Post Graduate Resident Dept of Dermatology, STD & Leprosy Command Hospital Air Force, Bangalore Digital Lecture Series : Chapter 27 CONTENTS Potassium hydroxide (KOH) examination Wet preparation Tzanck smear Tissue smear Dark Ground Microscopy Demonstration of scabies mite Woods Light examination Slit skin smear Staining for AFB(L) Gram stain Dermatoscopy Patch Test Skin biopsy and Histopathology MCQs Photo Quiz Potassium hydroxide (KOH) examination KOH - dissolves keratin of skin, hair and nails but not the fungal elements Indicated in suspected fungal infections (dermatophytosis, candidiasis and pityriasis versicolor). Steps : Scrapings taken from active margins of skin lesions, infected hair and proximal most part of involved nail (sub ungual). Skin specimen placed in a drop of 10% KOH on a clean glass slide and covered with a cover slip. Place hair and nail specimens in a test tube or small vial of 10% KOH. Allow to stay for min for skin; overnight for nails & hair and then place a drop on a slide as in step 2. Press the slide between two folds of filter paper to remove excess of KOH. Examine the slide with 10x & then 40x with condenser lowered and iris diaphragm closed to allow minimum light. Branching septate hyphae dermatophytosis Short hyphae with spores- P. versicolor Ectothrix spores and hyphae around hair shaft Budding yeast with thin filaments-Candida Utilized for diagnosis of trichomonal infestation of genital tract and for bacterial vaginosis Steps : Swabs of vaginal mucosa and vaginal pool or scraping of suspected skin area Place 1 drop of saline on the slide and add the specimen Cover the specimen with a cover slip to exclude air bubbles Examine - 10x objective for epithelial cells, flagellate organisms or clue cells Wet Preparation Trichomonas vaginalis- pear shaped, jerky movements Bacterial Vaginosis -Diagnosis is made if >20% of cells are clue cells* AND two of the following three criteria are met (Amsel's Criteria) : Discharge is thin and homogeneous Sample smells fishy when mixed with potassium hydroxide ("whiff test) Vaginal pH is >4.5 * Clue cells are epithelial cells of the vagina that get their distinctive stippled appearance by being covered with bacteria. Wet Preparation Trichomonas vaginalisClue cells of Gardnerella-bacterial vaginosis Diagnosis of the pemphigus group of autoimmune bullous diseases and mucocutaneous herpes virus infections. Steps : Viral infection - Gentle scraping of the base of a fresh blister with 15 no. blade after de roofing the blister. Blistering disorders- intact roof of a blister is opened along one side, folded back and the floor gently scraped Material is smeared on slide and air dried. Fixation- gentle heating/ methanol. Staining (Giemsa / Wrights)- diluted (1:10 )with distilled water, and the diluted solution is poured over the smear and kept for 15 minutes, wash and air dry. Examine under oil immersion. Tzanck smear Interpretation : Herpes infection-acantholytic cells and multinucleate giant cells. Vesicobullous disorders-only acantholytic cells. Tzanck smear Acanthoytic cell-pemphigus Multinucleated giant cell-herpes Tissue Smear Diagnosis of granuloma inguinale and molluscum contagiosum. Steps : Tissue from active edge of granulomatous ulcer is taken with toothed forceps; curetted material from suspected molluscum contagiosum lesion. Tissue is crushed between two slides. Smear is air dried and fixed and stained with Giemsa or Wrights stain Examine under oil immersion for Donovan bodies; under low and then high power for molluscum bodies(Henderson-Patterson bodies). Molluscum bodiesDonovan bodies Diagnosis of primary and secondary Syphilis by demonstration of Treponema pallidum Steps : Surface of ulcer is cleaned with saline or gently scraped if dry Serum is obtained by pressing base of lesion firmly between thumb and index finger with care to exclude blood Wet film is covered with a cover slip Examine under dark ground microscope Interpretation : Treponema pallidum appears as a slender, spiral organism showing corkscrew rotation, bending and flexion/extension movements. Dark ground microscopy Treponema pallidum - brightly illuminated organisms against a dark background. It is m wide and 6-16m long organism with 8-14 regular, tightly wound, deep spirals. Interpretation of DGI Dark field microscopy showed Treponema pallidum Samples are best obtained from a non-excoriated papule or burrow Classic scabies mite burrows appear as thin, short, gray-brown, wavy channels on the skin Steps : After applying mineral oil to the lesion, superficially shave or scrape the lesion with a No. 15 scalpel blade. Apply scrapings to a glass slide, cover with a coverslip Examine under the microscope with 4-10 X objective to identify the mite, its eggs or feces. Demonstration of Scabies mite Adult female scabies miteBurrows on the shaft of the penis Demonstration of Scabies mite ScybalaEggs Demonstration of Scabies mite Woods lamp is a low output mercury arc lamp covered by Woods filter(Barium silicate and 9% Nickel oxide) which emits Light of wavelength nm(peak 365 nm) It is used to detect fluorescence in the lesions and to differentiate between epidermal(enhanced by Woods lamp) and dermal pigmentation(unchanged) Steps involved : Examine in a dark room Skin or hair should be examined in a natural state Switch the Woods lamp on and wait for 1-2 minutes for the lamp to emit the correct wavelength. Hold the light 4 to 6 inches from skin or hair, and look for fluorescence or pigmentary change Woods light examination Interpretation of Woods lamp findings : Tinea capitis bluish green fluorescence (Microsporum species) and dull blue in Trichophyton schonenleinii. Other organisms do not fluoresce. Tinea versicolor yellow fluorescence. Erythrasma coral red fluorescence. Porphyria cutanea tarda bright red (urine). Pseudomonas infection green fluorescence. Malassezia folliculitis bluish white fluorescence. Scabies burrow dusted with tetracycline powder yellow fluorescence of the burrow. Woods lamp examination Blue-green fluorescence of T.capitis Coral red - Erythrasma Porphyria - bright red Yellow fluorescence- P. versicolor Specimen from skin lesions, ear lobules, eyebrow, nasal scrapings in leprosy; from suspected nodules/ulcers/plaques in cutaneous Leishmaniasis. Steps : Skin is cleaned with spirit. Skin is pinched to minimise bleeding. Incision with 15 no. blade, 5mm long, 3mm deep is made. Blood, tissue exudates are wiped off. Blade is turned at right angle to the line of incision and is scraped to obtained tissue fluid and pulp. Collected specimen smeared 8mm area, air dried and fixed by gentle heating. Incision site sealed with a wisp of cotton dipped in tincture benzoin or healex spray. Slit skin smear Steps : Stain- Modified Ziehl- Neelsen stain for AFB(L) Giemsa/Wright stain for LD bodies in Leishmaniasis Examine under oil immersion Interpretation : Modified Ziehl- Neelsen stain for AFB(L) Bacteriological index(BI)- number of living and dead bacilli, denotes density of bacilli in smears. Morphological index(MI)- percentage of uniformly stained bacilli out of total number of bacilli counted (200). Slit skin smear Bacteriological index (BI) 1 10 bacilli in 100 fields1+ 1 10 bacilli in 10 fields2+ 1 10 bacilli per field3+ 10 100 bacilli per field 1000 bacilli per field5+ > 1000 bacilli per field6+ AFB (L) seen BI of 6+ with globii Bacteriological index (BI) Staining of lepra bacilli Steps : Freshly prepared, filtered, strong Carbol fuschin stain is poured on a slide containing the fixed smears. Gently heated until fumes appear and the stain is left for 15 to 20 minutes. Wash under a gentle stream of running water. Smear is decolourised with 1% hydrochloric acid & 70% alcohol till no further pink colour comes out. Counterstain with 2% methylene blue for 2-3 minutes. Wash with water and air dry. Examine under oil immersion for AFB (L). Modified Ziehl-Neelsen stain (Acid fast stain) Staining of lepra bacilli Observation: Acid fast bacilli appear red against a blue background Live bacilli- solid stained, long slender with round ends Non viable bacilli- fragmented or granular in appearance Ziehl-Neelsen stain (Acid fast stain) LD bodies in Leishmaniasis Slit Skin smear for LD bodies Slit Skin Smear for LD bodies LD bodies are seen inside macrophages as oval bodies with a kinetoplast perpendicular to the centre of the LD body. Most widely used stain in bacteriology, discovered by the Danish scientist and physician Hans Christian Joachim Gram in Steps : Heat fixed smear of specimen is stained with 2% crystal violet solution for one minute. Pour Grams iodine over the slide for 1-2 minutes. Wash the smear with water. Decolorise with acetone or absolute alcohol for seconds. Wash the smear with water. Counterstain with dilute carbol fuchsin or safranin dye for 30 seconds. Wash the smear, air dry and examine under oil immersion. Gram Stain Gram positive- resist decolorization and retain the colour of the primary stain (violet) Gram negative- are decolorized by acetone/ alcohol, therefore take counterstain and appear red Primarily used to detect gonococci and H. ducreyi in STIs Gram Stain Gram negative diplococci with neutrophils- N.gonorrhoeae Gram positive cocci in bunches - Staphylococci Gram Stain Gram negative cocco- bacilli In chains - H.ducreyi Surface microscopy or epiluminescence microsopy. Allows visualization of pigmented cutaneous lesions in vivo upto reticular dermis. Dermatoscope generates a beam of light that falls on the cutaneous surface at an angle of 20. Light reflection is eliminated by placing fluids like immersion oil, mineral oil or olive oil. Visualization of dermatoscopic characteristics results from presence of melanin and hemoglobin in different skin layers. Link between clinical and histopathology features. Dermatoscopy Early diagnosis of skin melanoma, other pigmented skin lesions like seborrhoeic keratosis, pigmented BCC, hemangioma, nevus. Detailed examination of nail fold capillaries, Wickhams striae, scabies burrow. Scalp surface examination Androgenetic alopecia- Different hair diameters with miniaturisation. Telogen effluvium normal hair diameter with reduced density. Scarring alopecia- loss of follicular opening. Lichen planus- follicular hyperkeratosis. Dermatoscopy Multi-step diagnostic procedure has proven to be successful for pigmentary lesions of nails Differentiation of Melanocytic origin (longitudinal melanonychia) Non-melanocytic origin (non-continuous discoloration) - based on typical dermatoscopic criteria Dermatoscopy Early recurrence of vascular malformation after laser ablation Acquired Melanocytic Naevus Dermatoscopy images Nail fold capillariesAndrogenetic alopecia Dermatoscopy images A bedside diagnostic procedure for identification of suspected allergens in allergic contact dermatitis. Involves exposure of patients skin to potential allergens and observing its reaction. Principle : Re-exposure to the culprit allergen (drug) will elicit similar clinical reaction pattern in previously sensitized individuals. Uses : Allergic contact dermatitis, also useful in drug reactions. Methods of patch testing : Finn chamber or occlusive patch test disc used. Cleanse non hairy, upper back with spirit. Arm/thigh can also be used. Apply test units with control after marking sites. Patient instructed not to bathe or exercise strenuously. Remove the patches after 48 hrs, reading taken after 1 hr. Patch Test Results read at day 2 (day3 and day 7 if initial results are negative). 1- 2% of pure drug in petrolatum/ water/ alcohol is used. Controls are used for high predictive value of positive results and to exclude irritant reactions. Photo-patch test : Irradiation with ultraviolet (UV)-A (5 or 10 J/cm2) at 24 or 48 h done in drug induced photodermatitis, photo allergic/toxic reactions. Finn chamber- 8 mm diameter and 0.5 mm deep, made of stiff aluminum and placed on a strip of adhesive tape. Patch Test Patch Test Readings International Contact Dermatitis Research Group criteria Type of reactionScore No reaction0 Doubtful reaction- faint erythema? Weak positive- palpable erythema, infiltration, papules 1+ Strong positive -erythema, infiltration, papules, vesicles 2+ Extreme positive reaction- intense erythema and infiltration and coalescing vesicles 3+ Irritant reactionIR Finn Chamber Patch Test Positive patch test 2+ reading Types of skin biopsy : Excisional biopsy - removal of entire lesion. Incisional (Wedge biopsy) 12 x 5 mm- removal of small sample of lesion. Punch biopsy (4mm to 6 mm). Shave biopsy- for lesions confined to the epidermis or flat raised lesions. Procedure : Written informed consent. Local anesthesia 1 to 2% Lignocaine +/- Adrenaline injected around biopsy site. Incision along or parallel to RSTL. Sample sent in 10% Formalin; Michaels solution or Normal saline-for IF. Post procedure dressing, antibiotics, suture removal when indicated (face - 6/7 days; legs and back - 14 days). Skin Biopsy Early lesion for biopsy bullous disorder, ulcers, pustular lesions and vasculitis. Established lesion - Psoriasis, DLE, Leprosy. Include surrounding skin for comparison - morphea, anetoderma, malignancy. Scalp biopsy - 2 specimen( horizontal and vertical section) for studying hair follicles. Nail biopsy - Nail bed or nail matrix depending on disorder. Palms and soles - biopsy incision parallel to flexion creases. Choice of lesion and technique in skin biopsy Layers of skin Epidermis Stratum corneum Stratum lucidum ( only in palms & soles) Stratum granulosum Stratum spinosum Stratum basale Dermis Papillary dermis Reticular dermis Subcutaneous tissue Histopathology of Skin Cells in epidermis Keratinocytes - 90%, keratin filaments Melanocytes - 1 for every 10 to 14 basal cell, Melanocyte- Keratinocyte unit ( 1:36), Masson Fontana stain for melanin Langerhans cells - dendritic, Antigen presenting cell in St. spinosum, Gold stains > Silver stains Merkel cells - neural crest derivative, present in St. basale, role in touch sensation Hematoxylin and Eosin > Periodic Acid Shiff most commonly used stains in dermatopathology Histopathology of Skin Epidermal Changes Hyperkeratosis - thickening of stratum corneum (> 1/3rd of total thickness of epidermis), orthokeratosis-hyperkeratosis with normal st. corneum and granular layer e.g- Ichthyosis Parakeratosis - retention of nuclei in st. corneum, Ex- psoriasis, lichen nitidus Acanthosis - thickened st. malpighii (live layers of epidermis), psoriasis, Lichen simplex chronicus, warts Atrophy - thining of epidermis, Ex- steroid induced, lupus erythematosus Histopathology of Skin Epidermal Changes Spongiosis - intercelluar edema in St. malpighii, Eg- Eczema. Acantholysis - loss of cohesion between keratinocytes, Eg. Pemphigus, Herpes Simplex/ Zoster Exocytosis - migration of cells into epidermis in presence of spongiosis, Eg- Acute Eczema Epidermotrophism - Presence of mononuclear cells in epidermis in absence of spongiosis, Eg- Mycosis fungoidis Follicular plug - plugging of hair follicle with compact keratin, Eg- DLE, Acne vulgaris, Keratosis pilaris, PRP Histopathology of Skin Epidermal Changes Abscess - collection of neutrophils or eosinophils in epidermis/ dermis. Munros microabscess - neutrophilic collection in St. corneum. Kogojs spongiform pustule - collection of polymorphonuclear cells in St. granulosum (Psoriasis) Papillary tip microabscess - neutrophilic/ eosinophilic collection in tips of dermal papillae (Dermatitis herpetiformis) Degeneration Balloon degeneration - cells are swollen with fluid, appear balloon like with central nucleus, lower epidermis,e.g - HSV Reticular degeneration - rupture of keratinocytes, fusion of margins to form reticular network, upper epidermis, e.g -HZV Histopathology of Skin Dermal Changes Grenz Zone - A clear zone of normal dermis between the epidermis and pathological changers deeper in dermis, e.g- Lepromatous leprosy. Granuloma - collection of mature mononuclear phagocytes (macrophage/epithelioid cells) and its derivatives, associated with necrosis/inflammatory cells Tuberculoid Sarcoid naked granuloma Necrobiotic Xanthogranuloma Foreign body granuloma Histopathology of Skin Dermal Changes Fibrosis - increased collagen formation with increase in fibroblast number. Sclerosis - increased collagen, with homogenous and hyalinized appearance with decreased fibroblasts. Pigmentary incontinence - loss of melanin from basal layer and accumulation of melanin within melanophages due to basal cell degeneration. Interface dermatitis - lichenoid tissue reaction at DEJ characterized by basal cell vacuolization, apoptotic keratinocytes ( civatte body) obscuring DEJ, e.g. Lichen Planus Histopathology of Skin MCQs Q.1) An adult presents with 5 to 10 mm oval scaly hypopigmented macules over the chest and back. The diagnosis is A.Leprosy B.Lupus Vulgaris C.Pityriasis versicolor D.Pityriasis alba Q.2) Woods lamp is useful in the diagnosis of : A.Erythrasma B.Psoriasis C.Lichen Planus D.Tinea Corporis Q.3) Eczema herpeticum is associated with infection by: A.HSV B.CMV C.EBV D.VZV Q.4) A 28yr old presents with multiple grouped papulovesicular lesions on both elbows, knees, buttocks and upper back associated with severe itching. The most likely diagnosis is : A.Pemphigus vulgaris B.Dermatitis herpetiformis C.Bullous pemphigoid D.Herpes Zoster MCQs Q.5) A patient had seven irregular hyperpigmented macules on the trunk and multiple small hyperpigmented macules in the axillae and groin since early childhood. There were no other skin lesions. Which of these is the most useful investigation to come to a diagnosis? A.Slit lamp examination of eye B.Intra Ocular Pressure measurement C.Fundus examination D.Retinal artery angiography MCQs Q.6) What is your diagnosis? A.Pemphigus foliaceous B.Bullous pemphigoid C.Herpes Zoster D.Pemphigus vulgaris Photo Quiz Q.7) Direct microscopic examination of this organism can be done by obtaining a skin scraping from- A.Burrow B.Excoriation C.Nodule D.Keloid Photo Quiz Thank You!