Bedside investigations in dermatology

Dr. Sinni JainMD Dermatology

Jaipur

Summary

• KOH mount• Gram stain• Tzank smear• AFB stain• Slit skin smear• Dark groung

microscopy• Diascopy• Wood’s lamp

examination

• Patch testing• Intra dermal testing• Nikolsky’s sign • Grattage test• Auspitz sign• Dermoscopy • Skin biopsy• Immunofluoroscence

KOH Mount

Skin scraping for KOH examination

• Involves microscopic examination of stratum corneum to visualize fungal elements.

• KOH solution causes separation and destruction of the stratum corneum cells.

• This allows easy identification of exogenous materials such as hyphae and spores which are unaffected by the KOH solution.

Procedure-

• Swab the site with spirit• Scrap the lesion at active border with a 15 no. blade or

take hair/nail clipping• Add 1-2 drops of 10% KOH and put cover slip• Wait for 15-20 min. for the keratin to digest (overnight

for nail clipping).• nail involvement- scraping the affected sites at a

considerable depth. Scooping out the deeper keratinous matrix and mounting it in 20% potassium hydroxide yields better results

(For thick, hyperkeratotic specimens, leave the potassium hydroxide preparation for 'digestion' and 'clearing' for ½ to 2 h. This clearing time for nails and hairs may extend to 24-48 h. 

Indications –

• Dermatophytosis of the skin, hair and nails • Candidiasis• Tinea versicolor• Vaginosis • Tinea nigra• Demonstration of mites (Demodex folliculorum,

sarcoptes scabiei)• Demonstration of fungi from cutaneous lesions

of deep fungal infections ( cryptococcosis, blastomycosis, chromoblastomycosis)

Dermatophytes- multiple, refractile, branched, septate hyphae

Candidiasis- budding ovoid yeast cells and pseudohyphae

Tinea versicolor- hyphae with clusters of spores , often called “spaghetti and meatballs”

Chromoblastomycosis- sclerotic or muriform cells

Blastomycosis- refractile spherical cells with broad-based buds.

Cryptococcosi - capsulated forms

Vaginosis- clue cells

Scraping for scabies

• After applying a drop of mineral oil, the burrow is scraped with a 15 no. scalpel blade.

• Scraping transferred to glass slide and seen under microscope.

• Reveals mite, eggs or fecal pellets.

• Modifications of standard method-1.A 5 cm long and 2 cm wide scotch tape

(transparent cellophane tape) can be applied over the affected site, pressed firmly and removed.

The tape is then stuck on the surface of a glass slide and sent to the laboratory, where it is gently lifted and replaced after placing 3 to 4 drops of 10% potassium hydroxide solution.

The undersurface of the slide is warmed gently and examined under microscope

2. Parker's ink added to potassium hydroxide stains the fungal wall blue

3.  fluorochrome stain(calcofluor white) for rapid detection. Viewed under ultraviolet light, fungal structures display a brilliant apple-green or a ghostly blue-white color.

GRAM STAINING

Gram’s staining of exudates

Used to identify the organism in infected lesions.

Procedure- A thin layer of specimen is spread on a glass

slide, dried, and heat fixed to the glass.  The slide is flooded with 2% crystal violet and

allowed to stain for 30 seconds to two minutes and then gently rinsed off with water.

• Incubated in Gram’s iodine for > 30 seconds (iodine fixes the crystal violet to peptidoglycans of the Gram-positive cell wall).

• After rinsing off the Gram’s iodine with water, the slide is briefly decolorized with acetone.

• Then counterstained with dilute carbol fuschin for a few seconds, rinsed and air-dried.

Gram positive cocci (blue/purple) Gram negative bacilli (pink)

A Gram stain of mixed Staphylococcus aureus (Gram positive cocci) and Escherichia coli (Gram negative bacilli

TZANK SMEAR

• usesImmunobullous disordersCutaneous infectionsGenodermatoses Suspected tumors

TZANK SMEAR

Procedure-• After deroofing the blister, floor is scraped and

material smeared on a slide.• Stained with Wright's or Giemsa's stain. • For the cytodiagnosis of suspected tumors, any

crust should be removed from ulcerated tumors, and non-ulcerated tumors should be incised with a sharp, pointed scalpel (avoid undue bleeding). Sample of tumor is then obtained with either a blunt scalpel, and the tissue obtained is pressed between the two slides

Tzanck testINDICATIONS1.Cytodiagnosis of immunobullous disorders-a) P. vulgaris- Acantholytic cells (Tzanck cells). A typical Tzanck cell is a large round keratinocyte with a hypertrophic nucleus, hazy or absent nucleoli, and abundant basophilic cytoplasm. The basophilic staining is deeper peripherally on the cell membrane ("mourning edged" cells) due to the cytoplasm's tendency to get condensed at the periphery, leading to a perinuclear halo.Pemphigus vegetans- the cytologic features are identical but there are usually more inflammatory cells, particularly eosinophils.

In contrast to pemphigus vulgaris, the acantholytic cells in pemphigus foliaceus and pemphigus erythematosus often have a hyalinized cytoplasm that corresponds to the dyskeratosis seen in tissue sections 

b) Toxic epidermal necrolysis (TEN) and staphylococcal scalded skin syndrome (SSSS)TEN show necrotizing or degenerating basal cells with scattered inflammatory cells and fibroblasts while those from SSSS show dyskeratotic acantholytic cells with very few inflammatory cells.c) Bullous pemphigoid (BP), Stevens-Johnson syndrome (SJS) and erosive lichen planus  No acantholytic cells. The smear only serves to readily rule out pemphigus

2) Cytodiagnosis of cutaneous infectionsa) Herpes simplex, varicella, herpes zoster – The typical features include characteristic multinucleated syncytial giant cells and acantholytic cells. The cells appear as if they have been inflated ("ballooning degeneration") Intranuclear inclusion bodies b)  Molluscum contagiosum-  Intracytoplasmic molluscum bodies (Henderson-Patterson bodies)

c) Vaccinia, orf, milker's nodules and variola: Eosinophilic cytoplasmic inclusion called a "Guarnieri body", frequently surrounded by a clear halo.d) Leishmaniasis: Leishman-Donovan (LD) bodies

3) Cytodiagnosis of gendermatosesi)Hailey-Hailey disease: multiple acantholytic cells

ii)Darier’s disease - corps ronds & grains. "Corps ronds"-isolated keratinocytes with a round shape and an acidophilic cytoplasm, which is retracted from the nucleus and denser peripherally ("mantle cells"). Grains -small, hyaline, acidophilic ovoid bodies resembling pomegranate seeds.

3) Cytodiagnosis of cutaneous tumorsi)Basal cell epithelioma:clusters of basaloid cells which look like normal basal cells except for being a little larger and more deeply basophilic. ii) Squamous cell carcinoma: The two distinctive cytological features of squamous cell carcinoma are the tendency of cells to be isolated (absence of clusters), and pleomorphism.iii) Paget's disease: Paget's cells, occur singly or in small groups, and are round to oval cells with amphophilic, vacuolated cytoplasm and a hypertrophic nucleolated nucleus. They appear larger than keratinocytes. 

• iv) Erythroplasia of Queyrat: polyhedral, spindle-shaped and round cells with "poikilokaryosis" (nuclear polymorphism relating to size, shape and staining), practically diagnostic for this intraepithelial carcinoma.v) Mastocytoma- useful in children, in whom the need for biopsy may be obviated. Tzanck smear stained by 1% methylene solution for 1 minute shows plenty of mast cells, which are recognized by their irregular shape (triangular, polygonal, or pyriform) and metachromatic staining of granules (reddish purple).

vi) Histiocytosis X:Multinucleate atypical Langerhans cells appear as 12-15 mm sized cells with wide, pale, weakly eosinophilic or amphophilic, micro-vacuolated or granular cytoplasm and a large lobulated, convoluted, reniform or centrally grooved nucleus.

Multinucleate giant cells

Acantholytic cell

Handerson Patterson bodies Corps ronds & grains

AFB (Zeihl- Neelsen) staining

• PURPOSE: demonstration of acid-fast bacteria belonging to the genus 'mycobacterium‘

• PRINCIPLE: The lipoid capsule of the acid-fast organism takes up carbolfuchsin and resists decolorization with a dilute acid rinse. The lipoid capsule of the mycobacteria is of such high molecular weight that it is waxy at room temperature and successful penetration by the aqueousbased staining solutions (such as Gram's) is prevented.

AFB (Zeihl- Neelsen) staining

• PROCEDURE: 1. Air dry the smear.2. Carbol-fuchsin solution for 5 minutes 3. Wash in running tap water. 4. 20% sulphuric acid until light pink and color stops

running. 5. Wash in running tap water for 5 minutes6. methylene blue for 30 seconds.7. Rinse in water.8.. Dehydrate, clear, and coverslip.

• RESULTS: Acid-fast bacilli bright red …Background blue

• List of Acid Fast organisms (Other than Mycobacteria)

• Nocardia spp: Partial Acid Fast• Rhodococcus spp: Partial Acid Fast• Legionella micdadei: Partially acid fast in

tissue• Cyst of Cryptosporidium: Acid Fast• Cyst of Isospora: Acid Fast

Slit skin smear examination

• Most important laboratorial test to detect lepra bacilli in suspected Hansen’s patches and to classify the d/s.

• Role 1.Confirm diagnosis of leprosy2.Classify the disease3.Determine disease activity in a patient4.Assess progress of disease5.Follow-up patients on treatment

• SITES1.right ear lobe2.Forehead3.Chin4.Left buttock in men, left upper thigh in

women

Procedure-• Lesion is cleaned with spirit.• After pinching the skin b/t thumb and index

finger, a 5mm long and 2mm deep cut is made with sterile blade (Bard Parker No. 15)

• Base is scraped and the material is smeared (8-10mm) on a glass slide.

• After drying and heat fixing the smear, Ziehl-Neelsen staining is done.

• MORPHOLOGICAL INDEXPercentage of solid stained bacilli

calculated after examining 200 bacilli lying singly.

Bacilli are considered solid staining if- a)Entire organism is uniformly stainedb)Longitudinal sides are parallelc)Both ends are roundedd)Length is five times its width

Dark ground microscopy• Most specific and sensitive technique to diagnose

syphilis when an active chancre or condyloma lata is present

• The dark ground microscope creates a contrast between the object and the surrounding field, such that, the background is dark and the object is bright.

•  Special condenser is used, which prevents the transmitted light from directly illuminating the specimen. Only oblique scattered light reaches the specimen and passes onto the lens system causing the object to appear bright against a dark background 

Procedure-• Remove any scab or crust covering the lesion.• Remove any exudates with a gauze sponge.• Compress base of the lesion to promote accumulation of

tissue fluid on the surface.• Apply glass slide with a sterile bacterial loop to the

surface of the lesion.• Press a glass coverslip on the specimen and press it

down to remove any air bubbles.• Examine the slide immediately.

• Interpretations Treponema pallidum appear as brightly

illuminated objects against a dark background. 0.25-0.3µm wide and 6-16µm long organism with 8-14 regular, tightly wound, deep spirals.

It exhibits quick and abrupt movements. The organism rotates slowly along the longitudinal axis (corkscrew motion) accompanied by bending and twisting in the middle. 

• False positive- when oral spirochetes are not confirmed or when there is misinterpretation of the characteristic motility of genital spirochetes.

False negative- if insufficient exudates are taken, if the interval between sample collection and examination is too long, if the lesion is approaching natural resolution or in patients already on treatment with penicillin.

Diascopy • A refinement in which a piece of clear glass or

plastic is pressed against the skin while the observer looks directly at the lesion under pressure.

• The purpose of this procedure is to empty blood from the superficial vessels to determine if skin redness is due to blood within vessels (erythema) or extravasated into the skin (petechiae, purpura).

• The former will blanch with pressure, the latter will not.

Clinical Significance• telangiectasia (in which the central "feeder"

vessel may be distinguished)• petechiae and purpura• superficially dilated veins (venous lake,

varicosities)• granulomatous nodules such as sarcoidosis,

granuloma annulare, and lupus vulgaris (reveal a brownish-yellow "apple jelly" nodules)

Wood’s lamp examination

• Wood’s lamp is a mercury vapor ultraviolet lamp with a filter which is opaque to all wavelengths except those b/t 320 to 400 nm

• Peak at 365 nm

• emits long-wave UV radiation (UVR), also called black light, generated by a high pressure mercury arc fitted with a compound filter made of barium silicate with 9% nickel oxide, the “Wood's filter.”

• Fluorescence of normal skin is very faint or absent and is mainly due to constituents of elastin, aromatic amino acids and precursors or products of melanin

• Increase in pigmentation (eg melasma, postinflammatory pigmentation) to determine whether the pigmentation is epidermal (pigmentation enhanced by Wood lamp examination) or dermal (pigmentation unchanged by Wood lamp examination.

• Loss of pigmentation (eg vitiligo, ash-leaf macules in tuberous sclerosis, and hypomelanosis of Ito) to identify affected areas in light skinned people. Hypopigmented skin has sharper borders under black light and fluoresces bright blue-white. In contrast, areas of reduced blood flow are unchanged

• Pityriasis versicolor— yellowish- white• Malassezia folliculitis—hair follicles fluoresce bluish-white• Tinea capitis—. Microsporum species fluoresce blue-green (M

canis, M audouinii, M ferrugineum); Trichophyton schoenleinii fluoresces dull blue. Fungal infectiondue to other organisms does not fluoresce

• Erythrasma—coral-pink colour• Pseudomonas  infection - green• Acne fluoresces orange-red due to propionibacteria in hair follicles• Porphyria causes red-pink fluorescence of the skin (porphyria

cutanea tarda) 

T.capitis

Erythrasma

Vitiligo

Vitiligo

Patch testing

• Used to identify causes of allergic contact dermatitis.• Procedure-• Various patch test allergens (contained within small

metal chambers made of aluminium and plastic) are held against the skin using a hypoallergic tape. Finn chamber is commonly used (8mm diam & 0.5mm depth)

• Upper back/ arm/ thigh• Standard vehicle is white petrolatum• Polypropylene syringes are used to store allergens in

cool, dark place

• Remains on the skin for 48 hours during which the person cannot get the tape wet.

• Reading is taken half an hour after removal of patch.

• 2nd reading on day 4 to day 7 likely to be taken• PHOTOPATCH TEST- two identical sets of

substances are put on as described above. One set is exposed to some UV light. 

• Allergens used in patch testing include- metals (e.g. nickel), rubber, leather, hair dyes, formaldehyde, neomycin,fragrance, preservative etc..

• Erythema, infilteration, papules and vesicles indicate positive reaction.

GRADING

DESCRIPTION INTERPRETATION

- No erythema/ papule Negative reaction+-or ? Erythema only Doubtful + reaction+ Erythyma, mild

infiltration, discrete papules

Weak + reaction

++ Erythema, infiltration,papules & vesicles

Strong + reaction

+++ Intense erythema, coalescing vesicles

Extreme + reaction

IR Sharply demarcated erythema, epidermal necrosis

Irritant reaction

NT Not tested

False negative reactions•  delayed test reading. • the allergen concentration is too low to

elicit a response• the test site might have been inappropriate• the patient's skin is unresponsive by prior

sun exposure• concurrent immunosuppressive therapies • methodological flaws, such as insufficient

occlusion, early removal, non occlusion.

False positive reactions• Use of wrong test substance- when

substance is irritant in nature or used in higher concentration, contamination of test substance

• Hyperreactive skin/ excited skin syndrome/ angry back syndrome/ crazy back

• Artifact (scratching, otherwise irritating skin by patient)

Complications

• Severe reaction• Persistent positive reaction (> 1 month)• Anaphylaxis • Active sensitisation (positive after 10-14

days)• Focal flare• Depigmentation, scars, keloids (rare)

Intra dermal testing•  mainly indicated for the detection of

immediate (Type I hypersensitivity) and delayed type hypersensitivity (Type IV hypersensitivity) towards exogenous or endogenous antigens

• advisable to stop or avoid systemic steroids or immunosuppressive agents at least three days before the procedure

• PROCEDURE-injection of 0.1ml antigen into the superficial layer of the dermis through a fine-bore (26 or 27-G) needle.

• INTERPRETATIONRead at 48h usually (The lepromin test is

read at four weeks and depends on the formation of a granuloma, which is a measure of cell-mediated immunity.)

The size of the induration is more important than erythema while interpreting Type IV hypersensitivity.

• TUBERCULIN TEST- Induration more than 10 mm in diameter -positive less than 5 mm -negative.

between 6 mm and 9 mm are doubtful and could be because of an atypical mycobacterial infection

• A positive test indicates past or present infection with M. tuberculosis or BCG vaccination.

• induration of more than 15 mm is usually not due to BCG vaccination.

• A positive test does not indicate active infection except in children younger than two years.. 

Lepromin test

It is a prognostic test •helpful in classifying leprosy. •negative towards the lepromatous pole•Two types of antigens : Mitsuda lepromin and Dharmendra lepromin•The response after intradermal injection is typically biphasic, with an early Fernandez (within 48h) and a late Mitsuda (5-6 wks)reaction. Both responses are manifestations of CMI towards the antigen. 

Nikolsky’s sign

• elicited in blistering diseases to determine whether the epidermis is adherent to the underlying dermis.

• A finger or rounded object such as a pencil eraser is used to rub or rotate the skin with a mild shearing effect.

• Clinical Significance• Diagnostic possibilities include pemphigus and toxic

epidermal necrolysis, staphylococcal scalded skin syndrome• most easily produced when the epidermis is acantholytic• not diagnostic or absolutely specific.

Grattage test

• In psoriasis• On grattage, characteristic coherence of

scales seen as if one scratches a wax candle(‘signe de la tache de bougie), accentuation of scales on scrapping of psoriatic lesions

Auspitz sign

• Typical , but not diagnostic of psoriasis• When the thick white scale of psoriasis is

carefully scraped away from the surface of a plaque, tiny bleeding points may be seen in the underlying epidermis.

• These points are the vascular dermal papillae that have been traumatized by removal of the thin suprapapillary epidermis.

Dermoscopy • Dermoscope is a  Non-invasive, diagnostic tool which

visualizes subtle clinical patterns of skin lesions and subsurface skin structures not normally visible to the unaided eye.

• Skin surface microscope, epiluminescence microscope or episcope.

• Added advantages over magnifying glass:-1. inbuilt illuminating system2. higher magnification which can be adjusted3. ability to assess structures as deep as in the

reticular dermis4. ability to record images.

PRINCIPLE•  Transillumination of a lesion and studying it with a

high magnification to visualize subtle features.• Light incident on skin undergoes reflection, refraction,

diffraction and absorption. These phenomena are influenced by physical properties of the skin

• Most of the light incident on dry, scaly skin is reflected, but smooth, oily skin allows most of the light to pass through it, reaching the deeper dermis.

• Improve the visibility of subsurface skin structures- application of linkage fluids over the lesions- improves the translucency of skin.

• Various linkage fluids- oils (immersion oil, olive oil and mineral oil), water, an antiseptic solution and glycerin. 70% alcohol –best results in term of image clarity.

Skin biopsy

Process by which a part or whole of the suspected diseased tissue is obtained for microscopy and other investigation.

INDICATIONS Confirm clinical diagnosis Gauge prognosis For special investigations As a therapeutic modality

CONTRAINDICATION Bleeding diasthesis Active infection at the site Keloidal tendency TYPES Shave for exophytic growths Punch for endophytic growths Excisional for suspected malignancy and as

therapeuticapproach. Incisional for deeper lesions

Biopsy procedure : • Select proper site . • Intradermal or ring anaethesia • Sample is kept in formalin • Specimen must be labeled •  Topical antibiotic for one week should

be prescribed .

• Complications –1.Hypersensitivity to local anaesthesia2.Pain of local anaesthesia3.Bleeding4.Scarring 5.Infection

Immunofluoroscence • Technique for the detection of a wide variety of antigens in

tissues or on cells in suspension.• Types- • Direct immunofluorescence (DIF): one step procedure that

involves application of fluoresceinated antibodies to a frozen section of the skin, determines the deposition of immunoreactants in the patient's tissue.

• Indirect immunofluorescence (IIF): normal whole tissue is the substrate (usually monkey esophagus), requires two incubations. The patient's serum is layered on the substrate followed by application of fluoresceinated antibodies, detect circulating antibodies in the serum.

A modified IIF technique using the patient's own skin as a substrate known as immunomapping (antigen mapping) is used to determine the exact site of cleavage or abnormalities in the distribution of mutated structural proteins (normal, reduced, or lack of expression) in various forms of hereditary epidermolysis bullosa (EB).

• Complement fixation: After the patient's serum is layered on the substrate, a source of complement is added. Fluoresceinated anticomplement antibodies are then used to detect the presence of complement in the tissue, can detect small quantities of complement fixing antibodies.

• Immunoelectron microscopy (IEM): It can be performed in an analogous fashion to detect DIF or IIF. Instead of fluoresceinated antibodies, the antibodies are labeled with an enzyme, such as horseradish peroxidase or a heavy metal, such as colloidal gold, provides subcellular or ultrastructural localization of immunoreactants. This may be helpful in the differential diagnosis of subtypes of hereditary EB, where antigen mapping is not significant.

• Other variants of IF1.Salt split technique2.Antigen mapping3.Double staining method

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