Investigations
(Outline of common investigations in Dermatology)
Digital Lecture Series : Chapter 27
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-560007
CONTENTS
 Potassium hydroxide (KOH)
examination
 Slit skin smear
 Staining for AFB(L)
 Wet preparation
 Gram stain
 Tzanck smear
 Dermatoscopy
 Tissue smear
 Patch Test
 Dark Ground Microscopy
 Skin biopsy and Histopathology
 Demonstration of scabies mite
 MCQ’s
 Wood’s Light examination
 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 20 -30 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 sporesP. versicolor
Budding yeast with thin
filaments-Candida
Ectothrix – spores and
hyphae around hair shaft
Wet Preparation
 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.
‘Clue cells‘ of
Gardnerella-bacterial vaginosis
Trichomonas vaginalis
Tzanck smear
 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 / Wright’s)- 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.
Multinucleated giant cell-herpes
Acanthoytic cell-pemphigus
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 Wright’s stain
 Examine under oil immersion for Donovan bodies; under low and
then high power for molluscum bodies(Henderson-Patterson bodies).
Donovan bodies
Molluscum bodies
Dark ground microscopy
 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.
Interpretation of DGI
 Treponema pallidum -
brightly illuminated organisms
against a dark background.
 It is 0.25-0.3µm wide and 6-16µm
long organism with 8-14 regular,
tightly wound, deep spirals.
Dark field microscopy showed
Treponema pallidum
Demonstration of Scabies mite
 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
Burrows on the shaft
of the penis
Adult female scabies mite
Demonstration of Scabies mite
Eggs
Scybala
Wood’s light examination
 Woods lamp is a low output mercury arc lamp covered by Wood’s
filter(Barium silicate and 9% Nickel oxide) which emits Light of
wavelength 320-450 nm(peak 365 nm)
 It is used to detect fluorescence in the lesions and to differentiate
between epidermal(enhanced by Wood’s lamp) and dermal
pigmentation(unchanged)
Steps involved :
 Examine in a dark room
 Skin or hair should be examined in a natural state
 Switch the Wood’s 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
Wood’s lamp examination
Interpretation of Wood’s 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.
Coral red - Erythrasma
Blue-green fluorescence of
T.capitis
Yellow fluorescence- P. versicolor
Porphyria - bright red
Slit skin smear
 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).
Bacteriological index (BI)
1 – 10 bacilli in 100 fields
1+
1 – 10 bacilli in 10 fields
2+
1 – 10 bacilli per field
3+
10 – 100 bacilli per field
4+
100 – 1000 bacilli per field
5+
> 1000 bacilli per field
6+
Bacteriological index (BI)
AFB (L) seen – BI of 6+ with globii
Modified Ziehl-Neelsen stain (Acid fast stain)
 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).
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
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.
LD bodies in Leishmaniasis
Gram Stain
 Most widely used stain in bacteriology, discovered by the Danish
scientist and physician Hans Christian Joachim Gram in 1884.
Steps :
 Heat fixed smear of specimen is stained with 2% crystal violet solution
for one minute.
 Pour Gram’s iodine over the slide for 1-2 minutes.
 Wash the smear with water.
 Decolorise with acetone or absolute alcohol for 10- 30 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 positive cocci in
bunches - Staphylococci
Gram negative coccobacilli
In chains - H.ducreyi
Gram negative
diplococci with
neutrophilsN.gonorrhoeae
Dermatoscopy
 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 20⁰.
 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, Wickham’s 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 images
Acquired Melanocytic Naevus
Early recurrence of vascular
malformation after laser ablation
Dermatoscopy images
Androgenetic alopecia
Nail fold capillaries
Patch Test
 A bedside diagnostic procedure for identification of suspected
allergens in allergic contact dermatitis.
 Involves exposure of patient’s 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 Readings
International Contact Dermatitis Research Group criteria
Type of reaction
Score
No reaction
0
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 reaction
IR
Patch Test
Finn Chamber
Positive patch test 2+
reading
Skin Biopsy
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; Michael’s solution or Normal saline-for
IF.
 Post procedure dressing, antibiotics, suture removal when indicated
(face - 6/7 days; legs and back - 14 days).
Choice of lesion and technique in 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.
Histopathology of Skin
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, MelanocyteKeratinocyte 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, EgDLE, Acne vulgaris, Keratosis pilaris, PRP
Histopathology of Skin
Epidermal Changes
 Abscess - collection of neutrophils or eosinophils in epidermis/
dermis.
 Munro’s microabscess - neutrophilic collection in St. corneum.
 Kogoj’s 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
MCQ’s
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) Wood’s lamp is useful in the diagnosis of :
A. Erythrasma
B. Psoriasis
C. Lichen Planus
D. Tinea Corporis
MCQ’s
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
MCQ’s
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
Photo Quiz
Q.6) What is your diagnosis?
A.
B.
C.
D.
Pemphigus foliaceous
Bullous pemphigoid
Herpes Zoster
Pemphigus vulgaris
Photo Quiz
Q.7) Direct microscopic examination of
this organism can be done by
obtaining a skin scraping fromA.
B.
C.
D.
Burrow
Excoriation
Nodule
Keloid
Thank You!