Basic bedside investigations performed in dermatology OPD in routine practice by Dr. Sinni Jain

1. Bedside investigations in
dermatology
Dr. Sinni Jain
MD Dermatology
Jaipur

2. 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

3. KOH Mount

4. 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.

5. 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.

6. 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)

7. Dermatophytes- multiple, refractile,
branched, septate hyphae

8. Candidiasis- budding ovoid yeast
cells and pseudohyphae

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

10. Chromoblastomycosis- sclerotic or muriform cells

11. Blastomycosis- refractile spherical cells with broad-based
buds.

12. Cryptococcosi - capsulated forms

13. Vaginosis- clue cells

14. 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.

16. • 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.

17. GRAM STAINING

18. 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.

19. • 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.

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

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

22. TZANK SMEAR
• uses
Immunobullous disorders
Cutaneous infections
Genodermatoses
Suspected tumors

23. 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

24. Tzanck test
INDICATIONS
1.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

25. 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

26. 2) Cytodiagnosis of cutaneous infections
a) 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)

27. 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

28. 3) Cytodiagnosis of gendermatoses
i)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.

29. 3) Cytodiagnosis of cutaneous tumors
i)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.

30. • 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.

31. Multinucleate
giant cells

32. Acantholytic cell

33. Handerson Patterson bodies Corps ronds & grains

34. 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.

35. 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 minutes
6. methylene blue for 30 seconds.
7. Rinse in water.
8.. Dehydrate, clear, and coverslip.

36. • 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

38. 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 leprosy
2.Classify the disease
3.Determine disease activity in a patient
4.Assess progress of disease
5.Follow-up patients on treatment

39. • SITES
1.right ear lobe
2.Forehead
3.Chin
4.Left buttock in men, left upper thigh in women

40. 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.

42. • MORPHOLOGICAL INDEX
Percentage of solid stained bacilli calculated after
examining 200 bacilli lying singly.
Bacilli are considered solid staining if-
a)Entire organism is uniformly stained
b)Longitudinal sides are parallel
c)Both ends are rounded
d)Length is five times its width

43. 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

44. 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.

45. • 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.

46. • 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.

48. 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.

49. 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)

51. 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

52. • 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

53. • 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)

54. T.capitis
Erythrasma
Vitiligo

55. Vitiligo

56. 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

57. • 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.

58. • 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.

59. 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

61. 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.

62. 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)

63. Complications
• Severe reaction
• Persistent positive reaction (> 1 month)
• Anaphylaxis
• Active sensitisation (positive after 10-14 days)
• Focal flare
• Depigmentation, scars, keloids (rare)

64. 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.

65. • INTERPRETATION
Read 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.

66. • 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..

67. 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.

68. 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.

71. 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

72. 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.

74. 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 system
2. higher magnification which can be adjusted
3. ability to assess structures as deep as in the reticular dermis
4. ability to record images.

75. 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.

79. 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

80. 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

81. 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
.

83. • Complications –
1.Hypersensitivity to local anaesthesia
2.Pain of local anaesthesia
3.Bleeding
4.Scarring
5.Infection

84. 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).

85. • 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.

86. • Other variants of IF
1.Salt split technique
2.Antigen mapping
3.Double staining method

87. THANK YOU….