This presentation includes following diagnostic tests in dermatology,
WOOD’S LAMP, DERMATOSCOPY
DIRECT IMMUNOFLUORESCENCE
INDIRECT IMMUNOFLUORESCENCE
LUPUS BAND TEST
ANTI NUCLEAR ANTIBODY
POLYMERASE CHAIN REACTION
PATCH TEST
PHOTOPATCH TEST
TZANCK SMEAR
KOH MOUNT
2. • Majority of skin lesions can be diagnosed
with simple visual inspection with clinically
well trained eye and patient history
• Details seen by naked eye are limited in
magnification, depth and contrast
• Diagnostic tests that aid clinician in making
correct diagnosis
INTRODUCTION
4. WOOD’S LAMP
• First described in 1903
• Simple and Non invasive tool
• Diagnosis of
1. Infective dermatoses
2. Pigmentary dermatoses
3. Porphyrias
4. Skin tumors
5. WOOD’S LAMP - COMPONENTS
• High pressure mercury
vapor lamp
• Wood’s filter – Made of
Barium silicate and nickel
oxide
• Wavelength – 320 to 400
nm (UVA) – Peak 360 nm
• After absorption by a
substance or tissue,
emission of a longer
wavelength light results
in visible fluorescence
6. WOOD’S LAMP EXAMINATION
• Dark room
• Dark adapted eyes
• Warmed Lamp
• 4 to 5 inches distance from the lesion
• Materials that may give interfering
fluorescence should be removed
7. INFECTIVE DERMATOSES
Tinea capitis
(Microsporum)
Blue Green
Pteridine
Pityriasis
Versicolor
(Malazzesia furfur)
Yellowish white
Erythrasma
(Corynebacterium
minutissimum)
Coral Pink
Coproporphyrin III
Ecthyma
gangrenosum, Burns
(Pseudomonas)
Green
Pyoverdin
Pyocyanin
8. HYPOPIGMENTED DISORDERS
• Bright Blue white color
• Margins appear
accentuated due to
contrasting effect of
autofluorescence of
dermal collagen
• Diagnosis of
1. Vitiligo
2. Ash leaf macules
3. Differentiation between
nevus depigmentosus
from nevus anemicus
Vitiligo lesion on Wood’s Light
9. HYPERPIGMENTED DISORDERS
• Differentiates epidermal hyperpigmentation
from dermal hyperpigmentation
• Epidermal hyperpigmentation –
Accentuation of contrast from normal skin
• Dermal hyperpigmentation – No change in
contrast
10. PORPHYRIA
• Presumptive diagnosis of
Porphyria Cutanea Tarda
• Specimen – Teeth, Urine,
Stool, Blister Fluid, Red
Blood Cells
• Red Pink Fluorescence
• Intensified by addition of
dilute HCl
(Porphyrinogens are
converted into
porphyrins)
Red pink fluorescence in urine sample
of PCT compared with control sample
11. OTHER USES
Tumors
•Solar keratoses and
Bowen’s disease
•Red Fluorescence –
Photodynamic diagnosis
Acne Vulgaris
•Propionibacterium acnes
•Orange red fluorescence -
coproporphyrin
Scabies
•Demonstration of burrow
•By applying
fluorescein/Tetracycline
Chromhidrosis
•Stained Clothing
•Lipofuscin
Drugs
•Tetracycline deposition in
teeth and mepacrine in
nails
•Yellow Fluorescence
12. DERMATOSCOPE
• Skin surface microscope, epiluminescence
microscope or episcope
• Non invasive tool
• Subtle clinical pattern of clinical lesions
• Subsurface skin structures not visible to
naked eye
13. DERMATOSCOPY -PRINCIPLE
• Transillumination of a lesion
and studying it with high
magnification
• Light incident on skin
undergoes reflection,
refraction, diffraction and
absorption
• Dry skin – Reflection
• Smooth skin – Pass through
• Improved by linkage fluids
14. DESIGN
• Contact Plate – Made of
multicoated silicone
glass
• Achromatic lens system
– 10x or higher
magnification
• Inbuilt illuminating lamp
• Power supply
• Inbuilt Photography
system
15. TECHNIQUES
Contact
Glass plate
comes in contact
with lesion
Non
Contact
No contact with
glass plate and
skin
Decreased
illumination and
Poor resolution
No risk of spread
of nosocomial
infection
16. NEVI AND MELANOMA
BENIGN NEVI MELANOMA
Even Pigment pattern Disordered pigment pattern, variably
sized dots, blue grey areas
18. OTHER APPLICATIONS
• Diagnosis of Dermatofibroma, Darier’s
disease, Cicatrical alopecia, Seborrheic
keratosis and urticarial vasculitis
• Calculation of follicular density
• Monitoring adverse effects of potent topical
steroids
19. IMMUNOFLUORESCENCE
• Immunofluorescence is a technique allowing
the visualization of a specific protein or antigen
in tissue sections by binding a specific antibody
chemically conjugated with a fluorescent dye
such as fluorescein isothiocyanate (FITC)
• The specific antibodies are labeled with a
compound (FITC) that makes them glow in
apple-green color when observed
microscopically under ultraviolet light
20. DIRECT
IMMUNOFLUORESCENCE
• Primary antibody is
labelled with
fluorescent dye
• Tissue for diagnosis of
autoimmune
vesicobullous disorders
– Perilesional area
• Tissue for diagnosis of
vasculitis – Fresh lesion
itself
21. DIF - PROCEDURE
Liquid nitrogen/Michel’s medium
Cut in 4-6 micron
Wash with Phosphate Buffered Saline
Stain with Fluorescein labelled conjugates
Examine with Fluorescein microscope
IgG, IgM, IgA, C3 and fibrin
28. INDIRECT
IMMUNOFLUORESCENCE IN
PEMPHIGUS VULGARIS
• IgG is positive in 80-
90% patients
• IgG1 is the best
indicator
• Titres can be
correlated with disease
activity
False Positive
•SLE
•TEN
•Lichen Planus
•Lepromatous
Leprosy
•Thermal burns
29. INDIRECT
IMMUNOFLUORESCENCE IN
LICHEN PLANUS
• Used to demonstrate lichen planus specific
antigen (LPSA)
• LPSA is expressed in stratum granulosum
and stratum spinosum
• Found in 80% of patients with or without oral
lesions
30. LUPUS BAND TEST
• Deposition of
immunoglobulins and
complement components in
the skin of patients in lupus
erythematosus demonstrable
as a linear band at basement
membrane zone by direct
immunofluorescence
• Test is positive, when one or
more immunoreactants (IgG,
IgA, IgM, C3) are found at
dermoepidermal junction
• Exact site of biopsy whether
involved or uninvolved skin,
photoexposed or
photoprotected should be
specified
Positive Linear immunofluorescence
to IgG – Lupus band test
31. STAINING PATTERNS OF LBT
High Power
(Continuous)
Stippled
Normal skin in
SLE
Homogenous
Atrophic and
Hypertrophic skin
lesions
Thready
Acute edematous
erythematous
lesions
32. LUPUS BAND TEST
• SCLE – 60% patients have lesional lupus band.
Dust like pattern of IgG deposition
• DLE – 90% patients have lesional lupus band
• Sensitivity in SLE – 95%
• Specificity in SLE – 87%
• Three or more immunoreactants in
dermoepidermal junction in non lesional
sunprotected skin – Highly specific for SLE
• Non lesional positive LBT – Positive correlation
for developing Lupus nephritis in SLE
33. USES OF LBT
• Diagnosing LE in patients with cutaneous lesions –
Positive test in involved skin is very sensitive
indicator for LE
• Differentiates SLE from DLE (Both involved and
uninvolved skin in SLE and only in involved skin in
DLE)
• Diagnosing SLE in patients without cutaneous lesions
(Positive LBT in clinically normal skin - early
confirmation for SLE)
• Diagnosing SLE from other ANA positive disorders
• Prognostic importance
34. DIFFERENTIAL DIAGNOSIS OF
LUPUS BAND TEST
•Granular band/Homogenous band
•Vertically oriented fibres at dermoepidermal junctionPositive LBT
•Less intense, interrupted band
•No reactivity in sun protected skin
Health sun exposed
skin
•Sharply defined thin linear band at dermoepidermal junction
•Circulating antibodies against basement membrane antigenBullous Pemphigoid
•Fluorescence of dermoepidermal junction is less intense than
that of dermal blood vesselsPorphyria
•Less intense
•Focal or interrupted bandRosacea/PMLE
35. ANTINUCLEAR ANTIBODY
• Autoantibodies that bind to the contents of
cell nucleus
• Also known as Anti Nuclear factor
• ANA test detects autoantibodies in serum
• Detected by Indirect Immunofluorescence
and ELISA (Generic Assay and Antigen
Specific Assay)
36. ANTINUCLEAR ANTIBODY –
SUBTYPES
Anti Ro/SS-A
•Sjogren syndrome
•SLE
Anti La/SS-B
•Sjogren syndrome
•SLE
Anti Sm
•SLE
Anti u1RNP
•Mixed Connective
Tissue Disorders
Anti Scl-70
•Systemic sclerosis
Anti dsDNA
•SLE
Anti histone
•Drug induced
Lupus
Anti
centromere
•CREST syndrome
Two Subtypes
1. Auto antibodies to
Histone and DNA
2. Auto antibodies to
Extractable Nuclear
Antigens (ENA)
37. SENSITIVITY AND SPECIFICITY
OF ANA
ANA
CTD Sensitivity Specificity
SLE 93 57
Systemic
Sclerosis
85 54
Raynaud
Phenomena
64 41
Dermato-
myositis
61 63
SPECIFIC ANA IN SLE
Antibody Sensitivity Specificity
Anti dsDNA 57 97
Anti Smith 25-30 99
38. DETECTION OF ANA
Three parameters are evaluated
1. Pattern of Fluorescence
2. Substrate Used
3. Titer of a Positive test
41. SPECKLED STAINING
TYPE ANTIGEN CONDITION
Fine speckled Ro, La Sjogren’s syndrome, SLE
Coarse speckled
u1RNP Mixed Connective tissue disorder
Sm SLE
Atypical speckled Scl-70 Systemic Sclerosis – severe from
45. TYPES OF SUBSTRATE
• HEp-2 cells – Cultured
cells of laryngeal
squamous cell carcinoma
• Sera of some patients
with SLE may be
negative on animal
substrates (mouse
kidney or rat liver) but
positive on human
substrate (hep2 cell line)
Substrate
Animal
Mouse
Kidney
Rat Liver
Human
Hep 2 cell
lines
46. ANA TITER
• A titer is determined by repeating positive
test with serial dilutions until the test yields
a negative result
• Less than or equal to 1:40 – Negative
• More than 1:80 titer is significant
47. POLYMERASE CHAIN REACTION
• Molecular diagnosis technique
• Amplify a specific segment of DNA
• To detect extremely small amount of
pathogen
• Specimen – Skin biopsy, blood or other body
fluids
48. STEPS IN PCR
Primer Extension
Addition of new bases to primer catalysed by
DNA Polymerase
Annealing
Addition of forward and reverse primers
Denaturation
Thermal
denaturation
dsDNA into two
single strands
49. VARIANTS OF PCR
Real time PCR
•Identical
•Progress of
reaction is
monitored real
time
Reverse
Transcriptase PCR
•RNA is
converted to
cDNA
•cDNA is
subjected to PCR
50. APPLICATIONS IN
DERMATOLOGY
•Epidermolysis bullosa
•Ichthosiform syndromes
Genetic Disorders
•Bacterial, Fungal and Parasitic
•Herpes, Human Papilloma Virus
Infections
Cutaneous Neoplasms
•Scleroderma
•Alopecia areata
•Psoriasis
Autoimmune
inflammatory
syndromes
51. MERITS AND DEMERITS OF PCR
MERITS
• High Sensitivity
• High Specificity
DEMERITS
• False positive result
• Relative lack of availability
• High Cost
52. PATCH TEST
• Used for diagnosis of allergic contact
dermatitis
• Basis of the testing is to elicit an immune
response by challenging already sensitized
persons to defined amount of allergen and
assessing the degree of response
53. INDICATIONS FOR PATCH
TESTING
• Eczematous disorders where contact allergy
is suspected or to be excluded
• Eczematous disorders failing to respond to
treatment as expected
• Chronic hand and foot eczema
• Persistent or intermittent eczema of face,
eyelids, ear and perineum
• Varicose eczema
54. PATCH TEST MATERIALS
•Finn Chamber
•Aluminium - 8 mm diameter & 0.5 mm depth
•Tight apposition
Patch Test
Units
•Non – allergenic
•Non - IrritantTapes
•PetrolatumVehicles
•Suitable concentration
•Stored properlyAllergens
55. PATCH TEST PROCEDURE
0 hours
•Antigens
applied
0 hours
•Occluded
immediately
48 hours
•Occlusion
removed
48 hours
•Reading after
half an hour
96 hours
•Reading at
96 hours
56. PATCH TEST GRADING
GRADING DESCRIPTION PICTURE INTERPRETATION
- No erythema or papules Negative
+/- or ? Erythema only Doubtful Positive
+ Erythema, mild infiltration, discrete
papules
Weak Positive
++ Erythema, infiltration, papules and
vesicles
Strong Positive
+++ Intense erythema, coalescing vesicles Extreme Positive
IR Sharply demarcated erythema or
epidermal necrosis
Irritant reaction
NT Not Tested
57. FALSE POSITIVE AND FALSE
NEGATIVE
FALSE POSITIVE PATCH TEST
• Wrong Test Substance
• Excited Skin Syndrome/Angry
back Syndrome/Status
eczematicus – Occurs in
patients presenting with
multiple concomitant positive
reactions to diagnostic patch
tests for ACD in whom a
single repeated challenge
reveals some of them to be
non reproducible
• Artifact
FALSE NEGATIVE PATCH TEST
• Insufficient amount
• Non occlusion
• Corticosteroids
• Refractory state
58. PHOTOPATCH TEST
• Diagnosis of Photoallergic contact dermatitis
• Indication – Eczematous eruption
predominantly affecting light exposed sites
and history of worsening after sun exposure
59. PROCEDURE OF PHOTOPATCH
TESTING
0 hr
• Test material applied in duplicate
48 hr
• Only one set is read
• Irradiate allergens
• 5-15 J/m2 of UVA and covered again
96 hr
• Both sets are evaluated
60. INTERPRETATION OF
PHOTOPATCH TEST
IRRADIATED SITE NONIRRADIATED SITE INTERPRETATION
0 0 No Allergy
1-4 + 0 Photoallergy
1-4 + =1-4+ Contact allergy
1-4 + >1-3+ Contact dermatitis with photoaggravation
61. TZANCK SMEAR
• Simple and rapid
cytodiagnostic technique
• Blistering disease – Early
uninfected lesion
• For suspected ulcerative
tumor - Crusts removed
• For suspected non
ulcerative tumor – Incised
with pointed scalpel and
tissue obtained with blunt
scalpel or a small curette.
Tissue pressed between
two glass slides
FIXATIVES STAINS
Formalin
Glutaraldehyde
Formol-Denker
Solution
Giemsa
Hematoxylin and
Eosin
Wright
Methylene Blue
Papanicoaou
Toluidine Blue
62. TZANCK SMEAR - PROCEDURE
Open intact
roof of fresh
vesicle
Blot excess
fluid
Scrap the base
with scalpel
Fixation
(2 minutes)
Allow to air dry
Transfer
material to
Glass Slide
Stain with
Giemsa Stain
Examination
(after 15
minutes)
63. TZANCK CELL
• Acantholytic cell
• Large rounded keratinocyte
• 20 microns size
• Large Nuclear cytoplasmic
ratio
• Rim of eosinophilic cytoplasm
• Staining more deeper and
basophilic peripherally
(Mourning edged cells)
64. TZANCK SMEAR
DISEASE FINDINGS
Pemphigus vulgaris Acantholytic cells with hazy nucleoli
Pemphigus vegetans Similar but more eosinophils
Pemphigus foliaceous Hyalinized cytoplasm.
Hailey Hailey Disease Acantholytic cells with normal nucleoli
Bullous Pemphigoid Non specific. Scarcity of epithelial cells
and an abundance of leukocytes
particularly eosinophils with leukocyte
adherence
Staphylococcal Scalded Skin Syndrome Minimal or no inflammation,
dyskeratotic acantholytic cells
Steven Johnson Syndrome No acantholytic cells, but plenty of
leukocytes
Toxic Epidermal Necrolysis Necrotic Basal cells and Leukocytes
67. POTASSIUM HYDROXIDE
MOUNT
• To demonstrate evidence of fungal infection
of skin, hairs and nails
• Simple, reliable and results are rapidly
available
68. KOH MOUNT - PROCEDURE
•Skin – cleaned with alcohol, scraped with scalpel
•Hair – plucked with forceps
•Nail – undersurface of nail plate is scraped
Sample
Collection
•10% KOH (20% for nails) added and heated
•Hyperkeratotic specimens – left for half to 2 hours
•Nail – 24 to 48 hours
KOH mount
•Fungal spores or hyphae are detected
Microscopic
Examination
69. KOH MOUNT – MODIFIED
PROCEDURE
• Cellophane tape applied over affected site,
pressed firmly and removed
• Tape is then stuck on surface of a glass slide
• KOH mount done in laboratory
• Parker’s ink can be added to KOH to stain
fungal wall blue
• Fluorochrome stain such as calcofluor white
results in brilliant apple green or blue white
color of fungal structures under UV light
70. POTASSIUM HYDROXIDE
MOUNT - USES
Dermatophytic infections
•Skin, hairs and nails
•Retractile, long, branching and
septate hyphae are seen with our
without arthroconidiospores
Candidiasis
•Budding yeast cells and
pseudohyphae
Pityriasis versicolor
•Thick walled clustered spherical
yeast cells often with short
filaments
•Spaghetti and meat balls or
banana and grapes appearance
Other fungal infection
•Tinea nigra and deep fungal
infection
Scabies and other mites
•Sarcoptes scabiei
•Demodex folliculorum (in
rosacea like lesions)
Bacterial vaginosis
•Fishy odor when few drops of
KOH are added to vaginal
discharge
71. DERMATOPHYTOSIS – KOH
MOUNT
Regular septate hyphae Hyphae fragmenting to
form arthroconidia
Ectothrix – Brown pigment
delimits edge of hair
Endothrix – Entirely within
hair shaft