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Diagnostic tests in dermatology
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- 2. • Majority ofskin 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
- 3. WOOD’S LAMP DERMATOSCOPY DIRECT IMMUNOFLUORESCENCE INDIRECTIMMUNOFLUORESCENCE LUPUS BAND TEST ANTI NUCLEAR ANTIBODY POLYMERASE CHAIN REACTION PATCH TEST PHOTOPATCH TEST TZANCK SMEAR KOH MOUNT DIAGNOSTIC TESTS
- 4. WOOD’S LAMP • Firstdescribed 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) BlueGreen Pteridine Pityriasis Versicolor (Malazzesia furfur) Yellowish white Erythrasma (Corynebacterium minutissimum) Coral Pink Coproporphyrin III Ecthyma gangrenosum, Burns (Pseudomonas) Green Pyoverdin Pyocyanin
- 8. HYPOPIGMENTED DISORDERS • BrightBlue 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 • Differentiatesepidermal hyperpigmentation from dermal hyperpigmentation • Epidermal hyperpigmentation – Accentuation of contrast from normal skin • Dermal hyperpigmentation – No change in contrast
- 10. PORPHYRIA • Presumptive diagnosisof 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 keratosesand 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 surfacemicroscope, epiluminescence microscope or episcope • Non invasive tool • Subtle clinical pattern of clinical lesions • Subsurface skin structures not visible to naked eye
- 13. DERMATOSCOPY -PRINCIPLE • Transilluminationof 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 incontact 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 BENIGNNEVI MELANOMA Even Pigment pattern Disordered pigment pattern, variably sized dots, blue grey areas
- 17. PSORIASIS AND LICHEN PLANUS PSORIASISLICHEN PLANUS Regularly distributed dotted capillaries and surface scales Wickham stria on dermoscopy
- 18. OTHER APPLICATIONS • Diagnosisof 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 isa 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 antibodyis labelled with fluorescent dye • Tissue for diagnosis of autoimmune vesicobullous disorders – Perilesional area • Tissue for diagnosis of vasculitis – Fresh lesion itself
- 21. DIF - PROCEDURE Liquidnitrogen/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
- 22. DIRECT IMMUNOFLUORESCENCE PEMPHIGUS VULGARIS BULLOUSPEMPHIGOID Deposition of intercellular IgG in epidermis Linear basement membrane zone deposition of IgG
- 23. DIRECT IMMUNOFLUORESCENCE DERMATITIS HERPETIFORMIS LUPUSERYTHEMATOSUS Granular IgA deposition in dermal papilla Speckled basment membrane deposition of IgM
- 24. DIRECT IMMUNOFLUORESCENCE LICHEN PLANUS Bright fluorescenceof cytoid bodies with anti IgM
- 25. INDIRECT IMMUNOFLUORESCENCE • Identification of circulating immunoreactantsin body fluids • Secondary antibody labelled with fluorochrome is used to recognise primary antibody • Used for Diagnosis and Monitoring disease activity
- 26. IIF - PROCEDURE Substratesare allowed to react with serially diluted patient’s serum (Primary antibody) Wash with Phosphate Buffered Saline Stain with Fluorescein labelled conjugates (Secondary antibody) Wash with Phosphate Buffered Saline Examine with Fluorescein microscope
- 27. AUTOANTIBODIES BY IIF Homogenousantinuclear antibodies by IIF Coarse speckled pattern of anti RNP antibodies
- 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 OFLBT 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 LUPUSBAND 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 • Autoantibodiesthat 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 AntiRo/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 OFANA 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 Threeparameters are evaluated 1. Pattern of Fluorescence 2. Substrate Used 3. Titer of a Positive test
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- 40. HOMOGENOUS STAINING ANTIGEN CONDITION dsDNASLE Histones SLE Drug induced LE Mi-2 Steroid responsive dermatomyositis
- 41. SPECKLED STAINING TYPE ANTIGENCONDITION 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
- 42. PERIPHERAL (RIM) STAINING ANTIGENCONDITION dsDNA SLE
- 43. NUCLEOLAR STAINING TYPE ANTIGENCONDITION Speckled nucleolar RNA Pol I Diffuse systemic sclerosis Homogenous nucleolar PM-Scl Myositis scleroma overlap Clumpy nucleolar Fibrillarin Systemic sclerosis (Lung and heart, few joints)
- 44. CENTROMERE STAINING ANTIGEN CONDITION CENA,CENB, CENC Limited Scleroderma (CREST Syndrome)
- 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 • Atiter 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 PrimerExtension 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 Realtime 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 •Ichthosiformsyndromes Genetic Disorders •Bacterial, Fungal and Parasitic •Herpes, Human Papilloma Virus Infections Cutaneous Neoplasms •Scleroderma •Alopecia areata •Psoriasis Autoimmune inflammatory syndromes
- 51. MERITS AND DEMERITSOF PCR MERITS • High Sensitivity • High Specificity DEMERITS • False positive result • Relative lack of availability • High Cost
- 52. PATCH TEST • Usedfor 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 •FinnChamber •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 0hours •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 GRADINGDESCRIPTION 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 ANDFALSE 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 • Diagnosisof Photoallergic contact dermatitis • Indication – Eczematous eruption predominantly affecting light exposed sites and history of worsening after sun exposure
- 59. PROCEDURE OF PHOTOPATCH TESTING 0hr • 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 IRRADIATEDSITE 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 • Simpleand 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 • Acantholyticcell • 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 Pemphigusvulgaris 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
- 65. TZANCK SMEAR DISEASE FINDINGS HerpesSimplex, Varicella, Herpes zoster Ballooning multinucleate giant cells Molluscum contagiosum Henderson – Patterson bodies Leishmaniasis Leishman – Donovan bodies Darier’s disease Corps ronds and grains Basal cell carcinoma Basaloid cells Paget’s disease of breast Paget cells Mastocytoma Mast cells Histiocytosis Atypical Langerhan cells
- 66. TZANCK SMEAR Pemphigus Vulgaris– Acantholytic cell Pemphigus Foliaceous – Acantholytic cell Herpes – Multinucleated Giant cells Leishmaniasis – LD bodies
- 67. POTASSIUM HYDROXIDE MOUNT • Todemonstrate 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 Regularseptate hyphae Hyphae fragmenting to form arthroconidia Ectothrix – Brown pigment delimits edge of hair Endothrix – Entirely within hair shaft
- 72. CANDIDIASIS AND PITYRIASIS VERSICOLOR Candidiasis– Budding Yeast and Slender filaments Pityriasis Versicolor – Spaghetti and Meat ball appearance
- 73.