Immunofluorescence in dermatopathology

IF skin

1. Immunofluorescence in dermatology PRESENTER - Dr. Neha Sharma MODERATOR - Dr. Purnima

2. CONTENTS Immunofluoresence – types , technique, indications Bullous lesions – classification & brief overview of immunobullous disorder Antigen mapping in HEB Brief role of IF in other lesions

3. INTRODUCTION Immunofluorescence (IF) microscopy is a well established technique used for the detection of a wide variety of antigens in tissues or on cells in suspension Diagnostic immunopathology in dermatology started in 1963 with the description of the lupus band test (LBT),i.e. deposits of immunoglobulins and complement at the dermo-epidermal junction

4. In 1964, Beutner and Jordon used the indirect IF technique to demonstrate antibodies in the sera of Pemphigus patients Since then, the use of IF has become routine in the diagnosis of immunologically mediated diseases of the skin the demonstration of circulating antibodies such as autoantibodies

5. Normal skin histology Epidermal layers : 1. Stratum basale 2. Stratum spinosum 3. Stratum granulosum 4. Stratum corneum Dermal layers : 1. Papillary 2. Reticular Specialized cell of the epidermis : (non keratinocytes) : 1. Melanocytes click 2. Langerhans cells 3. Merkel cells Epidermal appendages : Hair follicles Sebaceous glands Sweat glands (eccrine and apocrine glands)

6. LAYERS OF EPIDERMIS

7. DESMOSOME COMPLEX

8. DERMO-EPIDERMAL JUNCTION

9. Direct Immunofluorescence (DIF)

10. Indirect Immunofluorescence (IIF)

11. Antigen Mapping ( Modified IIF)

12. Salt-split Technique ( Modified IIF)

13. Immunofluorescence Technique IF technique involves viewing of antigen–antibody complexes under ultraviolet microscope using corresponding antibodies tagged to a fluorochrome

14. The most important factors are: Preservation of substrate antigen Antibody conjugate Fluorescence microscopy system Staining and incubation Immunofluorescence Technique

15. SUBSTRATE ANTIGEN

16. Transportation of biopsy specimen Phosphate Buffered Saline (PBS) Michel’s Medium (MM ) : ammonium sulfate , N ethylmaleimide,Potassium citrate buffer, magnesium sulfate , distilled water Can preserve specimen upto 6 months pH 7- 7.2 Store at 4ºC Normal Saline - upto 24 hour

17. SUBSTRATE ANTIGEN(DIF) Skin Biopsy - either quick-frozen or placed in Michel’s transport medium for later quick freezing Quick freezing by isopentane liquid nitrogen is the most widely used method Unfixed cryostat section (2-5µm ) used These are mounted on a slide previouly coated with gelatin adhesive and dried Frozen tissue blocks can be stored in air tight plastic bag in low temperature cabinets at -70˚C or lower

18. SUBSTRATE ANTIGEN ( IIF) Monkey esophagus antigen : substrate for pemphigus ,pemphigoid antibody tests & herpes gestationis Rat urinary bladder epithelium : for the diagnosis of paraneoplastic pemphigus Others - guinea pig lip and esophagus, rabbit lip and esophagus and normal human skin (NHS) Human salt-split skin biopsies - to separate acquired autoimmune subepidermal bullous disorders

19. Sections of monkey esophagus prepared Used within 10 days to 2 weeks after killing and quick freezing Tissues and sections need to be stored at -70 °C to -80 °C Sections should be cut at 4 μ m

20. For antinuclear antibody (ANA) testing : HEp-2 cell substrates - cultured monolayered cell preparations of laryngeal SCC available commercially prefixed on glass slides Tests for anti-DNA antibodies : Crithidia luciliae as a substrate nonpathogenic protozoan with DNA both in the nucleus and the kinetoplast

21. Antineutrophil cytoplasmic antibody (ANCA) testing commercially prepared neutrophil preparations can be prepared in the lab (in house ethanol fixed neutrophil spots)

22. Handling of sera for indirect test About 3 ml of clotted blood used Hereditory EB- 2-5 ml EDTA blood for mutation analysis All sera should be refrigerated until tests are performed Repeated freezing and thawing should be avoided, since this causes a rapid loss of antibody activity Positive and negative control sera must be frozen in aliquots of a size adequate for single experiment EDTA blood should not be frozen

23. ANTIBODY CONJUGATE

24. ANTIBODY CONJUGATE Two types monospecific reagents used for direct staining of biopsies human anti-whole-IgG conjugates used for the IIF test of sera

25. Most commercial conjugates are supplied in a lyophilized form They require reconstitution with distilled water or in diluents The undiluted stock should be divided into volumes of 0.10–0.50 ml and stored frozen (−20 °C) until ready to be diluted The diluted conjugates should be stored at 4 °C and not refrozen

26. FLUOROCHROME

27. Compounds containing electrons which when irradiated with a light of a particular wavelength achieve an unstable higher energetic state On returning to the ground state as a spontaneous process, they emit light of a longer wavelength To function as labelers, they must possess chemical groups capable of forming covalent bonds with protein molecules, which emit high fluorescence in visible spectrum FLUOROCHROME

28. FLUOROCHROME Fluorochrome used are: 1. Fluorescein( FITC) 2. Rhodamine(TRITC) 3. Texas red 4. Phycoerytherin

29. Spectrophotometric and Spectrofluorometric analysis Absorption maximum Emission maximum FITC conjugate 495nm (blue) 525nm(apple green) TRITC conjugate 555nm(green) 580nm(orange red)

30. Staining and incubation

31. DIF

32. IIF

33. ANTIGEN MAPPING

34. SALT SPLIT SKIN TECHNIQUE (SST)

35. INDICATIONS FOR DIF

36. DIF is diagnostic (A) Bullous diseases Pemphigus(all forms) Pemphigoid(all forms) Herpes gestationis Dermatitis Herpetiformis Linear IgA bullous dermatosis Epidermolysis bullosa acquisita (B) Connective tissue diseases DLE SLE DIF : highly characteristic & some diagnostic value (A) Vascular diseases Allergic vasculitis HSP Essential mixed cryoglobulinemia Polyarteritis nodosa (B) Other diseases Porphyria cutanea tarda Other forms of porphyria Lichen Planus DIF : not diagnostic but only suggestive (A) Connective tissue diseases Mixed CTD Systemic Sclerosis Dermatomyositis Psoriasis

37. Biopsy techniques A 3- to 4-mm punch biopsy is generally adequate In autoimmune blistering diseases (AIBD) ,an inflammed but unblistered perilesional area is the ideal specimen Whenever possible biopsy a single fresh small blister including adjacent clinically uninvolved skin (perilesional)

38. For a large blister, biopsy edge of blister and adjacent uninvolved skin (perilesional) Cut the perilesional end and send for IF studies and the remaining blister for histology For uninvolved skin, a 3-mm punch biopsy is sufficient

39. SITE OF THE BIOPSY CHARACTERIZATION Mucosa Perilesional with apparently normal mucosa with intact epithelium Pemphigus or pemphigoid (Skin) Edge of lesion, second biopsy 3 mm from lesion Pemphigus or pemphigoid (Mouth) 1st biopsy 3mm from lesion, 2nd at edge Vasculitis PAN Very fresh lesions Deep skin biopsy Dermatitis herpetiformis normal skin about 3 mm. from the lesion Porphyria and pseudoporphyria dorsum of hand lesions as well as normal skin Systemic Lupus erythematosus both lesional and apparently normal skin in sun-exposed areas DLE lesional skin Lichen planus Inflammed mucosa and/or skin

40. BULLOUS DISORDERS

41. Blister - fluid-filled cavity formed within or beneath the epidermis Vesicle - <0.5 cm bulla- > 0.5 cm

42. Blistering/ vesicobullous disorders include several diseases may be hereditary in origin immunologically mediated secondary to infections inflammatory process

43. APPROACH Clinical history & classical features Age of onset, family history & drug history Nature of bullae- flaccid /tense Bulla spread sign & Nikolsky sign : to look for acantholysis Tzanck smear : acantholytic cells ; eosinophils; neutophils Histopathology : level of split & type of cellular infiltrate Immunofluorescence : both direct & indirect for autoimmune bullous disorders

44. FLACCID BULLAE TENSE BULLAE

45. Nikolsky's sign Slight mechanical pressure (by rubbing) is exerted on the skin → upper epidermal layer slips away from lower layer → separation of epidermis → blistering Test is positive on previously unaffected skin present in Pemphigus vulgaris , toxic epidermal necrolysis, staphylococcal scalded skin syndrome, bullous impetigo and Stevens-Johnson syndrome Not present in Bullous pemphigoid Bulla spread sign : refers to the extension of a blister to adjacent unblistered skin when pressure is put on the top of the bulla

46. Tzanck test Microscopic examination of scrapings from the base of a lesion to look for Tzanck cells Tzanck cells (multinucleated giant cells) are present in: Pemphigus vulgaris Herpes simplex type 1 (HSV-1) infection Varicella zoster virus infection (chickenpox or shingles) Cytomegalovirus HSV PEMPHIGUS VULGARIS

47. Mechanism of blister formation Spongiosis ECF accumulation form vesicle, bulla

48. Acantholysis desmosome rupture lead to separation of keratinocyte form acantholytic cells

49. Reticular/ballooning degeneration

50. Cytolysis

51. Basement membrane zone disruption/destruction results from primary structural deficiencies humoral and cellular immunologically mediated damage

52. Spongiosis Acantholysis Reticular degeneration Cytolysis Basement membrane zone destruction Eczematous dermatitis Pemphigus Viral infections Epidermolysis bullosa simplex Bullous pemphigoid Miliaria TAM Eczematous dermatitis (late stage) Epidermolytic hyperkeratosis Mucosal pemphigoid Pemphigus (early) Hailey-Hailey disease Friction blister Cicatricial pemphigoid Transient acantholytic dermatosis (one pattern) Darier disease Erythema multiforme (in part) Linear IgA dermatosis Irritant dermatitis Irritant dermatitis (some) Dermatitis herpetiformis Epidermolysis bullosa acquisita Epidermolysis bullosa letalis Epidermolysis bullosa dystrophica

53. CLASSIFICATION BULLAE

54. CLASSIFICATION

55. Subcorneal /Granular Spinous Suprabasal INTRAEPIDERMAL Miliaria crystallina SSSS Pemphigus foliaceus and variants Bullous impetigo IgA pemphigus Subcorneal pustular dermatosis Erythema toxicum neonatorum Transient neonatal pustular melanosis Acropustulosis of infancy Spongiotic dermatitis Friction blister (may extend into dermis) Miliaria rubra Incontinentia pigmenti IgA pemphigus Epidermolytic hyperkeratosis Hailey–Hailey disease Pemphigus vulgaris and variants Paraneoplastic pemphigus Darier disease

56. Basal Keratinocyte Necrosis, Cytolysis, or Damage Epidermal Basement Membrane Zone Destruction or Disruption (Lamina Lucida) Epidermal Basement Membrane Zone Destruction or Disruption ( Sublamina Densa ) SUBEPIDERMAL Epidermolysis bullosa simplex Thermal injury (some) Erythema multiforme Bullous pemphigoid Cicatricial pemphigoid Herpes gestationis Dermatitis herpetiformis Linear IgA dermatosis Epidermolysis bullosa letalis (junctional) Suction blister Thermal injury (some) Bullous SLE Epidermolysis bullosa acquisita Linear IgA dermatosis (IgA-mediated epidermolysis bullosa acquisita ) Epidermolysis bullosa dystrophica Porphyria cutanea tarda / pseudoporphyria DERMAL Penicillamine-induced blisters (iatrogenic) Bullous amyloidosis (primary systemic)

57. Subepidermal blisters with little inflammation Subepidermal blisters with lymphocytes Subepidermal blisters with eosinophils Subepidermal blisters with neutrophils Subepidermal blisters with mast cells Epidermolysis bullosa Porphyria cutanea tarda and pseudoporphyria Bullous pemphigoid (cell-poor variant) Toxic epidermal necrolysis Bullous amyloidosis Erythema multiforme Paraneoplastic pemphigus Bullous fixed drug eruption Bullous leprosy Bullous mycosis fungoides Wells’ syndrome Bullous pemphigoid Pemphigoid gestationis Arthropod bites (in sensitized individuals) Drug reactions Epidermolysis bullosa Dermatitis herpetiformis Linear IgA bullous dermatosis Mucous membrane pemphigoid Ocular cicatricial pemphigoid Localized cicatricial pemphigoid Deep lamina lucida (anti-p105) pemphigoid Anti-p200 pemphigoid Bullous urticaria Bullous acute vasculitis Bullous LE Sweet’s syndrome EBA Bullous urticaria pigmentosa

58. CLASSIFICATION( based on etiology )

59. Immuno-bullous

60. PEMPHIGUS PEMPHIGOIDS LAD EBA DH

61. PEMPHIGUS GROUP OF DISORDERS Autoimmune condition Characterised by vesicles and bullae due to antibody formation against cell adhesion molecules of keratinocyte

62. TYPES (a) Pemphigus vulgaris Pemphigus vegetans (reactive state) (b) Pemphigus foliaceus Pemphigus erythematosus (lupus-like variant) Fogo selvagem ( endemic variant) (c) Drug-induced pemphigus (d) IgA pemphigus (e) Paraneoplastic pemphigus (PNP)

63. DESMOSOME COMPLEX Desmosome complex contains Transmembrane: desmoglein desmocollins Cytoplasmic: plakoglobin, plakophilin & desmoplakin

64. Desmoglein Type 1- upper layer of epidermis throughout body very few in mucosa Ab Dsg1 – Pemphigus foliaceus, only skin involved Desmoglein type 2- simple epithelia & basal epidermis Desmoglein Type 3- mainly basal and suprabasal layers of mucosa

65. P.vulgaris P.vegetans P. foliaceous Drug induced P. IgA P. PNP Age group middle aged , elderly & rarely children Rare variant of P. Vulgaris 2 types Neumann Hallopeau middle age Variants Pemphigus erythematosus - has LE features Pemphigus herpetiformis resemble DH penicillamine, captopril, penicillin derivatives middle-aged & elderly two types: Subcorneal pustular dermatosis (SPD) Intraepidermal neutrophilic dermatosis (IEN) patients with underlying malignancy : thymoma, B –CLPD, sarcomas etc Site oral mucosa, scalp, midface, sternum, groin, pressure points intertriginous areas trunk, face, back Mucosa spared axilla and groin Lesions flaccid intraepithelial blisters , erosions, ulcerations of skin and mucous membrane Neumann type : vesicles/bulla rupture to form erosions Later : verrucous vegetations Hallopeau type : mild,present as pustule: dry, hyperkeratotic & fissured Well demarcated crusting , scaly exfoliative lesions PE : erythematous plaques and patches in butterfly distribution PH : severely pruritic papulo -vesicular lesions nonspecific morbilliform or urticarial eruption pruritic pustular eruption flaccid pustules on an erythematous base in annular arrangement six variants – Bullous pemphigoid like Lichen Planus like Erythema multiforme like Cicatricial pemphigoid like Pemphigus like GVHD like polymorphous eruption

66. P.Vulgaris P. Vegetans P. Foliaceous Drug induced P. IgA P. PNP M/E Earliest feature spongiosis (eosinophilic) suprabasal cleft formation acantholytic cells Downward growth of epidermal cells into dermal papilla give rise to villi Firmly attached basal cells to the basement membrane shows a tomb stone appearance Neumann : early lesions -similar to P.vulagris Older lesions: acantholysis ± papillomatosis of dermal papilla VEH Eosinophils Hallopeau : suprabasal acantholysis with acantholytic cells, pustules & eosinophilic abscess Verrucous epidermal hyperplasia acantholysis within or adjacent to granular layer subcorneal bulla with dyskeratotic granular keratinocytes Older lesions may show hyperkeratosis, acanthosis & parakeratosis . Early eruption nonspecific, spongiosis, parakeratosis variable dermal infiltrate . Well developed lesions identical to those of P. foliaceus P. vulgaris eosinophilic spongiosis SPD type subcorneal vesico -pustules/ pustules with minimal acantholysisIEN type Intraepi . vesico -pustules or pustules with variable no. neutrophils Suprabasal acantholysis dyskeratotic keratinocytes, variable epi. necrosis, vacuolar interface dermatitis ± lichenoid inflammation Dermal changes include supf PVLI D/D Hailey- hailey disease Transient acantholytic dermatosis/ grovers disease Pyoderma vegetans SSSS SCPD SPD type Sneddon –Wilkinson Pustular psoriasis P. Vulgaris

67. IF P.Vulgaris P. Vegetans P.Foliaceous Drug induced P. IgA P. PNP DIF lacelike IgG in the squamous intercellular Substance (95-100%) C3 (50–100%) squamous intercellular IgG (100%) squamous ICS IgG (full thickness) superficial portion of the epidermis (rarely) PE : squamous ICS IgG (75%) granular deposition of IgM and IgG (i.e., a positive lupus band test) at the DEJ PH : ICS IgG upper epidermis squamous ICS IgG (90%) squamous ICS IgA throughout the epidermis IIF results are positive in fewer than 50% of reported cases. In the (66). In the; however, further study is needed squamous ICS + immune reactant deposition at DEJ DEJ : granular complement Linear deposition of complement, IgG, IgM and granular deposition of complement and IgG IIF IgG autoantibody against Dsg 3 & Dsg 1 (80-90%) squamous ICS IgG Dsg 1 (80-90%) PE :Antinuclear antibodies (30-80%) PH : IgG against Dsg1 circulating squamous ICS IgG antibodies (70%) SPD type : IgA autoantibodies for desmocollin1 IEN type : Ab for desmoglein 1 or desmoglein 3 (50%) Circulating PNP antibodies against multiple antigens plakin , Dsg bind squamous keratinocytes of rat bladder epithelium

68. Pemphigus vulgaris A: intercellular edema with eosinophilic spongiosis leading to loss of intercellular bridges in the lower epidermis B: The suprabasal blister contains acantholytic cells, neutrophils, and eosinophils. dermal papillae lined by a single layer of basal keratinocytes, so-called villi C: row of tombstone D: lacelike squamous ICS deposition of IgG in the lower epidermis (direct immunofluorescence)

69. PEMPHIGUS VEGETANS

70. PEMPHIGUS FOLIACEOUS

71. IgA PEMPHIGUS

72. PARANEOPLASTIC PEMPHIGUS

73. PEMPHIGOID GROUP Autoimmune disease The IgG antibodies bind to two main antigens at the basement membrane : BPAg1 (BP230 most commonly) BPAg2 ( BP180 less often) Activate complement starting an inflammatory cascade causing the epidermis to separate from the dermis

74. DERMO-EPIDERMAL JUNCTION

75. BULLOUS PEMPHIGOID Most common subepidermal blister Occurs primarily in elderly tense bullae develop on normal or erythematous skin Oral lesions seen in 10-40% May occur in childhood – two types 1. Infantile BP – in 1st yr of life – in acral areas 2. Localised vulval BP- confined to vulva only

76. Target antigens – BPAg1/230 kDa and BPAg2/180kDa Abs are – IgG4 and IgG1 Abs to BPAg2 is more associated with oral lesions, less responsive to steroids and poor prognosis

77. HISTOPATHOLOGY Unilocular subepidermal blister Cell rich type – blister develop on erythematous skin eosinophils predominant cell in blister cavity and in dermis Cell poor type – blister develop on normal skin scant perivascular lymphocytic infiltrate with few eosinophils

78. IMMUNOFLUORESCENCE DIF - linear , homogenous deposition of IgG/C3 along BM IIF - identifies IgG antibodies that react with BMZ (70%) Salt-split skin technique - deposition found on the epidermal side of the blister

79. PEMPHIGOID GESTATIONIS aka herpes gestationis Rare, pruritic, vesicobullous dermatosis of pregnancy and puerperium Occasionally seen in association with H. mole or Choriocarcinoma Onset is in 2nd or 3rd trimester Subside within several weeks of delivery IgG1 class Ab (pemphigoid gestationis factor) is formed against a placental antigen This cross reacts with BP180 antigen of BM

80. HISTOPATHOLOGY Early lesion : marked edema of papillary dermis superficial and mid dermal perivascular infiltrate by lymphocytes, eosinophils and histiocytes Established blister subepidermal , contains similar inflammatory infiltrate within the cavity eosinophilic microabscesses may be formed in dermal papillae DIF – Linear pattern of C3/ IgG (30-40%) in BM zone Salt split skin – deposits in epidermal side

81. Cicatricial Pemphigoid/Mucosal Pemphigoid Characterized by a chronic course, scarring, and predilection for mucosal surfaces Most patients : elderly and male predilection seen Oral blisters are present (100%) ,ocular involvement (75%) & cutaneous involvement in 33% or less The cutaneous lesions are of two types: (a) an extensive eruption of bullae that heals without scarring (b) areas of erythema mainly on the face and scalp in which bullae erupt intermittently followed by atrophy and scarring

82. Histopathology Cutaneous lesions Subepidermal blister develops Neutrophils and lymphocytes predominate Eosinophils may or may not be numerous Lamellar fibrosis beneath the epidermis - hallmark Mucosal lesions lichenoid lymphocytic infiltrate in which neutrophils or eosinophils or both present

83. IMMUNOFLUORESCENCE DIF : linear IgG and C3 at the squamous BMZ in lesional and perilesional skin ( 80% of cases) IIF : salt-split human skin is used as substrate IgG may be localized only to the roof or to the base of the induced separation

84. LINEAR IgA BULLOUS DERMATOSES Subepidermal blistering disorder Two clinical variants – Chronic bullous dermatosis of childhood Adult IgA bullous dermatosis Target Ag – 97 kDa Ag (degradation products of 180kDa/BPAg2) Circulating IgA Abs are present in 70% of childhood disease and in 20% of adult cases

85. HISTOPATHOLGY & IMMUNOFLUORESCENCE Subepidermal blisters with neutrophil as predominant cell Indistinguishable from DH by presence of papillary microabscesses and neutrophils in light microscope DIF – homogenous linear pattern of IgA deposition along BM zone of non- lesional area

86. DERMATITIS HERPETIFORMIS Subepidermal blistering disorder – intensely pruritic papules and vesicles – bullae are uncommon Ass. with high incidence of gluten sensitive enteropathy – in 90% cases and also with internal cancers, esp intestinal lymphoma S ites- elbows, knees, shoulders, nape of neck O nset – early adult life

87. Target Ag – tissue transglutaminase ( tTG ) IgA Abs formed in gut binds with skin transglutaminase(TG) In skin 6 TG isoenzymes are present Gluten free diet – reversal of villous atrophy and skin lesions /also protective effect against development of lymphoma

88. HISTOPATHOLOGY Early lesions – collection of neutrophils and occasional eosinophils at tips of dermal papillae – papillary microabscesses f ibrin present at the tips of dermal papillae : necrotic appearance O lder lesions – subepidermal vesiculation occurs i nitially multilocular blisters due to interpapillary ridges – but after few days these attachments break down – formation of unilocular blister

89. IMMUNOFLUORESCENCE DIF - Granular / fibrillary/ thready deposits of IgA in dermal papillae of perilesional and uninvolved skin If DIF testing is negative – repeat test IIF – C irculating IgA antibodies that react against reticulin, smooth muscle endomysium, the dietary antigen gluten, bovine serum albumin and β- lactoglobin may be present detect anti endomysial antibodies (52-100%)

90. EPIDERMOLYSIS BULLOSA ACQUISITA Non inflammatory subepidermal bullae develop in areas subjected to minor trauma such as extensor surface of limbs Target antigen – EBA antigen 290 kDa type VII collagen – a major component of anchoring fibrils

91. HISTOPATHOLOGY Classic form – non inflammatory subepidermal blisters Bullous pemphigoid like – inflammatory blisters – mostly lymphocytes and neutrophils PAS stain- BM is split and most of the PAS positive material are in blister roof

92. IMMUNOFLUORESCENCE DIF – linear deposition of IgG / C3/ C5 along BM zone A useful clue to the presence of Abs targeting type VII collagen : presence of u-serrated pattern of linear IgG deposition Routine DIF cannot distinguish between EBA and bullous pemphigoid

93. SALT-SPLIT SKIN TECHNIQUE Abs bind to dermal floor deposited below lamina densa

94. Hereditary E pidermolysis B ullosa EB is a genetically heterogeneous mechanobullous disorder defined by fragility of the skin and mucous membranes more than 30 subtypes described so far consequence of mutations in genes coding for proteins involved in adhesion of epidermal keratinocytes to each other or to the underlying dermis

95. DIAGNOSIS Light microscopy is not very useful C onfirmed by immunofluorescence mapping (IFM) and/or electron microscopy Electron microscopy is very time consuming and expensive IFM : based on detection of structural proteins in the epidermis or DEJ using specific monoclonal antibodies

96. LUPUS ERYTHEMATOSUS

97. Multifaceted autoimmune disease that affects multiple organ systems Its clinical manifestations range from limited cutaneous involvement to fatal systemic illness

98. TYPES

99. Disease Lesional LBT(%) Nonlesional LBT (%) Chronic DLE 60-90 Subacute cutaneous LE 60-100 SLE 90-100 50-90

100. DIF

101. IIF Indirect immunofluorescence detects nuclear, homogeneous, rim, speckled and nucleolar patterns The fluorescence ANA test can be regarded as a specific marker for SLE in a rim pattern with a titer of 1:160 or higher It is indicative of the presence of anti–native or double-stranded (ds) DNA antibodies

102. IIFT Crithidia luciliae sensitive (anti-dsDNA) The haemoflagellate Crithidia luciliae is particularly suitable as a substrate in IIFT It contains a highly dense mass of circular dsDNA in its large mitochondrion BIOCHIPs coated with smears of Crithdia luciliae are incubated with diluted patient samples In the case of positive reactions , specific antibodies bind to the antigens. In a second step, the attached antibodies ( IgG ) are stained with fluorescein- labelled anti-human antibodies and made visible with the fluorescence microscope

103. Scleroderma, dermatomyositis and MCTDs DIF of lesional skin from patients with dermatomyositis and MCTDs may yield similar fluorescence results as in DLE In diffuse scleroderma, the frequency of positive LBT consisting of IgG or IgM class and/or in vivo ANA varies from 0 to 60%

104. Vasculitis DIF of skin biopsies taken from very fresh lesions display vascular deposits of IgM, C3, fibrinogen, and sometimes IgG A negative result does not exclude vasculitis HSP lesions : characterized by predominant deposition of IgA in the walls of upper dermal vessels Wegener’s granulomatosis : immune deposits can be detected in skin biopsies with IgG (most common) immunoreactant deposited in and around subepidermal blood vessels, but occasionally also along the BMZ

105. HSP IgA reactive granular (+++) deposits in the blood vessel walls in the upper dermis

106. IIF

107. LICHEN PLANUS DIF characterized by large, grouped and globular deposits of immunoglobulins and complement, i.e. civatte bodies(CB) The most important findings on DIF favoring the diagnosis of LP any immunoreactant deposit at CBs plus shaggy fibrinogen deposition, whether alone or combined with other immunoreactants at DEJ

108. In LP, CBs tend to be more numerous in number, form clusters or groups of 10 or more in the papillary dermis This cluster formation on DIF may be useful in distinguishing LP from LE (more linear arrangement) The positive yield of DIF in LP is in the range of 37–97%

109. Porphyria Cutanea Tarda The blisters are subepidermal , with preservation of the dermal papillae in the floor of the lesion (‘festooning ’). Hyaline material : PAS positive and diastase resistant, present in the walls of the small vessels in the upper dermis and sometimes in the basement membrane of the epidermis U sually no inflammatory infiltrate DIF : Homogenous deposits of IgG in dermal vessel walls and in BMZ (90–100%)

110. NEXT ACTIVITY : Dr. Ruby Journal club – 12/11/2018