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Immunofluorescence and its role in histopathology


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An in-depth review of the uses of immunofluorescence in histopathology

Published in: Health & Medicine

Immunofluorescence and its role in histopathology

  1. 1. Immunofluorescence and its role in Histopathology
  2. 2. •Brief history •Principle •Types of IF •IF microscope working principle •Drawbacks •Uses of IF in Histopathology
  3. 3. History • Sir George G. Stokes (1852), made the first observation that the mineral ‘fluorspar’ exhibits fluorescence when illuminated with ultraviolet light, and he coined the word "fluorescence“
  4. 4. • The microscope was devised in the early part of the twentieth century (1904) by August Köhler • M. Haitinger (1933) - first to stain histological specimens with fluorescent dyes • Coons et al. (1941): DIF – Pneumococcal Ag • Weller & Coons (1954): IDIF - Herpes Zoster Ag Coons
  5. 5. Introduction • Technique allowing the visualization of a specific protein or antigen in tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye
  6. 6. Principle of fluorescence • Property of certain molecules or fluorophores to absorb light at one wave length (excitation wavelength) and emit light at different wave length (emission wavelength) • Selective filters used for this purpose
  7. 7. 1. Energy is absorbed by the atom which becomes excited 2. The electron jumps to a higher energy level 3. Soon, the electron drops back to the resting state, emitting a photon (or a packet of light) - fluorescence
  8. 8. Immunofluorescence • Labeling of antibodies with fluorescent dyes • Immunofluorescent labeled tissue sections are studied using a fluorescence microscope
  9. 9. Types of immunofluorescence 1. Direct IF: • One step histological staining process for identifying in vivo antibodies that are bound to tissue antigen • Used to detect antigen in clinical specimens using specific fluorochrome labeled antibody
  10. 10. Types of immunofluorescence 2. Indirect IF: • Detect circulating antibodies in patient serum • The antigen on smear are made to react with specific unlabelled antibody • Anti-gamma globulins conjugated with fluorescein dye added
  11. 11. Direct vs. Indirect Immunofluorescence Direct IF Indirect IF Time Shorter procedure, single labelling step Longer, double labelling step Cost Expensive Relatively inexpensive Complexity Less steps Additional steps involved Flexibility Limited flexibility Greater flexibility Sensitivity Less High, amplification of the signal Species Cross- reactivity Minimised May cross-react with species other than the target
  12. 12. Immunofluorescence Microscopy • The “fluorescence microscope” refers to any microscope that uses fluorescence to generate an image
  13. 13. Immunofluorescence Microscopy Components of a fluorescence microscope are : 1. Light source: Xenon arc lamp or mercury- vapour lamp; LEDs and lasers 2. Excitation filter 3. Dichroic mirror (Dichroic beam splitter) 4. Emission filter Epifluorescence: excitation of the fluorophore and detection of the fluorescence are done through the same light path (i.e. through the objective)
  14. 14. Drawbacks and Limitations 1. Photo bleaching: fluorescence from the chromophore fades with time 2. Autofluorescence : Biological autofluorescence in mammalian cells, flavin coenzymes (NAD, FMN, NADH); washing with 0.1% sodium borohydride in phosphate-buffered saline prior to antibody incubation 3. Hg-vapour lamps , Xenon-arc lamps can explode!
  15. 15. Applications 1. Renal Biopsy 2. Skin biopsy 3. Muscle biopsy 4. Lung biopsy 5. Autoantibodies in serum and other body fluids 6. Hormones 7. Microbiology 8. Flow cytometry 9. FISH
  16. 16. Immunofluorescence in Histopathology
  17. 17. Tissue processing • Unfixed cryostat sections are the best • Unfixed tissue to be frozen within brief period of time can be placed on normal saline / PBS and transported to the lab in a sealed container • Tissue that will not be frozen for several hours is best preserved using a transport medium (Michel’s Medium) • Snap freezing in isopentane + liquid nitrogen (stored at – 80°C) *PBS: Phosphate-buffered saline
  18. 18. Michel’s Fixative • Transport medium • N-ethylmaleimide: anti autolytic agent • Ammonium sulfate: precipitate tissue-bound immunoglobulins without losing their antigenicity • Multiple washing in PBS to reverse the precipitation of immunoglobulins
  19. 19. Method for preparing tissue sections Remove from the transport medium and placed in biopsy wash solution for a minimum of 30 minutes Remove excess wash solution Chuck with frozen OCT and biopsy tissue should be left at −25°C for at least 30 minutes Cut in 5 µm sections Sections are checked at regular intervals for the presence of glomeruli; toluidine blue and light microscope Air dry for a minimum of 30 minutes Slides are kept in aluminium foil and stored at −20°C until ready for staining OCT: optimum cutting temperature compound
  20. 20. Staining procedure Section is circled on the slide Washed in phosphate buffered saline (PBS) for 5 minutes Removed from the wash tank and excess PBS removed Flooded with working-strength antibody or conjugated antibody and incubated for 30 minutes Washed in PBS for 10 minutes Removed from wash tank and excess PBS is removed Second stage conjugated antibody and incubate for 30 minutes Washed in PBS for 10 minutes Mounted in buffered glycerol using coverslips Store the slides at 4°C until review
  21. 21. IF staining on paraffin sections 3 micron sections Fix at 37 ̊C overnight or at 60 ̊C for 15 mins Sections down to water Rinse in tris buffer having pH 7.6 at 37 ̊C for 30 min Add 100 μl proteinase-k on the sections and keep at 37 ̊C for 20 min Stop enzymatic digestion with tris buffer at 4 ̊C for 40 mins Rinse in PBS for 10 mins Add FITC conjugated antibodies Incubate in a wet chamber at 4 ̊C for 30 mins Mount either with vector shield aqueous mounting media or PBG
  22. 22. IF in Renal Biopsy
  23. 23. • Standard procedure for establishing diagnosis in kidney disease in parallel with routine haematoxylin & eosin stained paraffin sections • Rapid and reliable • Portion of every examination of the renal biopsy • Correlation of light microscopy with immunofluorescence findings • Exclusive diagnoses: • IgA nephropathy • IgM nephropathy • C1q nephropathy • Monoclonal immunoglobulin deposition disease
  24. 24. • Should include a piece of cortex, approximately 20% of the biopsy, containing > 5 glomeruli • A routine antibody panel for immunofluorescence on native kidney biopsy: • IgA • IgM • IgG • C3 • C1q • Fibrinogen • ʎ light chains and κ light chains • Collagen IV alpha chains • Allograft renal biopsies: • C4d should be determined by an indirect IF method
  25. 25. Approach to the interpretation of IF findings in glomerular diseases • The precise diagnosis of glomerular diseases requires integration of immunopathological findings with light microscopic study and clinical data • Pattern and site of IF staining
  26. 26. Patterns of IF in Renal Biopsies 1. Linear glomerular deposits 2. Granular glomerular staining 3. Tubulo-interstitial staining 4. No or scanty glomerular immunostaining
  27. 27. Biopsy Interpretation • Slides are evaluated qualitatively and semi-quantitatively on a 0 – 4 + scale • none (0) • trace (0.5+) • mild (1+) • moderate (2+) • moderately severe (3+) • severe (4+) • Positive results • Distribution (e.g. glomerulus, tubulointerstitial, and/or blood vessels) • Glomerular localization (capillary ,mesangial, podocyte) • Pattern (linear, granular, nodular, etc.) • Intensity (1-4+) • Which antisera positive ( IgG, IgA….) • Negative result
  28. 28. Post-infectious glomerulonephritis • Post-streptococcal , Gp A β-hemolytic streptococci • Other infections: bacterial, viral, parasitic etc • Microscopy: • Global and diffuse • Enlarged, hypercellular glomeruli • Proliferation of endothelial and mesangial cells • Infiltration by leukocytes, both neutrophils and monocytes • Crescent formation (severe cases) • Red cell casts
  29. 29. • IF: • Coarse, granular staining of glomerular capillary loops with IgG and C3. • Less frequently IgM, IgA and early complement components (C1q, C4). • 3 immunostaining patterns: • “Garland-type”- staining of peripheral capillary loops for IgG and C3 • “Starry sky”- IgG and C3 irregular deposits (along capillary walls and in mesangium) • “Mesangial”- IgG and C3, predominantly C3 Post-infectious glomerulonephritis IgG C3 Mesangial deposits
  30. 30. Membranoproliferative GN • Thickening of the glomerular capillary wall (membrano) • Increase in the number of cells in the glomerular tuft (proliferative) • Mesangiocapillary glomerulonephritis • Two types: 1. Type 1: deposition of immune complexes (IgG and C3), subendothelial 2. Type 2: dense deposit disease (C3), intra-membranous • Microscopy: • Glomeruli are large and hypercellular, proliferation of cells in both mesangium and endocapillary with infiltrating leukocytes • “Lobular” appearance of glomeruli due to the proliferating mesangial cells and increased mesangial matrix • “Double contour” or “tram-track” appearance caused by “duplication” of the basement membrane
  31. 31. Lobular appearance of glomerulus Tram track appearance Type 1 MPGN Type 2 MPGN
  32. 32. Membranoproliferative GN • IF studies 1. Type 1: • Fine to coarse granular pattern along the glomerular capillaries • Strongly positive for IgG, C3 • C1q, C4 • Lobules stand out clearly as a negative zone 2. Type 2: • Intense staining for C3 • Glomerular capillary walls and mesangial regions (mesangial rings), • Usually diffuse and global • C3 is also detected in Bowman's capsule and along the TBMs
  33. 33. Newer Classification of MPGN based on DIF • Sethi et al. (2003) • 3 types 1. Immune complex mediated – IgA, IgG, IgM 2. C3 glomerulopathy – C3GN, DDD 3. Light Chain mediated – κ, λ • Easier classification system than EM • Targeted therapies are being developed in cases of C3 nephropathy
  34. 34. IgA Nephropathy (Berger Disease) • Diagnosis exclusively given by IF • Recurrent hematuria • Most common type of glomerulonephritis worldwide • Secondary IgA nephropathy in patients with liver and intestinal disease • Microscopy: • Lesions vary considerably • Glomeruli may be normal • Endocapillary proliferation • Mesangial widening • Segmental proliferation, sclerosis • Overt crescentic glomerulonephritis
  35. 35. IgA Nephropathy (Berger Disease) • IF: • Mesangial deposition of IgA, fibrinogen, C3 and properdin and lesser amounts of IgG or IgM • Granular staining of IgA in peripheral capillary loops is associated with more proliferative forms of IgA nephropathy
  36. 36. Membranous nephropathy • Diffuse thickening of the glomerular capillary BM • Accumulation of deposits containing Ig along the subepithelial side of the basement membrane • Primary (75%) or secondary • PLA₂R (Phospholipase A₂ receptor) ; epithelial cell membrane • Nephrotic syndrome • Microscopy: • Normal • Uniform, diffuse thickening of glomerular capillary wall • EM: electron dense deposits between the basement membrane and the overlying epithelial cells, flattening of podocyte foot processes • Spikes protruding from the GBM
  37. 37. Membranous nephropathy
  38. 38. Membranous nephropathy • IF: • Strong fine granular staining for IgG and less intense granular staining for C3 along capillary loops • IgM, IgA • The presence of IgA and C1q in the deposits has been proposed as a marker for SLE MGN
  39. 39. Crescentic glomerulonephritis (RPGN) • Associated with severe glomerular injury • Characterized by rapid and progressive loss of renal function associated with severe oliguria and signs of nephritic syndrome • Renal failure occurs within weeks to months • Three groups: on the basis of immunologic findings: 1. Type I : Anti-GBM antibody-mediated disease, Linear deposits of IgG and C3, Goodpasture syndrome; type IV collagen 2. Type II : Diseases caused by immune complex deposition, idiopathic, post-infectious glomerulonephritis, lupus nephritis, IgA nephropathy, and HSP 3. Type III : Pauci-Immune, lack of detectable anti-GBM antibodies or immune complexes, ANCA associated (c-ANCAs or p-ANCAs in the sera) systemic vasculitis
  40. 40. Crescentic glomerulonephritis (RPGN) Frequency of Immunopathological categories of Glomerulonephritis in Native Kidney Biopsies Evaluated by Immunofluorescence Microscopy
  41. 41. Crescentic glomerulonephritis (RPGN) • Microscopy: • Distinctive crescents • Fibrin strands in the crescents • Migration of monocytes and macrophages into the urinary space • Segmental glomerular necrosis, feature of Pauci immune RPGN • EM: Wrinkling of glomerular basement membrane with focal disruptions
  42. 42. Crescentic glomerulonephritis (Rapidly progressive) • IF: 1. Type I : Linear IgG and C3 staining of GBM. Fibrin positive in areas of necrosis, crescents, periglomerular interstitium, and in Bowman’s capsules 2. Type II : Granular immune deposits of variable immunoglobulins and complement components (mesangial, capillary loops) 3. Type III : Negative or very weak staining, fibrin positivity in glomerular necrosis, cellular crescents and in fibrinoid vascular necrosis Linear Fibrin
  43. 43. Lupus nephritis • Renal manifestations of SLE, 50% cases • Clinically, renal involvement may occur at any time in the course of SLE • Onset within the first year • Clinical features: asymptomatic urinary findings of microhematuria and mild proteinuria to full-blown nephrotic syndrome or rapidly progressive renal failure • Microscopy: • Extremely diverse • Glomeruli, tubules, interstitium, and blood vessels • Deposition of immune complexes in the mesangium, basement membrane and sometimes throughout the glomerulus • 6 subtypes
  44. 44. Lupus nephritis **International Society of Nephrology/. Renal Pathology Society (ISN/RPS)
  45. 45. Lupus nephritis • Microscopy: • Class I : no structural changes by light microscopy • Class II : mesangial cell proliferation, mesangial matrix expansion • Class III : less than 50% of all glomeruli, segmental or global, swelling and proliferation of endothelial and mesangial cells associated with leukocyte accumulation, capillary necrosis, and hyaline thrombi; extracapillary proliferation, crescents • Class IV : the most common and severe form, lesions similar to Class III, but involves > 50% of glomeruli • Class V : diffuse thickening of the capillary walls due to deposition of subepithelial immune complexes, increased production of basement membrane-like material • Class VI : sclerosis of more than 90% of the glomeruli, end stage renal disease, severe tubular atrophy, interstitial fibrosis, inflammation
  46. 46. Class I Class II Class III Class IV Class V Class VI
  47. 47. Lupus nephritis • IF studies: • Class I : delicate mesangial positivity for IgG • IF studies: • Class II : granular mesangial positivity of all three immunoglobulins and both complements (C1q and C3) (“full house” pattern)
  48. 48. Lupus nephritis • IF studies: • Class III : full house pattern as in class II, immune deposits also identified in tubular basement membranes, interstitial capillary walls, interstitial collagen, arterial intima, and media, Fibrinogen positivity • IF studies: • Class IV : same as class III but affecting > 50% glomeruli
  49. 49. Lupus nephritis • IF studies: • Class V : There are delicate subepithelial immune deposits staining for IgG with or without mesangial deposits
  50. 50. Lupus nephritis • IF studies: • Class VI : Sclerosis of more than 90 % glomeruli without residual activity. Small granular immune deposits in the thickened and sclerotic GBMs, in the fibrotic tubulointerstitial compartment, or in vessel walls
  51. 51. Focal Segmental Glomerulosclerosis • Most common cause of nephrotic syndrome in adults • Focal : sclerosis of < 50% glomeruli • Segmental : only a portion of the capillary tuft is involved • Primary or secondary; HIV, SCD • Epithelial damage with foot process effacement is the hallmark of FSGS • The recurrence of proteinuria after transplantation; within 24hrs • Microscopy: • Collapse of capillary loops • Increase in matrix • Hyalinosis • Lipid droplets and foam cells in sclerotic areas • Collapsing glomerulopathy - HIV
  52. 52. Focal Segmental Glomerulosclerosis • IF: • IgM and C3 may be present in the sclerotic areas and mesangium IgM C3
  53. 53. C1q nephropathy • Poorly understood entity • Jennette and Hipp in 1985 • Histological features resembling lupus nephritis • C1q nephropathy falls within the clinical-pathologic spectrum of FSGS • African – Americans in 20s • Light microscopy : variable histomorphology - no significant glomerular abnormality, mesangial proliferation, diffuse proliferative glomerulonephritis. FSGS is the most common pattern • ‘Wire loop’ lesions
  54. 54. C1q nephropathy • IF : • Immunofluorescence staining for C1q in a predominantly mesangial distribution • The pattern of staining is mesangial in all cases, often with a ‘comma shaped’ appearance • “Full house” staining for IgG, IgM, IgA, C3, and C1
  55. 55. IgM nephropathy • Idiopathic nephrotic syndrome both in children and adults • 1978 • Microscopy : • Diffuse and global distribution • Mesangial cell proliferation and mesangial expansion • IF : The presence of IgM as the sole or dominant immunoglobulin in the mesangial regions of the glomeruli; IgA, IgG, C3, C1q
  56. 56. Fibronectin Glomerulopathy • Unusual cause of adult-onset proteinuria and nephrotic syndrome • Familial disease; autosomal dominant • Histologically • Glomeruli show lobular accentuation • Mesangial and subendothelial deposits of pale homogenous material • Weakly PAS positive • Fuchsinophilic on trichrome • Negative for silver and Congo Red stains
  57. 57. Fibronectin Glomerulopathy • IF: • Negative: IgG, IgA, IgM, κ, λ, C3, Cq1, fibrinogen, and albumin • Diffuse, strong, granular-smudgy staining was observed in mesangial and subendothelial locations with fibronectin
  58. 58. Fibrillary glomerulonephritis • Rare, 0.6% of native renal biopsies • Rosenmann and Eliakim in 1977 • Renal insufficiency, nephrotic range proteinuria, and microhematuria • Diverse histologic pattern : DPGN, MPGN, MGN, DS • Deposition in glomeruli of fibrillary deposits (16 – 24 nm), mesangium, GBM, or both, which are Congo red – negative • IF : • Confluent mesangial and glomerular capillary wall staining for IgG (3+) and κ and λ light chain • κ staining is noted more often
  59. 59. Immunotactoid glomerulopathy • Rarer, 0.06% of native renal biopsies • Renal insufficiency, nephrotic range proteinuria • Larger microtubular deposits, usually >30 nm, non-fibrillary, hollow, arranged in stacks • Histologic pattern : MPGN, DPGN • IF : • Monoclonal IgG (IgG1) deposition with light chain restriction (predominantly κ) • IgM, IgA, C3 and C1q positivity also seen
  60. 60. Renal Involvement in Plasma Cell Dyscrasias 1. Light chain (myeloma) cast nephropathy 2. Acute tubulopathy (acute tubular damage or necrosis) 3. Inflammatory tubulointerstitial nephritis 4. Amyloidosis (light chain- AL or heavy chain- AH related) 5. Deposition diseases (LCDD/HCDD)
  61. 61. Light chain (myeloma) cast nephropathy • Most common cause of acute renal failure in patients with myeloma • Nephrotic range proteinuria, predominantly composed of light chains • Light Microscopy • Glomerular and vascular compartments are normal • Casts present in the distal nephron and collecting ducts • Crystals in tubules
  62. 62. Light chain (myeloma) cast nephropathy • IF: • The glomeruli and vasculature reveal no specific findings • Monoclonal (restricted) light chain staining of the casts is only seen when the casts have been formed acutely k staining ʎ staining
  63. 63. Light Chain mediated Acute Tubulopathy • Increased filtration of pathogenic light chains • Direct tubular damage • Secondary Fanconi syndrome • Vacuolization, apical blebbing, loss of surface microvilli, desquamation and fragmentation of the tubular cells • Tubular regeneration with mitotic figures • Fanconi syndrome, needle-like intracytoplasmic tubular inclusions may be identified with PAS and trichrome
  64. 64. Light Chain mediated Acute Tubulopathy • Immunofluorescence: • Monoclonal light chains detected in the cytoplasm of the tubular cells
  65. 65. Light Chain Deposition disease • Multisystem disease • LCDD is found in 50%-60% of patients with myeloma • Monoclonal light chains, κ > λ • Nodular glomerulosclerosis is most characteristic; PAS-positive • IF: • Glomerular capillary loop • Mesangial staining • Linear tubular staining
  66. 66. Transplant renal biopsy • Deposition of C4d, sensitive marker for the antibody-dependent humoral rejection • Presence of anti-donor antibodies • High prevalence of transplant glomerulitis/glomerulopathy, transplant endarteritis and high risk of graft dysfunction • Absence of immune deposits by immunofluorescence helps to differentiate transplant glomerulopathy from recurrent primary MPGN, FSGS
  67. 67. Transplant renal biopsy • Light Microscopy: • Glomeruli: inflammatory cells; neutrophils, mononuclear cells • Tubules: acute tubular injury (loss of brush borders, thinning of cytoplasm, paucity of nuclei), neutrophilic tubulitis • Interstitium: edema, scant mononuclear infiltrate • Vessels: neutrophils in peritubular capillaries, fibrinoid necrosis, transmural arterial inflammation, thrombosis
  68. 68. Transplant renal biopsy • Immunofluorescence study: • Intense positivity for C4d in the peritubular capillaries, homogenous ring like fashion
  69. 69. IF uses in Dermatopathology
  70. 70. CLASSIFICATION: 1. Sub-corneal blisters: very thin roof (stratum corneum) breaks easily • Bullous impetigo • Miliaria • Pemphigus foliaceus • Staphylococcal scalded skin syndrome Mucocutaneous Bullous Diseases
  71. 71. 2. Suprabasal blisters: thin roof (portion of epidermis) ruptures to leave denuded surface • Pemphigus vulgaris • Acute eczema • Varicella • Herpes simplex • Darier’s disease
  72. 72. 3. Subepidermal blisters: tense roof (entire epidermis) often remain intact • Bullous pemphigoid • Dermatitis herpetiformis • Erythema multiforme • TEN • Friction blisters
  73. 73. BPAG – Bullous pemphigoid antigens
  74. 74. • Mucocutaneous involvement • At least 7 subtypes caused by pathogenic IgG antibodies to intra- epidermal cell adhesion molecules (Dsg1 & 3) • Diagnosed by clinical presentation and skin biopsy • Sub-types:- • Pemphigus vulgaris • Pemphigus foliaceus • Pemphigus erythematosus • Pemphigus vegetans • Paraneoplastic pemphigus Pemphigus
  75. 75. • DIF: • Diagnostic test for pemphigus vulgaris • Fishnet like IgG in the squamous intercellular/cell surface areas in upto 95%; upto100% cases with active disease. • IDIF • Shows circulating IgG autoantibody in squamous intercellular substance in 80%- 90% of cases. Pemphigus
  76. 76. • Bullous pemphigoid is the most common immunobullous disease, affects the elderly • Sub-epidermal acantholytic blisters • Tense bullae • Autoantibodies against BPAG2, a component of the hemidesmosome • Early signs: • Dermatitis-like: dry or exudative discoid eczema • Urticaria-like: erythematous urticated plaques • Nonspecific: patchy erythema and/or dryness Bullous pemphigoid
  77. 77. • DIF • Peri-lesional skin show linear C3 deposition at the dermal epidermal junction in virtually 100% of cases & IgG in 65% to 95% • IDIF • Circulating anti-basement membrane IgG antibodies in 70% to 80%. IgA & IgM are observed in about 25% of cases Bullous pemphigoid
  78. 78. Salt-split skin IF studies • Used to distinguish between Sub-epidermal blistering diseases with similar DIF studies • Incubation of normal or patient skin in 1mol/L NaCl for 48-72 hrs, which splits the basement membrane at the level of lamina lucida • DIF Pemphigoid, IgG is present on the roof or on the roof & the floor • Localization to only the floor characteristic of Epidermolysis bullosa aquisita
  79. 79. Dermatitis herpetiformis • Mostly affects young adults but may present at any age as a chronic prurigo • Mainly affects scalp, elbows, buttocks, knees and shoulders • Signs include: • Papules • Vesicles • Crusted excoriations • Dermatitis herpetiformis (DH) is associated with gluten sensitive enteropathy in most cases (85%) • HPE: • accumulation of neutrophils (microabscesses) at the tips of dermal papillae
  80. 80. Dermatitis herpetiformis • DIF: • Granular deposits of IgA within the dermo-epidermal junction with concentration at the papillary tips • Both lesional & non-lesional skin • IDIF: • Circulating IgA antibodies that react against reticulin, smooth muscle endomysium, the dietary gluten
  81. 81. • EBA presents in adult life. • Autoimmune blistering disease, IgG autoantibodies directed against the NC1 domain of type VII collagen. • Tense vesicles and bullae (blood or pus-filled blisters) primarily on extensor surfaces of hands, knees, knuckles, elbows and ankles. • Skin biopsy shows the presence of a blister under the epidermis (sub- epidermal bulla) and an infiltrate of mixed inflammatory cells in the dermis. Epidermolysis Bullosa Aquisita
  82. 82. • DIF • Perilesional skin show linear deposition of IgG and C3 at the basement membrane zone • IgA & IgM may be present as well • IDIF • Circulating anti-basement membrane antibodies (IgG) in upto 50% • The use of salt- split skin technique lead to appropriate diagnosis in most cases. IgG is on the floor, beneath the lamina densa Epidermolysis Bullosa Aquisita
  83. 83. Lichen planus • “Pruritic, purple, polygonal, planar, papules, and plaques.” • Flat-topped with white lacelike pattern of lines referred to as Wickham striae • Self-resolving in 1-2 years duration • Characterized histologically by a dense, continuous infiltrate of lymphocytes along the dermoepidermal junction, a prototypic example of interface dermatitis
  84. 84. Lichen planus • Positive yield of DIF in LP - 75% • The fluorescent pattern shows shaggy fibrin deposition at the DEJ • Deposition of IgG, IgA and IgM to a lesser extent • Using IDIF reveals antibodies against cytoplasm of basal keratinocytes
  85. 85. Linear IgA Dermatosis • Mucocutaneous autoimmune disease, drug-induced or idiopathic • Subepidermal blisters, linear distribution of neutrophils along the dermal-epidermal junction • Papillary dermal edema
  86. 86. Linear IgA Dermatosis • DIF • Linear IgA and C3 along the BMZ in perilesional skin in 100% cases • Salt-Split skin study: • In lamina lucida type , IgA antibodies bind to the epidermal side of salt-split skin • In sub lamina densa type, such as IgA-mediated EBA, IgA antibodies bind to the dermal side of salt-split skin
  87. 87. Systemic lupus erythematosus • Lupus band test (DIF): • Show shaggy, particulate deposits of IgG or C3 at the dermo- epidermal junction. IgA & IgM deposits in 50%-60% cases • MAC at the DEJ is very specific • IDIF (95%): • ANAs, double-stranded DNA antibodies, Sm protein antibodies which are very specific
  88. 88. • Evidence of antibodies to type VII collagen via DIF or IDIF on salt- split skin • DIF • Demonstrate IgG, with or without IgA and IgM deposits at the basement membrane zone (BMZ) • A salt- split skin preparation using patient serum reveals localisation to the split floor, as in EBA Bullous Systemic Lupus Erythematous
  89. 89. Systemic lupus erythematosus • The hallmark of SLE is the production of autoantibodies • Antibodies recognize diverse nuclear and cytoplasmic components • Antinuclear antibodies (ANAs) 1. Antibodies to DNA 2. Antibodies to histones 3. Antibodies to non-histone proteins bound to RNA 4. Antibodies to nucleolar antigens • Four basic patterns recognized on IDIF: 1. Homogeneous or diffuse nuclear staining – chromatin and histones, double-stranded DNA 2. Speckled pattern - Sm antigen, ribonucleoprotein 3. Centromeric pattern - centromeres 4. Nucleolar pattern - antibodies to RNA 5. Rim or peripheral staining patterns: double-stranded DNA and sometimes to nuclear envelope proteins
  90. 90. Homogenous Centromeric pattern Speckeled Nucleolar
  91. 91. Vasculitides
  92. 92. Chapel Hill Consensus Conference (CHCC) 2012 1. Large vessel vasculitis (LVV) • Takayasu arteritis (TAK) • Giant cell arteritis (GCA) 2. Medium vessel vasculitis (MVV) • Polyarteritis nodosa (PAN) • Kawasaki disease (KD) 3. Small vessel vasculitis (SVV) • Antineutrophil cytoplasmic antibody (ANCA) associated Vasculitis (AAV) • Microscopic polyangitis (MPA) • Anti-glomerular basement membrane (anti-GBM) disease • IgA vasculitis (Henoch-Schönlein) (IgAV)
  93. 93. Chapel Hill Consensus Conference (CHCC) 2012 4. Variable vessel vasculitis (VVV) • Behcet’s disease (BD), Cogan’s syndrome 5. Single-organ vasculitis (SOV) • Cutaneous leukocytoclastic angiitis • Cutaneous arteritis • Primary central nervous system vasculitis • Others 6. Vasculitis associated with systemic disease • Lupus vasculitis 7. Vasculitis associated with probable etiology • Hepatitis C virus–associated Cryoglobulinemic vasculitis • Hepatitis B virus–associated vasculitis • Syphilis-associated aortitis
  94. 94. Vasculitides ANCA‐associated small vessel vasculitis MPA GPA Eosinophilic granulomatosis with polyangitis (CSS) Drug‐induced ANCA‐associated vasculitis Immune complex small vessel vasculitis Henoch – Schönlein purpura Cryoglobulinaemic vasculitis Lupus vasculitis Rheumatoid vasculitis Sjögren's syndrome vasculitis Hypocomplementaemic urticarial vasculitis Behcet's disease Goodpasture's syndrome Serum sickness vasculitis Drug‐induced immune complex vasculitis Infection‐induced immune complex vasculitis
  95. 95. Henoch – Schönlein purpura • Clinically, palpable purpura at the buttocks and lower extremities, abdominal pain, and hematuria • Light microscopy: • LCV, although the degree of vascular damage is milder • Immunofluorescence studies: • Demonstrate the deposition of IgA in capillary walls
  96. 96. Lupus vasculitis • Leukocytoclastic vasculitis, inflammation of the vessel wall, fibrinoid necrosis, extravasation of erythrocytes, granulomatous or lymphocytic inflammation • Small-vessel vasculitis in LV patients is predominately cutaneous • Immunofluorescence: • Immune complex deposition within the vessel wall - IgG, IgM, C3
  97. 97. Pauci immune vasculitides • ANCA associated vasculitis • Indirect IF studies
  98. 98. Granulomatosis with Polyangitis • Classic triad 1. Necrotizing and granulomatous inflammation of the upper and lower respiratory tracts 2. Glomerulonephritis 3. Systemic vasculitis • C-ANCA • Histopathology: • Necrotizing/leukocytoclastic small vessel vasculitis and granulomatous inflammation. • Minute foci of tissue necrosis, collagen degeneration, surrounded by histiocytes.
  99. 99. Granulomatosis with Polyangitis • IDIF studies • c-ANCA positive in 80% cases
  100. 100. Churg-Strauss Syndrome • Asthma, fever, hyper-eosinophilia • Classic pathologic triad: necrotizing vasculitis, eosinophilic tissue infiltration, and extravascular granuloma, extremely rare • Phases: • Prodromal phase: nonspecific symptoms of asthma and allergic rhinitis • Second phase: hypereosinophilia with eosinophilic pneumonitis or gastroenteritis • Third phase: systemic vasculitis, petechiae to extensive ecchymoses • Histopathology: • Typically leukocytoclastic vasculitis • Diffuse inflammatory exudate rich in eosinophils • Palisading necrotizing granuloma, radially arranged histiocytes, giant cells
  101. 101. Churg-Strauss Syndrome • IDIF studies • p-ANCA positivity in >70%
  102. 102. Polyarteritis Nodosa • Medium to small sized arteries • ‘Nodular protuberances’ along the course of medium-size muscular arteries • Fever, muscle weakness, acute abdominal crises, stroke, myocardial infarction, and mononeuritis multiplex, renal disease (75%) • Histopathology: • Panarteritis • Skin shows only small-vessel disease - necrotizing leukocytoclastic vasculitis • Destruction of the external and internal elastic laminae • Intimal proliferation and thrombosis lead to complete occlusion of the lumen with subsequent ischemia and ulceration
  103. 103. Polyarteritis Nodosa • IDIF studies • Positivity for both c-ANCA (55%) and p-ANCA (45%)
  104. 104. Muscle Biopsy • Various muscle dystrophy. • Duchenne and Becker muscle dystrophies • Dystrophin gene dystrophin • Link between the intracellular actin cytoskeleton and extracellular matrix via ‘Dystrophin Glycoprotein Complex’. (DGC) • Absence of Dystrophin: Membrane instability and render dystrophic muscle fibers highly susceptible to contraction-induced injury Normal DMD BMD
  105. 105. Lung Biopsy • Goodpasture's syndrome: • Counterpart of anti GBM disease in lung • IgG Ab against alveolar basement membrane
  106. 106. Lung Biopsy • Scleroderma: • Systemic sclerosis • Chronic systemic auto-immune disease • Hardening (sclero) of the skin (derma) • Damage to small blood vessels; activation of T lymphocytes; increased synthesis of collagen; and production of altered connective tissues • IF: nucleolar staining of septal capillary endothelium for IgG (intranuclear oligodot pattern) IgG
  107. 107. Lung Biopsy • Fibrosing pneumonitis: • Anti–beta-2 glycoprotein antibodies of the IgA isotype • Light microscopy: interstitial pneumonitis pattern of interstitial fibroplasia but with zones of hemorrhage and intra-alveolar fibrin deposition • IF: prominent septal capillary deposition for IgA
  108. 108. Lung Biopsy • Transplant rejection: • Light microscopy: extensive septal and intra-alveolar fibrin deposition accompanied by variable red cell extravasation; minimal inflammatory reaction • IF: prominent inter alveolar deposition for IgG with septal capillary and endothelial cell localization
  109. 109. Summary • Rapid and early diagnosis of various autoimmune systemic diseases • Resolving diagnostic dilemmas in certain renal diseases • Cost is a limiting factor
  110. 110. References • Heptinstall's Pathology of the Kidney (2-Volume Set), 6th ed • Robbins and Cotran Pathologic basis of disease 9th ed • Lever's Histopathology of the Skin, 9th ed • Bancroft Theory and Practice of Histological Techniques 7th 2012 • H. U. Zollinger M. J. Mihatsch. Renal Pathology in Biopsy Light, Electron and Immunofluorescent Microscopy and Clinical Aspects • Pierre Ronco,* Emmanuelle Plaisier,* Be ´atrice Mougenot,* and Pierre Aucouturier† ; Immunoglobulin Light (Heavy)-Chain Deposition Disease:From Molecular Medicine to Pathophysiology-Driven Therapy • Malgorzata wągrowska-danilewicz1, jan żeromski; Immunofluorescent evaluation of renal biopsy: Current point of view. • Indian journal of Nephropathology 2011; Oct- Dec; 21(4): 39-244 • J. Charles Jennette, MD; An Algorithmic Approach to Renal Biopsy Interpretation of Glomerular Diseases • Gisela Gaina et al; Muscular Dystrophies protein evaluation by western blot and immunofluorescence; University of Bucharest, molecular biology centre. • J. Paul Robinson PhD, Jennifer Sturgis BS and George L. Kumar PhD; IHC staining methods, fifth edition, DAKO • KH Mohan, Satish Pai et al; Department of skin and STD, KMC, Manipal, India; Techniques of Immunofluorescence and their significance • Vandana Mehta, Aarti Sarda, C. Balachandran; Lupus band test; on Saturday, December 19, 2015, IP:] • Javier Conde Vancells; Understand the difference between direct and indirect methods for immunofluorescence; Abcam • Valéria Aoki et al; An Bras Dermatol. 2010;85(4):490-500; Direct and indirect immunofluorescence; Department of Dermatology, Laboratory of Cutaneous Immunopathology, Faculty of Medicine, University of Sao Paulo (USP)- Sao Paulo (SP), Brazil.
  111. 111. Thank You