Immunofluorescence and its role in histopathology

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

1. Immunofluorescence and its role in Histopathology

2. Brief history Principle Types of IF IF microscope working principle Drawbacks Uses of IF in Histopathology

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. 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. 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. 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. Energy is absorbed by the atom which becomes excited 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. Immunofluorescence Labeling of antibodies with fluorescent dyes Immunofluorescent labeled tissue sections are studied using a fluorescence microscope

9. Types of immunofluorescence 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. 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. 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. I mmunofluorescence Microscopy The “ fluorescence microscope ” refers to any microscope that uses fluorescence to generate an image

13. I mmunofluorescence Microscopy Components of a fluorescence microscope are : L ight source: Xenon arc lamp or mercury-vapour lamp; LEDs and lasers Excitation filter D ichroic mirror (Dichroic beam splitter) E mission filter Epifluorescence : excitation of the fluorophore and detection of the fluorescence are done through the same light path (i.e. through the objective )

14. Drawbacks and Limitations Photo bleaching : fluorescence from the chromophore fades with time 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 Hg-vapour lamps , Xenon-arc lamps can explode !

15. Applications Renal Biopsy Skin biopsy Muscle biopsy Lung biopsy Autoantibodies in serum and other body fluids Hormones Microbiology Flow cytometry FISH

16. Immunofluorescence in Histopathology

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. 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. 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. 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. 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. IF in Renal Biopsy

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. 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. 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. Patterns of IF in Renal Biopsies Linear glomerular deposits Granular glomerular staining Tubulo-interstitial staining No or scanty glomerular immunostaining

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 a ntisera positive ( IgG, IgA….) Negative result

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. 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. Membranoproliferative GN Thickening of the glomerular capillary wall ( membrano ) Increase in the number of cells in the glomerular tuft ( proliferative ) Mesangiocapillary glomerulonephritis Two types: Type 1 : deposition of immune complexes (IgG and C3), subendothelial 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. Lobular appearance of glomerulus Tram track appearance Type 1 MPGN Type 2 MPGN

32. Membranoproliferative GN IF studies 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 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. Newer Classification of MPGN based on DIF Sethi et al. (2003) 3 types Immune complex mediated – IgA, IgG, IgM C3 glomerulopathy – C3GN, DDD Light Chain mediated – κ , λ Easier classification system than EM Targeted therapies are being developed in cases of C3 nephropathy

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. 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. 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. Membranous nephropathy

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. 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: Type I : Anti-GBM antibody-mediated disease, Linear deposits of IgG and C3, Goodpasture syndrome; type IV collagen Type II : Diseases caused by immune complex deposition, idiopathic, post-infectious glomerulonephritis, lupus nephritis, IgA nephropathy, and HSP 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. Crescentic glomerulonephritis (RPGN) Frequency of Immunopathological categories of Glomerulonephritis in Native Kidney Biopsies Evaluated by Immunofluorescence Microscopy

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. Crescentic glomerulonephritis (Rapidly progressive) IF: Type I : Linear IgG and C3 staining of GBM . Fibrin positive in areas of necrosis, crescents, periglomerular interstitium, and in Bowman’s capsules Type II : Granular immune deposits of variable immunoglobulins and complement components (mesangial, capillary loops) Type III : Negative or very weak staining, fibrin positivity in glomerular necrosis, cellular crescents and in fibrinoid vascular necrosis Linear Fibrin

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. Lupus nephritis **International Society of Nephrology/. Renal Pathology Society ( ISN / RPS )

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. Class I Class II Class III Class IV Class V Class VI

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. 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. Lupus nephritis IF studies: Class V : There are delicate subepithelial immune deposits staining for IgG with or without mesangial deposits

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. 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 E pithelial 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. Focal Segmental Glomerulosclerosis IF: IgM and C3 may be present in the sclerotic areas and mesangium IgM C3

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. 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. 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. 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. 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. 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 f ibrillary 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. 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. Renal Involvement in Plasma Cell Dyscrasias Light chain (myeloma) cast nephropathy Acute tubulopathy (acute tubular damage or necrosis ) Inflammatory tubulointerstitial nephritis Amyloidosis (light chain- AL or heavy chain- AH related ) Deposition diseases ( LCDD/HCDD )

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. 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. 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. Light Chain mediated Acute Tubulopathy Immunofluorescence: Monoclonal light chains detected in the cytoplasm of the tubular cells

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. 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. 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, f ibrinoid necrosis, transmural arterial inflammation , thrombosis

68. Transplant renal biopsy Immunofluorescence study: Intense positivity for C4d in the peritubular capillaries, homogenous ring like fashion

69. IF uses in Dermatopathology

70. CLASSIFICATION: Sub-corneal blisters: very thin roof ( stratum corneum ) breaks easily Bullous impetigo Miliaria Pemphigus foliaceus Staphylococcal scalded skin syndrome Mucocutaneous Bullous Diseases

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. 3 . Subepidermal blisters: t ense roof ( entire epidermis ) often remain intact Bullous pemphigoid Dermatitis herpetiformis Erythema multiforme TEN Friction blisters

73. BPAG – Bullous pemphigoid antigens

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. 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. 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. 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. 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. 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. 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. 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. DIF Perilesional skin show linear deposition of IgG and C3 at the basement membrane zone IgA & IgM may be p resent 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. 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 C haracterized histologically by a dense, continuous infiltrate of lymphocytes along the dermoepidermal junction, a prototypic example of interface dermatitis

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. Linear IgA Dermatosis Mucocutaneous autoimmune disease, drug-induced or idiopathic Subepidermal blisters, l inear distribution of neutrophils along the dermal-epidermal junction Papillary dermal edema

86. Linear IgA Dermatosis DIF L inear 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. 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. 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. Systemic lupus erythematosus The hallmark of SLE is the production of autoantibodies Antibodies recognize diverse nuclear and cytoplasmic components Antinuclear antibodies (ANAs ) Antibodies to DNA Antibodies to histones Antibodies to non-histone proteins bound to RNA Antibodies to nucleolar antigens Four basic patterns recognized on IDIF: Homogeneous or diffuse nuclear staining – chromatin and histones, double-stranded DNA Speckled pattern - Sm antigen, ribonucleoprotein Centromeric pattern - centromeres Nucleolar pattern - antibodies to RNA Rim or peripheral staining patterns : double-stranded DNA and sometimes to nuclear envelope proteins

90. Homogenous Centromeric pattern Speckeled Nucleolar

91. Vasculitides

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. 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. 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. 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. 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. Pauci immune vasculitides ANCA associated vasculitis Indirect IF studies

98. Granulomatosis with Polyangitis Classic triad Necrotizing and granulomatous inflammation of the upper and lower respiratory tracts Glomerulonephritis Systemic vasculitis C-ANCA Histopathology: Necrotizing/leukocytoclastic small vessel vasculitis and granulomatous inflammation. Minute foci of tissue necrosis, collagen degeneration, surrounded by histiocytes.

99. Granulomatosis with Polyangitis IDIF studies c -ANCA positive in 80 % cases

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. Churg-Strauss Syndrome IDIF studies p-ANCA positivity in >70 %

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. Polyarteritis Nodosa IDIF studies Positivity for both c-ANCA (55 % ) and p-ANCA (45 % )

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. Lung Biopsy Goodpasture's syndrome: Counterpart of anti GBM disease in lung IgG Ab against alveolar basement membrane

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. 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. 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. Summary Rapid and early diagnosis of various autoimmune systemic diseases Resolving diagnostic dilemmas in certain renal diseases Cost is a limiting factor

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; http://www.ijdvl.com on Saturday, December 19, 2015, IP: 117.232.59.20 ] 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. Thank You