Amyloidosis D iagnosis and C lassification in N ative K idney and R enal transplants

1. Amyloidosis Diagnosis and Classification in Native Kidney and Renal transplants Prof. Dr. B. Handan Özdemir Baskent University Ankara-Turkey

2. Definition Amyloidosis comprises a diverse group of systemic and local diseases Characterized by organ involvement by the extracellular deposition of fibrils composed of subunits of a variety of normal serum proteins

3. Physical Nature Amyloid fibril protein occurs in tissue deposits as rigid, non-branching fibrils 7-to 10 nm in dm When analysed by X-ray diffraction, the fibrils exhibit a characteristic cross Beta diffraction pattern

4. Chemical Nature • Pentagonal molecule • 95% Protein Fibril • 5% Glycoprotein P component

5. Geneticfactorsmay be involved in predisposingtothedevelopment of fibrillogenesisandamyloidosis • Cofactors such as amyloid P component may have an important role in the tissue deposition and resorption • Promoting fibrillogenesis • Stabilization of the fibrils • Binding to matrix proteins • Affecting metabolism and proteolysis of formed fibrils Act by Husby G. Clin Immunol Immunopathol 1994:70:2

6. What factors allow some proteins to aggregate into amyloid fibrils ? Patientswith AA amyloidosishavelevels of SAA protein no greaterthanthosepatients withinflammatorydiseaseswho do not haveamyloid Therefore some additional unknown stimulus is required for amyloid fibrils to form and precipate

7. In AL amyloidosisbiochemicalcharacteristics of thelightchain is important in determiningamyloidformation CastnephropathyversusAmyloidosis Certainlightchainsalsomay form highmolecularweightaggregates in vitro

8. Amyloid fibril protein nomenclature • The established amyloid fibril nomenclature is based on the chemical nature of the fibril protein • Which is designated protein A and followed by a suffix that is an abbreviated form of the parent or precursor protein name • For example, when amyloid fibrils are derived from immunoglobulin light chains, the amyloid fibril is AL and the disease is AL amyloidosis Amyloid, 2010; 17(3–4): 101–104

9. Amyloid fibril protein nomenclature • At least 25 different precursor proteins are known • They areassociated with variety of Inflammatory Immune Infectious Hereditary conditions

11. TYPING OF RENAL AMYLOIDOSIS • Typing of amyloid deposits isimportant because of the difference in their treatment strategies • Typing of the amyloid deposits can be performed withvarious techniques • The most definitive method used is IF or IHC staining of tissue using antibodies that are directed against known amyloid proteins

12. Amyloid Typing and Pitfalls • IHC typing of amyloidosis poses severalproblems and requires experience • Wide range of success in IHC amyloid typing, ranging from 38% to 87% was reported • IHC diagnosis of AA type is relatively reliable, • But there is a problem in the differentiation of AL andhereditary amyloidosis Kebbel A, Röcken C. Am J Surg Pathol. 2006;30:673

13. Amyloid Typing and Pitfalls • Commercial antibodies are raised against the constant regions of the Ig light chains • A subset of AL, in which amyloid fibrils are derived from a truncated light chain “containing only variable regions” will be nonreactive with commercial antibodies • Therefore, negativelight chain staining does notrule out AL amyloidosis

14. Immunopathology in Renal Amyloidosis • The typical antibody panel should include • Amyloid P component • Kappa & lambda Ig light chains • Amyloid A protein • Transthyretin • Fibrinogen • Beta-2 microglobulin • This panel allowed definitive typing of amyloid in 90% of kidney biopsies

15. Classification of Amyloidosis Systemic Amyloidosis Primary Amyloidosis Secondary Amyloidosis Localized amyloidosis Senile cerebral Senile cardiac Type 2 diabetes

16. Primary Systemic Amyloidosis SecondarySystemic Amyloidosis

17. Themostcommontype of amyloidosisdepends on thepopulationstudied • Inthe USA andthe Western World AL amyloidosis is themostprevelant, followedby AA amyloidosis • InTurkey AA amyloidosiswith an underlyingdisease of FMF is themostcommontype • Indevelopingcountries AA amyloidosis is far morecommonthan AL amyloidosis (Tbc !) Ozdemir AI. Am J Gastroenterol 1969; 51:311

18. AL Amyloidosis • Derived from the immunoglobulin light chain • Can occur alone or in association with • M. Myeloma • Malignant lymphomas • Macroglobulinemia Kyle RA et al N Engl J Med. 2006;354:1362

19. AL Amyloidosis • Light chain deposition disease has a similar pathogenesis with AL amyloidosis • Primary difference is that • Deposited light chain fragments do not form fibrils and do not engender deposition of amyloid cofactors

20. AL Amyloidosis • Wide spectrum of organ system involvement • The kidneys are commonly affected • Proteinuria • Edema and hypoalbuminemia • Mild renal dysfunction is frequent • Rapidly progressing renal failure is rare

21. Waxy appearance of intradermal amyloid deposition around the eye Peri-orbital haemorrhage (raccoon or panda eyes) Macroglossia showing teeth indentations Gross lymphadenopathy

22. AA Amyloidosis • The AA amyloid proteins resultfrom a proteolytic cleavage SAA protein • SAA is an acute-phase reactant produced by liver • Sustained high concentration of SAA prerequisite for AA amyloidosis Benditt EP et al. Ann N Y Acad Sci. 1982;389:183

23. AA Amyloidosis Acquired and hereditarydiseases can lead to AA amyloidosis Chronic inflammatory diseases Rheumatoid arthritis Inflammatory bowel disease FMF Bronchiectasis, Tuberculosis

24. AA Amyloidosis • Kidney is the most frequently affected target organ • The primary clinical manifestation is proteinuria • The underlying disease usually is longstanding • Active inflammation typically is presentwhen amyloidosis becomes evident

25. Hereditary Amyloidosis • Caused by deposition of geneticallyvariant proteins • Associated with mutationsin the genes for • Transthyretin • Apolipoprotein AI, • Apolipoprotein AII • Lysozyme • Fibrinogen A.

26. Hereditary Amyloidosis • Typically they associated with polyneuropathy and cardiac involvement but can affect kidneys • Renal deposits may be clinically silent • Isolated glomerular involvement with no amyloidin the tubules, interstitium, or vessels has beenfound to be characteristic of fibrinogen A • Renal failure develops rapidly Blood. 1997;90:799–805

27. RENAL AMYLOIDOSIS • Clinically evident renal involvement occurs mainly inAA and AL amyloidosis • The deposition of Abeta2M occurs in patients on prolonged dialysis • But diagnosis on the kidney biopsy is unexpected • Eight precursor fibrils are particularly important for kidney

29. RENAL AMYLOIDOSIS • The incidence of amyloid in patients with nephrotic syndromeor proteinuria was found in 2% to 12% of native renal biopsies N Engl J Med. 2003;349:583–596. Arch Pathol Lab Med. 2007;131:917–922 Amyloidosis without therapy usually progresses to endstagekidney disease Deposits may also regress Ozdemir BH. Transplant Proc. 2006;38:432–434.

30. Diagnosis of Amyloidosis • Can be very difficult • No blood test can diagnose or exclude amyloidosis • Usually relies on clinical suspicion • Possibility supported by • Underlying chronic inflammatory state – AA • Underlying plasma cell dyscrasia – AL • Family history - hereditary • Evidence of renal dysfunction

31. Detection of Renal Amyloid • In H&Estained sections, amyloidis recognized as amorphous hyaline and eosinophilic material • Weakly PAS positive

32. Detection of Amyloid • Amyloid do not stain by silver staining • Occasionally may stain with silver stains and show spicules (Jones silver)

33. Detection of Amyloid • Congo red is the gold standard • Slides must be examined under polarized light • Apple-green birefringent deposits is diagnostic

34. Detection of Amyloid • Caution is advised regarding ‘‘overinterpreting’’ collagen as amyloid • Because deposits of amyloid are frequently very focal and irregularly distributed in tissue sections • Multiple and thicker (5–10 m) sections may need to be examined Picken MM. Curr Opinion Nephrol Hypertens. 2007;16:196

35. Detection of Amyloid • Other stains, or techniques • Fluorescence • Thioflavin S and T • Methyl violet • Sulphonated Alcian blue • They are less specific Sen S. Arch Pathol Lab Med 2010: 134: 532 Curr Opinion Nephrol Hypertens. 2007;16:196

36. Electron Microscopy • Characterized by randomly disposed, rigid, nonbranching, variably long, 7-to 10nm-dm fibrils • Ultrastructural immunogold labelling can depict the precursor protein in amyloif fibrils

37. Electron Microscopy • Rarecasesexhibitmassiveaggregates of amyloidfibrils in subendothelium • Theyarranged in thightlypacked, electron-dense structuresand can be easilyconfusedwith • MPGN • Cryoglobulinemicglomerulopathy • Thesecasesareusuallyassociatedwithmonoclonalkappalightcahins

38. Gross Pathology of Renal Amyloidosis • Enlarged kidneys • Pale, waxy appearing cut surfaces • Increase in the weight of kidney Weight of thekidneydid not correlatewith Renalfunction The site of renalamyloiddeposition Inverselyproportionaltotheamount of amyloid

39. Microscopy of Renal Amyloidosis • Amyloid can be found in any of the renal compartment • The most common andthe earliest site of amyloid deposition in the kidney is the glomeruli • Glomerular amyloid formations begins in the mesangium • Extends into capillary walls

40. Microscopy of Renal Amyloidosis • Amyloid deposition in glomeruli may occur • Segmental • Diffuse mesangial • Nodular • Pure basement membrane patterns

41. A proposedhistopathologic classification Renal amyloidosis was divided into 6 classes Similarto the classification of SLE Sen S. Arch Pathol Lab Med 2010: 134: 532

42. Microscopy of Renal Amyloidosis • Early segmental deposits are small and confined to mesangium without creating nodularity • It is very easy to miss this early form

43. Microscopy of Renal Amyloidosis • In the diffuse form • The mesangium is uniformly expanded by deposit

44. Microscopy of Renal Amyloidosis • In the nodular form • Mesangium is asymmetrically expanded by amyloid • Distinguish from diabetic nephropathy • Other forms of nodular glomerulosclerosis

45. Microscopy of Renal Amyloidosis • Subepithelialamyloiddeposition Associatedwithspikes Can be seen at theperiphery of mesangialareas

47. Microscopy of Renal Amyloidosis • Rarely cresents can be seen Highlighting the fact that capillary wall rupture has occured

48. Microscopy of Renal Amyloidosis • Interstitial amyloid are seen in 50% cases • Generally begins in areas adjacent to blood vessels • Medullary amyloid deposits are more frequent

49. Microscopy of Renal Amyloidosis • AA amyloidosis • Certainmutants of transthyretin • May showamyloiddepositionlimitedtotheinterstitiumandmedulla • Suchpatientspresentwithrenalfailure not associatedwithproteinuria

50. Microscopy of Renal Amyloidosis • Renal vessels are often involved • Arteriolar deposits being most frequent • Followed by deposits in arteries, PTCs and veins