1. AMYLOIDOSIS III
HA MWAKYOMA, MD

2. Amyloid: Amyloidosis Amyloid Amyloidosis Histological Definition
= insoluble fibrous protein aggregations that share specific structual traits
Amyloidosis
= abnormal deposition of amyloid proteins in tissues or organs  cause disease
Histological Definition
extracellular proteinaceous deposits exhibiting cross-beta structure due to misfolding of unstable proteins
stains with Congo-Red & seen under polarised light

3. Definition cont--- Biophysical Definition
Any polypeptide which adopts a cross-beta polymerization (in vivo or in vitro)
Some may fail congo red birefringence test
Characterised by cross-beta quaterny structure

4. Amyloidosis Group of disorders which have in
common extracellular deposition of a similar appearing, insoluble, abnormal fibrils, causing organ dysfunction.
Fibrils are formed by the aggregation of misfolded normally soluble proteins.
Term amyloid first coined by Virchow in mid 19th century (meaning starch or cellulose).

5. Amyloidosis variety of proteinaceous materials ↓
abnormally deposited insidiously in tissue interstitium
spectrum of clinical disorders
(depends on various biochemical forms that are deposited via several different pathogenic mechanisms)
recognition depends on morphologic identification
(distinct staining & optical properties)
does not excite inflammatory response
(inert)
*Not a single disease, rather a group of diseases that share in common

6. Recognition of Amyloid
Organs (Macroscopic appearance)
Staining (Microscopic appearance)
Congo red
Iodine (plus H2SO4)
Haematoxylin & eosin
Thioflavin T
Immunohistochemical stains
Electron Microscope
Non-branching fibrils (β -pleated sheets)
Non-fibrillar pentagonal glycoprotein (P-component)
Proteoglycans

7. Biochemical Nature of Amyloid
Amyloid is not a single chemical entity:
3 major biochemical forms
Amyloid Light Chain (AL)
Amyloid-Associated (AA); Serum Amyloid-Associated (SAA)
Transthretin,A β Amyloid; Amyloid Precursor Protein (APP); A4
Several minor biochemical forms

8. Biochemical Nature of Amyloid-CONT
Morphological uniformity of amyloid in all cases:
95% non-branching fibrils (β -pleated sheets)
5% P-component & proteoglycans
Structural organization seen regardless of the clinical setting or chemical composition, responsible for distinctive staining & optical properties

9. Chemical Nature
About 23 different proteins are known to form amyloid fibrils, many of them circulate as constituents of plasma
All types of amyloid consist of a major fibrilar protein that defines the type of amyloid plus minor components:
Non-fibrillar glycoprotein: amyloid P and other glycoproteins: 5%-10%

10. Chemical Nature- CONT---
Fibril proteins: 95%
AL protein (amyloid light chain) is derived from plasma cells and contains immunoglobulin light chains;
AA (amyloid-associated) is a unique nonimmunoglobulin protein synthesized by the liver;
Aβ amyloid is found in the cerebral lesion of Alzheimer disease
transthyretin (protein normal mutated)
ß-2 microglobuline (MHC I molecule)

11. Biochemical Classification of Amyloid
Amyloid Light Chain (AL)
Consists of complete immunoglobulin light chains and/or NH2-terminal fragments of light chains
Produced by immunoglobulin-secreting cells
Deposition is associated with some form of monoclonal B-cell proliferation
Amyloid Associate (AA)
No structural homology to Ig’s
Derive from a larger serum precursor sythesised in the liver, called Serum Amyloid Associate (SAA) protein
Typically deposited in setting of chronic inflammatory states

12. Biochemical Classification of Amyloid-Cont---
β –Amyloid Protein (Aβ Amyloid)
Derived from a much larger glycoprotein, called Amyloid Precursor Protein (APP)
Constitutes core of cerebral plaques found in Alzheimer's Disease, and amyloid deposited in walls of cerebral blood vessels in AD patients.

13. Biochemical Classification of Amyloid
Amyloid Transthyretin (ATTR)
Tranthyretin (TTR) is a normal serum protein that binds & transports thyroxine & retinol
Mutant form (ATTR) differs by a single amino acid
ATTR deposited in a group of genetic disorders (Familial Amyloid Polyneuropathies)
Normal TTR may be deposited in heart of aged individuals (senile systemic amyloidosis)

14. Biochemical Classification of Amyloid- cont---
Amyloid β2-Microglobulin (Aβ2m)
β2-Microglobulin (β2m) is a normal serum protein (component of MHC-I molecules)
Identified as amyloid fibril subunit (Aβ2m) in amyloidosis that complicates course of patients on long-term haemodialysis
Typically deposited in setting of chronic inflammatory states
Other Examples
Amyloid deposits derived from diverse precursors such as hormones (procalcitonin) & keratin have also been reported

15. Clinical Classification of Amyloidos based onTissue distribution of amyloid deposits (local or systemic)
Generalised(Systemic)
Immunocytic dyscrasias (1° amyloidosis)
Chronic inflammatory disease (2° amyloidosis)
Hereditary syndromes
familial Mediterranean fever
neuropathic forms
Haemodialysis associated
Localised:
Senile cardiac
Senile cerebral
Endocrine
Isolated deposits

16. CLASSIFICATION OF AMYLOIDOSIS cont--
The absence or presence of preexisting
- Amyloid deposition may be either
PRIMARY
Idiopathic process without known antecedent (cause)
Tend to involve mesodermal tissues, most frequently affecting peripheral nerves, skin, tongue, joints, heart, and liver OR
SECONDARY
May follow some other conditions or diseases (see later)
Mainly affects parenchymatous organs, such as spleen, liver and adrenals

17. Pathogenesis of Amyloid
stimulus ↓
soluble precursor
insoluble fibrils
deposition in various tissues
deposition must be massive before clinical effects seen

18. Pathogenesis

19. GENERALIZED(SYSTEMIC) FORM:
Associated conditions:
Multiple myeloma
- may be primary or secondary
- the fibril type of protein deposited may be Amyloid light chain (AL) which is a precursor of lambda or kappa chain
THE SECONDARY AMYLOIDOSIS- conditions
Chronic inflammatory diseases
- the deposition of amyloid is secondary

20. Chronic inflammatory diseases cont---
Rheumatoid arthritis
Tuberculosis
Lung abscesses
Bronchiectasis
Dermatomyositis
Scleroderma
Crohn’s disease
Ulcerative colitis
Chronic osteomyelitis etc

21. GENERALIZED(SYSTEMIC) FORM:-chronic inflammato9ry disease
The fibril type of proteins deposited in chronic inflammatory diseases is of AA (amyloid associated) which is a precursor of SAA (serum amyloid associated protein)
Associated conditions cont—
Cancers:
- the deposition of fibril protein is secondary
- the fibril type of protein deposited is AA
Hodgkin’s disease

22. Cancers cont-- Renal cell carcinoma Associated condions cont –
Hemodialysis for chronic renal failure(CRF
- the amyloid fibril type of protein deposited is Beta2 – microglobulin which is a normal serum protein and component of MHC class 1 molecule

23. Reactive Systemic Amyloidosis
chronic inflammatory disease
(protracted cell injury occurring in a spectrum of infectious and non-infectious chronic inflammatory conditions) ↓
e.g. Rheumatoid arthritis; Ulcerative colitis
SAA
(acts like acute phase protein)
AA deposition
(esp. kidney, liver, spleen, intestines)

24. Primary systemic amyloidosis
Heredofamilial amyloidosis
Familial mediterranean fever
Familial amyloid polyneuropathy
Familial mediterranean fever:
- may be associated with deposition of AA fibrils
- is an autosomal recessive disorder
- is seen in individuals of Sephardic Jews, Armenian, and Arabic descent

25. Heredofamilial amyloidosis
Familial amyloid neuropathy (AF):
- the febril protein deposited is a MUTANT form of Transthyretin
- the deposition occurs in peripheral nerves in familial amyloid polyneuropathy
- it is an autosomal dorminant disorder
- occurs in different parts of the world (Sweeden, Japan, and USA)

26. Familial Amyloidosis Familial Mediteranean Fever
autosomal recessive condition ↓
febrile disorder of unknown cause
periodic attacks of fever & inflammation of serosal membranes
AA deposition
(chronic inflam)
widespread tissue involvement indistinguishable from reactive systemic amyloidosis
Neuropathic
autosomal dominant condition ↓
serum prealbumin
(protein precursor)
mutant variants (ATTR)
deposition of amyloid predominantly in peripheral & autonomic nerves
familial amyloidosis &
age-associated amyloid deposition

27. Systemic amyloidosis cont--
Amyloid associated with hemodialysis (AH).
Systemic amyloid deposition of beta 2-microglobulin (beta2-m)
Beta2-m is a normal serum protein, occuring as a complication in patients with long-term dialysis with chronic renal failure because this protein does not pass through conventional dialysis membranes.

28. Haemodialysis ↓ high serum concentrations in renal disease
β2-microglobuin ↓
high serum concentrations in renal disease
can’t be filtered via haemodialysis
amyloid deposition (Aβ2m)
synovium; joints; tendon sheaths
70% patients on haemodialysis

29. Localised Amyloidosis
Amyloid deposits limited to single organ or tissue.
Deposits may produce grossly detachable nodular masses or be evident only on microscopic investigation.
Nodular deposits of amyloid most often found in lung, larynx, skin, bladder, tongue & region around the eye.

30. Localised Amyloidosis-cont--
Frequently there are infiltrates of lymphocytes and plasma cells in the periphery of amyloid masses.
Some cases consists of AL protein, and may therefore represent a localized form of immunocyte-derived amyloid

31. LOCALIZED FORM Associated conditions: Age –related:-
Senile cardiac amyloidosis
- the amyloid fibril type of protein deposited in the heart of ederly is Transthyretin which is a normal serum protein that transports thyroxine and retino (Vitamin A)

32. LOCALIZED FORM Associated condition: Endocrine -Related:
- endocrine tumours:-
Medullary carcinoma of the thyroids
- the fibril type of protein deposited is Procalcitonin which is a precursor of calcitonin.
Islet cell tumour of the pancrease
- the fibril type of protein deposited is islet associated polypeptide protein (IAPP) found in islets of langerhans
- is common in patient with type II diabetes mellitus

33. LOCALIZED FORM Age –related:- Senile cardiac amyloidosis
- the amyloid fibril type of protein deposited in the heart of ederly is Transthyretin which is a normal serum protein that transports thyroxine and retino (Vitamin A)

34. LOCALIZED FORM Associated condition:
Senile cerebral amyloidosis (Alzheimer’s disease)
- the amyloid fibril type deposited is beta Amyloid protein which is a precursor of amyloid precursor protein (APP)

35. Amyloid of Ageing amyloid deposition occurring with ageing
senile cardiac amyloidosis ↓
asymptomatic or
severe cardiac dysfunction
restrictive cardiomyopathy & arrhythmias
senile cerebral amyloidosis ↓
amyloid deposition as plaques
Alzheimers disease

36. AL AMYLOIDOSIS: Amyloidosis related to monoclonal Ig light chains
AL-is derived from monoclonal Ig light chains, usually of lambda type
Is produced by abnormal clones of Ig-secreating plasma cells (B cells)
May be primary or may occur secondary to multiple myeloma or some other monoclonal gammopathy (immunocyte dyscrasia)
AL fibril is derived from circulating light chain by proteolytic cleavage and conversion to insoluble form

37. AL AMYLOIDOSIS cont---
The organ distribution is usually generalized (Systemic) and conforms to either primary or secondary patterns
May be associated with some other rare monoclonal gammopathy:-
Plasma cell/ immunocyte dyscrasias;
- solitary myloma (of bone or soft tissue)
- Waldestrom’s macroglobulinemia or
- heavy chain disease

38. AA AMYLOIDOSIS Is associated with inflammatory or infectious diseases.
Amyloid deposits have a systemic distribution
Amyloid deposits contain AA fibrils
AA fibrils are related to the non-immunoglobulin AA protein
Its serum protein precursor is SAA, which is an acute phase reactants sythesized in the hepatic cells.

39. AA AMYLOIDOSIS cont---
This is also called reactive or secondary amyloidosis
This form occurs mainly as a complication of long standing inflammatory diseases
Reactive-type amyloidosis also occur in association with CANCER such as Hodgkin’s disease and Renal cell carcinoma

40. Beta amyloid Protein:
Is deposited in the cerebral blood vessels in plaques of patients with senile cerebral amyloidosis and alzheimer’s disease

41. Organ involvement Heart Kidney Liver Polyneuropathy
Gastrointestinal tract
Soft tissue
Lung (rare)

42. Organ involvement Soft tissue involvement Muscle
Bleedings, impaired coagulation
Skin and vessels
Tongue
Lymph nodes
Spleen
Joints

43. Clinical Features
Clinical manifestations depend on particular sites or organs affected & the severity of the involvement.
Common nonspeciafic complaints: weakness; fatigue; weight loss
Eventually amyloidosis manifests 1 of several ways:
Renal disease
protienurea/nephrotic syndrome
often major cause of symptoms in reactive systemic amyloidosis
progression leads to renal failure (important cause of death in amyloidosis)

44. Clinical Features Hepatomegaly/Splenomegaly (Hepatosplenomegaly)
rarely causes significant clinical dysfunction
Cardiac abnormalities
cardiac arrhythmias (important cause of death in cardiac amyloidosis)
restrictive cardiomyopathies

45. THE HEART: Heart (Gross).
Amyloid of the heart may accompany systemic amyloid deposition or localized organ involvement (amyloid of aging).
Histologically, the deposits are located between the myocardial fibers or in the walls of the coronary arteries

46. THE SPLEEN: The Spleen:- (Gross).
Amyloid of the spleen has TWO different anatomical patterns.
The deposition of amyloid may be limited to the SPLENIC FOLLICLES
Moderately enlarged spleen dotted with gray nodules (so-called “Sago” spleen)

47. THE SPLEEN: cont---
Alternatively, amyloid deposits may spare the splenic follicles and mainly infiltrate the RED PULP SINUSES, producing a large, firm spleen mottled with waxy discolorations (“Lardaceous” spleen)

48. Clinical Effect on Spleen
Nodular Distribution ↓
amyloid laid down in white pulp of follicular arteries
organ full of firm grey nodules
like boiled sago grain
(sago-spleen)

49. Kidneys: (microscopic)
Glomeruli:
The mesangium and capillary basement membrane of the glomeruli are the most frequent renal sites of amyloid deposition
Other sites of amyloid deposition are arteriolar and arterial walls and peritubular interstitial tissue

50. Clinical Effect on Kidney
deposition between basement membrane & endothelial cells ↓
spread
epithelial cells of BM & mesangium
glomerular capillaries eventually obliterated
increased glomerular permeability
(proteinurea)
extends to peritubular CT
thickening of artreies & arterioles
ischaemia

51. THE LIVER: Liver:- (Gross) Is enlarged Pale, smooth surfaced
Firm in consistency
Microscopic:
Amyloid is deposited in spaces of Disse between the hepatocytes and vascular sinusoids leading to nutritional and pressure atrophy of hepatic cords.

52. Clinical Effect on Liver
deposition between walls of sinuses and liver cells ↓
vessels in portal tracts may be affected
atrophy of liver cells
liver functions rarely affected

53. Clinical Effect on GIT deposition in small vessels in submucosa
(from tongue & anus) ↓
may extend from vessels
plaque formation in submucosa or muscularis subserosa
malabsorbtion haemorrhage perforation/obstruction

54. Diagnosis & Prognosis May be suspected from clinical signs & symptoms.
Specific histopathological tests for definitive diagnosis
Biopsy with Congo red staining is most important tool in diagnosis
Biopsy taken from organ suspected to be involved
In suspected cases of AL amyloidosis, serum & urinary protein electrophoresis & immunoelectrophoresis should be performed.
Poor prognosis in generalized amyloidosis
Mean survival time after diagnosis: 1-3 years
AA amyloidosis prognosis depends on control of underlying cause

55. Macroscopy the affected organs are often enlarged (macrogllosy)
firm and have a waxy appearance
If the deposits are sufficiently large, painting the cut surface with iodine imparts a yellow colour that is transformed to blue violet after application of sulfuric acid.

56. Macroglossia
Extensive hypertrophy
with yellow amyloid deposits

57. Macroglossia

58. Shoulder pad sign

59. Periorbital Bleeding

60. Skin bleedings

61. Explanted Heart

62. DIAGNOSIS Diagnosis is established by demonstration of amyloid
material in tissue specimens
Amorphous, eosinophilc, hyaline, extracellular substance on light microscopy after HE staining
Apple green birifrangence under polarized light in Congo red staining
Positively (as do glycoproteins) – with periodic acid schiff (PAS)

63. DIAGNOSIS
Metachromatically – with crystal violet stain as is done on sulfated glycoaminoglycans
Cross-β-pleated sheet conformation observed by x-ray diffraction
Regular fibrillar structure as observed by electronic microscopy

64. Microscopy
HE: omogene, anhiste, pale eosinophilic material

65. Non-specifical stains
Trichrome:
green
PAS: +

66. Specifical Stains
Congo red: orange, apple green birifrangence under polarized light

67. Kongored Staining
Heart
Kidney
Heart

68. apple green birifrangence under polarized light

69. Specific Stains Thioflavin T or S fluorescense in UV
Violet of Paris metachromatic, violet

70. immunohistochimistry
Anticorps anti
- transthyretin,
- AA protein,
- kappa,
- lambda,
- ß 2 microglobuline

71. Fibrillar nature and beta pleated sheet configuration described by electron microscopy in 1959
nobranching rigid, fibrils of indefinite length and a diameter of approximately 7.5 to 10nm no

72. Amyloid Structural Features
Congo red

73. Structure of an amyloid fibril, depicting the β-pleated sheet structure and binding sites for the Congo red dye, which is used for diagnosis of amyloidosis.

74. Atomic Force Microscopy of Amyloid Fibrils
200
400
600 nm
Linear non branching aggregated fibrils with a diameter of nm and β pleated sheet conformation by x-ray crystallography
The β sheets consist of strands of polypeptides in a zig-zag formation. Contiguous β sheet polypeptide chains constitute a protofilament. Generally 4-6 protofilaments are wound around one another to form an amyloid fibril