sri siddhartha medical college, tumkur

1. Amyloidosis
from basics to recent advances
Moderator- Dr Chaithra
Presenter – Dr Dhanya A N

2. Contents
• Introduction
• Definition
• Structure
• Classification
• Pathogenesis
• Diagnosis
• Morphology
– Gross
– Microscopy
• Recent advances
• Summary

3. Introduction
• The name amyloid -
Rudolph Virchow in 19th
century.
• Amyloid means “starch
like” because it stained
violet with iodine and
sulphuric acid and thus
attributed this to its
cellulose or starch like
nature.

4. Definition
• It is a condition
associated with a number
of inherited and
inflammatory disorders in
which extracellular
deposits of fibrillar
proteins are responsible
for tissue damage and
functional compromise.
• Produced by aggregation
of misfolded proteins

5. Structure of amyloid protein
• Fibers are long,
• Straight or moderately curved
• Generally unbranched.
• Fibrils can appear as wavy filaments, rod shaped,
with a diameter ranging from 5 to 25 nm.

6. • Amyloid fiber are formed
by fibrillar subunits -
“protofilaments”.
• In mature fiber,
protofilaments can vary in
number (2-6) and can twist
one another forming an
hollow fibril core.

7. • Mature fibers which forms amyloid are
characterized by cross-beta sheet conformation

8. • Fibre showing four
fibrils (there can be as
many as six in each
fibre) wound around one
another with regularly
spaced binding of the
Congo red dye

9. Electron microscopic picture
Elongated, non branching, fibrillar filaments

10. Chemical nature of amyloid
• 95 % of amyloid consists of - fibril Proteins
• 5 % of amyloid consists of - proteoglycans and
glycosaminoglycans
– Heparin sulfate
– Dermatan sulfate
– Plasma protiens- serum amyloid P component
(SAP)

11. Types of amyloidosis
3 major types
1. AL ( amyloid light chain )
• Made up of immunoglobulin light chain
• Most of AL protein composed of λ light chain
and occasionally қ chain
• Secreted by a monoclonal population of
plasma cells, associated with plasma cell
tumors

12. 2. AA ( amyloid associated )
• Derived from non Ig protein, synthesized by
the liver
• Are formed by proteolysis of larger precursor
serum amyloid associated (SAA), synthesized
by liver
• SAA production increases in inflammatory
conditions in response to stimuli from various
proinflammatory cytokines, such as interleukin
IL-1, IL-6, and tumor necrosis factor (TNF)

13. 3. β- amyloid protein (Aβ)
• Constitute core of the cerebral plaques found
in Alzheimer disease
• Derived from proteolysis of larger trans
membrane glycoprotein, amyloid precursor
protein (APP), due to genetic mutation of APP
gene, on chromosome 21

14. Other types
1. Amyloid derived from Transthyretin (ATTR)
• Normal serum protein which transport thyroxine
and retinol.
• TTR gene on chromosome 18
• Mutated TTR (ATTR) deposited in familial
amyloid polyneuropathy, senile cardiac
amyloidosis

15. 2. Amyloid derived from β2 microglobulin
(Aβ2 )
• Component of MHC class 1 molecule and a
normal serum protein
• Seen in long term hemodialysis patients
• It is not effectively filtered during
hemodialysis and hence its levels are more in
these patients

16. 3. Amyloid of prion protein(Aprp)
• It is derived from precursor prion protein
• PrP gene on chromosome 20
• Mutated prion proteins are proteinacious
infectious particles lacking in RNA and DNA.
• In prion diseases misfolded prion proteins
aggregate in the extracellular space and acquire
the structural and staining characteristic of
amyloid protein.

17. 4. Amyloid from hormone precursor protein
• It includes amyloid derived from
– Calcitonin(Acal),
– Insulin(Ains),
– Prolactin(Apro),
– Lactoferrin(Alac)

18. Classification of amyloidosis
Clinicopathological
category
Associated diseases Major fibril protein Precursor protein
Systemic
amyloidosis
Immune dyscrasias
with amyloidosis /
Primary amyloidosis
Multiple myeloma
and other plasma
cell proliferation
AL Ig light chain ,
mostly λ type
Reactive systemic
amyloidosis/
secondary
amyloidosis
Chronic
inflammatory
conditions
AA SAA
Hemodialysis
associated
amyloidosis
Chronic renal
failure
Aβ2 m β2 microglobulin

19. Classification of amyloidosis
Clinicopathological
category
Associated diseases Major fibril protein Precursor protein
Hereditary
amyloidosis
Familial
Mediterranean
fever
AA SAA
Familial amyloidotic
neuropathies
ATTR transthyretin
Systemic senile
amyloidosis
ATTR transthyretin

20. Classification of amyloidosis
Clinicopathological
category
Associated diseases Major fibril protein Precursor protein
Localized
amyloidosis
Senile cerebral Alzheimers disease Aβ APP
Endocrine
- Medullary ca of
thyroid
- Islets of
langerhans
Type 2 DM
Acal
AIAPP
Calcitonin
Islet amyloid poly
peptide
Isolated atrial
amyloidosis
AANF Atrial natriuretic
factor

21. Pathogenesis
• Abnormal folding of proteins, which becomes
insoluble, aggregate and deposit as fibrils in
the extracellular tissue

22. • Normally, misfolded proteins are removed by
proteasomes intracellularly and by
macrophages extracellularly
• Any of these mechanism fail, amyloid fibril
deposition occurs

23. • The proteins that form amyloid
– Normal proteins – tendency to form improper
folding, and they do so when they are produced in
large amount .
– Mutated proteins - prone to misfolding and
aggregate to form fibrils

24. Primary amyloidosis (AL type)
• Associate with plasma cell disorder
• Systemic distribution
• Clonal proliferation of malignant plasma cell
Ig proteins(unpaired free қ , λ light chain
which has amyloidogenic potential)
amyloidosis ( in the tissues)

25. Reactive systemic amyloidosis (AA
type)
• Associated with
– Tuberculosis, Bronchiectasis
– Chronic osteomyelitis
– Rheumatoid arthritis
– Connective tissue disorders like ankylosisng
spondilitis
– Inflammatory bowel diseases
– Heroin abusers
– Solid tumors like RCC and hodgkins lymphoma

26. Reactive systemic amyloidosis (AA
type)
Chronic inflammatory conditions
IL-1, IL-6, TNF
SAA produced by liver
normally
Monocyte derived enzyme defect or
Enzymes genetically abnormal SAA
which resist degradation
Soluble end product
insoluble AA
molecule

27. Pathogenesis for Aβ type of
amyloidosis

28. Familial Mediterranean fever
• Autosomal recessive, an systemic
autoinflammatory disorder
• Activation of antigen-independent inflammatory
mechanisms, involving mediators and cells of the
innate immune system due to genetic mutation of
MEFV gene located on chromosome 16
• Mutated MEFV leads to the over secretion of
inflammatory cytokines
• As a result SAA will be produced and AA fibrils
are deposited in the tissues

29. Familial amyloidotic polyneuropathy
• Autosomal dominant
• Amyloid fibrils are made up of mutated TTR
• TTR gene is located on chromosome 18
• More than 100 mutations in the TTR protein
have been reported ,the mutations causes a
single nucleotide substitution in the codon of
TTR gene

30. Conti..
• Transthyretin (TTR) exists as a tetrameric
plasma transport protein.
• The tetrameric structure has surface receptors
for retinol-binding protein as well as binding
sites for thyroxine
• The misfolding of the protein, cytotoxic and
leads to inappropriate aggregation and
accumulation in a variety of organ systems

31. Conti..
• These TTR produced in the
– liver
– retinal pigment epithelium of the eye
– choroid plexus of the brain
– neurons
– peripheral nerve Schwann cells

32. Conti..
• Nerve biopsies (sural nerve) reveal amyloid
deposits in the endoneurial and epineurial
connective tissue, along with deposits in the
endoneurial and epineurial blood vessel walls.

33. HAEMODIALYSIS ASSOCIATED
AMYLOIDOSIS
• Seen in patients on long term hemodialysis for more
than 10 years
• These patients have high concentration of β2
microglobulin in the serum because it cannot be filtered
through dialysis membrane and gets accumulated in the
synovium, joints or tendon sheath.
• Patient often presents with carpel tunnel syndrome
because of amyloid deposition.

34. Localized Amyloidosis
a)Senile Cardiac(ATTR), isolated artial
amyloidosis (AANF)
b)Senile Cerebral(Aβ,APrP)
c)Endocrine Amyloidosis
d)Localised Tumour forming amyloid(AL)

35. Senile cardiac amyloidosis
• Individual > 70 yrs
• Mutated TTR (ATTR)
• In isolated cardiac amyloidosis – atrial
natriuretic peptides are deposited
• Causes restrictive cardiomyopathy

36. Senile Cerebral(Aβ,APrP)
• Spongiform Encephalopathies (APrP
Amyloid )
-Kuru
-Creutzfeldt-Jakob disease (CJD),
-Gerstmann-Straussler-Sheinker disease (GSS),
• Down’s syndrome(Aβ)
• Alzheimer’s Disease(Aβ)

37. Endocrine Amyloid(hormone
precursors)
• Microscopic deposits of localised amyloids incertain
endocrine lesions –
- Medullary carcinoma of thyroid -calcitonin
- Islet tumors of pancreas -Islet amyloid
polypeptide or
Amylin)
- Type 2 diabetes mellitus -Proinsulin(Ains)
- Pitutary amyloid -Prolactin(Apro)

38. Localized Tumor forming
amyloid(AL)
• Isolated tumor like formation of amyloid(AL)
deposits. Seen as nodular masses in-
– lungs,
– larynx,
– skin,
– urinary bladder,
– tongue, eye

39. Clinical features
Renal involvement
• Proteinuria
• Renal failure and uremia
Associated with multiple myeloma
• Serum and urine bence jones protein
• Bony lytic lesions
• Hypercalcemia

40. Conti..
Cardiac involvement
• Features of congestive heart failure
• Conduction defects like arrhythmias
• Restrictive cardiomyopathy
Gastrointestinal tract
• Tongue- macroglossia
• Stomach, duodenum – malabsorption
• Diarrhea
• Bleeding

41. conti..
Familial mediterranean fever
• Repeated attacks of fever lasting for 1-3 days
and resolve spontaneously
Familial amyloidotic polyneuropathy
• Numbness, Paresthesia, and pain
• Compression of peripheral nerves, especially
the median nerve within the carpal tunnel,
• Localized sensory changes

42. Conti..
Skin manifestations
• Periorbital Purpura
Bleeding diathesis
• Mechanisms include factor X deficiency due to
binding to amyloid fibrils primarily in the liver
and spleen
• Decreased synthesis of coagulation factors in
advanced liver disease;
• Amyloid infiltration into blood vessels.

44. Diagnostic approach
• Clinical features laboratory findings
• Lab findings
– Monoclonal protein study in serum by electrophoresis
– shows M band
– Bence jones proteins estimation
– Creatinine clearance – reduced
– Bony lytic lesion – x-ray
– ECG shows arrhythmias, low voltage wave seen
– Serum troponin T – increased
– Serum brain natriuretic peptide – increased
– Liver enzymes – AST, ALT, ALK elevated

45. Serum electrophoresis
Arrow indicates M band in gamma region , immunoglobulin light chain lambda type

46. Tissue biopsy
• Any of the following tissue,
– Abdominal fat pad aspirate
– Rectal biopsy
– Bone marrow biopsy
– Gingival biopsy - not routinely performed ,
uncomfortable to patient
• Specific organs biopsy

47. Abdominal fat aspiration
Abdominal fat pad aspiration ,congo red staining, apple green
birefringence

48. Staining characteristics
Specific stains
1)Congo red Red-pink stain that shows
typical green birefringence in
polarized light.
2)Methyl violet/
crystal violet Metachromatic -rose pink
3)Thioflavin Uv light - Fluorescence
Non specific stains
a) Toluidine blue- Orthochromatic pale blue
b) Alcian Blue Blue Green
c) PAS Pink

49. • H and E stain
showing dense
amorphous
eosinophilic
deposition in the
glomeruli

50. • Congo Red positivity
in diagnosing
amyloid deposition

51. • When stained with
Congo Red, the
sections show a
typical apple-green
birefringence under
polarized light

52. • Crystal violet stains
amyloid deposits pink
color, that are present
in both glomeruli and
vessels

53. • Glomeruli and vessels
appear diffusely
positive when stained
with fluorescent
Thioflavin T

54. H and E stain showing pink amorphous
deposit (arrow) around the distorted
myocytes
Toluidine blue stain shows pale purple-
blue staining around the distorted
myocytes

55. H and E of cardiac muscle showing
amorphous pink deposit
Alcian blue staining of same section
showing greenish blue color of the deposit

56. • Amyloid appears as
an amorphic,
eosinophilic, PAS
negative or scantly
positive,
extracellular
substance.

57. Determining the type of amyloid
Immunohistochemical –
• A panel of antibodies against four major
amyloid fibril proteins
• Includes serum AA protein, kappa light chain,
lambda light chain and transthyretin (TTR),
antibody to amyloid P (AP) component also
added to the panel of antibodies

59. Direct Immunofluorescence
microscopy
• show deposition of a monoclonal (lambda or
kappa) light chain in AL amyloidosis.
• Frozen sections were cut at 3-𝛍m
• Direct IF staining was performed using
fluorescein isothiocyanate conjugated primary
polyclonal rabbit anti-human antibodies to
lambda and kappa light chain

60. • Fluorescent microscopy is used, the antigen –
antibody complex absorbs UV light and emits
visible light.
• The intensity of staining was graded on a scale
from 0 to 3 (1+, mild staining; 2+ , moderate
staining; 3+, to strong staining; +/- indicated
trace staining)

61. IF for kappa light chain IF for lambda light chain
(1 + ) ( 3 + )

62. Indirect Immunoperoxidase
• Staining for AA type
• Sections were deparaffinized and rehydrated
• Sections were stained with primary polyclonal
rabbit antihuman amyloid A antibody
• Followed by goat anti-rabbit secondary
antibody
• counterstained with hematoxylin

63. Indirect Immunoperoxidase positive congo red showing apple green birefringence
for AA protein in glomeruli of kidney on polarized microscope of same section

64. Morphology
Kidney
• Can have both primary (AL) and secondary (AA)
types of deposits
• Gross – normal or enlarged in the early stage
shrunken in late stage- due to ischemia and
narrowing of blood vessels
• microscopy – amyloid deposits seen
– Glomeruli
– Interstitial peritubular region
– Arteries and arterioles

65. H and E
Congo red

66. Heart
Gross
• Firm, rubbery
• Chambers are normal or dilated
• Wall thickened
• Nodule resembling drips of wax in endocardium
Microscopy
• On H and E amorphous pink hyaline material
seen around the myocytes

67. H and E Congo red

68. Spleen (sago spleen, lardaceous
spleen)
Always an expression of systemic amyloidosis
Gross
• Mild to moderate splenomegaly
• Cut section, pale wax like deposition, resemble
sago grains
Microscopically
• Deposits involve walls of splenic sinuses,
small arteries, connective tissue.

69. H and E Congo red

70. Liver
Seen in systemic amyloidosis
Gross
• Marked hepatomegaly
• Cut section –pale waxy appearance
Microscopy -
• Deposition first at space of disse
• Encroaches adjacent liver parenchyma
• Around the blood vessels and sinusoids

71. Congo red
H and E

72. Brian
Commonly associate with alzheimers disease
Gross
• Variable degree of cortical atrophy
• Widening of cerebral sulci
• Enlarged ventricles secondary to atrophy
Microscopically
• Plaque are focal, arranged around the central
amyloid core

74. Gastrointestinal tract
• Common sites are the second part of the
duodenum, the stomach and colorectum and
the esophagus
• Deposition seen in muscularis mucosae,
submucosa, and muscularis propria leads to
polypoid protrusions and thickening of the
valvulae conniventes l/t obstruction

75. H and E of duodenum showing pink
amorphous deposition in lamina propria
Congo red stain showing amyloid in
lamina propria and around
blood vessels

76. Medullary carcinoma thyroid
H and E stain, pink amorphous deposit
between the tumor cells of medullary
carcinoma thyroid
Apple green birefringence for congo red stain
seen under polarized microscope

77. Islets of pancreas
H and e stain, pink amorphous deposit
around the islets of pancreas
Congo res stain, red deposit around
the islets of pancreas

78. Bone marrow biopsy in multiple
myeloma
Bone marrow biopsy stained with PAS (left) shows pink deposition around the sinusoids
And also shows malignant plasma cells > 30 % and few multinucleated giant cells
Same section stained with congo red (right) shows red color around the sinusoids

79. Conti..
Strong positive staining with λ light chain immunoglobulin antibody
(immunoperoxidase with hematoxylin counterstain

80. Recent advances in amyloidosis

81. Laser microdissection with mass
spectrometry
• Tissue is fixed in formalin and processed in
routine method and paraffin blocks are
prepared
• Sections of 10𝛍 thickness are prepared and
stained
• With the help of software, area is selected for
microdissection

82. • Microdissected fragments were digested into
tryptic peptides and analyzed by liquid
chromatography Mass Spectrometry

83. Laser microdissection

84. Immuno-gold electron microscopy
• This is particularly useful for AL amyloidosis
• Its one of the staining technique used in
electron microscopy
• The high electron density of the gold particles
are used to identify the target molecule
• These will increase the electron scatter to give
high contrast dark spots
• Different sized gold probes are available
ranging from 1-40 nm

85. SAP scintigraphy (SAP scan)
• In 1987 Sir Mark Pepys invented
• Professor Philip Hawkins developed it for
routine clinical use.
• This technique shows the distribution and
amount of amyloid in the organs throughout
the body without the need for biopsies
• The basis for the test is injection of a small
amount of radio-labelled SAP which binds
on amyloid deposits throughout the body.

86. • The deposited radio labeled SAP transmits a
radioactive signal.
• This signal is picked up by a detector, gamma
camera. All body parts where radioactive
signal is detected will contain amyloid deposits

87. SAP scan showing amyloid deposit strongly in liver and sleep (left) and
Same person SAP scan (right) showing reduced amyloidosis after treatment

88. SAP scintigraphy (SAP scan)

89. Genetic testing
• Mutations have been identified in all of the
hereditary amyloidosis, such as
– TTR gene on chromosome 18
– APP gene on chromosome 21
– PrP gene on chromosome 20
– Apo A-1 gene on chromosome 11
– Apo A II on chromosome 1
– Fibrinigen A alpha on chromosome 4
– Gelsolin gene on chromosome 9
– Cystatin C gene on chromosome 20

90. Conti..
• Patient’s peripheral blood collected
• Genes that are routinely sequenced in the
laboratory by PCR
• Determine the mutated genes

91. Diagnostic criteria for AL type
• Mayo Clinic and the International Myeloma
Working Group Presence of all of the
following four criteria
1. Presence of an amyloid-related systemic
syndrome.
2. Positive amyloid staining by Congo red in
any tissue or the presence of amyloid fibrils
on electron microscopy

92. Conti..
3. Evidence that the amyloid is light chain-
related established by direct examination of the
amyloid using spectrometry-based proteomic
analysis or immunoelectron microscopy.

93. Conti..
4. Evidence of a monoclonal plasma cell
proliferative disorder (eg, presence of a serum or
urine M protein, abnormal serum free light
chain, or clonal plasma cells in the bone marrow)

94. Diagnostic algorithm
clinical features
lab findings
biopsy ( abdominal fat)
congo red stain

95. Conti..
congo red
negative positive
specific organ biopsy
positive
typing of amyloidosis

96. Conti..
typing of amyloidosis
immnohistochemistry
immunoelectron microscopy
immunofloroscenc
laser microdissection and mass spectrometry
SAP scan
genetic tests

97. Prognosis
• Serious disease with high mortality
• Overall median survival rate after diagnosis is
< 2 years in most of the cases
• Patient with coexistent multiple myeloma has
poor prognosis
• Survival time depends on the type of
predominantly involved organ
• Cardiac involvement is the major determinant
of survival prognosis – major cause of death

98. Summary
• Rare, uncommon disease with poor long term
survival.
• Associate with hereditary and inflammatory
conditions.
• Symptoms are vague and its necessary to
diagnose and to type the amyloidosis.
• Diagnosis is evolved with many recent
advance techniques.

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104. Thank you