2. OVERVIEW:-
INTRODUCTION.
DIFFERENTIATION OF AMYLOID.
PROPERTIES OF AMYLOID PROTEINS.
RARE FORMS OF PROTEINS.
PATHOGENESIS.
CLASSIFICATION.
MORPHOLOGICAL APPEARANCE.
HISTOLOGICAL FEATURES.
AMYLOIDOSIS IN DIFFERENT ORGANS IN DIFFERENT CLINICAL FORMS.
CLINICAL FEATURES.
DIAGNOSIS OF AMYLOIDOSIS.
DEMONSTRATION OF AMYLOID.
PROGNOSIS.
REFERENCES.
3. INTRODUCTION:-
Amyloidosis is a condition associated with a number of disorders in
which extracellular deposits of fibrillar proteins are responsible for
tissue damage and dysfunction.
These abnormal fibrils are produced by the aggregation of
misfolded proteins.
These fibrils binds wide variety of proteoglycans, glycosaminoglycan
including heparan sulfate and dermatan sulfate , plasma proteins,
Notably serum amyloid P.
4. INTRODUCTION:-
The presence of these abundant charged sugar groups
mainly starch in these absorbed proteins give staining
characteristics that resembles to starch
(amylose),Therefore these fibrils are called amyloid.
Progressive accumulation-it encroaches on and produces
pressure atrophy of adjacent cells.
5. DIFFERENTIATION OF AMYLOID:-
WITH LIGHT MICROSCOPE:
H &E STAIN: It appears as an amorphous, eosinophilic,
hyaline, extracellular substance.
It is difficult to differentiate amyloid from other hyaline
substance. A wide variety of histochemical techniques
applied for identification
Most widely used is: CONGO RED STAIN:-Appears as pink or
red color to tissue deposits.
In polarising microscopy:-Apple green birefringence
(diagnostic feature).
6.
7. PROPERTIES OF AMYLOID PROTEINS:-
A) PHYSICAL :-
Electron microscopy : continuous, nonbranching fibrils with diameter
of approx 7.5 to 10 nm.
x-ray crystallography and infrared spectroscopy :cross linked beta sheet
conformation.
8. B)CHEMICAL :-
It is 95% of fibrils proteins,5% p – component , other
glycoproteins.
Three forms:
a)AMYLOID LIGHT CHAIN(AL)
b)AMYLOID ASSOCIATED(AA)
C ) β- AMYLOID (Aβ)
9. A) AMYLOID LIGHT CHAIN:-
It is composed of immunoglobin light chains , amino-
terminal fragments of light chains , or both.
It is produced from free Ig light chains secreted by
monoclonal plasma cells.
Its deposition is associated with certain plasma cell
tumors.
10. B)AMYLOID ASSOCIATED (AA) PROTEIN:-
It is derived from unique non- Ig protein made by the liver.
The AA protein found in the fibrils is created by proteolysis of a
larger
precursor called SAA(SERUM ASSOCIATED AMYLOID).
Non- Ig protein synthesized in liver and circulates in the blood ,
bound to high density lipoproteins.
SAA is increased in inflammatory states as part of acute phase
response .
This form of amyloid protein is associated with chronic
inflammation and called as secondary amyloidosis.
11. C) β- AMYLOID (Aβ) :-
This constitutes core of cerebral plaques in Alzheimer disease.
Derived by proteolysis of much larger transmembrane glycoprotein –
amyloid precursor protein.
This amyloid deposited in walls of cerebral blood vessels.
12. RARE FORMS OF PROTEINS:-
TRANSTHYRETIN:-
It is a normal serum protein that binds and transports
thyroxine and retinol.
Mutant forms (fragments) from amyloid in a group of
genetically determined disorders referred as FAMILIAL
AMYLOID POLYNEUROPATHIES.
Unmutated forms may also deposit as amyloid in heart of
aged individuals as SENILE SYSTEMIC AMYLOIDOSIS.
β macroglobulin:-
component of MHC class-I molecules ,normal serum
protein ,Identified as the major component of a form of
Aβ2 amyloid deposits around joints or soft tissues of
patients on long term hemodialysis.
13. PATHOGENESIS:-
Normally, misfolded proteins, degrade intracellularly in
proteosomes or extracellular tissues by macrophages.
It appears that in amyloidosis, these mechanisms fail
leading to accumulation of a misfolded protein outside
cells.
Proteins forming amyloid are of two categories:-
Normal proteins that have inherent tendency to misfold-
and aggregate when produced in large amounts.
Mutant proteins prone to misfolding and subsequent
aggregation.
14.
15. CLASSIFICATION OF AMYLOIDOSIS:-
Amyloid classified into systemic (generalized), involving
several organs systems ,or it may be localized to a single
organ (heart).
On clinical it is subclassified into
a)PRIMARY AMYLOIDOSIS:-
when associated with plasma cell proliferations.
b)REACTIVE SYSTEMIC AMYLOIDOSIS(SECONDARY
AMYLOIDOSIS):-
when occurs as a complications of underlying chronic
inflammatory process.
c)HEREDITARY OR FAMILIAL AMYLOIDOSIS:-
with distinctive patterns of organ involvement.
16. A)PRIMARY AMYLOIDOSIS :-
It is of AL type.
Associated with clonal proliferation of plasma cells that
synthesize an Ig light chain that is prone to form amyloid.
Best defined is occurrence of systemic amyloidosis in 5-
15% of individuals with multiple myeloma.
Malignant plasma cells – secrete free unpaired λ or κ light
chains(referred to as BENCE-JONES PROTEINS).
Due to their small molecular weight free light chains are
deposited as amyloid.
17. A)PRIMARY AMYLOIDOSIS :-
Most patients with AL amyloid do not have classic
multiple myeloma or any overt B cell neoplasm.
classified as primary amyloidosis , because clinical
features derived from deposition of amyloid without any
other associated diseases.
Monoclonal immunoglobulins or free light chains or both
can be seen serum or urine.
But there is increase in number of plasma cells in bone
marrow
There is underlying monoclonal proliferation of Ig
producing cells(monoclonal gammopathy).
18. B)REACTIVE SYSTEMIC AMYLOIDOSIS
(secondary amyloidosis) :-
Amyloid deposits in this pattern are systemic in distribution
and are composed of AA subtype
Previously -secondary amyloidosis –secondary to chronic
inflammation(like Tuberculosis , bronchiectasis).
Now- Reactive systemic amyloidosis secondary to rheumatoid
arthritis , ankylosing spondylitis , inflammatory bowel disease.
Most common is rheumatoid arthritis.
Intravenous drug use(Heroin)- chronic skin infections associated
with “skin popping”.
In association with solid tumors- renal cell carcinoma and
Hodgkin lymphoma.
19. B)REACTIVE SYSTEMIC AMYLOIDOSIS
(secondary amyloidosis) :-
Inflammation(long standing inflammation)
IL-6, IL-1
INCREASE SAA synthesis by liver cells
SAA Degraded to soluble end products (by marcophages ).
IN AMYLOIDOSIS:-
Chronic inflammation Incomplete breakdown of SAA INSOLUBLE
AA.
20. C)HEREDOFAMILIAL AMYLOIDOSIS:-
It is rare.
It is best studied and most common in autosomal recessive
condition called Familial Mediterranean fever(FMF).
FMF is an autoinflammatory syndrome associated with
excess production of cytokine IL-1 .
Clinical features: Fever, inflammation of serosal surfaces
like pleuritis , peritonitis , synovitis.
The gene that FMF encodes a protein called pyrin –that
activates inflammasomes and production of mainly IL-1
and lead increased production SAA.
The amyloid seen in this is AA type.
21. C)HEREDOFAMILIAL AMYLOIDOSIS:-
In contrast to FMF, a group of autosomal dominant familial
disorders is characterized by deposition of amyloid in
peripheral and autonomic nerves .
In disorders like familial amyloidotic neuropathies and
cardiac amyloidosis fibrils are made up of mutant form of
transthyretin that leads to aggregation and misfolding and
resistant to proteolysis.
22. HEMODIALYSIS ASSOCIATED
AMYLOIDOSIS:-
It is seen in long term hemodialysis for renal failure can
develop amyloidosis as result of deposition of β2
macroglobulin.
This protein is present in high concentrations in serum of
persons with renal disease.
This could not be filtered through dialysis membranes.
Accumulation in joints, muscle, tendons or ligaments –
presented as carpal tunnel syndrome.
23. LOCALIZED AMYLOIDOSIS:-
Limited to single organ or tissue.
Grossly ,detectable as nodular masses or be evident only
on microscopic examination
Nodular deposits are encountered in lung ,larynx , skin,
urinary bladder,tongue and the regions around eyes.
Frequently, there are infiltrates of lymphocytes and
plasma cells in the periphery of these amyloid masses.
At least in some cases it consists of AL protein.
Al may represent as localized form of plasma cell derived
amyloid.
24. ENDOCRINE AMYLOID:-
Microscopic deposits of localized amyloidosis may be seen in
endocrine tumors-
Medullary thyroid carcinoma (MTC),
Pancreatic islet tumor and pheochromocytoma ,
Islets of Langerhans in T2DM.
Amyloidogenic proteins are derived from polypeptide
hormones (MTC) proteins and from unique proteins (islet
amyloid polypeptide).
In MTC, presence of amyloid is a diagnostic feature.
26. AMYLOID OF AGING :-
Senile systemic amyloidosis.
Predominantly heart is involved – senile cardiac
amyloidosis (previously).
Symptoms of restrictive cardiomyopathy and arrhythmias.
Amyloid is derived from normal TTR, mutant TTR
accumulation also predominantly involves heart.
27.
28. MORHOLOGY:-
In amyloidosis secondary to chronic inflammatory
disorders- kidneys, liver, spleen, lymph nodes, adrenals,
and thyroid as well as many other tissues are typically
affected.
It more often involves heart ,git ,respiratory tract,
peripheral nerves , skin and tongue.
In familial Mediterranean fever- wide spread
involvement- kidneys, blood vessels, spleen, respiratory
tract and (rarely) liver.
29. GROSS APPEARANCE :-
GROSSLY, when amyloid accumulates in larger amounts,
The organs is frequently enlarged
The tissue appear gray with a waxy , firm consistency.
30. HISTOLOGICAL FEATURES:-
The amyloid deposition is always extracellular and beings
in between the cells, adjacent to basement membranes.
As the amyloid accumulates, it encroaches on the cells
surrounding and destroying them.
In the form associated with plasma cell proliferation,
perivascular and vascular deposits are common.
31. HISTOLOGICAL FEATURES:-
HISTOLOGICALLY-the amyloid deposition is always
extracellular and beings in between the cells,
adjacent to basement membranes.
As it accumulates, it encroaches on the cells
surrounding and destroying them.
In the form associated with plasma cell proliferation,
perivascular and vascular deposits are common.
32. Diagnosis:-
The histologic diagnosis of amyloid is based almost
entirely on its staining characteristics.
Congo red dye- under light microscopy- imparts a pink or
red color to amyloid deposits.
Under polarized light - the Congo red-stained amyloid
shows apple-green birefringence.
Congo red staining amyloid deposits - bright red
appearance under ultraviolet light on fluorescent
microscopy (Congo red fluorescence).
34. DIAGNOSIS:-
CONFIRMATION, can be obtain by electro microscopy
which reveals amorphous non-oriented thin fibrils.
AA, AL, and ATTR types of amyloid can also be
distinguished by specific immunohistochemical staining.
Positive for anti-ATTR, salivary gland
biopsy
6E10 Anti-Amyloid Beta staining
35. AMYLOIDOSIS PATTERN OF DIFFERENT ORGANS
IN DIFFERENT CLINICAL FORMS:-
KIDNEY:-
Most common and most serious form of organ
involvement.
Grossly, the kidneys may be of normal size and color.
In advanced cases, shrunken because of ischemia caused
by vascular narrowing induced by the deposition of
amyloid within arterial and arteriolar walls.
36. KIDNEY:-
Amyloid deposits seen in predominantly in glomeruli,
interstitial peritubular tissue, arteries, and arterioles are also
affected.
The glomerular deposits first appears as subtle thickenings of
the mesangial matrix , accompanied by uneven widening of the
basement membranes of the glomerular capillaries.
In time the mesangial deposits and deposits along the
basement membranes cause capillary narrowing and distortion
of the glomerular vascular tuft.
In advanced stages- capillary lumen are obliterated and
glomerulus shows confluent masses or interlacing broad ribbons
of amyloid.
38. SPLEEN:-
May be inapparent grossly or may cause moderate to marked
splenomegaly (up to 800 g).
Two patterns of deposition are seen:-
The deposits are limited to the splenic follicles, producing
tapioca-like granules on GROSS INSPECTION -SAGO SPLEEN.
In another pattern ,It involves the walls of the splenic sinuses
and connective tissue framework in the red pulp. Fusion of the
early deposits gives rise to large, map-like areas of
amyloidosis, designated as LARDACEOUS SPLEEN.
40. LIVER:-
The deposits may be inapparent grossly or may cause moderate
to marked hepatomegaly.
Amyloid appears first in the space of Disse and then
progressively encroaches on adjacent hepatic parenchymal cells
and sinusoids.
In time, deformity, pressure atrophy, and disappearance of
hepatocytes occur, causing total replacement of large areas of
liver parenchyma.
Vascular involvement is frequent.
Even with extensive involvement, liver function is usually
preserved.
42. AMYLOID DEPOSITS IN WALLS OF THE
BLOOD VESSELS AND SINUSOIDS.
Congo red reveals pink-red deposits
of amyloid in the walls of blood
vessels and along sinusoids.
Under Polarizing microscope
43. HEART:-
Amyloidosis of heart may occur in any form of systemic
amyloidosis.
It is also the major organ involved senile systemic amyloidosis.
Grossly- enlarged and firm, but more often- no significant
changes
The deposits begin as focal subendocardial accumulations and
within the myocardium between the muscle fibers.
Expansion of these myocardial deposits , pressure atrophy of
myocardial fibers.
when amyloid are subendocardial deposits ,the conduction
system may be damaged accounted for ECG abnormalities.
45. OTHER ORGANS:-
Nodular deposits in tongue cause macroglossia ,giving rise to
the designation tumor forming amyloid of the tongue.
Respiratory tract may involves focally or diffusely from the
larynx to the smallest bronchioles.
In Brain it present as Senile neuritic plaques in paitents with
Alzheimer disease.
Amyloidosis of peripheral and autonomic nerves is a feature of
familial polyneuropathies.
Long-term hemodialysis patients deposition of amyloid - carpal
ligament of the wrist(carpal tunnel syndrome) also in joints.
47. CLINICAL FEATURES:-
Asymptomatic or depend on magnitude and site of
deposits.
Initially clinical features are non- specific symptoms –
weakness, weight loss, light headness, syncope.
Renal involvement:-
Proteinuria may be severe enough to cause nephrotic
syndrome.
Progressive obliteration of glomeruli in advance cases –
renal failure and uremia.
Renal failure – death.
48. CLINICAL FEATURES:-
Cardiac amyloidosis:- may present as
Insidious congestive heart failure,
Serious effects affects are conduction disturbances,
arrhythmias.
occasionally -Restrictive cardiomyopathy.
Chronic pericarditis.
49. CLINICAL FEATURES:-
Gastrointestinal amyloidosis can be asymptomatic .
Amyloidosis of tongue may cause slurred speech and
difficulty in swallowing.
Deposits of stomach and intestine: malabsorption and
diarrhoea.
Vascular amyloidosis:-
Fragile vessels – bleeding.
AL amyloid binds to and inactivates factor X- bleeding
leading to life threatening bleeding disorder.
50. DIAGNOSIS OF AMYLOIDOSIS:-
THE DIAGNOSIS OF AMYLOIDOSIS DEPENDS ON
THE HISTOLOGIC DEMONSTRATION OF AMYLOID
DEPOSITS IN TISSUES.
Biopsy is the gold standard for demonstration
of amyloid.
Most common sites- kidney for renal
involvement.
Cardiac involvement- endomyocardial biopsy.
Rectal or gingival tissues i/c/o systemic
amyloidosis.
Abdominal fat aspirate is highly specific
helps diagnosis of systemic amyloidosis.
51. DEMONSTRATION OF AMYLOIDOSIS :-
H&E : amorphous, pale eosinophilic, extracellular, faintly
refractile substance.
Congo red: pink red color, apple green birefringence,
congo red fluorescence.
Sirius red F3B: intense red and green birefringence; no
fluorescence.
Previously: crystal violet method, methyl green .
Fluorescence dyes: Thiofalvine T.
52. DEMONSTRATION OF AMYLOIDOSIS :-
AL amyloidosis suspected cases these tests should be performed
Serum protein electrophoresis
Urine protein
Immuno-electrophoresis
Bone marrow aspiration- monoclonal plasmacytosis even in
absence of overt myeloma ,so Scintigraphy with radio-labelled
serum amyloid P – rapid and specific test.
As, it binds to amyloid deposits and reveals their presence.
54. PROGNOSIS:-
The prognosis of generalized amyloidosis is poor.
In AL amyloidosis have an overall median survival
of 2 years after diagnosis, and the prognosis is
even poor with myeloma –associated AL
amyloidosis.
Reactive amyloidosis some better depends to
some extent on the control of the underlying
conditions.
55. REFERENCES:-
1. Kumar, V., Abbas, A. K., Aster, J. C., & Perkins, J. A.
(2018). Robbins pathologic basis of disease 10th South Asia
: Elsevier:259-265.
2. Linder,J.,Damjanov,I.,Anderson’s Pathology.10thed;South
Asia:Elsevier 2014;1:448-456.
3. . Mohan,H., Textbook of Pathology; 8th ed; Jaypee
Brothers Med. Publishers; 2019;152-162.
