Amyloidosis in brief

1. The micrograph shows amyloid (extracellular fluffy pink material) and
abundant lipofuscin (yellow granular material).

2. Amyloid
positivity with
Congo red
stain.
Polarized
microscopy
reveals apple-
green
birefringence
for amyloid

3. AMYLOIDOSIS
PRESENTER- DR ZUBIY SUHA
MODERATOR- DR PRINCY SOMAN

4. OBJECTIVES-
◦ Introduction
◦ Physical properties
◦ Chemical properties
◦ Pathogenesis
◦ Classification
◦ Morphology
◦ Diagnosis
◦ Stains
◦ Summary
◦ References

5. AMYLOID
• Amyloid is the eosinophilic amorphous extracellular insoluble misfolded
fibrillar protein.
• Deposition of this extracellular amyloid protein in various organs and tissues is
known as Amyloidosis.
• Different diseases of various etiopathogenesis may show amyloidosis

6. PHYSICAL PROPERTIES
ELECTRON MICROSCOPY
◦ The fibrils are arranged in random
array and are nonbranching
◦ Insoluble
◦ Linear
◦ Rigid
◦ 7-10 nm diameter.

7. PHYSICAL PROPERTIES
ON X-RAY CRSTALLOGRAPHY
AND INFRA RED
SPECTROSCOPY
• Each fibril consists of β pleated
sheet polypeptide chains
• Congo red dye binds to these fibrils
and produces classic apple green
birefringences under polarized light

8. CHEMICAL PROPERTIES
Amyloid composed of 2 main types
of amyloid
Fibril protein - 95% of amyloid
material consists of fibril proteins.
Non-fibrillar component- 5%- P
component with other proteins like
apolipoprotein, glycosaminoglycans
Chemically, 2 major forms of
amyloid fibrils-AL
and AA

14. AMYLOIDOSIS MORPHOLOGY
◦ Amyloidosis of different organs show
variation in morphologic pattern,
general features are-
◦ GROSS-affected organ is large, grey,
waxy, and rubbery (firm in
consistency)
◦ MICROSCOPY- deposits are always
extracellular, begins between cell close
to the basement membrane and are
amorphous, eosinophilic

15. AMYLOIDODIS OF
KIDNEY
◦ Most common and serious form
◦ GROSS- kidneys maybe normal sized,
enlarged or shrunken in advance cases
because of ischemia
◦ C/S- pale, waxy, translucent
◦ MICROSCOPY- amyloid deposit
primarily in glomeruli, but arteries,
arterioles and peritubular tissue are
also affected

16. AMYLOIDODIS OF SPLEEN
◦ 2 patterns of deposition
◦ SAGO SPLEEN- MC the amyloid
deposition is limited to splenic
follicles, resulting in gross appearance
of moderately enlarged spleen dotted
with gray nodules
◦ LARDACEOUS SPLLEN- Amyloid
involves the walls of splenic sinuses
and connetive tissue framework in the
red pulp. Fusion of early deposits give
rise to large, map like areas of
amyloidosis

17. AMYLOIDODIS OF LIVER
• It may cause enlargement,
pale, waxy and firm.
• Histologically, amyloid
deposits first appear in
the space of Disse.(space
between hepatocytes and
sinusoidal endothelial cells)
• Normal liver function is
usually preserved.

18. AMYLOIDODIS OF HEART
• It may produce arrhythmias due
to deposition in conduction system.
• In localized form, deposits seen in left
atrium
• Amyloid first deposits in subendocardial
areas of atrium.
• Most common cause of death is cardiac
failure.
◦ GROSS- heart is enlarged and firm
◦ Epi/endocardium and valves show tiny
nodules
◦ MICROSCOPY- focal subendocardial
accumulations, in primary form, deposits
are seen around myocardial fibres in ring
forms also known as ring fibres

19. AMYLOIDODIS OF
BRAIN
◦ Diseases A/W senile cerebral amyloidosis
are Alzheimers disease, Downs syndrome,
Creutzfeldt Jakob disease, kuru, mad cow
disease
◦ Alzheimer's disease and other
neurodegenerative disorders belong to
family of protein misfolding diseases,
characterized by protein self-aggregation
and deposition
◦ In vivo detection of amyloid plaques and
neurofibrillary tangles in the brain enables
early identification of AD

20. When to suspect amyloidosis…..?
◦ Nephrotic range proteinuria with or without renal insufficiency
◦ Unexplained kidney failure
◦ Non-dilated cardiomyopathy
◦ Peripheral or autonomic neuropathy
◦ Hepatomegaly or splenomegaly
◦ Malabsorption

21. Diagnosis
◦ Iodine staining of amyloid
◦ -means starch like - painting on C/s with iodine – yellow color,
which is transformed to blue violet after application of sulfuric
acid(acidifies iodine) –
◦ This method was used to demonstrate cellulose or starch

22. DIAGNOSIS
◦ Presence of amyloid-
-Evaluation of organ involvement
(in-vivo test and imaging)
-Tissue biopsy and its histology
-Congo red staining
◦ Type of amyloid-
-Immunohistochemistry
◦ Mutation type
- amino acid sequence analysis Amyloid AAntibody Immunohistochemistry on
a FFPE Kidney Tissue

23. TISSUE BIOPSY
◦ Subcutaneous fat aspiration(provides
enough material from aspiration)rectal
biopsy
◦ Gums
◦ Bone marrow
◦ Others- kidneys, nerves, heart, liver
◦ Organ biopsy- if subcutaneous fat
investigation did not provide diagnosis
◦ Kidney biopsy to determine the cause
of nephrotic syndrome

24. Amyloidosis leads to…..

25. CONGO RED STAIN
Steps of staining
• Deparaffinize.
• Pass through graded alcohol to bring in water
• Rinse in distilled water: 10–15 dip
• Stain by Mayer’s haematoxylin
• Blueing by running tap water
• Wash in distilled water
• Alkaline alcohol sodium chloride solution: 20
min
• Alkaline Congo red: 20 min
• Rapid dehydration
• Clear in xylene
• Mount.
Principle- Congo red intercalates between the
parallel fibrils of the amyloid protein and forms a
non-polar hydrogen bond

26. CONGO RED- Results:
◦ With the light microscope, amyloid
deposits are red to pink-red, nuclei are
blue
◦ Amyloid deposits show an “apple-
green” birefringence with the
polarizing microscope.
◦ Nuclei of inflammatory cells, and
granular basophilic debris associated
with vacuoles, stain dark blue/black
Renal AA amyloidosis

27. THIOFLAVINE T STAIN
• Thioflavine T is a very sensitive technique.
Principle- This cationic benzothiazole dye increases in
fluorescence upon binding to the stacked β sheets
of amyloid fibrils
• Not a specific stain for amyloid.
Result-
• Fluorescence microscope : Bright yellow fluorescence

28. OTHER STAINS
•Metachromatic techniques
methyl crystal violet
crystal violet
methyl green
-imparts rose pink color.
• Low sensitivity
• Lack specificity
◦ • Van gieson- khakhi color
◦ • PAS- pink

29. OF VARIOUS AMYLOID
STAININGSUMMARY METHODS

30. Identify ?
Seen in ?
Type of amyloid ?
Neurofibrillary
tangles
Alzheimers
disease
A beta amyloid

31. First described by-
Term first used by-based on color after
staining with iodine
Later recognized as
protein by-
Rudolf Virchow
Karl von Rokitansky
Friedrich August
Kekule

32. REFERENCES
 Kumar V, Abbas A, Aster JC, Deyrup AT, editors. Robbins & Kumar Basic Pathology.
11th ed. Philadelphia, PA: Elsevier - Health Sciences Division; 2022.
 Suvarna KS, Layton C, Bancroft JD. Bancroft’s theory and practice of histological
techniques. 8th ed. London, England: Elsevier Health Sciences; 2018.
 Muchtar E, Dispenzieri A, Magen H, Grogan M, Mauermann M, McPhail ED, et al.
Systemic amyloidosis from A (AA) to T (ATTR): a review. J Intern Med.
2021;289(3):268–92.
 Picken MM. The pathology of amyloidosis in classification: A review. Acta Haematol.
2020;143(4):322–34