AMYLOIDOSIS

Amyloidosis in brief

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

2. A myloid 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

9. LOCAL VARIANTS AL (Locally produced monoclonal Ig)- urogenital, skin, eyes, respiratory AANF(abnormal atrial natriuretic factor)-atria Medin -aortic amyloidosis in elderly Insular amyloid polypeptide/amylin insulinoma- diabetic mellitis

10. 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

11. 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

12. 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

13. 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.

14. 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

15. 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

16. 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

17. 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

18. 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 A Antibody Immunohistochemistry on a FFPE Kidney Tissue

19. 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

20. Amyloidosis leads to…..

21. 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

22. CONGO RED STAIN BY HIGHMAN

23. 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

24. 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

25. 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

26. OF VARIOUS AMYLOID STAININGSUMMARY METHODS

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

28. 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

29. 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