Amyloidosis

Definition of amyloidosis
physical and chemical nature
mechanism and components
classification
staining characteristics
diagnosis

1. AMYLOIDOSIS EMANI APARNA SRINIVAS COLLEGE OF PHARMACY

2. A medical condition resulting from aggregation of extracellularly deposited abnormal proteins called amyloid fibrils that cause damage to organs and tissues. These fibrils are insoluble, linear, rigid and measures approximately 7.5 to 10mm in width.

3. PHYSICAL AND CHEMICAL NATURE OF AMYLOID Physical Nature : 7.5 -10 nm in diameter, cross- Beta pleated sheet. The fibrils are delicate, randomly dispersed, non-branched and having indefinite length. Chemical nature : 95% Protein Fibril + 5% Glycoprotein P component - pentagonal molecule

4. MECHANISM OF FORMATION Amyloid fibrils arise from misfolded proteins. Alpha helix to beta pleated sheet Proteins are deposited extracellularly Proteins aggregate and form fibrils called amyloid fibrils. Misfolded proteins may result from point mutations. Deposited as localized vs systemic -localized; close to cells producing it. -Systemic; distant sites from these cells producing these abnormal proteins

5. FIBRIL COMPONENTS AL PROTIEN: Fibril protein is derived from immunoglobin light chain, which includes amino-terminal segment of the immunoglobulin light chain and part of C region. Al fibril proteins are produced from immunoglobulin-secreting cells and is seen in association with plasma cell dyscrasias and in primary systemic amyloidosis. Al fibril protein is derived from lamba light chain than kappa

6. AA PROTIEN: Composed with molecular weight of 8.5-kD which is derived from larger precursor protein in the serum called SAA with the molecular weight of 12.5kD. SAA circulates in association with HDL3 . SAA is an acute reactant protien synthesized in the liver its level being high in inflammatory and traumatic conditions.

7. NON FIBRILLAR COMPONENTS AMYLOID P component: Synthesised in the liver Derived from circulating serum amyloid P component, A glycoprotien resembling the normal serum α 1-glycoprotien and is PAS positive. On electron microscopy it appears like pentagonal profile or doughnut –shape profile with external diameter of 9 mm and internal diameter of 4mm.. APOLIPOPROTIEN: Regulator of lipoproptien metabolism Increases the risk of alzehimers precursor protien ( APP). SULFATED GYLCOAMINOGYCANS (GAG) : These are the constituents of matrix protiens ; particularly associated is heparan sulfate in all types of tissue amyloid .

8. CLASSIFICATION OF AMYLOIDOSIS Systemic amyloidosis Primary amyloidosis Secondary amyloidosis Localized amyloidosis Senile cerebral Senile cardiac Endocrine amyloidosis 4. Localised tumour forming amyloid

9. Primary amyloidosis: Consist of fragment of light chain 5-15% of the patients with multiple myeloma develop AL type amyloid systemic amyloidosis. Organs: heart, skin, GIT, skin, mucous membrane. Secondary amyloidosis: AA amyloid. Secondary amyloidosis is associated with chronic inflammatory states ie both infectious and non –infectious inflammatory conditions are associated with tissue destruction like tuberculosis, leprosy, inflammatory bowel disease and autoimmune diseases. Organs: liver, kidneys, spleen, adrenal glands .

10. Localised amyloidosis: 1. Senile cardiac amyloidosis : age of 70 years. Amyloid deposits are seen in the heart and aorta. 2.Senile cerebral amyloidosis: Deposition of amyloid material in the walls of cerebral blood vessels. Majority of population over age 70 yrs of age. 3 . Endocrine amyloidosis: microscopic deposition of amyloid results in the tumors of endocrine glands. Eg: medullary carcinoma of thyroid in which amyloid is derived from the precursor of hormone i.e procalcitonin.

11. 4. Localised tumour forming amyloid (AL): Isolated tumour like formation of amyloid deposits are seen. Eg: lungs, skin, tongue.

12. STAINING CHARACTERISTICS OF AMYLOID STAIN IN GROSS: LUGOL’S IODINE imparts purple colour to the amyloid -containing area on dilution with sulphuric acid turns blue. Starch-like property of amyloid is due to AP component , Glyco-protien present in all forms of amyloid. H & E: Amyloid by light microscopy with haematoxylin and eosin staining appears as extracellular, homogenous, structures and esinonophlic hyaline material especially in relation to blood vessels. Deposits are small they are difficullt to detect by routine H and E stains. Few hyaline deposits may also take pink colour.

13. H & E staining in renal biopsy

14. METACHROMATIC STAINS ( ROSANILINE DYES) Amyloid has property of metachromasia ie . Dye reacts with amyloid and undergoes a colour change. Metachromatic stains employed are rosaniline dyes such as methyl violet and crystal violet which imparts rose-pink colouration to amyloid deposits. CONGO RED AND POLARISED LIGHT: Amyloid has affinity for congo red . This can been used for gross and microscopic sections ; amyloid of all types stains red colour. If stained section is viewed in polarised light, the amyloid characteristically shows apple-green birefringence due to cross- β pleated sheet configuration of amyloid fibrils. This stain can be used to distinguish AL and AA amyloid After prior treatment with permanganate or trypsin on the section, congo red stain is repeated – Congo red positivity- primary amyloid Congo red negativity- secondary amyloid

15. CONGO RED STAINING OF CARDIAC BIOPSY CONTAINING AMYLOID VIEWED UNDER POLARISED LIGHT

16. FLUORESCENT STAINS: Fluorscent stain thioflavin -T binds to amyloid and fluoresce yellow under ultraviolet light i.e amyloid emits secondary fluorescence. IMMUNOHISTOCHEMISTRY: Anti –AP for confirmation of presence of amyloid of all types and anti-AA anti-Lambda, Anti-Kappa antibody stains for fibril protien of specific types by positive immunoreactivity .

17. NON-SPECIFIC STAINS: STANDARD TOLUIDINE BLUE: This method gives orthochromatic blue colour to amyloid which under polarizing microscopy produces blue dark birefringence. ALCIAN BLUE: It imparts blue-green colour to amyloid positive areas and is used for mucopolysaccaride content in amyloid. PERIODIC ACID SCHIFF(PAS): It is used for demonstration of carbohydrate content of amyloid.

18. DIAGNOSIS OF AMYLOIDOSIS BIOPSY EXAMINATION: Biopsy of an affected organ is likely to offer the best results Eg: kidney biopsy in case of dialysis Fine needle aspiration of abdominal subcutaneous fat followed by congo red staining and polarising microscopic examination for confirmation . It is very simple and useful technique with excellent result.

19. INVIVO CONGO RED TEST: A known quantity of Congo red dye may be injected intravenously in living patient. If amyloidosis is present the dye gets bound to amyloid deposits and its levels in blood rapidly decline. This test is not popular due to risk of anaplylaxis to the injected dye.

20. OTHER TEST: PROTIEN ELECTROPHOROSIS IMMUNOELECTROPHOROSIS BONE MARROW ASPIRATION