Pathologic Diagnosis in Amyloidosis JungSun Kim MD Ph

Pathologic Diagnosis in Amyloidosis Jung-Sun Kim, MD Ph. D Department of Pathology Samsung Medical Center Sungkyunkwan University School of Medicine

Amyloidosis • A heterogeneous group of diseases of diverse origin • Caused by extracellular deposition of misfolded, insoluble aggregated proteins with a characteristic -pleated sheet configuration

Structure of Amyloid • Crystallography: Cross- -pleated sheet conformation • EM: Continuous nonbranching fibrils with a diameter of 7. 5 to 10 nm

Amyloid Fibril Formation Dember, Kidney Int 2005; 68: 1377– 90

Amyloid Proteins and their Precursors in Human Westermark et al. , Amyloid 2007; 14: 179 -83

Amyloid Proteins and their Precursors in Human Westermark et al. , Amyloid 2007; 14: 179 -83

Amyloid Proteins and their Precursors in Human Westermark et al. , Amyloid 2007; 14: 179 -83

Classification of Amyloidosis Clinicopathologic Category Associated Diseases Major Fibril Protein Chemically Related Precursor Protein AL Immunoglobulin light chains, chiefly λ type AA SAA SYSTEMIC (GENERALIZED) AMYLOIDOSIS Multiple myeloma and other Immunocyte dyscrasias with amyloidosis monoclonal plasma cell (primary amyloidosis) Proliferations Reactive systemic amyloidosis Chronic inflammatory (secondary amyloidosis) Conditions Hemodialysis-associated amyloidosis Chronic renal failure Aβ 2 m β 2 -microglobulin HEREDITARY AMYLOIDOSIS Familial Mediterranean fever AA SAA Familial amyloidotic neuropathies (several types) 　 ATTR Transthyretin SENILE SYSTEMIC AMYLOIDOSIS 　 ATTR Transthyretin LOCALIZED AMYLOIDOSIS Senile cerebral Alzheimer disease A APP Endocrine Medullary carcinoma of thyroid Islets of Langerhans Isolated atrial amyloidosis Type 2 diabetes 　 A Calcitonin AIAPP Islet amyloid peptide AANF Atrial natriuretic factor

Detection of Amyloid • Congo red stain: the gold standard – Salmon-pink color in bright field – Apple-green birefringent deposits under polarized light – Good fixation, strong light source in a darkened room, thicker sections, multiple sections

Detection of Amyloid • Electron microscopic demonstration of the fibrillar nature of deposits

Detection of Amyloid • Thioflavin S & T • Methyl violet • Sulphonated Alcian blue – less sensitive – less specific

Amyloid Typing • Need to have stringent criteria • Indistinguishable on LM or EM • Most direct (but not applicable in reality) – Mass spectrometry – Amino acid sequencing • In clinical setting – immunostaining

Amyloid Typing • Immunohistochemistry: – Currently the standard – Kappa, Lambda, Amyloid A, Transthyretin for primary screening Kappa Lambda Amyloid A

Amyloid Typing • Immunohistochemistry: – Limitation • Nonreactive with a truncated light chain • Limited antibody panel missing a number of hereditary amyloidoses • Background staining : “locking-in” of serum proteins during fixation

Amyloid Typing • Immunofluorescence – Cleaner background – Higher Sensitivity – Limitation: requirement of frozen tissue Collins et al. , Cardiovasc Pathol 2009; 18: 205 -16

Amyloid Typing • Combined Congo red and IHC • Immuno-electron microscopy • Western blotting

The Choice of Tissue Specimen: Fat as a Source of Tissue for Amyloid Detection • Biopsy of a clinically affected organ • Subcutaneous fat pad biopsy: less invasive screening test • To judge systemic versus local amyloidosis

Biopsy-Proven Amyloidosis in SMC (2000. 1 – present; by Congo red or EM) • • • Kidney: 37 Heart: 35 Gastrointestinal tract: 34 Skin and soft tissue: 13 Respiratory tract: 12 Bone (marrow): 11 Liver, pancreatobiliary tract: 7 Thyroid: 5 (medullary carcinoma) Thymus, Urinary bladder, Seminal Vesicles, Peripheral Nerve, Eye, Lacrimal gland

Amyloid Typing in Kidney Biopsy by Immunohistochemistry • 23 Kidney biopsies in which IHC/IF stainings were done – 6 AA type (Amyloid A (+); kappa/lambda (+/-) – 17 AL type (Amyloid A (-): kappa/lambda (+)) • 1 kappa • 6 lambda • 10 not determined EM: non-branching fibrils

Amyloid Typing in Heart Biopsy by Immunohistochemistry Patient Age Sex kappa lambda amyloid A Type 1 51 M P N N AL kappa 2 72 M P N N AL kappa 3 50 F P WP N AL kappa 4 58 F N P N AL lambda 5 74 M WP P N AL lambda 6 64 M WP P ND AL lambda 7 60 M WP P N AL lambda 8 57 M WP P N AL lambda 9 71 M WP WP N AL 10 58 M WP WP N AL N, negative; ND, not done; P, positive; WP, weak positive

Summary • Detection of Amyloid – Congo Red staining – EM • Diagnosis of the Amyloid type – Immunohistochemistry – Immunofluorescent staining

HE Congo Red Kappa Lambda Amyloid A