Liver Pathology • Why the Stain Is Done Which, Why, How……Really? • How the Stain Is Interpreted Pitfalls, Technical Aspects
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Current Issues in Surgical Pathology 2014 Outline • Which stains Special stains in liver pathology • Why the stain is done Which, why, how……Really? • How the stain is interpreted Pitfalls, technical aspects Sanjay Kakar, MD • Really University of California, San Francisco Reflex use of special stains Process Role Principle Special stains: liver pathology Iron hematoxylin Nuclear stain Works well in acidic solutions Red dye: Stains cytoplasm, Intermediate • Trichrome Acid fuchsin muscle molecular weight, • Iron (Biebrich scarlet) stain both collagen chromotrope 2R and muscle • PAS-diastase Polyacid Removes red dye Large molecules (phospho- from collagen • Reticulin tungstic acid) • Copper Blue/green dye: Stains collagen Large molecule Methyl green dye: stains only • Other: elastic, PAS, bile Fast Green collagen Aniline Blue Masson: sequential staining, Gomori: single step 1 Pale staining, no nuclear staining Trichrome stain • Why Staging: viral hepatitis, steatohepatitis Diagnosis of steatohepatitis Regression of cirrhosis Fibrosis vs. necrosis Recognizing unsuspected amyloidosis • How Interpretation and pitfalls Steatosis Steatohepatitis: essential features mild inflammation AASLD/NASH Clinical Research Network • Steatosis • Inflammation • Hepatocellular injury Ballooned hepatocytes Pericellular fibrosis 2 Steatosis Pericellular fibrosis Steatosis vs. steatohepatitis • Disease progression • Treatment Steatohepatitis guidelines 3 Overstained trichrome Pitfall in staging - histiocytic aggregate Chronic venous outflow obstruction Trichrome stain • Staging: viral hepatitis • Steatohepatitis • Regression of cirrhosis • Fibrosis vs. necrosis 4 Alcoholic cirrhosis with regression Cirrhosis regression • Thin fibrous septa with perforations • Prominent vessels and ductular reaction disappear • Nodularity may persist Wanless, Arch Pathol Lab Med, 2000 Friedman, Hepatology 2006 Chang, Hepatology, 2010 Thin septa: no shunting vessels or ductular reaction Regression: perforated fibrous septa 5 42 M with jaundice,hepatomegaly x 4 wks Live in the moment : Stephanie Page ALT, AST >1000 U/L Ultrasound: cirrhosis Repeat trichrome Dark: portal collagen, Light: necrosis 6 Orcein stain: no elastic fibers in necrotic area Amyloid: pale deposits Globular amyloid deposits: subtle on HE stain Globular amyloid: highlighted by trichrome → → → 7 Special stains: liver pathology Perls iron stain (not Perl’s) • Trichrome • K ferrocyanide + HCl • Iron • Ferric ferrocyanide (Prussian blue) • PAS-diastase • Max Perls: German pathologist • Reticulin • Copper • Other: elastic, PAS, bile Entombment of Christ: Peter van der Werff, 1709 Starry Night: van Gogh 8 Normal iron regulation Iron stain • Why Distinguish from other pigments Semiquantitative analysis • How Patterns of hepatic iron overload Grading of iron overload Hepcidin • Activity depends on iron stores • Binds ferroportin • Genetic/acquired Hepcidin Ferroportin Transferrin • Increased iron Dietary Hemolysis Fig: Textbook of Liver Pathology: Kakar, Fig: Textbook of Liver Pathology: Kakar, Ferrell, Eds. Chapter by M Torbenson Ferrell, Eds. Chapter by M Torbenson 9 Iron storage Primary Pattern of siderosis Mechanism HFE Hepatocellular HFE gene hemochromatosis Starts periportal mutation Storage form Distribution Non-HFE Mostly hepatocellular Non-HFE Ferritin hemochromatosis Some: macrophages mutations Iron oxyhydroxide and Virtually all cells apoferritin Trace amounts in the plasma Hemosiderin Secondary Pattern of siderosis Mechanism Aggregates of iron Reticuloendothelial system Hemolysis, multiple Macrophages Excess iron oxyhydroxide crystals including Kupffer cells transfusions from RBC without apoferritin Chronic diseases Macrophages Excess iron in macrophages Hemosiderin Ferritin blush Iron stain: interpretation • Grading of iron overload • Patterns of hepatic iron overload 10 Modified Scheuer grading scheme Deugner-Turlin grading scheme Grade Definition Grade 0 Granules absent or barely discernible at 400x Grade 1 Granules discernible at 250x Grade 2 Granules discernible at 100x Grade 3 Granules discernible at 25x Grade 4 Masses visible at 10x or naked eye Iron grading: simple method Iron: quantitative analysis Can be performed from paraffin embedded tissue Grade Extent of iron Allows correlation with H&E morphology Minimal <5% Normal iron 10-36 µmol/g of liver tissue Mild 5-33% Mild increase Up to 150 µmol/g of liver tissue Moderate 34-67% Moderate 151-300 µmol/g of liver tissue Marked 68-100% Marked >300 µmol/g of liver tissue • Separate grade: hepatocellular, Kupffer cell Hepatic iron index µg iron per gram dry weight of liver/55.846 • Hepatocellular: periportal vs. random patient's age >1.9: suggests hemochromatosis (non-cirrhotic) 11 Iron stain: interpretation History • Grading of iron overload • 35/M with obesity • Patterns of hepatic iron overload • Elevated serum ferritin • Liver biopsy: steatohepatitis Periportal hepatocellular siderosis Periportal hepatocellular siderosis 12 Iron overload in NASH Periportal siderosis • 20-50% serum ferritin elevated • HFE hemochromatosis • 15-60% increased hepatic iron • Non-HFE hemochromatosis • Secondary iron overload Distribution Interpretation Steatohepatitis Kupffer or hepatocellular Secondary mild/moderate, random • Rare conditions Hepatocellular, periportal HH or secondary Porphyria cutanea tarda Hereditary aceruloplasminemia Diagnosis History HFE 282Y homozygous • 55/M with cirrhosis • Steatohepatitis • No HFE mutation • HFE hemochromatosis with mild • No known etiology periportal hepatocellular siderosis, no portal based fibrosis Significance of iron overload or HFE mutations in progression of steatohepatitis is not clear 13 HII>2, heterogeneous iron overload Cirrhosis with siderosis • Non HFE hemochromatosis • Secondary siderosis in cirrhosis of another etiology Hemochromatosis Siderosis in cirrhosis Genetics Liver biopsy Clinical Ludwig, Gastroenterology, 1997 presentation rd th (n=447, HII>1.9) Type 1 Autosomal recessive Hepatocytes 3 or 4 decade (HFE HH) C282Y homozygous, Liver, pancreas, Hereditary hemochromatosis 100% C282Y /H63D heart, skin, joints st Alpha-1-antitrypsin deficiency 28% Type 2 Autosomal recessive Hepatocytes 1 three decades (Juvenile HH) Hemojuvelin (2A) or More severe Cryptogenic cirrhosis 19% hepcidin (2B) disease than HFE Alcoholic cirrhosis 14% HH Type 3 Autosomal recessive Hepatocytes Similar to HFE HH Chronic hepatitis B, hepatitis C 18%, 7% Transferrin receptor type Intermediate PBC, PSC 1% each 2 mutation between HFE HH and juvenile HH st th th Type 4 Autosomal dominant 1 subtype: 4 or 5 decade • Marked siderosis can occur in the absence of HH Ferroportin mutation hepatocytes Severity varies • Siderosis rare in biliary diseases nd 2 subtype: with type of • Siderosis is an adverse risk factor* Kupffer cells mutation *Brandhagen, Hepatology, 2000 14 HFE HH: Homogeneous distribution Siderosis: periseptal, stroma, endothelial cells Image: Dr. Linda Ferrell Bile duct siderosis Cirrhosis: HH or secondary siderosis Hereditary Cirrhosis with marked hemochromatosis secondary siderosis Homogeneous distribution Heterogeneous Siderosis in bile ducts, Generally absent stroma, endothelial cells HFE mutation (in HFE HH) Not present Diagnosis: Cryptogenic cirrhosis with secondary iron overload 15 Collapse with ductular reaction with siderosis: often nonspecific -High grade dysplastic lesions -50% develop HCC on follow-up Image: Dr. L Ferrell Iron stain: role of the pathologist Iron stain: role of the pathologist Clinical setting Interpretation Clinical setting Interpretation HFE C282Y homo Extent of iron HFE not known Raise possibility of HH C282Y/H63D Extent of fibrosis Periportal siderosis, or HFE other mutations Extent of iron moderate to marked No risk for HFE HH hepatocellular iron Chronic viral hepatitis Recommend HFE testing Steatohepatitis Possible disease progression Cirrhosis Possible poor prognosis 16 Special stains: liver pathology PAS-diastase stain • Trichrome Glycogen, other carbohydrates • Iron • Periodic acid converts –OH component • PAS-diastase to aldehyde • Combines with Schiff reagent: magenta • Reticulin complex • Copper • Diastase digests glycogen • Other: elastic, PAS, bile A1AT deficiency Mallory hyaline PAS-D stain • Why Alpha-1-antitrypsin deficiency Highlight macrophages Glycogen (with PAS stain) Giant mitochondria Highlights basement membrane • How Pitfalls Interpretation 17 A1AT deficiency Giant mitochondria A1AT deficiency Incomplete digestion Immunohistochemistry: alpha-1-antitrypsin 50/F with cirrhosis, obese, serum A1AT normal 18 PAS-D stain Cytoplasmic globules Sweet Spot: Robert Langford Contemplating Space: Sandra Wilson 19 Alpha-1-antitrypsin deficiency Alpha-1-antitrypsin deficiency • Normal allele PiMM Challenges in diagnosis (clinical) • Homozygous state (PiZZ) • Uncommon disease Chronic hepatitis and cirrhosis • Can occur in the absence of child hood • Heterozygous state (PiMZ) symptoms and lung disease Significance unclear • Serum levels unreliable Progression of fibrosis in other liver diseases Homozygous (PiZZ) Heterozygous (PiMZ) Alpha-1-antitrypsin deficiency Challenges in diagnosis (pathologic) • Cytoplasmic globules can be subtle • PAS-D: periportal location • Globules not specific for diagnosis Vascular etiologies Acute hepatitis • PiZZ vs. PiMZ cannot be distinguished on biopsy Gold standard for diagnosis: Protease inhibitor phenotyping 20 Mild portal inflammation Resolving hepatitis: PAS-D stain highlights macrophages Fig 7.3 Cytoplasmic inclusions History • 40/M with renal transplant