1. How many portal tracts are present in the biopsy?

   • Typically, a minimum of 4-6 portal tracts​ are required to be considered "adequate"

   • For transplant livers, we ideally want to see a minimum of 10 portal tracts

2. Is the architecture of portal tracts in relation to central veins normal or abnormal?

3. Evaluate each of the structures in every single portal tract: (portal vein, hepatic artery, bile duct)

   • Does the vein represent  approximately 75% of the portal tract? (this is a normal finding)

   • Is every artery paired with a bile duct that is approximately the same size?

     • Are bile ducts missing or damaged in any of the portal tracts?  (see inflammation section below)?​​​

     • Is there a bile ductular reaction (increased # of small (proliferating) bile ducts at the periphery of the portal tract)?

4. Is there any inflammation within the portal tract? (if YES, answer the questions below)

   • How many of the portal tracts are affected? (Rare, Few, Most, All)

   • Does the inflammation affect the entire portal tract, or only specific structures? Are any of these structures atypical/destroyed by the inflammation?

     • Portal vein (endothelialitis)​

     • Hepatic artery (arteritis)

     • Bile ducts (ductopenia, epithelial atypia, ductular reaction) 

   • Do the inflammatory cells completely expand the portal tract?

   • Does any inflammation "flow out" of the portal tract and into the adjacent hepatocytes? 

     • NOTE: This is also referred to as "piecemeal necrosis" or "interface hepatitis"​

   • Which inflammatory cell type(s) are present?​

     • ​Lymphocytes​

     • Plasma cells

     • Neutrophils

     • Eosinophils

     • Histiocytes/macrophages

   • Are there any granulomas identified?

     • Do they appear to affect a particular structure?​

5. Assess the liver lobules (hepatocytes)

   • Is there any necroinflammatory activity?​ How severe?

     • Piecemeal necrosis? (Interface hepatitis)​

     • Coagulative necrosis? (around the central veins/ ischemic type)

     • Apoptosis? (Hepatitis C)

   • Is there cholestasis?

   • Is there steatosis? (aka fatty, bubbly, vacuolated) 

     • Microvesicular? (small vacuoles in cytoplasm, nucleus is in center) 

     • vs. Macrovesicular? (large cytoplasmic vacuole that pushes the nucleus to the side- looks like a signet ring)

     • What percentage of the biopsy is affected?

       • Typically this is assessing MACROvesicular ​

     • Is there steatohepatitis? (steatosis plus any combination of the following...)

       •  inflammation (often PMNs)

       • ballooned/large hepatocytes

       • Mallory bodies/Mallory hyaline (intermediate filaments within cytoplasm​ of injured hepatocytes & chemotactic for PMNs)

       • +/- fibrosis

       • The differential diagnosis = alcoholic steatohepatitis (ASH), non-alcoholic steatohepatitis (NASH)- obesity, diabetes, drugs

         • ASH vs. NASH cannot be DDx on histology alone! ​

6. Are special stains required? What do they show? (if performed)

   • Masson's Trichrome:

     • Assess the degree of fibrosis and overall architecture​ 

     • BLUE/GREEN = Collagen

     • Normally, portal tracts have a small amount of collagen and central veins have a thin, fibrous rim

     • ASH/NASH: Zone 3 peri-cellular/sinusoidal fibrosis ("Spidery fibrosis")

   • Iron:

     • Increased in hemochromatosis, chronic blood transfusion, iron overloaded state, and others

     • Useful to DDx iron/hemosiderin (POS) from lipofuscin or bile pigment which stain similarly on routine H&E, but neg on iron

   • PAS-D:

     • The diastase (D) will digest glycogen in hepatocytes to help highlight any Alpha 1 antitrypsin globules

     • Also stains ceroid-laden Kupffer cells which may suggest recent lobular injury when numerous

   • Orcein

     • Helps visualize elastic fibers

       • Elastic fibers within fibrous septa indicate chronicity (rather than collapse)​

     • Stains Copper binding protein (Wilson's dz)

       • Copper binding protein within zone 1 hepatocytes may suggest cholestasis​

     • Stains Hepatitis B surface antigen

   • Reticulin

     • Assess the architecture and thickness of hepatocyte plates/"cords" 

       • Normally, each "plate" is only 1 hepatocyte/cell layer thick between the black staining reticulin fibers

       • Diffuse "twin-cell" plates = Regenerative hyperplasia

     • ​

     • Helps to assess the thickness of each hepatocyte "cord"

     • Nodular distortion is often more obvious with a reticulin stain

     • This stain is especially helpful in patients with portal hypertension but a non-cirrhotic liver on histology or trichrome stains

​​

NOTE: At many institutions, Trichrome (Masson's), PAS-D and Iron stains are routinely performed on all "medical liver biopsies" or when there is hepatitis (especially when this is a first time liver biopsy on a patient). We typically perform additional stains on a case-by-case basis for transplant liver biopsies that aim to assess for transplant rejection depending on the histologic findings, how long it has been since their transplant, and previous biopsy results. Often, we do not perform these routine stains on liver biopsies performed to evaluate for possible metastatic disease, but instead workup any tumor to determine whether it is metastatic vs. primary hepatobiliary tumor. This is by no means "set in stone" and each case should be assessed individually to ensure that the proper diagnosis is reached.

​​