A minimum of 3, but preferably 5, laparoscopic or surgical biopsy specimens from 2-3 liver lobes should be obtained for histopathology and placed in formalin (see below). The diagnostic accuracy of liver biopsies increases with the size of the biopsy specimens and the number of lobes sampled.

Wedge biopsies are preferred over punch biopsies. Peripheral wedge biopsies should be 1-2cm deep to account for changes caused by capsular fibrosis at the edge. If punch biopsy samples are taken, they should be obtained from the center rather than the edge of the lobe, as the periphery of the lobe may be affected by fibrosis.

In addition, if indicated, obtain 1 fresh sample for aerobic and anaerobic culture (can be very small) and 1 fresh sample for quantitative copper analysis.

For histopathology samples, if there are gross differences in the areas sampled, submit each in a different container labeled with the location; otherwise, submit all liver specimens together in one container.

If laparoscopic or surgically obtained biopsies are not possible, needle biopsies, Tru-Cut or Menghini, can be performed:

• Obtain at least 5 samples for histopathology, 1 for bacterial culture if indicated (can be very small or just a portion of a needle biopsy), and at least 2 for mineral analysis (see below).
• Medium- and large-sized dogs: use a 14G needle
• Smaller dogs and sometimes cats: use a 16G needle
• Needle biopsy specimens should be at least 1cm long, but ideally 2cm, to ensure adequate sampling depth.
• If the samples obtained are not ideal, submit anyway.
• If there is a focal lesion, sample from the periphery AND the center.
• Be careful not to crush samples with forceps.
• Do not set specimens on paper, cloth, or tongue depressors.
• Needle biopsies should be placed in mesh cassettes to prevent fragmentation; non-compressive sponges can be used in the cassettes if they do not compress or perforate the biopsy specimens.

If mineral analysis is desired (e.g. concern for copper associated hepatopathy):

• Sample the most normal-appearing areas to avoid regions of fibrosis and nodular regeneration.
• Submit liver biopsy(ies) in an empty metal-free and rubber-free glass or plastic tube (rubber can cause zinc contamination); microcentrifuge tubes work well.
• A minimum mass of 50mg is recommended for mineral analysis.
  • Approximate wet tissue masses collected from selected surgical biopsy instruments are:
    • Stryker Double Action Biopsy Clamp (5.0 mm diameter) – 100 to 200 mg
    • Acu Punch (6 mm) – 90 to 180 mg (dependent on depth of punch)
    • TruCut (14 G) – 20 to 25 mg (This is the amount collected if the notch in the instrument is full. Thus, two samples are required for optimal sample size, if the notch is full; more if it is only partially full.)
    • Note: when ultrasound-guided needle biopsies are submitted directly for mineral analysis, they may contain other tissues such as skeletal muscle, which can result in a falsely low copper value. Fibrotic or regenerative areas can also result in low copper values.
• Refrigerate or freeze sample until shipment. Ship with a cold pack.
• Mineral analysis can be performed on fresh, formalin-fixed, or formalin-fixed paraffin-embedded samples.
• Mineral analysis should always be performed in conjunction with assessment/grading of copper staining on histopathology samples.
• The specimen should be kept moist but not saturated.
• Bile can also be collected in a red top tube for culture.
• If the gallbladder is removed, a fresh portion of the wall placed in a red top tube with 0.5ml of sterile saline is the best sample for culture. Culture swabs may be submitted as well.
• Refrigerate the sample until shipment. Ship the specimen that day with a cold pack for overnight delivery.
• Clearly label all samples and submit together with a submission form that includes a thorough, but relevant, history including clinicopathologic and gross findings and where the samples were taken.

If bacterial infection is of concern, submit one liver sample in a red top tube with 0.5ml of sterile saline for culture.

If a liver lobectomy is performed to remove a mass, submit the entire lobe.

• Inking the surgical margin is very helpful before placing the lobe in formalin.
• If the lobe/mass is large, it may be helpful to take a 0.5-1.0 cm wedge section from the center of the mass and another from the periphery of the mass that includes normal adjacent liver (if present) and place those wedge sections in a separate biopsy container to ensure adequate fixation of the mass and better diagnosis. Be careful not to cut through any of the margins so that accurate margin assessment can still be performed.
• Then, to help the fixative penetrate the remaining large specimen, slices can be made every 1-2 cm throughout the lobe, as long as they are NOT made through a surgical margin and the lobe is kept intact.
• Place the large liver lobe/mass specimen in a separate leakproof container and fill with formalin. A 10:1 formalin to tissue ratio is ideal but, realistically, may not always be possible for large specimens. Large screw top plastic containers, such as those from medications, are good options.
• Place all formalin containers in one or more Ziploc bags and package with absorbent material during shipping.