Corresponding to other laboratory tests, the quality of histopathology reporting is directly related to the quality of the sample received. This document will explain common pitfalls in sampling and shipment of histopathology samples to help you avoid introducing pre-analytical artefacts.

Sampling

• In particular when taking small biopsies, even with careful sampling, crush artefact cannot always be prevented. In addition, small biopsies may not always be fully representative of the underlying lesion. To mitigate this, we recommend taking multiple samples to increase the chance of obtaining representative tissue sections. For example, one study on gastrointestinal biopsy sampling reported a minimum of 6 mucosal biopsy specimen of adequate quality from the duodenum of cats are required for a reliable histopathological evaluation. For optimal reporting, 10-15 gastrointestinal biopsies per site (excluding ileum) are recommended¹.
• If you are using electrocautery, be aware that this causes coagulation artefact at the borders of the samples and may therefore impact histologic margin assessment (if required).
• Transportation can cause fragmentation, especially with friable samples. To protect such samples during transit, it is advisable to use histology cassettes, which can be ordered via VPG via your local laboratory.
• Avoid using foam inlays for fresh tissue, as they can cause crush artifact.

Histology cassettes

 

Inking

• If histologic margin assessment is required, inking by the surgeon is very helpful for tissue orientation and to ensure that the tissue border on a histologic slide represents the surgeon-cut edge created at surgery (any samples that require histologic margin assessment that do not arrive in the lab pre-inked will be inked in the lab)
• For best inking results, blot the tissue dry, apply surgical ink to the desired area, with a brush or cotton swab, wait for five minutes for the ink to dry, and then immerse the tissue in formalin.

‘Formalin pigment’ (acid hematin)

 

Fixation

• To achieve optimal fixation, use 10% neutral-buffered formalin (NBF). The buffering agent reduces the formation of “formalin pigment,” which can interfere with accurate analysis. The histology containers that you can order via your local laboratory are already prefilled with 10% NBF.
• Never freeze the sample before fixation or send it in alcohol or saline. These methods are not suitable and will cause significant tissue artefact.
• Ideally, maintain a 10:1 formalin-to-sample volume ratio during fixation. However, if this is not feasible, ensure that the sample is at least covered with formalin and send it to the lab as soon as possible.
• We recommend a fixation time of 24 to 48 hours. Prolonged fixation in particular of large samples and samples rich in adipose tissue (mammary strips) or blood (spleens) without cutting the sample further will not improve fixation in the centre.
• For larger samples, carefully consider cutting them to enhance formalin penetration. However, exercise caution to avoid destroying the lesion or the margins that need assessment. For example, a large elliptical skin excision with a mass can be incised from the skin side into the mass without cutting through the mass or the deep and horizontal tissue borders

Incision into mass without cutting through tissue borders to enhance formalin penetration

 

• If a container of adequate size is not available, samples can be halved. For large and elongate samples, such as mammary strips, cutting the sample into multiple sections is acceptable. Cutting a single mass into more than 2 sections should be a last resort, as it can make accurate margin assessment challenging. If you have surgical ink, apply the ink to the sample prior to sectioning to preserve the ability to perform accurate margin assessment. Any post-surgical alteration of a sample should always be noted down in detail on the submission form and containers must be labelled accordingly.

Sectioning a mammary strip

 

Shipment

• Make sure no needles or other sharp materials are left in the sample/ histology container, as this is a significant health and safety risk for our staff!
• Always use containers of appropriate size to prevent impairing fixation and deforming the sample. Squeezing samples into containers that are too small can adversely affect margin assessment.
• Please do not use unsuitable containers (sharps containers, Tupper ware) or containers that put our staff at risk (e.g. glass containers).
• Ensure that the container is leak-proof, shatter-proof, and include absorbent material in the shipment bag if possible.
• Use individual containers for each sample or site to allow for correct sample/ site identification throughout the process; label the containers and make notes on the submission form accordingly.
• Keep cytology samples separate from histology samples, as formalin fumes can cause severe artefact in cytology sample.

 

Example of well-packaged histology sample

Shipment of large samples

For large samples that do not fit in any suitable containers and cannot be cut into smaller sections (such as whole legs) consider:

• Is margin assessment required for this sample? For example in a leg amputation where the lesion is located at or below the carpus/ tarsus, margin assessment is likely not relevant, as ‘clear’ margins are expected. In those cases, the portion of the sample with the lesion can be cut out and fixed and submitted as usual.
• Note that margin tissue could also be submitted separately and clearly labelled as such.
• Can parts of the tissue that are not relevant for the histologic assessment be discarded? For example in leg amputations with the lesion high on the leg, the lower extremity can be cut off and the remaining sample fixed and submitted as usual.
  • Keep any tissue that has been cut off in the practice in formalin until the final report has been issued.

If the whole sample is required for histologic assessment:

• Fix the whole sample in a container of sufficient size with an adequate amount of formalin for 24-48h. If the day of shipment would fall on a Friday, then keep the sample in formalin over the weekend and ship the sample on Monday. Consider if incisions can be made that facilitate better formalin penetration without impairing any necessary margin assessment (see above).
• Once fixed, wrap the sample in formalin-soaked gauze/ tissue papers, then double-bag in plastic bags of adequate size; consider adding absorbent material (tissue papers/ cotton wool) inside the second bag to prevent leakage.
• Sending samples fresh without formalin-fixing them first should be an absolute last resort! Please contact VPG Histology prior to sending a fresh sample.

Sampling and shipment of spleens

• Spleens have a thick capsule and are often markedly congested, both of which hampers proper formalin fixation. To enhance fixation, either several incisions through the capsule above a mass and in ‘normal’ areas of the spleen, at approximately 2cm intervals.

Entire spleen submitted to VPG Histology. Incisions can be made at approximately 2cm intervals (green lines) through normal spleen and masses to improve fixation.

 

• If the whole spleen cannot be sent/ you wish to send incisional biopsies, please consider the following
  • It is critical that representative sections are taken from the transition zone between the mass and normal spleen. Splenic masses commonly rupture: sampling of the haematoma or necrosis often does not reveal the underlying cause of rupture.
  • Neoplasia (including haemangiosarcoma) may not be diagnosed if only the subcapsular haematoma is sampled or if fewer than 5 sections are taken.
    • In fact, in one study², the likelihood of missing a haemangiosarcoma is 32% and 17% if only 1 or 2 sections, respectively, are examined.
    • If 5 sections are examined the likelihood of missing haemangiosarcoma decreases to 5%.
    • If 10 sections are examined, the likelihood of missing haemangiosarcoma decreases to 1%.
  • Consequently, we recommend taking 5 representative sections from the transition area between the mass and normal spleen and 1 additional representative section from the normal spleen.
    • Please take care to sample the transition area between a mass and ‘normal’ spleen, otherwise it is possible that only reactive lesions (e.g. the central haematoma in a haemangiosarcoma) are represented in the samples.

Obtaining representative sections from a spleen. Take a minimum of 5 sections between the transition zone of the mass and normal spleen (green boxes). Also take at least one section of normal spleen (orange box).

 

Sampling of the hepatobiliary tree

Biopsy of the hepatobiliary tree is often required for investigation of hepatobiliary disease. Techniques for biopsy of the hepatobiliary tree include needle biopsy (Tru-cut biopsy), laparoscopic biopsy (cup forceps), surgical biopsy (e.g. wedge or guillotine biopsy), and liver lobectomy. Gall bladder sampling most often involves cholecystectomy, but incisional biopsies may also be taken.

Current guidelines for investigation of hepatobiliary disease recommends a minimum of 12-15 portal tracts be available for histologic examination. Artefacts introduced during sampling, such as crush, shearing, or puncture of the liver by sponges in cassettes, can reduce the number of intact lobules and inhibit histologic interpretation.

To maximise the pathologist’s ability to achieve a diagnosis and to have adequate tissue for additional tests, multiple needle biopsies (>4, ideally from a 14 gauge needle) or laparoscopic, cup forceps biopsies (2-4) are required.

• Multiple lobes should be sampled and labelled according to the lobe from which they were obtained (if it can be identified). If specific lesions are sampled (for example masses), these should be placed in a separate pot and labelled.
• Biopsies for histology should be placed in a labelled pot containing 10% neutral buffered formalin at a ratio of 10:1 (formalin : tissue).
  • Put needle biopsies in a mesh cassette, taking care not to crush the samples. Avoid using sponges, which can puncture the tissue and introduce artefact.

Example of a mesh cassette

Large biopsies, including partial or complete lobectomies, may not fix fully. To enhance fixation, incise the lesional areas such as masses.

• If margin assessment is required, avoid incising the margin. If you have histologic marking ink at your clinic, consider applying the ink to the margin prior to placing the sample in formalin and/or incising it.
  • Note: Staples must be cut out of the tissue before it can be processed. Consequently, if surgical margins are stapled, this may interfere with histologic margin assessment.

Liver lobe with 2 masses (yellow stars). To improve fixation, incise the lobe at approximately 2cm intervals. Avoid the surgical margins.

Sampling of patients with suspected canine chronic hepatitis

Laparoscopic biopsies are preferred for sampling patients with suspected chronic hepatitis. However, needle biopsies may also provide sufficient tissue for diagnosis, if enough biopsies are obtained. The current ACVIM consensus guidelines for investigation of canine chronic hepatitis recommend obtaining  biopsies for histology, culture, and copper quantification³.

• Histology: 3 laparoscopic biopsies or 4+ full length, 14 gauge (2cm long) needle biopsies, placed in labelled pot(s) of 10% neutral buffered formalin
  • Copper staining of formalin-fixed liver biopsies is recommended for patients with suspected chronic hepatitis, to allow for assessment of copper accumulation in relation to the pathologic changes. Copper staining may be performed as part of the initial histologic assessment or can be requested after the histology has been performed.
• Copper quantification: 1 laparoscopic biopsy or 1 full length, 14 gauge (2cm long) needle biopsy in a sterile, liquid free, labelled pot
  • Note that copper quantification requires approximately 20-40mg of liver (wet weight). Insufficient amounts of tissue may yield inaccurate results. This equates to approximately 1 full length 14 gauge (2cm long) needle biopsy or ½ of a 5mm laparoscopic biopsy specimen. A full length 18 gauge needle biopsy provides only 3-5mg of tissue.
• Culture: 1 laparoscopic biopsy in a sterile, liquid-free, labelled pot
  • In some cases (for example, sampling of liver abscesses), a swab may be indicated. These should be placed in the appropriate transport medium, labelled, refrigerated until transport, and transported to the laboratory as quickly as possible.

Sampling of patients with suspected biliary disease

In patients with suspected biliary disease (including cats with cholangiohepatitis), evaluation of bile, bile “sludge”, cholecystoliths/choledocholiths, and/or the gall bladder may be indicated in addition to liver histology

• Culture can be performed on fluid, bile sludge and/or stones, and gall bladder and liver (swabs or tissue).
  • These should be placed in the appropriate transport medium or a sterile container. Culture samples should be refrigerated until they are sent via courier to the appropriate laboratory, as quickly as possible.
• Cytology specimens of the liver and/or bile should be placed in a separate parcel from formalin samples because formalin fumes can alter cellular morphology, limiting cytologic interpretation.
• The gall bladder has a thick wall, which can slow the rate of formalin penetration and inhibit fixation. To expedite fixation, it can be incised. If margin assessment is required, avoid cutting into the relevant margin.

 

Sampling of patients with suspected lymphoma

Formalin fixation can inhibit PCR including PARR (PCR for antigen receptor rearrangement), which may be recommended by the pathologist to investigate for a clonal lymphocyte population and the diagnosis of lymphoma.

• If lymphoma is suspected clinically (particularly in cats with suspected lymphocytic cholangitis/cholangiohepatitis or small cell lymphoma), consider placing one biopsy sample in saline for PARR, if needed.

 

Submission form and relevant patient history

• You can book in any collected samples onto Pathport at: https://pathport.thevpg.co.uk
  • Alternatively, downloadable versions of our submission forms are available on our website at: https://thevpg.co.uk/downloads/
• Along with the submission form containing the animal details, for optimal reporting, it is essential to provide the relevant patient history. Include a description of the lesions, their distribution, timeframe of occurrence/ progression, international travel history, and any previous or ongoing treatment, particularly antibiotic and steroid usage.
• Include any relevant photos, imaging results, blood results, and cytology results from other labs, as they can aid in accurate interpretation. Photos can be uploaded directly into Pathport, emailed to [email protected], or printed and affixed to the submission form.
• Specify your clinical differential diagnoses and the specific questions you want the histopathology report to address.

 

Interpreting the Pathology Report

We will always strive to give you a definitive diagnosis. However, in some cases, for various reasons this may not be possible. In those cases we will provide an opinion on the most likely diagnosis and possible differential diagnosis and discuss further diagnostic tests, if applicable. In particular in cases with some uncertainty regarding the diagnosis, it is important to compare to the clinical picture, list of clinical differential diagnoses and results of other tests (e.g. imaging) that you may have done. We are always happy to discuss any you may have regarding the histopathology report.

For more information on our histopathology services, including comprehensive guides on sampling spleens and the hepatobiliary tree, please visit our website or contact us by calling 0117 951 1283 or emailing [email protected].

 

References:

1. Willard MD, Mansell J, Fosgate GT, et al. Effect of sample quality on the sensitivity of endoscopic biopsy for detecting gastric and duodenal lesions in dogs and cats. J Vet Intern Med. 2008;22:1084-1089.)
2. Herman EJ (2019). Understanding the efficiency of splenic hemangiosarcoma diagnosis using Monte Carlo simulations. Vet Pathol 56(6): 856-859 doi:10.1177/0300985819868732
3. Webster CRL, Center SA, Cullen JM, et al. ACVIM consensus statement on the diagnosis and treatment of chronic hepatitis in dogs. J Vet Intern Med. 2019; 33: 1173–1200. https://doi.org/10.1111/jvim.15467