Introduction

Taking a biopsy sample of diseased tissue for histopathological assessment is one of the most commonly performed diagnostic procedures in small animal practice. Considerable effort and expense are often expended in obtaining such biopsy specimens, particularly if the procedure involves incision or excision of a lesion under general anaesthesia. For the benefit of the animal and the client, it therefore makes sense to do everything possible to maximize return from the submission of biopsy specimens. This short presentation reviews the key stages in sample submission from the perspective of the diagnostic histopathologist.

What Sample?

The clinician must make the fundamental decision as to the optimum sample to collect from a lesion arising within the individual patient—and this may differ between patients for a variety of reasons (e.g., anaesthetic risk, client budget, desired speed of result). In general, pathologists prefer the largest sample possible—so an excisional sample is preferable to an incisional biopsy, Tru-Cut® core biopsy or fine needle aspiration (FNA). FNA of a skin mass is a sensible rapid preoperative procedure when performed in-house, but where cytological samples are sent to a diagnostic laboratory from a mass that is likely to be excised surgically no matter what the outcome, it may make sense to simply undertake the surgical procedure and submit the excised specimen.

Fixing the Sample

Tissue samples for histopathological assessment should be fixed in 10% neutral buffered formalin. A tenfold excess of formalin to sample volume is required for adequate fixation. Formalin penetrates into tissue at the rate of 5 mm per 24 hours. Where possible, an entire specimen should be submitted (e.g., if splenectomy is undertaken the entire spleen rather than a selected portion), but where size precludes sending the entire sample (e.g., expense of postal costs or safety of postal submission) the most representative area of the lesion to include a junction with normal tissue, should be sent. Dermatologists often deliberately submit a biopsy of normal skin in parallel with lesional tissue. Sending an entire sample will allow the assessment of multiple margins. If possible, you should avoid incising samples or dividing them into multiple portions. It is better that the pathologist does this and allows further fixation time in the laboratory; however, bisecting a relatively large specimen will enable more effective fixation.

Samples should always be submitted in purpose-designed, wide-mouthed biopsy pots. A fresh sample may readily be squeezed through the neck of a pill bottle, but once fixed, often the only way to remove the sample is by smashing the container. Portions of tissue that are likely to fold or curl during fixation (e.g., samples of intestine) may be pinned to board so that they fix in an optimum position. Very small endoscopic biopsy samples may benefit from being fixed by being layered within purpose-designed ‘sponges’ in histocassettes or in purpose-designed mesh bags. This will avoid unnecessary trauma to the delicate samples by being shaken within the formalin pot.

Where margins are to be assessed (i.e., for tumours) it is important that you are able to indicate to the pathologist the orientation of the excisional sample within the animal so that specific margins can be identified. The use of photographs or sutures placed on the specimen can achieve this; alternatively, the use of purpose-designed inks can mark particular margins. These surgical inks are resistant to histological processing and should appear on the final stained section.

Postal samples should be packaged according to local regulations—generally surrounded by layers of absorbent material, within a sealed plastic bag and an appropriate outer container. Fixed tissues are generally regarded as being ‘category B’ biological samples and the outer envelope should bear the designation UN3373.

Sending the Clinical History

One of the greatest frustrations to the diagnostic pathologist is receiving no or minimal clinical history or sometimes not even the signalment data of the patient. Most diagnostic pathology laboratories provide submission forms that detail the required information. It goes without saying that you might provide the most detailed clinical history, but unless the pathologist can read the handwriting this effort is wasted! Quite simply, the more information you provide, the more helpful the pathologist can be. It is wonderful when submission forms are accompanied by digital photographs of the patient—showing the lesions in situ. Dermatologists seem particularly adept at using this technology. The clinician should also be aware that some pathologists and some laboratories have particular expertise in dealing with specific sample types (e.g., endoscopic biopsies of gastrointestinal mucosa, skin or bone marrow biopsies), and it may be that on occasion a specialist laboratory might be used for a particular case.

Be Patient

Most diagnostic histopathology laboratories offer a 24-hour turn-around for routine biopsy submissions, but the clinician should appreciate that non-standard samples require more complex processing and it will therefore take longer to generate a result. A large (e.g., orange-sized) or very soft tissue sample may require additional fixation in the laboratory before processing or a ‘long process’ over several days rather than overnight. Tissues including areas of bony metaplasia or samples including bone (e.g., digit removal, bone tumours) will require prolonged decalcification before the tissue is soft enough to process and section. Decalcification of bone may take many weeks.

Understand the Language of the Pathologist

Once the pathology report is sent to you, do take the time to read and understand the complete report. Many clinicians will read only the ‘bottom line’ (i.e., the diagnosis) or the ‘comments’ section of a report and skip the actual descriptive report. Much information is conveyed in that description, particularly assessment of marginal infiltration or metastatic activity. Pathologists do use a very specific language to convey degrees of certainty (and this has been studied and published) and you should understand the way that your pathologist uses terms such as ‘consistent with,’ ‘not inconsistent with,’ ‘probable,’ ‘likely’ and others.

Communicate with Your Pathologist

This is the most important of all. If anything is unclear or the pathological report does not match your clinical expectations then do not be afraid to telephone your pathologist to discuss the case. This exercise is of mutual benefit. The pathologist should be able to advise on whether further sectioning of the gross specimen is justified, or whether special histochemical, immunohistochemical or molecular studies might be performed. For some infectious agents it is now possible to take samples from the wax-embedded tissue for PCR diagnosis and molecular tests for cancer characterization are now routinely available. Many pathology laboratories can now readily provide digital images of the histopathology to help your understanding. Finally, no pathologist will be offended if you ask for a second opinion on a slide and should be able to advise on an appropriate colleague to provide that opinion.

References

1.  Cade S. Sending diagnostic samples: your questions answered. In Practice. 2010;32:312–314.

2.  Stidworthy M, Priestnall S. Getting the best results from veterinary histopathology. In Practice. 2011;33:252–260.