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Histopathology: What you need to know in practice

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Annika Herrmann

Board Certified Anatomic Pathologist

Histopathology processing is a crucial and intricate procedure that involves multiple steps. Throughout each step, there is a risk of introducing artefacts that can affect the accuracy of the results. In this blog, we will provide you with essential tips to help you mitigate these risks and ensure reliable histopathological analysis.

Sampling

When dealing with small tissue samples, crush artefacts can occur, even with careful sampling. To minimize this issue, we recommend taking multiple samples. This approach increases the chances 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 with an optimum of 10-15 biopsies. (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.)

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 from VPG via your local laboratory.

Avoid using foam inlays for fresh tissue, as they can lead to crush artifacts.

Crush artefact
Histology cassettes

For complex samples like legs or jaws, it can be beneficial to the lab if the surgeon marks the sample for orientation and highlights regions of particular interest (for example the area of the lesion, if not obvious, or an area of specific concern regarding adequate margins). Blot the tissue dry, apply surgical ink to the desired area, wait for five minutes, and then immerse the tissue in formalin.

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 VPG 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 artefact.

Ideally, maintain a 10:1 formalin-to-sample volume ratio during fixation. However, if that is not feasible, ensure that the sample is at least covered with formalin.

We recommend a fixation time of 24 to 48 hours. Prolonged fixation 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.

Shipment of Sample

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.

Ensure that the container is leak-proof and shatter-proof. If possible, include absorbent material in the packing materials surrounding the container.

Use individual containers for each sample or site to allow for correct sample/ site identification throughout the process.

Keep cytology samples separate from histology samples, as formalin fumes can cause severe artefact in cytology samples.

For large samples, fix them in formalin for 24 hours in a container of sufficient size. Blot dry and place them in a plastic bag, then insert the bag into a shipment bag. It is advisable to double-bag for added security.

Alternatively, if the sample is too large to fit into one container, cut it into pieces that fit into two or more containers. Make detailed notes or draw a diagram to ensure accurate assessment, especially when margins need to be evaluated. If margins are not important, sending representative samples is a good alternative.

Spleens often have a thick capsule that hampers proper formalin penetration. To enhance fixation, either make incisions through the capsule if fixing for 24 hours in practice or sample five regions from the periphery of a discrete mass (along with a section of “normal” spleen). This strategy significantly improves the detection probability of haemangiosarcoma, if present, to 95%. 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.

‘Formalin pigment’ (acid hematin)
An example of a well packaged sample

Submission Form and Relevant Patient History

Along with the submission form containing the animal details, it is essential to provide the relevant patient history. Include a description of the lesions, their distribution, timeframe, 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.

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, 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 discrepancies.

By following these guidelines and ensuring proper handling, fixation, and shipment of samples, you can enhance the accuracy and reliability of histopathological analysis.

For more information on our histopathology services please visit our website or contact us by calling 0117 951 1283 or emailing [email protected].