Corresponding to other laboratory tests, the quality and accuracy 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 such as crush artefact pictured below.

Fixation

• Always use 10% neutral-buffered formalin (NBF) to achieve optimal fixation.
• The buffering agent reduces the formation of “formalin pigment,” which can interfere with accurate analysis.

‘Formalin pigment’ (acid hematin)

• The histology containers that you can order via your local laboratory are already prefilled with 10% NBF (except larger 300ml+ containers)
• Never freeze the sample before fixation or send it in alcohol or saline. These methods are not suitable and will cause significant tissue artefact. In severe instances, a diagnosis will not be achieved due to these artefacts.
• 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 but the length of time the samples should be left to fix at the surgery will depend on the size/type of tissue and method of transportation to the laboratory
• If the sample is still not fixed when it arrives at the VPG, we will section and allow it to remain in formalin until it is completely fixed. We will notify you when this occurs if it affects our published TAT.

Submission form and relevant patient history

• 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, 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.
• We have two urgent streams HURGE (same day urgent) and NURGE (next day urgent). Please mark accordingly and note the charges associated with prioritising samples.

Packing for sending to the laboratory

• 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 could break in transit (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 samples.
• Please ensure that you adhere to the correct requirements when packing your samples your specimens Packaging and transport requirements for patient samples – UN3373 – GOV.UK (www.gov.uk)
• If you need assistance with customs documentation, please contact our histopathology department directly.

Example of well-packaged histology sample

Safety

• There are risks associated with handling formalin, please ensure you are familiar with your site COSHH assessment before proceeding and dispose of any unused formalin correctly.
• It is important that you keep containers closed and only handle only in well-ventilated areas, wear designated PPE and handle any spills immediately.
• 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

Sampling small biopsies

• When taking small biopsies, even with careful sampling it is easy to introduce crush artefact.
• 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 specimens 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 recommended (1).

• 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 requested).
• 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.

Histology cassettes

• Avoid using foam inlays for fresh tissue, as they can amplify the formation of crush artifact. For small samples, instead use a mesh cassette.

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.

Large samples

• NB Prolonged fixation 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, consider carefully cutting them to enhance formalin penetration. 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. For samples which do not require margin assessment, such as spleens with large masses, make serial cuts approximately 1cm in width throughout the length of the mass and extending into the adjacent, macroscopically normal tissue.

Incision into mass without cutting through tissue boarders 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. Any post-surgical alteration of a sample should always be noted down in detail on the submission form and containers must be labelled accordingly.
• Note that during fixation and transport, tissues shrink and shift. It can be challenging for the lab to re-orient the sample and fit the pieces back together. To minimise this, place samples in large enough formalin pots so that the sample does not “mould” into the shape of the pot. You can use sutures to tack down fat and muscle into the correct orientation. If you have surgical marking ink, apply it to the relevant margins prior to cutting the sample and placing it into formalin.

Sectioning a mammary strip

Shipment of large samples

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

• Is margin assessment required for this sample?

For example, in a whole 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, till the final report has been issued.

If the whole large 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. Contact the Histology laboratory prior to sending a sample fresh and we can advise you on the shipment methods. Never send fresh samples on a Friday!
• To make sure diagnosis of the lesion is not impaired by poor fixation due to sample size, an incisional biopsy of the lesion (avoiding cutting into any surgical margins) can be provided in a separate container along with the main sample. Unless other additional histology sample sites are sent, this does not incur an extra cost.

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 make one or several incisions through the capsule above a mass and in ‘normal’ areas of the spleen.
• If the whole spleen cannot be sent or you wish to send incisional biopsies, please consider the following:
• Larger splenic masses, in particular haemangiosarcomas are prone to develop extensive haemorrhage and necrosis to a point where neoplastic tissue is to a vast extent replaced by a haematoma. Because of this, neoplastic tissue may be very difficult to find in the histologic sections.
• The likelihood of detecting a haemangiosarcoma in the spleen compared to the number of sections examined has been evaluated in a paper by Herman et al.
• According to this paper, if only one or 2 sections of a splenic mass are examined, the likelihood of missing a haemangiosarcoma is 32% and 17%, respectively.
• If 5 sections from a splenic mass are examined, the likelihood of detecting haemangiosarcoma increases to 95%.
• Therefore, we would recommend sending 5 separate samples from large splenic masses, and ideally a section of ‘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.

Laboratory limitations

Some pre-analytical causes that may lead to inconclusive reports or prevent histopathological analysis:

We understand that many different factors are involved in deciding which methods are applied in obtaining biopsies for histopathology. Some sampling methods are associated with significant risks of leading to (partially) inconclusive reports or even prevent (meaningful) histopathological analysis.

This does not mean that these methods should never be applied; often taking the best possible samples is restricted by the individual circumstances of the case. This makes it even more important to be aware of at least some of the causes that may lead to impaired processing and reporting and manage expectations with the client upfront.

• There is some limitation associated with the size of samples. The smaller the biopsy, the higher the likelihood that the sample is not fully representative or insufficient features can be observed for a conclusive diagnosis.
• Very small biopsies (<1-2 mm), in particular from friable tissue (even if protected in a histology cassette), is at risk of disintegrating during transport or processing of the sample, in which case no report can be provided. If we receive samples that we perceive are at risk of being non-diagnostic for this reason, we will phone you and advise accordingly.
• Electrocautery causes coagulation artefact in the tissue; in small samples, this can impair the histologic assessment. In larger tumour excisions, this may interfere with the histologic margin assessment.
• Staples must be cut out of the tissue before it can be processed. If surgical margins are stapled this may interfere with the histologic margin assessment.
• We understand that sending crusts in isolation without associated skin biopsies), nails that have come off or tissues that have been excreted (e.g. coughed or sneezing out) or chewed off seem like an easy, non-invasive and inexpensive way to obtain histopathological results. However, please be aware, that in crusts and nails, the histopathological features are in most cases unspecific and will not allow for diagnosis of a cause/ disease process. Excreted or chewed off tissue is often necrotic or potentially autolytic, which may prevent meaningful histological analysis. Often, for these types of samples, follow-up histologic biopsies are required.
• We sometimes receive foreign material to investigate the nature of the material. This is sometimes possible to some extent (e.g. we can recognise general plant origin, cotton swabs, etc.), but often the result is inconclusive. Note that plastic and metal foreign bodies cannot be processed.

Protocols for unfixed, decalcification and zoonotic samples

In order to provide the best diagnostic service, a small number of our samples may be held back for various reasons. We will keep you informed of any delays via email

TB risk samples: sample requires fixing in formalin for 10d in total to guarantee inactivation of zoonotic pathogens.

Other zoonotic risk samples (including Brucella) are fixed for one extra day.

Unfixed samples: usually only a day or two delay. Tissues must be fully fixed to allow processing. Required steps for optimal fixation are taken in the lab.

Bone/teeth: for tissues containing calcified components an additional decalcification step is required. The time required for decalcification is difficult to predict and depends on the amount and density of the calcified structures in the tissue. As a general rule, we advise an additional 7 days till samples can be processed.

Further testing

We routinely perform a large number of special stains where required for a complete report at no extra charge.

Our pathologists may recommend ancillary tests to assist with diagnosis – these could include PARR, other PCR tests, IHC or FISH analysis, which can be ordered with our admin team. All extra charges for additional test are listed in our price list.

We keep our wet tissue for 5 weeks and our blocks and slides indefinitely, so if you require any ancillary testing at a later stage, this is also always possible.

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 diagnoses 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 questions you may have regarding the histopathology report.

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

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References:

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.)

Herman EJ (2019). Understanding the efficiency of splenic hemangiosarcoma diagnosis using Monte Carlo simulations. Vet Pathol 56(6): 856-859 doi:10.1177/0300985819868732