Sampling and packaging techniques for histology samples, and how to deal with unusual biopsy specimens

Surgical biopsies taken for histological examination should be fixed in 10 percent neutral buffered formalin (NBF) which is usually obtained in prepared pots. These can be obtained through NationWide Laboratories or other medical supply companies. Maximum tissue thickness to allow optimal penetration of fixative is 10mm.

Always include a completed submission form containing a relevant clinical history, including signalment, reason for presentation (eg was the lesion the reason for presentation or was the lesion found incidentally) and location/description of lesion (eg size of enlarged lymph nodes).

Sample packaging

Use appropriate zip-locked or biohazard bags for packaging samples, and use a separate bag for each submission/case.

Cytology slides should never be packaged in the same bag as formalin-fixed tissue. The slides should be placed in slide mailers in a separate bag to avoid contamination from formalin fumes as this can cause cellular changes which can affect the reading of the cytology. If you indicate on the submission forms that there is linked cytology and histology, then we know to look out for it.

The histology container must be enclosed in a sealed plastic bag with enough absorbent material to account for any potential leaks. Count the number of submissions and write the total number on the bag label. Ensure your completed submission form is placed in the side pocket of the biohazard bag or firmly attached to the bag so that the sample and form can’t be separated. Do not place multiple forms and samples in the same bag.

This should then be placed in an additional outer bag or container. NationWide Laboratories offers a locked box courier service so please contact us for more information.

Unusual biopsy specimens

Most biopsy specimens arrive at the histology laboratory in good order and can be easily orientated and sectioned; however, some samples are not straightforward and can provide frustrating results. Here is a quick guide to some of the more common problems. As always, if you have any queries, please call our office and they will be happy to direct you to a pathologist or member of the histology laboratory staff who can advise you.

Most biopsy specimens arrive at the histology laboratory in good order and can be easily orientated and sectioned; however, some samples are not straightforward and can provide frustrating results

Large specimens

Take multiple representative samples of up to 10mm from different areas of the lesion. We will examine up to four specimens for the same price. If you want to send the whole sample you can slice through the tissue using parallel slices which do not quite reach the bottom of the sample. This means that we can reconstruct the sample in the lab and orientate it better. Such samples really need to be fixed in very large volumes of fixative.

If the quantity of fixative required is too large and costly, the sample can be sliced and fixed for 24 hours at your practice before being wrapped in wet paper towel or swabs, double bagged in two zip-lock plastic bags and sent to our laboratory.

Margins

The outer surface can be inked, using surgical ink, to outline the margins. Allow the ink to begin to dry before immersing in fixative. Rinsing the inked sample in a weak acetic acid solution prior to fixing has been found to help the ink stay on the sample.

Margins can be tagged using different colours, lengths or numbers of sutures. Please do not under any circumstance use needles to mark margins. This is a safety issue for our laboratory staff.

If submitting separate biopsies please submit in separately labelled pots. Tissues can shrink and change texture and colour when fixed, making it very difficult to differentiate multiple tissues from the same pot.

Small or friable specimens

Small or friable specimens such as endoscopic samples, punch biopsies and needle core biopsies are best placed in cell safe or screen cassettes which we can supply on request. Please pre-soak the cassettes in NBF before putting the sample inside. This helps to prevent the tissue sticking to the foam material inside. Pre-labelling the cassettes using a No. 2encil before immersion in NBF means that multiple cassettes can be placed in the same pot. Samples can be fixed free in NBF; however, although we endeavour to sieve these samples, some fragments can be left behind – particularly if they are placed in the same pot as larger biopsies.

Submission of samples on gauze or card can cause samples to rip when removing for processing.

A single line drawn along the centre of skin punch biopsies in the direction of hair growth prior to biopsy can greatly assist us when orientating these samples.

Eyes

Enucleated eyes can be submitted whole in 10 percent NBF. There is no need to section the eye or inject the fixative as this causes significant artefactual damage.

Bone

Bone biopsies can be difficult to obtain and frustrating to interpret. This is because of the high risk of fracture during the biopsy procedure and, histologically, due to the presence of large quantities of reactive periosteum in most lesions, regardless of pathogenesis. A diagnosis of reactive hyperplasia is often of little clinical help. To try to alleviate this frustration, it is useful to take as large a sample as possible and/or to take multiple biopsies with some from the centre of the lesion (which we don’t often ask for). This is more likely to demonstrate the true lesion rather than reactive hyperplasia. Multiple biopsies reduce the risk of obtaining only haemorrhage or necrotic tissue. Fixation in 10 percent NBF is adequate for routine diagnostic biopsies.

It is useful to take as large a sample as possible and/or to take multiple biopsies with some from the centre of the lesion… This is more likely to demonstrate the true lesion rather than reactive hyperplasia

Claws

Claws need to be softened and phalangeal bone needs to be decalcified, so biopsies from claws and digits will take longer to report. The digital site is naturally very restrictive, so any expansile mass will cause similar clinical signs: abnormal nail growth, swelling, pain, lameness and lysis of the third phalanx. For this reason, amputation of one or more phalanges is the biopsy technique of choice. This includes inflammatory conditions of the nail bed, such as lupoid onychitis.

Punch biopsy techniques have been described in the literature; however, these can be very difficult to orientate in the lab and can also result clinically in permanent disfigurement of the claw.

If possible, an affected dew claw can be sacrificed. In most cases we will issue a preliminary report based on a soft tissue biopsy taken in the histology laboratory. In many cases this may reveal a definitive diagnosis; however, in some cases we may need to wait for the final decalcified or softened sections before concluding the diagnosis or to establish excision margins.

When can I expect my results?

Routine histopathology results are available within two to five working days of receipt at our histology laboratory in Newton Abbot, Devon. This variation takes account of the need for further fixation of very fresh samples, sample size and receipt of samples over weekends.

Occasionally the process takes slightly longer if there is a Bank Holiday or if the sample is particularly large or complex and requires further dissection and fixation. Large samples such as mammary strips may require further fixation. Samples which contain a lot of blood, such as spleens, can also take longer to fix. Samples containing a lot of keratin, such as claws and keratinised cysts, may require some surface softening or surface decalcification, and this may add 24 hours to the reporting time. Of course, samples containing bone will require full decalcification which can take up to 14 days. Very thick samples such as large jaw bones may take longer. We will always endeavour to contact you about these samples so please do not hesitate to call the laboratory to discuss your requirements.

We are also pleased to offer urgent rapid histology reporting within two working days at a small additional charge. In these cases, the sample must only measure up to 5mm³ and be adequately fixed on arrival at the laboratory. Tissue which requires more fixation or decalcification or samples which arrive at the laboratory on a Friday cannot be fast tracked.