Guidelines for Submitting Specimens to
Specimen preparation for submission
Wherever possible, the whole sample should be submitted in formalin. For tumours, this allows the assessment of the entire tumour specimen including surgical margins. If the tissue is large, then it may be more appropriate to submit representative sections from the tissue rather than attempting to send the entire specimen. For instance when sampling splenic masses, which are often very large, it is possible to send a number of representative portions from the periphery of the mass, avoiding areas of haemorrhage or necrosis.
If only part of the tumour is being submitted, then avoid necrotic or haemorrhagic areas e.g.: the centre of an ulcerated mammary gland mass, as these areas often lack sufficient cellular detail for a diagnosis to be made. Ideally, samples should not be thicker than 10mm, to allow adequate formalin penetration.
Identification of tumour margins
The meaningful interpretation of the surgical margins of a tumour mass by us as pathologists is unfortunately not quite as straightforward as you might imagine! To assure that the pathologist is examining the ‘true’ or most important surgical margins of a specimen, particularly in an anatomically-complex site, it is helpful for the surgical margins to be marked. There are a number of different ways that this can be done.
1. Inking of margins
Although we ink the margins for every specimen that we receive where a tumour is suspected, it is sometimes helpful to identify margins prior to submission, that you as a surgeon are particularly interested in. Painting the specimen is the most reliable method of margin identification and using different colours to paint different surgical margins, can enable specific margins to be identified and individually assessed. While there are many different types and colours of commercially available surgical inks available, the cheapest and easiest methods is to use one or more different colours of Indian ink.
To ink the margins, the tissue is first patted dry with paper towels to remove the surface blood. The tissue is then painted as appropriate with undiluted Indian ink over the margin surfaces, for instance over the subcutaneous fat over the deep border of a skin tumour. The tissue should be painted with a large soft brush, rather than being immersed in the ink, as ink can otherwise percolate through small fissures rather than remaining on the tissue surface. The painted tissue should then be immersed in 10% acetic acid (we use white vinegar from the supermarket in the lab, and re-use it multiple times) to stop the ink from being washed off in the formalin. The tissue can then be patted with a paper towel to remove the excess ink/acetic acid and then placed into formalin for submission to the lab. For the pathologists at the microscope, prior inking of the tissues can make a huge difference to how easy it is identify the ‘real’ surgical margins from false margins.
2. Suture-tagging of margins
Place sutures at the appropriate margins that are of particular importance eg: a margin that grossly is close to normal tissue, or at an anatomically important site. This method works less well for broad tumour fronts or very large specimens, or where multiple surgical margins are being assessed. Because the sutures have to be removed prior to processing, it is often impossible to identify the specific margins again on the wet tissue should further sections be requested.
3. Separate surgical margins
Marginal tissue is taken from the tissue bed that is left behind in the animal and this tissue is submitted separately as representing the tissue margins. This is a good way of submitting margins particularly for large samples, reducing the chances of processing/trimming errors.
Biopsy specimens should be submitted in 10% formal saline or neutral buffered formal saline. This is usually made up by diluting formalin (37-40% formaldehyde) at a ratio of 1 part formalin to 9 parts water. Buffering the solution will reduce histological artefacts, especially in blood-rich tissues such as the spleen.
The formula for making up 1 litre of 10% formal saline is:
Saline solution: 900ml
Formalin (Formaldehyde 37-40% solution): 100ml
The formula for making up 1 litre of 10% neutral buffered formal saline is:
Formalin (Formaldehyde 37-40% solution) :100 ml
Water (tap water is fine): 900 ml
Sodium dihydrogen phosphate dehydrate: 4.5 g
Sodium phosphate, (anhydrous): 6.5g
Packaging the samples
The maximum amount of formalin that should be sent by Royal Mail or courier is 250ml. Samples should be submitted at a ratio of 10:1 formalin to tissue volume.
When they need to be individually identified, for instance punch biopsies taken from different parts of the body, then we prefer to receive specimens in separate containers. We generally find that skin biopsies that are sent on labelled pieces of card fall off the card during transit! Alternatively, small samples can be placed in individually labelled plastic biopsy cassettes and then the cassettes placed together in the same formalin container. Please contact the lab if you would like us to send you some cassettes.
Minimising the air pocket by filling the sample container to just below the lid, will help prevent fragmentation of small delicate samples, such as Tru-cut biopsies, during transit.
If you are sending a large sample, then fix it in a large container of formalin at the practice for 24 hours prior to submission. After 24 hours fixation, take out the specimen and then wrap in formalin-soaked paper towels, place in a sealable plastic bag or container with a sealable lid. Double bag with cotton wool between the bags to absorb any formalin that might leak out.
On no account should glass bottles be submitted as these are a hazard to the postal workers and to our staff in the laboratory. Tupperware boxes, sharps boxes, milk bottles, etc., are not suitable containers and when we have received these in the past they have invariably leaked!!
1. All specimen containers or bags should be tightly sealed and wrapped with an absorbent material such as cotton wool. The amount of cotton wool used should be sufficient to absorb all of the formalin that might leak out if the specimen container becomes damaged.
2. The primary container/bag and the associated absorbent material should then be sealed tightly in another plastic bag. The form should then be separately attached to the outer bag.
3. The entire package should then be placed in a padded envelope for posting.
4. Submission forms and postage paid labels are available and can be downloaded and printed off from here.