Sample Collection & Fixation for Histological Examination

Follow the guidelines below to ensure that you receive maximum benefit from each sample collected and submitted for histopathological examination

• Take histology samples as soon as possible and transfer to fixative as soon as practical. Carcasses should be stored in a refrigerator. DO NOT FREEZE; the ice crystals will disrupt histology.
• Use only fresh supplies of buffered formaldehyde solution of the correct concentration (4% in phosphate buffered saline, pH 7.2 to 7.4. Stock solutions of formaldehyde are typically 40%, pH 3 or 4). Non-buffered or the wrong concentrations of formaldehyde cause poorer fixation and precipitate formalin pigment in tissues, which can confuse analysis. In tropical conditions, fixative should be kept cool to prevent deterioration.
• Use enough fixative. The volume of fixative should be ten times the volume of the tissue to be fixed. Samples should be added to fixative, and not vice versa, so that they do not stick to the side of containers. If samples are to be sent by post and weight is an issue, it is better to place the samples in a large volume of fixative for 24 hours, then transfer to a small volume for transport.
• Samples thicker than 0.5–1 cm will not fix adequately at their centre, because the formaldehyde cannot penetrate through. Where an obvious lesion is observed, take a slice through this, and a separate slice of the “normal” organ, rather than squeezing the whole organ into a pot.
• If entire animals, such as fish, are to be sent, open up the body cavities with a single ventral midline incision, to allow access by fixative.
• When submitting large pieces of tissue, partially slice them at 0.5 to 1 cm intervals to allow fixative penetration but so that the receiving pathologist can reconstruct them. The heart and brain are the only exceptions. The heart should be submitted whole if possible, particularly if cardiovascular disease is suspected. Open the chambers and wash out clotted blood to assist fixation. The whole heart enables valves, conduction system and adaptive changes to be assessed. Brain is also best removed whole and fixed by immersion. Brain tissue requires fixation before it can be sliced systematically to detect subtle lesions.
• Muscle, skin and nerve biopsies may require special treatment.
• For samples requiring bacterial or fungal cultures, virus isolation or molecular testing such as PCR, formalin-fixed samples are not appropriate, and additional samples should be taken in parallel. Please contact the office pathologists (01535 692000) if in doubt.
• Alternatives to formalin fixation exist and where situations prohibit formalin use, our pathologists can advise of workable alternatives.