Selection, Collection & Submission of Samples for Histopathology

By Moges Woldermeskel, DVM, PhD, DACVP

Histopathology is a powerful and inexpensive diagnostic method. Submission of the right specimen in the right fixative is crucial for microscopic evaluation of biopsy specimens and organ or tissue samples from necropsies. Properly selected, appropriately collected and preserved specimens are very helpful in establishing an accurate diagnosis.

Sample selection and collection:

During sample collection, a representative specimen of the gross lesion observed at necropsy or biopsy should be carefully selected. The specimen should include the typical lesion, active margin, and adjacent unaffected/normal tissue. Collecting cores of lesions, which may contain only necrotic debris, often renders the specimen unsuitable for diagnosis.

Handle the specimens gently to avoid compression artifacts, crushing or tearing by forceps (at collection). Improper handling mechanically disrupts tissue architecture and impairs microscopic evaluation. While performing skin punch biopsies, go perpendicular to the skin surface and deep enough to include the underlying subcutaneous fat. Avoid shaving, cleaning and scrubbing. Clipping hair may be sufficient. Very small samples removed by laser or electrocautery in most cases are unsuitable for histopathological evaluation and definitive diagnosis due to heat coagulation artifact (coagulation necrosis).

Occasionally there may be no apparent gross lesion at necropsy. If doubtful about which tissue to collect, include tissues from all major organs, intestines and brain (preferably whole brain). If a grossly visible lesion is found, include the adjacent normal appearing tissue during collection.

In collecting specimens from the gastrointestinal tract (gut), select at least three different intestinal sites of 1–2 cm (about 1 inch) in length. Ingesta in the gut may impair proper fixation. Use fixative to gently rinse the ingesta out of lumen (do not use water). Mucosa is the most commonly affected layer of the gastrointestinal tract and an intact mucosa is essential for evaluation. It also is prone to disruption during handling. Therefore, handle the tissue gently and make sure the formalin gains access to the intestinal lumen for adequate fixation.

Sample preservation and fixation:

If specimens from multiple sites or lesions are to be submitted, identify each specimen by site or size and place them in a separate container.

Specimens approximately 1 cm thick preserved in 10% buffered neutral formalin are the most appropriate for best results. The specimen should be completely immersed in ample amounts of fixative. Saline solutions, ethanol or isopropanol should not be used as fixatives and can render the biopsy useless. A tissue to fixative ratio of 1:10 is the appropriate minimum volume (fixative 10 times the volume of the tissue).

Larger specimens (greater than 1–2 cm in diameter) should be cut into smaller slices of about 1 cm in diameter to facilitate easy penetration by the fixative. This renders better preservation of tissue architecture and cellular detail. Some specimens, such as eye, should be fixed whole. When collecting smaller pieces from a large specimen, include specimens from areas representing lesions of different colors, consistencies, and apparently normal areas. Submit the whole neoplasm (if relatively small) from biopsies with surgical margins included.

To avoid autolysis, fix the specimen as soon as possible after collection. Avoid refrigerating or freezing specimens fixed in formalin. Formalin begins to freeze at about 40˚F and may damage tissues. Addition of 1 ml of 95–100 percent ethanol to 9 ml of 10 percent buffered neutral formalin helps to prevent this. Fixing the specimen at least 24 hours before shipment may help prevent sample deterioration if the fixative is lost by leakage during shipment.

Specimens should be transported in just enough formalin to keep them moist after previous 24 hours fixation in an adequate volume of formalin. Avoid glass containers, which often break during shipment.

Forcing large specimens into narrow-mouthed bottles mechanically distorts and disrupts the tissue. Fresh tissue may easily be placed into narrow-mouthed bottles, but are often difficult to remove after fixation and may require breaking the container, which is hazardous to laboratory personnel. Do not reuse specimen containers. This helps to avoid residual tissue from previous specimens and confusion from additional potentially incorrect patient information on the container.

Labeling the container and completing the submission form:

Label the container properly with owner’s and/or patient’s name, site of collection, veterinary practice/veterinarian, etc., for definite sample identification.

Clinical history complements microscopic evaluation and is crucial for full and accurate interpretation of the microscopic findings. Therefore, fully complete the submission form and specify each site on the body for each specimen if multiple tissues from different locations are submitted.

Indicating the age, species and sex of the patient involved is useful for obtaining a definitive diagnosis, as some lesions/diseases may be more common in or specific to a given breed, age, sex, or location on the body.

In summary, for better results:

  1. Select a representative sample of the grossly observed lesion.
  2. Collect the appropriate size (1 cm thick) representative specimen containing the active part of the lesion and apparently normal adjacent tissue.
  3. Handle the specimens gently—avoid collection artifacts.
  4. Preserve the specimen in adequate fixative (1:10 tissue:fixative ratio).
  5. Label the container properly.
  6. Fill the submission forms completely and provide the clinical history.

Carefully selected, appropriately collected and an adequately fixed representative specimen of a lesion with adequate clinical history are essential steps in obtaining a meaningful diagnosis.

If you have further questions, please contact the Athens or Tifton Diagnostic Laboratories.

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