Contributors: Vetstream Ltd, Michael Day

 Species: Feline   |   Classification: Techniques

Introduction Requirements Preparation Procedure Aftercare Outcomes Further Reading

Introduction

  • Standard technique to support diagnosis of inflammatory, proliferative, reactive and neoplastic conditions within tissue.
  • Biopsy refers to the collection of a sample of intact tissue for examination of tissue microarchitecture and structural change. This is distinct from cytopathology which is based on assessment of individual cells or groups of cells derived from a tissue lesion.
Print off the owner factsheet on Biopsy Biopsy to give to your client.

Uses

  • Confirmation of diagnosis or rule-outs.
  • Biopsies are taken to:
    • Establish a specific diagnosis.
    • Eliminate other clinical diagnoses.
    • Follow the course of the disease.
    • Confirm the completeness of excision of a tumor by evaluation of tumor margins. Additionally, biopsy evalutes the potential for metastatic spread of a neoplasm by evaluating vascular or lymphatic invasion.
  • Biopsy specimens are used for several clinical purposes including:
    • Histological diagnosis involving examination of hematoxylin and eosin-stained sections of tissue by light microscopy. 
    • Special tests:
      • Application of histochemical stains to tissue - for example to detect organisms (eg Gram stain Staining techniques: Gram stain, periodic acid Schiff stain Staining techniques: Periodic acid/schiff, Grocott stain or ZN stain Staining techniques: Ziehl-Neelson) or evaluate the presence of collagen, fibrin, amyloid etc.
      • Immunohistochemistry/immunofluorescence  Immunohistochemistry (IHC) - for example to specifically identify micro-organisms, to phenotype neoplastic cells, determine the nature of extracellular matrix tissue or identify deposits of immunoglobulin or complement within tissue lesions.
      • Fresh biopsy tissue may be submitted directly for microbial culture.
      • Molecular diagnostics for example the use of polymerase chain reaction (PCR PCR (Polymerase chain reaction)) for detection of infectious agent, or clonality testing to determine whether a lymphoid infiltration is reactive or neoplastic.
    • Types of biopsy include:

Advantages

  • Can be the most effective way to establish a definitive diagnosis.
  • Facilitates assessment of prognosis and treatment.
  • Excisional biopsy may result in cure if the biopsy procedure removes the lesion.

Disadvantages

  • Invasive in some cases and requires local or general anesthesia General anesthesia: overview.
  • Correct tissue is not always accessible without complex procedures.
  • Minor interference has been suggested to possibly exacerbate some neoplastic and reactive conditions although the evidence for this actually occurring is limited.
  • Biopsy is a relatively slow diagnostic procedure - tissue samples require processing, so turn-around time is generally 24 hours.

Technical Problems

  • Accessibility.
  • Biopsy by endoscopic biopsy instruments often too small and superficial to be useful.

Alternative Techniques

Decision Taking

Criteria for choosing test

  • Confirmation of suspected diagnosis or rule out of differential diagnoses.

Risk assessment

  • Minimal but specific organs may have greater or lesser risks.

Requirements

Personnel

Veterinarian expertise

  • Careful judgment to get best specimen of diagnostic tissue.
  • Surgical or endoscopic procedure to obtain sample.

Anesthetist expertise

Materials Required

Minimum equipment

  • Suitable biopsy method/equipment as indicated.
  • Fixatives:
    • For most purposes 10% neutral buffered formalin.
    • For possible electron microscopic examination, fixation in glutaraldehyde is preferable. 
    • For microbial culture no fixation is required. 
    • For cryosectioning, tissue should be snap-frozen as soon as possible after collection, generally by freezing surrounded by cryoprotectant medium such as OCT (Optimal Cutting Temperature).
  • Biopsies may be used for microbiological examination (eg by swabbing) or impression cytology before fixation.

Other requirements

Fixatives

  • Fixatives require a finite time to exert their effects. This is dependent on the type and size of the specimen (and the extent of penetration required) but is seldom less than 24-36 h. For a large tissue mass, bisecting the lesion can increase the speed and efficiency of fixation. Pathologists are justifiably reluctant to process specimens at an earlier stage and more rapid results may require frozen sections. Very small endoscopic biopsies may require relatively less fixation. For immunohistochemical procedures on fixed tissue samples, there is now evidence that prolonged fixation is not ideal and that samples should be processed as soon as possible after fixation is achieved.
  • Neutral buffered formalin (10%) is used routinely:
    • At least 10x volume of fixative to specimen must be present.
  • Frozen sections are sometimes used to obtain a very rapid result:
    • Only indicated in very select cases as tissue morphology is inferior to formalin-fixed material.
    • Requires close proximity to, and liaison with, the laboratory which will provide detailed specific instructions on sample submission.
  • Immunohistochemical procedures can be carried out on the same formalin-fixed tissue submitted for routine light microscopic evaluation, however some antibodies used in immunohistochemistry (particularly monoclonal antibodies) will only work when applied to fresh-frozen cryosections. Most pathology laboratories will offer specific immunohistochemical testing or profiles and will advise on tissue samples required for these procedures. If in doubt, it is best to check with the Laboratory.

Preparation

Pre-medication

Site Preparation

  • All are sterile but surgical scrub should not be performed on cutaneous lesions because the procedure may induce significant secondary inflammation.
    DO NOT SHAVE or vigorously clean the lesion prior to biopsy.
  • Sterilize around lesion rather than over it:
    • Owners may be somewhat surprised by the lack of sterilization but careful instruction should avoid any subsequent complication.

Other Preparation

Procedure

Approach

Step 1 - Biopsy types

  • In all cases, supply full details of the lesion distribution/location, size/shape, color/consistency and if submitting only part of a lesion, describe the overall context of the portion submitted. If in doubt, liaise with laboratory to ensure correct selection and orientation of specimen(s). 
    It is essential to provide the pathologist with as much clinical detail as is allowed by space on the submission form.

Punch biopsy 

  • A core of tissue removed with a commercially available disposable biopsy punch Biopsy punch - 6 mm .
  • Available in several different sizes:
    • The standard skin punch used for small animals is 6 mm in diameter. Smaller samples may not process effectively and can be harder to interpret.
    • Pathologists are often significantly hindered by having very small specimens, which may not be fully representative of the diseased tissue.   

Wedge biopsy

  • Outward facing wedge with only a small amount of normal tissue may be more sensible in some circumstances.
  • Important portion is the abnormal tissue and the small segment of normal tissue provides the pathologist with an orientation of the lesion and an example of the normal tissue for comparison.
  • A separate biopsy of adjacent normal tissue may be sent if requested by the pathologist.

Excision biopsy 

  • Used when normal and abnormal tissue is required in the sample or when the lesion is so small that biopsy of a portion is difficult.  
  • Used when an entire lesion present, eg a vesicle or pustule, is likely to be adversely affected by partial biopsy.
  • Indicated when malignancy and dissemination of tumour may follow partial biopsy - clear documentation of this effect is limited.

Hollow needle (TruCut) biopsy

  • Useful for core biopsy of masses and other organs, eg kidney, liver  Biopsy: hepatic, and lung   Tru-cut biopsy needle   Biopsy: trucut sampling 02   Biopsy: trucut sampling 03 .
  • Effective for remote/internal organs under ultrasonographic guidance.
  • Minor skin damage with core of tissue obtained.
  • Good biopsy samples can be obtained with experience.
  • Provides more architectural information than FNA.

Core Procedure

Step 1 - Biopsy techniques

Wedge or excision biopsy

  • Wedge of tissue is obtained by full thickness incisions with a suitable scalpel blade through the abnormal tissue to include a small rim of normal skin on the wider (outer) edge. 
  • An excisional biopsy is similar in execution but involves the removal of the whole lesion which may involve more surgery if it is large.
  • An elliptical incision with an appropriate margin for the type of tissue is made with a suitable scalpel to include all tissue down to the cutaneous muscle.
  • The specimen should not be gripped with sharp or crushing instruments.
  • For wedge and excisional samples, sutures, staples or adhesives may be used to close the wound depending on the size and nature of the defect.
  • An accurate diagrammatic orientation of the biopsy should be made immediately to assist the pathologist.

Punch biopsy

  • Disposable skin punch of varying diameter (usually 6 mm diameter  Biopsy punch - 6 mm ) is used to remove a core of abnormal tissue consisting of epidermis, dermis, subcutaneous fat and panniculus muscle.
  • Punch is used to incise the skin without significant distortion.
  • Specimen can be easily and significantly traumatized by rat-tooth forceps or by excessive pressure/squeezing by forceps and these should therefore not be used.
  • A 25G needle is used to carefully lift the specimen so that the underlying attachments can be cut with the scalpel blade (not scissors).
  • Remove specimen from the site without damaging it and place it in fixative.
  • A specimen of adjacent normal skin may be obtained in a similar fashion for comparative purposes.
  • In cases where hair follicle interpretation is important, placing samples on card to indicate direction of hair growth facilitates interpretation of hair follicle architecture.

Impression smears

  • Obtained by pressing a clean (new) glass slide onto the surface of a suitably representative lesion.
  • Slides are air-dried immediately and stained appropriately before being examined.
  • Some mass lesions may require to be incised before the smear is taken from the cut surface.

Step 2 - Specimen handling

  • Biopsy samples of skin Biopsy: skin or muscle Biopsy: muscle which might become distorted during fixing should either be placed onto a small square of cardboard for about 1 min to adhere to the cardboard before being placed in fixative or should be pinned gently to a piece of card in its natural shape.
  • Sample specimen with sterile swab for bacteriology and fungal culture if indicated.
  • Place sample in appropriate fixative solution.

Aftercare

Immediate

Monitoring

  • Aftercare depends on tissue sampled and specific risks of the organ involved, eg biopsy of skin lesions is usually well tolerated and complications are rare.
  • Hepatic or pulmonary biopsy using a hollow needle system may cause hemorrhage and so careful monitoring for 24 h is essential.
  • Antibiotic treatment may be indicated.

Analgesia

  • Nil usually required.

Antimicrobial therapy

  • May consider use locally for skin and systemic for internal biopsies.

Special precautions

  • Liver biopsy must be delayed until blood clotting is tested.

Potential complications

  • Depend on organ involved:
    • Lung biopsy may cause serious pulmonary bleeding.
    • Hepatic Biopsy: hepatic biopsy may cause bleeding.
    • Failure to identify the liver may result in intestinal puncture and associated risks of peritonitis Peritonitis.
  • Measures to improve the quality of the results from tissue biopsy include:
    • Do not stretch the skin during biopsy.
    • This causes distortion both on a macroscopic and microscopic scale.
    • Use a scalpel, not scissors. This eliminates crushing artifacts along the line of incision.
    • Try to avoid over-handling of the specimen and use fine plain forceps very gently or, preferably, a fine hypodermic needle to remove/handle the specimen.
    • Try to reduce specimen size to <1 cm to allow rapid and proper fixation.
    • Large samples should be serially cut into pieces <1 cm and preserved in numbered separate containers with a diagram to help the pathologist orientate the specimen correctly.
    • Avoid TOO SMALL specimens. Samples <4-6 mm in diameter are probably barely adequate and are particularly liable to artifactual distortions from stretching, compression or crushing during sampling and may be difficult to handle when fixed.
    • Multiple sections are useful from large lesions and in some circumstances the complete specimen should be sent in a suitable container with at least 10x the specimen volume of fixative. Solid intact specimens require to be cut open and advice should be obtained from the pathologist as to the best way of handling such a specimen.
    • Remember that specimens will need to be removed from the container. A narrow-mouthed container (such as a glass pill bottle) means that the specimen has either to be handled roughly or the container needs to be broken to get the fixed specimen out. Therefore, a suitable wide mouthed inert, clean container must always be used. Most histopathology laboratories will supply suitable containers containing an appropriate volume of formalin for use with variably sized tissue samples.
    • Samples which have to sent by post for which there is no urgency can be fixed for a suitable time before being drained of fixative prior to posting. A day or two without the fixative will not usually harm the specimen - remember though to ensure that it is packed suitably to prevent desiccation, eg surrounded by formalin-soaked tissues in an airtight plastic bag. The pathologist will need to know that the sample has no fixative.
      Remember that there may be specific local (legal) requirements regarding the shipment of biological samples by post to diagnostic laboratories.

Outcomes

Complications

  • Peritonitis Peritonitis or other local infection.
  • Exacerbation of the neoplastic conditions.
  • Dissemination of infective organisms along biopsy tract. Although this is often discussed as a potential complication of biopsy (or cytology), in fact there are very few documented cases of this ever occurring and the risk is considered minimal.

Prognosis

  • Good in all cases if planned properly.

Further Reading

Publications

Refereed papers