Species: Canine | Classification: Techniques
- Skin histopathology is a valuable aid to differential and definitive diagnosis of many skin disorders.
- Definitive diagnosis of certain skin diseases.
- Categorization of skin disease.
- Exclusion of some diseases.
- Provides useful information.
- May not be diagnostic in all cases.
- Sutures, dehiscence.
- Most animals require sedation.
- Nasal, facial and footpad lesions: may require short-acting general anesthesia.
- Must be obtained early in course of disease before chronic inflammatory changes occur.
- Exercise caution if:
- Bleeding disorders or concurrent medication, eg aspirin Acetyl salicylic acid or anticoagulants Heparin Warfarin affecting bleeding.
- Immunosuppressed animals: rare (may be wound healing problem).
- Local anesthesia Local anesthesia: overview : injecting lidocaine with adrenalin near extremities and into patients with circulatory disorders (maximum 1 ml of 2% lidocaine Lidocaine /5 kg bodyweight).
- Artifacts may cause difficulty in interpretation.
- Representative tissue must be submitted.
- 5-20 min (depending on the site).
- 5-20 min.
Criteria for choosing testSituations in which biopsy is indicated
- Deep lesions, especially if infectious organisms are suspected, eg atypical mycobacteria.
- Masses or any possible neoplastic lesions.
- Persistently ulcerated lesions.
- Conditions failing to respond to therapy (generally within 3 weeks).
- Unusual or atypical dermatoses.
- Conditions suspected which require therapy which may be expensive, of extended duration or hazardous.
- Dermatosis diagnosed by biopsy, eg immune-mediated skin disease.
Stop topical or systemic glucocorticoid therapy 2-4 weeks before biopsy.
If secondary bacterial pyoderma administer course of systemic antibiotic therapy before biopsy.
- Sedation or general anesthesia: routine checks
- If known bleeding disorder: caution.
- Veterinary specialist dermatohistopathologist.
- Sterile surgical instruments.
- Local anesthetic (lidocaine Lidocaine ) preferably without adrenalin.
Adrenalin can cause histological artifacts in vasculature.
- 25-gauge needles and syringe.
- Disposable biopsy punch, eg 6 mm.
- Scalpel blade or 4-8 mm biopsy punch.
- Suture material.
- Small-toothed forceps.
- Wooden tongue depressors or cardboard.
- 10% neutral buffered formalin for most samples. For some types of immunohistochemistry protocols, samples should be snap frozen rather than fixed in formalin.
- Select primary lesions (macules, papules, pustules, vesicles, bullae, nodule and tumors).
- Avoid excoriated sites.
- Clip hair very gently, if necessary.
Do not cleanse or prepare site in any way.
OrSedation and assistant holding.
- Punch biopsy: allows rapid sampling of multiple sites with minimal suturing.
- 6-8 mm biopsy punches recommended, 4 mm reserved for difficult areas such as lips periorbital and muzzle.
- Wedge biopsy: usually requires sedation or general anesthesia; indicated for:
- Removing whole nodules or masses.
- Fragile lesions, eg large pustules, vesicopustules, hemorrhagic bullae.
- When larger samples are neccessary.
Step 1 - Select site
- Biopsy at junction of normal and abnormal skin, ensure wedge orientation is correct.
The lesion should be at one pole of ellipse and normal tissue at other because samples are cut longitudinally during processing.
Step 2 - Marking site
- Mark site of biopsy with indelible marker to prevent inadvertent sampling of non-blocked areas.
- Place 4 dots equidistant from site or encircle site.
Step 3 - Local anesthesia
- Using a syringe with 25-gauge needle introduce lidocaine 2% Lidocaine into subcutaneous tissue directly beneath lesion.
- For minimal patient discomfort, gently reposition the needle (without removing) to deposit anesthetic in fanlike pattern.
1 ml of lidocaine per site is adequate and maximum 1 ml/5 kg bodyweight.
- Wait 1-5 min before performing biopsy.
Step 1 - Punch biopsy
- Place punch directly over lesion to be sampled.
- Rotate gently in one direction while gentle downwards pressure is exerted.
Do not rotate back and forth because this may cause sample damage by shearing forces.
- Continue until punch 'drops' into subcutaneous tissue.
- Gently remove specimen with a pair of small toothed forceps or mosquito hemostats.
- Grasp only by hair stubble, subcutaneous fat or edge of dermal-epidermal junction.
Excessive manipulation can make specimen useless.
- Blot excess blood.
- Use scalpel blade to cut sample plug from small pedicle of subcutaneous tissue and fat to which it is attached.
Step 2 - Wedge biopsy
- Excise ellipse of tissue using a surgical blade.
- Do not exceed 2 cm in width or length because larger specimens may not fix adequately.
- Grasp one end with forceps when freeing section from subcutaneous tissues.
Step 3 - Specimen preservation
- Place subcutaneous side of biopsy on small piece of tongue depressor or cardboard to prevent curling during fixation.
- Allow to stand for 30-60 seconds.
- Place in 10% neutral buffered formalin as soon as biopsy has adhered, ensuring volume of fixative is at least x10 volume of sample.
- Allow specimen to fix for 24 hours.
- If immunohischemistry is needed, samples should be transferred into 70% ethanol after 24 h.
Step 1 - Suture biopsy site
- Standard skin suturing technique.
Step 2 - End sample
- Use specialist veterinary dermatohistologist.
- Include details of history (including medication), duration and progression of disease, clinical appearance, sites biopsied and differential diagnosis.
- Only if immunosuppressed animal.
- Hemorrhage in animals with bleeding disorders, taking aspirin or anticoagulants (stop 1-2 weeks before biopsy).
- Delayed wound healing in immunosuppressed patients + those on corticosteroids.
- Lidocaine toxicity particulary if injected near extremities and into patients with circulatory disorders (maximum 1 ml of 2% lidocaine/5 kg bodyweight).
Reasons for Treatment Failure
Poor biopsy sampling
- Improper site selection.
- Improper preparation, eg surgical scrubbing.
- Intradermal local anesthetic injection.
- Shearing of specimen by blunt punch or poor technique.
- Squeeze artifacts due to crushing with forceps.
- Hemorrhage due to inadequate blotting can obscure pathologist's view of tissue.
- Dehydration due to drying in air: specimen must be placed in formalin within 1-2 minutes.
- Shrinkage, curling and folding due to failure to use wooden or cardboard splints.
- Recent references from PubMed and VetMedResource.
- Rizzo L B, Ritchey J W, Higbee R G et al (2004) Histologic comparison of skin biopsy specimens collected by use of carbon dioxide or 810-nm diode lasers from dogs. JAVMA 225 (10), 1562-1566 PubMed.
Other sources of information
- Ihrke P J (1996)Bacterial Skin Disease in the Dog - a Guide to Canine Pyoderma.Veterinary Learning Systems. pp 59-61 (Biopsy technique with special reference to pyoderma).
- Moriello K A & Mason I S (eds) (1995)Handbook of Small Animal Dermatology. 1st edn. Pergamon. pp 31-33 (Step by step proceedure with photographs.)
- Muller G Het al(1995)Muller and Kirk's Small Animal Dermatology.5th edn. Philadelphia: W B Saunders. pp 111-118 (Detailed account).