Getting the Best From Skin Biopsies
World Small Animal Veterinary Association World Congress Proceedings, 2007
Sonya Bettenay1, BVSc, FACVSc, DECVD; Julie Yager2, BVSc, PhD
1Tierdermatologie Oberhaching, Germany; 2Professor, Ontario Veterinary College, University of Guelph, Canada

Histopathology can be very useful in establishing a diagnosis of skin disease including neoplasia.

The definitive diagnosis which can be made by skin biopsy include:

 Neoplasia (type and whether benign or malignant)

 Infectious agents (bacterial, fungal, parasitic)

 Allergic (although the specific type of allergy may not be identified)

 Endocrine (both for focal and generalised alopecia)

 Drug related/immune mediated disease

When Should We Perform a Skin Biopsy?

 Any skin lesion which appears unusual to the clinician should be biopsied.

 A biopsy should also be considered if lesions fail to respond to empirical therapy.

 Any lesion which appears to be serious or severe. Ulceration especially of the mucocutaneous areas, widespread lesions, painful conditions.

 Diseases which need intensive or expensive treatment, or treatment which has possibly severe adverse effects needs to have a firmly established diagnosis. This will frequently require a biopsy.

 Neoplasia should be considered in patients with nodular diseases.

One of the other major reasons to perform a skin biopsy is to rule out other diagnoses. "I think this is an allergy but ...". In this situation the biopsy report of 'chronic hyperplastic dermatitis with mononuclear perivascular infiltrate'--while not confirming allergy--has at least ruled out the common infectious agents and unusual dermatoses. A supportive pathologic diagnosis interpreted in conjunction with the clinical impressions may be just as useful as a confirmatory diagnosis. One should--however--never perform a biopsy as "the first line of investigation". Even in an acute case with severe multifocal acutely erupting ulcerative lesions the first line of investigation is to perform a thorough clinical work-up and to formulate a list of differential diagnoses.

Biopsy Technique

Selection of the Site

Selection of the site requires careful examination of the entire dog for the most representative samples, the identification of the primary and secondary lesions present and the formation of a differential diagnosis list prior to biopsy. With the exception of a solitary nodule it is recommended that multiple tissue samples are taken. A good number to choose is 6. Try to find 6 sites which represent the range of lesions present. Whenever there is alopecia, try to take a "normal, haired, non-alopecic" piece from an area on the dorsal--not the ventral- trunk.

Select primary lesions if present, choose a representative range of lesions and above all they should be taken and handled carefully.

Depigmenting lesions should be biopsied in an area of active depigmentation i.e., grey colour rather than the final stage, i.e., white.

Alopecia should be biopsied in the centre of the worst area as well as in junctional and normal areas. The junctional site may help us identify dermatophytes or a special type of follicular inflammation. However it is most important to take the centre of the most alopecic area!

Ulcers may be biopsied if one suspects an infectious process, however IF one chooses to do this, then it is always best to use an elliptical biopsy and include the transition from ulcer to completely normal skin. Do not expect a pathologist to be able to describe more than an ulcer if an ulcer is biopsied. If the ulcer is associated with an immune mediated disease of the epidermis (for example pemphigus vulgaris) a biopsy confirmation / diagnosis is only possible if the epidermis is also present!

Excoriated areas are not typically useful. The crusts and destruction of the self trauma tend to mask the underlying pathology process.

Preparation of the Site

With the exception of the excision biopsy of nodules, no surgical preparation of the site should be employed. Even topical application and air drying of alcohol may alter the epidermis. If crusts are present, these should be left on the skin. If they are accidentally dislodged they should still be placed in the formalin and a note 'please cut in crusts' should be added to the request form. Wrapping the crust pieces in a small piece of lens cleaning paper (not tissue!) can be helpful, but the formalin must be encouraged to "soak through" to reach the crusts. Do not simply throw them in and leave the packet floating on the surface. Crusts may contain micro-organisms or acantholytic cells which will help to obtain a diagnosis. Infection as a result of this lack of surgical preparation is almost never seen.

Drawing an orientation line for alopecic lesions is a simple and incredibly useful concept. Normally, when skin is sectioned in the laboratory the technician looks for the line of the follicles and tries to cut along this. With alopecic lesions there are no hair shafts sticking up above the skin to allow any orientation by the pathology technician. The result is that many biopsies are sectioned so that follicles are in cross--cut or so called "donut" cut and not in the nice longitudinal orientation which is needed to adequately assess the hair follicle.

Wedge/Ellipse versus Punch

There are two commonly employed biopsy techniques in veterinary medicine, the punch and the wedge/ellipse biopsy. The latter is commonly employed as an excisional technique when removing solitary nodules. It is also the sampling technique of choice with vesicles, suspected cases of panniculitis and when biopsying the edge of a lesion. This allows correct orientation of the lesion by the pathology technician and protects the fragile vesicles. Even when one can see the fat at the bottom of a punch biopsy, the amount is insufficient to properly evaluate the panniculus when it is processed. Punch biopsies are not recommended for lesions involving deep dermis and panniculus.

The punch biopsy is quick, relatively atraumatic and usually employed with suspected infectious, inflammatory and endocrine dermatoses. Disposable biopsy punches are readily available in 4, 6 and 8mm diameter sizes. They can be autoclaved and reused several times without greatly affecting their sharpness. The 8mm punch is preferred and is suitable for all truncal lesions (even in cats and Chihuahuas!) The 6mm punch is preferred for most dog noses and footpads.

The 4mm punch is very small and should only be used for small cat and Chihuahua noses and footpads.

Punch Biopsies

The overlying hair is clipped and gently removed. If crusts are present it may be less traumatic to use scissors than electric clippers. If a general anaesthetic is employed (as indicated for nasal or footpad biopsies!), no further preparation is necessary. If the biopsy is performed under manual restraint or with sedation (we use xylazine at 0.4mg/kg IV), then the subcutaneous injection of 1 or 2 mls of Xylocaine (or prilocaine which does not sting as much when injected) without adrenaline will usually provide adequate local anaesthesia. If administered subcutaneously with the needle entry point outside the proposed biopsy area there should be no disruption to the biopsy. Allow time for the local anaesthetic to have effect. The punch is then held at right angles to the surface of the skin and gently placed over the selected lesion. Firm continuous pressure is applied and the punch is rotated in one direction until a sufficient depth has been reached to free the dermis from its underlying attachment. The punch is removed and any blood carefully blotted. The section of tissue is grasped at the base--which should be the panniculus--and subcutaneous attachments severed. Under no circumstances should the dermis or epidermis be grasped with forceps as this leads to 'crush artifact'. Crushed tissue may be misinterpreted as scarring at best and at worst renders the sample worthless. The tissue is rolled on gauze to gently blot the blood from its surface. In case of a thin sample (say from abdominal skin), it should then be placed--panniculus down--onto a rigid piece of cardboard or broken tongue depressor. This prevents the tissue from curling when placed in the formalin optimising the interpretation by the pathologist. The 'unit' of tissue and cardboard is placed into 10% formalin (tissue side down, so that the tissue is in contact with the formalin!) and allowed to fix for 4 hours minimum prior to sectioning. The volume of formalin required is approximately 10 times the volume of the sample. While this is not typically a problem with small punch biopsies, it is a common mistake with larger excisional biopsies and frequently makes the centre of large pieces unable to be interpreted by the pathologist--as the tissue which is not in contact with the formalin undergoes autolysis. Nodules should be sectioned into 1cm thick pieces to allow adequate penetration of the formalin into the centre of the lesion.

Submission of Biopsy Samples

Because the skin can only react in a limited number of ways the information submitted with the biopsy sample is an invaluable aid to the pathologists. Many dermatoses are diagnosed using a combination of the signalment (age, breed, sex), clinical presentation (distribution, type of primary lesions, if present), history (in particular previous response to therapy) and supportive histopathology.

If the list of clinical differentials fails to correlate with the histopathology then the pathologist will be able to review the sections thoroughly, to look for subtle clues. If the submission clearly states that a deep infectious process is suspected and that the patient has already failed to respond to an appropriate trial antibiotic therapy--or if for example a deep tissue culture has already been performed, then special stains are more likely to be ordered to further investigate the possibility of unusual infectious organisms. If an immune mediated dermatosis is the major clinical differential diagnosis listed and convincing supportive evidence is not seen on the first pathology sections then recuts may reveal this. These will not be likely to be ordered when the pathologist first reads the biopsy without the additional clinical input.

Classical textbook histopathology is seen occasionally, but as in the clinical situation not all of the textbook features are always present. Careful completion of an appropriate--dermatology oriented--skin biopsy request form will greatly improve the chances of the pathologist helping in the establishment of a diagnosis in these 'grey zone" cases.

The Differential Diagnosis List

A differential diagnosis list is important with any clinical case but it is essential with dermatologic cases. Seborrhoea or draining tracts can be the result of a wide range of disease processes. This list is important for the clinician to ensure that they have considered the options and obtained as much information from both pet and owner as possible and necessary prior to taking the biopsy. It is also important for the pathologist (as discussed above).

A good example is a case of pododermatitis with draining tracts. The clinical presentation as described could be the result of any number of disease processes. Some history clues which could be important in this case include:

 Breed, conformation, activities compatible with foreign bodies

 A history of pruritus prior to the development of the interdigital lesions which in turn would be compatible with an underlying hypersensitivity process

 Systemic signs which may be compatible with an underlying endocrinopathy

 Negative skin scrapings for demodicosis

 Lack of response to corticosteroid or antibiotic therapy

The biopsy procedure in this case will typically require a general anaesthestic. It is important that all the samples which may be necessary are taken at that time--in particular deep tissue cultures in access where one suspects a deep infectious process. Impression cytology of the deep cut surface of the biopsy can also sometimes be extremely helpful in differentiating unusual, undifferentiated tumours.

Speaker Information
(click the speaker's name to view other papers and abstracts submitted by this speaker)

Sonya Bettenay, BVSc (Hons), FACVSc, DECVD
Tierdermatologie Oberhaching
Germany

Julie Yager, BVSc, PhD
Ontario Veterinary College


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