Cytology and Biopsies for the Practitioner: Part 2
WSAVA 2002 Congress
C. Guillermo Couto, DVM, ACVIM
College of Veterinary Medicine, The Ohio State University
Columbus, Ohio, USA

When a specimen for a cytologic diagnosis cannot be obtained or is inconclusive, the clinician needs to obtain a sample for histopathologic diagnosis. Histopathologic evaluation of a tumor is also vital to monitor response to treatment.

Biopsy Techniques

Specimens for histopathologic evaluation can be obtained by using one of several techniques (Table 1). Incisional biopsies are typically used in patients with large masses that are difficult to resect in their entirety by means of a relatively simple surgical procedure (e.g.; large subcutaneous sarcoma or mast cell tumor; metastatic liver nodule), or in patients in which a general anesthetic to excise the mass may result in a high risk of complications. Excisional biopsies are usually reserved for patients in which it as easy to resect the mass in its entirety as it is to obtain an incisional biopsy (e.g.; superficial skin masses; lung lobe mass; splenic mass). In these cases, in addition to obtaining a diagnosis, the procedure may be the only therapy required. Endoscopic biopsies areused for superficial lesions of the gastrointestinal, respiratory, or lower urinary tract. Obviously, the mass has to be in an anatomic location that allows for endoscopic examination. The main disadvantage of endoscopic biopsies is that oftentimes they are too superficial to include the main lesion. For example, in dogs and cats with alimentary lymphoma, a diagnosis of "lymphoplasmacytic gastroenteritis" is frequently obtained when the submucosal lymphomatous nodule is missed by the biopsy instrument, and a superficial mucosal biopsy which contains increased numbers of mononuclear inflammatory cells is obtained

Sample Handling and Processing

Specimens obtained for histopathologic evaluation need to be handled carefully to avoid inducing artifacts (e.g.; crushing from hemostats, coagulation necrosis from cautery). In our clinic, we routinely make imprints of the specimens on a glass slide, so that the samples can immediately be evaluated cytologically. This is accomplished as detailed in Figure 1.

Samples for histopathology need to be fixed in the proper type and volume of fixative, and accompanied by a detailed signalment and clinical summary of the patient. The fixative of choice is 10% buffered formalin, used at the ratio of 9 parts of fixative per one part of tissue. Because formalin penetrates only 5 to 10 mm, the samples should be at most 2 cm thick. If the specimen is larger, it should be sliced at 8-10 mm intervals, without cutting through the bottom of the sample, to allow for better fixation. For best results, tissue samples should be fixed for a minimum of 24 hours. Also, samples from different areas (e.g.; a dog or cat with multiple skin masses) should be submitted in separate vials to assure that the clinician will be able to determine which tumor originated form a specific anatomic area, once he/she receives the pathology report.

If possible, the whole specimen should be submitted to a veterinary pathologist. This will allow for evaluation of the so-called surgical margins (i.e.; whether there was tumor left on the surgical site). The presence of tumor extending to the margins of the excision usually suggests that there will be local tumor regrowth, and that a second surgical procedure or adjuvant therapy (i.e.; radio- or chemotherapy) is indicated. If a mass is too large to be properly fixed, it should either be handled as described above, or several portions of the mass (preferably those that are morphologically different) should be included for evaluation (if possible, the deep and lateral margins should be included in different containers). If the surgeon believes that in a given area of the mass he/she did not obtain good surgical margins, he/she can tag the specific area by using suture material.

Interpretation of Biopsy Reports

Ideally, every biopsy report should provide the clinician with a definitive diagnosis. However, this is not always possible. As discussed above, artifacts induced by the surgical technique or by improper fixation, or a biopsy specimen that is not representative of the lesion may preclude the pathologist from generating a definitive diagnosis. In dogs and cats with poorly differentiated or anaplastic malignancies, the pathologist may not be able to further classify the lesion.

In addition to providing a histopathologic diagnosis, the pathologist should also "grade" the neoplasm. The grading system routinely used consist of 3 categories: well-differentiated neoplasm (grade 1); intermediately differentiated neoplasm (grade 2), and poorly differentiated neoplasm (grade 3). The histopathologic grading usually correlates well with the biological behavior of the tumor (i.e.; grade 1 tumors are "less malignant" than grade 3 tumors). in addition, most grade 1 tumors respond poorly to chemotherapy, so grading can be used indirectly for treatment planning. Other terms that are frequently included in pathology reports are listed in Table 2.

Table 1. Indications for Different Biopsy Techniques




Large mass or mass difficult to resect; patient in which general anesthesia posses a major risk;


Small superficial masses (e.g.; skin tumors); large localized masses easy to resect (e.g.; lung lobe mass)


Superficial gastrointestinal, respiratory, or lower urinary tract mass or lesion/s

Table 2. Terms Commonly Used in Pathology Reports



Grade 1/well-differentiated

Malignant tumor with low metastatic potential; usually poorly responsive to chemotherapy

Grade 2/moderately differentiated

Variable metastatic potential; usually somewhat responsive to chemotherapy

Grade 3/poorly differentiated

Malignant tumor with an aggressive metastatic behavior; usually responsive to chemotherapy


See grade 3/poorly differentiated

Incompletely excised

Tumor cells extend to the edges of the surgical excision; recurrence common; usually requires 2nd surgery or adjuvant therapy

High mitotic index

Usually correlates with grade 3 (i.e.; aggressive biologic behavior and good response to chemotherapy)

Vascular/lymphatic invasion

Tumor cells are found in vascular/lymphatic channels; suggestive of aggressive metastatic behavior

Speaker Information
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C. Guillermo Couto, DVM, ACVIM
College of Veterinary Medicine
The Ohio State University
Columbus, Ohio, USA

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