1 How to obtain the perfect biopsy
Obtaining a diagnosis is one of the most important steps in the management of the cancer patient. Obtaining a biopsy before the surgical procedure is performed is best clinical practice in the majority of cases as it provides a pre-treatment diagnosis, helps the clinician plan the surgery and can provide the owner with a more accurate prognosis. There are a number of methods of obtaining samples from the tumour and the choice is based on a number of factors including:
Four months previously, the dog had presented with a 2-cm mass located in the region of the left hip. The veterinary surgeon had excised the mass; however, the owners declined histopathology so the diagnosis was not determined. The mass gradually recurred and the dog was referred for further evaluation and treatment (Fig. 1.1).
As the mass had been previously undiagnosed and had recurred, any future treatment would be dependent on an accurate diagnosis. If surgical excision was indicated, definitive surgery can then be planned. An excisional biopsy in this case would not be indicated. Options for obtaining a diagnosis, therefore, include cytological sampling, needle-core or incisional biopsy. Using a punch biopsy may not be an appropriate choice in this case due to the location of the mass in the deeper subcutaneous tissues. Therefore, in this case a fine needle aspirate was opted for initially as the procedure was the least invasive of all the biopsy options, inexpensive, easy to perform and results were made available rapidly.
Figure 1.3 Case 1.1 Step 2. Insert needle into mass with syringe attached and apply negative pressure to the syringe. The needle can be redirected within the mass but aspiration should be ceased if fluid is observed within the hub of the needle
Figure 1.4 Case 1.1 Step 2 can be carried out alternatively using the capillary method. Here a needle is used without a syringe to obtain a needle core sample of cells by swiftly moving the hub of the needle backwards and forwards within the mass
Figure 1.5 Case 1.1 Step 3. Release negative pressure whilst the needle is within the mass and remove the syringe with the needle attached. Aspirate air into the syringe and reattach needle (or for the capillary sampling method attach a sterile, air-filled syringe)
Figure 1.7 Case 1.1 Step 5. Prepare the slide by placing another slide on top of the aspirated material and gently slide the two slides away in opposite directions. The slide is then air dried and stained
For masses that are very vascular or that contain fluid cavities the capillary method may improve diagnostic yield by reducing blood or fluid contamination and increasing the relative cellularity of the sample. Similarly, the syringe method may be more suitable for solid tumours, such as a soft tissue sarcoma.
The smears should be air dried and the sample should be thin enough to dry within 1 minute. The best cytology stains to use in clinical practice are the Romanowsky stains (such as Wright’s stain, Giemsa stain and May-Grünwald-Giemsa stain) as these provide clear detail of both the nuclear and cytoplasmic structures. They are relatively quick to prepare and these stains will also stain bacteria if they are present. The ‘rapid’ stain kits such as ‘Diff-Quik’ are highly useful and obviously extremely convenient but it is important to realize that these may not always stain the granules within mast cells clearly, thereby leading to potential confusion in the diagnosis. In such a situation, Toludine blue stain will identify mast cell granules. The use of Toludine blue may be especially useful in poorly differentiated mast cell tumours in which the granularity can be low.
Mast cells tumours are classified as being a round cell tumour, which means they exfoliate as discrete round cells with clear margins and a rounded nucleus. Other tumours in the round cell category include lymphoma, histiocytoma, plasmacytoma and transmissible venereal tumour (which does not occur naturally in the UK but may theoretically be seen in imported dogs). Mast cells contain azurophilic granules in their cytoplasm which stain a crimson-purple colour with Romanowsky stains, therefore usually making their identification relatively straightforward but the number of granules present in different tumours can vary considerably depending on the tumour grade. In the hands of an experienced cytologist, aspirates can be helpful to indicate the tumour grade, as low-grade mast cell tumours usually have well-defined and uniform nuclei with a high number of granules in each cell. The more poorly differentiated the tumour is, then the fewer the number of cytoloplasmic granules there are, the more prominent the nucleoli are and the degree of variation in cell size (anisocytosis) and nuclear size (ansiokaryosis) increases. The use of silver staining of the nucleolar organizing regions (‘AgNOR staining’) can also be used to help predict the grade of a tumour on aspirate samples, thereby adding further detail and diagnostic use from a simple aspirate. However, histopathological analysis will still be required to obtain accurate grading information.
For this case a fine needle aspirate provided an accurate diagnosis although providing a definitive grade for this tumour type is not possible by cytology alone as explained above. However, by submitting the entire tumour after excision, a tumour grade can be assigned and the completeness of surgical excision evaluated. In this and similar cases, knowledge of the tumour type preoperatively allows careful surgical planning. Additionally, appropriate staging of the disease can be performed by means of thoracic radiographs, local lymph node assessment and hepatic and splenic ultrasound examination. The mass was excised with 2-cm lateral margins and one fascial plane deep and the wound deficit was repaired using a local transposition flap (Figs 1.9 and 1.10).