DOES THIS PATIENT HAVE CANCER? USING IN-HOUSE CYTOLOGY TO HELP YOU MAKE THIS DIAGNOSIS. Joyce Obradovich, DVM, Diplomate, ACVIM (Oncology) Animal Cancer & Imaging Center, Canton, Michigan Almost every veterinary clinician has the tools in their practice to perform one of the easiest and least expensive diagnostic tests available to preliminarily rule in or rule out a cancer diagnosis. Fine needle aspiration (FNA) and cytology is an excellent non-invasive tool for every day clinical practice. FNA of superficial masses rarely requires anesthesia and is usually a quick, outpatient procedure. Techniques used are easy and the risk is low. Accurate cytologic evaluation, however, requires expertise, and the cytologist should understand their own limitations. At the primary care level, FNA and cytology are excellent for informing your client if their pet may or may not have cancer. The success of cytology also depends on obtaining a representative specimen, proper application to a glass slide, good quality staining, and the use of a quality microscope. 1 Cytology is useful for evaluating cutaneous and subcutaneous masses, lymphadenopathy, and thoracic and abdominal effusions. With ultrasound guided fine needle aspiration, cytology is also useful for evaluating abdominal masses, prostatic masses, organomegaly (liver, kidney, spleen), mediastinal masses, and peripherally located lung masses. When cytology is inconclusive, a tissue biopsy is usually necessary to make a definitive diagnosis Almost any suspected tumor can be aspirated easily and safely. Some masses are more risky to aspirate including: Splenic masses that appear cavitated (cystic or blood filled) as there is a risk of intracavitary bleeding. Bladder masses: risk of transplantation of the tumor cells along needle tracts needs to be considered. Lung masses: if deeper in the chest, the risk of pneumothorax increased. Limitations of cytology include: Samples that contain individual or small groups of cells, they do not show the tumor tissue in it s normal architecture as does a biopsy. You can often tell the main group of cancer that the main belongs to (i.e. sarcoma, carcinoma, or round cell tumor) but it may be difficult without biopsy to confirm the specific type: for example, a bone aspirate may show sarcoma cells, but it is usually not possible to differentiate osteosarcoma from chondrosarcoma. Certain types of tumors do not exfoliate well (e.g. sarcomas). Differentiation between benign and malignant can be tough in certain cases (scar tissue, for example). When evaluating cytology, certain criteria of malignancy must be met in order to make a presumptive or definitive diagnosis. These criteria are widely described in the literature and include the following: High N:C ratio (nuclear: cytoplasmic) Anisocytosis and pleomorphism of the cell. Anisonucleoleosis (large, variably shaped multiple nucleoli)
Multinucleation Anisokaryosis (varying nuclear size) Coarse nuclear chromatin Abnormal mitotic figures (uneven divisions) Nuclear molding related to rapid growth of cells. This slide shows a clump of cells that meets the criteria of malignancy. There is prominent anisocytosis, anisokaryosis, and anisonucleoleosis present. The arrow points to an example of nuclear molding due to the rapid growth of the malignant cells. Stain quality is important. If you perform cytology but do not feel comfortable reading the slides in house, you at least need to stain some of the slides you make to be sure that you have a diagnostic sample of good quality. There is nothing more frustrating for a pet owner than to wait several days or longer for a cytology result, only to be told the sample was non-diagnostic. The suggested procedure for Diff Quik stain is: Fixative: 60-120 seconds (especially for suspected mast cell tumors advise at least 120 seconds); Solution 1: 30-60 seconds; Solution 2: 5-60 seconds. Rinse under cold tap water or distilled water for 15 seconds. Examine the stain quality with a low power objective eosinophilia or basophilia can be enhanced by returning to either solution 1 or 2. 1 A B The slide on the left (A) is from a lymph node, but the sample is too hemodiluted to read. The slide on the right (B) is from the same lymph node, but the quality is excellent. There are three basic cell categories in cancer that can be appreciated cytologically: carcinomas, sarcomas, and round cell tumors:
Carcinomas: a malignancy arising from epithelial cells (skin and linings). The cells tend to clump together on cytology. There are tight junctions (or desmosomes) present that cause cell to cell adherence. In general, cells are large and round with distinct, intact cytoplasmic borders and the nuclei tend to be round to oval in shape. Carcinoma Sarcomas: cells of mesenchyma origin (cells that develop into connective tissue, bone, muscle, etc.). From the Greek sarx meaning flesh. On cytology, cells tend to exfoliate individually or in small groups rather than clumped like carcinomas. Cells are plump or fine spindle shaped, with often indistinct cytoplasmic borders. Cells are loosely arranged with an abundant extracellular matrix. Samples are often poorly cellular. The nuclei round to elliptical. Sarcoma Round Cell Tumors: include such tumors as lymphoma, plasma cell tumors, mast cell tumors, malignant melanoma (sometimes considered melanosarcoma), histiocytoma and TVT (transmissible vetereal tumor). Round cell tumors exfoliate individually having distinct cytoplasmic borders. Samples are usually moderately to very cellular. The nuclei are round to indented.
Malignant Melanoma Mast Cell Tumor Plasma Cell Tumor Lymphoma (considered a round cell tumor) is a very common cancer diagnosis in clinical practice. A presumptive diagnosis is very helpful in providing the owner with a quick and easy answer. Care must be taken not to over interpret lymph nodes. Keep in mind that a reactive lymph node will have lymphoblasts present, but the majority of lymphocytes will be more normal in size. In addition, plasma cells and occasional mast cells are seen in the reactive node. With lymphoma, small cell or low grade lymphomas can be difficult to diagnosis with cytology alone. The full architecture of the node needs to be evaluated (lymph node removal) or more specialized testing such as PCR or immunocytochemistry performed on the cytology slides. In higher grade lymphomas, finding a neutrophil in the smear is helpful as a size comparison. Normal lymphocytes should be smaller than a lymph node, whereas lymphoma cells will all be larger than lymphocytes. The following is a guideline for describing lymphoma. Protocol to Read Lymphoma: 2 Estimate the mitotic index by looking at 5 cellular fields under 40X or 50X objective. o Low = 0-1 o Moderate = 2-3 o High > 3 per Determine cell size compared to an erythrocyte o Small = 1-1.5 x RBC o Medium = 2-2.5 x RBC
o Large > 3 x RBC Nuclear shape and location: o Round: circular with no indentation o Irregularly round: round with few indentations or convolutions o Convoluted: several deep indentations o Clefted: Single deep indentation o Central vs. eccentric Nucleoli: o Single vs. multiple o Large vs. small o Indistinct vs. prominent o Central vs. marginal or peripheral placement o Relationship to each other (varying sizes and shapes) Describe Cytoplasm by amount and color (also note presence of Golgi zone or granulation) o Scant (small rim around nucleus) o Moderate o Abundant (nearly 2x size of nucleus o Pale light basophilia or clear o Moderate basophilia color between pale and dark blue o Deep basophilia royal blue or darker Tumor grade is morphologically based on cell size and mitotic index: o Low grade: low mitotic index and small cell size o High grade: moderate or increased mitotic index and medium or large size 1. Meyer DJ: The Acquisition and Management of Cytology Specimens. In Atlas of Canine and Feline Cytology. WB Saunders, Philadelphia, 2001, pp, 1-17. 2. Raskin RE: Lymphoid System. In Atlas of Canine and Feline Cytology. WB Saunders, Philadelphia, 2001, pp 119.