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Veterinarian Technician February 2006 (Vol 27, No 2) Focus: Oncology

Basic Overview of Oncologic Cytology

by Michelle Miller, BS, CVT

    CETEST This course is approved for 0.5 CE credits

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    Key Points

    • Understanding the type of tissue to be examined is helpful in choosing the most appropriate method of sample collection.
    • Romanowsky-type stains are commonly used in cytology because they are inexpensive and easy to use.
    • Although specimens are often sent to a reference laboratory, technicians should be familiar with the different types of tumor cells.

    In both private and specialty practices, veterinary technicians often encounter patients that present with unusual "lumps" and "bumps" that may be in­flammatory or neoplastic in origin. Veterinary technicians play an increasingly important role in the collection and preparation of specimens from these patients. Although it is common practice to send samples to reference laboratories for evaluation, the ability to collect and process samples in-house has many advantages.

    Performing cytology in-house brings additional revenue to a practice and further utilizes the skills of veterinary technicians, making them more valuable to a practice. Although there are many ways to obtain samples for cytologic evaluation, the following information is directed at techniques that can be performed by veterinary technicians. Collection methods such as abdominocentesis,1,2 punch biopsy, excisional or incisional biopsy,3 and bone marrow aspiration4 should be performed by the attending veterinarian; therefore, these methods are not discussed in this article. Numerous sources are available that explain these procedures.

    Sample Collection and Preparation

    In cytology, the first and most important step is appropriate collection and handling of the specimen. The goal is to preserve as many cells as possible. Cancerous cells are often fragile, and in order to cytologically identify these cells properly, they must be handled carefully during processing. Understanding the type of tissue to be examined is helpful in choosing the most appropriate method of sample collection.

    Solid Samples

    The three techniques that technicians can use to obtain cytologic samples from solid masses are fine-needle aspiration (FNA), impression smears, and skin scrapings.

    Fine-Needle Aspiration

    When obtaining samples using FNA, a needle is used to gather samples from both firm and soft masses. The two techniques used are aspiration and nonaspiration (fine-needle capillary sampling).

    The aspiration technique5,6 is used to obtain cells from firm, solid, rigid masses using negative pressure. To collect a viable sample, wipe the area to be sampled with an alcohol pad as if performing a venipuncture or giving a vaccination. Attach an 18- to 25-gauge needle to a 3- to 20-ml syringe. The size of the needle depends on the size and consistency of the mass being aspirated.5 For example, some soft tissue sarcomas require an 18-gauge needle, whereas lymph node aspirates require little more than a 22-gauge needle. A 22-gauge needle is most commonly used. Syringe size is best determined by what is most comfortable to the user's hand size and the consistency of the mass being aspirated. The denser the mass, the larger the syringe used. Usually, 6- to 12-ml syringes and 1- to 1-1/2-inch needles7 are used for small animal patients.

    Insert the needle with the attached syringe directly into the mass and retract the syringe about 1/2 to 1 ml to create negative pressure. Gently redirect the needle to gather cells from different areas inside the mass while continuing to apply negative pressure to the syringe. If blood is seen in the syringe at any time, immediately release the negative pressure to avoid dilution of aspirated cells with blood cells. Remember that too much negative pressure causes cell lysis, which will damage the cytomorphology of the sample. When an adequate amount of cells has been collected, release suction in the syringe and then withdraw and disconnect the needle.

    For additional flexibility, intravenous extension tubing can be connected between the needle and the syringe. This method offers additional support for the operator and is helpful in the event of patient movement.8

    To apply the specimen to the slide, fill the syringe with air and reattach the needle. Standing directly over the clean slide, push the air through the syringe and onto the slide. Repeat this process onto new slides until the contents of the needle hub have been emptied. Be sure to have the needle close enough to the slides to prevent spraying the contents onto the table.

    When contents contain thicker material, the sample should be prepared using the compression smear (squash) technique.5,6 With the specimen emptied onto the middle of a clean slide, gently place a second clean slide on the specimen, perpendicular to the original slide. The weight of the slide should be sufficient to spread out thick, tenacious material into a monolayer. Sometimes, a small amount of pressure is needed to allow the sample to spread. It is important to compress and slide at the same time, keeping the slides flat. In one quick motion, pull the slides apart, keeping your elbows at an equal level as the slides are pulled back to avoid adding additional pressure to the original slide. Label the slides and let them air-dry before staining.

    Other methods of preparing smears from aspirates of solid masses include the modified compression smear and the starfish preparation technique. The modified compression smear technique5 results in fewer ruptured cells. Once the sample is expelled onto the glass microscope slide, place another slide on top of the sample. Rotate the top slide 45°, and then lift it directly upward.5 The starfish preparation technique5,6 is used to obtain a thick layer of tissue fluid around the cells. It is performed by expelling the sample onto a glass microscope slide. Then, using the point of the needle, drag the sample in several directions, producing a "starfish" shape. Although minimal damage is done to fragile cells, cells are not easily spread and may be difficult to identify under a microscope.6

    Collecting specimens using the nonaspiration technique5is similar to using the aspiration technique but results in less blood contamination from vascular tissues. This technique is used to collect specimens from soft masses (e.g., lymph nodes) and more vascular masses (e.g., thyroid tumors). With the nonaspiration technique, collection of cells is facilitated through the shearing of cells (removal of cells from a mass using a parallel motion). Prepare the area with an alcohol pad. Insert the needle directly into the mass without a syringe attached. Redirect the needle several times and then withdraw it from the mass. Attach an appropriately sized syringe filled with air and empty the contents of the needle hub onto a clean slide. Repeat this process until the needle hub is clear. Use the compression smear technique as previously described to complete slide preparation. Then label the slides and allow them to air-dry before staining. If the sample is too viscous for the compression smear technique, the fluid sample method (discussed below) may be more appropriate.

    Impression Smear (Touch Imprint)

    With solid lesions and masses that are exposed and/or ulcerated, it is best to get a sample directly from the site, using an impression smear (touch imprint).6 Gently flush the exposed area using the wound lavage technique. Attach a 35- to 60-ml syringe to one port of a three-way stopcock and an 18-gauge catheter to the second port. The desired pressure for wound lavage is 7 psi, which is achieved through the 18-gauge catheter.9 Using a catheter instead of a needle is recommended because of the damage that could occur if a needle disconnected from the three-way stopcock. The third port of the three-way stopcock should be connected via a microdrip fluid line to a 250-ml bag of 0.9% sodium chloride. Following wound lavage, it is important to blot dry the area with sterile gauze to remove blood, tissue, and debris; this will help relieve the inflammatory load present and make it easier to expose tissue that is of diagnostic value. Gently press a clean slide onto the lesion. Several slides may be needed, depending on the size of the lesion. Label the slides and allow them to air-dry before staining.

    Skin Scraping

    Skin scraping5 is a technique that is used most often for firm external lesions or during necropsy or surgery. Although these samples are difficult to collect, the technique is rewarding because a high number of cells can be obtained for evaluation. First, clean the area with 0.9% sodium chloride and blot it dry. Using a sterile #10 surgical blade, hold the cutting surface perpendicular to the lesion and pull back ("scrape") several times toward your body in quick motions. Transfer the scrapings to clean slides and use the compression smear method to prepare the slides. Then label and air-dry the slides before staining.

    Fluid Samples

    Fluid samples can be obtained through many different techniques, including abdominocentesis and thoracocentesis. These methods are performed by the veterinarian and are discussed in great detail in other sources.1,2 Endotracheala and transtrachealb washes and bronchoalveolar lavageb are other methods that can be used to obtain fluid samples; discussion of these procedures is beyond the scope of this article.

    After obtaining a fluid sample, save a small amount of the sample in an EDTA tube in case further diagnostics are required. The contents of an EDTA tube should be examined within 2 hours of collection. For fluid samples that appear clear (indicating low cellularity), the sample should be spun down and a clean pipette used to pull cells from the bottom for examination.

    Once the fluid sample has been collected, technicians should prepare a fluid or diluted slide similar to the method used to prepare a peripheral blood film. Although not always possible, the goal is to obtain a sample with a clear, feathered edge. Place a drop of the specimen at one end of a glass microscope slide. Holding another slide at a 40° angle to the original slide, pull back the sample until it covers the base of the spreader slide. Gently push the spreader slide forward for the length of the bottom slide, leaving a smear with a feathered edge.5 The line smear technique5 is most commonly used for fluid samples when cellularity is low. Use the same method as with a blood smear technique, but finish by bringing the spreader slide directly up, creating a line of cells at the end of the sample.6

    Staining

    When staining samples for examination, several options are available. The Romanowsky-type stains are most commonly used because they are inexpensive as well as easy to maintain and use.7,8 These stains (e.g., Wright's, Giemsa, and Diff-Quik [Baxter Diagnostics, McGraw Park, IL]) are excellent for staining cytoplasm and adequate for staining the nucleus. In addition, mast cell granules do not stain consistently with Diff-Quik; therefore, Giemsa or new methylene blue stains can be used if these lesions are suspected. Although most commercial stain kits will include recommended staining times, some adaptations may be needed, depending on the type and thickness of the sample. For example, thin samples require less stain, which can be achieved by using a shorter staining time, whereas thicker samples require longer staining times.

    New methylene blue stain can be used to clearly visualize the nuclei1 and nucleic material; however, it often does not stain the cytoplasm. When using new methylene blue stain, place one to two drops of stain onto the air-dried sample. Cover the area completely with a coverslip and examine promptly. Although this method is useful for immediate examination of the specimen's nucleic material, the stain is not permanent and cytoplasmic staining is minimal.7

    Although each stain has its own unique recommendations for use that should be followed carefully, it is sometimes necessary to make adjustments according to the individual sample. Common problems with Romanowsky-type stains include excessive pink or blue staining, artifacts (e.g., latex powder, lubrication), and uneven staining. Excessive pink or blue staining can be remedied by increasing or decreasing, respectively, staining times. To decrease the risk of uneven staining, make sure the sample is consistent, with little variation in thickness.

    Evaluating the Specimens

    After the slide has been prepared and stained, the specimen is ready to be evaluated. Because of the experience and knowledge needed to evaluate these samples, many clinics and even specialty hospitals prefer to send them to a reference laboratory for definitive diagnosis. Before the slides are sent out, however, they are often viewed in-house. Technicians who are given the opportunity to view the samples will learn more about cytology and become more involved with the process. Technicians who work in a facility that specializes in oncology may often have the opportunity to view slides; if this is the case, in addition to the clinical pathologist who makes the diagnosis, several individuals may view the slides.

    Types Of Tumor Cells

    Even if specimens are sent to a reference laboratory, technicians should be familiar with the different types of tumor cells. The type of tumor is determined based on the cell of origin; some tumors have more than one cell of origin. There are three categories of tumors based on the cell of origin.

    Epithelial Cell Tumors

    Epithelial cell tumors are most likely to involve glandular tissue or tissue surfaces or linings.10 When aspirated, these cells have a high cellularity and typically exfoliate in sheets (i.e., the cells have adjoining cell borders).6,10 The cells are large and round to polygonal with distinct cell borders and moderate amounts of cytoplasm.10 The nucleus is round to oval-shaped with a smooth to slightly coarse chromatin pattern that becomes coarser as the malignant potential increases.6 Malignant epithelial cell tumors often have an increased nucleus:cytoplasm ratio.

    Adenoma (e.g., anal sac and sebaceous adenomas) is a benign example of this type of tumor. Malignant examples are classified as carcinomas and include adenocarcinoma (e.g., nasal, renal, anal sac, and mammary tumors), squamous cell carcinoma (e.g., skin, nasal, and oral tumors), salivary gland carcinoma, and transitional cell carcinoma.

    Patients with epithelial cell tumors commonly present with open, inflamed wounds with or without a secondary infection. Therefore, it is best to obtain a cytologic sample by impression smear, using the technique described earlier, to avoid contamination of inflammatory cells.

    Mesenchymal Tumors

    Mesenchymal tumors, also known as spindle cell tumors, are derived from connective tissue spindle cells.6 Connective tissue cells are known for continuously producing extracellular material. Mesenchymal tumor cells exfoliate in low numbers and usually exfoliate individually10; when viewed under a microscope, the cells often appear as "streams." The cells are small- to medium-sized with a distinct spindle shape, thicker in the middle and tapered at both ends. A cytoplasmic tail may be seen going away from the nucleus in one or two directions.6

    The nucleus of a mesenchymal tumor cell is round and has a fine, lacy chromatin pattern.6 When stained with Romanowsky-type stains, the cytoplasm stains light to medium blue, giving way to indistinct borders. With the increase in malignant potential, there is an increase in visibility of nucleoli, which are not typically seen in nonneoplastic spindle cells. The nuclear material also retains a coarser pattern.

    An example of a benign mesenchymal tumor is a fibro­ma, whereas malignant forms include osteosarcoma, soft tissue sarcoma (e.g., fibrosarcoma), nerve sheath tumor, and leiomyosarcoma. Patients with malignant mesenchymal tumors have varying clinical signs, depending on the location and invasiveness of the tumor.

    Round Cell Tumors

    A round cell tumor is a special type of mesenchymal tumor that is most often derived from blood or tissue leukocytes. These tumors are the most variable and diverse of the three types of tumors. On cytologic evaluation, the cells appear generally small to medium in size and are usually round.11 The nuclear makeup varies, depending on each individual cell type. Although round cell tumors do not have the cell-to-cell contact of epithelial cell tumors, their cellularity in most aspirates is high, giving off the impression of "sheets" or "clusters" of cells. When visually assessing the cells, it is important to determine whether cell borders are really "adjoining" (as discussed earlier) or whether the cells are simply crowded together because of high cellularity, so as not to confuse them with other tumor cell types.

    Both benign and malignant forms of round cell tumors are commonly seen in veterinary medicine. Cutaneous histiocytoma is a benign round cell tumor. Malignant round cell tumors include lymphosarcoma, mast cell tumor, and transmissible venereal tumor. Melanomas are sometimes considered to be malignant round cell tumors; however, they have characteristics of both epithelial and mesenchymal tumors.11 Because round cell tumors are so varied, clinical signs can be very different from one patient to another.

    Common round cell tumors encountered in companion animal practice include the following:

    • Lymphoma (malignant lymphoma or lymphosarcoma) is one of the most common cancers in dogs.12 Clinical signs may include enlarged lymph nodes and increased calcium values in peripheral blood; however, many dogs show no clinical signs. The sample should be obtained using FNA. Normal lymph node aspirates will yield small lymphocytes, with occasional lymphoblasts, plasma cells, and macrophages.13 A sample containing 50% lymphoblasts is consistent with a finding of lymphoma.13 A lymphoblast, or immature lymphocyte, is a large cell that contains a small amount of dark-blue staining cytoplasm around a large, round nucleus. Nucleoli are often seen in the nucleus.14 Lymphoglandular bodies may be seen in a sample that is indicative of lymphoma; they are similar in size and shape to platelets and are cytoplasmic fragments.13 Lymphoma can take on varied forms; therefore, it is recommended that samples be sent to a reference laboratory for a definitive diagnosis.
    • Mast cell tumors are common in dogs and cats.15 Clinical signs depend on the location and to some degree the appearance of the tumor. Most mast cell tumors are solitary and may or may not be ulcerated lesions.15 Samples should be obtained using FNA. The well-differentiated mast cells found in mast cell tumors are easy to distinguish under the microscope by the purple-staining granules. Mast cells also release histamine and other material that attract eosinophils. Although normal mast cells in tissue appear very similar, identifying an abnormally high number of mast cells aspirated from a mass can be helpful in determining malignancy.

    Conclusion

    In today's ever-changing world of veterinary medicine, technicians are encouraged to be knowledgeable and proficient in many areas, especially in sample collection and preparation. Technicians who are able to properly collect, handle, and stain cytologic specimens are an invaluable asset to the practice.

    Bibliography

    Nelson RW, Couto CG: Cytology, in Small Animal Internal Medicine, ed 2. St. Louis, Mosby, 1998, pp 1093-1098.

    Raden MJ, Wellman ML: Interpretation of Canine and Feline Cytology. Wilmington, DE, Ralston Purina Company, Purina Clinical Science Handbook, 2001.

    Reagan WJ, Saunders TG, DeNicola DB: Veterinary Hematology — Atlas of Common Domestic Species, ed 1. Ames, Iowa State Press, 1998.

    Tyler RD, Cowell RL, Meinkoth JH: Cutaneous and subcutaneous lesions: Masses, cysts, ulcers, and fistulous tracts, in Cowell RL, Tyler RD, Meinkoth JH (eds): Diagnostic Cytology and Hematology of the Dog and Cat, ed 2. St. Louis, Mosby, 1999, 21-46.

    1. Shelly SM: Body cavity fluids, in Raskin RE, Meyer DJ (eds): Atlas of Canine and Feline Cytology. Philadelphia, WB Saunders, 2001, pp 187-205.

    2. Mann FA: Acute abdomen: Evaluation and emergency treatment, in Bonagura JD (ed): Kirk's Current Veterinary Therapy XIII: Small Animal Practice. Philadelphia, WB Saunders, 2000, pp 160-164.

    3. Ogilvie GK, Moore AS: Skin biopsy, in Managing the Canine Cancer Patient: A Practical Guide to Compassionate Care. Yardley, PA, Veterinary Learning Systems, in press.

    4. Littlewood JD: Diseases of blood and blood-forming organs, in Dunn J (ed): Textbook of Small Animal Medicine. London, WB Saunders, 1999, pp 765-784.

    5. Cowell RL, Dorsey KE: Cytologic examination, in Hendrix CM (ed): Laboratory Procedures for Veterinary Technicians, ed 4. St. Louis, Mosby, 2002, pp 453-471.

    6. Tyler RD, Cowell RL, Baldwin CJ, Morton RJ: Introduction, in Cowell RL, Tyler RD, Meinkoth JH (eds): Diagnostic Cytology and Hematology of the Dog and Cat, ed 2. St. Louis, Mosby, 1999, pp 1-19.

    7. Fisher DJ: Laboratory procedures: Cytologic examination, in Pratt PW (ed): Principles & Practice of Veterinary Technology, ed 1. St. Louis, Mosby, 1998, pp 210-215.

    8. Meyer DJ: The acquisition and management of cytology specimens, in Raskin RE, Meyer DJ (eds): Atlas of Canine and Feline Cytology. Philadelphia, WB Saunders, 2001, pp 1-17.

    9. Fassig SM: Management of wounds, fractures, and other injuries, in Pratt PW (ed): Principles & Practice of Veterinary Technology, ed 1. St. Louis, Mosby, 1998, pp 428-429.

    10. Raskin RE: General categories of cytologic interpretation, in Raskin RE, Meyer DJ (eds): Atlas of Canine and Feline Cytology. Philadelphia, WB Saunders, 2001, pp 19-33.

    11. Ogilvie GK, Moore AS: Clinical cytology and neoplasia, in Managing the Canine Cancer Patient: A Practical Guide to Compassionate Care. Yardley, PA, Veterinary Learning Systems, in press.

    12. Vail DM, MacEwen EG, Young KM: Canine lymphoma and lymphoid leukemias, in Withrow SJ, MacEwen EG (eds): Small Animal Clinical Cytology, ed 3. Philadelphia, Saunders, 2001, pp 558-559.

    13. Duncan JR: The lymph nodes, in Cowell RL, Tyler RD, Meinkoth JH (eds): Diagnostic Cytology and Hematology of the Dog and Cat, ed 2. St. Louis, Mosby, 1999, pp 97-102.

    14. Rakich PM, Latimer KS: Cytology, in Latimer KS, Mahaffey EA, Prasse KW (eds): Veterinary Laboratory Medicine: Clinical Pathology, ed 4. Ames, IA, Duncan & Prasse, 2003, pp 304-318.

    15. Thamm DH, Vail DM: Mast cell tumors, in Withrow SJ, MacEwen EG (eds): Small Animal Clinical Cytology, ed 3. Philadelphia, WB Saunders, 2001, pp 261-268.

    aFor more information about performing an endotracheal wash procedure, see page 30 of the January 2006 issue.

    bFor more information about performing a transtracheal wash or bronchoalveolar procedure, visit the Free Downloads section of VetTechJournal.com and download the handout.

    References »

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    CETEST This course is approved for 0.5 CE credits

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