Sample Collection and Preparation

Aspiration Technique: When one is aspirating peripheral lymph nodes, the site of aspiration is prepared as for an injection. Suitable fine-needle aspirates can be collected using a 21- to 25-gauge needle attached to a 5-ml or larger syringe. Small (5-ml) syringes can be used when aspirating lymph nodes because lymph node cells exfoliate easier than those of many other body tissues. For collection, the lymph node is held stationary between the operator’s thumb and forefinger. With syringe attached, the needle is inserted into the enlarged lymph node. The syringe plunger is rapidly withdrawn two thirds to three fourths of the barrel length to create sufficient negative pressure to aspirate lymphoid cells.

If the node is large enough to redirect the needle without fear of the needle’s exiting the node, negative pressure is maintained while the needle is redirected. Otherwise, negative pressure should be released before the needle is redirected and then negative pressure reapplied. If the needle exits the node while negative pressure is being applied, artifacts (eg, aspiration of subcutaneous fat and/or various skin layers) may be seen on cytologic smears. Also, if only a small sample is collected, it may be aspirated into the syringe barrel and lost for cytologic evaluation.

When tissues are aspirated for cytologic evaluation, the tissue of least resistance is aspirated into the needle and/or syringe. If a vessel is ruptured and blood is aspirated, continued aspiration results in excessive blood contamination. This dilutes the sample and impairs cytologic evaluation. Therefore, once blood enters the syringe and/or needle, negative pressure should be released and the needle redirected, or the aspiration procedure should be repeated with a new syringe and needle, depending on the degree of blood contamination.¹

When the sample appears in the hub of the needle or if the needle has been redirected three or four times but the sample is still not visible in the syringe or hub of the needle, negative pressure is released and the needle is withdrawn from the node. The needle is removed from the syringe and a few milliliters of air (2 to 4) is drawn into the syringe barrel. The needle is replaced on the syringe and the plunger is rapidly depressed, forcing some of the aspirated contents onto a clean, dry glass slide. The sample must be smeared into a monolayer without causing excessive cell rupture (Fig. 7-2). Because lymphocytes, especially lymphoblasts, are fragile and rupture easily, special care must be taken. A combination smear procedure is depicted in Fig. 1-2. This requires some technical skill but can be mastered easily with minimal practice.

Fig. 7-2 Lymph node aspirate with all nucleated cells stripped of cytoplasm.
Nuclear chromatin of many cells forms strands across the slide. Smears with excessive cell rupturing cannot be evaluated cytologically. (Wright’s stain; original magnification 250X)

Nonaspiration Technique (Capillary Technique, Stab Technique): A 22-gauge needle is attached to a 3- to 10-ml syringe that has been prefilled with air. The node to be sampled is held firmly to aid penetration and to help direct the needle. The needle/syringe apparatus is grasped as if holding a throwing dart. The needle (attached to the syringe) is introduced into the mass. The needle is moved rapidly back and forth through the mass and along the same plane five to six times. There is no need to aspirate, since the cells are collected by shearing and capillary action. The needle is removed from the mass, and the material is expelled onto a clean glass microscope slide by rapidly depressing the plunger. Smears are made by one of the techniques described in Chapter 1.

Generally, one collects only enough material to make one smear. Therefore, the procedure should be repeated two or three times in different sites of the node or in different nodes to have adequate slide numbers and areas to evaluate.

Staining

Several types of stains have been used for cytologic examination of lymph node aspirates. The most commonly used are the Romanowsky stains (Wright’s, Giemsa, Diff-Quik). These stains are inexpensive, readily available to the practicing veterinarian, and easy to prepare, maintain, and use. They stain organisms and the cytoplasm of cells excellently. Nuclear and nucleolar detail usually is sufficient for differentiating neoplasia from inflammation and for evaluating neoplastic cells for cytologic evidence of malignancy.

Smears to be stained with Romanowsky stains should be air dried. Air drying partially preserves (“fixes”) the cells and causes them to adhere to the slide so they do not fall off the slide during the staining procedure.

Commercially available Romanowsky stains include Diff-Quik, Dip-Stat, and various other “quick” Wright’s stains. Most, if not all, Romanowsky stains are acceptable for staining lymph node preparations. The variations among the different Romanowsky stains should not cause a problem once the evaluator has become familiar with the stain used routinely.

The staining procedure recommended by the stain’s manufacturer should be followed in general but adapted to the type and thickness of smear being stained and to the evaluator’s preference. In general, the thinner the smear, the less immersion time needed in the stain. The thicker the smear, the more immersion time needed in the stain. For this reason, thick smears, such as smears of neoplastic lymph nodes, may require the recommended immersion time in stain be doubled or tripled. Each person tends to have different preferred staining characteristics. By trying variations in the recommended time intervals for the stains, the evaluator can establish immersion times to produce the preferred staining characteristics.¹

Lymph Node Cell Types

A variety of cell types can be seen in lymph node aspirates. These include small lymphocytes, medium lymphocytes, large lymphocytes (lymphoblasts), plasma cells, macrophages, mast cells, neutrophils, eosinophils, inflammatory giant cells, and metastatic cancer cells. These cell types are identified by their morphologic characteristics.

Small Lymphocytes

Small lymphocytes are ≤9 μ in diameter (recognizably smaller than neutrophils) and have a scanty amount of clear to light blue cytoplasm that may contain a few azurophilic (red-purple) granules (Fig. 7-3). The nucleus is round to oval, usually indented, with dense clumps of nuclear chromatin in a coarse, smudged pattern. Nucleoli are not visible.

Fig. 7-3 Lymph node aspirate.
Note numerous small lymphocytes, a plasma cell (broad arrow), a basophil, and eosinophils. Free granules from ruptured eosinophils also are present (narrow arrow). (Wright’s stain; original magnification 330X)

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