Selection of Syringe and Needle

FNBs are collected with a 22- to 25-ga needle and a 3- to 20-ml syringe. The softer the tissue, the smaller the needle and syringe used. It is seldom necessary to use a needle larger than 22-ga for aspiration, even for firm tissues. When needles larger than 22-ga are used, tissue cores tend to be aspirated, resulting in a poor yield of free cells. Also, larger needles tend to cause greater blood contamination.

The size of syringe used is influenced by the consistency of the tissue being aspirated. Softer tissues, such as lymph nodes, often can be aspirated with a 3-ml syringe. Firm tissues, such as fibromas and squamous-cell carcinomas, require a larger syringe to maintain adequate negative pressure (suction) for collection of a sufficient number of cells. A 12-ml syringe is a good choice if the texture of the tissue is unknown.

Preparation of the Site for Aspiration

If microbiologic tests are to be performed on a portion of the sample collected, or a body cavity (peritoneal and thoracic cavities, joints, etc.) is to be penetrated, the area of aspiration is surgically prepped. Otherwise, skin preparation is essentially that required for a vaccination or venipuncture. An alcohol swab can be used to clean the area. If the samples are being collected using ultrasound guidance, it is important to avoid the use of ultrasound gel, substituting alcohol as a contact agent instead. Ultrasound gel stains pink with commonly used cytology stains. Even a small amount of ultrasound gel picked up as a contaminant when the needle passes through the skin is enough to completely obscure the cells and render a slide nondiagnostic.

Aspiration Procedure

With the standard aspiration method of FNB, the mass is stabilized with one hand while the needle, with syringe attached, is introduced into the center of the mass (Figure 1-1). Strong negative pressure is applied by withdrawing the plunger to about threefourths the volume of the syringe (Figure 1-2). If the mass is sufficiently large and the patient sufficiently restrained, negative pressure can be maintained while the needle is moved back and forth repeatedly, passing through about two thirds of the diameter of the mass. With large masses, the needle can be redirected to several areas within the mass to increase the amount of tissue sampled. Alternatively, several different areas of the mass can be sampled with separate collection attempts. Care should be taken to not allow the needle to exit the mass while negative pressure is being applied because this can result in either aspiration of the sample into the barrel of the syringe (where it may not be retrievable) or contamination of the sample with tissue surrounding the mass.

Figure 1-1 Aspiration technique of FNB. The mass is stabilized with one hand while the needle is introduced into the center of the mass. The hand holding the syringe is used to pull back on the plunger, creating negative pressure.

(Courtesy Oklahoma State University teaching files.)

Figure 1-2 Fine-needle aspiration from a solid mass. After the needle is within the mass (A), negative pressure is placed on the syringe by rapidly withdrawing the plunger (B), usually one half to three fourths the volume of the syringe barrel. The needle is redirected several times while negative pressure is maintained, if this can be accomplished without the needle’s point leaving the mass. Before the needle is removed from the mass, the plunger is released, relieving negative pressure on the syringe (C).

The negative pressure should not be applied for more than a few seconds in any one area. Often, there will be no material visible in the syringe or in the hub of the needle even though an adequate sample has been obtained. With excessive force or prolonged application of negative pressure, disruption of blood vessels will eventually occur and the sample will be contaminated with peripheral blood, diluting the tissue cells and rendering the sample nondiagnostic.

After several areas are sampled, the negative pressure is released and the needle is removed from the mass and skin. The needle is removed from the syringe and air is drawn into the syringe. The needle is replaced onto the syringe and some of the tissue in the barrel and hub of the needle is expelled onto the middle of a glass microscope slide by rapidly depressing the plunger. When possible, several preparations should be made, as described later in this chapter (“Preparation of Slides”).

Nonaspiration Procedure (Capillary Technique, Stab Technique)

Many people prefer to collect FNB without the application of negative pressure, and this technique can yield samples of equal or better quality than those obtained with the standard aspiration technique.^4,5 The nonaspiration technique works well for most masses, especially those that are highly vascular.^1,4 This technique is similar to the standard fine needle aspiration biopsy technique, except no negative pressure is applied during collection. The procedure is performed using a small-gauge needle on a 5- to 12-ml syringe. The barrel of the syringe is filled with air prior to the collection attempt to allow rapid expulsion of material onto a glass slide. The syringe is grasped at or near the needle hub with the thumb and forefinger (much like holding a dart) to allow for maximal control (Figure 1-3). The mass to be aspirated is stabilized with a free hand, and the needle is inserted into the mass. The needle is rapidly moved back and forth in a stabbing motion, trying to stay along the same tract. This allows cells to be collected by cutting and tissue pressure. Care must be taken to keep the needle tip within the mass to prevent contamination with surrounding tissue. The needle is then withdrawn and the material in the needle is rapidly expelled onto a clean glass slide, and a smear is made using one of the techniques listed later in this chapter (see “Preparation of Slides”). Having the syringe pre-filled with air allows the sample to be expelled onto a slide more quickly, thereby helping to avoid desiccation (drying out) of the collected cells and coagulation of the sample. Generally, material sufficient for only one smear is collected. If possible, it is optimal to perform multiple collection attempts at various sites within the mass to increase the chance of obtaining diagnostic material and to ensure a representative sampling of the lesion.

Figure 1-3 Nonaspiration technique of FNB. The syringe is held at or near the needle hub with the thumb and forefinger. Note that the syringe is prefilled with air. The free hand is used to stabilize the mass. This technique allows greater control over movement of the needle.

(Courtesy Oklahoma State University teaching files.)

Collection Tip

Make and submit multiple slides—This is one of the most important things that can be done to increase the diagnostic yield. Small-gauge needles are used for collecting cytologic specimens and the procedure is usually relatively painless. It takes less time to perform several collection attempts and prepare multiple slides when the animal is first presented than to repeat a procedure after finding the slide(s) to be nondiagnostic, often after the animal has already been discharged from the hospital. This is particularly important if sedation/anesthesia is required for collection. It is optimal to stain and briefly examine one or two slides to ensure that they are adequately cellular while the patient is still in the hospital (or before animal is recovered, if anesthesia/sedation is required). If the slides stained are not cellular, additional collection attempts can be performed immediately.

There are many reasons why any one slide may be nondiagnostic. The slide may not have any diagnostic cells because the needle missed the lesion during collection (geographic miss) (Figure 1-4) or may have been in a nonrepresentative portion of the lesion (e.g., an area of inflammation or necrosis within a neoplasm (Figure 1-5). In addition, some lesions simply do not exfoliate cells well. Even if adequate cells were collected, many times the cells do not spread out well and the slides are too thick to evaluate (especially common with lymph node aspirates) or all of the cells are ruptured during smear preparation (Figure 1-6). Even in the hands of clinicians who are highly experienced in sample collection, it is not unusual to evaluate multiple slides from a single lesion and have all but one of the slides be nondiagnostic for one reason or another.

Figure 1-4 Geographic miss. Sometimes the needle is not in the area containing representative tissue of the lesion during sample collection. This is common in obese animals where the lesion may be surrounded by abundant subcutaneous fat.

(Courtesy Oklahoma State University teaching files.)

Figure 1-5 Samples collected from a prostatic carcinoma with areas of necrosis. A, Most slides were from aspirates of necrotic areas and contain predominantly necrotic cellular debris (black arrows). A single partially intact cell is present (blue arrow). These slides would be nondiagnostic. B, One of the aspiration attempts sampled a nonnecrotic area and the resulting slides contained numerous intact cells allowing a diagnosis to be made. This demonstrates the importance of sampling multiple sites of a mass.

(Courtesy Oklahoma State University teaching files.)

Figure 1-6 Images from an aspirate of a reactive lymph node. This sample was nondiagnostic because all of the cells have been ruptured due to excessive downward pressure being applied during sample preparation. A, The linear streaks of material represent nuclear chromatin of ruptured cells. B, Ruptured cells often appear to have “comet tails” all going the same direction.

(Courtesy Oklahoma State University teaching files.)

If possible, a minimum of four to five slides, representing collection attempts from several sites within the lesion, should be submitted from any lesion. If some of the samples appear to be excessively thick or if little to no material is apparent on the slides, make additional slides. With multiple slides the odds are better that at least one of them will be of diagnostic quality.

If multiple masses are sampled, always use a new needle and syringe with each mass. If this is not done, slides from one mass may be contaminated with cells left in the needle from previous collection attempts.

Collection Tip

Avoid blood dilution—Blood contamination (hemodilution) is another common cause of nondiagnostic slides. FNB with aspiration will collect the tissue of least resistance. If blood vessels within the lesion have been ruptured, the tissue of least resistance will be peripheral blood. Once significant blood contamination has occurred, it is difficult to salvage the sample. Additional collection attempts using a clean syringe and needle should be performed.

The two major causes of blood contamination are the use of too large a needle (<22-ga) and prolonged aspiration. Larger-bore needles do not usually collect more cells, but are more likely to rupture small blood vessels. As said before, material is often not visible in the syringe during sample collection, despite adequate numbers of cells being present within the needle. Any time material is visible in the hub of the needle, the collection procedure should be stopped and slides made immediately.

Some lesions are highly vascular, making it difficult to avoid blood contamination, even with good collection technique. In these cases, use of a nonaspiration technique may result in less blood contamination and more tissue cells for evaluation.

Collection Tip

Don’t be timid—Other reasons for poor cellularity of a sample are inadequate negative pressure (aspiration technique) and slow or shallow needle passages (nonaspiration technique). Needle passages should be quick and of sufficient length (although the size of the mass may limit the length of the needle pass).