Chapter 1

Sample Collection and Preparation

James H. Meinkoth, Rick L. Cowell, Ron D. Tyler and Rebecca J. Morton

Evaluation of cytologic samples has become well established as a method of obtaining a diagnosis of lesions in a wide variety of tissues. Cytology and histopathology will likely always remain complementary diagnostic procedures, reflecting a trade-off between the lower degree of invasiveness of sample collection and more rapid turnaround time with cytology and the increased amount of information available from the ability to evaluate tissue architecture with histopathology. However, the ever-increasing availability of advanced imaging techniques has resulted in an increased reliance on cytopathology to evaluate focal lesions of internal organs, which previously could not be reliably sampled. As clinicians have increased their use of this diagnostic modality and cytopathologists have become more experienced with the wider variety of lesions and tissues sampled, the spectrum of disease processes that can be identified by cytology and the reliability and precision of the diagnoses for lesions of many tissues have increased.

Other than the experience of the cytopathologist evaluating the samples, one of the major factors determining the diagnostic value of cytologic specimens is the quality of the sample. The diagnostic yield of cytology is noticeably higher in the hands of clinicians who have a great deal of experience with obtaining cytologic specimens. With histologic specimens, once the tissue sample is collected and placed in an appropriate amount of formalin, laboratory technicians handle the remainder of sample preparation. With cytology, the clinician is faced with the responsibility of not only collecting an adequately representative specimen but also preparing the slides that are to be examined and, often, staining of the slides as well. Because the cells to be examined are not grossly visible during sample collection and slide preparation, it is often difficult to tell whether an adequate specimen has been obtained at the time of the sampling procedure.

Collection and preparation of cytologic specimens is definitely a skill gained only through experience and refinement of technique based on the results obtained. Many clinicians (and owners) are understandably frustrated when a sample submitted is determined to be nondiagnostic. Fortunately, an understanding of some basic principles of sample collection and familiarity with some of the more common pitfalls related to cytologic sample preparation can increase the odds of a diagnostic result.1–5

Methods of Sample Collection

Several methods of collecting samples for cytologic analysis exist. The indications for each are outlined in Table 1-1.

TABLE 1-1

Indications for Various Methods of Sample Collection

Collection Method	Indications for Uses	Comments
Fine-needle biopsy (aspiration or nonaspiration method)	Masses (surface or internal)	Best method for cutaneous or subcutaneous masses because it avoids surface contamination
	Lymph nodes
	Internal organs	Best method for minimally invasive sampling of internal organs or masses
	Fluid collection
Impression smear	Exudative cutaneous lesions	Most useful for identification of infectious organisms
		May yield only surface cells and contamination (problem with ulcerated tumors)
	Preparation of cytology samples from biopsy specimens	With biopsy specimens, it is imperative to blot excess blood from sample
		Impression smears of biopsy specimens must be made before exposure of biopsy sample to formalin
Scraping	Used with flat cutaneous lesions that are not amenable to fine-needle biopsy	With dry cutaneous lesions (e.g., ringworm), it is important to scrape sufficiently to obtain some blood or serum to help cells stick to slide
	Preparation of cytology samples from poorly exfoliative biopsy specimens
Swab		Generally used only when anatomic location not amenable to collection by other means
	Vaginal smears
	Fistulous tracts	With fistulous tracts, most useful in classifying type of inflammatory response and identifying infectious organisms

Fine-needle biopsy (FNB) can be performed using a standard syringe and needle with or without aspiration (as described later). This is the best overall method for sampling any cutaneous mass or proliferative lesion.¹ FNB allows collection of cells from deep within the lesion, avoiding surface contamination with inflammatory cells and organisms that often plague impression smears, swabs, or scrapings. Surface cells are often poorly preserved and may show artifacts related to cellular aging and exposure to secondary inflammation responses, especially with ulcerated masses. These changes can make evaluation of the significance of cellular atypia more difficult. A classic example of this is masses of the urinary bladder. Samples collected by traumatic catheterization often contain cells that show significant degeneration and artifact from aging and prolonged exposure to urine (Figure 1-1). Conversely, samples collected via FNB from deep within the lesion are typically well preserved and easier to evaluate (see Figure 1-1, A). FNB is also the only practical technique for sampling of subcutaneous or internal organs or masses.

Figure 1-1 Photomicrograph of samples collected from transitional cell carcinoma.
A, Sample collected by fine-needle biopsy of the mass. The cells are well preserved allowing for examination of nuclear and cytoplasmic detail. B, Sample collected by traumatic catheterization. These samples typically collect superficial cells that show marked changes resulting from cellular aging and exposure to urine. Nuclear degeneration is noted as a homogenous light pink-purple color as well as fragmentation with numerous clear spaces evident (arrows). (Courtesy Oklahoma State University Teaching Files)

Selection of Syringe and Needle

FNBs are collected with a 22- to 25-gauge needle and a 3- to 20-mL syringe. The softer the tissue, the smaller are the needle and syringe used. It is seldom necessary to use a needle larger than 22-gauge for aspiration, even for firm tissues. When needles larger than 22-gauge 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. The size of the syringe is not as critical when the samples are collected using the nonaspiration technique.

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 1-2). Strong negative pressure is applied by withdrawing the plunger to about three fourths the volume of the syringe (Figure 1-3). 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 1-2 Aspiration technique of fine-needle biopsy.
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 1-3 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, no material will be 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 one end of a glass microscope slide by rapidly depressing the plunger. When possible, several preparations should be made, as described later in this chapter (see “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.4–6 The nonaspiration technique works well for most masses, especially those that are highly vascular.¹ This technique is similar to the standard FNB aspiration 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 1-4). 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 in an attempt to stay along the same tract, similar to the action of a sewing machine. 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 prefilled 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.⁶

Figure 1-4 Nonaspiration technique of fine-needle biopsy.
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.)

Some perform the nonaspiration technique with a needle only with no attached syringe. This may allow for even greater control of the placement and movement of the needle, although the syringe must then be attached after sample collection to expel the material from the needle. Another variation which has been recommended for ultrasound-guided collection is to have an intravenous fluid extension set placed in between the needle and the syringe.⁶ This allows freedom of movement of the needle with one hand during the collection procedure. The syringe can be hung over the shoulder during collection, and then the other hand can be used to quickly expel the material onto the slide.

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.

Collection Tips

Impression Smears

Impression smears can be made from ulcerated or exudative superficial lesions (Figure 1-8) or tissue samples collected at surgery or necropsy (Figure 1-9). Impression smears from superficial lesions often yield only inflammatory cells even if the inflammation is a secondary process; neoplastic cells may not exfoliate in exudates or impression smears of ulcerated masses. If possible, FNB of tissue under the ulcerated or exudative area should be collected in addition to the impression smears. Inserting the needle at a nonulcerated area will help reduce contamination during collection. Impression smears of exudates or ulcers are most beneficial for determining if bacterial or fungal organisms are present. Keep in mind that bacteria may reflect only a secondary bacterial infection.

Figure 1-8 Ulcerative, exudative lesions on the face of a cat.
This lesion is well suited for impression smears. Slides from these lesions revealed inflammatory cells and many Sporothrix organisms. (Courtesy Oklahoma State University teaching files.)

Figure 1-9 Impression smear of tissue removed at surgery.
The tissue is trimmed so that a fresh surface is created for making the impression smear. If normal tissue surrounding a mass has been excised, it is important to be sure that the tissue is cut through the area of interest. (Courtesy Oklahoma State University teaching files.)

Ulcerated areas should be imprinted before they are cleaned. The lesion should then be cleaned with a saline-moistened surgical sponge and reimprinted or scraped.

To collect impression smears from tissues collected during surgery or necropsy, the tissue should first be cut so that a fresh surface for imprinting is created (see Figure 1-9). Next, the excess blood and tissue fluid should be removed from the surface of the lesion being imprinted by blotting with a clean absorbent material (Figure 1-10). Excessive blood and tissue fluids inhibit tissue cells from adhering to the glass slide, producing a poorly cellular preparation. Also, excessive fluid inhibits cells from spreading and assuming the size and shape they usually have in air-dried smears. After excess blood and tissue fluids have been blotted from the surface of the lesion, the surface of the lesion is touched (pressed) against the middle of a clean glass microscope slide and lifted directly up (Figure 1-11). This should be repeated several times so that several tissue imprints are present on the slide. If the excess blood has been adequately removed, the tissue will stick somewhat to the slide and will appear to peel off the slide, if removed slowly. Properly made slides will have slightly opaque areas at the areas of the impressions but should not have excessively thick areas of blood (Figure 1-12).