The success of an FNA procedure depends on the nature of the lesion (e.g., size, location, and consistency), the skill of the aspirator, and the availability of rapid on-site evaluation. Large palpable lesions can be aspirated by a pathologist, and small deeply seated lesions are usually aspirated by the radiologist under image guidance. Usually, a 22- to 25-gauge needle is used, and two to three needle passes are made. Aspirated materials are expressed onto several glass slides and smeared out.

Two types of stains are typically performed: Diff-Quik staining using air-dried smears and Papanicolaou staining using smears fixed in 95 % ethanol or Carnoy’s solution. Both stains are complementary for cytologic diagnosis. Diff-Quik staining preferably highlights cytoplasmic details and extracellular or background contents, whereas Papanicolaou staining allows better visualization of nuclear characteristics such as the nuclear membrane, chromatin, and nucleoli. In some laboratories, liquid-based preparations (e.g., ThinPrep and SurePath) may be used.

Cells retained in the needle hub are rinsed into cell-preservative medium (e.g., RPMI-1640 medium) and usually spun down to make cell block or cytospin slides, depending on the size of the pellet after centrifugation. However, if a hematopoietic malignancy is suspected on the basis of clinical or cytologic findings and a flow cytometric immunophenotyping is anticipated, fresh cells should be preferentially collected and suspended in cell-preservative medium containing fetal bovine serum.

Rapid on-site evaluation ensures high diagnostic accuracy by assessing sample adequacy and determining triage strategy. It is usually performed by an on-site pathologist who correlates the cytologic findings of direct smears (Diff-Quik-stained smears, with or without Papanicolaou-stained smears) with the clinical and radiologic findings (so-called triple test ).

Key factors to determine during on-site rapid evaluation in stepwise fashion:

During the triple test, detailed clinical and radiologic information should be obtained via chart review and communication with caregivers. In addition to the age, sex, clinical history, and current presentation of the patient, it is important to obtain radiologic information about the current lesion. For example, for a pulmonary lesion, it is helpful to know the size, location (central vs. peripheral and upper lobe vs. lower lobe), number (solitary vs. multiple), margin (well circumscribed vs. poorly defined), and other information (such as the presence of a cavitary component or hilar lymphadenopathy and a history of smoking). The presence of hilar lymphadenopathy and a smoking history favor a primary lung carcinoma over metastasis. For a patient with a history of breast carcinoma, it is helpful to know the primary cancer type (ductal, lobular, or others), the histologic and nuclear grade, the lymph node status and hormone receptor status, and the interval duration from the primary cancer diagnosis to the current FNA and location of the new lesion (locoregional vs. distant site). Abnormal laboratory (serum) findings may also provide useful diagnostic clue of some tumors, for example, CA19.9 in pancreatic adenocarcinoma, AFP in hepatocellular carcinoma and some germ cell tumors, CA125 in ovarian serous carcinoma, calcitonin in medullary thyroid carcinoma, and PSA in prostatic adenocarcinoma. Common conditions should be considered first before considering rare scenarios.

Sample adequacy is determined on the basis of a triple test. If a sample contains material representative of the target lesion found in clinical or radiologic assessment, the sample is considered “adequate.” If a sample is scant in cellularity or contains fibrotic tissue that is incompatible with the clinical or radiologic findings or shows significant cellular distortion or artifacts, it is deemed to be “inadequate.” In any case with a mismatched triple test, additional material should be requested.

Any FNA sample that appears to be adequate during on-site evaluation should be triaged properly to ensure that a definitive yet informative diagnosis can be rendered. The strategy of sample triage relies on the cytologic features of the direct smears during rapid on-site evaluation using low and high magnification (Table 2.1). The general features of malignancy are listed in Table 2.2. Sample triage may need to request additional aspirates for making a cell block which allows for better architectural assessment and potential immunostaining, for microorganism cultures if the lesion appears to be infectious in nature, for flow cytometric immunophenotyping if non-Hodgkin lymphoma is suspected, for cytogenetic and molecular studies if small blue cell tumor in pediatric patients is encountered, or targeted therapy is possibly applied and for requesting a concurrent core needle biopsy if sarcoma or Hodgkin lymphoma is suspected (Fig. 2.1). Cytologic features on smears that are suggestive of each of the “Big 4” categories are summarized below.

General Features of Carcinoma (Fig. 2.2)

General Features of Melanoma (Fig. 2.3)