Cytology may be helpful for differentiating mucinous from non-mucinous cysts through identifying mucin-producing cells (Table 4.1). When positive, the characteristic cytology of SCNs is that of cellular sheets of low-cuboidal, glycogen-containing cells without cellular atypia. The cytoplasm is clear and without vacuoles and intracellular cytoplasmic inclusions. SCNs demonstrate positive immunostaining for cytokeratins AE ₁ and AE ₃ and positive staining with the periodic acid-Schiff reaction (Fig. 4.1) [30–33].

In contrast, low-grade MCNs are characterized by honeycomb sheets and clusters of columnar, mucin-containing cells with rare, small, papillary sheets [34, 35]. In addition, MCNs have abundant mucin in their background, which differentiates MCNs from SCNs [35]. Because of the extreme heterogeneity of the epithelial lining of MCNs, one must remember that there may be marked discrepancies between the cytologic typing and the subsequent histologic diagnosis of these neoplasms. Importantly, however, the degree of cytologic atypia has been shown to be predictive of malignancy [36]. Also cytology may diagnose malignant cystic lesions (e.g., cystadenocarcinoma) by demonstrating malignant cells or cells with high-grade atypia (dysplasia) in the aspirated cystic fluid [37].

IPMNs are characterized on FNA cytology by the presence of papillary clusters lined by columnar, mucin-containing cells, usually with some degree of atypia [38]. Although low-grade MCNs may have a few papillary clusters, the papillary projections in MCNs are usually not as tall, abundant, and striking as the clusters observed in IPMNs. One study suggested the presence of hemosiderin-laden macrophages in the aspirated fluid as a finding supporting the diagnosis of SCN [39]. In this study, hemosiderin-laden macrophages were identified in 11 of the 21 cases of SCNs (52 %) but in only 2 % of IPMNs and MCNs and in only 9 % of pseudocysts (Fig. 4.1e); at the current time, however, the presence of hemosiderin-laden macrophages is not considered a reliable diagnostic feature of SCN and can only serve as a surrogate marker to suggest the diagnosis of SCN. In contrast to PCNs, FNA of pancreatic pseudocysts yields a “dirty” material with macrophages and other inflammatory cells, proteinaceous precipitates, and calcified debris.

Macroscopically, the aspirated fluid of SCNs is typically thin, clear, and without mucin, but on occasion may be bloody [40] (Table 4.1). In contrast, the aspirated fluid in mucinous neoplasms is thick, viscid, and of a mucinous nature; the mucinous nature of the fluid can often be appreciated grossly in the endoscopy suite when smears are made. A typical analysis of the aspirated cystic fluid would include biochemical testing (for mucin, tumor markers, and amylase) and potentially for molecular analysis.

EUS-guided transluminal FNA is a well-tolerated and safe procedure when performed by an experienced operator. EUS, however, remains a specialized examination limited to a few centers with the necessary equipment and especially within the obligate experience of the endoscopist. Analysis of the cystic fluid depends on the ability of local cytologic and laboratory testing and is not available routinely. Potential complications of needle aspirations of cystic fluid include bleeding due to vascular injury (clinically relevant bleeding, <1 %; self-limiting intracystic hemorrhage ~6 %), pancreatitis (~1–3 %), infection (<1 %), and, at least in theory, the seeding of malignant cells along the tract of the needle [36, 55, 56]. Periprocedural antibiotics are used commonly to decrease the risk of introduced intracystic infection. Most endoscopists also tend to remove as much fluid as possible to decrease the theoretic risk of bacterial inoculation of the fluid.

Consideration should be given to the size of the lesion and the size of the individual “cysts,” because aspirates may be very limited for small lesions which might preclude or complicate cyst analysis and cytology. A small (<1 ml) volume of the aspirates may be due to the high viscosity of the aspirated fluid (due to the presence of mucin) in MCNs or to the small size of the cysts in the cystic lesion of SCNs [57]. Moreover, the size of the cystic lesion is of clinical importance when deciding about the indication to perform EUS-guided FNA. There is no uniform agreement in relation to the cutoff size for EUS-guided aspiration. Some investigators have proposed a cutoff diameter of 1.5 cm [58, 59]; these groups rarely perform EUS-guided FNA in smaller lesions. A size of >1.5 cm was chosen based on the likelihood that it would allow the aspiration of an adequate volume of fluid for analysis [59]. Others use a threshold of 2.0 cm [60, 61], while some groups do not quote specific size criteria [62, 63]. A few centers advocate EUS in all patients with asymptomatic cysts [64, 65].

Some reports are very enthusiastic about the value of the combination of EUS and FNA in predicting which lesions require resection, with reported sensitivities and specificity of 97 and 100 %, respectively [34]; in contrast, other studies report less convincing results. In a large, prospective, multicenter trial, cyst fluid cytology had a high specificity (83 %) but a low sensitivity (35 %) for distinguishing mucinous vs. non-mucinous cysts [43]. In this study, the sensitivity for diagnosing malignancy was only 22 % [43]. Others have reported diagnostic accuracy of EUS-guided FNA ranging only between 10 and 60 % [31, 33, 66]. Genevay et al. re-reviewed the cytology slides of 112 patients with histologically confirmed, mucinous cysts of the pancreas. They found that high-grade atypia (dysplasia) in the epithelial cells had a specificity of 85 % and sensitivity of 72 % for predicting malignancy in mucinous cysts [37]. Pais et al. reported that EUS-guided FNA cytology proved helpful with a sensitivity, specificity, and accuracy for the diagnosis of malignancy of 75, 91, and 86 % respectively [46]. The reported variations in diagnostic accuracy likely are due to differences in the sampling technique, the experience of the endoscopist or cytopathologist, and the thoroughness with which clinicopathologic correlation was accomplished.

The low cellularity of the aspirated pancreatic cyst fluid is a major limitation of FNA cytology for the differentiation between the different types of PCNs. As a result, cytologic examination of the cyst fluid is often non-diagnostic. In the study by Huang et al. [31], 32 % of the 28 cases were classified initially as “non-diagnostic specimens” or as having “no malignant cells.” Even in re-aspirated specimens, the interpretations were usually unchanged. In contrast, when such samples are positive, the specificity is high [31, 67]. As reported by Al-Haddad et al. [68], brushing the cyst wall during FNA increases the diagnostic yield of EUS-guided FNA; in their study of 37 patients with a pancreatic cystic lesion, the sensitivity of cyst fluid FNA for detecting intracellular mucin was 23 % but increased to 62 % by brushing the cyst wall [68].

The accuracy of preoperative differential diagnosis can also be increased by obtaining image-guided “mini-biopsies” (Tru-Cut or core tissue biopsies) from the solid component of a cystic neoplasm or from the wall of the cyst (cyst wall puncture) [69, 70]. Recently, although the technology to allow an endoscopic, Tru-Cut core tissue biopsy has been developed, it is not yet available widely. Early data with EUS-guided Tru-Cut biopsies of PCNs have shown very promising results. In the study by Levy et al., 5 of 6 SCNs were identified correctly on a Tru-Cut biopsy [71]. In the study by Belsley et al. [30], both biopsies performed with the Tru-Cut technique were diagnostic, without any procedural complications reported.

Apart from the sampling error, “contamination” of the aspirates from the normal (mucus-producing) epithelium of the gastrointestinal tract during the passage of the needle may pose another problem in interpreting the results of EUS-guided, FNA cytology (Fig. 4.1d). Such an error may result in the misdiagnosis of a SCN as a mucinous neoplasm. Indeed, caution should be taken not to misinterpret mucin or mucin-producing cells as the mucinous material or epithelial cells of a MCN [72]. Mucin from the gastrointestinal tract tends to be thinner, wispier, and devoid of degenerated epithelial cells and the inflammation more typical of MCN [67, 72]. Unfortunately, this distinction is not always easy, because the features can overlap. Cell block material and ancillary tests (i.e., stains for glucagon, cystic levels of carcinoembryonic antigen (CEA), amylase activity, viscosity, etc.) may help to clarify the differential diagnosis, but only if the results are truly informative. Moreover, epithelial cells from the GI tract tend to form large, cohesive, monolayered sheets consisting of uniform, columnar cells without cytologic atypia and often with a luminal edge. These columnar cells usually do not contain abundant cytoplasmic mucin, and thus the cell border is not as prominent as that in MCN cells. Incarcerated, mucin-producing goblet cells are a constant feature of incidentally sampled duodenal epithelium; also, the openings of the crypts of Lieberkuhn or the pits may be seen in some cell groups [31, 67, 72]. In contrast, MCN cells are characterized by a mucin-rich columnar epithelium with thick mucin in the background. Although MCN cells may appear extremely bland, they often exhibit at least focally some cytologic atypia and architectural complexity [72].

From a practical point of view, it should be emphasized that when the imaging features of the cystic lesion are virtually diagnostic, FNA can be omitted, and the lesion should then be managed appropriately (see below). FNA should also be omitted when the cystic lesion is symptomatic, because in this case, resection is clearly indicated. FNA should probably be entertained only when its results may change the therapeutic plan, e.g., when high-quality, cross-sectional imaging reveals non-diagnostic findings or when the clinical and morphologic characters of the cystic lesion have changed during follow-up [73]. Another potential indication of FNA is when a non-operative approach is considered for a presumed SCN not diagnosed confidentially on cross-sectional imaging. In this case, if the results of FNA analysis of the cystic fluid are compatible with a MCN, the conservative approach should be reevaluated.