Which technique should be used when preparing/processing samples? Smear cytology, liquid-based cytology, cell block, or histologic preparations?

Diagnostic performance differs by cytologic or histologic sample preparations (smear, rapid cytology, liquid-based cytology, cell block, and histology) and by staining methods [49]. Ideally, samples from solid pancreatic tumors should be examined by both histological and cytological evaluation.

Direct smear facilitates rapid staining and cytological diagnosis; thus, is an essential step in processing EUS-FNA specimens from pancreatic tumors. The assistant nurse places the stylet into the needle channel to extrude the aspirated sample onto the slide. Appropriate quantities of acquired sample material should be mounted on the slide for inspection of optical properties of the optimal specimen. Applying large quantities of the sample at once can lead to thick smears (with cells obscured within clusters) or clotting artifacts, while watery or small quantities of sample do not smear well on the slide causing air-drying artifacts [50]. Insufficient sample quantities, which cannot pick up the whitish component, can be processed by the cytospin method. In this method, the sample should first be immersed in saline and then centrifuged at 2000-3000 rpm for 2-3 minutes before being applied to the slide [51]. This technique involves separating the sample into multiple small aliquots as well as treatment with a hemolytic agent and mucus softener. This is favorable as it allows for optimal sample preparation with adequate cellularity and minimal artifacts.

When smear specimens are made using two slide glasses, usually one slide is prepared using the conventional air-dried method for rapid cytology with Diff-Quik, whereas the other slide is fixed in ethanol for later staining using Papanicolaou and HE stain [52]. Diff-Quik, a rapid version of Giemsa and Papanicolaou staining, allows for rapid cytological analysis in under a minute, even with watery samples containing an abundance of exfoliated cells [50].

Almost all false-negative cytologic results are due to errors in sampling, preparation of the sample, and interpretation of the sample. For this reason, liquid-based cytology (LBC) is an effective technique for sample preparation which can be manipulated automatically within only 2-4 minutes per sample after the acquisition of cytologic materials [53]. The results of comparisons between the diagnostic performance of LBC and smears for pancreatic cancer are conflicting. While Siddiqui et al. found LBC to be superior [54] (LBC vs. smear; 91% vs. 58%), Qin et al. found the techniques to be similar [55] (LBC vs. smear; 73.3% vs. 70%), and others found LBC to be inferior [56-58] (LBC vs. smear; 61.7-75.0% vs. 91.6-97.9%). However, there was a consensus that LBC specimens showed clearer backgrounds. LBC has the advantage of reducing false-negative results because it provides better specimen preservation, a clearer background with less mucin, necrotic material, and inflammatory cells, and higher cellularity, by reducing artifacts and extracellular elements compared with conventional smear cytology. Furthermore, prepared samples from LBC can be used for genetic analysis and IHC to provide further cytologic information.

The cell block is an effective technique that overcomes the disadvantages of conventional smear cytology and can lead to a definite diagnosis through IHC and molecular assays. Various cell block techniques have been developed over time: the traditional manual, involving rinsing the sample with 50% ethanol; the sodium alginate method, involving fixing the sample in 10% formalin and 1% sodium alginate; and the novel Cellient Automated Cell Block System (Hologic Inc, Marlborough, MA, Mass). All methods involve embedding the collected cell pellets in paraffin and cutting thin 3-5 µm sections before staining [59]. Sample prepared by cell block can be applied to IHC and specific molecular assays to differentiate between malignant and benign lesions and to determine tumor phenotypes. In one study [60] comparing the diagnostic abilities of the cell block and smear cytology using 33 pancreatic tumors or lymph node samples acquired through EUS-FNA, the cell block technique with IHC was superior to smear cytology in regard to sensitivity (92% vs 60%, p=0.02) and accuracy (94% vs 61%, p=0.003).

Recent technical and instrumental improvements in EUS-FNB for pancreatic solid tumors have enabled adequate histologic sampling, even using 22- or 25-gauge needles. According to a recent network meta-analysis [61], two RCTs reported that 25-gauge FNB was superior to 25-gauge FNA (RR, 1.17; 95% CI, 1.00-1.36) [20] and that 22-gauge FNB was more predominant than 25-gauge FNA (RR, 4.56; 95% CI, 2.49-8.35) [62] in regards to optimal histologic core procurement. They also reported that the 22-gauge FNB and 22-gauge FNA needles (RR, 1.01; 95% CI, 0.89-1.15) did not differ in terms of optimal histologic core procurement. IHC and molecular assays can be applied more easily to FNB samples than to samples from cytology or cell block. Ideally, EUS-guided tissue acquisition of pancreatic tumors should routinely include a histological evaluation by biopsy in addition to cytology.