Methods of tissue preparation after Endoscopic Ultrasound guided fine-needle aspiration without rapid on-site assessment: results of a randomized study.

The participating subjects underwent standard endoscopic ultrasound (EUS) examination with a linear echo-endoscope (GF180, Olympus, alpha 10, Japan) by an experienced endo-sonographer, who had performed over 5000 EUS procedures. All procedures were performed with anaesthetist-assisted propofol sedation. Once the mass was identified and confirmed to be at least 1cm in size, three passes of EUS FNA were performed without the presence of rapid on-site examination (ROSE). The choice of needle size (25G, 22G or 20G) and type (fine-needle aspiration (FNA) or fine-needle biopsy (FNB)) was at the discretion of the endo-sonographer. The material derived from each FNA pass was prepared by one of the following 3 tissue preparation techniques: (i) smearing plus cell-block (CS+CB), where material is divided to either CS or CB; (ii) cell-block alone (Pure CB); where material is only used in CB; (iii) direct histology alone (DH). The order of tissue preparation technique was randomized, which was generated by a computer program. The tissue preparation was performed outside the procedure room to blind the endo-sonographer on which specimen processing technique was used. The specimens were sent to the dedicated GI cytopathologist, where the specimens were processed and examined as outlined below. Techniques of tissue preparation: 1. Cytological smears: Using the stylet, 2 drops of specimen from the EUS FNA needle were expelled onto 2 pathology slides (1 drop per slide). The material on the slides was smeared, then fixed with alcohol and air-dried. The smears were done by the investigator who had dedicated training in cytological smearing technique. The remaining material in the FNA needle was expelled in Hank’s solution with 1ml of normal saline for cell-block preparation (see below for description). In the pathology laboratory, the slides were stained using Papanicolaou stain and examined by the dedicated cytopathologist. 2. Cell-block preparation: This technique involved expulsion of contents from the EUS FNA needle into Hank’s solution with 1ml of normal saline, and the specimen was then sent to the laboratory within 2 hours for processing. The contents were centrifuged, removed and fixed into formalin solution. The specimen was centrifuged again and formalin solution was removed before Agar gel was added. This was then embedded in Paraffin wax and cut at 4 microns to create slides. The slides were examined by the dedicated cytopathologist 3. Direct histology: This technique involved expressing the EUS FNA contents directly into Formalin and sending to the pathology laboratory for routine histological processing. 4µm sections were cut from paraffin embedded tissue blocks and stained routinely with Haematoxylin and Eosin. Special stains and immunohistochemistry were done when additional tests were required for diagnostic purposes.