Preparation of EMR solution. The EMR solution we used consists of three components: the physiological (saline) solution, in which the visual contrasting aid - the sodium salt of [4-(alpha-(4-diethylaminophenyl)-5-hydroxy-2,4disulfophenyl-methylidene)-2,5cyclohexadiene-1-ylidene]diethyl-ammonium hydroxide inner salt (PATENTEBLAU V sol. inj. 2ml/50mg, GUERBET, France)6 and the colloid modulator of velocity - a Hydroxyethyl starch (HES) (VOLUVEN® sol. inf. Then, 1x500 ml, Fresenius Kabi, Bad Homburg, Germany)7 was dissolved. We prepared a mixture of EMR composition by mixing 500 ml of isotonic saline solution with 1 ml of colour constituent (PATENTEBLAU V) followed by diluting with a HES drawn up into the syringe with Combi-Stopper (syringe bung) in the ratio 3:7 under aseptic conditions and apyrogenic (dilution closed path) in a laminar flow hood.
The selection of suitable patients and administration protocol. From March 1st to June 30th, 2014, 62 patients (19 females and 43 males of average age 56,8 and 61,1 years, respectively) were indicated for EMR at the Department of Gastroenterology of the Central Military Hospital.
The patients were selected for EMR either directly at the Gastroenterology Department or following doctor referral at another department of gastroenterology in Slovakia due to a preventive examination. The patients were diagnosed during routine colonoscopy with sessile or semisessile adenoma of the colon and rectum (diagnosis group ICD-10 codes D 12.0, D12.2-D12.8). All 62 patients undergoing polypectomy at The Department of Gastroenterology of the Central Military Hospital during this period were administered the new EMR solution. Exclusion criteria (EMR contraindication) were thrombocytes (PLT) less than 50x109/l, prothrombin time ratio (INR) more than 1.4, discontinuation of anticoagulant or dual antiaggregant therapy less than seven days before EMR. The number of patients (sample size n = 62) who underwent polypectomy with EMR intervention was typical for a 4-month period at the hospital. There were no other selection and/or exclusion criteria applied. In other words, all patients within the period were administered the new EMR composition, with probability p = 1.0.
was obtained from all patients. The procedure was approved by the Ethical Committee of the Central Military Hospital SNP Ružomberok, Slovakia, according to valid Slovak law.
The administration protocol followed the routinely used procedure and was performed in accordance with the relevant guidelines and regulations. The only change in the standard application protocol was replacing the EMR composition consisting of a physiological (saline) solution, PATENTEBLAU V, and adrenaline with the new composition.
During the endoscopic session, the close neighbourhood of suspected tissues was injected by submucosal injection via the endoscopic channel in a 1–20 ml volume (depending on neoplasm size), leading to bolus and subsequent volume and colour changes. The elevated polyps were removed using a polypectomic loop, and removed tissue was analysed for histology. As a rule, several polyps became visible with the formed stem lasting several minutes and were removed in a single step during the same endoscopic session.
Cell line model. The human cancer cell line HCT116 (large intestine) was purchased from American Type Culture Collection (ATCC, CCL-247™) and cultured in RPMI 1640 growth medium (Biosera, Kansas City, MO, United States). The growth medium was supplemented with 10% foetal bovine serum (FBS) and 1x HyClone™ Antibiotic/Antimycotic solution (GE Healthcare, Little Chalfont, UK) and maintained in an atmosphere containing 5% CO2 in humidified air at 37°C. Before experiments, the viability of cells was analyzed by trypan blue assay. The cell line was authenticated by the ATCC Laboratory Authentication Service using Sanger sequencing. ATCC declared no Mycoplasma contamination. Before experiments, cell specimens were tested again for Mycoplasma contamination by DNA staining and fluorescence microscopy visualization with negative results.
For experiments, cells were seeded in 96-well low-density plates and maintained in complete culture medium for 24 hours. The cells were then starved in saline solution without nutrients for 24 hours, mimicking the patient preparation before EMR surgery.
The initial cell culture was grown to 10 thousand cells per well, forming plaques. During starvation, some of the cells detached and floated freely in the medium. We removed free-floating cells with the part of liquid media, and the removed volume was then restored by adding the same volume of saline solution. Even though the saline solution detaches part of the starving cells, a significant fraction of the starving cells remain attached to the density plate after adding the saline solution.
Adding the contrasting composition. The EMR composition was applied for two groups of cells – nonstarving and after 24 hours starving in saline solution. After 24 hours, the nonstarving cells adhered to the density plate, while in starving cells, the free-floating cells had to be removed, and the media was replenished by saline solution first. In both cases, the last step of the procedure consisted of replacing the saline medium with an EMR composition. For starving cells, approximately half of the volume of saline was replaced by EMR. Subsequently, we prepared a set of substances without a colour constituent. In all cases, the cells exposed to the solution were followed by 10 minutes of live video flow on a Cytation 3 Cell Imaging multimode sensor (BioTek Instruments, Inc.) and evaluated visually for cell count, cell volume, and cell shape change.