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 (Patent Blue V, solution for injection 50 mg/2 ml, GUERBET, France)6 and the colloid modulator of velocity –HES, (also known as hydroxyethyl starch)(VOLUVEN solution for infusion, 1 x 500 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 color constituent (Patent Blue 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 semi sessile adenoma of the colon and rectum (diagnosis group ICD-10 codes D 12.0, D12.2-D12.8) with adenomas of size ranking from 5 to over 40 mm in its largest dimension. Exclusion criteria for polypectomy (EMR contraindication) were thrombocytes (PLT) less than 50 × 109/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. All patients within the period which qualified for EMR were administered the new EMR composition.
Informed consent 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 law8.
In all cases, the submucosal injection solution was prepared ahead in 10 ml syringes, containing either isotonic saline, saline with epinephrine or saline with Methylene Blue or Patent Blue V. The new EMR composition was prepared in the same 10 ml syringe form with composition as specified above.
The administration protocol followed the routinely used procedure and was in accordance with the relevant guidelines and regulations. The only change in the standard application protocol was replacing the syringes containing a physiological (saline) solution, Methylene Blue/Patent Blue V, and epinephrine with the syringes containing new composition.
During the endoscopic session, the close neighbor of suspected tissues was injected by submucosal injection via the endoscopic channel leading to bolus formation in submucosa and subsequent volume and color changes in the area. Depending on the size of initially observed polyp, the amount of injected EMR solution was 1–3 ml for small, 5 mm diameter polyps, the larger lesions eg, 40 x 50 mm surface required 50–80 ml of EMR solution. There were no noticeable differences in volumes of EMR solution compared to the typical volumes of EMR solutions applied before. When new polyps were elevated and became visible in the field of view, the additional EMR solution was applied, when necessary. The elevated polyps were removed using a polypectomic loop, and removed tissue was analyzed for histology. As a rule, several polyps became visible lasting several minutes and were removed in a single step during the same endoscopic session. En-bloc resection rate was not significantly different for the new solution. Generally, polyps below 20 mm size were resected en-bloc, while above that size, piece-meal resection was more typical. In some cases, polyps of about 30 mm size could be removed en-bloc.
Cell line model
The human colorectal carcinoma cell line HCT116 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 h. The cells were then starved in saline solution without nutrients for 24 h, 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 remains 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 h starving in saline solution. After 24 h, 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. The estimated volume of the saline solution replaced by EMR composition was in 100 µl range. Subsequently, we prepared a set of substances without a color constituent. In all cases, the cells exposed to the solution were followed by 10 min of live video flow on a Cytation 3 Cell Imaging multimode sensor (BioTek Instruments, Inc.) and visually evaluated for cell count, cell volume, and cell shape change.