Sample Size Calculations
Sample size calculations were performed assuming a mean anastomosis construction time of 20 minutes with a standard deviation of 1 minute, based on previous studies, (13, 14, 24) an expected 10% decrease in anastomosis time, 90% power and a 0.05 alpha (https://clincalc.com/stats/samplesize.aspx). Calculations determined a minimum of 5 anastomosis per group were required.
Collection of Jejunal Specimens
Jejunal segments were collected from horses slaughtered at commercial abattoirs using captive bolt. Within 45 minutes of euthanasia, the gastrointestinal tract was examined to confirm there were no gross abnormalities present. The jejunum was then harvested and ingesta evacuated from the lumen. Jejunal segments were submerged into ice water for transport and stored at 4˚C until utilized.
The jejunum was divided into 40 cm segments. Segments were labelled consecutively from oral to aboral, and randomly allocated into four groups: anastomosis with 15 cm Frimand Duo-Grip (FR) needle holders (Group 1), anastomosis with 16 cm Mayo-Hegar (SMH) needle holders (Group 2), anastomosis with 20.5 cm Mayo-Hegar (LMH) needle holders (Group 3); or control Group (no anastomosis performed; Group 4).
Anastomosis Construction
Single layer end-to-end jejunojejunal anastomoses were performed on the Group 1, 2 and 3 segments using a simple interrupted Lembert pattern as described elsewhere(41). A 3-0 monofilament absorbable suture (72% glyconate; Monosyn, B.Braun Surgical, Rubi, Spain) on a 26 mm taper needle was used by the same right-handed ECVS Diplomate with each of three randomly allocated needle holders (Figure 1):
1.) 15 cm Frimand Duo-Grip (FR; Stille Surgical Instruments, Torshälla, Sweden),
2.) Stille 16 cm Mayo-Hegar (SMH; Stille Surgical Instruments, Torshälla, Sweden) and
3.) Stille 20.5 cm Mayo-Hegar (LMH; Stille Surgical Instruments, Torshälla, Sweden).
Jejunal segments were sharply transected using Metzenbaum scissors. Stay sutures were placed at the mesenteric and antimesenteric borders of each portion of transected jejunal segment to allow the assistant to apply tension to appose the jejunal portions as required during the anastomosis. Suture bites were taken approximately 2-3 mm from the incised edge. An Adson Brown tissue forceps was used to manipulate tissue and suture needle in a no touch technique. The assistant cut suture tails as directed by the surgeon.
Each anastomosis construction was timed and videoed for later review. The time from first contact of needle with jejunum to the suture tail of last suture being cut was recorded as “anastomosis construction time”. The number of sutures placed were recorded for each anastomosis, and the anastomosis construction time was divided by the number of sutures placed to calculate the “time per suture”. The cadaver the jejunal segment originated from and session in which the anastomosis was performed were considered to be potential confounders, so were also recorded.
Biomechanical Testing
Bursting pressures of group 1, 2, 3 and 4 segments were determined using a previously described technique(14, 15, 22). Immediately after the anastomosis was performed, the jejunal segments were individually submerged in a bath of room temperature water. An irrigation pump (Karl Storz, Hamou Endomat 26331120) with fluids coloured with green food dye (Queen, Green Food Colouring) was connected to one end of the jejunal segment and secured with a zip tie at 15 cm from the anastomosis, with 5 cm excess jejunum left on the non-pressurized side of the zip tie. The other end of the jejunal segment was connected to a blind-ended pressure monometer (Livingstone, Aneroid blood pressure sphygmomanometer), again 15 cm from the anastomosis with 5cm excess jejunum external to the zip tie (Figure 2). Control segments were secured with 30 cm segments between the connection for the fluids and monometer, leaving 5 cm excess jejunum either side of the pressurized segment.
The green coloured fluid was progressively pumped into the jejunal lumen whilst intraluminal pressures were recorded until the anastomosis or intestinal segment failed. Maximum pressure reached prior to failure was recorded as “bursting pressure” in mmHg and the location of egressing fluid at that point was recorded.
Statistical Analysis
Descriptive statistics for the cadavers, jejunal segments, number of anastomoses in each group, number of sutures placed and time between euthanasia and testing were calculated and reported as mean ± standard deviation (SD).
Normality of the anastomosis construction time and bursting pressure was determined using Kolmogorov-Smirnov and Shapiro-Wilk tests. Mean anastomosis construction time and bursting pressure were compared between the 3 groups using generalized linear models and then recalculated with additional variables added as random effects. Comparison of the marginal means of the adjusted and unadjusted models were used to identify confounders; confounding was confirmed if the adjusted model marginal means varied from the unadjusted model by >20%.
Pearson’s correlation was used to determine correlation between anastomosis construction time and bursting pressure.
Statistical analyses were performed using R studio software (RStudio Version 1.2.5001, Boston, MA) and SPSS (IBM Corp. Released 2016. IBM SPSS Statistics for Windows, Version 24.0. Armonk, NY: IBM Corp.). P <0.05 was considered significant for all statistical tests.