Metabolism, infl ammation and postoperative time are the key to early diagnosis of anastomotic leak

Objective: The aim of the study was to fi nd laboratory samples for early diagnosis of anastomotic leak. Summary background data: Anastomotic leakage after rectal cancer surgery is a severe complication with high mortality. Outcome is highly dependent on early diagnosis. Methods: 29 patients were investigated postoperatively after having undergone low anterior resection due to cancer recti. Patient outcomes were divided into three groups: Anastomotic leak in 7 patients, other complications in 9 patients and 13 patients who were free of complications. Patients were monitored every 6th hour with blood and intraperitoneal samples in order to identify laboratory markers for early detection of anastomotic leakage. An anastomotic leak index was created, a scoring system where points count for values higher than reference values of CRP and interleukin 6 in blood and intraperitoneal lactate, lactate/ pyruvate ratio and interleukin 6 were measured at 18, 24, 42 and 48 hours postoperatively. Results: Signifi cant differences between groups were found regarding CRP, Interleukin 6, fi brinogen and D-dimer in blood. Intraperitoneal differences were found not only in lactate and lactate/pyruvate ratio measured by microdialysis, signifi cant differences in interleukin 6, interleukin 10 and tumour necrosis factor-α could also be demonstrated between the groups. The anastomotic leak index had a sensitivity and specifi city of 86% (p=0.0007). Conclusions: The most important factor was time after operation. No laboratory parameter in itself could predict an anastomotic leak but the anastomotic leak index was a useful tool in the monitoring and assessment of clinical outcome. Mini abstract: Patients with anastomotic leak after rectal surgery were monitored with higher intraperitoneal cytokines and lactate/pyruvate ratio. The results suggest intraperitoneal microdialysis combined with blood samples of CRP and IL 6 as a feasible method for early diagnosis of anastomotic leak. Research Article Metabolism, infl ammation and postoperative time are the key to early diagnosis of anastomotic leak Daniel T Jansson1, Ioannis Oikonomakis2, Ida E U Hall Strand3, Adrian D Meehan4 and Kjell S Jansson2* 1Medical University of Gdańsk, Poland 2Department of Surgery, Örebro University Hospital, Södra Grev Rosengatan, 701 85 Örebro, Sweden 3Department of Medicine, Skaraborg Hospital, Lidköping, Sweden 4Department of Geriatric, Örebro University Hospital, Örebro University, Örebro, Sweden Received: 23 September, 2019 Accepted: 24 October, 2019 Published: 25 October, 2019 *Corresponding author: Kjell Jansson, MD, Associate Professor, Department of Surgery, Örebro University Hospital, Södra Grev Rosengatan, 701 85 Örebro, Sweden, Tel: +46196021000; E-mail:


Introduction
Anastomotic leakage after colorectal surgery is a common and feared complication. The incidence of anastomotic leakage in Sweden 2015 reported by the Regional Cancer Center (RCC) was 7%-12% (depending for instance on hospital) and postoperative 30 days mortality was 1%-6% (depending for instance on the age of the patient). Surgical results have improved in recent years primarily because of the early diagnosis of anastomotic leakage but the diagnosis of anastomotic leakage remains diffi cult and is often discovered at a late stage, not seldom at reoperation. Early in the postoperative phase, Computer Tomography (CT) with an anal enema is often utilized for leakage detection. CRP can be useful in diagnosis but only fi rst after day four or fi ve postoperatively [1,2].
Intraperitoneal cytokines IL 6 and IL 10 have been reported to predict anastomotic leakage in the fi rst postoperative day, while blood IL6 was higher, it was nevertheless not predictive of anastomotic leakage [19]. In a systematic review and metaanalysis of seven articles it was found that peritoneal IL 6 and TNF- are signifi cantly associated with colorectal anastomotic leakage and their monitoring might lead to early detection of leakage [24].
The aim of the study was to identify feasible biomarkers for the early diagnosis of anastomotic leakage after rectal surgery. In the leak group there were 7 patents (5 men, 2 women) median age 71 years (range 51-89), median anastomotic level 5 cm (range [3][4][5], and all patients received preoperative radiation 5x5 Gy. Peroperatively, 4 patients were also operated with a loop ileostomy, 1 patient transversostomy and 2 patients did not receive any divergent stoma (Table 1).

Patients
In the free group, 13 patients (4 men, 9 women) with a median age 69 years (range 53-86), median anastomotic level 5.25cm (range 3-9). Nine patients received 5x5 Gy preoperative radiation and 1 patient was given 2x25 Gy, while the remaining 3 patients did not receive any preoperative radiation treatment.
Peroperatively, 6 patients were also operated with a loop ileostomy, 2 patients with transversostomy and 5 patients did not receive any divergent stoma ( Table 1).
The "other" complications group included 9 patients (1 man 8 women) with a median age of 67 years (range 29-80), and a median anastomotic level 5cm (range 4-10). All 9 patients were given 5x5 Gy preoperative radiation treatment. Peroperatively, 4 patients were also operated with a loop ileostomy and 5 patients did not receive any divergent stoma (Table 1).
Intraperitoneal fl uid was collected by an intraperitoneal drainage, inserted peroperatively. The fi rst sample was collected six hours postoperatively and collection was continued every 6 th hour. The collected samples included IL 6, IL 10 and TNF-.
All blood samples and intraperitoneal cytokines were analyzed at the Clinical Department of Laboratory Medicine at the University Hospital of Örebro.
Intraperitoneal lactate and pyruvate were collected and analyzed using microdialysis equipment (M-dialysis AB, Stockholm, Sweden), thereby allowing the lactate/ pyruvate ratio to be determined. Before closing the abdomen, a microdialysis catheter M-dialysis 62 was introduced intraperitoneally through a small incision in the abdominal wall with an M-dialysis needle and placed free-fl oating in the intraperitoneal cavity. The catheter was fi xed to the skin with a suture to minimize the risk of unintentional extraction. Samples were continuously collected from the intraperitoneal fl uid every second hour from 2-60 hours postoperatively in microvials from the microdialysis catheters and immediate analysis was performed in the analyzer.

Microdialysis
The microdialysis catheter is a 0.9 mm thin, double lumen concentric plastic tube with a 30 mm semi-permeable tubular membrane (cut off at 20.000 Dalton) at its distal end. An M-dialysis 62 gastrointestinal catheter with 210 mm shaft and 30 mm membrane was used in the peritoneal cavity. Physiologic perfusion fl uid T1 was pumped at a rate of 0.3 μl/min from an M-dialysis 106 microdialysis pump through the outer tube of the catheter and fl owed underneath the membrane, where the exchange between the intraperitoneal fl uid and the perfusion fl uid took place. At the tip the fl uid entered the inner tube through a small hole, fl owed backwards and was fi nally collected in a microvial. The perfusate equilibrates with molecules in the intraperitoneal fl uid. In this way, microdialysis monitors substances supplied from the blood as well as substances originating from cell metabolism. A microvial with the microdialysis sample takes 7 minutes to be analyzed for glucose, pyruvate, lactate and glycerol. The lactate/pyruvate ratio was calculated in the analyzer.

Cytokines
The cytokines, TNF-, IL 6 and IL 10 were analyzed from intraperitoneal fl uid, which was collected from an 18 French pelvic drain, and intravenous blood every 6 th hour until 48 th postoperative hours. Samples were determined using an enzyme-labelled chemiluminiscent sequential immunometric assay upon an Immulite ® instrument (DPC, Los Angeles, California, USA) according to the manufacturer's instructions.

Statistical analysis
In this study, metabolic samples from microdialysis, intraperitoneal lactate and pyruvate were collected every 2 nd hour. Due to the large amount of data, we present the median value and interquartile range from 2 nd to 6 th hour and soon.
Due to large variations over time in the leakage group, statistical analysis was conducted using Kruskal-Wallis test (leak, free and other) and the Wilcoxon rank sum test (leak and free) for variables ascertained every 6 th hour in every analysis. P values <0.05 were regarded as signifi cant. All statistical analyses were performed in Statistix 8.
The study was approved by the local Ethics Committee and, after informed consent, patients were included in the study.

Results
Blood cytokines (Table 2) TNF- (tumor necrosis factor ): Anastomotic leak group started preoperatively at 5.6, the free group at 6.7 and other complications group at 6.0 ng/L. Initially increasing levels peaked at 42 hours at 10.9, 7.3 and 10.4 respectively, and no signifi cant differences between the groups were seen.

IL 6 (interleukin 6)(Figure 1):
Preoperative low values (5.8, 2.4, 6.9ng/L) increased quickly and an irregular pattern was observed, whereby the leak group peaked after 30 hours (947 ng/l), the free group peaked after 12 hours (195ng/l) and other complication group peaked after 6 hours (648ng/l). Signifi cant differences in Wilcoxon rank sum test comparing leak and free groups were seen after 12, 18, 24, 30 and 42 hours respectively. Signifi cant differences comparing all three groups in Kruskal Wallis test was noted after 12, 18, 24 and 30 hours.

IL 10 (interleukin 10):
The low levels before operation (4.0, 4.0, and 4.0ng/L) increased rapidly, where all three groups peaked after the 6 th postoperative hour (27.4, 15, and 16.9ng/l), with parallel declines thereafter. No signifi cant differences between groups could be detected.

Various/others blood samples (Table 2)
Fibrinogen: Preoperative values in the three groups started at 4.0, 3.3 and 4.2g/L respectively. Values increased in all three groups to 6.0, 5.6 and 6.8g/l after 54 hours. The increase was faster in the leakage group, leading to signifi cant differences at 12, 24 and 30 hours when using the Wilcoxon rank sum test comparing leak and free groups, whereas, the Kruskal Wallis test showed signifi cant differences after 12 and 30 hours when comparing all three groups. Despite initial increases, no signifi cant difference could be detected between the groups.
Albumin: Preoperative values were 33.1, 34.5 and 36.6 g/L respectively. Values decreased during the fi rst 18 hours (24.0, 24.6, and 25.2g/l). In the free and other groups, slight increases were noted, while values in the leak group continued to decrease to 22.4 g/l at the end of the study period, but this difference was not signifi cant at any time.
C3d (complement factor C3d): Very small differences in preoperative values between the groups as well as postoperatively, varying between 6.0 and 6.4. Figure 2): A small difference, but not signifi cant, was noted preoperatively (16,6, and 6mg/L in the respective groups). Levels increased in all groups but a faster and higher increase was seen in the leakage group. After 12 hours, levels were 41, 10 and 15 mg/L, after 24 hours levels were 160, 74 and 85mg/l, after 36 hours 250, 162 and 203mg/L. After 48 hours levels had increased to 306mg/L in the leakage group, 146mg/L in the free group and 203mg/L in the other complication group. Signifi cant differences were noted in the Wilcoxon rank sum test between leak and free groups after 12, 24, 30, 36, 42 and 48 hours. Signifi cant differences were found while comparing all three groups with the Kruskal Wallis test at 12, 24, 42 and 48 hours.

Intraperitoneal metabolism (Table 2)
Lactate: (Figure 4) Starting values 6 hours postoperatively Anastomotic leak index: After 18, 24, 42 and 48 hours postoperatively, four measurements were calculated using fi ve metabolites (the fi fth metabolite, IL6, was measured in both blood and intraperitoneal fl uid) ( Table 3). The Anastomotic Leak Index was constituted by fi ve metabolites, namely, serum CRP and IL 6, intraperitoneal IL 6, lactate and the lactate/ pyruvate ratio. The cytokine IL 6 was only registered after 18 and 24 hours in both blood and intraperitoneally while CRP, lactate and the lactate/pyruvate ratio were registered at all four time-periods. Abnormal CRP values were observed  higher than 70 after 18 hours, higher than 150 after 24 hours and higher than 250 after 42 and 48 hours. Blood IL 6 was higher than 400ng/L at both 18 and 24 hours while abnormal levels of intraperitoneal IL 6 should reach levels higher than 150000 after 18 hours and higher than 130000 after 24 hours. Intraperitoneal lactate should be higher than 3.5 after 18 hours and higher than 4.0 after 24, 42 and 48 hours to be considered as abnormal. An abnormal lactate/pyruvate ratio over 20 was registered at all 4 time-periods.
Sixteen separate registrations of the metabolites were recorded. For every abnormal value (higher than reference) one point was added, potentially giving a total maximal index score of sixteen. A score of 5 or more for a patient gave a sensitivity and specifi city for anastomotic leak of 0.86 in this study. Reference values are presented in Table 3.
When scoring the 29 patients in this study, the leak group had a median score of 11 (Q1=5, Q3= 15), while free and other group had a median score of 1 (Free: Q1= 0, Q3=2, other: Q1= 0, Q3=4). Using the Wilcoxon rank sum test when comparing leak-free and leak-free + other groups according to the Anastomotic Leak Index, clear statistical signifi cance could be gained (p=0.0007), similarly when comparing the leak-freeother in the Kruskal Wallis test scoring system (p=0.0007).

Discussion
This study analyzed several metabolic, infl ammatory and organ dysfunctionalities every 6 th hour in related samples in three groups after anterior resection due to cancer recti. Three groups have been studied: anastomotic leak group (leak), complication free group (free) and other complications group (other). The study showed that time after operation, together with specifi c metabolic and infl ammatory biomarkers are the most important indicators for the early detection of anastomotic leak.
Cytokines showed similar reactive patterns, being more pronounced intraperitoneally, and generally showing high response rates directly postoperatively, with a subsequent rapid decline. The most suitable cytokine for early diagnosis of anastomotic leakage seems to be IL6 in blood and intraperitoneal fl uid, but TNF- and IL 10 intraperitoneally also seem to have some prognostic advantages in the early diagnosis of anastomotic leakage [25,26].
Intraperitoneal metabolic sampling with microdialysis provides a promising diagnostic strategy. Analyzing lactate and lactate/pyruvate ratio offers several advantages such as a stable sample due to the size of the holes in catheter membrane of 20 kDa, which do not allow the perfusion of enzyme lactate dehydrogenase (143 kDa) into the sample. In addition, continuous sampling over 20 minutes and a rapid analysis within in 7 minutes makes the method highly useful in the critical periods postoperatively. In the present study, the microdialysis catheter had been placed free-fl oating around the small intestines and demonstrated, as in other studies, that a lactate/pyruvate ratio over 20 is pathological [3,5,6].
Intraperitoneal lactate values in this study over 3.  microdialysis method is expeditious, allowing for more or less concurrent measurements.
Today, most patients with a low anterior resection receive a divergent stoma, but many of these patients are not offered the opportunity to discuss care options. Subsequently, many patients have problems with discomfort, thus leading to reduced quality of life and increased health care costs. Based on the results of this study, we suggest that a divergent stoma should not be performed as the primary surgical strategy. Rather