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Introduction
The first studies in laparoscopic surgery (LS) reported a higher tumor recurrence rate1,2. However, as subsequent randomized studies were conducted and surgeons overcame the learning curve inherited by a new surgical technique, numerous benefits of LS were established. Among the short-term advantages, the most outstanding ones are the faster return of intestinal motility, which leads to an earlier return to a normal diet; the reduction of post-operative pain, which translates into a lower need for analgesia; and finally, quick recovery of the patient, which results in a shorter length of stay. In the long term, identical rates of tumor recurrence and patient survival have been showed in open versus LS3-6.
In parallel to the development of LS, the multimodal rehabilitation programs emerged. These programs are also known as Fast-track protocols, enhanced recovery after surgery (ERAS) protocols, or Enhanced Recovery Protocols (ERP)7,8. These protocols aim to achieve an early recovery of patients after surgery, minimizing stress and comorbidities, and reducing hospital stay, with the consequent decrease in waiting lists and hospital costs.
This study aims to understand the immune response that occurs after open and laparoscopic intervention within ERPs. Evidence-based medicine supports the use of laparoscopic approaches in colorectal cancer surgery. However, clear evidence that this surgical technique provides greater benefits than open surgery (OS) is still lacking. By studying the immune response of patients within these protocols will help to homogenize the sample, so that the patients treated for oncological pathology start from the best possible physiological conditions.
In this work, it has been hypothesized that advantages observed in LS may arise due to a better-preserved immune system in this procedure with respect to OS. Therefore, immune parameters have been collected to compare the immune response between both surgical approaches. Not only the immune response between both surgical approaches has been compared but also the evolution over time of each parameter has been analyzed.
Materials and methods
Patient selection
A prospective non-randomized study including patients diagnosed with operable colon cancer and included in an ERP was carried out. Patients underwent major abdominal surgery for a colorectal cancer process, either by laparotomy or laparoscopy, in the General and Digestive Surgery Department of the Hospital Lozano Blesa (Zaragoza, Spain). The sampling was conducted between June 1, 2013, and June 15, 2017.
A surgical team composed of surgeons, anesthetists, and nurses trained in ERP was established. The ERP established by the Spanish Multimodal Rehabilitation Group was followed.
The surgeons were trained in both laparoscopic and open colorectal surgery. Surgical technique was decided by this surgical team according the patient’s clinical history to provide them the best possible treatment. Right hemicolectomy, left hemicolectomy, sigmoid colectomy, and anterior resection were the surgical procedures included in this study, being considered as the most standardized and less aggressive techniques. The same anesthetic procedure by intravenous analgesia was followed in all cases. Patients’ exclusion criteria are shown in table 1.
Table 1 Exclusion criteria of patient selection
| Patients classified as ASA IV |
| Patients with uncompensated cardiorespiratory disease |
| Patients with chronic and acute renal impairment |
| Patients with altered preoperatory parameters |
| Patients with chronic hepatic impairment |
| Patients with situations of uncorrected intraoperative hypovolemia |
| Patients with generalized sepsis |
| Patients with coagulopathies |
| Patients with increased intracranial pressure (ICP) |
| Patients with a known allergy to anesthetics |
| Patients with needing a perioperative transfusion |
| Chronic opiate users |
ASA: American society of anesthesiologists.
Finally, 148 patients were included in the study, 74 undergoing OS, and 74 LS. The study was approved by the Aragon Institutional Review Board (IRB number: C.P.-C.I. PI13/0087). A written informed consent was provided by all the patients included in this study.
Blood sampling
Blood samples were collected from patients before the surgical intervention (POD0) and at 24 (POD1) and 72 h (POD3) post-surgery. C-reactive protein (CRP) parameter was performed from serum samples taken from patients by venipuncture. To this end, IMMAGE® Immunochemistry Systems (Beckman Coulter) were used. Human physiological values are 0.02-0.61 mg/dl. Higher values indicate a systematic inflammatory response.
Hemogram was performed in a DxH 800 Hematology Analyzer (Beckman Coulter) from whole blood in EDTA anticoagulant obtained by venipuncture. Blood parameters measured were white blood cell (WBC) count (WBC, 4-11 mil/mm3), hematocrit (HCT, 36-45%), neutrophil percent (NE, 40-75%), lymphocyte percent (LY, 20-45%), monocyte percent (MO, 2-10%), eosinophil percent (EO, 0-5%), and basophil percent (BA, 0-2%).
Serum samples were obtained by venipuncture and collected to determine immunoglobulins IGA (68-378 mg/dl), IGG (768-1632 mg/dl), and IGM (60-263 mg/dl). The nephelometry technique was carried out by IMMAGE® Immunochemistry Systems (Beckman Coulter). The nephelometer BN II System (Siemens) was used to determine IGE (0-180 mg/dl). The end-point nephelometry was carried out from serum samples obtained by venipuncture.
The determination of all the antibodies was automatically carried out in a Cytomics FC500 flow cytometer (Beckman Coulter), following the manufacturer’s instructions for the treatment of the samples from the analysis of whole blood in anticoagulant heparin obtained by venipuncture. The percentage of total T lymphocytes (CD3, 65-80%), T-helper (Th) cells (CD4, 40-50%), T cytotoxic (Tc) cells (CD8, 26-30%), B lymphocytes (CD19, 10-15%), CD4/CD8 (1,5-2), and natural killer (NK) cells (CD56, 5-10%) were obtained.
Statistical analysis
Statistical analysis was performed using the SPSS software package, with p < 0.05 indicating statistical significance.
The Chi-square test and Fisher test were used to show the relationship between independent qualitative variables, while the McNemar test or Crochan’s Q test were applied if the variables were found to be related. The Kolmogorov–Smirnov test was used to determine whether the variables complied with normality criteria, whereas the Mann–Whitney U-test and Student’s t-test were used to compare the means of independent variables. The means obtained at the different sample-processing times were collated using the Wilcoxon test when 2 times were compared, and the Friedman tests when all 3 times were compared.
Results
Clinical and demographic characteristics
No statistically significant differences (p > 0.05) were found between the study groups with respect to the clinical and demographic aspects studied (Table 2). More complications were not observed in OS and although not significantly, operation time and length of stay were longer in the OS group. In addition, a statistically significant difference (p < 0.005) was observed in the meantime of patients who remained in hospital, with those suffering complications being hospitalized for longer (32.29 days; SD = 28.09) than those who did not (7.83 days; SD = 3.98), irrespective of the intervention group.
Table 2 Clinical and demographic variables according to the type of surgery
| OS (n = 74) | LS (n = 74) | p-value* | |
|---|---|---|---|
| Sex (male) | 61 (82.43) | 47 (63.51) | 0.143 |
| Age (years) | 69.33 (10.42) | 66.98 (10.38) | 0.416 |
| Weight (Kg) | 82.23 (12.33) | 76.97 (10.15) | 0.140 |
| Height (cm) | 166.55 (7.49) | 164.23 (7.45) | 0.365 |
| Body Mass Index | 29.83 (4.03) | 28.53 (2.85) | 0.155 |
| ASA | 0.746 | ||
| II | 32 (43.24) | 35 (47.30) | |
| III | 42 (56.76) | 39 (52.70) | |
| Cancer stage (TNM Classification) | |||
| Stage II | 34 (45.95) | 30 (40.54) | 0.803 |
| Stage III | 40 (54.05) | 44 (59.46) | |
| Operation type | |||
| Right hemicolectomy | 31 (41.90) | 19 (25.68) | |
| Left hemicolectomy | 15 (20.27) | 11 (14.86) | |
| Sigmoidectomy | 4 (5.40) | 35 (47.30) | |
| Anterior resection | 24 (32.43) | 9 (12.16) | |
| Complications | 31 (41.89) | 22 (29.73) | 0.511 |
| Operation time (min) | 206.15 (31.11) | 190.27 (22.51) | 0.121 |
OS: open surgery, LS: laparoscopic surgery, ASA: American Society of Anesthesiologists.
Data showed as n (%) for qualitative variables and as mean value (SD) for quantitative variables.
Pro-inflammatory marker
Higher CRP significantly levels were obtained on POD1 and POD3 in the OS group compared to LS group, 10.63 (SD = 4.5) and 13.77 (SD = 9.31) compared to 5.28 (SD = 1.60) and 7.20 (SD = 5.73), respectively (Table 3). CRP followed the same evolution in both groups, increasing significantly their values over time, remaining in all the post-operative days higher in OS than in LS.
Table 3 CRP levels (mg/dl) according to the type of surgery (mean ± SD) in the 3 different moments
| POD0 | POD1 | POD3 | p (global) | p (POD0-POD1) | p (POD0-POD3) | p (POD1-POD3) | |
|---|---|---|---|---|---|---|---|
| CRP | |||||||
| OS | 1.40 (1.99) | 10.63 (4.5) | 13.77 (9.31) | < 0.001 | < 0.001 | < 0.001 | 0.395 |
| LS | 0.84 (0.91) | 5.28 (1.60) | 7.20 (5.73) | < 0.001 | < 0.001 | < 0.001 | 0.291 |
| p | 0.132 | < 0.001 | < 0.001 |
POD0: day if intervention; POD1: post-operative day 1; POD3: post-operative day 3, CRP: C-reactive protein.
Immune parameters
HEMATOCRIT AND WBCS
The evaluation of blood parameters according to the type of surgery (Fig. 1) depicted significant differences when comparing OS and LS on POD1. In the OS group, the percentage of lymphocytes was lower (9.61% vs. 13.53%, p = 0.005), while the percentage of neutrophils was higher (81.59% vs. 77.43%, p = 0.011).
Figure 1 Evolution of hematocrit and white blood cells according to the type of surgery (mean0 ± SD). A: hematocrit levels. B: white blood cells levels. C: neutrophils levels. D: lymphocyte levels. E: monocytes levels. F: eosinophil levels. G: basophils levels. *Statistical significance (p < 0.05) between groups (OS: open surgery; LS: laparoscopic surgery). *Statistical significance (p < 0.05) between a POD and the previous (within LS). *Statistical significance (p < 0.05) between a POD and the previous (within OS). Statistical significance (p < 0.05) between POD3 and the POD0 (within LS). Statistical significance (p < 0.05) between POD3 and the POD0 (within OS).
It can be observed in the graphs how the levels of the hematocrit, lymphocytes, monocytes, eosinophils, and basophils are higher in the case of LS according the time, whereas the values of WBC and neutrophils remain lower, indicating a less aggressive innate immune response of the laparoscopic approach to the surgical act. In fact, the percentage variation was used to compare the evolution of the parameters between the two intervention groups. No statistically significant differences between them were obtained, but a trend toward restoring baseline levels of the following parameters in LS group with respect to OS group on POD3-POD0 was observed: hematocrit (−7.48% vs. −8.8%), lymphocytes (−29.99% vs. −37.06%), monocytes (0.38% vs. −3.5%), eosinophils (63.38% vs. 196.64%), and basophils (−4.7% vs. −20.78%).
IMMUNOGLOBULINS
The comparison between the two groups did not show any significant difference in the immunoglobulin levels for the periods studied (Fig. 2). There were no statistically significant differences in the evolution of immunoglobulins according to the type of surgical intervention. Besides, most of levels of immunoglobulins remained within the biological reference interval, pointing to a preserved humoral immunity in both cases.
LYMPHOCYTE PROFILE
As it is shown in figure 3, when comparing LS versus OS, a statistically significant higher percentage of B-lymphocytes was found in LS (14.12% vs. 11.05%, p = 0.023) on POD1, but a lower percentage of NK cells (10.58% vs. 14.33%, p = 0.034) on POD3.
Figure 3 Lymphocyte profile according to the type of surgery (mean ± SD). A: T cells levels. B: T-helper cells levels. C: T cytotoxic levels. D: CD4/CD8 ratio. E: B cells levels. F: NK cell levels. *Statistical significance (p < 0.05) between groups (OS: open surgery; LS: laparoscopic surgery). *Statistical significance (p < 0.05) between a POD and the previous (within LS). *Statistical significance (p < 0.05) between a POD and the previous (within OS). ◾Statistical significance (p < 0.05) between a POD and the POD0 (within LS). ◾Statistical significance (p < 0.05) between POD3 and the POD0 (within LS). Statistical significance (p < 0.05) between POD3 and the POD0 (within OS).
Analyzing the evolution of the parameters over time, a significant difference in the case of the NK cells on POD3-POD0 (p = 0.026) was found. An increase of almost 30% was observed in the OS group, while there was a decrease of 10.51% in the LS group. It was also observed that the percent variation on the interval POD3-POD0 for all parameters (except CD3) - while not significant - was lower in the LS group compared to the OS group. This suggests that the immune global change produced in the lymphocyte populations after laparoscopy surgery is inferior to that produced by OS.
Discussion
In this study, we compared immune response due to open and LS in oncological patients undergoing colorectal surgery. All patients were included within the same ERP to optimize their physical, physiological, and psychological conditions. This fact allows attributing the effects obtained in changes in the immune response mainly to the surgical approach, and not to other individual characteristics factors. Indeed, no significant demographic or clinical differences were obtained in the comparison groups (open vs. laparoscopy).
Many works9-11 have focused on the study of the systemic inflammation response produced by the surgical act. Pro-inflammatory markers such as CRP or interleukin (IL)-6 as well as different acute phase proteins have been studied, concluding that LS produces a lower innate response than OS, especially in the first post-operative hours. In this work, the systemic inflammation marker CRP was analyzed. CRP levels were significantly increased over time (p global < 0.001) in both OS and LS, suggesting that an inflammatory response occurs in both cases. However, CRP levels were significantly higher in OS, not only on POD1 but also on POD3, indicating a more aggressive inflammatory response.
In this work, hematocrit levels were used to evaluate intraoperative bleeding. A significant reduction was observed in both surgical groups, without statistically significant differences among them. However, it was observed higher hematocrit levels in the LS group, which could suggest a lower risk of surgical and post-operative hemorrhages, as it was also observed in the Evans et al. study12.
The results obtained regarding leukocyte levels and their populations partially agree with the ones reported by Huang et al.13 and Fujii et al.14 An increase in leukocytes and neutrophils levels as well as a reduction in lymphocyte levels in both groups was also observed. However, the authors observed greater lymphocyte levels in the LS group on POD4, while, in this study, these values have arisen on POD1. Fujii et al.14 also observed an increase in leukocytes and a decrease in the lymphocyte levels on POD1, without finding differences between groups except for the case of leukocytes (higher in OS vs. LS). However, they studied more post-operative days, verifying that the leukocyte levels returned to their baseline levels on POD7 in both groups. In our study, the maximum post-operative time studied was 72 h (POD3). Thus, this tendency to recover leukocytes or neutrophils baseline levels could not be observed. Nevertheless, a certain trend to recover earlier baseline levels of the rest of lymphocyte populations has been observed. Furthermore, it seems that LS group recovers earlier baseline levels than OS group. These findings pointed to an immune system suppression caused by surgery. The higher lymphocytes levels found in LS versus OS indicate that this immune system suppression is less pronounced in LS, since the reduction in lymphocytes is lower compared to OS. This fact would support studies that have reported preserved cell-mediated immunity in LS15-17.
In addition, the highest neutrophil and CRP levels observed in OS would support a greater inflammatory response produced by this surgical technique, especially in the short-term (on POD1), as Ramanathan et al.18, Gustafsson et al.19, and among others20-22 have reported.
The analysis of NK cells showed significantly lower levels in LS on POD3. This fact could suggest a lower anti-tumor capacity of the laparoscopic approach. Studies that have included NKs23 did not observe significant differences between OS and LS. However, Huang et al.13 reported a pronounced post-operative decrease of NK cells in OS. Nevertheless, data did not show significantly statistically differences. Furthermore, NK cells recovered their baseline levels on POD7 in both surgical approaches. Within ERP, Wichmann et al.24 also observed a NK cell suppression after surgery. However, they did not compare OS versus LS, but conventional care versus ERP. Long-term patient survival data should be collected to assess this effect more accurately.
In the case of Tc lymphocytes, when studying evolution over time, a significant reduction was observed in OS group on POD1-POD0 and POD3-POD0. This may suggest a major immunosuppression in OS. Nevertheless, CD4/CD8 ratio did not show significant variations. Thus, immunosuppression does not seem to have any influence. On the other hand, although there are no differences in the evolution of Th and Tc lymphocytes in LS, the CD4/CD8 ratio showed a significant increase on POD1, what could indicate a greater preservation of cellular immunity in the LS recent post-operative period.
With respect to humoral immunity, lower B lymphocyte levels on POD1 were observed in OS versus LS. This fact could suggest a major immunosuppression in OS, as other studies have been suggested25,26. Despite of this, no difference in the immunoglobulins levels between groups was observed. Therefore, a suppression of the humoral response in OS cannot be reported.
Conclusions
As a synthesis of all the immune parameters analyzed, it has not been possible to establish a different pattern of immune response between OS and LS. However, a greater organic aggression and inflammatory response in OS has been observed, especially in the short-term period. In addition, the evolution of the immune parameters studied over time has shown a trend toward recovering earlier their pre-operative levels, as well as minor immune changes in the laparoscopic approach. This fact points to a better-preserved immune system in LS. However, further studies should be designed, to assess this hypothesis.
