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. 2019 Oct 29;14(10):e0224562. doi: 10.1371/journal.pone.0224562

Volatile anesthetics versus total intravenous anesthesia in patients undergoing coronary artery bypass grafting: An updated meta-analysis and trial sequential analysis of randomized controlled trials

Xue-feng Jiao 1,2,3,4, Xue-mei Lin 3,5, Xiao-feng Ni 1,2,3,4, Hai-long Li 1,2,3, Chuan Zhang 1,2,3, Chun-song Yang 1,2,3, Hao-xin Song 1,2,3, Qiu-sha Yi 1,2,3,4, Ling-li Zhang 1,2,3,*
Editor: Martina Crivellari6
PMCID: PMC6818786  PMID: 31661512

Abstract

Background

The benefits of volatile anesthetics in coronary artery bypass grafting (CABG) patients remain controversial. We aimed to conduct an updated meta-analysis to assess whether the use of volatile anesthetics during CABG could reduce mortality and other outcomes.

Methods

We searched eight databases from inception to June 2019 and included randomized controlled trials (RCTs) comparing the effects of volatile anesthetics versus total intravenous anesthesia (TIVA) in CABG patients. The primary outcomes were operative mortality and one-year mortality. The secondary outcomes included the length of stay in the intensive care unit (ICU) and hospital and postoperative safety outcomes (myocardial infarction, heart failure, arrhythmia, stroke, delirium, postoperative cognitive impairment, acute kidney injury, and the use of intra-aortic balloon pump (IABP) or other mechanical circulatory support). Trial sequential analysis (TSA) was performed to control for random errors.

Results

A total of 89 RCTs comprising 14,387 patients were included. There were no significant differences between the volatile anesthetics and TIVA groups in operative mortality (relative risk (RR) = 0.92, 95% confidence interval (CI): 0.68–1.24, p = 0.59, I2 = 0%), one-year mortality (RR = 0.64, 95% CI: 0.32–1.26, p = 0.19, I2 = 51%), or any of the postoperative safety outcomes. The lengths of stay in the ICU and hospital were shorter in the volatile anesthetics group than in the TIVA group. TSA revealed that the results for operative mortality, one-year mortality, length of stay in the ICU, heart failure, stroke, and the use of IABP were inconclusive.

Conclusions

Conventional meta-analysis suggests that the use of volatile anesthetics during CABG is not associated with reduced risk of mortality or other postoperative safety outcomes when compared with TIVA. TSA shows that the current evidence is insufficient and inconclusive. Thus, future large RCTs are required to clarify this issue.

Introduction

Coronary artery bypass grafting (CABG) is one of the most common surgeries performed on patients with coronary artery disease [1]. In America alone, nearly 400,000 CABG surgeries are performed every year [2], and the associated mortality is reported to be 2 to 3% [3].

The anesthesia scheme for CABG commonly consists of intravenous anesthetics only, or a combination of volatile anesthetics and intravenous anesthetics. A great deal of experimental and clinical evidence has demonstrated that volatile anesthetics have cardioprotective effects [4,5], even if administered for only a short time [6]. These effects have been attributed to multiple mechanisms, including modulation of gene expression, mitochondrial function, signaling pathways, G-protein-coupled receptors, and potassium channels [7].

However, the benefits of volatile anesthetics in CABG patients is an intensely disputed topic. In 2006, two meta-analyses of randomized controlled trials (RCTs) suggested that volatile anesthetics were not associated with reduced mortality when compared with total intravenous anesthesia (TIVA) in CABG surgery [8,9]. On the other hand, meta-analyses published later have shown a reduced mortality associated with volatile anesthetics in CABG surgeries [10–14]. Finally, both the guideline from the American College of Cardiology/American Heart Association (ACCF/AHA) and the guideline from the European Association for Cardio-Thoracic Surgery (EACTS) have recommended the use of volatile anesthetics in patients undergoing CABG surgery [15,16]. However, several recent, well-conducted, large-scale RCTs did not show reduced mortality by volatile anesthetics [17–19]. These new RCTs have since doubled the number of trial participants, which may lead in the future to reversals in the results of previous meta-analyses.

Thus, in order to evaluate the most recent available evidence, we performed an updated meta-analysis of RCTs to test the hypothesis that the use of volatile anesthetics during CABG does not result in lower mortality than the use of TIVA. We also aimed to examine the effects of volatile anesthetics on lengths of stay in the intensive care unit (ICU), lengths of stay in hospital, and other postoperative safety outcomes. Moreover, we used trial sequential analysis (TSA) to determine whether the currently available evidence was sufficient and conclusive.

Materials and methods

This meta-analysis was conducted in accordance with the Cochrane Handbook as well as the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [20,21] (S2 Table). In addition, we deposited our protocol in protocols.io; the identifier is dx.doi.org/10.17504/protocols.io.7y2hpye.

Search strategy

We searched Pubmed, Embase (Ovid), the Cochrane Library, and three Chinese databases, the China Knowledge Resource Integrated Database (CNKI), the VIP Database and the Wanfang Database (up to June 2019). We also searched ongoing clinical trial databases, such as “http://clinicaltrials.gov” and “http://www.controlled-trials.com/”. The search terms included the following: volatile anesthetics, halothane, sevoflurane, desflurane, isoflurane, enflurane, methoxyflurane, ether, and coronary artery bypass (S1 Table). Moreover, the references of included studies were manually checked for identifying additional relevant studies.

Inclusion criteria

Inclusion criteria with reference to the PICOS criteria were as follows: (1) Participants: patients undergoing general anesthesia for CABG. (2) Intervention: an anesthesia plan that included a volatile anesthetic (such as halothane, sevoflurane, desflurane, isoflurane, enflurane, or ether) without restrictions in times and doses of administration. (3) Comparison: TIVA (such as propofol, fentanyl, sufentanil, midazolam, thiopental, or etomidate). (4) Outcomes: the primary outcomes were operative mortality and one-year mortality. The secondary outcomes included length of stay in ICU, length of stay in hospital, and postoperative safety outcomes (myocardial infarction, heart failure, arrhythmia, stroke, delirium, postoperative cognitive impairment, acute kidney injury, the use of intra-aortic balloon pump (IABP), and the use of other mechanical circulatory support). Eligible studies should report at least one of the above outcomes. According to the Society of Thoracic Surgeons, operative mortality is defined as “① all-cause mortality occurring during the hospitalization in which the operation was performed, even beyond 30 days; and ② all-cause mortality occurring after hospital discharge, but within 30 days of the operation [22]”. (5) Study design: RCT.

Exclusion criteria

Exclusion criteria were as follows: (1) CABG combined with valve surgery; (2) epidural anesthesia included in the anesthetic plan; (3) lack of outcome data; (4) full-text not available; (5) study not published in English or Chinese.

Study selection

Two independent reviewers (XF Jiao and XF Ni) screened the titles and abstracts to determine potentially relevant initial studies. They then evaluated the full-text articles of potentially relevant studies for eligibility. Reviewers resolved disagreements by consensus, and, if necessary, asked for a third reviewer’s adjudication.

Data extraction

Two independent reviewers (XF Jiao and XF Ni) extracted data from each included study using a pre-piloted data extraction form. This included general study information, general patient characteristics, sample sizes, interventions, comparisons, and outcomes. Postoperative safety outcomes occurring during hospitalization or within 30 days of operation were extracted for meta-analysis.

Risk of bias in individual studies

Two independent reviewers (XF Jiao and XF Ni) evaluated the risk of bias in each selected study using the Cochrane risk of bias tool. This tool consists of seven items: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting, and other biases. According to the Cochrane Handbook for Systematic Reviews of Interventions, we assessed the risk of bias per outcome across trials as: low risk of bias (if the seven items were all evaluated as at low risk of bias); unclear risk of bias (if one or more items were evaluated as at unclear risk of bias); high risk of bias (if one or more items were evaluated as at high risk of bias) [23].

Quality of evidence

We used the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach to assess the overall level of evidence strength of each outcome [24]. By considering five categories of limitations (risk of bias, inconsistencies, indirectness, imprecisions, and reporting biases), the GRADE approach provided a rating of quality of evidence (high, moderate, low, or very low) for each outcome [25].

Data synthesis and analysis

For dichotomous data, we extracted the numbers of events and total numbers for each group and adopted the risk ratio (RR) with 95% confidence interval (CI) as the effect measure. For continuous data, we extracted mean and standard deviation (SD), and adopted mean difference (MD) with 95% confidence interval (CI) as the effect measure. If the included studies only provided the median, range and/or the first and third quartiles, we first transformed these data to sample means and SDs through published formulas [26]. If there were more than one volatile anesthetics or TIVA groups in the included studies, these were combined for the pooled analyses. I-squared (I2) test was used to assess heterogeneity. If heterogeneity was acceptable (I2 ≤ 50%), a fixed effect model was used. If heterogeneity was significant (I2>50%), a random effects model was used and sensitivity analysis was conducted by excluding one or more studies at a time to assess stability of results. Moreover, if ten or more studies were included, a funnel plot was used to assess publication bias. Statistical analyses were conducted by RevMan 5.3.

For all primary and secondary outcomes, we conducted TSA to reduce the risk of random errors. TSA is a method which calculates the required information size (RIS) for meta-analysis, constructing both the trial sequential monitoring boundaries for benefit or harm, and the futility boundary before reaching RIS. The risk of type Ⅰ error was set at 5% with a power of 80%. For dichotomous outcomes, we initially calculated RIS to detect a 20% relative risk reduction (RRR), as this value is believed to represent a reasonable intervention effect in most therapeutic protocols [27]. If the boundary RIS was ignored due to too little information use, we then tested for a 30% RRR. We used the median event proportion of TIVA groups (excluding zero-event trials) as the control group event proportion. For continuous outcomes, we calculated RIS based on empirical estimation from TSA software [27]. TSA was conducted with the TSA viewer version 0.9.5.10 Beta (www.ctu.dk/tsa).

Results

Search results

A total of 4289 records were identified by the initial search. After selection, 89 RCTs that met our inclusion criteria were included in this meta-analysis (Fig 1).

Fig 1. Flow diagram of study selection (PRISMA format).

Fig 1

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Characteristics of the included studies

The 89 included RCTs comprised 14,387 patients, including 7,719 patients anesthetized with volatile anesthetics and 6,668 patients with TIVA. These RCTs took place in a variety of settings: 40 RCTs in Asia, 32 RCTs in Europe, 11 RCTs in North America, 4 RCTs in Australia, 1 RCT in South America, and 1 RCT in Africa. Most RCTs were performed on patients undergoing on-pump CABG surgery, while 27 RCTs were performed on patients undergoing off-pump CABG surgery. Among the included RCTs, 14 were three-arm studies and 3 were four-arm studies. The volatile anesthetic used in the included RCTs varied: 39 RCTs used sevoflurane, 23 RCTs used isoflurane, 8 RCTs used desflurane, 6 RCTs used enflurane, and 13 RCTs used more than one volatile anesthetics. Detailed characteristics of the included RCTs are shown in S4 Table.

Risk of bias in individual studies

The risk of bias assessment of the included RCTs is shown in S1 Fig. We determined the following features to be at high risk of bias: random sequence generation in 1 RCT; blinding of participants and personnel in 8 RCTs; blinding of outcome assessment in 2 RCTs; incomplete outcome data in 1 RCT; other biases in 2 RCTs. Overall, 5 (6%) RCTs were rated at low risk of bias, 12 (13%) RCTs were rated at high risk of bias, and the remaining RCTs were rated at unclear risk of bias.

Primary outcomes

Operative mortality

A total of 44 RCTs reported operative mortality. There was no statistically significant difference between the volatile anesthetics and TIVA groups in operative mortality (RR = 0.92, 95% CI: 0.68–1.24, p = 0.59), and no heterogeneity (I2 = 0%) (Fig 2).

Fig 2. Forest plot of operative mortality.

Fig 2

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TSA showed that the accrued information size (n = 11,567) was 35% of RIS (n = 33,174). The cumulative Z-curve crossed neither the trial sequential monitoring boundary nor the futility boundary, indicating that current evidence was insufficient and inconclusive (Fig 3).

Fig 3. Trial sequential analysis of operative mortality.

Fig 3

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The risk of type Ⅰ error was set at 5% with a power of 80%. The variance was calculated from the data obtained from the included trials. The relative risk reduction (RRR) was set at 20%.

The quality of evidence was rated as moderate for this outcome (S5 Table).

One-year mortality

Five RCTs reported one-year mortality. There was no statistically significant difference between the volatile anesthetics and TIVA groups in one-year mortality (RR = 0.64, 95% CI: 0.32–1.26, p = 0.19), with moderate heterogeneity (I2 = 51%) (Fig 4). Sensitivity analysis did not change the result for one-year mortality (RR = 0.92, 95% CI: 0.68–1.25, p = 0.61, I2 = 0%) (Table 1).

Fig 4. Forest plot of one-year mortality.

Fig 4

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Table 1. Sensitivity analysis.
Outcome Number of RCTs Number of patients MD or RR 95% CI p-value I2 Model
One-year mortality 4 5539 0.92 0.68–1.25 0.61 0% Fix
Length of stay in ICU 32 7345 -0.01 h -0.04–0.01 h 0.38 36% Fix
Length of stay in hospital 29 7535 -0.16 d -0.28– -0.04 d 0.008 26% Fix
Postoperative cognitive impairment 6 5565 1.37 0.94–1.98 0.1 36% Fix
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RCTs, randomized controlled trials; MD, mean difference; RR, relative risk; CI, confidence interval; I2, statistical heterogeneity; ICU, intensive care unit; h, hour; d, day.

TSA showed that the accrued information size (n = 5,953) was 27% of RIS (n = 22,248). The cumulative Z-curve crossed neither the trial sequential monitoring boundary nor the futility boundary, indicating that current evidence was insufficient and inconclusive (Fig 5).

Fig 5. Trial sequential analysis of one-year mortality.

Fig 5

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The risk of type Ⅰ error was set at 5% with a power of 80%. The variance was calculated from the data obtained from the included trials. The relative risk reduction (RRR) was set at 20%.

The quality of evidence was rated as low for this outcome (S5 Table).

Secondary outcomes

Length of stay in ICU

A total of 43 RCTs reported length of stay in ICU, which was shorter in the volatile anesthetics group than in the TIVA group (MD = -4.14 h, 95% CI: -5.63– -2.66 h, p<0.00001), with high heterogeneity (I2 = 98%) (Table 2). After sensitivity analysis, there was no statistically significant difference between the volatile anesthetics and TIVA groups in length of stay in ICU (MD = -0.01 h, 95% CI: -0.04–0.01 h, p = 0.38), with mild heterogeneity (I2 = 38%) (Table 1).

Table 2. Results of meta-analysis for secondary outcomes.
Outcome Number of RCTs Number of patients MD or RR 95% CI p-value I2 Model
Length of stay in ICU 43 8980 -4.14 h -5.63– -2.66 h <0.00001 98% Random
Length of stay in hospital 34 9267 -1.22 d -1.81– -0.62 d <0.0001 95% Random
Postoperative safety outcomes
Myocardial infarction 28 8574 0.94 0.73–1.21 0.64 0% Fix
Heart failure 4 461 0.39 0.08–2.01 0.26 0% Fix
Arrhythmia 29 3084 0.89 0.77–1.03 0.11 0% Fix
Stroke 2 5353 1.46 0.76–2.81 0.25 0% Fix
Delirium 3 5582 0.96 0.71–1.29 0.78 0% Fix
Postoperative cognitive impairment 8 5792 1.20 0.74–1.94 0.46 59% Random
Acute kidney injury 5 5706 0.98 0.79–1.22 0.88 0% Fix
The use of IABP 6 5587 0.67 0.29–1.54 0.34 0% Fix
The use of other mechanical circulatory support 4 5458 0.73 0.33–1.63 0.45 40% Fix
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RCTs, randomized controlled trials; MD, mean difference; RR, relative risk; CI, confidence interval; I2, statistical heterogeneity; ICU, intensive care unit; h, hour; d, day; IABP, intra-aortic balloon pump.

TSA showed that the accrued information size (n = 8,980) was 13% of RIS (n = 68,085). The cumulative Z-curve crossed neither the trial sequential monitoring boundary nor the futility boundary, indicating that current evidence was insufficient and inconclusive (S2 Fig).

The quality of evidence was rated as very low for this outcome (S5 Table).

Length of stay in hospital

A total of 34 RCTs reported length of stay in hospital, which was shorter in the volatile anesthetics than in the TIVA group (MD = -1.22 d, 95% CI: -1.81– -0.62 d, p <0.0001), with high heterogeneity (I2 = 95%) (Table 2). After sensitivity analysis, the length of hospital stay in the volatile anesthetics group was shorter than in the TIVA group (MD = -0.16 d, 95% CI: -0.28– -0.04 d, p = 0.008), with mild heterogeneity (I2 = 26%) (Table 1).

TSA showed that the accrued information size (n = 9,267) exceeded RIS (n = 3,945), indicating that the current evidence was sufficient to reach a firm conclusion (S3 Fig).

The quality of evidence was rated as low for this outcome (S5 Table).

Myocardial infarction

A total of 28 RCTs reported myocardial infarctions. There was no statistically significant difference between the volatile anesthetics and TIVA groups in the incidence of myocardial infarctions (RR = 0.94, 95% CI: 0.73–1.21, p = 0.64), without heterogeneity (I2 = 0%) (Table 2).

TSA showed that the accrued information size (n = 8,574) was 44% of RIS (n = 19,590). The cumulative Z-curve crossed the futility boundary, indicating that current evidence was sufficient to reach a firm conclusion (S4 Fig).

The quality of evidence was rated as high for this outcome (S5 Table).

Heart failure

A total of 4 RCTs reported heart failures. There was no statistically significant difference between the volatile anesthetics and TIVA groups in the incidence of heart failures (RR = 0.39, 95% CI: 0.08–2.01, p = 0.26), without heterogeneity (I2 = 0%) (Table 2).

TSA showed that the accrued information size (n = 461) was 5% of RIS (n = 8,908). The cumulative Z-curve crossed neither the trial sequential monitoring boundary nor the futility boundary, indicating that current evidence was insufficient and inconclusive (S5 Fig).

The quality of evidence was rated as moderate for this outcome (S5 Table).

Arrhythmia

A total of 29 RCTs reported arrhythmias. There was no statistically significant difference between the volatile anesthetics and TIVA groups in the incidence of arrhythmias (RR = 0.89, 95% CI: 0.77–1.03, p = 0.11), without heterogeneity (I2 = 0%) (Table 2).

TSA showed that the accrued information size (n = 3,084) exceeded RIS (n = 2,897), indicating that the current evidence was sufficient to reach a firm conclusion (S6 Fig).

The quality of evidence was rated as high for this outcome (S5 Table).

Stroke

A total of 2 RCTs reported strokes. There was no statistically significant difference between the volatile anesthetics and TIVA groups in the incidence of strokes (RR = 1.46, 95% CI: 0.76–2.81, p = 0.25), without heterogeneity (I2 = 0%) (Table 2).

TSA showed that the accrued information size (n = 5,353) was 10% of RIS (n = 52,433). The cumulative Z-curve crossed neither the trial sequential monitoring boundary nor the futility boundary, indicating that the current evidence was insufficient and inconclusive (S7 Fig).

The quality of evidence was rated as moderate for this outcome (S5 Table).

Delirium

A total of 3 RCTs reported delirium. There was no statistically significant difference between the volatile anesthetics and TIVA groups in the incidence of delirium (RR = 0.96, 95% CI: 0.71–1.29, p = 0.78), without heterogeneity (I2 = 0%) (Table 2).

TSA showed that the accrued information size (n = 5,582) exceeded RIS (n = 3,286), indicating that the current evidence was sufficient to reach a firm conclusion (S8 Fig).

The quality of evidence was rated as moderate for this outcome (S5 Table).

Postoperative cognitive impairment

A total of 8 RCTs reported postoperative cognitive impairment. There was no statistically significant difference between the volatile anesthetics and TIVA groups in the incidence of postoperative cognitive impairment (RR = 1.20, 95% CI: 0.74–1.94, p = 0.46), with moderate heterogeneity (I2 = 59%) (Table 2). Sensitivity analysis did not change the result for postoperative cognitive impairment (RR = 1.37, 95% CI: 0.94–1.98, p = 0.1, I2 = 36%) (Table 1).

TSA showed that the accrued information size (n = 5,792) was 52% of RIS (n = 11,082). The cumulative Z-curve crossed the futility boundary, indicating that the current evidence was sufficient to reach a firm conclusion (S9 Fig).

The quality of evidence was rated as low for this outcome (S5 Table).

Acute kidney injury

A total of 5 RCTs reported acute kidney injury. There was no statistically significant difference between the volatile anesthetics and TIVA groups in the incidence of acute kidney injury (RR = 0.98, 95% CI: 0.79–1.22, p = 0.88), without heterogeneity (I2 = 0%) (Table 2).

TSA showed that the accrued information size (n = 5,706) was 55% of RIS (n = 10,385). The cumulative Z-curve crossed the futility boundary, indicating that the current evidence was sufficient to reach a firm conclusion (S10 Fig).

The quality of evidence was rated as high for this outcome (S5 Table).

The use of IABP

A total of 6 RCTs reported the use of IABPs. There was no statistically significant difference between the volatile anesthetics and TIVA groups in the use of IABPs (RR = 0.67, 95% CI: 0.29–1.54, p = 0.34), without heterogeneity (I2 = 0%) (Table 2).

TSA showed that the accrued information size (n = 5,587) was 41% of RIS (n = 13,493). The cumulative Z-curve crossed neither the trial sequential monitoring boundary nor the futility boundary, indicating that the current evidence was insufficient and inconclusive (S11 Fig).

The quality of evidence was rated as moderate for this outcome (S5 Table).

The use of other mechanical circulatory support

A total of 4 RCTs reported the use of other mechanical circulatory support. There was no statistically significant difference between the volatile anesthetics and TIVA groups in the use of other mechanical circulatory support (RR = 0.73, 95% CI: 0.33–1.63, p = 0.45), with mild heterogeneity (I2 = 40%) (Table 2).

TSA showed that the accrued information size (n = 5,458) was 64% of RIS (n = 8,590). The cumulative Z-curve crossed the futility boundary, indicating that the current evidence was sufficient to reach a firm conclusion (S12 Fig).

The quality of evidence was rated as moderate for this outcome (S5 Table).

Publication bias

No significant publication bias was found with respect to operative mortality (Fig 6).

Fig 6. Funnel plot of operative mortality.

Fig 6

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Discussion

Main findings

To our knowledge, this is the most comprehensive meta-analysis comparing the effects of volatile anesthetics versus TIVA in patients undergoing CABG surgery and is the first study to investigate these effects using the TSA method. Our conventional meta-analysis suggested that there were no statistically significant differences in operative mortality, one-year mortality, myocardial infarction, heart failure, arrhythmia, stroke, delirium, postoperative cognitive impairment, acute kidney injury, the use of IABP, and the use of other mechanical circulatory support between the volatile anesthetics and TIVA groups after CABG surgery. However, volatile anesthetics were associated with reductions in length of stay in ICU and hospital when compared with TIVA. TSA showed that the results for length of stay in hospital, myocardial infarction, arrhythmia, delirium, postoperative cognitive impairment, acute kidney injury, and the use of other mechanical circulatory support were conclusive, while the results for operative mortality, one-year mortality, length of stay in ICU, heart failure, stroke, and the use of IABP were inconclusive.

Compared with previous meta-analyses and RCTs

Our meta-analysis did not reveal reductions in mortality by the use of volatile anesthetics in CABG surgery, a result which was similar to those of meta-analyses published in 2006. However, the meta-analyses published after 2006 were not consistent with our results. For example, the meta-analyses of EI Dib et al. (2017), Uhlig et al. (2016), Zangrillo et al. (2015), Landoni et al. (2013) and Landoni et al. (2007) all showed that volatile anesthetics could reduce mortality when compared with TIVA in CABG survey [10–14]. The meta-analyses of EI Dib et al. (2017) and Landoni et al. (2007) also indicated that volatile anesthetics could reduce the risk of myocardial infarction when compared with TIVA in CABG surgery [10,14]. Moreover, the meta-analyses of Cai et al. (2014) indicated that volatile anesthetics could reduce the incidence of acute kidney injury when compared with TIVA in CABG survey [28]. These inconsistencies may be due to differences in the numbers of included RCTs and patients. In previous meta-analyses, the largest sample size was 58 RCTs with 6105 patients [10], while the smallest sample size was only 10 RCTs with 1600 patients [28]. However, our meta-analysis included 89 RCTs with 14,387 patients, much larger numbers than those of any previous meta-analyses. Moreover, we further performed TSA to assess the adequacy of current evidence and the likelihood of future RCTs altering the present conclusions. Thus, our meta-analysis provides more robust and precise estimations of these outcomes.

Recently Landoni et al. published a well-designed, multicenter RCT encompassing 5400 patients, which was much larger than any of the previous RCTs on this topic [17]. This largest RCT failed to reveal any benefits of volatile anesthetics in CABG patients, which was quite contrary to the results of several previous meta-analyses. However, whether this RCT had enough statistical power to counter the results of previous meta-analyses and end further discussions about the benefit of volatile anesthetics was not known. Thus, we performed an updated and comprehensive meta-analysis on this topic. After pooling all the available evidence, the majority of our results were similar to the results of Landoni et al.; however, the results for length of stay in ICU and hospital were not consistent with the results of Landoni et al. Furthermore, our TSA results indicated that current evidence for mortality as well as several other postoperative safety outcomes was insufficient and inconclusive, and more RCTs would be required to further clarify this issue. Thus, although of large sample size, the Landoni et al. RCT is not convincing enough to end any further discussion on this topic.

Heterogeneity

In our meta-analysis, the heterogeneities associated with length of stay in ICU and hospital were relatively high. One possible reason was that some of the included RCTs provided the median, range, and/or the first and third quartiles for these outcomes. Transformation of these data to sample means and SDs for meta-analysis may have introduced errors, thus causing heterogeneity between studies [26]. Other reasons may include variations in ethnicity, age, severity of disease, treatment regimens, operation types and anesthesia schemes. It is noteworthy that the heterogeneities may also be attributed to variations in settings, hospitals, medical insurance systems, economy levels and cultural differences. However, these were not analyzed in our meta-analysis due to insufficient data. We used a random effects model to account for these heterogeneities and found shorter lengths of both ICU stay and hospital stay in the volatile anesthetics than in the TIVA group. Furthermore, we conducted sensitivity analysis to reduce these heterogeneities, after which the result for length of stay in ICU was not statistically reliable. In addition, even though the result for length of stay in hospital was statistically reliable after sensitivity analysis, it amounted to only a 0.16-day reduction by volatile anesthetics, which was of little clinical value. Thus, the results concerning length of stay in ICU and hospital should be interpreted cautiously.

Quality of evidence

According to GRADE, only three outcomes (myocardial infarction, arrhythmia, and acute kidney injury) in our meta-analysis were rated as of high quality. Of the other outcomes, six (operative mortality, heart failure, stroke, delirium, the use of IABP, and the use of other mechanical circulatory support) were rated down to moderate quality, three (one-year mortality, length of stay in hospital, and postoperative cognitive impairment) were rated down to low quality, and one (length of stay in ICU) was rated down to very low quality. The reasons for rating down the quality of evidence for the above outcomes included imprecisions revealed by TSA, significant heterogeneity, lack of or unclear blinding, and large differences between point estimates. Overall, in accordance with the primary outcomes, we have low confidence that the use of volatile anesthetics is superior to TIVA for patients undergoing CABG surgery [29].

Implications for practice

Nowadays, volatile anesthetics have seen widespread use in CABG surgery. Particularly in America, nearly all patients undergoing CABG surgery receive an anesthesia plan containing volatile anesthetics [15]. Furthermore, both the ACCF/AHA and the EACTS guidelines give a class IIa or class I recommendation for the use of volatile anesthetics in CABG patients [15,16]. However, our results suggest that although the current evidence is insufficient and inconclusive, the use of volatile anesthetics may not be superior to TIVA in CABG patients. Thus, based on our results, the widespread use of volatile anesthetics in CABG surgery and the current guidelines regarding this topic may be not appropriate.

Strengths and limitations

The strengths of our meta-analysis include the large sample size, the comprehensive outcomes, the use of multiple sensitivity analyses, the assessment of reliability of current evidence by TSA, and the assessment of quality of evidence for each outcome. On the other hand, our meta-analysis also has certain limitations. First, we only focused on patients who underwent CABG surgery. Thus, our results cannot be extrapolated to patients undergoing other types of cardiac surgery. Second, the majority of the included RCTs had an unclear risk of bias, with only five (6%) RCTs having a low risk of bias. Third, we only included RCTs published in English or Chinese, which might result in a language bias. Fourth, our meta-analysis was heavily influenced by the Landoni et al. RCT [15]. For example, with respect to primary outcomes, this large RCT contributed a weight of evidence of 38.8% for operative mortality and 52.5% for one-year mortality, which were quite high compared to the number of included studies.

Conclusions

Conventional meta-analysis suggests that the use of volatile anesthetics during CABG is not associated with reduced risks of operative mortality, one-year mortality, and postoperative safety outcomes (myocardial infarction, heart failure, arrhythmia, stroke, delirium, postoperative cognitive impairment, acute kidney injury, the use of IABP and the use of other mechanical circulatory support) when compared with TIVA. TSA shows that the current evidence for mortality as well as several other postoperative safety outcomes (heart failure, stroke, and the use of IABP) is insufficient and inconclusive. Thus, the use of volatile anesthetics may not be superior to TIVA for CABG patients. Future large RCTs are still needed to clarify this issue.

Supporting information

S1 Table. Search strategy in PubMed.

(DOCX)

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S2 Table. PRISMA 2009 checklist.

(DOCX)

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S3 Table. Reference list of included RCTs.

(DOCX)

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S4 Table. Characteristics of the included studies.

(DOCX)

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S5 Table. Grade evidence profile for each outcome.

(DOCX)

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S1 Fig. Risk of bias for each included RCT.

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S2 Fig. Trial sequential analysis of length of stay in intensive care unit (ICU).

The risk of type Ⅰ error was set at 5% with a power of 80%. The variance was calculated from the data obtained from the included trials. The mean difference reduction was set at -0.05 h.

(TIF)

Click here for additional data file. (766.3KB, tif)
S3 Fig. Trial sequential analysis of length of stay in hospital.

The risk of type Ⅰ error was set at 5% with a power of 80%. The variance was calculated from the data obtained from the included trials. The relative risk reduction (RRR) was set at 20%.

(TIF)

Click here for additional data file. (830KB, tif)
S4 Fig. Trial sequential analysis of myocardial infarction.

The risk of type Ⅰ error was set at 5% with a power of 80%. The variance was calculated from the data obtained from the included trials. The mean difference reduction was set at -1.18 d.

(TIF)

Click here for additional data file. (807.1KB, tif)
S5 Fig. Trial sequential analysis of heart failure.

The risk of type Ⅰ error was set at 5% with a power of 80%. The variance was calculated from the data obtained from the included trials. The relative risk reduction (RRR) was set at 30%.

(TIF)

Click here for additional data file. (707.6KB, tif)
S6 Fig. Trial sequential analysis of arrhythmia.

The risk of type Ⅰ error was set at 5% with a power of 80%. The variance was calculated from the data obtained from the included trials. The relative risk reduction (RRR) was set at 20%.

(TIF)

Click here for additional data file. (562.7KB, tif)
S7 Fig. Trial sequential analysis of stroke.

The risk of type Ⅰ error was set at 5% with a power of 80%. The variance was calculated from the data obtained from the included trials. The relative risk reduction (RRR) was set at 30%.

(TIF)

Click here for additional data file. (427.5KB, tif)
S8 Fig. Trial sequential analysis of delirium.

The risk of type Ⅰ error was set at 5% with a power of 80%. The variance was calculated from the data obtained from the included trials. The relative risk reduction (RRR) was set at 20%.

(TIF)

Click here for additional data file. (422.6KB, tif)
S9 Fig. Trial sequential analysis of postoperative cognitive impairment.

The risk of type Ⅰ error was set at 5% with a power of 80%. The variance was calculated from the data obtained from the included trials. The relative risk reduction (RRR) was set at 20%.

(TIF)

Click here for additional data file. (816.1KB, tif)
S10 Fig. Trial sequential analysis of acute kidney injury.

The risk of type Ⅰ error was set at 5% with a power of 80%. The variance was calculated from the data obtained from the included trials. The relative risk reduction (RRR) was set at 20%.

(TIF)

Click here for additional data file. (498.5KB, tif)
S11 Fig. Trial sequential analysis of the use of intra-aortic balloon pump (IABP).

The risk of type Ⅰ error was set at 5% with a power of 80%. The variance was calculated from the data obtained from the included trials. The relative risk reduction (RRR) was set at 20%.

(TIF)

Click here for additional data file. (492.8KB, tif)
S12 Fig. Trial sequential analysis of the use of other mechanical circulatory support.

The risk of type Ⅰ error was set at 5% with a power of 80%. The variance was calculated from the data obtained from the included trials. The relative risk reduction (RRR) was set at 20%.

(TIF)

Click here for additional data file. (512.6KB, tif)

Acknowledgments

We thank Group of People with Highest Risk of Drug Exposure of International Network for the Rational Use of Drugs, China and Evidence-based Pharmacy Committee of Chinese Pharmaceutical Association for helping to coordinate the authors to review earlier manuscript drafts and provide valuable comments.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This study was supported by Natural Science Foundation of China: Evidence based establishment of evaluation index system for pediatric rational drug use in China (No. 81373381), and National Science and Technology Major Project: Construction of pediatric new drug clinical evaluation technology platform (No. 2017ZX09304029) to LZ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  • 1.Alexander JH, Smith PK. Coronary-Artery Bypass Grafting. N Engl J Med. 2016;375(10):e22. [DOI] [PubMed] [Google Scholar]
  • 2.Benjamin EJ, Virani SS, Callaway CW, Chamberlain AM, Chang AR, Cheng S, et al. Heart Disease and Stroke Statistics-2018 Update: A Report From the American Heart Association. Circulation. 2018;137(12):e67–e492. 10.1161/CIR.0000000000000558 [DOI] [PubMed] [Google Scholar]
  • 3.Moazzami K, Dolmatova E, Maher J, Gerula C, Sambol J, Klapholz M, et al. In-Hospital Outcomes and Complications of Coronary Artery Bypass Grafting in the United States Between 2008 and 2012. Journal of cardiothoracic and vascular anesthesia. 2017;31(1):19–25. 10.1053/j.jvca.2016.08.008 [DOI] [PubMed] [Google Scholar]
  • 4.Warltier DC, Pagel PS, Kersten JR. Approaches to the prevention of perioperative myocardial ischemia. Anesthesiology. 2000;92(1):253–259. 10.1097/00000542-200001000-00038 [DOI] [PubMed] [Google Scholar]
  • 5.Pagel PS, Crystal GJ. The Discovery of Myocardial Preconditioning Using Volatile Anesthetics: A History and Contemporary Clinical Perspective. Journal of cardiothoracic and vascular anesthesia. 2018;32(3):1112–1134. 10.1053/j.jvca.2017.12.029 [DOI] [PubMed] [Google Scholar]
  • 6.Meco M, Cirri S, Gallazzi C, Magnani G, Cosseta D. Desflurane preconditioning in coronary artery bypass graft surgery: a double-blinded, randomised and placebo-controlled study. Eur J Cardiothorac Surg. 2007;32(2):319–325. 10.1016/j.ejcts.2007.05.005 [DOI] [PubMed] [Google Scholar]
  • 7.Pagel PS. Myocardial protection by volatile anesthetics in patients undergoing cardiac surgery: a critical review of the laboratory and clinical evidence. Journal of cardiothoracic and vascular anesthesia. 2013;27(5):972–982. 10.1053/j.jvca.2012.10.011 [DOI] [PubMed] [Google Scholar]
  • 8.Yu CH, Beattie WS. The effects of volatile anesthetics on cardiac ischemic complications and mortality in CABG: a meta-analysis. Canadian journal of anaesthesia. 2006;53(9):906–918. 10.1007/BF03022834 [DOI] [PubMed] [Google Scholar]
  • 9.Symons JA, Myles PS. Myocardial protection with volatile anaesthetic agents during coronary artery bypass surgery: A meta-analysis. British journal of anaesthesia. 2006;97(2):127–136. 10.1093/bja/ael149 [DOI] [PubMed] [Google Scholar]
  • 10.El Dib R, Guimaraes Pereira JE, Agarwal A, Gomaa H, Ayala AP, Botan AG, et al. Inhalation versus intravenous anaesthesia for adults undergoing on-pump or off-pump coronary artery bypass grafting: A systematic review and meta-analysis of randomized controlled trials. Journal of clinical anesthesia. 2017;40:127–138. 10.1016/j.jclinane.2017.05.010 [DOI] [PubMed] [Google Scholar]
  • 11.Landoni G, Greco T, Biondi-Zoccai G, Nigro Neto C, Febres D, Pintaudi M, et al. Anaesthetic drugs and survival: a Bayesian network meta-analysis of randomized trials in cardiac surgery. British journal of anaesthesia. 2013;111(6):886–896. 10.1093/bja/aet231 [DOI] [PubMed] [Google Scholar]
  • 12.Uhlig C, Bluth T, Schwarz K, Deckert S, Heinrich L, De Hert S, et al. Effects of Volatile Anesthetics on Mortality and Postoperative Pulmonary and Other Complications in Patients Undergoing Surgery: A Systematic Review and Meta-analysis. Anesthesiology. 2016;124(6):1230–1245. 10.1097/ALN.0000000000001120 [DOI] [PubMed] [Google Scholar]
  • 13.Zangrillo A, Musu M, Greco T, Di Prima AL, Matteazzi A, Testa V, et al. Additive Effect on Survival of Anaesthetic Cardiac Protection and Remote Ischemic Preconditioning in Cardiac Surgery: A Bayesian Network Meta-Analysis of Randomized Trials. PloS one. 2015;10(7):e0134264 10.1371/journal.pone.0134264 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Landoni G, Biondi-Zoccai GG, Zangrillo A, Bignami E, D'Avolio S, Marchetti C, et al. Desflurane and sevoflurane in cardiac surgery: a meta-analysis of randomized clinical trials. Journal of cardiothoracic and vascular anesthesia. 2007;21(4):502–511. 10.1053/j.jvca.2007.02.013 [DOI] [PubMed] [Google Scholar]
  • 15.Hillis LD, Smith PK, Anderson JL, Bittl JA, Bridges CR, Byrne JG, et al. 2011 ACCF/AHA Guideline for Coronary Artery Bypass Graft Surgery: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Circulation. 2011;124(23):e652–735. 10.1161/CIR.0b013e31823c074e [DOI] [PubMed] [Google Scholar]
  • 16.Sousa-Uva M, Head SJ, Milojevic M, Collet JP, Landoni G, Castella M, et al. 2017 EACTS Guidelines on perioperative medication in adult cardiac surgery. Eur J Cardiothorac Surg. 2018;53(1):5–33. 10.1093/ejcts/ezx314 [DOI] [PubMed] [Google Scholar]
  • 17.Landoni G, Lomivorotov VV, Nigro Neto C, Monaco F, Pasyuga VV, Bradic N, et al. Volatile Anesthetics versus Total Intravenous Anesthesia for Cardiac Surgery. The New England journal of medicine. 2019;380(13):1214–1225. 10.1056/NEJMoa1816476 [DOI] [PubMed] [Google Scholar]
  • 18.Hofland J, Ouattara A, Fellahi JL, Gruenewald M, Hazebroucq J, Ecoffey C, et al. Effect of Xenon Anesthesia Compared to Sevoflurane and Total Intravenous Anesthesia for Coronary Artery Bypass Graft Surgery on Postoperative Cardiac Troponin Release: an International, Multicenter, Phase 3, Single-blinded, Randomized Noninferiority Trial. Anesthesiology. 2017;127(6):918–933. 10.1097/ALN.0000000000001873 [DOI] [PubMed] [Google Scholar]
  • 19.Likhvantsev VV, Landoni G, Levikov DI, Grebenchikov OA, Skripkin YV, Cherpakov RA. Sevoflurane Versus Total Intravenous Anesthesia for Isolated Coronary Artery Bypass Surgery With Cardiopulmonary Bypass: A Randomized Trial. Journal of cardiothoracic and vascular anesthesia. 2016;30(5):1221–1227. 10.1053/j.jvca.2016.02.030 [DOI] [PubMed] [Google Scholar]
  • 20.Higgins JP, Green S. Cochrane Handbook for Systematic Reviews of Interventions Version 5.1.0. Available from: www.cochrane.org/training/cochrane-handbook
  • 21.Moher D, Liberati A, Tetzlaff J, Altman DG. Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. J Clin Epidemiol. 2009; 62(10):1006–12. 10.1016/j.jclinepi.2009.06.005 . [DOI] [PubMed] [Google Scholar]
  • 22.Overman DM, Jacobs JP, Prager RL, Wright CD, Clarke DR, Pasquali SK, et al. Report from the Society of Thoracic Surgeons National Database Workforce: clarifying the definition of operative mortality. World J Pediatr Congenit Heart Surg. 2013;4(1):10–12. 10.1177/2150135112461924 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Higgins JP, Altman DG, Gotzsche PC, Juni P, Moher D, Oxman AD, et al. The Cochrane Collaboration's tool for assessing risk of bias in randomised trials. BMJ. 2011;343:d5928 10.1136/bmj.d5928 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Atkins D, Best D, Briss PA, Eccles M, Falck-Ytter Y, Flottorp S, et al. Grading quality of evidence and strength of recommendations. BMJ. 2004;328(7454):1490 10.1136/bmj.328.7454.1490 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Balshem H, Helfand M, Schunemann HJ, Oxman AD, Kunz R, Brozek J, et al. GRADE guidelines: 3. Rating the quality of evidence. J Clin Epidemiol. 2011;64(4):401–406. 10.1016/j.jclinepi.2010.07.015 [DOI] [PubMed] [Google Scholar]
  • 26.Wan X, Wang W, Liu J, Tong T. Estimating the sample mean and standard deviation from the sample size, median, range and/or interquartile range. BMC Med Res Methodol. 2014;14:135 10.1186/1471-2288-14-135 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Thorlund K, Engstrøm J, Wetterslev J, Brok J, Imberger G, Gluud C. User manual for Trial Sequential Analysis (TSA). Available from: http://www.ctu.dk/tsa/files/tsa_manual.pdf
  • 28.Cai J, Xu R, Yu X, Fang Y, Ding X. Volatile anesthetics in preventing acute kidney injury after cardiac surgery: a systematic review and meta-analysis. The Journal of thoracic and cardiovascular surgery. 2014;148(6):3127–3136. 10.1016/j.jtcvs.2014.07.085 [DOI] [PubMed] [Google Scholar]
  • 29.Guyatt GH, Oxman AD, Kunz R, Vist GE, Falck-Ytter Y, Schunemann HJ, et al. What is "quality of evidence" and why is it important to clinicians? BMJ. 2008;336(7651):995–998. 10.1136/bmj.39490.551019.BE [DOI] [PMC free article] [PubMed] [Google Scholar]

Decision Letter 0

Martina Crivellari

16 Sep 2019

PONE-D-19-23473

Volatile anesthetics versus total intravenous anesthesia for patients undergoing coronary artery bypass grafting: An updated meta-analysis of 90 randomized controlled trials

PLOS ONE

Dear Dr Zhang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

This manuscript is an appropriately and well written structured meta-analysis. The topic is interesting and has clinical relevance. Numerous previous meta-analyses were conducted on the topic so the findings are not surprising. The multicenter large randomized clinical trial by Giovanni Landoni and colleagues recently published on NEJM, stopped for futility after 50% of the proposed patients were enrolled, should stop any other discussion on this specific topic. Evidence provided by a randomized study is certainly stronger then the results of a meta-analysis, and the findings of the current meta-analysis are influenced by the results of Landoni et al.

Please put in evidence on your limitations these comments, and answer to the question on the review section. 

The grammar should be correct by an english native speaker. 

In the manuscript N2O is included among the "volatiles". I think this is a procedural mistake. I suggest you to change the title or alternatively remove these studies.   

There are not conflicts between the reviews. 

We would appreciate receiving your revised manuscript by October 6 th.

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Comments to the Author

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Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: General comments:

Current manuscript is an appropriately structured meta-analysis with 35 pages; 2 tables; 4 figures and 24 references. In general, the manuscript is well-written, but there is some grammar unpunctuality. The topic of this meta-analysis is interesting and have a great clinical relevance, however numerous previous meta-analyses were conducted on the topic. The novelty of the findings of current study is based on the results of one recently published big multinational mRCT (MYRIAD).

Specific revision comments:

-Major

ABSTRACT

- Few grammar inaccuracies and unpunctuality

METHODS

- Postoperative safety outcomes were collected on the longest follow-up available. Perhaps, it can add an additional bias, because adverse events occurring after several months are not necessarily due to the previous anaesthetic technique.

- Trial Sequential Analysis (TSA) would improve the value of the study.

RESULTS

- Most of the included RCTs are small and therefore, none of the patients died in the operative period. Basically, three study gives the main result and one study have a weight of 38.1%, which is quite high compared to the number of included studies.

- In 1-year mortality one study gives more than 50% of the results.

- Number of included studies in the final analyses can be confusing because only 45 and 5 RCTs were included in the main analyses.

DISCUSSION

- Discussion of the high heterogeneities are insufficient. Authors should describe the potential underlying causes.

Reviewer #2: The authors conducted a meta-analysis of randomized controlled clinical trials examining the efficacy of volatile anesthetics compared with total intravenous anesthesia to provide myocardial protection against ischemic injury in patients undergoing coronary artery surgery. A total of 90 trials involving 14,598 patients were included in the analysis. The results indicate that use of volatile anesthetics was not associated with improvements in outcome compared with total intravenous anesthesia. The findings are not surprising in the least. Giovanni Landoni and colleagues recently published a large prospective multicenter randomized clinical trial in which volatile anesthetics and total intravenous anesthesia were directly compared in patients undergoing coronary artery surgery; the study was stopped for futility after 50% of the proposed 10,000 patients were enrolled (NEJM, 2019). This definitive clinical trial effectively ended any further discussion about the so-called “cardioprotective” actions of volatile anesthetics that were demonstrated repeatedly in many laboratory studies during the past two decades. It is not clear to this reviewer why yet another meta-analysis is needed after this clinical trial, as the evidence provided by any meta-analysis is certainly not as strong as that provided by a well-conducted large scale randomized prospective clinical trial. The findings of the current meta-analysis are undoubtedly heavily influenced by the results of Landoni et al, as they should be. The current work is conducted appropriately using PRISMA guidelines and its data are interpreted correctly. The manuscript is fairly well written, although there are a few spelling and grammatical errors present that the authors should correct.

Specific Comments

P 3 L 68 and P 14 L 287: Several meta-analyses did not support this hypothesis (see references #5 and #7) for details.

P 3 L 74: Given the size and quality of the study of Landoni et al, did the authors think that their meta-analysis would somehow reach conclusions that truly different from the NEJM trial?

P 4 L 79: What is the hypothesis of the current work? This should be explicitly stated.

P 4 L 82: Please state that PRISMA guidelines were followed at this point of the text.

**********

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Reviewer #1: Yes: Nagy Ádám

Reviewer #2: No

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PLoS One. 2019 Oct 29;14(10):e0224562. doi: 10.1371/journal.pone.0224562.r002

Author response to Decision Letter 0

10 Oct 2019

This rebuttal letter responded to each point raised by the academic editor and reviewers, and was uploaded as a separate file labeling 'Response to Reviewers'. We specified the changes made to address each point, indicated their locations in the response (in brackets), and highlighted the changes in the file labeling 'Revised Manuscript with Track Changes' (in red font). This letter was divided into three parts: “Response to the Comments of Academic Editor”, “Response to the Comments of Reviewer #1”, and “Response to the Comments of Reviewer #2”. The details were as follows:

Response to the Comments of Academic Editor

1. The multicenter large randomized clinical trial by Giovanni Landoni and colleagues recently published on NEJM, stopped for futility after 50% of the proposed patients were enrolled, should stop any other discussion on this specific topic. Evidence provided by a randomized study is certainly stronger then the results of a meta-analysis, and the findings of the current meta-analysis are influenced by the results of Landoni et al.

Please put in evidence on your limitations these comments, and answer to the question on the review section.

Reply: Our meta-analysis was heavily influenced by the Landoni et al. RCT. This was an important limitation of our study, and we added this issue to our limitations section (Page 23, Line 459-462, in red font). Moreover, in order to answer to the question on the review section, we discussed the reasons why our meta-analysis is needed, and the differences between the results of our meta-analysis and the Landoni et al. RCT (Page 20-21, Line 396-409, in red font). The details were as follows:

“Landoni et al. RCT encompassed 5400 patients, which was much larger than any of the previous RCTs on this topic. This largest RCT failed to reveal any benefits of volatile anesthetics in CABG patients, which was quite contrary to the results of several previous meta-analyses. However, whether this RCT had enough statistical power to counter the results of previous meta-analyses and end further discussions about the benefit of volatile anesthetics was not known. Thus, we performed an updated and comprehensive meta-analysis on this topic. After pooling all the available evidence, the majority of our results were similar to the results of Landoni et al.; however, the results for length of stay in ICU and hospital were not consistent with the results of Landoni et al. Furthermore, our TSA results indicated that current evidence for mortality as well as several other postoperative safety outcomes was insufficient and inconclusive, and more RCTs would be required to further clarify this issue. Thus, although of large sample size, the Landoni et al. RCT is not convincing enough to end any further discussion on this topic.”

2. The grammar should be correct by an English native speaker.

Reply: The grammar has already been corrected by an English native speaker, and we provided the certificate of English editing in the online submission system.

3. In the manuscript N2O is included among the "volatiles". I think this is a procedural mistake. I suggest you to change the title or alternatively remove these studies.

Reply: This is our mistake. We have removed the studies using N2O or xenon as the anesthetic. After carefully checking the 90 included RCTs, one RCT using xenon as the anesthetic was removed, and none of the RCTs used N2O as the anesthetic. Thus, after removing, 89 RCTs were remained for our meta-analysis (Page 9, Line 177, 183-184, in red font). Consequently, the results for operative mortality, length of stay in ICU, length of stay in hospital, myocardial infarction, arrhythmia, delirium, and acute kidney injury were changed (Page 10-17, Line 202-204, 237-242, 249-254, 275-278, 294-297, 311-313, 329-332, Figs 2 and 4, Tables 1 and 2, in red font).

4. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

Reply: We have made changes to our financial disclosure, and included our updated statement in the cover letter. Our financial disclosure statement was updated as follows:

“This study was supported by Natural Science Foundation of China：Evidence based establishment of evaluation index system for pediatric rational drug use in China (No. 81373381), and National Science and Technology Major Project: Construction of pediatric new drug clinical evaluation technology platform (No. 2017ZX09304029). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” (in red font)

5. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Reply: We have deposited our protocol in protocols.io; the identifier (DOI) is dx.doi.org/10.17504/protocols.io.7y2hpye. Moreover, we added the DOI link to the Methods section of our revised manuscript (Page 5, Line 89-90, in red font).

6. Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Reply: In accordance with the above requirements, we prepared and uploaded 'Response to Reviewers', 'Revised Manuscript with Track Changes', and 'Manuscript' separately.

7. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Reply: We have checked our manuscript carefully on the basis of the PLOS ONE style templates, and tried our best to ensure that our manuscript met PLOS ONE's style requirements.

8. We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

We apologize that this was not previously requested and look forward to receiving your revised manuscript,

Reply: We removed the funding information from our manuscript. Moreover, in the online submission system, we updated our Funding Statement as follows:

“This study was supported by Natural Science Foundation of China：Evidence based establishment of evaluation index system for pediatric rational drug use in China (No. 81373381), and National Science and Technology Major Project: Construction of pediatric new drug clinical evaluation technology platform (No. 2017ZX09304029). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

Response to the Comments of Reviewer #1

1. ABSTRACT - Few grammar inaccuracies and unpunctuality

Reply: The grammar has been corrected by an English native speaker, and we provided the certificate of English editing in the online submission system.

2. METHODS - Postoperative safety outcomes were collected on the longest follow-up available. Perhaps, it can add an additional bias, because adverse events occurring after several months are not necessarily due to the previous anaesthetic technique.

Reply: In order to reduce this bias, we changed our Methods as: “Postoperative safety outcomes occurring during hospitalization or within 30 days of operation were extracted for meta-analysis.” (Page 6, Line 129-130, in red font). Consequently, in our conventional meta-analysis, the results for arrhythmia, postoperative cognitive impairment, and the use of other mechanical circulatory support were changed (Page 13-18, Line 265, 271, 294-297, 318-321, 347-350, Tables 1 and 2, in red font).

3. METHODS - Trial Sequential Analysis (TSA) would improve the value of the study.

Reply: We performed trial sequential analysis (TSA) to determine whether the currently available evidence was sufficient and conclusive (Page 4, Line 82-84, in red font). The methods of TSA could be seen on Page 8, Line 162-173 (in red font), and the results of TSA could be seen on Page 10-12, 15-18, Line 205-208, 223-226, 243-246, 255-256, 279-281, 288-291, 298-299, 305-308, 314-315, 324-326, 333-335, 341-344, 351-353 (in red font), Figs 3 and 5, S2-S12 Figs. In summary, TSA showed that the results for length of stay in hospital, myocardial infarction, arrhythmia, delirium, postoperative cognitive impairment, acute kidney injury, and the use of other mechanical circulatory support were conclusive, while the results for operative mortality, one-year mortality, length of stay in ICU, heart failure, stroke, and the use of IABP were inconclusive (Page 19, Line 370-375, in red font). Moreover, when considering the TSA results, the quality of evidence for our outcomes was also changed (Page 10-11, 15-16, 18, 22, Line 209, 227, 292, 309, 345, 428-439, S5 Table, in red font).

4. RESULTS - Most of the included RCTs are small and therefore, none of the patients died in the operative period. Basically, three study gives the main result and one study have a weight of 38.1%, which is quite high compared to the number of included studies. In 1-year mortality one study gives more than 50% of the results.

Reply: Our meta-analysis was heavily influenced by three large-scale RCTs, especially the Landoni et al. RCT. This was an important limitation of our study, and thus we added this issue to our limitations section (Page 23, Line 459-462, in red font). Moreover, we also discussed the differences between the results of our meta-analysis and the Landoni et al. RCT, and the reasons why our meta-analysis is needed (Page 20-21, Line 396-409, in red font).

5. RESULTS - Number of included studies in the final analyses can be confusing because only 44 and 5 RCTs were included in the main analyses.

Reply: We included RCTs that reported at least one of our outcomes (primary outcomes or secondary outcomes). According to this criteria, a total of 89 RCTs were included. For primary outcomes, 44 RCTs were included for operative mortality, and 5 RCTs were included for one-year mortality. While the remaining RCTs only reported the secondary outcomes.

6. DISCUSSION - Discussion of the high heterogeneities are insufficient. Authors should describe the potential underlying causes.

Reply: We described the potential underlying causes on Page 21, Line 412-419 (in red font). One possible reason was that some of the included RCTs provided the median, range, and/or the first and third quartiles for these outcomes. Transformation of these data to sample means and SDs for meta-analysis may have introduced errors, thus causing heterogeneity between studies [1]. Other reasons may include variations in ethnicity, age, severity of disease, treatment regimens, operation types and anesthesia schemes. It is noteworthy that the heterogeneities may also be attributed to variations in settings, hospitals, medical insurance systems, economy levels and cultural differences.

References: 1. Wan X, Wang W, Liu J, Tong T. Estimating the sample mean and standard deviation from the sample size, median, range and/or interquartile range. BMC Med Res Methodol. 2014;14:135.

Response to the Comments of Reviewer #2

1. Giovanni Landoni and colleagues recently published a large prospective multicenter randomized clinical trial in which volatile anesthetics and total intravenous anesthesia were directly compared in patients undergoing coronary artery surgery; the study was stopped for futility after 50% of the proposed 10,000 patients were enrolled (NEJM, 2019). This definitive clinical trial effectively ended any further discussion about the so-called “cardioprotective” actions of volatile anesthetics that were demonstrated repeatedly in many laboratory studies during the past two decades. It is not clear to this reviewer why yet another meta-analysis is needed after this clinical trial, as the evidence provided by any meta-analysis is certainly not as strong as that provided by a well-conducted large scale randomized prospective clinical trial.

Given the size and quality of the study of Landoni et al, did the authors think that their meta-analysis would somehow reach conclusions that truly different from the NEJM trial?

Reply: In the Discussion section, we discussed the reasons why our meta-analysis is needed, and the differences between the results of our meta-analysis and the Landoni et al. RCT (Page 20-21, Line 396-409, in red font). The details were as follows:

“Landoni et al. RCT encompassed 5400 patients, which was much larger than any of the previous RCTs on this topic. This largest RCT failed to reveal any benefits of volatile anesthetics in CABG patients, which was quite contrary to the results of several previous meta-analyses. However, whether this RCT had enough statistical power to counter the results of previous meta-analyses and end further discussions about the benefit of volatile anesthetics was not known. Thus, we performed an updated and comprehensive meta-analysis on this topic. After pooling all the available evidence, the majority of our results were similar to the results of Landoni et al.; however, the results for length of stay in ICU and hospital were not consistent with the results of Landoni et al. Furthermore, our TSA results indicated that current evidence for mortality as well as several other postoperative safety outcomes was insufficient and inconclusive, and more RCTs would be required to further clarify this issue. Thus, although of large sample size, the Landoni et al. RCT is not convincing enough to end any further discussion on this topic.”

2. There are a few spelling and grammatical errors present that the authors should correct.

Reply: The spelling and grammatical errors have been corrected by an English native speaker, and we provided the certificate of English editing in the online submission system.

3. P 3 L 68 and P 14 L 287: Several meta-analyses did not support this hypothesis (see references #5 and #7) for details.

Reply: It is our mistake. We carefully checked the previous published meta-analyses and found that two meta-analyses2,3 published in 2006 did not support this hypothesis. Thus, we modified P 3 L 68 as follows:

“However, the benefits of volatile anesthetics in CABG patients is an intensely disputed topic. In 2006, two meta-analyses of randomized controlled trials (RCTs) suggested that volatile anesthetics were not associated with reduced mortality when compared with total intravenous anesthesia (TIVA) in CABG surgery [2,3]. On the other hand, meta-analyses published later have shown a reduced mortality associated with volatile anesthetics in CABG surgeries.” (Page 3-4, Line 65-70, in red font)

Moreover, we modified P 14 L 287 as follows:

“Our meta-analysis did not reveal reductions in mortality by the use of volatile anesthetics in CABG surgery, a result which was similar to those of meta-analyses published in 2006. However, the meta-analyses published after 2006 were not consistent with our results.” (Page 19, Line 377-380, in red font)

References:

2. Yu CH, Beattie WS. The effects of volatile anesthetics on cardiac ischemic complications and mortality in CABG: a meta-analysis. Canadian journal of anaesthesia = Journal canadien d'anesthesie. 2006;53(9):906-918.

3. Symons JA, Myles PS. Myocardial protection with volatile anaesthetic agents during coronary artery bypass surgery: A meta-analysis. British journal of anaesthesia. 2006;97(2):127-136.

4. P 4 L 79: What is the hypothesis of the current work? This should be explicitly stated.

Reply: Our hypothesis was that the use of volatile anesthetics during CABG does not result in lower mortality than the use of total intravenous anesthesia (TIVA). This hypothesis was explicitly stated on Page 4, Line 78-80 (in red font).

5. P 4 L 82: Please state that PRISMA guidelines were followed at this point of the text.

Reply: We stated as: “This meta-analysis was conducted in accordance with the Cochrane Handbook as well as the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.” (Page 4-5, Line 87-89, in red font)

Attachment

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Decision Letter 1

Martina Crivellari

17 Oct 2019

Volatile anesthetics versus total intravenous anesthesia in patients undergoing coronary artery bypass grafting: An updated meta-analysis and trial sequential analysis of randomized controlled trials

PONE-D-19-23473R1

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Acceptance letter

Martina Crivellari

21 Oct 2019

PONE-D-19-23473R1

Volatile anesthetics versus total intravenous anesthesia in patients undergoing coronary artery bypass grafting: An updated meta-analysis and trial sequential analysis of randomized controlled trials

Dear Dr. Zhang:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. Search strategy in PubMed.

    (DOCX)

    Click here for additional data file. (16.6KB, docx)
    S2 Table. PRISMA 2009 checklist.

    (DOCX)

    Click here for additional data file. (26.4KB, docx)
    S3 Table. Reference list of included RCTs.

    (DOCX)

    Click here for additional data file. (30.4KB, docx)
    S4 Table. Characteristics of the included studies.

    (DOCX)

    Click here for additional data file. (24.6KB, docx)
    S5 Table. Grade evidence profile for each outcome.

    (DOCX)

    Click here for additional data file. (17.6KB, docx)
    S1 Fig. Risk of bias for each included RCT.

    (TIF)

    Click here for additional data file. (1.5MB, tif)
    S2 Fig. Trial sequential analysis of length of stay in intensive care unit (ICU).

    The risk of type Ⅰ error was set at 5% with a power of 80%. The variance was calculated from the data obtained from the included trials. The mean difference reduction was set at -0.05 h.

    (TIF)

    Click here for additional data file. (766.3KB, tif)
    S3 Fig. Trial sequential analysis of length of stay in hospital.

    The risk of type Ⅰ error was set at 5% with a power of 80%. The variance was calculated from the data obtained from the included trials. The relative risk reduction (RRR) was set at 20%.

    (TIF)

    Click here for additional data file. (830KB, tif)
    S4 Fig. Trial sequential analysis of myocardial infarction.

    The risk of type Ⅰ error was set at 5% with a power of 80%. The variance was calculated from the data obtained from the included trials. The mean difference reduction was set at -1.18 d.

    (TIF)

    Click here for additional data file. (807.1KB, tif)
    S5 Fig. Trial sequential analysis of heart failure.

    The risk of type Ⅰ error was set at 5% with a power of 80%. The variance was calculated from the data obtained from the included trials. The relative risk reduction (RRR) was set at 30%.

    (TIF)

    Click here for additional data file. (707.6KB, tif)
    S6 Fig. Trial sequential analysis of arrhythmia.

    The risk of type Ⅰ error was set at 5% with a power of 80%. The variance was calculated from the data obtained from the included trials. The relative risk reduction (RRR) was set at 20%.

    (TIF)

    Click here for additional data file. (562.7KB, tif)
    S7 Fig. Trial sequential analysis of stroke.

    The risk of type Ⅰ error was set at 5% with a power of 80%. The variance was calculated from the data obtained from the included trials. The relative risk reduction (RRR) was set at 30%.

    (TIF)

    Click here for additional data file. (427.5KB, tif)
    S8 Fig. Trial sequential analysis of delirium.

    The risk of type Ⅰ error was set at 5% with a power of 80%. The variance was calculated from the data obtained from the included trials. The relative risk reduction (RRR) was set at 20%.

    (TIF)

    Click here for additional data file. (422.6KB, tif)
    S9 Fig. Trial sequential analysis of postoperative cognitive impairment.

    The risk of type Ⅰ error was set at 5% with a power of 80%. The variance was calculated from the data obtained from the included trials. The relative risk reduction (RRR) was set at 20%.

    (TIF)

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    S10 Fig. Trial sequential analysis of acute kidney injury.

    The risk of type Ⅰ error was set at 5% with a power of 80%. The variance was calculated from the data obtained from the included trials. The relative risk reduction (RRR) was set at 20%.

    (TIF)

    Click here for additional data file. (498.5KB, tif)
    S11 Fig. Trial sequential analysis of the use of intra-aortic balloon pump (IABP).

    The risk of type Ⅰ error was set at 5% with a power of 80%. The variance was calculated from the data obtained from the included trials. The relative risk reduction (RRR) was set at 20%.

    (TIF)

    Click here for additional data file. (492.8KB, tif)
    S12 Fig. Trial sequential analysis of the use of other mechanical circulatory support.

    The risk of type Ⅰ error was set at 5% with a power of 80%. The variance was calculated from the data obtained from the included trials. The relative risk reduction (RRR) was set at 20%.

    (TIF)

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    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (29.7KB, docx)

    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.

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