Objectives
This is a protocol for a Cochrane Review (intervention). The objectives are as follows:
This review aims to look at the benefits and harms of MIS in kidney transplant recipients.
Background
Description of the condition
Kidney transplantation is the best treatment for patients with end‐stage kidney disease (ESKD). It prolongs life (Wolfe 1999) and increases the quality of life for these patients (Dew 1997; Dobbels 2007). Every year about 79,000 kidney transplants are performed worldwide, of which 3000 are performed in the UK. Despite that, there remains a discrepancy between available organs for transplantation and patients on the waiting list because of increasing demand.
Description of the intervention
Minimally invasive surgery (MIS) has been a key innovation in surgery over the last 20 years, improving outcomes and becoming the standard of care in many areas. Within kidney transplantation, it has transformed the living donor nephrectomy operation, significantly improving outcomes. However, kidney transplantation has not changed significantly since it was first performed over 50 years ago until recently.
MIS techniques are methods developed to access body cavities, organs and tissues using smaller skin incisions. They have been shown to improve outcomes for many surgical procedures by reducing post‐operative morbidity (Gandaglia 2014) and by promoting a faster return to normal function after surgery (Nicholson 2010). These techniques include small incision open surgery, laparoscopic surgery and robotic surgery.
Small incision open surgery has variously been described as mini‐incision kidney transplantation and minimal access kidney transplantation. This involves open transplantation with an incision limited to 10 cm or less. The first reported series used an 8 cm incision (Oyen 2006) followed by 10 cm (Park 2008) and a 5 cm incision (Kacar 2013) with the kidney placed in the retroperitoneal space and all three anastomoses performed. An 8 cm incision with anastomoses performed with the kidney extracorporeal, then placed inside the abdomen, has also been reported (Brockschmidt 2014).
Laparoscopic surgery for kidney implantation also uses a reduced incision of about 7 cm for insertion of the kidney with an additional 3 to 4 ports for laparoscopic instruments (Rosales 2010; Modi 2011; Modi 2012; Modi 2013). The main differences with open surgery being the anastomoses are all performed intracorporeally with laparoscopic instruments. An alternative technique using the vagina to insert the kidney has also been described (Modi 2015).
Robotic‐assisted kidney transplantation is the latest development of minimally invasive surgery for kidney transplantation (MIKT). This procedure uses a small incision of about 7 cm to introduce the kidney with 4 or 5 laparoscopic ports. The key differences from laparoscopic transplantation are that the laparoscopic instruments are controlled remotely from a console by the surgeon and the instruments' design allows for an improved range of movement. This process has itself evolved from when it was first reported with a small open incision to the side of the lower abdomen followed by the robot being used to complete the anastomoses through this incision (Hoznek 2002) to retroperitoneal placement of the kidney through a similar incision but the anastomoses were completed transabdominally using the robot arms (Tsai 2014). A transabdominal technique has been developed (Abaza 2014; Boggi 2011; Giulianotti 2010) and standardised (Menon 2014a; Menon 2014b,; Sood 2014; Sood 2016). Variations of this technique are now being used worldwide (Breda 2016; Doumerc 2015; Frongia 2015; Tugcu 2016).
How the intervention might work
Patients with ESKD are more vulnerable to the stresses of surgery due to chronic uraemia and multiple systemic illnesses with multiple studies demonstrating increased complications and mortality in this group of patients undergoing surgery (Bechtel 2008; Drolet 2010; Gajdos 2013; Labrousse 1999; Schneider 2009). Kidney transplantation is conventionally performed with a large abdominal incision which is painful and at risk of complications and it poses a significant challenge to the body's homeostatic mechanisms. Minimally invasive surgery reduces the incision size which aids faster recovery with less pain. This has been demonstrated in donor nephrectomy surgery (Nicholson 2010; Wilson 2011). These benefits may also be relevant to ESKD patients undergoing transplantation.
Why it is important to do this review
A key issue arising from MIKT is the effect on the kidney allograft. Some MIKT techniques prolong the re‐warming time due to the anastomoses. Results suggest that this may reduce the eGFR in the early post‐transplant period when compared to open kidney transplant with no difference in function at 1 and 2 year follow‐up (Modi 2013). The long‐term consequences for the graft beyond this period are unknown. There is some evidence that prolonged anastomosis time is associated with an increased rate of delayed graft function and longer hospital stay in deceased donor kidney transplants (Marzouk 2013), this may raise concerns with regard to MIKT from living donors. The effects of pneumoperitoneum on the transplant kidney have also been questioned with suggestions that higher intra‐abdominal pressures may reduce allograft perfusion during surgery with negative consequences for the allograft based on experience with other studies of both human (Koivusalo 1998) and animal (Lindberg 2003) models. There is now some limited evidence regarding the effects of patient positioning and pneumoperitoneum on the outcome for MIKT (Parikh 2013).
Other important concerns for surgeons are issues with developing the appropriate skill sets required with minimally invasive surgery. It is not known how transferable skills from open surgery are to laparoscopic and robotic surgery. The learning curve is an area of interest and has been investigated in other specialties (Lucereau 2016) and might present a challenge to the transplant surgeon. Robotic surgery also has additional costs associated with acquiring equipment, skills and training. The machines are a scarce item in great demand which creates issues with access to them that may have implications for the transplant surgeon.
MIKT promises to offer significant benefits to kidney transplant recipients. A reduction in surgical site infections has already been noted in obese patients undergoing robotic MIKT (Oberholzer 2013) and further evidence suggests that morbidly obese patients derive equivalent benefit and complications from robotic surgery compared to open kidney transplantation (Garcia‐Roca 2016). Smaller incisions with reduced analgesia requirement are a key benefit which may affect length of stay and associated co‐morbidity, hospital costs and the patient experience.
Objectives
This review aims to look at the benefits and harms of MIS in kidney transplant recipients.
Methods
Criteria for considering studies for this review
Types of studies
All randomised controlled trials (RCTs) and quasi‐RCTs (RCTs in which allocation to treatment was obtained by alternation, use of alternate medical records, date of birth or other predictable methods) looking at minimally invasive surgical techniques for kidney transplant recipients.
Non‐randomised studies such as case‐control studies, cohort studies and case series will be included. These must have recipient demographics and detailed surgical techniques with stratified outcomes for each surgical technique (mini‐incision open, laparoscopic or robotic) that are reported in a standardized manner.
Non‐randomised studies will be included because this intervention is relatively new in kidney transplantation and there may not be any or sufficiently good quality RCTs available. Non‐randomised studies may be the only source of data for this intervention and it is important to be able to quantitatively assess the available evidence in a robust manner to help address concerns about this technique. This may provide the rationale for conducting future RCTs to provide the best evidence for this intervention.
Studies that do not report on any of our primary outcomes will be excluded.
Types of participants
Inclusion criteria
All patients undergoing kidney transplantation for ESKD by minimally invasive surgical techniques are eligible for inclusion in this review as well as patients undergoing conventional open kidney transplantation in one arm of a study involving other patients with MIS transplantation.
Exclusion criteria
Any patients in studies where conventional open kidney transplantation is the only intervention.
Types of interventions
Studies that include at least one of the following interventions will be included.
Mini‐incision open kidney transplantation
Laparoscopic kidney transplantation
Robotic kidney transplantation
The interventions will be considered in either single intervention studies or as part of multiple intervention studies involving MIS kidney transplantation.
Types of outcome measures
Outcome measures to be included are the established standard for reporting in kidney transplantation
Primary outcomes
Kidney function (GFR and creatinine at 1 year and latest reported)
Delayed graft function
Primary non‐function.
Secondary outcomes
Mortality (30 day, 1 year and overall)
Graft loss (1 year post transplant and overall)
Infections (all, surgical site, chest, urinary tract, viral, fungal)
Acute rejection
Pain
Incision length (in centimetres)
Incision hernia
Length of hospital stay
Warm ischaemia time(primary, rewarming time)
Operative time (total operative time and anastomosis time).
Search methods for identification of studies
Electronic searches
We will search the Cochrane Kidney and Transplant Specialised Register through contact with the Information Specialist using search terms relevant to this review. The Specialised Register contains studies identified from the following sources:
Monthly searches of the Cochrane Central Register of Controlled Trials (CENTRAL)
Weekly searches of MEDLINE OVID SP
Handsearching of kidney‐related journals and the proceedings of major kidney conferences
Searching of the current year of EMBASE OVID SP
Weekly current awareness alerts for selected kidney and transplant journals
Searches of the International Clinical Trials Register (ICTRP) Search Portal and ClinicalTrials.gov.
Studies contained in the Specialised Register are identified through search strategies for CENTRAL, MEDLINE, and EMBASE based on the scope of Cochrane Kidney and Transplant. Details of these strategies, as well as a list of handsearched journals, conference proceedings and current awareness alerts, are available in the Specialised Register section of information about Cochrane Kidney and Transplant.
See Appendix 1 for search terms used in strategies for this review.
We will also search MEDLINE (OVID) and EMBASE (OVID) for non‐randomised studies.
Searching other resources
Reference lists of review articles, relevant studies and clinical practice guidelines.
Letters seeking information about unpublished or incomplete trials to investigators known to be involved in previous studies.
Data collection and analysis
Selection of studies
The search strategy described will be used to obtain titles and abstracts of studies that may be relevant to the review. The titles and abstracts will be screened independently by two authors, who will discard studies that are not applicable, however studies and reviews that might include relevant data or information on trials will be retained initially. Two authors will independently assess retrieved abstracts and, if necessary the full text, of these studies to determine which studies satisfy the inclusion criteria.
Data extraction and management
Data extraction will be carried out independently by two authors using standard data extraction forms. Studies reported in non‐English language journals will be translated before assessment. Where more than one publication of one study exists, reports will be grouped together and the publication with the most complete data will be used in the analyses. Where relevant outcomes are only published in earlier versions these data will be used. Any discrepancy between published versions will be highlighted.
Assessment of risk of bias in included studies
The following items will be independently assessed by two authors using the risk of bias assessment tool (Higgins 2011) (see Appendix 2).
Was there adequate sequence generation (selection bias)?
Was allocation adequately concealed (selection bias)?
-
Was knowledge of the allocated interventions adequately prevented during the study?
Participants and personnel (performance bias)
Outcome assessors (detection bias)
Were incomplete outcome data adequately addressed (attrition bias)?
Are reports of the study free of suggestion of selective outcome reporting (reporting bias)?
Was the study apparently free of other problems that could put it at a risk of bias?
For non‐randomised studies, risk of bias will be assessed by two authors using the Newcastle‐Ottawa Scale (NOS) to reach an overall judgement of risk of bias (Appendix 3; Appendix 4) (Wells 2012).
Measures of treatment effect
For dichotomous outcomes (delayed graft function, primary non‐function, mortality, graft loss, acute rejection and incision hernia) results will be expressed as risk ratio (RR) with 95% confidence intervals (CI). Where continuous scales of measurement are used to assess the effects of treatment (kidney function (GFR), pain, incision length, length of hospital stay, warm ischaemia time, operative time), the mean difference (MD) will be used, or the standardised mean difference (SMD) if different scales have been used.
Unit of analysis issues
In studies with multiple intervention groups, only groups relevant to the review will be included (Higgins 2011). Otherwise it is expected that there will be minimal unit of analysis issues as participants can only be allocated to one intervention as part of a study.
Dealing with missing data
Any further information required from the original author will be requested by written correspondence (e.g. emailing and/or writing to corresponding author/s) and any relevant information obtained in this manner will be included in the review. Evaluation of important numerical data such as screened, randomised patients as well as intention‐to‐treat, as‐treated and per‐protocol population will be carefully performed. Attrition rates, for example drop‐outs, losses to follow‐up and withdrawals will be investigated. Issues of missing data and imputation methods (for example, last‐observation‐carried‐forward) will be critically appraised (Higgins 2011).
Assessment of heterogeneity
We will first assess the heterogeneity by visual inspection of the forest plot. We will quantify statistical heterogeneity using the I2 statistic, which describes the percentage of total variation across studies that is due to heterogeneity rather than sampling error (Higgins 2003). A guide to the interpretation of I2 values will be as follows.
0% to 40%: might not be important
30% to 60%: may represent moderate heterogeneity
50% to 90%: may represent substantial heterogeneity
75% to 100%: considerable heterogeneity.
The importance of the observed value of I2 depends on the magnitude and direction of treatment effects and the strength of evidence for heterogeneity (e.g. P‐value from the Chi2 test, or a confidence interval for I2) (Higgins 2011).
Assessment of reporting biases
If possible, funnel plots will be used to assess for the potential existence of small study bias (Higgins 2011).
Data synthesis
Data will be pooled using the random‐effects model but the fixed‐effect model will also be used to ensure robustness of the model chosen and susceptibility to outliers.
Data from RCTs and non‐randomised studies will be analysed and reported separately.
Subgroup analysis and investigation of heterogeneity
Subgroup analysis will be used to explore possible sources of heterogeneity for participants and interventions. Heterogeneity among participants could be related to age and renal pathology, subgroup analysis based on body mass index (BMI) will be performed. This is because one of the key indications for minimally invasive transplantation is obesity (Oberholzer 2013). Heterogeneity in treatments could be related to prior agent(s) used and the agent, dose and duration of therapy. Subgroup analysis of each intervention will be performed (Mini‐open, laparoscopic and robotic surgery). Adverse effects will be tabulated and assessed with descriptive techniques, as they are likely to be different for the various agents used. Where possible, the risk difference with 95% CI will be calculated for each adverse effect, either compared to no treatment or to another agent.
Sensitivity analysis
We will perform sensitivity analyses in order to explore the influence of the following factors on effect size.
Repeating the analysis excluding unpublished studies
Repeating the analysis taking account of risk of bias, as specified
Repeating the analysis excluding any very long or large studies to establish how much they dominate the results
Repeating the analysis excluding studies using the following filters: diagnostic criteria, language of publication, source of funding (industry versus other), and country.
'Summary of findings' tables
We will present the main results of the review in 'Summary of findings' tables. These tables present key information concerning the quality of the evidence, the magnitude of the effects of the interventions examined, and the sum of the available data for the main outcomes (Schünemann 2011a). The 'Summary of findings' tables also include an overall grading of the evidence related to each of the main outcomes using the GRADE (Grades of Recommendation, Assessment, Development and Evaluation) approach (GRADE 2008). The GRADE approach defines the quality of a body of evidence as the extent to which one can be confident that an estimate of effect or association is close to the true quantity of specific interest. The quality of a body of evidence involves consideration of within‐trial risk of bias (methodological quality), directness of evidence, heterogeneity, precision of effect estimates and risk of publication bias (Schünemann 2011b). We plan to present the following outcomes in the 'Summary of findings' tables.
Kidney function (GFR) at 1 year
Delayed graft function
Mortality (30 days and 1 year) and graft loss (within 1 year post transplant): these outcomes will be amalgamated into one for the summary of findings tables as numbers are expected to be low and they both represent the most critical quality marker for kidney transplantation
Infections (all sites)
Acute rejection
Pain
Length of hospital stay.
History
Protocol first published: Issue 7, 2017
Notes
June 2021: This protocol for a Cochrane Review has been withdrawn by Cochrane Kidney and Transplant. Unable to maintain contact with author team.
Acknowledgements
We wish to thank the referees for the comments and feedback during the preparation of this protocol.
Appendices
Appendix 1. Electronic search strategies
| Database | Search terms |
| CENTRAL |
|
| MEDLINE |
|
| EMBASE |
|
Appendix 2. Risk of bias assessment tool: randomised studies
| Potential source of bias | Assessment criteria |
|
Random sequence generation Selection bias (biased allocation to interventions) due to inadequate generation of a randomised sequence |
Low risk of bias: Random number table; computer random number generator; coin tossing; shuffling cards or envelopes; throwing dice; drawing of lots; minimisation (minimisation may be implemented without a random element, and this is considered to be equivalent to being random). |
| High risk of bias: Sequence generated by odd or even date of birth; date (or day) of admission; sequence generated by hospital or clinic record number; allocation by judgement of the clinician; by preference of the participant; based on the results of a laboratory test or a series of tests; by availability of the intervention. | |
| Unclear: Insufficient information about the sequence generation process to permit judgement. | |
|
Allocation concealment Selection bias (biased allocation to interventions) due to inadequate concealment of allocations prior to assignment |
Low risk of bias: Randomisation method described that would not allow investigator/participant to know or influence intervention group before eligible participant entered in the study (e.g. central allocation, including telephone, web‐based, and pharmacy‐controlled, randomisation; sequentially numbered drug containers of identical appearance; sequentially numbered, opaque, sealed envelopes). |
| High risk of bias: Using an open random allocation schedule (e.g. a list of random numbers); assignment envelopes were used without appropriate safeguards (e.g. if envelopes were unsealed or non‐opaque or not sequentially numbered); alternation or rotation; date of birth; case record number; any other explicitly unconcealed procedure. | |
| Unclear: Randomisation stated but no information on method used is available. | |
|
Blinding of participants and personnel Performance bias due to knowledge of the allocated interventions by participants and personnel during the study |
Low risk of bias: No blinding or incomplete blinding, but the review authors judge that the outcome is not likely to be influenced by lack of blinding; blinding of participants and key study personnel ensured, and unlikely that the blinding could have been broken. |
| High risk of bias: No blinding or incomplete blinding, and the outcome is likely to be influenced by lack of blinding; blinding of key study participants and personnel attempted, but likely that the blinding could have been broken, and the outcome is likely to be influenced by lack of blinding. | |
| Unclear: Insufficient information to permit judgement | |
|
Blinding of outcome assessment Detection bias due to knowledge of the allocated interventions by outcome assessors. |
Low risk of bias: No blinding of outcome assessment, but the review authors judge that the outcome measurement is not likely to be influenced by lack of blinding; blinding of outcome assessment ensured, and unlikely that the blinding could have been broken. |
| High risk of bias: No blinding of outcome assessment, and the outcome measurement is likely to be influenced by lack of blinding; blinding of outcome assessment, but likely that the blinding could have been broken, and the outcome measurement is likely to be influenced by lack of blinding. | |
| Unclear: Insufficient information to permit judgement | |
|
Incomplete outcome data Attrition bias due to amount, nature or handling of incomplete outcome data. |
Low risk of bias: No missing outcome data; reasons for missing outcome data unlikely to be related to true outcome (for survival data, censoring unlikely to be introducing bias); missing outcome data balanced in numbers across intervention groups, with similar reasons for missing data across groups; for dichotomous outcome data, the proportion of missing outcomes compared with observed event risk not enough to have a clinically relevant impact on the intervention effect estimate; for continuous outcome data, plausible effect size (difference in means or standardised difference in means) among missing outcomes not enough to have a clinically relevant impact on observed effect size; missing data have been imputed using appropriate methods. |
| High risk of bias: Reason for missing outcome data likely to be related to true outcome, with either imbalance in numbers or reasons for missing data across intervention groups; for dichotomous outcome data, the proportion of missing outcomes compared with observed event risk enough to induce clinically relevant bias in intervention effect estimate; for continuous outcome data, plausible effect size (difference in means or standardized difference in means) among missing outcomes enough to induce clinically relevant bias in observed effect size; ‘as‐treated’ analysis done with substantial departure of the intervention received from that assigned at randomisation; potentially inappropriate application of simple imputation. | |
| Unclear: Insufficient information to permit judgement | |
|
Selective reporting Reporting bias due to selective outcome reporting |
Low risk of bias: The study protocol is available and all of the study’s pre‐specified (primary and secondary) outcomes that are of interest in the review have been reported in the pre‐specified way; the study protocol is not available but it is clear that the published reports include all expected outcomes, including those that were pre‐specified (convincing text of this nature may be uncommon). |
| High risk of bias: Not all of the study’s pre‐specified primary outcomes have been reported; one or more primary outcomes is reported using measurements, analysis methods or subsets of the data (e.g. sub‐scales) that were not pre‐specified; one or more reported primary outcomes were not pre‐specified (unless clear justification for their reporting is provided, such as an unexpected adverse effect); one or more outcomes of interest in the review are reported incompletely so that they cannot be entered in a meta‐analysis; the study report fails to include results for a key outcome that would be expected to have been reported for such a study. | |
| Unclear: Insufficient information to permit judgement | |
|
Other bias Bias due to problems not covered elsewhere in the table |
Low risk of bias: The study appears to be free of other sources of bias. |
| High risk of bias: Had a potential source of bias related to the specific study design used; stopped early due to some data‐dependent process (including a formal‐stopping rule); had extreme baseline imbalance; has been claimed to have been fraudulent; had some other problem. | |
| Unclear: Insufficient information to assess whether an important risk of bias exists; insufficient rationale or evidence that an identified problem will introduce bias. |
Appendix 3. Risk of bias assessment for non‐randomised studies: Newcastle Ottawa Scale Form
Note: A study can be awarded a maximum of one star for each numbered item within the Selection and Outcome categories. A maximum of two stars can be given for Comparability.
Selection
-
Representativeness of the exposed cohort
truly representative of the average _______________ (describe) in the community *
somewhat representative of the average ______________ in the community *
selected group of users (e.g. nurses, volunteers)
no description of the derivation of the cohort
-
Selection of the non exposed cohort
drawn from the same community as the exposed cohort *
drawn from a different source
no description of the derivation of the non exposed cohort
-
Ascertainment of exposure
secure record (e.g. surgical records) *
structured interview *
written self‐report
no description
-
Demonstration that outcome of interest was not present at start of study
yes *
no
Comparability
-
Comparability of cohorts on the basis of the design or analysis
study controls for _____________ (select the most important factor) *
study controls for any additional factor * (This criteria could be modified to indicate specific control for a second important factor.)
Outcome
-
Assessment of outcome
independent blind assessment *
record linkage *
self‐report
no description
-
Was follow‐up long enough for outcomes to occur
yes (select an adequate follow up period for outcome of interest) *
no
-
Adequacy of follow‐up of cohorts
complete follow‐up: all subjects accounted for *
subjects lost to follow‐up unlikely to introduce bias: small number lost; < 15% follow‐up, or description provided of those lost *
follow‐up rate < 85% and no description of those lost
no statement
Appendix 4. Risk of bias assessment for non‐randomised studies: Newcastle Ottawa Scale
Coding manual for cohort studies
Selection
1. Representativeness of the exposed cohort
Item is assessing the representativeness of exposed individuals in the community, not the representativeness of the sample of women from some general population. For example, subjects derived from groups likely to contain middle class, better educated, health oriented women are likely to be representative of postmenopausal oestrogen users while they are not representative of all women (e.g. members of a health maintenance organisation (HMO) will be a representative sample of oestrogen users. While the HMO may have an under‐representation of ethnic groups, the poor, and poorly educated, these excluded groups are not the predominant users users of oestrogen).
Allocation of stars as per rating sheet
2. Selection of the non‐exposed cohort
Allocation of stars as per rating sheet.
3. Ascertainment of exposure
Allocation of stars as per rating sheet.
4. Demonstration that outcome of interest was not present at start of study
In the case of mortality studies, outcome of interest is still the presence of a disease/incident, rather than death. That is to say that a statement of no history of disease or incident earns a star.
Comparability
1. Comparability of cohorts on the basis of the design or analysis
A maximum of 2 stars can be allotted in this category. Either exposed and non‐exposed individuals must be matched in the design and/or confounders must be adjusted for in the analysis. Statements of no differences between groups or that differences were not statistically significant are not sufficient for establishing comparability. Note; If the relative risk for the exposure of interest is adjusted for the confounders listed, then the groups will be considered to be comparable on each variable used in the adjustment. There may be multiple ratings for this item for different categories of exposure (e.g. ever versus never, current versus previous or never) Age = , Other controlled factors =.
Outcome
1. Assessment of outcome
For some outcomes (e.g. fractured hip), reference to the medical record is sufficient to satisfy the requirement for confirmation of the fracture. This would not be adequate for vertebral fracture outcomes where reference to X‐rays would be required.
Independent or blind assessment stated in the paper, or confirmation of the outcome by reference to secure records (X‐rays, medical records, etc.)
Record linkage (e.g. identified through ICD codes on database records)
Self‐report (i.e. no reference to original medical records or X‐rays to confirm the outcome)
No description.
2. Was follow‐up long enough for outcomes to occur
An acceptable length of time should be decided before quality assessment begins (e.g. 5 years for exposure to breast implants).
3. Adequacy of follow‐up of cohorts
This item assesses the follow‐up of the exposed and non‐exposed cohorts to ensure that losses are not related to either the exposure or the outcome. Allocation of stars as per rating sheet.
Contributions of authors
Draft the protocol: RU
Study selection: RU, ZA
Extract data from studies: RU, ZA, PC, PG
Enter data into RevMan: RU, PG
Carry out the analysis: RU, ZA
Interpret the analysis: RU, ZA
Draft the final review: RU
Disagreement resolution: NM, NK
Update the review: RU
Sources of support
Internal sources
No sources of support provided
External sources
No sources of support provided
Declarations of interest
None known
Edited (no change to conclusions)
References
Additional references
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