Zinc supplementation of parenteral nutrition in newborn infants

Abstract

This is a protocol for a Cochrane Review (Intervention). The objectives are as follows:

The primary objectives are to determine the effectiveness and safety of parenteral zinc supplementation with regards to morbidity and mortality in parenterally fed term and preterm infants.

Secondary objectives are to determine:

1. Which infants (within predefined subgroups: gestational age ‐ term, preterm, very preterm, extremely preterm; birthweight ‐ low birth weight, very low birth weight, extremely low birthweight; and, infants at greater risk of zinc deficiency due to specific illnesses) would benefit most from zinc supplementation;

2. The optimal dose of parenteral zinc supplementation: lower (< 450 μg/kg per day for preterm infants and < 250 μg/kg for infants less than 3 months) versus higher dose (≥ 450 μg/kg per day for preterm infants and ≥ 250 μg/kg per day for infants less than 3 months) zinc supplementation; and

3. The optimal timing of parenteral zinc supplementation: early (commenced at < 2 weeks of age) versus late (commenced at ≥ two weeks of age).

Background

Description of the condition

Zinc is an important trace element for protein, carbohydrate and lipid metabolism; neurological development; immune function and tissue growth (Aggett 2000; Friel 1994; Krebs 2014). Cells with rapid turnover demand the highest concentrations of zinc, notably skin, gastrointestinal, immunological, neurological, and, in the developing infant, skeletal cells.

The prevalence of zinc deficiency varies and is frequently unknown. The World Health Organization (WHO) estimated 20% of the world's population to be zinc deficient (WHO 1973). It is estimated that up to 82% of pregnant or lactating women worldwide may have inadequate zinc intakes (Caulfield 1998). Serum zinc is considered the best biochemical indicator of zinc status within a population. A prevalence of greater than 20% of low zinc concentrations is indicative of a significant elevated risk within a population (Krebs 2014).

Infants and toddlers are vulnerable to zinc deficiency due to their rapid growth rate and high demand for tissue synthesis. Reduced zinc intake occurs in infants who receive a combination of human milk and primarily plant based complementary foods that are low in zinc, particularly in populations with increased consumption of foods high in phytates (cereals, vegetables) which restrict the bioavailability of zinc. Estimated fetal accumulation of zinc ranges from 240 μg/day at 26 weeks' gestation to 675 μg/day at 36 weeks' gestation, averaging about 250 μg/kg per day across gestations (Friel 1994; Shaw 1979). Low birth weight infants, both preterm and small for gestational age, are at high risk of zinc deficiency (Agarwal 2013; Friel 1984; Krebs 2014). Several factors contribute to zinc deficiency in preterm infants including reduced zinc accumulation during the third trimester, use of parenteral nutrition (PN) without sufficient zinc supplementation, net intestinal zinc loss from the immature or diseased gastrointestinal tract, increased demand due to catch‐up growth and insufficient intake of zinc in breast milk once feeds are established (Aggett 2000; Dauncey 1977; Friel 1994; Lockitch 1983; Shaw 1979). Although colostrum is relatively high in zinc, the zinc content of breast milk falls substantially over time resulting in a negative zinc balance in preterm infants fed human milk (Arnaud 1995; Dauncey 1977; Nassi 1974; Shaw 1979).

Zinc levels collected from cord blood or within 96 hours of birth are negatively correlated with gestational age and birth weight (Lockitch 1983; Perveen 2002). Cord zinc levels are reported to be a mean ± standard deviation (SD) 13 ± 3 μmol/L (88 ± 20 μg/dL) (Kasperek 1977; Perveen 2002), and serum zinc levels (mean ± SD) reach a nadir of 9.7 ± 0.7 μmol/L (65 ± 5 μg/dL) at five months of age (Ohtake 1977), lower than reported in children over five years of age and adults who have a mean of approximately 15 μmol/L (100 μg/dL) (Brown 1998; Kasperek 1977). Preterm infants fed human milk have a negative zinc balance up to the 60th day of life. Zinc levels decrease with postnatal age (Dauncey 1977; Lockitch 1983; Shaw 1979), and have been reported to reach a nadir in preterm infants at six to 12 weeks of age (Altigani 1989; Dauncey 1977).

Clinical signs of zinc deficiency do not usually present until approximately three months of age (Altigani 1989), may be difficult to detect and may be associated with other nutrient deficiencies (Krebs 2014). Mild to moderate zinc deficiency is characterised by stunted growth, deficits in immune function, and altered integrity and function of the gastrointestinal tract (Krebs 2014). Zinc deficiency may be associated with deficits in attention, and motor and cognitive development in children (Black 1998), although there is no convincing evidence from randomised controlled trials that zinc supplementation of infants or children improves motor or mental development (Gogia 2012). This suggests that associations between zinc deficiency and altered development, behavioural and cognitive function may be due to concomitant macronutrient and micronutrient deficiencies. Subclinical features include oxidative stress and a proinflammatory state (Krebs 2014). In its severe form, characteristic signs of zinc deficiency include; periorificial and periacral dermatitis, alopecia and diarrhoea (Krebs 2014). Zinc deficiency may also be associated with tremor, irritability, jitteriness, hoarse cry, stomatitis, glossitis, paronychia, nail dystrophy and increased susceptibility to infection (Aggett 2000). Very rare forms of inherited zinc deficiency exist including acrodermatitis enteropathica (reduced intestinal zinc absorption) and transient neonatal zinc deficiency (low breast milk zinc content), characterised by periorificial and acral dermatitis, alopecia and diarrhoea, which may also be responsive to oral zinc supplementation (Kambe 2015).

Description of the intervention

PN is an intravenous solution used in the neonatal intensive care unit to meet the fluid, energy, protein, vitamin and mineral requirements of infants unable to be fed enterally. Preterm infants often endure prolonged periods of low enteral intakes due to illness, feed intolerance and necrotising enterocolitis (NEC). When the administration of PN is considered necessary for infants unable to tolerate oral feeding and nutrients, supplementation with zinc, as well as all other essential elements are often included (Zlotkin 1983). Preterm infants need a higher zinc intake than term infants because of their rapid growth equating to 450 μg/kg per day to 500 μg/kg per day to match in utero accretion rates (Friel 1994). Standard PN trace element preparations may not supply this amount so additional zinc may need to be added for the preterm infant or infants with high zinc losses from diarrhoea, stomal losses or severe skin disease (Koletzko 2005). The European Society of Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) recommendations are for an intravenous intake of 250 μg/kg per day for infants less than three months of age, 100 μg/kg per day for infants more than three months of age and 50 μg/kg per day for children (maximum of 5.0 mg/day). Zinc is the only trace element recommended as an additive to PN solutions for people on short term PN.

How the intervention might work

Preterm infants, especially extremely preterm and small for gestational age infants, are at increased risk of mortality, impaired postnatal growth, sepsis, chronic lung disease, NEC, retinopathy of prematurity and abnormal neurodevelopmental outcomes (Bolisetty 2014; Bolisetty 2015). A relationship between higher nutrient intake, improved postnatal growth and more optimal neurodevelopmental outcome at 18 months has previously been reported for preterm infants (Lucas 1990). Zinc supplementation may have the capacity to improve immune function and the integrity of the skin and gastrointestinal mucosa in preterm infants, reducing the incidence of nosocomial sepsis and NEC, and improving enteral feed tolerance. Similarly, improved epithelial cell repair and postnatal growth might reduce the incidence and severity of chronic lung disease. Enteral feeds with human milk are insufficient to create a normal zinc balance in preterm infants. Recommended enteral intake of zinc for preterm infants are 1.1 mg/kg per day to 2.0 mg/kg per day with the zinc to copper molar ratio in infant formulae should not exceed 20 (Agostoni 2010). Supplementation of PN with zinc reduces this deficit and may prevent clinical and biochemical zinc deficiency.

Several Cochrane Reviews have assessed the efficacy of zinc supplementation in various populations. Zinc supplementation of pregnant women in low income settings has been shown to reduce preterm birth but there was no difference in the incidence of low birthweight infants (Ota 2015). Zinc supplementation in infants and children was found to reduce the incidence and prevalence of pneumonia, the leading cause of death in children (Lassi 2010). Zinc supplementation of infants and children with diarrhoea and persistent diarrhoea in areas where the prevalence of zinc deficiency is high was found to reduce the duration of diarrhoea, although the effect has not been shown in infants below six months of age (Lazzerini 2013). These high level evidence findings suggest zinc supplementation has a potential role in reducing susceptibility to and severity of infection in newborn infants.

Why it is important to do this review

Zinc deficiency is very common worldwide and, although actual prevalence figures are not known, it is estimated that approximately 20% of the world population are zinc deficient (Brown 2004;Krebs 2014). WHO estimates that approximately 800,000 deaths per year are related to zinc deficiency, and that over 50% of these are infants and children under five years of age. Low birth weight infants, both preterm and small for gestational age, are at high risk of zinc deficiency (Agarwal 2013; Friel 1984). Zinc replacement has been shown to be beneficial in specific populations including pregnant women in reducing preterm delivery (Ota 2015), and in infants and children for preventing pneumonia (Lassi 2010), and reducing the duration of diarrhoea (Lazzerini 2013). Zinc supplementation of parenteral intake has the potential to prevent zinc deficiency and improve the outcomes of low birth weight and ill newborn infants.

Objectives

The primary objectives are to determine the effectiveness and safety of parenteral zinc supplementation with regards to morbidity and mortality in parenterally fed term and preterm infants.

Secondary objectives are to determine:

1. Which infants (within predefined subgroups: gestational age ‐ term, preterm, very preterm, extremely preterm; birthweight ‐ low birth weight, very low birth weight, extremely low birthweight; and, infants at greater risk of zinc deficiency due to specific illnesses) would benefit most from zinc supplementation;

2. The optimal dose of parenteral zinc supplementation: lower (< 450 μg/kg per day for preterm infants and < 250 μg/kg for infants less than 3 months) versus higher dose (≥ 450 μg/kg per day for preterm infants and ≥ 250 μg/kg per day for infants less than 3 months) zinc supplementation; and

3. The optimal timing of parenteral zinc supplementation: early (commenced at < 2 weeks of age) versus late (commenced at ≥ two weeks of age).

Methods

Criteria for considering studies for this review

Types of studies

Randomised and quasi‐randomised controlled trials, and cluster randomised trials where the unit of randomisation is a group of participants. We will exclude cross‐over trials.

Types of participants

Newborn infants receiving PN not enrolled on the basis of zinc status.

Types of interventions

1. Parenteral zinc supplementation compared to placebo or no treatment.

2. Early (commenced at less than two weeks of age) versus late (commenced at two weeks of age or more) parenteral zinc supplementation.

3. Higher (450 μg/kg per day or greater for preterm infants and 250 μg/kg per day or greater for infants less than three months of age) versus lower (less than 450 μg/kg per day for preterm infants and less than 250 μg/kg per day for infants less than three months of age) parenteral zinc supplementation.

Trials that specifically examine other types of trace element supplementation (e.g. copper, selenium, magnesium) in addition to zinc will be analysed separately, according to the type of trace element.

For example:

1. zinc supplementation plus copper supplementation versus placebo;

2. zinc supplementation plus selenium supplementation versus placebo;

3. zinc supplementation plus magnesium supplementation versus placebo.

We will exclude trials that have additional macronutrient supplementation which is not the same in both groups.

Types of outcome measures

Primary outcomes

1. Mortality prior to hospital discharge (latest time reported 36 weeks' postmenstrual age or greater).

2. Neurodevelopmental disability at 18 months' postnatal age or greater defined as a neurological abnormality including any one of the following:

   1. cerebral palsy on clinical examination;

   2. developmental delay more than two SDs below population mean on a standardised test of development;

   3. blindness (visual acuity less than 6/60); or

   4. deafness (any hearing impairment requiring amplification) at any time after term corrected.

3. Clinical zinc deficiency (as defined by author ‐ including any of the following; periorificial and periacral dermatitis, alopecia and diarrhoea, supported by the presence of low zinc levels or response to treatment) (Krebs 2014) (incidence up to latest time reported).

Secondary outcomes

1. Biochemical zinc deficiency (serum zinc greater than two SD below mean for postnatal age ‐ see Shaw 1979) at latest time reported up to discharge from hospital.

2. Late onset sepsis (positive bacterial culture in cerebrospinal fluid, sterile urine or blood greater than 48 hours of age).

3. Necrotising enterocolitis (NEC) (Bell's grade greater than 1) (Bell 1978).

4. Chronic lung disease (respiratory support or oxygen requirement at or beyond 36 weeks' postmenstrual age) (Shennan 1988).

5. Days on respiratory support (mechanical ventilation, continuous positive airway pressure, high flow nasal cannula, oxygen).

6. Days on oxygen.

7. Intraventricular haemorrhage (any or severe (grade 3 or 4)) (Papile 1978).

8. Periventricular leukomalacia (cystic) (de Vries 1992).

9. Term magnetic resonance imaging brain abnormalities graded as normal, mild, moderate or severe (Inder 2003).

10. Retinopathy of prematurity (any or severe (grade 3 or 4)) (ICROP 2005).

11. Postnatal growth failure (weight less than 10th percentile near term corrected age or at discharge).

12. Days of hospitalisation.

13. Infant growth: up to age one month; at latest time measured (definition = from one month to time of discharge); to follow‐up beyond 12 months:

    1. weight gain (g/kg per day);

    2. linear growth (cm/week);

    3. head circumference (cm/week).

14. Change of standardised growth: up to age one month; at latest time measured (definition = from one month to time of discharge); to follow‐up beyond 12 months:

    1. change in weight z‐score;*

    2. change in length z‐score;

    3. change in head circumference z‐score.

15. Individual components of neurodevelopment at 18 months' postnatal age or greater:

    1. cerebral palsy on clinical examination;

    2. developmental delay more than two SDs below population mean on a standardised test of development;

    3. blindness (visual acuity less than 6/60); or

    4. deafness (any hearing impairment requiring amplification) at any time after term corrected.

16. Neurodevelopment performance at 18 months' postnatal age or greater: developmental or intelligence quotients on a validated, standardised test.

*A z‐score is defined as the deviation of an observed value for an individual from the median value of the reference population, divided by the SD of the reference population (WHO 1995).

Search methods for identification of studies

Electronic searches

We will conduct a comprehensive search including: Cochrane Central Register of Controlled Trials (CENTRAL, current issue) in the Cochrane Library; MEDLINE via PubMed (1966 to current); Embase (1980 to current) and CINAHL (1982 to current) using the following search terms: (zinc OR trace element* OR micronutrient*) AND parenteral*, plus database‐specific limiters for randomised controlled trials and neonates (see Appendix 1 for the full search strategies for each database). We will not apply language restrictions.

We will search clinical trials registries for ongoing or recently completed trials (ClinicalTrials.gov (clinicaltrials.gov); the WHO International Trials Registry and Platform (www.whoint/ictrp/search/en/), and the ISRCTN Registry (www.isrctn.com/)). We will also search proceedings of the American Pediatric Society ‐ Pediatric Academic Societies annual meetings, European Society for Paediatric Research, Perinatal Society of Australia and New Zealand (PSANZ) (psanz.com.au/) and the Society for Parenteral and Enteral Nutrition for abstracts.

Searching other resources

We will perform additional searches using reference lists from other reviews and assessed studies, as well as seeking the advice of expert informants.

Data collection and analysis

We will use the criteria and standard methods of Cochrane and the Cochrane Neonatal Review Group. Two review authors will independently perform data extraction using a structured proforma and resolve differences by discussion. We will contact study authors to request additional data from unpublished studies in abstract form if required.

Selection of studies

Two review authors will independently assess the eligibility of all studies for inclusion in this review. Where there is uncertainty about inclusion of the study, we will retrieve the full text. We will resolve differences by consensus.

Data extraction and management

We will extract data independently using Review Manager 5 (RevMan 2014). We will resolve differences by consensus.

Assessment of risk of bias in included studies

We will assess risk of bias for each study as outlined in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2011), using the following criteria:

1. random sequence generation: selection bias (biased allocation to interventions) due to inadequate generation of a randomised sequence;

2. allocation concealment: selection bias (biased allocation to interventions) due to inadequate concealment of allocations prior to assignment;

3. blinding of participants and personnel: performance bias due to knowledge of the allocated interventions by participants and personnel during the study;

4. blinding of outcome assessment: detection bias due to knowledge of the allocated interventions by outcome assessors;

5. incomplete outcome data: attrition bias due to amount, nature or handling of incomplete outcome data;

6. selective reporting: reporting bias due to selective outcome reporting;

7. other bias: bias due to problems not covered elsewhere in the table.

See Appendix 2 for more detailed description of risk of bias for each domain.

Measures of treatment effect

We will carry out statistical analysis using the standard methods of the Cochrane Neonatal Review Group.

Dichotomous data

We will report dichotomous data using risk ratios (RR) with 95% confidence intervals (CI). We will calculate risk difference (RD) and number needed to treat for an additional beneficial outcome (NNTB) or for an additional harmful outcome (NNTH) with 95% CIs if there is a statistically significant difference in RR.

Continuous data

We will report continuous data using mean difference (MD) with 95% CI.

Unit of analysis issues

The unit of randomisation will be the intended unit of analysis and we expect this to be individual infants. Cluster‐randomised controlled trials will be eligible.

Cluster‐randomised trials

We plan to include cluster‐randomised trials in the analyses along with individually randomised trials. We intend to analyse them using the methods described in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2011), using an estimate of the intracluster correlation coefficient (ICC) derived from the trial (if possible) or from another source. If ICCs from other sources are used, we intend to report this and conduct sensitivity analyses to investigate the effect of variations in the ICC. If we identify both cluster‐randomised trials and individually randomised trials, we plan to synthesise the relevant information. We will consider it reasonable to combine the results from both if there is little heterogeneity between the study designs, and if we considered interaction between the effect of the intervention and the choice of randomisation unit to be unlikely.

Dealing with missing data

We will obtain missing data from the authors when possible. Where missing data cannot be obtained, we will examine the effect of excluding trials with substantial missing data (e.g. greater than 10% losses) in sensitivity analyses.

We will attempt to overcome potential bias from missing data (greater than 10% losses) using one or more of the following approaches:

1. whenever possible, contact the original investigators to request missing data;

2. performing sensitivity analyses to assess how sensitive the results are to reasonable changes in the assumptions that are made (e.g. the effect of excluding trials with substantial missing data (greater than 10% losses);

3. addressing the potential impact of missing data (greater than 10% losses) upon the findings of the review in the discussion section.

Assessment of heterogeneity

We will use Review Manager 5 to assess the heterogeneity of treatment effects between trials (RevMan 2014). We will use the following.

1. The Chi² test, to assess whether observed variability in effect sizes between studies is greater than would be expected by chance. Since this test has low power when the number of studies included in the meta‐analysis is small, we will set the probability at the 10% level of significance.

2. The I² statistic to ensure that pooling of data is valid. We will grade the degree of heterogeneity as less than 25% = none, 25% to 49% = low, 50% to 74% = moderate and 75% or greater = high heterogeneity.

We will assess the source of the heterogeneity using sensitivity and subgroup analyses, looking for evidence of bias or methodological differences between trials where there is evidence of apparent or statistical heterogeneity.

Assessment of reporting biases

We will assess reporting/publication bias by visual inspection of funnel plot asymmetry where there are 10 or more studies included in a meta‐analysis. If we find significant asymmetry in the funnel plot, we will report this in the corresponding results.

Data synthesis

We will perform statistical analyses according to the recommendations of the Cochrane Neonatal Review Group (neonatal.cochrane.org/en/index.html). We will analyse all infants randomised on an intention‐to‐treat basis. We will analyse treatment effects in the individual trials. We will use a fixed‐effect model in the first instance to combine the data. For any meta‐analyses, for categorical outcomes, we will calculate typical estimates of RR and RD, each with the 95% CI; for continuous outcomes, we will calculate the MD if outcomes are measured in the same way between trials, and standardised mean difference (SMD) to combine trials that measured the same outcome but used different scales. We plan to analyse and interpret individual trials separately when we judge meta‐analysis to be inappropriate.

Certainty of evidence

We will use the GRADE approach, as outlined in the GRADE Handbook 2013 (Schünemann 2013), to assess the certainty of evidence for the following (clinically relevant) outcomes: mortality prior to hospital discharge; neurodevelopmental disability at 18 months postnatal age or greater; clinical zinc deficiency; biochemical zinc deficiency: NEC; chronic lung disease and postnatal growth failure (weight less than 10th percentile near term corrected age or at discharge).

Two review authors will independently assess the certainty of the evidence for each of the outcomes. We will consider evidence from randomised controlled trials as high certainty but downgrade the evidence one level for serious (or two levels for very serious) limitations based upon the following: design (risk of bias), consistency across studies, directness of the evidence, precision of estimates and presence of publication bias. We will use the GRADEpro Guideline Development Tool (GRADEpro 2014) to create a 'Summary of findings' table to report the certainty of the evidence.

The GRADE approach results in an assessment of the certainty of a body of evidence in one of four grades.

1. High: we are very confident that the true effect lies close to that of the estimate of the effect.

2. Moderate: we are moderately confident in the effect estimate: the true effect is likely to be close to the estimate of the effect, but there is a possibility that it is substantially different.

3. Low: our confidence in the effect estimate is limited: the true effect may be substantially different from the estimate of the effect.

4. Very low: we have very little confidence in the effect estimate: the true effect is likely to be substantially different from the estimate of effect.

Subgroup analysis and investigation of heterogeneity

Where there are sufficient data from two or more studies corresponding to any of the prespecified subgroups to be combined in a meta‐analysis, we will explore potential sources of clinical heterogeneity through the following a priori subgroup analyses/

Comparison 1. Parenteral zinc supplementation compared to placebo or no treatment

Gestation:

Term (37 weeks' gestation or greater); preterm (32 weeks' gestation or greater to 36⁶ weeks' gestation); very preterm (28 weeks' gestation or greater to 31⁶ weeks' gestation); extremely preterm (less than 28 weeks' gestation).

Birthweight:

Low birth weight 1500 g or greater to less than 2500 g; very low birth weight 1000 g or greater to less than 1500 g; extremely low birth weight less than 1000 g.

Infants at risk of zinc deficiency:

Infants with coexisting disease associated with increased zinc requirements (e.g. infants with gastrointestinal or severe skin disease; Koletzko 2005).

Enteral zinc intake by one month of age:

Above or below recommended requirements of 1.1 mg/kg per day to 2.0 mg/kg per day (Agostoni 2010).

Comparison 2. Early versus late supplementation of parenteral zinc

Timing and duration of parenteral zinc supplementation

Commenced before two weeks of age versus two weeks of age or greater (Koletzko 2005).

Comparison 3. Higher versus lower parenteral zinc supplementation

Parenteral dose

Lower dose (less than 450 μg/kg per day) versus higher dose (450 μg/kg per day or greater) for preterm infants.

Lower dose (less than 250 μg/kg per day) versus higher dose (250 μg/kg per day or greater) for infants less than three months of age (Koletzko 2005).

Sensitivity analysis

We will explore methodological heterogeneity using sensitivity analyses if sufficient data are available. We will perform sensitivity analyses through excluding trials of lower quality, based on a lack of any of the following: allocation concealment, adequate randomisation, blinding of treatment and less than 10% loss to follow‐up. We will explore for heterogeneity between studies that compared zinc supplementation versus placebo compared with zinc supplementation versus treatment.

Appendices

Appendix 1. Search strategies

(zinc OR trace element* OR micronutrient*) AND parenteral*

Combine with database‐specific terms:

The Cochrane Library

AND (infant or newborn or neonate or neonatal or preterm or preterm or very low birth weight or low birth weight or VLBW or LBW)

PubMed

AND ((infant, newborn[MeSH] OR newborn OR neonate OR neonatal OR preterm OR low birth weight OR VLBW OR LBW or infan* or neonat*) AND (randomized controlled trial [pt] OR controlled clinical trial [pt] OR randomized [tiab] OR placebo [tiab] OR drug therapy [sh] OR randomly [tiab] OR trial [tiab] OR groups [tiab]) NOT (animals [mh] NOT humans [mh]))

Embase

AND (infant, newborn or newborn or neonate or neonatal or preterm or very low birth weight or low birth weight or VLBW or LBW or Newborn or infan* or neonat*) AND (human not animal) AND (randomized controlled trial or controlled clinical trial or randomized or placebo or clinical trials as topic or randomly or trial or clinical trial)

CINAHL

AND (infant, newborn OR newborn OR neonate OR neonatal OR preterm OR low birth weight OR VLBW OR LBW or Newborn or infan* or neonat*) AND (randomized controlled trial OR controlled clinical trial OR randomized OR placebo OR clinical trials as topic OR randomly OR trial OR PT clinical trial)

Appendix 2. 'Risk of bias' tool

1. Random sequence generation Selection bias (biased allocation to interventions) due to inadequate generation of a randomised sequence.
Criteria for a judgement of 'Low risk' of bias.	The investigators describe a random component in the sequence generation process such as: referring to a random number table; using a 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 equivalent to being random.
Criteria for a judgement of 'High risk' of bias.	The investigators describe a non‐random component in the sequence generation process. Usually, the description would involve some systematic, non‐random approach, for example: sequence generated by odd or even date of birth; sequence generated by some rule based on date (or day) of admission; sequence generated by some rule based on hospital or clinic record number. Other non‐random approaches happen much less frequently than the systematic approaches mentioned above and tend to be obvious. They usually involve judgement or some method of non‐random categorisation of participants, for example: allocation by judgement of the clinician; allocation by preference of the participant; allocation based on the results of a laboratory test or a series of tests; allocation by availability of the intervention.
Criteria for a judgement of 'Unclear risk' of bias.	Insufficient information about the sequence generation process to permit judgement of 'Low risk' or 'High risk'.
2. Allocation concealment Selection bias (biased allocation to interventions) due to inadequate concealment of allocations prior to assignment.
Criteria for a judgement of 'Low risk' of bias.	Participants and investigators enrolling participants could not foresee assignment because 1 of the following, or an equivalent method, was used to conceal allocation: central allocation (including telephone, web‐based and pharmacy‐controlled randomisation); sequentially numbered drug containers of identical appearance; sequentially numbered, opaque, sealed envelopes.
Criteria for a judgement of 'High risk' of bias.	Participants or investigators enrolling participants could possibly foresee assignments and thus introduce selection bias, such as allocation based on: 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 non­opaque or not sequentially numbered); alternation or rotation; date of birth; case record number; any other explicitly unconcealed procedure.
Criteria for a judgement of 'Unclear risk' of bias.	Insufficient information to permit judgement of 'Low risk' or 'High risk'. This is usually the case if the method of concealment is not described or not described in sufficient detail to allow a definite judgement (e.g. if the use of assignment envelopes is described, but it remains unclear whether envelopes were sequentially numbered, opaque and sealed).
3. Blinding of participants and personnel Performance bias due to knowledge of the allocated interventions by participants and personnel during the study.
Criteria for a judgement of 'Low risk' of bias.	Either: 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.
Criteria for a judgement of 'High risk' of bias.	Either: 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.
Criteria for a judgement of 'Unclear risk' of bias.	Either: insufficient information to permit judgement of 'Low risk' or 'High risk'; the study did not address this outcome.
4. Blinding of outcome assessment Detection bias due to knowledge of the allocated interventions by outcome assessors.
Criteria for a judgement of 'Low risk' of bias.	Either: 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.
Criteria for a judgement of 'High risk' of bias.	Either: 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.
Criteria for a judgement of 'Unclear risk' of bias.	Either: insufficient information to permit judgement of 'Low risk' or 'High risk'; the study did not address this outcome.
5. Incomplete outcome date Attrition bias due to amount, nature or handling of incomplete outcome data.
Criteria for a judgement of 'Low risk' of bias.	Any 1 of: 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 (mean differences or standardised mean differences) among missing outcomes not enough to have a clinically relevant impact on observed effect size; missing data have been imputed using appropriate methods.
Criteria for a judgement of 'High risk' of bias.	Any 1 of: 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 (mean differences or standardised mean differences) 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.
Criteria for a judgement of 'Unclear risk' of bias.	Either: insufficient reporting of attrition/exclusions to permit judgement of 'Low risk' or 'High risk' (e.g. number randomised not stated, no reasons for missing data provided); the study did not address this outcome.
6. Selective reporting Reporting bias due to selective outcome reporting.
Criteria for a judgement of 'Low risk' of bias.	Either: the study protocol is available and all the study's prespecified (primary and secondary) outcomes that are of interest in the review have been reported in the prespecified way; the study protocol is not available but it is clear that the published reports include all expected outcomes, including those that were prespecified (convincing text of this nature may be uncommon).
Criteria for the judgement of 'High risk' of bias.	Any 1 of: not all the study's prespecified primary outcomes have been reported; ≥ 1 primary outcome is reported using measurements, analysis methods or subsets of the data (e.g. subscales) that were not prespecified; ≥ 1 reported primary outcome is not prespecified (unless clear justification for their reporting is provided, such as an unexpected adverse effect); ≥ 1 more outcome of interest in the review is reported incompletely so that they cannot be entered in a meta‐analysis; the study report did not include results for a key outcome that would be expected to have been reported for such a study.
Criteria for the judgement of 'Unclear risk' of bias.	Insufficient information to permit judgement of 'Low risk' or 'High risk'. It is likely that most studies will fall into this category.
7. Other bias Bias due to problems not covered elsewhere in the table.
Criteria for a judgement of 'Low risk' of bias.	The study appears to be free of other sources of bias.
Criteria for the judgement of 'High risk' of bias.	There is ≥ 1 important risk of bias. For example, the study: has a potential source of bias related to the specific study design used; or has been claimed to have been fraudulent; or has some other problem.
Criteria for the judgement of 'Unclear risk' of bias.	There may be a risk of bias, but there is either: Insufficient information to assess whether an important risk of bias exists; or Insufficient rationale or evidence that an identified problem will introduce bias.

What's new

Date	Event	Description
17 November 2016	Amended	Incorporated editors feedback

Contributions of authors

AT, LJ and DO wrote the protocol.

Sources of support

Internal sources

• No sources of support supplied

External sources

• Australian Satellite of the Cochrane Neonatal Review Group, Australia.

  Supported by NH&MRC grant ACT9100101

• National Institute for Health Research, UK.

  Editorial support for Cochrane Neonatal has been funded with funds from a UK National Institute of Health Research Grant (NIHR) Cochrane Programme Grant (13/89/12). The views expressed in this publication are those of the authors and not necessarily those of the NHS, the NIHR, or the UK Department of Health.

• Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, USA.

  Editorial support of the Cochrane Neonatal Review Group has been funded with Federal funds from the Eunice Kennedy Shriver National Institute of Child Health and Human Development National Institutes of Health, Department of Health and Human Services, USA, under Contract No. HHSN275201600005C

Declarations of interest

None identified.

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