Early erythropoietin for preventing red blood cell transfusion in preterm and/or low birth weight infants

Title

Reviewers

Dates

Contact reviewer

Contribution of reviewers

Internal sources of support

External sources of support

Text of review

Synopsis

Abstract

Background

Objectives

Secondary objectives:
Subgroup analyses of low (< 500 IU/kg/week) and high (> 500 IU/kg/week) doses of EPO and, within these subgroups, analyses of the use of low (< 5 mg/kg/day) and high (> 5 mg/kg/day) doses of supplemental iron, in reducing red blood cell transfusions in these infants.

Search strategy

Selection criteria

Data collection & analysis

Main results

Reviewers' conclusions

Background

EPO and iron effectively stimulate erythropoiesis. Plasma erythropoietin (EPO) levels in neonates are lower than those of older children and adults. Brown and colleagues reported that between two and thirty days of life the mean EPO concentration was 10 mIU/ml as compared to 15 mIU/ml in concurrently studied adults (Brown 1983). A low plasma EPO level is a key reason that nadir hematocrit values of preterm infants are lower than those of term infants (Stockman 1986; Dallman 1981). Low plasma EPO levels provide a rationale for use of EPO in the prevention or treatment of anaemia of prematurity. Studies in newborn monkeys and sheep have demonstrated that neonates have a large volume of distribution and more rapid elimination of EPO, necessitating the use of higher doses than required for adults (Ohls 2000). A recent systematic review of EPO administration noted a wide range of doses used, from 90 to 1400 IU/kg/week (Kotto-Kome 2004). Side effects reported in adults include hypertension, bone pain, rash and rarely seizures. Only transient neutropenia has been reported in neonates (Ohls 2000).

The primary goal of EPO therapy is to reduce transfusions. Most transfusions are given during the first three to four weeks of life. The larger or stable preterm infants who respond best to EPO receive few transfusions. ELBW infants, who are sick and have the greatest need for RBC transfusions shortly after birth, have not consistently responded to EPO. This suggests that EPO is a more effective erythropoietic stimulator in more mature neonates. ELBW neonates are more likely to need transfusions even if their erythropoiesis is stimulated (Kotto-Kome 2004). In addition, ELBW neonates have a smaller blood volume and the relatively larger phlebotomy volumes that are required during hospital stay often necessitate "early" transfusions. In contrast "late" transfusions are more often given because of anaemia of prematurity (Garcia 2002). Most preterm infants who require blood transfusions will receive their first transfusion in the first two weeks of life (Zipursky 2000). Reducing the number of RBC transfusions reduces the risk of transmission of viral infections and may reduce costs. Frequent RBC transfusions may be associated with retinopathy of prematurity (Hesse 1997) and bronchopulmonary dysplasia.

Preterm infants need iron for erythropoiesis. As neonatal blood volume expands with rapid growth, infants produce large amounts of haemoglobin. Several studies have observed a decrease in serum ferritin concentration (an indication of iron deficiency) (Finch 1982) - during erythropoietin treatment. The use of higher, more effective doses of erythropoietin might be expected to increase iron demand and the risk of iron deficiency (Genen 2004). Iron supplementation during erythropoietin treatment has been observed to reduce the risk of the development of iron deficiency (Shannon 1995). The range of iron doses used in EPO treated infants is between 1 mg/kg/day to 10 mg/kg/day (Kotto-Kome 2004).

The efficacy of EPO in anaemia of prematurity has recently been systematically reviewed (Vamvakas 2001; Garcia 2002; Kotto-Kome 2004). Vamvakas et al concluded that there is extreme variation in the results, and until this variation is better understood, it is too early to recommend EPO as standard treatment for the anaemia of prematurity (Vamvakas 2001). Garcia et al concluded that administering EPO to VLBW neonates can result in a modest reduction in late erythrocyte transfusions and that this effect is dependent on the dose of EPO used (Garcia 2002) . Kotto-Kome et al concluded that if EPO is begun in the first week of life, a moderate reduction can be expected in the proportion of VLBW neonates transfused. The reduction is less significant for early transfusion than for late transfusion (Kotto-Kome 2004).

EPO has recently been found to have important non-hematopoietic functions in the brain and other organs during development (Juul 2002). Administration of EPO could potentially have a neuro-protective effect in preterm infants, especially in perinatal asphyxia (Juul 2002, Dame 2001). This aspect of EPO use in neonates has not been systematically reviewed.

It is likely that additional studies of EPO in preterm or LBW infants have been published since the reviews noted above. We performed a series of Cochrane reviews on the use of EPO in preterm infants including: 'Early administration of erythropoietin (EPO) (starting in infants ≤ 7 days of age) vs. placebo/no treatment' (this review), 'Late EPO (starting in infants > 7 days of age) vs. placebo/no treatment' and 'Early vs. late EPO' (as per previous definitions). The cutoff of ≤ 7 days of age for early and > 7 days for late treatment with EPO, although somewhat arbitrary, was chosen based on previously published meta-analyses (Garcia 2002; Kotto-Kome 2004) to allow us to compare the results between our reviews and previously published reviews.

This review concerns early administration of EPO (starting in infants ≤ 7 days of age). The main rationale for such EPO therapy is to reduce exposure of neonates to red blood cell transfusion and its associated risks. Between 60% and 100% of preterm infants are transfused before three weeks of age (Shannon 1995; Juul 1999; Zipursky 2000) and EPO administered during this period might decrease the need for RBC transfusions (Brown 1990; Kotto-Kome 2004). Several studies have concentrated on the effectiveness of administering EPO, beginning in the first week of life, in reducing or eliminating these "early" transfusions. We conducted a systematic review to evaluate all available studies where EPO was begun during the first week of life to assess the effect on erythrocyte transfusions.

Objectives

Secondary objectives: Subgroup analyses were performed within this review for low (< 500 IU/kg/week) and high (> 500 IU/kg/week) doses of EPO, and the amount of iron supplementation; none, low (≤ 5 mg/kg/day) and high (> 5 mg/kg/day).

Criteria for considering studies for this review

Types of studies

Types of participants

Types of interventions

Types of outcome measures

SECONDARY OUTCOMES:
1. The total volume (ml/kg) of blood transfused per infant
2. Number of transfusions per infant
3. Number of donors to whom the infant was exposed
4. Mortality during initial hospital stay (all causes of mortality)
5. Retinopathy of prematurity (any stage and stage > 3)
6. Proven sepsis (clinical symptoms and signs of sepsis and positive blood culture for bacteria or fungi)
7. Necrotizing enterocolitis (NEC) (Bell's stage II or more) or (stage not reported)
8. Intraventricular haemorrhage (IVH); all grades (we included in this group results from studies that did not define the grade) and grades III and IV
9. Periventricular leukomalacia (PVL); cystic changes in the periventricular areas
10. Length of hospital stay (days)
11. Bronchopulmonary dysplasia (BPD) (supplementary oxygen at 28 days of age or at 36 weeks post menstrual age with or without compatible X-ray; we included an additional group in which the age at BPD was not stated)
12. Sudden infant death after discharge
13. Neutropenia
14. Hypertension (not a pre-specified outcome)
15. Long term outcomes assessed at any age beyond one year of age by a validated cognitive, motor, language, or behavioural/school/social interaction/adaptation test
16. Post hoc analysis: Any side effects reported in the trials. (It is not possible to predict every side effect that can occur with a certain intervention. However, it is important that 'new side-effects' are reported)

Search strategy for identification of studies

Methods of the review

For studies identified as abstracts, the primary author would be contacted to obtain further information if the full publication was not available. The quality of included trials was evaluated independently by the review authors using the following criteria:

Blinding of randomisation
Blinding of intervention
Blinding of outcome measure assessment
Completeness of follow up

There are three potential answers to these questions yes, no, cannot tell.

The statistical methods included (typical) relative risk (RR), risk difference (RD), number needed to treat to benefit (NNTB) or number needed to treat to harm (NNTH) for dichotomous outcomes and weighed mean difference (WMD) reported with 95% confidence intervals (CI). A fixed effects model was used for meta-analysis.

Heterogeneity tests including the I squared (I²) statistic were performed to assess the appropriateness of pooling the data.

Subgroup analyses were performed within this review for low (< 500 IU/kg/week) and high (> 500 IU/kg/week) doses of EPO, and no iron, low (≤ 5 mg/kg/day) and high (> 5 mg/kg/day) doses of supplemental iron by any route (co-intervention). Any amount of iron given i.v. was classified as high dose iron.

Two post-hoc analyses to try and explain the between study heterogeneity for the primary outcome 'Use of one or more red blood cell transfusions' were conducted. In the first post-hoc analysis we divided the studies into two groups 'High quality studies' and 'Studies of uncertain quality'. In the second post-hoc analysis, we analyzed the results for the three studies in which most of the neonatal intensive care units enrolling patients used satellite units of red blood cells for transfusion.

Description of studies

Twenty-three studies enrolling 2074 infants were included. The studies were performed in 18 countries (Austria, Belgium, Chile, China, France, Germany (FRG and GDR), Greece, Italy, Mexico, New Zealand, Poland, Singapore, South Africa, Switzerland, Turkey, the UK, the US). Seven studies were excluded (see Characteristics of excluded studies).

All studies fulfilled our inclusion criteria of a gestational age < 37 weeks and birth weight < 2500 g. Inclusion of infants in the studies was based on either gestational age or birth weight or a combination. The highest cut-off for birth weight was 1800 g and the highest cut-off for gestational age was 35 weeks (Chang 1998). The lowest cut-off for birthweight was 401 g (Ohls 2001A). Most studies used an upper cut-off for birth weight of 1500 g and a gestational age of 32 - 33 weeks.

EPO was administered subcutaneously (s. c.) or intravenously (i.v.) or in a combination of i.v. followed by s. c. when i.v. access was no longer available. The dose ranged from 70 IU/kg/week (Obladen 1991) to 2100 IU/kg/week (Haiden 2005). The duration of EPO treatment ranged from two weeks (Ohls 1995; Ohls 1997) to nine weeks (Maier 2002) or to discharge from hospital (several studies).

Many different EPO preparations were used; EPREX 2000, Santa-Farma-Gurel, Istanbul (Arif 2005), Eprex, Cilag, Italy (Carnielli 1998), Cilag A.G., Zug, Switzerland (Soubasi 1993; Soubasi 1995; Soubasi 2000), Eprex 4000, Cilag de Mexico SA de CV (Lima-Rogel 1998), Eprex; Janssen-Cilag, Auckland, New Zealand (Meyer 2003), Recormon, Boehringer (Avent 2002; Lauterbach 1995), NeoRecormon, F. Hoffman-La Roche, Basel, Switzerland (Maier 2002), Epoetin beta, Boehringer-Mannheim, GmbH, Germany (Maier 1994; Obladen 1991), Kirin Brewery, Co., Ltd., Japan (Chang 1998), unnamed product (Carnielli 1992;Ohls 1995; Ohls 1997; Ohls 2001A; Ohls 2001B; Romagnoli 2000; Yeo 2001), Erypo, Janssen-Cilag pharmaceuticals, Vienna, Austria (Haiden 2005; Meister 1997), Eritropoyetina del Laboraorio Andromaco (Salvado 2000).

Previous donor exposure was an exclusion criterion in one study (Arif 2005). Maier et al (Maier 1994) included 28 infants (23%) in the EPO group and 17 (14%) in the control group, who had received red blood cell transfusions prior to study entry. Maier et al (Maier 2002) reported that 24 (32%) of the infants in the early EPO group and 22 (31%) in the control group were exposed to donor blood before they entered the study. The authors of the remaining studies reported their specific exclusion criteria, but did not list prior transfusion as an exclusion criterion. We assumed that infants who had received prior red blood cell transfusions were included.

Details for the transfusion guidelines are reported in Additional Tables (Table 01 Transfusion guidelines). As noted in the table, transfusion guidelines were based on various Hct and/or Hgb levels. In addition, researchers used many other criteria such as need for assisted ventilation, supplemental oxygen, age of the infant, weight gain, clinical condition, physiological or biochemical signs thought to be associated with anemia. We were unable to categorize the different guidelines in a few groups that could be meaningfully used for secondary analyses.

Transfusion guidelines were reported to be in place in all but one study (Chang 1998). Lima-Rogel et al. (Lima-Rogel 1998) referred to the 3^rd Spanish edition of 'Care of the high-risk neonate' by Klaus and Fanaroff for the guidelines they adhered to (Klaus 1987). We were not able to locate that book, but in the 3^rd English edition of the book, we could not find transfusion guidelines for preterm infants (Klaus 1986).

In the study by Carnielli et al (Carnielli 1998), all infants received dedicated units of red blood cells. In one of the studies by Ohls et al. (Ohls 1997), it is stated that "In some instances a new donor would be used each day for the newborn intensive care unit (University of Florida) and in other instances a unit would be dedicated to a single infant for the life of the unit (University of New Mexico and University of Utah)". These two studies did not report on our primary outcome of 'Use of one or more red blood cell transfusions'. In the study by Maier et al (Maier 2002), 12 of the 14 centers used satellite packs of the original red cell pack to reduce donor exposure. In the two studies by Ohls et al (Ohls 2001A; Ohls 2001B) it is noted that "Whenever possible designated donor units that were capable of providing at least four transfusions were assigned to each infant (available in six of the eight participating centers)". In a secondary (post hoc) analysis we combined the results of these three studies.

Iron was administered in all studies. In most studies, both the EPO and the control groups received iron, but often the dose was lower in the control groups. In three studies (Carnielli 1992; Carnielli 1998; Romagnoli 2000), the infants in the control groups did not receive iron.

Included Studies:

Arif 2005 was a single centre study performed in Istanbul, Turkey.

• Objective: To determine whether EPO would prevent anemia of prematurity and reduce the need for transfusion.
• Population: Preterm infants < 33 weeks GA, birth weight < 1500 g, seven days old, not having previously had blood transfusion.
• Intervention: The EPO group received EPO 200 IU/kg s. c. from the seventh day of life and continued twice weekly (low dose) for 6 weeks. Placebo was not given to the control group. Both groups received iron (3 - 5 mg/kg per day orally) (low dose).
• Outcomes assessed: Use one or more red blood cell transfusions, mortality, NEC, ROP, BPD, neutropenia, side effects.

• Objective: To evaluate the effectiveness of early treatment with EPO (both high and low dose) in comparison to conventional treatment with packed red blood cell transfusions in the management of anaemia of prematurity in a country with limited resources.
• Population: Preterm infants < 7 days of age, in room air or requiring up to 30% oxygen at study entry with birth weight between 900 and 1500 g. Infants were stratified by weight < 1250 g and > 1250 g.
• Intervention: One group received EPO 400 IU/kg s. c. three times a week (high dose), a second group received EPO 250 IU/kg three times a week (high dose). All infants received a therapeutic dose of 6 mg/kg (high dose) elemental iron orally every day, it was increased to 8 - 10 mg/kg (high dose iron) if the hypochromic cells became 20 per cent or more.
• Outcomes assessed: Use of one or more red blood cell transfusions, total volume (ml/kg) of blood transfused per infant, number of blood transfusions per infant, mortality and sepsis.

• Objective: to determine whether early administration of high dose of rHuEPO and iron to premature infants would be well tolerated and would reduce the need for blood transfusions.
• Population: Preterm infants with birth weight ≤ 1750 g and a GA ≤ 32 weeks, two days old.
• Intervention: The EPO group received EPO 400 IU, three times weekly (high dose) i.v. or s. c. and iron 20 mg/kg once a week i.v. (high dose iron) from second day of life until discharge. The control group did not receive either EPO or iron.
• Outcome assessed: Number and volume of transfusions, number of donor exposure, mortality during hospital stay, neutropenia and hospital stay in days.

• Objective: To determine whether iron supplementation would enhance erythropoiesis in preterm infants treated with EPO.
• Population: Birth weight ≤ 1750 g and GA ≤ 32 weeks, two days old.
• Intervention: The EPO + iron group received 400 IU/kg EPO three times a week (high dose) and 20 mg/kg/week of i.v. iron (high dose). The EPO + no iron group received EPO, 400 IU/kg three times a week (high dose) without iron (no iron); infants in the control group received no treatment or placebo
• Outcomes assessed: Number of donor exposures, BPD, IVH, sepsis, ROP, days on ventilator, days of oxygen, days in hospital, days to regain birthweight and weight gain from birth to 8 weeks.

• Objective: To assess the efficacy and the optimum dose of EPO on the anaemia of prematurity.
• Population: Infants with GA ≤ 35 weeks, birth weight ≤ 1800 g , age one day and no Rh or ABO incompatibility
• Intervention: Infants in EPO group one received EPO 150 IU/kg three times a week for six weeks. Infants in EPO group two received EPO 250 IU/kg three times a week for six weeks and infants in the control group received no treatment. Infants in group three did not receive any EPO treatment. All infants received oral iron 20 mg/kg/day (high dose) from day seven after birth
• Outcomes assessed: use of one or more red blood cell transfusions, hypertension and side effects.

• Objective: The aim of the study was to evaluate the effect of EPO therapy on platelet reactivity and thrombopoiesis in extremely low birth weight infants.
• Population: Preterm infants with BW < 800 g and GA < 32 weeks, < 8 days old.
• Intervention: The EPO group received 300 IU/kg/day of EPO i.v. (as long as i.v. access was available), or 700 IU/kg three times per week (2100 IU/kg/week, high dose) and iron dextran 1.5 mg/kg/day i.v. or iron polymerase complex 9 mg/kg/day orally (high dose).
• Outcomes assessed: Use of one or more red blood cell transfusions, number of donors, mortality, NEC, PVL, IVH (grade I - II), IVH grade III - IV), hospital stay, BPD (age not stated), ROP (stage I - II), ROP (stage III - IV).

• Objective: To evaluate the role of EPO in the treatment of anemia of prematurity.
• Population: Preterm infants with GA < 35 weeks and birthweight </= 1500 g, seven days old.
• Interventions: Infants in EPO group I received EPO 100 IU/kg twice a week between days 7 and 37 (low dose), and infants in EPO group II received 400 IU/kg twice weekly (high dose) during the same time period. The control group received no treatment or placebo. Both EPO groups and the control group received 10 mg/kg/week of iron i.v. (high dose)
• Outcomes assessed: Total volume (ml/kg) of blood transfused between days 7 and 37.

• Objective: To determine the efficacy of EPO in very low birth weight newborns less than 72 hours of age.
• Population: infants with birth weight between 750 and 1500 g, < 72 hours old.
• Intervention: Infants in the EPO group received EPO 150 IU/kg/d (high dose) during the first 6 weeks and infants in the control group received placebo. Both groups received iron 4 mg/kg/day (low dose)
• Outcomes assessed: Number of transfusions per group, sepsis, NEC, IVH and BPD.

• Objective: To determine whether EPO would prevent anaemia and reduce the need for transfusion in infants with very low birth weights.
• Population: Infants with birth weights 750 to 1499 g, and three days old.
• Intervention: The EPO group received 250 IU of EPO/kg i.m. three times per week (750 IU/kg/week, high dose). The treatment continued until day 40 to 42, for total of 17 doses. Infants in the control group did not receive placebo but adhesive tape was placed on both thighs, which remained there until next visit. Oral iron 2 mg/kg/day was started on day 14 in all infants (low dose).
• Outcomes assessed: Use of one or more red blood cell transfusions, number of transfusions per infant, mortality, ROP, sepsis, NEC, IVH all grades, neutropenia, hypertension, side effects, weight gain and costs.

• Objective: To investigate whether EPO reduces need the need for transfusion in ELBW infants and to determine the optimal time for treatment
• Population: Infants with birth weight between 500 and 999 g. three to five days old.
• Intervention: The EPO group received EPO 250 IU/kg, i.v. or s. c. three time a week (high dose) starting on day three to five of life, for nine weeks. The control group received sham-injections. Enteral iron 3 mg/kg was given to all infants from days three to five (low dose) and was increased at days 12 to 14 to 6 mg/kg/day (high dose) and to 9 mg/kg/day (high dose) at days 24 to 26 of life.
• Outcomes assessed: Use of one or more red blood cell transfusions, Number of donors the infant was exposed to, number of transfusions per infant, mortality during hospital stay, NEC, IVH, PVL, ROP, BPD, growth, days in oxygen, days in NICU and days in hospital.

• Objective: To evaluate the effect of EPO on circulating hematopoietic progenitor cells in anaemic premature infants.
• Population: Preterm infants with birth weights of 750 to 1499 g, at an age of five to ten days.
• Intervention: The EPO group received EPO 300 IU/kg s. c. three times a week (high dose) for four weeks. The control group did not receive the drug or placebo. Oral iron administration was started with a dose of 6 mg/kg/day (high dose) and increased after two weeks to 8 mg/kg/day (high dose). The control group received iron alone.
• Outcomes assessed: Cumulative volume of blood transfused/kg.

• Objective: To comprehensively identify preterm infants likely to require blood transfusion and to investigate the effectiveness of EPO in this high risk subgroup.
• Population: Preterm infants with gestation < 33 weeks and birth weight < 1700 g, at an age of 48 hours.
• Intervention: Infants in the treatment group received EPO at 1200 IU/kg/week s. c. in three divided doses until three weeks of age and then the dose was reduced to 600 IU/kg/week, until 34 weeks corrected GA or for a minimum of 3 weeks. Infants in the control group received sham treatment. Both group received elemental iron. 21 infants in control group received sham treatment, to avoid subcutaneous injection. Iron at a dose of 6 mg /kg/day (high dose) was given to the EPO group once they had attained a postnatal age of two weeks and receiving at least 50% energy intake orally. Those in control group received 2 mg/kg/day iron (low dose) from the same age in a more dilute preparation so that an equivalent volume was given.
• Outcomes assessed: Use of one or more red blood cell transfusions and number of donors the infant was exposed to.

• Objective: To investigate whether treatment with EPO reduces the anaemia of prematurity and thus the need for transfusion by one third in preterm infants.
• Population: Preterm infants with GA 28 - 32 completed weeks, three days old.
• Intervention: The EPO group received EPO 30 IU/kg (low dose) every third day from fourth to 25 th day of life. The control group did not receive study drug or placebo. Iron treatment 2 mg/kg (low dose) orally was started on day 14, if there was no feeding intolerance
• Outcomes assessed: Use of one or more red blood cell transfusions, total volume of blood transfused per infant, mortality, chronic lung disease, IVH, NEC, BPD and hypertension.

• Objective: To determine whether administering EPO to ill VLBW infants, beginning on the first or second day of life, would reduce blood transfusions and be cost-effective.
• Population: Infants less than 48 hours of age with birth weight 750 and 1500 g and GA > 27 weeks.
• Intervention: The EPO group received EPO 200 IU/kg/day (high dose) i.v. for 14 consecutive days. The control group received similar volume of 0.9% saline solution in similar fashion as placebo. Infants in both groups received iron, 2 mg/kg/day (low dose) orally, when they were taking 70 ml/kg per day enterally, which was increased to 6 mg/kg per day (high dose) when the infants were receiving more than 100 ml/kg per day of feeds.
• Outcomes assessed: Use of one or more red blood cell transfusions, total volume of blood transfused per infant, number of transfusions per infant, neutropenia, thrombocytopenia, neutrophilia, NEC and IVH.

• Objective: To evaluate the effect of EPO on the transfusion requirements of preterm infants weighing 750 g or less.
• Population: Infants with birth weight 750 g or less and 72 hours of age or younger.
• Intervention: The EPO group received EPO 200 IU/kg/day (high dose) i.v., for 14 consecutive days. The control group received placebo as an equivalent volume of diluent in similar fashion. All infants received 1 mg/kg/day iron dextran in TPN solution during treatment period (high dose)
• Outcomes assessed: Total volume of blood transfused per infant, number of transfusions per infant, mortality, sepsis, IVH, BPD and ROP.

• Objective: To evaluate the effects of early EPO therapy on the transfusion requirements of preterm infants below 1250 g.
• Population: Infants with birth weight between 1001 g to 1250 g, GA < 32 weeks and between 24 and 96 hours of age at the time of study entry.
• Intervention: The EPO group received 400 IU/kg EPO 3 times weekly (high dose) i.v. or s. c. when i.v. access was not available. The placebo/control group received sham s. c. injections when i.v. access was not available. Treatment was continued until discharge, transfer, death or 35 completed weeks corrected gestational age. EPO treated infants received a weekly i.v. infusion of 5 mg/kg iron dextran (high dose) until they had an enteral intake of 60 ml/kg/day. Placebo/control infants received 1 mg/kg iron dextran (high dose) once a week, administered in a similar manner. Once infants in both groups had enteral intake of 60 mg/kg/day, they were given iron at a dose of 3 mg/kg/day (low dose). The dose was gradually increased to 6 mg/kg/day (high dose) depending on enteral intake.
• Outcomes assessed: Use one or more red blood cell transfusions, mean number of erythrocyte transfusions per infant, number of donors to whom the infant was exposed, total volume of blood transfused per infant, late onset sepsis, mortality, chronic lung disease, ROP, severe IVH, NEC >Bell's stage II, BPD, neutropenia and hypertension. In a follow-up study of the 72 EPO treated and 70 placebo-control infants surviving to discharge follow-up data at 18 to 22 months' corrected age were collected on 51 EPO -treated infants (71%) and 51 placebo/controls (73%). The outcomes assessed were growth, psycho-motor development, rehospitalization and transfusions.

• Objective: To evaluate the effects of early EPO therapy on the transfusion requirements of preterm infants below 1000 g.
• Population: Infants with birth weight 401 to 1000 g, GA < 32 weeks and between 24 and 96 hours of age at the time of study entry.
• Intervention: Intervention: The EPO group received 400 IU/kg EPO 3 times weekly (high dose) i.v. or s. c. when i.v. access was not available. The placebo/control group received sham s. c. injections when i.v. access was not available. Treatment was continued until discharge, transfer, death or 35 completed weeks corrected gestational age. EPO treated infants received a weekly i.v. infusion of 5 mg/kg iron dextran (high dose) until they had an enteral intake of 60 ml/kg/day. Placebo/control infants received 1 mg/kg iron dextran (high dose) once a week, administered in a similar manner. Once infants in both groups had enteral intake of 60 mg/kg/day, they were given iron at a dose of 3 mg/kg/day (low dose) . The dose was gradually increased to 6 mg/kg/day (high dose) depending on enteral intake.
• Outcomes assessed: Use of one or more red blood cell transfusions, mean number of erythrocyte transfusions per infant, number of donors to whom the infant was exposed, total volume of blood transfused per infant, late onset sepsis, mortality, chronic lung disease, ROP, severe IVH, NEC >Bell's stage II, BPD, neutropenia and hypertension. In a follow-up study of the 72 EPO treated and 70 placebo control infants surviving to discharge follow-up data at 18 to 22 months' corrected age were collected on 51 EPO -treated infants (71%) and 51 placebo/controls (73%). The outcomes assessed were growth, psychomotor development, rehospitalization and transfusions.

• Objective: To evaluate whether EPO and iron supplementation increase the risk of retinopathy of prematurity.
• Population: Infants with gestational age 30 weeks and those with GA 31 - 34 weeks with respiratory distress syndrome, seven days old.
• Intervention: The EPO group received EPO 300 IU/kg s. c., three times a week (high dose) and Iron 1 mg/kg/day i.v. (high dose). The control group did not receive EPO, placebo or iron.
• Outcomes assessed: Use of one or more red blood cell transfusions, number of blood transfusions per infant, ROP, NEC, IVH >garde II, BPD at 28 days, and sepsis.

• Objective: To asses the benefits of early EPO administration to reduce the requirement of blood transfusion in very low birth weight infants.
• Population: Newborn infants with birth weight under 1500 g. Treatment started before 12 days of age (mean age EPO group 7.75 days, control group 7.96 days).
• Intervention: The EPO group received EPO 200 IU/kg s. c. three times a week (high dose), during four weeks. The control group received similar volume of isotonic saline solution in similar fashion. All infants received oral iron at a dose of 3 mg/kg/day (low dose).
• Outcomes assessed: Number of transfusions per infant, sepsis, IVH and days on ventilator.

• Objective: To assess whether EPO treatment is safe and reduces the need for transfusion.
• Population: Infants with GA </= 31 weeks, birthweight </= 1500 g, age one to seven days, no history of haemolytic disease and clinically stable.
• Intervention: The EPO group received 150 IU/kg/dose of EPO twice a week (low dose) during 4 weeks. The control group received a placebo. From the 15 th day of life iron was started at 3 mg/kg/day (low dose) in all infants.
• Outcomes assessed: Use of one or more red blood cell transfusions, number of transfusions per infant, mortality, sepsis, neutropenia, weight gain, hospital stay.

• Objective: Follow up of VLBW infants after EPO treatment.
• Population: Infants with GA ≤ 31 weeks, birth weight ≤ 1500 g, age one to seven days, no history of haemolytic disease and clinically stable.
• Intervention: The EPO 300 group received EPO 150 IU/kg twice weekly (low dose) and the EPO 750 group received EPO 250 IU/kg three times a week (high dose). The control group did not receive any study drug or placebo. All infants received oral elemental iron, 3 mg/kg per day from day 15 of life (low dose)
• Outcomes assessed: Use of one or more red blood cell transfusions, number of blood transfusions per infant, mortality, follow up to one year of age, weight gain and hospital stay.

• Objective: To investigate the effect of EPO on oxygen affinity and adequate oxygen delivery to the tissues of stable preterm infants.
• Population: Infants with GA ≤ 31 weeks and birth weight ≤ 1300 g with clinical stability at the time of entry. Although the authors do not state the age at entry, we assumed the age to be seven days from a graph (fig 6.) in the publication.
• Intervention: The EPO group received 200 IU/kg every alternate day (high dose) s. c. The control group did not receive EPO or placebo. The infants received oral iron at a dose of 12 mg/kg/day (high dose) in the EPO group and 4 mg/kg/day (low dose) in the control group.
• Outcomes assessed: Use of one or more red blood cell transfusions and number of transfusions per infant.

• Objective: To study efficacy, safety and cost effectiveness of EPO in reducing transfusion needs in VLBW infants.
• Population: VLBW infants with GA ≤ 33 weeks, age five days.
• Intervention: The EPO group received EPO 250 IU/kg/dose s. c. three times a week (high dose) from day five to day. Infants in the control group did not receive a placebo. Infants in both groups received elemental iron, 3 mg/kg/day (low dose) orally from day 10 and increased to 6 mg/kg/day (high dose) when full feeds were well tolerated.
• Outcomes assessed: Use one or more red blood cell transfusions, mean number of erythrocyte transfusions per infant, total volume of blood transfused per infant, mortality, ROP, Sepsis, NEC, BPD, hypertension, BPD, and costs.

Methodological quality of included studies

Results

Further analyses were conducted including studies that used a high dose of EPO (> 500 U/kg/week) or a low dose of EPO (< 500 U/kg/week)

Outcome 01.02: Use of one or more red blood cell transfusions [high dose of EPO (> 500 U/kg/week)]

A total of 15 studies enrolling 1432 patients testing a high dose of EPO (Outcome table 01.02) reported on this outcome. A high dose of EPO significantly reduced the proportion of infants who received one or more red blood cell transfusions [typical RR 0.79 (95% CI 0.74, 0.86); typical RD -0.14 (95% CI -0.18, -0.09); NNT 7 (95% CI 6, 11)]. There was statistically significant heterogeneity for this outcome [RR (p< 0.0010; I² 62.4%); RD (p = 0.0006; I² 62.9%)].

A subgroup analysis for a high dose of EPO in combination with a high dose of iron (Outcome table 01.02) was conducted. A total of 12 studies enrolling 1067 infants reported on this outcome. A high dose of EPO with a high dose of iron significantly reduced the proportion of infants, who received one or more red blood cell transfusions [typical RR 0.84 (95% CI 0.77, 0.92); typical RD -0.11 (95% CI -0.16, -0.06); NNT 9 (95% CI 6, 17)]. The test for heterogeneity was statistically significant [RR (p = 0.03; I² = 50.3%); RD (p = 0.02; I² = 50.1%)].

A total of three studies enrolling 365 infants testing a high dose of EPO and a low dose of iron (Outcome table 01.02) reported on this outcome. A high dose of EPO and a low dose of iron significantly reduced the proportion of infants, who received one or more red blood cell transfusions [typical RR of 0.66 (95% CI 0.55, 0.80); typical RD -0.23 (95% CI -0.33, -0.14); NNT 4 (95% CI 3, 7)]. There was statistically significant heterogeneity for this outcome [RR (p = 0.02; I² = 75.2%); RD (p = 0.02; I² = 74.5%)].

Outcome 01.03: Use of one or more red blood cell transfusions [low dose of EPO (<500 U/kg/week)]

A total of three studies including 192 patients testing a low dose of EPO (Outcome table 01.03) reported on this outcome. A low dose of EPO did not demonstrate a significant reduction in the proportion of infants, who received one ore more red blood cell transfusions [typical RR 0.80 (95% CI 0.60, 1.07); typical RD of -0.10 (95% CI -0.22, 0.02). There was statistically significant heterogeneity for this outcome [RR (p = 0.07; I² = 69.6%); RD (p = 0.10; I² = 55.8%)].

Subgroup analysis for a low dose of EPO in combination with a high dose of iron (Outcome table 01.03) was conducted. One study enrolling 30 infants reported on this outcome.
In this study there were no outcomes in either group and the RR was not estimable and the non-significant RD was 0.00 (95% CI -0.12, 0.12).

Two studies enrolling 162 infants testing the effectiveness of a low dose of EPO in combination with a low dose of iron (Outcome table 01.03) reported on this outcome. A low dose of EPO in combination with a low dose of iron did not significantly reduce the proportion of infants, who received one or more red blood cell transfusions [typical RR was 0.80 (95% CI 0.60, 1.07), the typical RD was -0.12 (95% CI -0.26, 0.03). There was statistically significant heterogeneity (p = 0.07; I² = 69.6%) for RR and borderline statistically significant heterogeneity for RD (p = 0.17; I² = 48.0%)

Only one study included a group that received no iron (Carnielli 1998), however this study did not report on the primary outcome of interest 'Use of one or more red blood cell transfusions'.

SECONDARY OUTCOMES:

Outcome 01.04: The total volume (ml/kg) of red blood cells transfused per infant

A total of six studies enrolling 515 infants reported on the total volume of red blood cells transfused per infant. The significant typical WMD was a reduction of 6 ml/kg of blood transfused (ml/kg) per infant (95% CI -11, - 1). There was statistically significant heterogeneity for this outcome (p = 0.02; I² = 63.0%).

Carnielli et al (Carnielli 1998) reported on the mean (95% CIs) volume of blood (ml/kg) transfused for the three groups; EPO + iron 16.7 (4.9 - 28.6); EPO only 20.1 (6.2 - 34.2) and the control group 44.4 (29.0 - 59.7) (EPO vs. control, p = 0.028; EPO + iron vs. control, p = 0.009) (p-values according to authors).

Lauterbach et al (Lauterbach 1995) reported that infants treated with 800 IU/kg/week required statistically significantly lower volume (ml/kg) of packed erythrocytes in comparison to untreated infants, both between days seven and 37 of life (18.6 ml vs. 46.8 ml) and between day seven of life and the day of discharge (35.8 ml vs. 94.2 ml); (p < 0.04 for both comparisons).

Maier 2002 reported on the mean (SD) volume of blood transfused as ml/kg/day; early EPO group 0.7 (1.2) and control group 1.1 (1.2), (p-value not provided). Meister reported on the median (first and third quartile) volume of blood transfused as ml/kg/day; EPO group 0 (0, 0.47) and the control group 0.86 (0.5, 1.1).

Outcome 01.05: Number of red blood cell transfusions per infant

The results from 13 studies enrolling 1115 infants reported on the number of red blood cell transfusions per infant. The significant typical WMD for number of red blood cell transfusions per infant was -0.27 (95% CI -0.42,-0.12). There was statistically significant heterogeneity for this outcome (p = 0.002, I² = 61.5%)

Carnielli et al (Carnielli 1998) reported on the mean (95% CIs) number of red blood cell transfusions for the three groups; EPO + iron 1.0 (0.28 - 1.18); EPO only 1.3 (0.54 - 2.06) and the control group 2.9 (1.84 - 3.88), (control vs EPO, p = 0.065) and (control vs. EPO + iron, p = 0.035) (p-values are according to the authors).

Avent et al (Avent 2002) reported the median and range of number of transfusions across three groups; low dose EPO group 0 (0-1), high dose EPO 0 (0-2) and Control group 0 (0-4); p = 0.03 across the three groups. Haiden et al (Haiden 2005) reported on the number of transfusions; EPO group 2 (0-15), control group 4.5 (0-12) (not statistically significant according to the authors).

Outcome 01.06: Number of donors to whom the infant was exposed

Two studies enrolling 188 infants reported on this outcome in means and SDs. The significant typical WMD for number of donors to whom the infant was exposed was -0.63 (-1.07, -0.19). There was no statistically significant heterogeneity for this outcome (p = 0.59; I² = 0%).

Carnielli et al (Carnielli 1992) reported that the number of donor exposures ranged from 0 - 5 in the EPO group and 0 - 6 in the control group (p-value not provided). Haiden et al. (Haiden 2005) reported on this outcome in a similar fashion; EPO group number of donors 1 (0-10), control group 3 (0-5) (not statistically significant according to the authors).

Outcome 01.7: Mortality during initial hospital stay (all causes of mortality)

A total of 13 studies enrolling 1485 infants reported on this outcome. Mortality was not significantly altered by the use of EPO [typical RR; 0.90 (95% CI 0.66, 1.22); typical RD -0.01 (95% CI -0.04, 0.02)]. There was no statistically significant heterogeneity for the outcome (RR p = 0.96; I² = 0%; RD p = 0.98; I² = 0).

Outcome 01.08: Retinopathy of prematurity (any stage or stage not stated by authors)

A total of 10 studies enrolling 1425 infants reported on retinopathy of prematurity. We obtained unpublished data from the study by Maier (Maier 2002) on the highest grade of ROP recorded during the study among examined survivors. EPO increased (borderline significance) ROP (any stage or stage not stated by authors) [typical RR; 1.18 (95% CI 0.99, 1.40; p = 0.06); typical RD; 0.04 (95% CI 0.00, 0.08); p = 0.06)]. There was no statistically significant heterogeneity for this outcome [RR (p = 0.22; I² = 24.2%; RD (p = 0.10, I² = 39.4%)].

Outcome 01.09: Retinopathy of prematurity (stage >3)

A total of six studies enrolling 930 infants reported on severe ROP (stage > 3). EPO significantly increased retinopathy of prematurity (stage >3), [typical RR; 1.71 (94% CI 1.15, 2.54; typical RD; 0.05 (95% CI 0.01, 0.09); NNTH; 20 (95% CI 11, 100 ]. There was no statistically significant heterogeneity for this outcome for RR (p = 0.82; I² = 0%), but there was statistically significant heterogeneity for RD (p = 0.0007; I² = 76.4%).

Ohls 1997 reported no differences in ROP (stage 3 or greater) rates between groups (data not provided).

Outcome 01.10: Proven sepsis (clinical symptoms and signs of sepsis and positive blood culture for bacteria or fungi)

Ten studies including 1162 infants reported on this outcome. EPO did not significantly change the rates of proven sepsis [typical RR 0.92 (95% CI 0.74, 1.13); typical RD - 0.02 (95% CI -0.06, 0.03)]. There was no statistically significant heterogeneity [RD (p = 0.77; I² = 0%) or RD (p = 0.66; I² = 0%)].

Outcome 01.11: Necrotizing enterocolitis (NEC) (stage not reported)
No study stated the stage of NEC reported. We included any outcome stated as NEC in this analysis.
Ten studies reporting on 1471 infants were included. EPO did not significantly change the rates of NEC [typical RR; 1.02 (95% CI 0.69, 1.51); typical RD 0.00 (95% CI -0.02, 0.03)]. There was no statistically significant heterogeneity for this outcome [RR (p = 0.86; I² = 0%); RD (p = 0.67; I² = 0%)].
Ohls 1995 reported no differences in NEC rates between groups (data not provided)

Outcome 01.12: Intraventricular haemorrhage (IVH); all grades

Many authors did not state the grade of IVH. We included in this outcome studies in which the grade was not stated and excluded IVH grades III-IV. A total of 8 studies including 744 infants reported on this outcome. EPO did not significantly change the rate of IVH (all grades), [typical RR; 0.99 (95% CI 0.70, 1.40); typical RD 0.00 (95% CI -0.05, 0.05)]. There was no statistically significant heterogeneity for this outcome [RR (p = 0.91; I² = 0%); RD (p = 0.87; I² = 0%)].

Ohls 1995 and Ohls 1997 reported no differences in IVH rates between groups (data not provided)

Outcome 01.13: Intraventricular haemorrhage (IVH); grades III and IV

A total of five studies enrolling 801 infants reported on this outcome. EPO did not significantly change the rate of IVH (grade III and IV), [typical RR; 1.13 (95% CI 0.64, 1.99); typical RD 0.01 (95% CI -0.02, 0.04)]. There was no statistically significant heterogeneity for this outcome [RR (p = 0.74; I² = 0%); RD (p = 0.67; I² = 0%)].

Outcome 01.14: Periventricular leukomalacia (PVL); cystic changes in the periventricular areas

Two studies enrolling 185 infants reported on PVL. EPO did not significantly change the rate of PVL, [typical RR was 0.92 (95% CI 0.27, 3.10); typical RD 0.00 (95% CI -0.06, 0.05)]. There was no statistically significant heterogeneity for this outcome [RR (p = 0.45; I² = 0%); RD (p = 0.54; I² = 0%)].

Outcome 01.15: Length of hospital stay (days)

A total of four studies enrolling 375 infants reported on the length of hospital stay. EPO did not significantly change length of hospital stay [typical WMD; 0.77 (95% CI -4.63, 6.16)]. There was no statistically significant heterogeneity for this outcome (p = 0.78; I² = 0%).

Avent el al (Avent 2002) reported the median and range (days) for hospital stay across three groups; low dose EPO 32 (5-54), high dose EPO 32 (16-74) and control group 30 (14-46); p = 0.10 across the three groups.

Haiden et al (Haiden 2005) reported on the hospital stay (days, median and range) EPO group 97 (59 - 162) and control group 89 (77 -157) (not statistically significant according to authors).

Maier 2002 reported on the median (quartiles) for hospital stay; early EPO group 87 (73 - 107, control group 87 (69 - 108).

Outcome 01.16: Bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) (supplemental oxygen at 28 days of age) (Outcomes table 01.16).

Two studies enrolling 330 infants reported on the use of supplemental oxygen at 28 days. EPO did not significantly change the rate of BPD (supplemental oxygen at 28 days of age), [typical RR; 1.27 (95% CI 0.90, 1.80); typical RD; 0.07 (95% CI -0.03, 0.16)]. There was no statistically significant heterogeneity for this outcome for RR (p = 0.12; I² = 58.2%) but for RD (p = 0.07; I² = 69.3%).

Ohls 1995 and Ohls 1997 reported no differences in BPD rates between groups (data not provided).

Bronchopulmonary dysplasia (BPD) (supplemental oxygen at age 36 weeks postmenstrual age) (Outcomes table 01.16).

Three studies enrolling 435 infants reported on the use of supplemental oxygen at 36 weeks postmenstrual age. EPO did not significantly change the rate of BPD (supplementary oxygen at age 36 weeks postmenstrual age), [typical RR;1.00 (95% CI 0.78, 1.29); typical RD 0.00 (95% CI -0.08, 0.08). There was no statistically significant heterogeneity for this outcome [RR (p = 0.86; I² = 0%); RD (p = 0.86; I² = 0%)].

Bronchopulmonary dysplasia (BPD) (age at diagnosis not stated) (Outcomes table 01.16).

A total of five studies enrolling 528 infants reported on this outcome. EPO did not significantly change the rate of BPD (age at diagnosis not stated), [typical RR; 0.98 (95% CI 0.61, 1.56); typical RD 0.00 (95% CI -0.05, 0.05). There was no statistically significant heterogeneity for this outcome [RR (p = 0.74; I² = 0%); RD (p = 0.67; I² = 0%)].

Sudden infant death after discharge (no outcomes table)

No study reported on this outcome

Outcome 01.17: Neutropenia

Nine studies including 982 infants reported on neutropenia. The non-significant typical RR was 0.81 (95% CI 0.53, 1.24); typical RD - 0.01 (95% CI -0.05, 0.02). There was no statistically significant heterogeneity for this outcome; RR (p = 0.61; I² = 0%); RD (p = 0.35; I² = 10.3%).

Outcome 01.18: Hypertension

A total of six studies enrolling 762 infants reported on hypertension. In five studies there were no outcomes in either the treatment or the control groups. Therefore, these five studies did not provide any information to the typical RR estimate. The RR (for one study) was 3.02 (95% CI 0.12, 73.52). All 6 studies are included in the typical RD; - 0.00 (95% CI -0.01, 0.02). There was no statistically significant heterogeneity for this outcome; RR (not applicable); RD (p = 1.00; I² = 10.3%).

Outcome 01.19: Long term outcomes assessed at any age beyond one year of age by a validated cognitive, motor, language, or behavioural/school/social interaction/adaptation test

Mental Developmental Index (MDI) < 70 at 18-22 month's corrected age (Outcomes table 01.19)

One study reported on this outcome in 90 children following EPO treatment. The RR was 0.88 (95% CI 0.49, 1.57); RD -0.04 (955 CI; -0.24, 0.15). These findings were not statistically significant.

Outcome 01.20: Psychomotor Developmental Index (PDI) < 70 at 18 - 22 months corrected age

One study reported on this outcome in 90 children following EPO treatment. The RR was 2.33 (95% CI 0.98, 5.53); RD 0.18 (95% CI 0.01, 0.35). These findings were not statistically significant.

Outcome 01.21: Cerebral palsy at 18-22 months corrected age

One study reported on this outcome in 99 children following EPO treatment. The RR was 1.06 (95% CI 0.46, 2.45); RD 0.01(-0.14, 0.16). These findings were not statistically significant.

Outcome 01.22 Any neurodevelopmental impairment at 18-22 month's corrected age

One study reported on this outcome in 99 infants following EPO treatment. The RR was 0.97 (95% CI 0.62, 1.51); RD -0.01 (-0.21, 0.18). These findings were not statistically significant.

Any side effects reported in the trials (No outcomes table)

Side effects were specifically reported not to have occurred in the following trials (Carnielli 1992, Chang 1998, Lauterbach 1995, Lima-Rogel 1998, Maier 1994, Meister 1997, Ohls 1995).

Outcome 01.23: Use of one or more red blood cell transfusions (secondary analysis - based on perceived study quality)

In a post hoc analysis to try and explain the between study heterogeneity for the primary outcome we divided the studies into two groups 'High quality studies' and 'Studies of uncertain quality. There were no substantial differences between either the point estimates for the effect size for the two groups nor was there any real differences in the degree of heterogeneity when all studies were analyzed together or in two separate groups.

Outcome 01.24: Use of one or more red blood cell transfusions (secondary analysis - based on NICUs using mostly satellite units of red blood cells)

In a second post hoc analysis to try and explain the between study heterogeneity for the primary outcome, we analyzed the results for the three studies in which most of the neonatal intensive care units used satellite units of red blood cells for transfusion. A total of three studies enrolling 435 infants reported on this outcome. The use of EPO in combination with dedicated red blood cell transfusion units did not significantly reduce the use of one or more red blood cell transfusions, [typical RR 0.91 (95% CI 0.81, 1.01; typical RD; -0.07 (95% CI -0.15, 0.01). There was no statistically significant heterogeneity for this outcome (RR; p = 0.52, I² = 0%; RD; p = 0.68, I² = 0%).

A funnel plot for the primary outcome 'Use of one or more red blood cell transfusions' was asymmetric, with a relative absence of smaller studies not having a protective effect (see Additional figures - Figure 01).

Discussion

In only one study (Arif 2005) did the authors specifically state that infants were not eligible to enter the study if they previously had received a red blood cell transfusion. In two studies it was stated that infants were included if they had received prior red blood cell transfusions; the rates varied between 14 and 32% (Maier 1994; Maier 2002). Although often not stated, it is likely that infants who had received blood transfusion prior to study entry were not excluded, as this was not mentioned as an exclusion criterion. All studies except one followed guidelines (with tremendous variation between studies) for red blood cell transfusions (see additional table - Table 01).

The use of early EPO in preterm infants (n = 2074) has been extensively studied. This review provides evidence that early administration of EPO significantly reduces the 'Use of one or more blood transfusions' following study entry, with a low NNTB of 8 and a narrow 95 % CI of 6 to 11. From our results, we cannot make a recommendation with regards to the best combination of high or low dose EPO and high or low dose of iron. We had arbitrarily set a cutoff of < 5 mg/kg/day of oral intake of iron for low and high dose of iron. When we conducted the review, we discovered that several studies started with i.v. administration of iron in variable doses, and we considered any i.v. dose of iron as a high dose. Early EPO significantly reduces the total volume (ml/kg) of red blood cells transfused, the number of red blood cell transfusions per infant and the number of donor exposures. For these outcomes, the effect sizes were small and of limited clinical importance.

There was statistically significant heterogeneity for the primary outcome, as well as for two important secondary outcomes (the 'Total volume of blood transfused per infant' and 'Number of transfusions per infant'). In an attempt to explore the reason for the between study heterogeneity, we performed a post-hoc analysis for the primary outcome. We divided the studies into two groups; 'High quality studies' (studies with concealed allocation and the use of a placebo or sham injections) and 'Studies of uncertain quality' (studies in which those criteria could not be ascertained). There were no substantial differences between either the point estimates for the effect size for the two groups, nor was there any real differences in the degree of heterogeneity when all studies were analyzed together or in two separate groups by quality. In our late EPO review (Aher 2006a), some of the heterogeneity could be explained by the same exercise. There could be other explanations for the heterogeneity, such as differences in dosing regimens for EPO and iron, EPO preparations, blood sampling, indications for transfusion (the rates of transfusions in the control groups varied), use of non-invasive monitoring, general standards of care and baseline characteristics among the infants enrolled. In an additional post-hoc analysis, we analyzed the results from three multicenter studies in which most of the centers used satellite packs of red blood cells for multiple transfusions to the same infant. The use of EPO in combination with dedicated red blood cell transfusion units did not significantly reduce the use of one or more red blood cell transfusions. There was no statistically significant heterogeneity for this outcome.

The use of red blood cells from satellite-bag-equipped dedicated units decreases donor exposure in low birth weight infants (Lee 1995). In a single center report, the red blood cell transfusion guidelines for infants with birth weight < 1000 g were changed three times (in 1989, 1991, and 1995) to become more restrictive (Maier 2000). The changes were made in association with the introduction of new EPO trials. Since 1990, the primary red blood cell pack was divided into three to four satellite packs. The median number of transfusions decreased from seven in the first period to two in the third period. Donor exposure decreased from five to one, and the blood volume transfused decreased from 131 to 37 ml/kg. The authors explained the changing transfusion practices to be due to several factors; stricter transfusion guidelines, increased adherence to transfusion guidelines, efforts to reduce sampling loss, and EPO therapy. The authors suggested that "not using transfusions to replace defined blood volume loss had the highest impact on reduced transfusions" (Maier 2000).

The importance of the marked reduction in the primary outcome in this review is limited by the fact that any donor exposure was likely not avoided, as many infants had required red blood cell transfusions prior to study entry. We assume that in most studies, infants who had received blood transfusions were not excluded, as most studies reported specific exclusion criteria that did not include prior red blood cell transfusions. It is unlikely that either the statistically significant reduction of < 1 (WMD - 0.63) donors to whom the infant was exposed, or the 6 ml/kg per infant (WMD - 6.03) reduction in total volume of blood transfused is of clinical importance.

With the exception of ROP, there were no statistically significant reductions/increases in the many secondary neonatal outcomes that we included a priori in this systematic review. There was a strong trend for increased risk of ROP (any stage reported) with the use of early EPO , which reached statistical significance for ROP stage > 3. With so many secondary outcomes included, this could be a chance finding. Only one study had as its primary objective to "Evaluate whether EPO and iron supplementation increase the risk of retinopathy of prematurity" (Romagnoli 2000). In that study, there was a statistically significantly increased risk of ROP following EPO treatment. The authors speculated that iron supplementation could be a contributing factor. In our early vs. late EPO review (Aher 2006b), we noted an increase in ROP with early EPO treatment, but not in the late EPO review (Aher 2006a). It may be that the infant is at greatest risk if EPO is administered early, starting in the first week of life. In an observational study, Rudzinska 2002 from Poland reported an increased risk of ROP following early vs. late treatment with EPO.

Manzoni 2005 reported data on 695 neonates with birth weights < 1500 g who were admitted between 1997 and 2004. Threshold ROP occurred in 31.4 percent (54 of 172) of infants < 1000 g who received erythropoietin therapy, as compared with 19.6 percent (22 of 112) of those who did not receive erythropoietin (p =0 .01 in univariate analysis, p = 0.04 in multivariate analysis; p-values according to authors). The authors suggested that erythropoietin is an additional, independent predictor of severe threshold ROP in infants < 1000 g (Manzoni 2005). In a retrospective case control analysis of 85 very low birth weight infants, Shah et al (Shah 2005) found no difference in the rate of ROP between EPO and control infants. However, they noted a significant weak positive correlation between the duration of EPO treatment and development of threshold ROP (Shah 2005). In an analysis of data from a neonatal network in South America, Musante 2006 et al found that treatment with EPO independently increases the risk of ROP and severe ROP. The incidence and severity of ROP may depend on the dose of EPO (Liu 2006). In a murine-normoxia-induced proliferative retinopathy model, Morita 2003 et al implicated EPO as a key factor in the development ROP, especially in the development of neovascularization. The authors suggested a therapeutic possibility of EPO and vascular endothelial growth factor (VEGF) inhibitors in the treatment of ROP (Morita 2003). Watanabe et al (Watanabe 2005) studied vitreous EPO levels in 73 adult patients with proliferative diabetic retinopathy and found that the median level was significantly higher (p < 0.001) than in 71 patients without diabetes. They suggested that EPO is a potent ischemia-induced angiogenic factor that acts independently of VEGF during retinal angiogenesis in proliferative diabetic retinopathy (Watanabe 2005).

These studies support an association between early EPO and ROP. Therefore, our finding of an increased risk of ROP following early administration of EPO should be taken seriously. Previous systematic reviews (including fewer studies than in our reviews) have not included ROP (or other common neonatal outcomes) as outcome measures (Vamvakas 2001, Garcia 2002, Kotto-Kome 2004). Those reviews noted similar effect sizes for transfusion needs and also reported on statistically significant between study heterogeneity.

In the analysis of 'Retinopathy of prematurity (stage > 3)' (outcome table 01.09), four of the five included studies used a combination of high EPO and high iron doses. In two studies, the iron dose was higher in the EPO treated group (Ohls 2001A; Ohls 2001B); in one study the control group did not receive iron (Romagnoli 2000); and in one study iron was provided i.v. in the EPO group from the initiation of therapy whereas the control group received oral iron from the 15th day of life. In one (low EPO) study (Maier 1994) both groups received the same amount of iron (2 mg/kg/day). It is therefore possible that the higher doses of iron was the cause of or contributed to the higher rates of ROP (stage > 3) in the EPO treated infants in these trials.

Any future studies of EPO should include ROP as an outcome measure of importance and data-monitoring/safety committees should be provided with this information on an ongoing basis. We will try to obtain unpublished data from the authors of published studies. It is likely that the outcome of ROP has been recorded in study protocols, or as most studies are of small sample size, could be obtained from patients' charts. Until more information is available, either from published studies or ongoing studies regarding ROP, we do not recommend any new trials of early EPO, especially in view of the limited benefits identified in this extensive review.

Meanwhile it is important that neonatal intensive care units develop practice guidelines to limit blood losses and donor exposure in neonates. The use of satellite packs and conservative transfusion guidelines reduces the exposure to multiple donors during the hospital stay. The need for red blood cell transfusions is linked to the loss of blood from sampling for laboratory testing.

Overall, early EPO provides very limited clinical benefits. It is associated with an increased risk for ROP stage >/= 3 and therefore its use is not recommended.

Reviewers' conclusions

Implications for practice

Implications for research

Acknowledgements

Potential conflict of interest

Characteristics of included studies