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PEDIATRICS Vol. 105 No. 5 May 2000, pp. 1066-1072
, **,,
,,, and
From the * Clinical Research Area, Bio Sidus S. A.;
Department of Pediatrics, Sanatorio de la Trinidad; § Department of
Neonatology, Clínica y Maternidad Suizo-Argentina;
Department of Neonatology, Instituto Médico de Obstetricia;
¶ Department of Neonatology, Sanatorio Otamendi; # Department of
Neonatology, Clínica del Sol; ** Department of Neonatology,
Sanatorio Jockey Club; and Department of Neonatology,
Clínica Independencia, Buenos Aires, Argentina.
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ABSTRACT |
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Top
Abstract
Methods
Results
Discussion
Conclusion
References
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Objective. The administration of recombinant human erythropoietin (rHuEPO), started after the first 2 weeks of life, reduces the transfusion requirement in premature infants. However, its use throughout the first 2 weeks of life, when anemia results predominantly from phlebotomy losses, remains controversial. We investigated whether early use of rHuEPO would reduce the total transfusion requirement and/or the number of transfusions throughout the first 2 weeks of life.
Methods. We randomized 114 infants with birth weight (BW) <1250 g to receive rHuEPO (1250 units/kg/week; IV; early group: n = 57) or placebo (late group: n = 57) from day 2 to day 14 of life; subsequently, all the patients received rHuEPO (750 units/kg/week, subcutaneously) for 6 additional weeks. All infants were given oral iron (6 mg/kg/day) and folic acid (2 mg/day).
Results. The early group showed higher hematocrit and reticulocyte counts than the late group in the first 3 weeks of life, but there was no difference in the total number of transfusions (early: 1.8 ± 2.3 vs late: 1.8 ± 2.5 transfusion/patient) or the transfusion requirement throughout the first 2 weeks of life (early: .8 ± 1.1 vs late: .9 ± 1.3) could be demonstrated. In infants with BW <800 g and total phlebotomy losses >30 mL/kg (n = 29), a lower number of transfusions was received by infants in the early group, compared with late group, from the second week to the end of the treatment (early: 3.4 ± 1.1 vs late: 5.4 ± 3.7 transfusion/patient). No clinical adverse effects were observed. Thrombocytosis was detected during the treatment with rHuEPO in 31% of the infants.
Conclusions. In the whole population, the early administration of rHuEPO induced a rise of reticulocyte counts, but not enough to reduce the transfusion requirement. The most severely ill infants (BW <800 g and phlebotomy losses >30 mL/kg) seemed to benefit from early use of rHuEPO, and this deserves additional study. Key words: erythropoietin, anemia, prematurity, transfusion, thrombocytosis.
An inadequate erythropoietin (EPO) response is a
principal patophysiologic feature of the anemia of
prematurity.1-6 Because the erythroid progenitors
(burst-forming unit-erythroid and colony-forming
unit-erythroid) of preterm infants are responsive to EPO in
vitro,7-10 multiple studies have attempted to reduce the
transfusion requirements of preterm infants through the use of
exogenous human recombinant erythropoietin (rHuEPO).11-27 Methodologic differences, regarding dose schedules, age at the beginning treatment, duration of therapy, entry criteria, and utilization of placebo, have made the interpretation of results difficult. Nevertheless, data from controlled trials published since
1990 support the conclusion that rHuEPO at doses over 500 units/kg/week, started after 15 days of life, reduces the transfusion requirement of very low birth weight (VLBW)
infants.17,18,20,22,25,28 However, the use of rHuEPO
throughout the first 2 weeks of life, when the need for transfusions
results predominantly from the amount of phlebotomy losses, remains
controversial.16,18,19,21,2326-28
Because of this, we decided to perform a study to evaluate whether
early treatment (day 2-3 of life) with rHuEPO in premature infants
with birth weight (BW) <1250 g: 1) decreases the number of
transfusions during the first 2 weeks of life; and 2) is more effective
than late treatment (day 15) to decrease the total transfusion requirement.
All infants born at 7 private hospitals of Buenos Aires,
Argentina, were included if they met the established criteria. The trial was approved by ethics and medical education committees of each
participating hospital and by the National Drugs, Foods, and Technology
Administration and was performed under the Good Clinical Practice
guidelines. The parents gave written informed consent before entering
patients into the protocol.
Calculation of Sample Size
A sample size of 100 was calculated to detect a difference of
20% between groups in the number of transfusions, with a power of 80%
and significance of .05. Because of the anticipated mortality rate of
20%, a total enrollment of 120 patients was planned.
Inclusion and Exclusion Criteria
Infants were included if they met the following criteria: 1) BW
<1250 g; and 2) gestational age (GA) <32 weeks. Exclusion criteria
were: 1) major congenital malformations; 2) chromosomal anomalies; 3)
hemolytic and/or hemorrhagic disease; 4) intrauterine infections; 5)
systemic hypertension; and 6) neutropenia (absolute neutrophil count
[ANC] under 1.5 × 109 per liter). No
patient was excluded based on the severity of the disease.
Patients
From July 1996 to October 1997, 120 infants were enrolled in the
study. Six infants with significant protocol violations were excluded.
Therefore, 114 infants remained available for the statistical analysis.
Treatment
At birth, through a central randomized process, infants were
assigned to 2 groups. The early group received rHuEPO (1250 units/kg/week, divided in 5 doses, by slow intravenous (IV) infusion in
5-10 minutes), starting before 72 hours of life, and until day 14 of life. We decided to use IV administration because all patients had an
IV access in place during the first 2 weeks of life, and serum rHuEPO
concentrations and clearance are similar for IV and subcutaneous
routes of administration.26 The late group received
placebo throughout this period. Starting on the third week of life,
both groups received rHuEPO in accordance with our standard protocol of
750 units/kg/week, divided in 3 doses, subcutaneously, during 6 weeks.
All patients in both groups were given oral iron (6 mg/kg/day, as
ferrous sulfate) and folic acid (2 mg/day), starting as soon
as enteral feedings were initiated and continuing during the entire
treatment period.
The rHuEPO (Hemax) was manufactured by the pharmaceutical laboratory
Bio Sidus S. A. (Buenos Aires, Argentina). Identical vials
containing equal volumes of either rHuEPO or placebo (human seroalbumin: 2.5 mg/ampule) were prepared and shipped to the
investigators. Placebo and rHuEPO were indistinguishable before and
after reconstitution. Parents, investigators, and nurses were unaware
of each patient's treatment group.
Transfusions
Indications for transfusions followed criteria described in the
US and Canadian collaborative study,22 slightly modified
(Table 1). Patients were transfused with
packed red blood cells at 15 mL/kg, administered in 2 to 3 hours.
TABLE 1
METHODS
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Abstract
Methods
Results
Discussion
Conclusion
References
Transfusion Criteria
Monitoring
Respiratory rate, heart rate, blood pressure, number of apneic episodes, weight gain, protein-caloric balance, and neurologic status were daily recorded. The total volume of blood withdrawn and the number and volume of blood transfusions were daily recorded from birth to the end of the study. Laboratory monitoring included: 1) complete blood cell count with reticulocyte and platelet counts twice weekly; 2) weekly liver and renal function tests; and 3) serum ferritin concentrations every 2 weeks. All the patients were followed for at least 6 months after discharge from the hospital.
Statistical Analysis
Student's t test was used to compare continuous
variables, and 
2 test for noncontinuous
variables. Repeated measures analysis of variance was used to compare
profiles throughout time, with the Greenhouse and Geisser adjustment of
the significance level, and Bonferroni tests as postanalysis of
variance. Systat software was used (Systat, Evanston, IL).
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RESULTS |
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Results
Discussion
Conclusion
References
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Patients Characteristics
Infants had GA of 27.8 ± 2.4 (mean ± standard deviation [SD]) weeks and BW of 944 ± 212 g. The groups were comparable for GA, BW, weight at the beginning of treatment, survival, prenatal administration of steroids, and incidences of respiratory distress syndrome, sepsis, bronchopulmonary dysplasia, patent ductus arteriosus, intraventricular hemorrhage, and necrotizing enterocolitis (Table 2). The volume of phlebotomy losses during the first 2 weeks, as well as throughout the 8 weeks of study, was not different between the groups (Table 3).
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Ten patients died during the study (4 in the early group and 6 in the late group) from sepsis (5), necrotizing enterocolitis (4), and grade IV intraventricular hemorrhage (1). Four additional infants (3 in the early group and 1 in the late group) died after the end of the study (3 from sepsis and 1 from necrotizing enterocolitis).
Hematologic Values
Figure 1 shows reticulocyte counts and hematocrits. Entry values were similar for reticulocyte counts (early: 6.4% ± 1.1%; late: 5.5% ± .8%) and for hematocrit (44.1% ± 1.1% and 43.8% ± 1.3%). Reticulocyte counts were significantly higher (P < .05) in the early group than in the late group at 7, 11, and 14 days of treatment, preceding the differences in hematocrit values, which were different (P < .05) on days 11, 14, and 21 of treatment. Thereafter, no differences between the groups were observed.
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Transfusion Requirement
All transfusions administered met transfusion criteria. The total transfusion requirement was the same for both groups. No differences in the number of infants transfused, number of transfusions per infant, or volume of packed erythrocytes transfused between groups could be demonstrated either during the first 2 weeks or throughout the 8 weeks of the study (Table 3). The number of transfusions per patient transfused was also similar during the first 2 weeks (2.0 ± .9 vs 1.9 ± 1.2 for early and late groups, respectively) and throughout the complete period of treatment (3.1 ± 2.3 vs 2.8 ± 2.6).
We next analyzed the subgroup of infants with BW <800 g and phlebotomy losses >30 mL/kg (n = 29). This group was arbitrarily selected because they constitute the sickest subpopulation of premature infants; only 3 additional infants in this weight category required <30 mL/kg of blood sampled. Patients from early and late groups were comparable for demographics as well as clinical severity and volume of phlebotomy losses. The mean GA was 25.7 ± 1.9 weeks (early: 26.3 ± 2.0 weeks; late: 25.7 ± 1.3 weeks) and the mean BW was 660 ± 73 g (early: 671 ± 76 g; late: 640 ± 66 g). The number of transfusions was significantly lower in the early group than in the late group from the second week to the end of the study (P < .05; Fig 2). The total transfusion requirements were 3.4 ± 1.1 and 5.4 ± 3.7 transfusions per infant for the early and late groups, respectively.
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Iron
Oral iron administration was started at 7.4 ± 3.0 days in the early group and at 7.3 ± 2.7 days in the late group. Figure 3 shows the pattern of serum ferritin levels. Values were similar at entry in both groups (232 ± 34 and 213 ± 20 ng/mL for early and late groups, respectively). Ferritin values increased in the late group during the first 2 weeks of treatment but remained stable in the early group during the same period; as a result, values at day 14 of treatment were significantly different between both groups (P < .01). Subsequently, serum ferritin decreased similarly in both groups; values by the end of the study were 132 ± 30 ng/mL and 110 ± 17 ng/mL for early and late groups, respectively.
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Growth
The infants in the early and late groups had equivalent caloric intakes and rates of growth. The weight gain during the study was similar for both groups (early: 791 ± 392 g; late: 785 ± 389 g). The mean weights at the end of the treatment were 1663 ± 536 g and 1691 ± 542 g, respectively.
Adverse Effects
No clinical adverse effects attributable to rHuEPO, oral iron, or folic acid administration were observed. No case of sudden infant death syndrome was reported after discharge (follow-up period: 9-24 months).
The incidence of ANC under 1.0 × 109/L (13%) or under .5 × 109/L (2.6%) was similar for both groups. Neutropenia resolved spontaneously in every case; no patient developed infections attributable to neutropenia. Thrombocytosis (platelet count over 500 × 109/L) was detected at some point during the treatment with rHuEPO in 31% of our population (18 infants in the early group and 17 infants in late group); this finding was transient and not associated with thrombosis or any other clinical manifestation. Platelets count showed no significant difference between initial and final values for both groups.
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DISCUSSION |
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Abstract
Methods
Results
Discussion
Conclusion
References
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VLBW infants are 1 of the main groups of patients requiring red blood cells transfusions: 60% to 100% of VLBW infants receive multiple transfusions, mostly during the first 2 weeks of life.12,22,2528-36 The use of rHuEPO throughout this period, when anemia results predominantly from the amount of blood sampled for laboratory tests, has not been adequately evaluated. A few published trials have suggested its usefulness, but the interpretation of these results remains controversial. Carnielli et al16 administered rHuEPO (1200 units/kg/week), from the second day of life, to 22 premature infants with BW <1750 g. They showed a decreasing number of transfusions per patient, from 3.1 in the control group to .8 in the treated group. The elevated BW of the study population (1328 g), exclusion of sickest infants, liberal transfusion criteria adopted, absence of a placebo group, and lack of administration of iron to the control infants influence these results. The European cooperative study21 randomized 241 infants with BW from 750 to 1500 g to receive rHuEPO (750 units/kg/week) from day 3 to day 42 or no treatment; the number of transfusions per patient diminished from 1.25 in the control group to .8 in the rHuEPO treated group. However, in this trial, there was no placebo group, and infants who required assisted ventilation were withdrawn from the study. Ohls et al23 administered rHuEPO (1400 units/kg/week) or placebo to 20 VLBW infants (BW: between 750 and 1250 g) from day 1 to day 14 of life, decreasing the transfusion requirement from 1.4 to .2 transfusions/patient. Recently, the same authors27 randomly assigned 28 infants with BW <750 g, in the first 72 hours of life, to receive either rHuEPO (1400 units/kg/week) or placebo for 14 days. During the 21-day study, the rHuEPO group received 4.7 transfusions/patient versus 7.5 transfusions/patient in the placebo group. The small size of the study populations and short time of monitoring the need for transfusion (3 weeks) limit the importance of the results of both trials.
To date, no trial comparing early versus late administration of rHuEPO has been reported. In our study, the early administration was associated with higher reticulocyte counts and hematocrits compared with late administration. However, this response was not sufficient to decrease the need for transfusion; the number of transfusions received during the first 2 weeks, as well as the total transfusion requirement, were similar for both groups.
The transfusion requirement is strongly correlated with the amount of phlebotomy losses and lower BW22,29,37,38; as a result, the smallest and sickest infants need more frequent transfusions. Therefore, although it was not originally designed as a primary outcome of the study, we compared the transfusion requirement in the subgroup of premature infants with BW <800 g and total phlebotomy losses >30 mL/kg. Infants in the early group showed a significantly lower need for transfusion from the second week to the end of the study, compared with the late group. At the end of the therapy period in this subgroup of tiny, sick infants, the total number of transfusions per patient was 3.4 in the early versus 5.4 in the late group.
It is important to emphasize the low average number of transfusions (1.8/patient) in our premature infants from both groups, during their first 8 weeks of life. This observation becomes more relevant in view of the facts that many of these infants were severely ill and that no patients were excluded based on the severity of the disease. This number of transfusions is much lower than the data reported in previous publications,27,29,31,32,34,39 including our own previous estimates when this protocol was designed. We suggest that this low transfusion requirement results from the combination of several factors: 1) use of rHuEPO; 2) adherence to very strict transfusion criteria followed from birth in all infants, including the fact that transfusions were not administered to replace phlebotomy losses; and 3) efforts to diminish the volume of blood sampled: the minimal amount of blood needed for micromethods was withdrawn, the number of routine tests was minimized, and many laboratory tests were replaced by noninvasive monitoring.
The studies published to date have administered a wide range of oral or parenteral iron doses11-27; Shannon et al22 stated that dose and route of iron supplementation were important concerns for future trails, particularly if therapy with rHuEPO begins early. We decided to start oral iron administration (6 mg/kg/day) as soon as enteral feedings were initiated. As expected, ferritin values declined gradually throughout the rHuEPO treatment period but not enough to drop to inadequately low levels. The pattern of ferritin concentrations in both groups during the first 2 weeks was similar to that reported by Ohls et al.27 We did not observe any clinical adverse effect attributable to iron administration. Therefore, we believe that our protocol for early oral iron supplementation is adequate to support an erythropoietic response to rHuEPO.
Some previous studies reported neutropenia as a consequence of rHuEPO treatment in VLBW infants,11-14 and experimental results support this observation.40-43 However, most current reports have failed to demonstrate a significant decrease of the ANC related to treatment.15-19,22,25 Furthermore, the study by Shannon et al22 and our own previous report25 showed a decrease of ANC during the study periods in infants receiving placebo. The incidence of neutropenia detected in this trial (13%) is similar to that published in the literature for VLBW premature infants without rHuEPO administration.13,17,44
In 31% of patients in both groups, we detected platelets count over 500 × 109/L at some point during the treatment with rHuEPO. To our knowledge, thrombocytosis over 500 × 109/L has only been occasionally described for premature infants receiving rHuEPO11,13,18; most trials have reported slight or no increase of platelet count in these VLBW infants, regardless of whether they were receiving rHuEPO.12,16,1719-24,26,27 Because all infants received rHuEPO, we have no comparative group by which to state that this phenomenon is secondary to the rHuEPO therapy. However, unexplained thrombocytosis also occurs frequently in iron deficiency anemia, a disease with very high levels of circulating EPO.45 The explanation for this observation is not clear, but it is tempting to speculate that the thrombocytosis found in our patients is attributable to the structural and functional similarities between EPO and thrombopoietin (TPO). The EPO and TPO genes evolve from a common ancestor, and their molecules show strong sequence similarity.4647-49 In addition, TPO acts in synergy with EPO to stimulate the growth of erythroid progenitor cells.50 The administration of TPO to mice treated with chemotherapy and/or radiotherapy induces a clear response of the reticulocyte counts.51 Inversely, Dessypris et al41 reported an increase of marrow megakaryocyte progenitor cells, as well as of peripheral platelets count, after 2 weeks of treatment with rHuEPO in patients with chronic renal failure.
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CONCLUSION |
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Top
Abstract
Methods
Results
Discussion
Conclusion
References
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We summarize that no benefit derives from the early instead of late use of rHuEPO in all patients. Therefore, we believe that our results do not justify the routine early administration of rHuEPO to all preterm infants with BW <1250 g as standard medical practice. In contrast, infants with BW <800 g, sick enough to require >30 mL/kg of blood sampling, tend to decrease their need for transfusion when they start to receive rHuEPO during the first 3 days of life. Although the early use of rHuEPO seems to be indicated for this population of sick and extremely low BW infants, additional studies specially designed for this population are needed to confirm our findings.
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APPENDIX |
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The following institutions and workers are members of the Private Hospitals Neonatal Network:
Steering Committee: H. Donato (Clinical Research Area, Bio Sidus S. A.), N. Vain (Department of Pediatrics, Sanatorio de la Trinidad), and P. Rendo (Clinical Research Area, Bio Sidus S. A.).
Participating Centers (listed in order of infants enrolled):
Consultant in Statistics: L. Marangunich (Clinical Research Area, Bio Sidus S. A.)
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ACKNOWLEDGMENTS |
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This work was supported by a grant from Bio Sidus S. A. Laboratory.
We thank Dr Robert D. Christensen for his critical review of the manuscript. We are indebted to Maria Laura Devincenzi for administrative support, to the nurses and doctors who cared for the patients, and to the parents who enrolled their infants in this trail.
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FOOTNOTES |
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The institutions and coworkers of the Private Hospitals Neonatal Network are listed in the "Appendix."
Conflict of interest: Dr Donato is Pediatrics Consultant for the Clinical Research Area of Bio Sidus S. A. Laboratory; and Dr Rendo is Chief of the Clinical Research Area of Bio Sidus S. A. Laboratory.
This article was presented in part at the Platform Session, Clinical Trials in Perinatal Medicine, at the annual meeting of the Society for Pediatric Research; May 5, 1998; New Orleans, LA.
Received for publication Mar 24, 1999; accepted Aug 2, 1999.
Reprint requests to (H.D.) Clinical Research Area, Bio Sidus S. A., Tarija 4243-1254, Buenos Aires, Argentina. E-mail address: h.donato{at}biosidus.com.ar
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ABBREVIATIONS |
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EPO, erythropoietin; rHuEPO, recombinant human erythropoietin; VLBW, very low birth weight; BW, birth weight; GA, gestational age; ANC, absolute neutrophil count; IV, intravenous; TPO, thrombopoietin; SD, standard deviation.
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REFERENCES |
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Results
Discussion
Conclusion
References
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(recombinant human erythropoietin) on the need for transfusion in very-low-birth-weight infants.
N Engl J Med
1994;
330:1173-1178 This article has been cited by other articles:
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  R. K. Ohls Transfusions in the Preterm Infant NeoReviews, September 1, 2007; 8(9): e377 - e386. [Abstract] [Full Text] [PDF]
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 N. Haiden, J. Schwindt, F. Cardona, A. Berger, K. Klebermass, M. Wald, C. Kohlhauser-Vollmuth, B. Jilma, and A. Pollak Effects of a Combined Therapy of Erythropoietin, Iron, Folate, and Vitamin B12 on the Transfusion Requirements of Extremely Low Birth Weight Infants Pediatrics, November 1, 2006; 118(5): 2004 - 2013. [Abstract] [Full Text] [PDF]
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R. Bierer, M. C. Peceny, C. H. Hartenberger, and R. K. Ohls Erythropoietin Concentrations and Neurodevelopmental Outcome in Preterm Infants Pediatrics, September 1, 2006; 118(3): e635 - e640. [Abstract] [Full Text] [PDF]
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N. Haiden, K. Klebermass, F. Cardona, J. Schwindt, A. Berger, C. Kohlhauser-Vollmuth, B. Jilma, and A. Pollak A Randomized, Controlled Trial of the Effects of Adding Vitamin B12 and Folate to Erythropoietin for the Treatment of Anemia of Prematurity Pediatrics, July 1, 2006; 118(1): 180 - 188. [Abstract] [Full Text] [PDF]
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R. K. Ohls, R. A. Ehrenkranz, A. Das, A. M. Dusick, K. Yolton, E. Romano, V. Delaney-Black, L.-A. Papile, N. P. Simon, J. J. Steichen, et al. Neurodevelopmental Outcome and Growth at 18 to 22 Months' Corrected Age in Extremely Low Birth Weight Infants Treated With Early Erythropoietin and Iron Pediatrics, November 1, 2004; 114(5): 1287 - 1291. [Abstract] [Full Text] [PDF]
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 A Pathak, P Roth, J Piscitelli, and L Johnson Effects of vitamin E supplementation during erythropoietin treatment of the anaemia of prematurity Arch. Dis. Child. Fetal Neonatal Ed., July 1, 2003; 88(4): F324 - F328. [Abstract] [Full Text] [PDF]
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M P Meyer, E Sharma, and M Carsons Recombinant erythropoietin and blood transfusion in selected preterm infants Arch. Dis. Child. Fetal Neonatal Ed., January 1, 2003; 88(1): F41 - 45. [Abstract] [Full Text] [PDF]
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M. P. Meyer, H. Donato, P. Rendo, L. Prudent, N. Vain, R. K. Ohls, and R. A. Ehrenkranz Erythropoietin and Preemies Pediatrics, December 1, 2002; 110(6): 1253 - 1255. [Full Text] [PDF]
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P. Raupp;, H. Donato, N. Vain, and P. Rendo Folate and vitamin B12 supplementation in very low birth weight infants treated with erythropoietin: a cautionary note. Pediatrics, July 1, 2001; 108(1): 217 - 217. [Full Text]
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Y Nako, A Tachibana, T Fujiu, T Tomomasa, and A Morikawa Neonatal thrombocytosis resulting from the maternal use of non-narcotic antischizophrenic drugs during pregnancy Arch. Dis. Child. Fetal Neonatal Ed., May 1, 2001; 84(3): 198F - 200. [Abstract] [Full Text]
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