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Erythropoietin is present in the cerebrospinal fluid of neonates
Erythropoietin is present in the cerebrospinal fluid of neonates Sandra E. Juul, MD, Jenny Harcum, RN, Yan Li, MD, a n d Robert D. Christensen, MD From the Division of Neonatology, Department of Pediatrics, University of Florida College of Medicine, Gainesville
Erythropoietin (Epo) was measured by enzyme-linked immunosorbent assay in 80 cerebrospinal fluid (CSF) samples to determine whether Epo is present in the CSF of infants, CSF Epo concentrations correlate with age, and CSF Epo concentrations correlate with Epo therapy. Epo was present in the CSF of normal neonates. CSF Epo concentrations correlated negatively with increasing age. Recombinant Epo therapy did not affect CSF Epo concentrations, although values ranged somewhat higher in this group. (J Pediatr 1997;130:428-30)
Erythropoietin was originally thought to have effects specific to cells of erythroid lineage.1 It has now been shown in animals that functional Epo receptors are not unique to hematopoietic cells but are also present on cells of neural origin and on endothelial cells.2-4 In the mouse, Epo and its receptor are found in the central nervous system during fetal life but are no longer present at birth. 4, 5 A functional role for Epo within the rodent CNS is suggested by studies showing hypoxia-mediated Epo synthesis in primary cultures of astrocytes and in whole animal experiments. 3, 6 Preliminary data from our laboratory suggest that Epo and Epo receptors also exist within the CNS of firstand second-trimester human fetuses. 7'8 Our objectives for the present study were to determine whether Epo is present in the cerebrospinal fluid of preterm and term neonates, to determine whether CSF levels of Epo change with gestational and postnatal age, and to determine whether treatment of infants with recombinant Epo increases these levels.
Supported by grants MCAP RR-00083 and HL-44951 from the National Institutes of Health and by a grant from the Children's Miracle Network. Submitted for publication Dec. 23, 1995; accepted Sept. 5, 1996. Reprint requests: Sandra Juul, MD, Division of Neonatology, University of Florida College of Medicine, PO Box 100296, J. Hillis Miller Health Center, Gainesville, FL 32610-0296. Copyright © 1997 by Mosby-Year Book, Inc. 0022-3476/97/$5.00 + 0 9/21/77829
428
METHODS CSF specimens. Spinal fluid was obtained from 50 neonates ranging in gestational age from 24 weeks to term, Twenty-three of these neonates were treated with rEpo at the discretion of the attending neonatologist. Two neonates had meningitis and were considered separately. Thirty additional spinal fluid samples from patients aged 1 month to 60 years of age, none of whom were receiving rEpo treatment, were assayed. All samples were obtained from the clinical laboratory of Shands ,Teaching Hospital after patients had undergone spinal taps that were clinically indicated. The investigators did not influence these clinical decisions in any CSF Epo rEpo
Cerebrospinalfluid Erythropoietin Recombinant erythropoietin
way. This study was done with the approval of the University of Florida Institutional Review Board as an exempted protocol. Spinal fluid characteristics. Cell counts, protein and glucose concentrations, and the presence of infection were documented for each spinal fluid sample. The third or fourth tube of collected CSF was used for Epo analysis. All infants who weighed less than 1500 gm at birth underwent cranial ultrasound examinations by 1 week of age as part of routine care in our neonatal intensive care unit. Ultrasound examinations were repeated sequentially if abnormalities were found, or at 1 month of age if the infant was normal initially.
The Journal ~f Pediatrics Volume 130, Number 3
Blood samples. Five infants had serum available for Epo assay from blood that had been drawn at the time of the lumbar puncture. Epo treatment. Infants were treated with rEpo (Epogen), at a dosage of either 200 U/kg per day given intravenously for 4 hours in a protein-containing solution, as a continuous infusion in the hyperalimentation solution, or 400 U/kg given subcutaneously on Monday, Wednesday, and Friday. Epo assay. Epo concentrations in the CSF were assayed with the Quantikine IVD human Epo enzyme-hnked immunosorbent assay (R&D Systems, Minneapolis, Minn.). The standard curve was done in duplicate by using control solutions, with a third standard curve done in human CSF to determine the reliability of the assay in CSF. Human CSF did not affect the reliability of the assay. Aliquots (100 pl) of spinal fluid were assayed in duplicate. Variability was less than 2%. The specificity of the assay is greater than 98%, with no difference in detection between rEpo and endogenous Epo. Statistical analysis. An unpaired Student t test was used to compare CSF Epo concentrations in control versus treated infants. RESULTS Epo was present in the spinal fluid of premature and term neonates in the absence of rEpo treatment, meningitis, or intracranial hemorrhage (Figure). In the neonates not receiving rEpo treatment (control infants), the mean spinal fluid Epo concentration was 7.6 + 1.4 mU/ml (±SEM), with a median of 5.0 mU/ml and a range of less than 0.6 (undetectable) to 21.0 mU/ml. The CSF concentrations in two term infants were not included in this statistical analysis but are shown on the Figure by filled triangles. One of these infants had presumed meningitis on the basis of hypoglycorrhacia and increased protein and neutrophils in the spinal fluid, but results of the CSF culture were negative because of the antibiotic pretreatment. The other infant had manifestations of severe congenital cytomegalovirus infection, including microcephaly, intracranial calcifications, and multisystem involvement. We found no significant relationship between CSF Epo concentrations and cell count or protein or glucose concentrations in the CSF. Only one infant had an intracranial hemorrhage: (CSF Epo concentration of 2.0 mU/ml) detected before the lumbar puncture. Five infants had had blood samples drawn at the time of their lumbar punctures. Serum Epo concentrations in these samples ranged from 1.0 to 56.0 mU/ml, with concomitant CSF Epo levels ranging from less than 0.6 to 12.0 mU/ml. There was no correlation between blood and CSF Epo concentrations. Thirty CSF samples were obtained from individuals rang-
Juul et al.
429
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Age in Weeks
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Figure. Epo concentrations in the spinal fluid of 80 subjects. Control individuals (no rEpo therapy) are identified by open circles, infants receiving treatment with rEpo are denoted by open triangles, and the two patients with meningitis (neither received rEpo) are shown by filled triangles. Erythropoietin concentrations are reported in millinnits per milliliter on the y-axis. Corrected age is plotted in weeks along the x-axis up to term gestation (denoted by arrow). After term gestation, age is plotted in years.
ing in age from 1 month to 60 years. The Figure shows the distribution of spinal fluid Epo levels. The mean CSF Epo concentration in this group was 2.8 ± 1.2 mU/ml, with a median of 0.6 and a range from undetectable to 32 mU/ml. All individuals older than 5 months of age had levels of less than 3.0 mU/ml. As a group, individuals older than 1 month of age had CSF Epo concentrations lower than preterm and term neonates (p = 0.01). To determine whether rEpo treatment affected the concentrations of CSF Epo, the spinal fluid Epo concentrations of neonates treated versus those not treated with rEpo were compared (Figure). The mean CSF Epo concentrations were 9.9 ± 2.4 mU/ml (range, <0.6 to 51.4 mU/ml) and 7.6 ± 1.4 mU/ml (range, <0.6 to 21.0 mU/ml), respectively. The mean corrected age of treated infants was less than that of untreated infants (29.6 vs 34.5 weeks). No infants treated with rEpo had meningitis, and all had normal cranial ultrasound findings. Four infants received two spinal taps during their hospitalization, and all of them had a lower CSF Epo concentration on the second tap. The mean CSF concentration on the
430
Juul et aI.
first was 5.8 mU/ml, failing to a mean of 4.0 mU/ml on the second. DISCUSSION Epo is a 34,000-dalton glycoprotein that, on the basis of size, is unlikely to cross the blood-brain barrier in the absence of a cartier molecule. It is possible, however, that in the presence of an extremely immature blood-brain bartier, asphyxia, acidosis, or meningitis, or after an intraventricular hemorrhage, the blood-brain barrier might be more likely to permit the transfer of large molecules such as Epo. We observed that Epo was present in the CSF of normal preterm and term infants in the absence of pathologic CNS changes. The concentration of Epo within the CSF decreases with age. This correlation between CSF Epo concentration and increasing age is analogous to findings in mice. The physiologic role of Epo within the developing CNS is unclear. We speculate that its effects might be neurotrophic. This speculation is supported by experiments demonstrating improved cholinergic neuronal function in the presence of Epo and improved cholinergic septal neuronal survival after fimbriafornix transections in the presence of Epo. 9 Serum Epo levels in premature infants have been reported to be between 5 and 307 mU/ml. 1° Epo concentrations in the serum samples that we tested fell within this range, with no correlation between serum and CSF concentrations, suggesting that Epo measured in the CSF was endogenous to the CNS. Peak serum Epo concentrations in premature infants treated with rEpo at the same dosage per kilogram and with the same routes of delivery that we use have ranged between 177 --- 29 and 400 --- 64 mU/mi. 11 We found no difference in CSF Epo concentrations between treated and untreated infants, suggesting that the higher circulating Epo levels in the treated infants did not affect the CNS Epo concentrations. The correlation between serum Epo and CSF Epo remains an open question, however, because of the small number of serum samples tested. It was interesting that the child with congenital cytomegalovirus infection had the highest CSF Epo concentration in our series (68.0 mU/ml). The explanation for this is not clear; possibilities include transfer of circulating Epo across an altered blood-brain barrier, increased production of Epo, or diminished clearance of Epo within the CNS. Because we did not have a simultaneous blood value to compare to the CSF value, we are unable to speculate regarding the source of the CSF Epo in this case. Administration of rEpo to certain groups of preterm infants can be both efficacious and economical, resulting in a reduction in the need for erythrocyte transfusions, a pro-
The Journal of Pediatrics March 1997
cedure known to carry a risk of infections. 12 In short-term studies, no adverse effects of rEpo administration have been identified in this population. It is unlikely, on the basis of our study, that rEpo crosses the blood-brain barrier in normal premature ilffants, and it is not clear whether the CNS effects of rEpo, should it cross the blood brain-barrier, would be harmful or beneficial. No studies have assessed the neurodevelopmental effects of rEpo administration to preterm infants. Perhaps our detection of Epo receptors, 7 and now Epo itself, in the CNS of preterm neonates provides an additional reason for conducting such studies. REFERENCES
1. Sawyer ST, Krantz SB, Sawada K. Receptors for erythropoietin in mouse and human erythroid cells and placenta. Blood 1989;74:103-9. 2. Anagnostou A, Liu Z, Steiner M, et al. Erythropoietin receptor mRNA expression in human endothelial cells. Proc Natl Acad Sci U S A 1994;91:3974-8. 3. Masuda S, Okano M, Yamagishi K, Nagao M, Ueda M, Sasaki R. A novel site of erythropoietin production. J Biol Chem 1994; 269:19488-93. 4. Liu Z-Y, Chin K, Noguchi C. Tissue specific expression of human erythropoietin receptor in transgenic mice. Dev Biol 1994; 166:159-69. 5. Yasuda Y, Nagao M, Okano M, et al. Localization of erythropoietin and erythropoietin-receptor in postimplantation mouse embryos. Development, Growth and Differentiation 1993;35: 711-22. 6. Tan CC, Eckardt K-U, Firth JD, Ratcliffe PJ. Feedback modulation of renal and hepatic erythropoietin mRNA in response to graded anemia and hypoxia. Am J Physiol 1992;263:F47481. 7. Li Y, Juul SE, Morris-Wiman JA, Calhoun DA, Christensen RD. Erythropoietin receptors are expressed in the central nervous system of mid-trimester human fetuses: a pilot study. Pediatr Res 1996;40:376-80. 8. Juul S, Li Y, Calhoun D, Christensen R. Erythropoietin and its receptor are expressed in the central nervous system of first- and second-trimester human fetuses [abstract]. Pediatr Res 1996; 39:219A. 9. Konishi Y, Chui D-H, Hirose H, Kunishita T, Tabira T. Trophic effect of erythropoietin and other hematopoietic factors on central cholinergic neurons in vitro and in vivo. Brain Res 1993; 609:29-35. 10. Yamashita H, Ktddta J, Ohga S, Nakayama H, Akazawa K, Ueda K. Serum erythropoietin levels in term and preterm infants during the first year of life. Am J Pediatr Hematol Oncol 1994;16:213-8. 11. Ohls RK, Veerman M, Christensen RD. Phannacokinetics and effectiveness of recombinant erythropoietin administered to preterm infants by continuous infusion in parenteral nutrition solution. J Pediatr 1996;128:518-23. 12. Shannon KM, Keith JF III, Mentzer WC, et al. Recombinant human erythropoietin stimulates erythropoiesis and reduces erythrocyte transfusions in very low birth weight preterm infants. Pediatrics 1995;95:1-8.
Erythropoietin (Epo) was measured by enzyme-linked immunosorbent assay in 80 cerebrospinal fluid (CSF) samples to determine whether Epo is present in the CSF of infants, CSF Epo concentrations correlate with age, and CSF Epo concentrations correlate with Epo therapy. Epo was present in the CSF of normal neonates. CSF Epo concentrations correlated negatively with increasing age. Recombinant Epo therapy did not affect CSF Epo concentrations, although values ranged somewhat higher in this group. (J Pediatr 1997;130:428-30)
Erythropoietin was originally thought to have effects specific to cells of erythroid lineage.1 It has now been shown in animals that functional Epo receptors are not unique to hematopoietic cells but are also present on cells of neural origin and on endothelial cells.2-4 In the mouse, Epo and its receptor are found in the central nervous system during fetal life but are no longer present at birth. 4, 5 A functional role for Epo within the rodent CNS is suggested by studies showing hypoxia-mediated Epo synthesis in primary cultures of astrocytes and in whole animal experiments. 3, 6 Preliminary data from our laboratory suggest that Epo and Epo receptors also exist within the CNS of firstand second-trimester human fetuses. 7'8 Our objectives for the present study were to determine whether Epo is present in the cerebrospinal fluid of preterm and term neonates, to determine whether CSF levels of Epo change with gestational and postnatal age, and to determine whether treatment of infants with recombinant Epo increases these levels.
Supported by grants MCAP RR-00083 and HL-44951 from the National Institutes of Health and by a grant from the Children's Miracle Network. Submitted for publication Dec. 23, 1995; accepted Sept. 5, 1996. Reprint requests: Sandra Juul, MD, Division of Neonatology, University of Florida College of Medicine, PO Box 100296, J. Hillis Miller Health Center, Gainesville, FL 32610-0296. Copyright © 1997 by Mosby-Year Book, Inc. 0022-3476/97/$5.00 + 0 9/21/77829
428
METHODS CSF specimens. Spinal fluid was obtained from 50 neonates ranging in gestational age from 24 weeks to term, Twenty-three of these neonates were treated with rEpo at the discretion of the attending neonatologist. Two neonates had meningitis and were considered separately. Thirty additional spinal fluid samples from patients aged 1 month to 60 years of age, none of whom were receiving rEpo treatment, were assayed. All samples were obtained from the clinical laboratory of Shands ,Teaching Hospital after patients had undergone spinal taps that were clinically indicated. The investigators did not influence these clinical decisions in any CSF Epo rEpo
Cerebrospinalfluid Erythropoietin Recombinant erythropoietin
way. This study was done with the approval of the University of Florida Institutional Review Board as an exempted protocol. Spinal fluid characteristics. Cell counts, protein and glucose concentrations, and the presence of infection were documented for each spinal fluid sample. The third or fourth tube of collected CSF was used for Epo analysis. All infants who weighed less than 1500 gm at birth underwent cranial ultrasound examinations by 1 week of age as part of routine care in our neonatal intensive care unit. Ultrasound examinations were repeated sequentially if abnormalities were found, or at 1 month of age if the infant was normal initially.
The Journal ~f Pediatrics Volume 130, Number 3
Blood samples. Five infants had serum available for Epo assay from blood that had been drawn at the time of the lumbar puncture. Epo treatment. Infants were treated with rEpo (Epogen), at a dosage of either 200 U/kg per day given intravenously for 4 hours in a protein-containing solution, as a continuous infusion in the hyperalimentation solution, or 400 U/kg given subcutaneously on Monday, Wednesday, and Friday. Epo assay. Epo concentrations in the CSF were assayed with the Quantikine IVD human Epo enzyme-hnked immunosorbent assay (R&D Systems, Minneapolis, Minn.). The standard curve was done in duplicate by using control solutions, with a third standard curve done in human CSF to determine the reliability of the assay in CSF. Human CSF did not affect the reliability of the assay. Aliquots (100 pl) of spinal fluid were assayed in duplicate. Variability was less than 2%. The specificity of the assay is greater than 98%, with no difference in detection between rEpo and endogenous Epo. Statistical analysis. An unpaired Student t test was used to compare CSF Epo concentrations in control versus treated infants. RESULTS Epo was present in the spinal fluid of premature and term neonates in the absence of rEpo treatment, meningitis, or intracranial hemorrhage (Figure). In the neonates not receiving rEpo treatment (control infants), the mean spinal fluid Epo concentration was 7.6 + 1.4 mU/ml (±SEM), with a median of 5.0 mU/ml and a range of less than 0.6 (undetectable) to 21.0 mU/ml. The CSF concentrations in two term infants were not included in this statistical analysis but are shown on the Figure by filled triangles. One of these infants had presumed meningitis on the basis of hypoglycorrhacia and increased protein and neutrophils in the spinal fluid, but results of the CSF culture were negative because of the antibiotic pretreatment. The other infant had manifestations of severe congenital cytomegalovirus infection, including microcephaly, intracranial calcifications, and multisystem involvement. We found no significant relationship between CSF Epo concentrations and cell count or protein or glucose concentrations in the CSF. Only one infant had an intracranial hemorrhage: (CSF Epo concentration of 2.0 mU/ml) detected before the lumbar puncture. Five infants had had blood samples drawn at the time of their lumbar punctures. Serum Epo concentrations in these samples ranged from 1.0 to 56.0 mU/ml, with concomitant CSF Epo levels ranging from less than 0.6 to 12.0 mU/ml. There was no correlation between blood and CSF Epo concentrations. Thirty CSF samples were obtained from individuals rang-
Juul et al.
429
7060-J
v
50E t--
~ 400 0
~- a0"E-
0
V V
LIJ
o
u_ co 20O
0
o
o Do
10-
0
........
-15
-10
I ....
-5
rw-,-I
0
Age in Weeks
....
5
i01t,
10
~,~-~
60
Age in Years Term
Figure. Epo concentrations in the spinal fluid of 80 subjects. Control individuals (no rEpo therapy) are identified by open circles, infants receiving treatment with rEpo are denoted by open triangles, and the two patients with meningitis (neither received rEpo) are shown by filled triangles. Erythropoietin concentrations are reported in millinnits per milliliter on the y-axis. Corrected age is plotted in weeks along the x-axis up to term gestation (denoted by arrow). After term gestation, age is plotted in years.
ing in age from 1 month to 60 years. The Figure shows the distribution of spinal fluid Epo levels. The mean CSF Epo concentration in this group was 2.8 ± 1.2 mU/ml, with a median of 0.6 and a range from undetectable to 32 mU/ml. All individuals older than 5 months of age had levels of less than 3.0 mU/ml. As a group, individuals older than 1 month of age had CSF Epo concentrations lower than preterm and term neonates (p = 0.01). To determine whether rEpo treatment affected the concentrations of CSF Epo, the spinal fluid Epo concentrations of neonates treated versus those not treated with rEpo were compared (Figure). The mean CSF Epo concentrations were 9.9 ± 2.4 mU/ml (range, <0.6 to 51.4 mU/ml) and 7.6 ± 1.4 mU/ml (range, <0.6 to 21.0 mU/ml), respectively. The mean corrected age of treated infants was less than that of untreated infants (29.6 vs 34.5 weeks). No infants treated with rEpo had meningitis, and all had normal cranial ultrasound findings. Four infants received two spinal taps during their hospitalization, and all of them had a lower CSF Epo concentration on the second tap. The mean CSF concentration on the
430
Juul et aI.
first was 5.8 mU/ml, failing to a mean of 4.0 mU/ml on the second. DISCUSSION Epo is a 34,000-dalton glycoprotein that, on the basis of size, is unlikely to cross the blood-brain barrier in the absence of a cartier molecule. It is possible, however, that in the presence of an extremely immature blood-brain bartier, asphyxia, acidosis, or meningitis, or after an intraventricular hemorrhage, the blood-brain barrier might be more likely to permit the transfer of large molecules such as Epo. We observed that Epo was present in the CSF of normal preterm and term infants in the absence of pathologic CNS changes. The concentration of Epo within the CSF decreases with age. This correlation between CSF Epo concentration and increasing age is analogous to findings in mice. The physiologic role of Epo within the developing CNS is unclear. We speculate that its effects might be neurotrophic. This speculation is supported by experiments demonstrating improved cholinergic neuronal function in the presence of Epo and improved cholinergic septal neuronal survival after fimbriafornix transections in the presence of Epo. 9 Serum Epo levels in premature infants have been reported to be between 5 and 307 mU/ml. 1° Epo concentrations in the serum samples that we tested fell within this range, with no correlation between serum and CSF concentrations, suggesting that Epo measured in the CSF was endogenous to the CNS. Peak serum Epo concentrations in premature infants treated with rEpo at the same dosage per kilogram and with the same routes of delivery that we use have ranged between 177 --- 29 and 400 --- 64 mU/mi. 11 We found no difference in CSF Epo concentrations between treated and untreated infants, suggesting that the higher circulating Epo levels in the treated infants did not affect the CNS Epo concentrations. The correlation between serum Epo and CSF Epo remains an open question, however, because of the small number of serum samples tested. It was interesting that the child with congenital cytomegalovirus infection had the highest CSF Epo concentration in our series (68.0 mU/ml). The explanation for this is not clear; possibilities include transfer of circulating Epo across an altered blood-brain barrier, increased production of Epo, or diminished clearance of Epo within the CNS. Because we did not have a simultaneous blood value to compare to the CSF value, we are unable to speculate regarding the source of the CSF Epo in this case. Administration of rEpo to certain groups of preterm infants can be both efficacious and economical, resulting in a reduction in the need for erythrocyte transfusions, a pro-
The Journal of Pediatrics March 1997
cedure known to carry a risk of infections. 12 In short-term studies, no adverse effects of rEpo administration have been identified in this population. It is unlikely, on the basis of our study, that rEpo crosses the blood-brain barrier in normal premature ilffants, and it is not clear whether the CNS effects of rEpo, should it cross the blood brain-barrier, would be harmful or beneficial. No studies have assessed the neurodevelopmental effects of rEpo administration to preterm infants. Perhaps our detection of Epo receptors, 7 and now Epo itself, in the CNS of preterm neonates provides an additional reason for conducting such studies. REFERENCES
1. Sawyer ST, Krantz SB, Sawada K. Receptors for erythropoietin in mouse and human erythroid cells and placenta. Blood 1989;74:103-9. 2. Anagnostou A, Liu Z, Steiner M, et al. Erythropoietin receptor mRNA expression in human endothelial cells. Proc Natl Acad Sci U S A 1994;91:3974-8. 3. Masuda S, Okano M, Yamagishi K, Nagao M, Ueda M, Sasaki R. A novel site of erythropoietin production. J Biol Chem 1994; 269:19488-93. 4. Liu Z-Y, Chin K, Noguchi C. Tissue specific expression of human erythropoietin receptor in transgenic mice. Dev Biol 1994; 166:159-69. 5. Yasuda Y, Nagao M, Okano M, et al. Localization of erythropoietin and erythropoietin-receptor in postimplantation mouse embryos. Development, Growth and Differentiation 1993;35: 711-22. 6. Tan CC, Eckardt K-U, Firth JD, Ratcliffe PJ. Feedback modulation of renal and hepatic erythropoietin mRNA in response to graded anemia and hypoxia. Am J Physiol 1992;263:F47481. 7. Li Y, Juul SE, Morris-Wiman JA, Calhoun DA, Christensen RD. Erythropoietin receptors are expressed in the central nervous system of mid-trimester human fetuses: a pilot study. Pediatr Res 1996;40:376-80. 8. Juul S, Li Y, Calhoun D, Christensen R. Erythropoietin and its receptor are expressed in the central nervous system of first- and second-trimester human fetuses [abstract]. Pediatr Res 1996; 39:219A. 9. Konishi Y, Chui D-H, Hirose H, Kunishita T, Tabira T. Trophic effect of erythropoietin and other hematopoietic factors on central cholinergic neurons in vitro and in vivo. Brain Res 1993; 609:29-35. 10. Yamashita H, Ktddta J, Ohga S, Nakayama H, Akazawa K, Ueda K. Serum erythropoietin levels in term and preterm infants during the first year of life. Am J Pediatr Hematol Oncol 1994;16:213-8. 11. Ohls RK, Veerman M, Christensen RD. Phannacokinetics and effectiveness of recombinant erythropoietin administered to preterm infants by continuous infusion in parenteral nutrition solution. J Pediatr 1996;128:518-23. 12. Shannon KM, Keith JF III, Mentzer WC, et al. Recombinant human erythropoietin stimulates erythropoiesis and reduces erythrocyte transfusions in very low birth weight preterm infants. Pediatrics 1995;95:1-8.