Journal of Perinatology (2014) 34, 116–119 & 2014 Nature America, Inc. All rights reserved 0743-8346/14 www.nature.com/jp

ORIGINAL ARTICLE Estimating the nucleated red cell ‘emergence time’ in neonates

RD Christensen1, DK Lambert1 and DS Richards1,2

OBJECTIVE: The time between onset of fetal hypoxia and first appearance of nucleated red blood cells (NRBCs) in the blood can conceptually be divided into two periods; (1) the ‘ (EPO) generation time’, which previous fetal studies suggest is 4 to 5 h, and (2) the ‘NRBC emergence time’. In this study, we estimated the latter as the time required for NRBC to appear in the blood after administering a dose of recombinant EPO. STUDY DESIGN: This was a retrospective analysis of data from a multihospital healthcare system (Intermountain Healthcare). Data were included only for neonates born X34 weeks gestation between the dates 1 January 2005 and 31 October 2012 and only if they received a dose of darbepoetin during their neonatal intensive care unit stay and had one or more complete counts (CBCs) obtained during the 3-day period before the dose was given and one or more CBCs in the 7-day period after the dose. RESULT: The study involved 31 neonates who received 34 doses of darbepoetin. Seven doses were 4 mgkgÀ 1 and twenty-seven doses were 10 mgkgÀ 1. Twenty-six CBCs were obtained during the 24-h period following the darbepoetin dose and none had NRBC identified. NRBC first appeared in the blood between 24 and 36 h after the dose. Recipients of the higher dose generally had a higher peak NRBC count but the NRBC ‘emergence time’ did not appear to depend on dose. CONCLUSION: Following fetal hypoxia, transcription and translation of the EPO gene result in an elevation in plasma EPO concentration. Previous fetal studies suggest this process requires 4 to 5 h. The present studies suggest that, following the increase in plasma EPO, NRBC emerge into the circulation in X24 h. If this model serves as a reasonable estimate, it suggests that neonates with an elevated NRBC count at birth had the onset of hypoxia at least 28 to 29 h before birth.

Journal of Perinatology (2014) 34, 116–119; doi:10.1038/jp.2013.113; published online 12 September 2013 Keywords: hypoxia; NRBC; erythropoietin; darbepoetin

INTRODUCTION ‘EPO generation time’, and (2) the time for EPO to induce an An abnormally high concentration of nucleated red blood cells elevation in NRBC, the ‘NRBC emergence time’. In this study, we (NRBCs) in the circulation of a term or late-preterm neonate has estimated the NRBC emergence time by determining the time- been proposed as a biomarker of fetal hypoxia.1–8 The rationale interval between recombinant erythropoietin (rEPO) administra- involves low oxygen tensions in the fetus stimulating hypoxia- tion to term or late-preterm neonates and the subsequent inducible factor (HIF)-2a, in a dose-dependent manner, appearance of NRBC in their blood. accelerating transcription of the EPO (erythropoietin) gene, resulting in elevated EPO concentrations in the blood, followed by EPO-mediated release of immature erythrocytes, including MATERIALS AND METHODS normoblasts, from the marrow or liver into the circulation.9–11 Information was collected as a deidentified data set from Intermountain At least two problems have been found with this concept. First, Healthcare records. NRBC values were electronically retrieved from accurate reference ranges for NRBC counts of neonates have been neonates with a date of birth from 1 January 2005 to 31 October 2012 who had one or more doses of darbepoetin alpha (Amgen, Thousand Oaks, lacking. Thus, recognizing a NRBC count as ‘elevated’ is inexact. CA, USA) administered during their hospital stay. This data set was reduced Second, the timing is not known between onset of human fetal to include only cases that had at least one CBC performed in the 3 days hypoxia and emergence of NRBC into the circulation. Unless before the darbepoetin dose was given and one or more additional CBC in this interval can be estimated, the link between an in utero the 7 days after dosing. The data set was further reduced by focusing hypoxic event and a subsequent elevation in NRBC count remains exclusively on neonates X34 weeks gestation at birth. indefinite. Blood samples were run on a Beckman Coulter Hematology Analyzer We recently published reference ranges for NRBC counts of (Beckman Coulter, Fullerton, CA, USA). Automated cell counts were neonates, using multihospital data, and we judge these to be of performed in accordance with the hematology analyzer manufacturer’s sufficient quality to assuage the first objection.12,13 The present instructions, in accordance with Intermountain Healthcare Laboratory Service standard operating procedures, by certified medical technologists. study was aimed at the second problem; quantifying the interval Values were expressed as NRBC per ml blood.12 between onset of fetal hypoxia and emergence of NRBCs into the Gestational age was determined by obstetrical assignment unless circulation. This interval consists of two periods; (1) the time changed by the neonatologist based on physical examination and between onset of fetal hypoxia and an increase in plasma EPO, the neurodevelopmental findings. The dose of darbepoetin per kg body

1Department of Women and Newborns, Intermountain Healthcare, Salt Lake City, UT, USA and 2Department of Obstetrics and Gynecology, University of Utah School of Medicine, Salt Lake City, UT, USA. Correspondence: Dr RD Christensen, NICU 4th Floor, McKay-Dee Hospital Center, 4401 Harrison Boulevard, Ogden, UT 84403, USA. E-mail: [email protected] Received 14 May 2013; revised 8 July 2013; accepted 5 August 2013; published online 12 September 2013 NRBC ‘emergence time’ RD Christensen et al 117 8000 Received Darbepoetin during NICU stay 10 g/kg n = 495 7000 4 g/kg 6000

5000 L  4000

Did not have a NRBC Had one or more NRBC NRBC / 3000 result prior and after results prior and after Darbepoetin Darbepoetin administration administration 2000

1000

n= 275 n=220 0 0 24487296120144168 Hours after Darbepoetin Administration Figure 2. Nucleated (NRBC) counts during the 7-day period after 34 doses of darbepoetin given to 31 neonates. The hour ‘zero’ was recorded as the time of the darbepoetin dose, which was GA < 34 weeks GA ≥ 34 weeks either 4 or 10 mgkgÀ 1 at the preference of the attending at birth at birth neonatologist.

n =189 n =31 À 1 Figure 1. Explanation of the data set used in this study. NICU, 10 mgkg dose. However, the ‘emergence time’ appeared to be neonatal intensive care unit; NRBC, . similar with the two doses. weight was recorded to seek for a dose–response relationship. Statistical DISCUSSION analysis used Microsoft Excel (Microsoft Corporation, Redmond, WA, USA). In 2003, the American College of Obstetricians and Gynecologists The Intermountain Healthcare Institutional Review Board approved the and the American Academy of Pediatrics jointly released the study as a retrospective, de-identified, data-only investigation. publication, ‘Neonatal Encephalopathy and Cerebral Palsy: Defin- ing the Pathogenesis and Pathophysiology’.14 The report concludes that most cases of neonatal encephalopathy and RESULTS cerebral palsy occur during a period before (not during) labor and During the period studied, 495 neonatal intensive care unit delivery. In the years preceding the publication of that admissions received one or more doses of darbepoetin (Figure 1). monograph, Green,1 Buonocore2 and Meadow15 had reported Of these, 220 neonates had one or more CBCs within the 3 days that an elevated NRBC in a neonate at birth appeared to be an before the darbepoetin administration and also within the 7 days index of encephalopathy. The 2003 monograph states that ‘NRBC following the dose. Thirty-one were term or late preterm neonates counts to determine the timing of neurologic injury should be (X34 weeks gestation at birth) and these comprised the study considered investigational’. After the writing of that monograph, group (Figure 1). These 31 received 34 doses of darbepoetin. The we established reference intervals for NRBC of neonates, organi- 31 were 34 to 42 weeks gestation at birth (38±2 weeks, mean zed into gestational and postnatal ages.12,13 Thus, elevated NRBC ±s.d.), birth weights were 3051±754 g, 71% were born by counts at birth, those exceeding the 95% upper reference interval, cesarean section, 74% were white, 68% male and 23% were small can now be recognized with confidence. In the present study, for gestational age. The dose of darbepoetin was given on a we provide data relevant to the timing of hypoxemia, when NRBC median day of life 3 (range 1 to 87). The indications for dosing are elevated at birth. Concordant with the conclusion of the were not stated in the records but the likely intention was to keep monograph, our data suggest that if the NRBC count is elevated at the high enough to prevent a RBC transfusion. birth, hypoxemia likely occurred in utero a day or so earlier. Conditions among those dosed in the first days after birth We speculated that the time between onset of fetal hypoxia included placental abruption, prolapsed cord or tight nuchal cord and emergence of NRBC into the circulation could be estimated (n ¼ 11), and meconium aspiration (n ¼ 7). Those dosed later in the by adding two intervals: (1) the time required for a fetus to neonatal intensive care unit course had respiratory distress and a endogenously generate EPO after hypoxic stimulation, plus (2) the risk for (n ¼ 11), or re-admission to the neonatal intensive time between EPO appearance in fetal plasma and the emergence care unit with respiratory syncytial virus and anemia (n ¼ 2). At the of NRBC into the fetal blood. The time needed to generate EPO discretion of the attending neonatologist, seven doses were after hypoxic stimulation involves a series of molecular events. written at 4 mgkgÀ 1 and 27 were 10 mgkgÀ 1. HIFs are transcription factors that respond to abnormally low NRBC counts obtained during the 7-day period following each oxygen at the cellular level. HIF-2a regulates EPO production in dose of darbepoetin are shown in Figure 2. During the first 24 h the fetal liver.16 Inadequate delivery of oxygen to the fetal liver after dosing, 26 CBCs were obtained, and all had zero or negligible results in a rapid inhibition of a prolyl-hydroxylase, which uses NRBC counts. The ‘NRBC emergence time’ was judged as the oxygen as a cosubstrate. This inhibition leads to translocation of interval between darbepoetin dose and the first appearance of HIF-2a to the nucleus where the alpha units heterodimerize and NRBC in the blood. This interval appeared to be between 24 and bind to the hypoxia response elements in the regulatory elements 36 h. Not all neonates had an elevation in NRBC count detected. In of the EPO gene. This binding induces a 41000-fold increase in general, peak NRBC counts were higher in those receiving the EPO transcription and translation, significantly raising circulating

& 2014 Nature America, Inc. Journal of Perinatology (2014), 116 – 119 NRBC ‘emergence time’ RD Christensen et al 118 EPO levels. This process takes about 4 to 5 h, which is consistent Future prospective clinical studies and animal models will likely with studies of fetal sheep, where Widness et al. observed an generate clearer and more definite time-intervals than those we increase in plasma EPO 4 to 5 h after initiating fetal hypoxia.17 have estimated. However, at present, we maintain our data Adding the 4 to 5 h needed for hypoxia to generate EPO, to the suggest that in term and late-preterm fetuses the time between ‘NRBC emergence time’ might give a reasonable estimate of the onset of hypoxia and appearance of NRBC in the blood is not less time-interval between fetal hypoxia and appearance of NRBC in than 28 to 29 h. Therefore, if an elevated NRBC count is found at the blood. In the present study, we estimated the NRBC birth, the implication is that hypoxia occurred at least 28 to 29 h emergence time by analyzing CBCs from term and late-preterm previously. neonates who had received a dose of rEPO for clinical care. We found that the interval between rEPO dosing and NRBC elevation was not shorter than 24 h. Thus, adding a 4- to 5- h EPO CONFLICT OF INTEREST generation time to a 24-h ‘NRBC emergence time’ suggest that a The authors declare no conflict of interest. period of not less than 28 to 29 h is required between onset of fetal hypoxia and elevation of NRBC in the blood. EPO has a variety of actions, all mediated through binding to specific EPO cell-surface receptors.18 EPO is required for survival, ACKNOWLEDGEMENTS proliferation and differentiation of erythroid progenitor cells, but We thank Josef T Prchal, MD, Departments of Internal Medicine, Genetics, and no EPO receptors are present once RBC reach the mature, , University of Utah School of Medicine, for critically reviewing the enucleated stage. The time required between EPO binding to manuscript, and Vickie L Baer, RN, Intermountain Healthcare Research, for assistance erythroid progenitor cells and emergence of clonally derived with data management. . mature erythrocytes into the blood is about 1 week,18,19 thus this action of EPO could not explain the increase in NRBC we observed 24 h after darbepoetin dosing. 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& 2014 Nature America, Inc. Journal of Perinatology (2014), 116 – 119