PEDIATRICS Vol. 105 No. 6 June 2000, pp. 1286-1291

Thrombopoietin in the Thrombocytopenic Term and Preterm Newborn

Timothy S. E. Albert, MD^*, Y. Gloria Meng, PhD, Paul Simms, MS, Robert L. Cohen, MD, and Roderic H. Phibbs, MD^*

From the ^* Department of Pediatrics and Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California; and  Departments of BioAnalytical Technology and Molecular Oncology, Genentech, Inc, South San Francisco, California.

	ABSTRACT

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Objectives.  Neonatal thrombocytopenia occurs commonly in neonatal intensive care units. The role of the thrombopoietin (Tpo) system in normal neonatal platelet regulation and neonatal thrombocytopenia is not well understood. The purpose of our study was to: 1) determine the normal Tpo level at birth in healthy nonthrombocytopenic term (NTT) and nonthrombocytopenic preterm (NTP) infants and in infants born to women with preeclampsia; and 2) measure Tpo levels in infants during and after the resolution of thrombocytopenia. Characterizing Tpo levels in the healthy and thrombocytopenic newborn is an important step in furthering our understanding of the pathophysiology of neonatal thrombocytopenia.

Methods.  This study is comprised of 2 parts. For the first part, cord blood was obtained at birth from both term (gestational age [GA]: 38-42 weeks) and preterm (GA: 25-36 weeks) infants. If birth platelet levels were 140 × 10³/µL and the infant fit criteria for being normal, or if the infant was born to a women with preeclampsia, Tpo levels were measured. For the second part, serial Tpo levels and concomitant platelet counts (Plts) were measured in both preterm and term infants during a period of marked thromboctyopenia (Plt < 100 × 10³/µL) until its resolution (Plt  140 × 10³/µL).

Results.  Median cord blood Tpo levels from NTP infants (n = 35) were higher than those of NTT infants (n = 32; 95 pg/mL vs 48 pg/mL, respectively). In addition, preterm infants born to women with preeclampsia (n = 11) had lower Tpo levels than NTP infants with a similar GA (<41 pg/mL vs 95 pg/mL). For infants with marked thrombocytopenia, median Tpo levels during thrombocytopenia were similar between term (n = 12) and preterm (n = 14) groups (223 pg/mL and 179 pg/mL, respectively), with the majority of individuals showing a decrease in Tpo with resolution of thrombocytopenia. Within each group, there was large variability in the Tpo response to thrombocytopenia.

Impression.  These data show that the Tpo system is intact in NTP and NTT neonates. Preeclampsia may be an example of a disorder that perturbs this system. The great variability in Tpo levels seen in infants during thrombocytopenia may be related to the mechanism of thrombocytopenia. The finding that, in general, Tpo levels decreased with resolution of thrombocytopenia is consistent with what has been described in adults and children.  Key words:  thrombopoietin, neonatal thrombocytopenia, blood platelets, umbilical cord blood, newborn.

Thrombocytopenia (platelet count [Plt] < 150 × 10³/µL) occurs in nearly 1% of all live births,¹ and in nearly 1 of 4 newborns admitted to neonatal intensive care units.² Infants who develop severe thrombocytopenia (Plt < 100 × 10³/µL) have increased neurologic morbidity and mortality.^3,4

Neonatal thrombocytopenia has been associated with maternal, fetal, and neonatal conditions, some of which include severe infection,⁴ maternal pregnancy-induced hypertension,^5,6 birth asphyxia,² and immune mechanisms.^1,2 Some studies have suggested that low Plts were a consequence of increased platelet destruction² or decreased platelet production.⁴ More recent evidence suggests that reduced numbers of circulating megakaryocyte progenitor and precursor cells at birth may play a role in thrombocytopenia in preterm infants.⁶

Thrombopoietin (Tpo) is the well-characterized hematopoietic factor that is thought to be the primary regulator of megakaryocytopoiesis and thrombopoiesis.⁷ Studies of Tpo in healthy adults show small but measurable levels of circulating Tpo.^8,9 Tpo levels may be elevated in thrombocytopenic adults and children, with the degree of Tpo elevation seeming to be dependent on the mechanism of thrombocytopenia.^10-12

Characterizing Tpo levels in the healthy and thrombocytopenic newborn is an important step in furthering our understanding of the pathophysiology of neonatal thrombocytopenia. Recently Murray et al¹³ and Sola et al¹⁴ published data showing Tpo levels in both normal and thrombocytopenic neonates. In this article, we present our own series in which we: 1) measured Tpo levels at birth in nonthrombocytopenic term (NTT) and nonthrombocytopenic preterm (NTP) infants and in preterm infant born to women with preeclampsia; and 2) determined the plasma Tpo levels in term and preterm infants during thrombocytopenia and after its resolution.

	METHODS

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This study was performed at the University of California, San Francisco Medical Center with the approval of the committee on human research.

Cord Blood Tpo Levels at Birth

Tpo levels and Plts were measured in umbilical cord blood prospectively acquired from 2 groups of nonthrombocytopenic neonates: term (gestational age [GA] range: 38-42 weeks) and preterm (GA range: 36 weeks). The blood was obtained immediately after birth from a doubly clamped section of umbilical cord by aspiration into unheparinized syringes with subsequent transfer into tubes for measurement of Tpo concentration or Plt. Infants were included in either group if they had: 1) Plt at birth  140 × 10³/µL, 2) a 5-minute Apgar score 7, 3) no major congenital anomalies, 4) a birth weight appropriate for GA (within 2 standard deviations of normal), and 5) showed no signs of infection or thrombocytopenia during the first week of life. When an accurate Plt measurement was not available from the cord blood, subjects were included if they had a normal peripheral blood Plt measurement obtained <2 hours after birth. All infants from pregnancies complicated by preeclampsia or chorioamnionitis were excluded from analysis as normal nonthrombocytopenic neonates. Cord blood Tpo values for preterm infants born to women with preeclampsia (PP) were analyzed separately and included all infants regardless of birth Plt.

Tpo Levels in Thrombocytopenic Neonates

Infants admitted to the University of California, San Francisco intensive care nursery have Plts measured at admission. Infants at risk for developing or who develop thrombocytopenia have their Plt measured daily. All infants who had marked thrombocytopenia (Plt < 100 × 10³/µL) within their first 2 weeks of life were eligible for enrollment. If parental consent for study entry was given, peripheral blood samples for Tpo analysis were obtained prospectively during the period of thrombocytopenia (Plt < 140 × 10³/µL), and after its resolution (Plt  140 × 10³/µL, not attributable to a platelet transfusion). Tpo samples were obtained in association with measurement of Plt. All of these newborns were followed until spontaneous resolution of thrombocytopenia and/or discharge.

Tpo Analysis

Blood for Tpo analysis was collected into tubes anticoagulated with sodium citrate. Plasma was obtained by centrifugation and then stored at 70°C until analysis. Tpo levels were measured using a monoclonal antibody sandwich enzyme-linked immunosorbent assay (ELISA), which has been described.¹⁵ Briefly, ELISA plates were coated with monoclonal antibody (4G5) to human Tpo at 5 µg/mL (Genentech, Inc, South San Francisco, CA) in 50 mM of carbonate buffer (pH: 9.6) at 4°C overnight and then blocked with .5% bovine serum albumin (pH: 7.2) at room temperature for 1 hour. Two and one half-fold serial dilutions of standards (1.6-400 pg/mL full length human Tpo produced in mammalian cells) and 4 twofold serial dilutions of samples (final dilution range: 1:5 to 1:40) in phosphate-buffered saline, containing .5% bovine serum albumin, .05% polysorbate 20, were incubated on the plate for 2 hours. Bound Tpo was detected using biotinylated monoclonal antibody 14G11 (Genentech, Inc) to Tpo, followed by peroxidase-labeled streptavidin (Amdex, Copenhagen, Denmark) and 3,3',5,5'-tetramethyl benzidine (Kirkegaard and Perry Laboratories, Gaithersburg, MD) as the substrate. The assay range for Tpo in serum or plasma is 41 to 4000 pg/mL using a minimum 1:10 sample dilution. This assay gives results similar to those previously reported by receptor-based ELISA.¹²

Data Analysis and Statistics

Results are expressed as median (range) or mean ± standard deviation unless otherwise stated. Comparisons between groups were made using either the Mann-Whitney U test or unpaired Student's t test, and within groups, by a paired t test analysis. Tpo concentrations less than the detection limit (<41 pg/mL) were assigned the value 40 pg/mL in the analysis of the data from the thrombocytopenic neonates. Similar results were obtained when they were assigned the value zero. If multiple Tpo measurements were obtained from a subject during thrombocytopenia or after resolution, the Tpo level obtained closest to the platelet nadir or at the highest measured Plt after resolution was used for analysis, respectively. A P < .05 was considered statistically significant.

	RESULTS

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Tpo Levels at Birth in Nonthrombocytopenic Neonates and Infants Born to Women With Preeclampsia

Cord blood samples were obtained from 78 newborns ranging in GA from 25 to 42 weeks. Table 1 shows clinical details of the NTT and NTP neonates, and a smaller group of PP infants. Birth Plt was similar between the NTT and NTP groups, but lower in the PP group (P  .05). In the PP group, 3 of 11 infants had birth Plts < 140 × 10³/µL. The mean arterial and venous cord blood gases (pH, partial pressure of carbon dioxide, and partial pressure of oxygen) of the NTT and NTP groups were similar to those reported for vigorous newborns¹⁶ (data not shown).

Figure 1 shows individual cord blood Tpo concentrations for each of the 3 groups. Tpo levels were below the sensitivity of our assay in 12 (38%) NTT, 7 (20%) NTP, and 6 (55%) of the PP infants. The median (range) plasma Tpo concentration was significantly lower in NTT infants (48 pg/mL [<41-257 pg/mL]), than in NTP infants (95 pg/mL [<41 to 276 pg/mL]; P = .003). In addition, birth plasma Tpo levels were lower in the PP infants (<41 pg/mL [<41-164 pg/mL], than in the NTP neonates; P = .03). No relationship between plasma Tpo concentration and Plt was found for any group. In addition, there was no difference in intragroup Tpo levels between infants who had Plts measured from cord blood or peripheral blood at birth. The median Tpo concentrations were not different between male and female infants. There were 5 sets of twins and 1 set of triplets. Tpo concentrations differed between siblings by 15% to 68% (average: 34%), with this difference being greater than the intraassay variation of our Tpo assay.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Individual plasma Tpo levels obtained from cord blood at birth are shown for NTT, NTP, and PP infants. The median Tpo level for each group is marked with a horizontal bar. The median Tpo levels for NTT and PP infants were significantly lower than NTP newborns. Both the median Tpo level for the PP group and individual samples with Tpo concentrations less than the detection limit (<41 pg/mL) were plotted as 40 pg/mL.

Tpo Levels in Thrombocytopenic Neonates

Plasma samples were obtained from 14 preterm (GA: 26-36 weeks) and 12 term (GA: 37-42 wks) neonates who had marked thrombocytopenia (Plt < 100 × 10³/µL) within their first 2 weeks of life. The time course of thrombocytopenia was similar between groups. The median day of life for developing marked thrombocytopenia was day 2, with the platelet nadir occurring on day 4. Seventeen infants were followed until resolution of thrombocytopenia (day 12). Table 2 provides clinical data on all patients studied. Plts measured during the first day of life (range: 5-310 × 10³/µL) and at the nadir (range: 5-95 × 10³/µL) were similar between groups. Preterm infants were more likely to have received an exchange transfusion than were term infants. There were no other differences in clinical factors.

The median Tpo concentration for the term group (n = 12) during thrombocytopenia was 223 pg/mL (<41-892 pg/mL), whereas that of the preterm group (n = 14) was 179 pg/mL (<41-624 pg/mL; P = .49). Four infants had measured Tpo >500 pg/mL during thrombocytopenia (n = 2 term and n = 2 preterm), whereas 8 infants had measured Tpo <100 pg/mL during thrombocytopenia (n = 2 term and n = 6 preterm; Fig 2). Tpo levels were measured an average of 2 days after the platelet nadir (range: 2 to 11 days) for both groups. The mean Plt when Tpo levels were measured during thrombocytopenia was similar between groups (term: 73 ± 41 × 10³/µL and preterm: 83 ± 32 × 10³/µL). Tpo levels during thrombocytopenia did not correlate with the Plt when the sample was drawn, the severity of thrombocytopenia (as determined by the platelet nadir), the day of life when the measurement was made, or the time samples were obtained after platelet nadir. Tpo levels were not different in platelet-transfused versus -nontransfused infants.

View larger version (9K): [in this window] [in a new window]   Fig. 2.   Plasma Tpo concentrations during thrombocytopenia (Plt < 140 × 103/µL) are shown for individual term and preterm infants. The median Tpo level for each group is marked with a horizontal bar. One infant in the term group and 2 in the preterm group had Tpo levels less than the detection limit of our assay (<41 pg/mL). For these 3 subjects, the Tpo level is plotted as 40 pg/mL.

A subset of the term (n = 7) and preterm (n = 10) infants had Tpo levels measured after resolution of thrombocytopenia. Figure 3 shows Tpo values during and after thrombocytopenia for individual subjects in both groups. Six of 7 term infants and 6 of 10 preterm infants had a decrease in Tpo with resolution of thrombocytopenia. Paired t test analysis showed a significant decrease in Tpo level with resolution of thrombocytopenia in the term group (P = .04), with a tendency toward a significant decrease in the preterm group (P = .08). Four of the 5 infants who did not show a decrease in Tpo with resolution of thrombocytopenia had increases in Tpo that were greater than the variation in our assay. Of these, all 4 were preterm and 3 were born to women with preeclampsia.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Tpo concentrations during thrombocytopenia (Plt < 140 × 103/µL) and after its resolution (Plt  140 × 103/µL) are shown for a subset of the term and preterm infants from whom both measurements were obtained. The lines connect samples obtained from the same individual.

Tpo Response to Thrombocytopenia for Individual Infants

Serial Tpo measurements were obtained in some subjects. For some infants, Tpo levels showed a reciprocal relationship with Plt, peaking near the nadir and decreasing as Plt increased. For others, Tpo levels seemed to stay unchanged, or even increase, as platelet levels returned to normal. Figure 4 illustrates the diverse Tpo responses to thrombocytopenia and its resolution.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Time course of Tpo concentration (filled squares) and Plt (open circles) in 3 infants from whom multiple Tpo samples were obtained and who showed a diverse Tpo response to thrombocytopenia. All subjects had their first Tpo level measured during marked thrombocytopenia (Plt < 100 × 103/µL) and their last Tpo level measured after resolution (Plt > 140 × 103/µL). Infants A and C had evidence of birth asphyxia. Infants B and C had exchange transfusions for treatment of erythroblastosis fetalis.

	DISCUSSION

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Tpo is the primary regulator of platelet production. This study shows Tpo levels at birth in nonthrombocytopenic neonates and follows the Tpo response in a series of thrombocytopenic infants. In general, both term and preterm newborns had higher Tpo levels during thrombocytopenia than after its resolution. There was great variability in the Tpo response to thrombocytopenia for both groups, which is consistent with what has been seen in adults and children.

Cord blood Tpo levels in nonthrombocytopenic infants at birth were greater in preterm than term infants (95 pg/mL vs 48 pg/mL, respectively). The NTT newborns had similar Tpo levels to those reported for normal, healthy adults^8,9; however, they were lower than those reported in 1 other series of healthy term newborns.¹⁷ In agreement with studies by Murray et al¹³ and Sola et al,¹⁴ there was no relationship between Tpo level and Plt. Our data are in disagreement with these studies, however, in our observation of a difference in Tpo level between term and preterm infants. Tpo levels in their studies were closer to that of our preterm infants. The reason for these differences is not readily apparent but may be related to the number of infants studied, clinical characteristics of the newborns, or time of sampling after birth. In the study by Sola et al,¹⁴ their data for all nonthrombocytopenic infants were obtained from infants admitted to a neonatal intensive care unit at an undefined period of time after birth. Our findings combined with other studies showing preterm infants to have higher levels of megakaryocytes and megakaryocyte precursors at birth than term infants^18,19 suggest a more highly active megakaryopoietic and thrombopoietic system in the premature neonate.

Hypertensive disorders of pregnancy are known to increase the risk of developing neonatal thrombocytopenia.⁵ In addition, platelets from infants born to women with preeclampsia are qualitatively different from those of healthy newborns.²⁰ By measuring cord blood Tpo levels in infants born to women with preeclampsia, we hypothesized we would see an example of a disorder that would perturb the normal Tpo system. Our finding that Tpo levels are reduced in premature infants born to women with preeclampsia compared with nonpreeclamptic preterm infants supports a perturbation of the Tpo system in this disorder. We speculate that this is a potential mechanism for the increased tendency for thrombocytopenia in infants of preeclamptic women. The finding that a hematopoietic factor may be altered in preeclampsia is not new, as attested by a recent study that found numerous other hematopoietic factor levels to be altered in fetuses from preeclamptic women.²¹

The infants in this study had a similar course of thrombocytopenia as has been found in infants admitted to other intensive care nurseries.^2,6 Median Tpo levels during thrombocytopenia were similar between the term and preterm groups (223 pg/mL and 179 pg/mL, respectively) and were similar to that seen by Sola et al.¹⁴ The majority of individuals from both groups also showed a decrease in Tpo with resolution of thrombocytopenia, which is similar to that which has been seen in both adults and children.^10-12 Interestingly, the 3 premature infants who where born to women with preeclampsia and followed until resolution of thrombocytopenia showed increases in Tpo levels with resolution of thrombocytopenia. Because of the small number of infants in this group, no generalizations can be made other than that this finding further suggests an unusual hematopoietic disturbance in the preeclamptic newborn.

The great variability in the Tpo response to thrombocytopenia could not be explained by differences in Plts when the samples were drawn or the severity of thrombocytopenia (as determined by the platelet nadir). This finding is consistent with the concept that Tpo levels are not directly regulated by platelet mass in the newborn, which is similar to the case for adults and children.^11,12,22 The great variability in the Tpo response to thrombocytopenia that we and Sola et al¹⁴ observed supports the hypothesis that the mechanism of thrombocytopenia influences the degree of Tpo elevation in the neonate. In adults, the greatest levels of Tpo are seen in those individuals with deficiencies of megakaryocytes and their precursors, whereas processes that lead to thrombocytopenia attributable to increased platelet destruction do not increase Tpo.^8,10 In our study, we could find no clear relationship between any proposed cause of thrombocytopenia (eg, exchange transfusion) and the Tpo response (data not shown). This is not surprising, however, because our study was not designed to analyze for this relationship, and infants in this study likely had multiple causes for their thrombocytopenia (see Table 2).

Serial sampling of a few subjects generally showed a similar Tpo response to thrombocytopenia as seemed to occur to the group as a whole; namely, those infants who responded to thrombocytopenia with elevations in Tpo showed a decrease with resolution of thrombocytopenia. We did not see a uniform pattern to the decrease, however, with some infants showing decreases only with large increases in Plt (see Fig 4A) and others showing a drop in Tpo level before a major increase in the platelet level (see Fig 4C). Infant B (see Fig 4B) exemplified a case in which there was no apparent relationship between Tpo level and Plt. This infant, who was preterm (GA: 30 weeks) and who we speculate developed thrombocytopenia secondary to an exchange transfusion for erythroblastosis fetalis, did not have characteristics that were unique to our study or that would help by themselves to explain the infants' lack of an elevation in Tpo.

This study was designed to answer preliminary questions about the Tpo system in neonates. These data show that the Tpo system is intact in NTT and NTP neonates. Our finding of low Tpo levels in preterm infants exposed to maternal preeclampsia may be an example of a perturbation of this system. The Tpo response to thrombocytopenia is highly variable in both preterm and term neonates, with the response likely related to the mechanism of thrombocytopenia. Because of the multiple potential causes of neonatal thrombocytopenia, a large study with many subjects will be necessary before we can more fully understand the relationship between plasma Tpo concentration and thrombopoiesis.

	ACKNOWLEDGMENTS

This study was supported in part by Grant MO1RR01271 from the National Institutes of Health.

We thank Nancy Newton, RN, BSN, and the other neonatal clinical research nurses for their assistance at the University of California, San Francisco, and Lisa Caris for her assistance on the enzyme-linked immunosorbent assay at Genentech, Inc.

	FOOTNOTES

Received for publication May 20, 1999; accepted Oct 14, 1999.

Reprint requests to (R.H.P.) Division of Neonatology, University of California, San Francisco, Box 0734, San Francisco, CA 94131. E-mail: rphibbs{at}itsa.ucsf.edu

	ABBREVIATIONS

Plt, platelet count; Tpo, thrombopoietin; NTT, nonthrombocytopenic term; NTP, nonthrombocytopenic preterm; GA, gestational age; PP, preterm infants born to women with preeclampsia; ELISA, enzyme-linked immunosorbent assay.

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