Martha C. Sola

Plasma thrombopoietin concentrations in thrombocytopenic and non-thrombocytopenic patients in a neonatal intensive care unit

Thrombocytopenia is a frequent occurrence in the neonatal intensive care unit (NICU), but the role of thrombopoietin (Tpo) in the pathophysiology is unknown. We obtained serial plasma Tpo concentrations in 20 thrombocytopenic neonates in our NICU, and performed bone marrow studies in 15. The initial Tpo levels ranged from undetectable (<41 pg/ml) to 1112 pg/ml and did not correlate with gestational age or platelet count. Neonates with decreased marrow megakaryocytes did not have plasma Tpo levels as high as those reported in adults, particularly in small for gestational age infants (Tpo < 300 pg/ml). In 14/15 neonates followed until resolution, the Tpo concentration decreased as the platelet count increased.

Platelet transfusions in the neonatal intensive care unit:factors predicting which patients will require multiple transfusions

BACKGROUND: Previous studies suggest that recombinant thrombopoietin (rTPO) will increase platelet production in thrombocytopenic neonates. However, the target populations of neonates most likely to benefit should be defined. Studies suggest that rTPO will not elevate the platelet count until 5 days after the start of treatment. Therefore, the neonates who might benefit from rTPO are those who will require multiple platelet transfusions for more than 5 days. This study was designed to find means of prospectively identifying these patients.

EVALUATION AND TREATMENT OF THROMBOCYTOPENIA IN THE NEONATAL INTENSIVE CARE UNIT

Thrombocytopenia is a very frequent problem among sick neonates, affecting up to 35% of all infants admitted to the NICU. Although multiple clinical conditions have been causally associated with neonatal thrombocytopenia, the cause of the thrombocytopenia is unclear in up to 60% of affected neonates. This article provides neonatologists with a practical approach to the thrombocytopenic neonate, with an emphasis on conditions that could be life-threatening or could have significant implications for further pregnancies. An overview of the current therapeutic modalities is also presented, including a discussion of the possible use of recombinant thrombopoietic cytokines to treat certain groups of thrombocytopenic neonates.

Epidemiologic and Outcome Studies of Patients Who Received Platelet Transfusions in the Neonatal Intensive Care Unit

STUDY DESIGN: We conducted a historic cohort study of neonates who received platelet transfusions at the National Institute of Perinatology, Mexico City, from January 1997 to May 2000. We obtained descriptive and outcome data, and assessed demographic and laboratory means of predicting “good candidates” for a future recombinant thrombopoietin (rTpo) trial.RESULTS: A minority of the transfused patients (11.4%) received only one transfusion; the majority (88.6%) received multiple transfusions. Neonates who received one or more platelet transfusions were more likely to die (24.5% mortality) than neonates who received no platelet transfusions (3.7% mortality). Regression analyses indicated that the presence of liver disease was the best predictor of a “good candidate” for rTpo administration.CONCLUSION: The majority of neonates in our institution who receive platelet transfusions receive multiple, not single, transfusions. Receiving any platelet transfusion is a marker for high risk of death. Neonates with liver disease who receive platelet transfusions might be a reasonable group for a phase I rTpo trial.

Dose–response relationship of megakaryocyte progenitors from the bone marrow of thrombocytopenic and non-thrombocytopenic neonates to recombinant thrombopoietin

Megakaryocyte (MK) progenitors from the marrow of adults undergo dose‐dependent clonogenic proliferation in response to recombinant thrombopoietin (rTpo). It is unknown whether progenitors from the marrow of thrombocytopenic neonates display rTpo dose‐dependent proliferation and whether they are more or less sensitive to rTpo than progenitors from non‐thrombocytopenic neonates or adults. To assess this, we cultured marrow from four thrombocytopenic and four non‐thrombocytopenic neonates, and from six healthy adults, in a serum‐free system in the presence of increasing concentrations of rTpo (0–100 ng/ml). Marrow from the thrombocytopenic and non‐thrombocytopenic neonates generated three times more MK colonies/105 light density cells (129 ± 39 and 167 ± 30 respectively) than marrow from adults (54 ± 30, P < 0·0001) at a rTpo concentration of 50 ng/ml. Neonatal and adult samples had a rTpo dose‐dependent increase in MK colonies. However, neonates reached a maximal number of colonies at a rTpo concentration of 10 ng/ml, compared with 50 ng/ml in adults, resulting in a larger area under the rTpo dose–response curve for neonatal progenitors (P = 0·0047). Neonates also generated more large MK colonies than the adults (24% vs. 2% at 100 ng/ml).

Thrombopoietin (Tpo) in the fetus and neonate: Tpo concentrations in preterm and term neonates, and organ distribution of Tpo and its receptor (c-mpl) during human fetal development

Little is known about thrombopoietin (Tpo) production in human fetuses and neonates. As a step toward determining whether Tpo is relevant to platelet production in the fetus and neonate, we hypothesized that: (1) like other cytokines, Tpo is present in the cord blood in higher concentrations than in adult plasma; (2) Tpo and its receptor (c-mpl) are expressed in fetuses at, and following, 5-6 weeks post-conception (when platelet production begins); and (3) the sites of Tpo and c-mpl production in the fetus are similar to those of adults. We quantified Tpo, by ELISA, in the plasma of 50 adults, as well as in the umbilical cord plasma of 50 preterm and term infants. We also characterized, by RT-PCR, the organ distribution of Tpo and c-mpl during fetal development (at 8 and 16 weeks). Tpo concentrations were measurable (> or =41 pg/ml) in only two of the 50 adult samples (44 and 46 pg/ml), but in 24 of the 50 cord plasma samples (of the 24 samples, the median was 62 pg/ml; mean+/-SD, 80+/-39 pg/ml). Tpo levels did not correlate with either gestational age or platelet count at birth. Similarly to adults, in the fetal tissues, Tpo transcripts were found in all organs tested, but the most dense bands were from liver. C-mpl transcripts were also predominantly from liver. We conclude that: (1) Tpo is present in higher concentrations in cord plasma than in venous plasma of adults; (2) Tpo and c-mpl transcripts are detected in human fetuses as early as the onset of platelet appearance; and(3) Tpo and c-mpl have a similar organ distribution in fetuses and adults.

A bone marrow biopsy technique suitable for use in neonates

Thrombocytopenia and neutropenia are common among neonates in intensive care units. Bone marrow aspirations are sometimes performed as part of their evaluation. However, marrow biopsies have not been reported from living neonates. Since architecture and cellularity cannot generally be accurately assessed from marrow aspirates, we devised a biopsy technique which we successfully applied to five cytopenic neonates (three with severe persistent thrombocytopenia and two with idiopathic neutropenia). This technique used a 19 gauge, half‐inch Osgood needle to obtain bone marrow clots from the tibias of small preterm neonates which enabled the assessment of marrow cellularity and architecture. On the basis of our initial experience we have ceased using the traditional bone marrow aspiration technique in neonates and now use this technique exclusively.

Pharmacokinetics, Pharmacodynamics, and Safety of Administering Pegylated Recombinant Megakaryocyte Growth and Development Factor to Newborn Rhesus Monkeys

Thrombocytopenia is common among sick neonates. Certain groups of thrombocytopenic adults respond favorably to the administration of recombinant thrombopoietin or to pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a recombinant human polypeptide that contains the receptor-binding N-terminal domain of thrombopoietin. The effectiveness and safety of such treatment in neonates, however, have not been reported. The purpose of the present study was to determine the biologic activity and safety of PEG-rHuMGDF administration to newborn rhesus monkeys. Eight monkeys were divided into four groups and treated subcutaneously with 0.00, 0.25, 1.00, or 2.50 μg/kg once daily for 7 d. Complete blood counts, serum chemistries, clotting panels, and MGDF levels were followed serially, and hematopoietic progenitor cell assays were performed on bone marrow aspirates before the first dose and again on d 8. Pharmacokinetic evaluations were performed on the animals that received the highest dose of PEG-rHuMGDF. All monkeys had normal growth during the study period, and all chemistries, clotting studies, and blood pressure measurements were normal. The peak serum MGDF concentration occurred at 3 h, and the half-life was 8.4 to 13.0 h. As in adult rhesus monkeys, platelet counts in the treated neonates began to rise on d 6, peaked on d 11, and returned to baseline by d 23. The two highest doses generated an 8- to 12-fold increase in platelets, whereas those treated with 0.25 μg/kg had a 6-fold increase. Other hematologic parameters measured were unaffected. Thus, newborn monkeys responded to doses of PEG-rHuMGDF that were similar to or smaller than (per kilogram body weight) those that are effective in adult animals and did so without obvious short-term toxicity.

The relationship between hematocrit and bleeding time in very low birth weight infants during the first week of life.

OBJECTIVES: The bleeding time is a measurement of platelet and capillary interaction following a small standardized cutaneous incision. In adults, anemia causes a prolongation of the bleeding time, and we hypothesized that the same would be true in very low birth weight (VLBW) infants during their first week of life.STUDY DESIGN: Template bleeding times, using the Surgicutt Newborn device, were performed on 20 VLBW weight infants ≤7 days old, before, and again following a clinically ordered erythrocyte transfusion.RESULTS: Neonates who had pretransfusion hematocrits ≤0.28 l/l had longer bleeding times, which fell 164±25 seconds (mean±SD; p<0.0001) following transfusion. Patients with pretransfusion hematocrits >0.28 l/l had no significant reduction in bleeding time following transfusion.CONCLUSIONS: In VLBW infants, during their first week of life (the time when their risk of intraventricular hemorrhage is greatest), a low hematocrit is associated with a significant prolongation in the bleeding time.

Developmental Differences in Megakaryocyte Maturation Are Determined by the Microenvironment

Historically, physicians have attributed delayed platelet engraftment following umbilical cord blood transplant to decreased numbers of stem cells in cord blood compared with adult bone marrow. However, recent studies suggest that delayed platelet engraftment may be caused by an intrinsic inability of neonatal stem cells to produce mature, polyploid megakaryocytes. We tested this hypothesis by transplanting adult bone marrow and newborn liver hematopoietic stem and progenitor cells from transgenic mice expressing green fluorescent protein into myeloablated wild‐type recipients and comparing the size and ploidy levels of megakaryocytes that developed in adult transplant recipients. Transplanted stem and progenitor cells, regardless of their source, gave rise to megakaryocytes that were larger than normal adult megakaryocytes as early as 7 days post‐transplant. However, megakaryocytes that developed after transplant of neonatal stem and progenitor cells were significantly smaller than those derived from adult stem and progenitor cells. Furthermore, megakaryocytes derived from neonatal cells had lower ploidy values than megakaryocytes derived from adult cells at 18 days post‐transplant, when ploidy could first be reliably measured in the bone marrow. These differences in size and ploidy disappeared by 1 month post‐transplant. The largest megakaryocytes developed in the spleen. These results suggest that, in the mouse, the microenvironment is responsible for some of the maturational differences in size and ploidy between neonatal and adult megakaryocytes. Furthermore, neonatal and adult megakaryocyte progenitors also have cell‐intrinsic differences in the way they engraft and respond to thrombocytopenic stress. These differences may contribute to the delay in platelet engraftment that frequently complicates cord blood transplants.