Matthew A. Saxonhouse

Neonatal thrombocytopenia: What we do and don't know

The evaluation and management of thrombocytopenia is a frequent challenge for neonatologists, as it affects 22-35% of infants admitted to the neonatal intensive care unit. Multiple disease processes can cause neonatal thrombocytopenia, and these can be classified as those inducing early thrombocytopenia (< or =72 h of life) and those inducing late-onset thrombocytopenia (>72 h). Most cases of neonatal thrombocytopenia are mild to moderate, and do not warrant intervention. In approximately 25% of affected neonates, however, the platelets count is <50 x 10(9)/L, and therapy with platelet transfusions is considered to decrease the risk of hemorrhage. The existing evidence to establish platelet transfusion triggers in neonates is very limited, but it suggests that transfusing platelets to non-bleeding neonates with platelet counts >50 x 10(9)/L does not decrease the risk of intraventricular hemorrhage (IVH), and that 30 x 10(9)/L might be an adequate threshold for stable non-bleeding neonates. However, adequately powered multi-center studies are needed to conclusively establish the safety of any given set of neonatal transfusion guidelines.

Neonatal Neutrophils with Prolonged Survival Exhibit Enhanced Inflammatory and Cytotoxic Responsiveness

Apoptosis is critical to the resolution of inflammation, as it promotes the removal of neutrophils (PMN) by the reticuloendothelial system. In contrast, PMN persistence characterizes the early stages of chronic inflammation. Adult PMN with delayed senescence retain some functionality, although this has not been described for neonatal PMN. We hypothesized that neonatal PMN with prolonged survival retain cytotoxic and inflammatory function. To test one aspect of inflammatory function, we determined surface CD11b expression on 0-h and 24-h PMN after chemotactic formyl-methionine-leucine-phenylalanine (fMLP) stimulation. Although fMLP induced a greater percentage up-regulation of CD11b on 0-h adult PMN, this was similar between nonapoptotic cord blood and adult PMN at 24 h. Furthermore, percentage up-regulation of CD11b was more robust for 24-h than for 0-h cord blood PMN. In contrast, there was no difference in responsiveness between 0-h and 24-h adult PMN. In studies of cytotoxic potential, we determined the expression of reactive oxygen intermediates (ROI) in phorbol 12-myristate 13-acetate–stimulated cord blood and adult PMN at 0 h and in 24-h nonapoptotic PMN, using the dihydrorhodamine 123 assay. Stimulated cord blood PMN generated more ROI than did adult PMN at both 0 h and 24 h; in addition, ROI levels in 24-h cord blood PMN were similar to those of 0-h adult PMN. We conclude that PMN with prolonged survival retain specific cytotoxic and inflammatory functions, and these are enhanced in cord blood PMN. We speculate that neonatal PMN with prolonged survival have the functional capacity to contribute to the pathogenesis of inflammatory disorders.

The Evaluation and Management of Neonatal Coagulation Disorders

Neonatal hemostatic abnormalities can present diagnostic and therapeutic challenges to the physician. Developmental deficiencies and/or increases of certain coagulation proteins, coupled with acquired or genetic risk factors, can result in a hemorrhagic or thromboembolic emergency. The timely diagnosis of a congenital hemorrhagic or thrombotic disorder can avoid significant long-term sequelae. However, due to the lack of randomized clinical trials addressing the management of neonatal coagulation disorders, treatment strategies are usually empiric and not evidence-based. In this chapter, we will review the neonatal hemostatic system and will discuss the most common types of hemorrhagic and thrombotic disorders. Congenital and acquired risk factors for hemorrhagic and thromboembolic disorders will be presented, as well as current treatment options. Finally, suggested evaluations for neonates with either hemorrhagic or thromboembolic problems will be reviewed.

Effects of Sepsis on Neonatal Thrombopoiesis

We serially evaluated the effects of sepsis and/or necrotizing enterocolitis (NEC) on neonatal thrombopoiesis, using a panel of tests that included platelet counts, thrombopoietin concentrations (Tpo), circulating megakaryocyte progenitor concentrations (CMPs), and reticulated platelets (RPs). Variables analyzed included sepsis type, time after onset of sepsis, platelet counts, and gestational (GA) and postconceptional ages (PCA). Twenty neonates were enrolled. Ten had Gram-negative, six had Gram-positive, and four had presumed sepsis. Four neonates had NEC stage II or higher, and six developed thrombocytopenia. Overall, septic neonates had significantly elevated Tpo concentrations and circulating megakaryocyte progenitors. The highest Tpo levels were associated with Gram-negative or presumed sepsis. RP percentages were increased only in neonates with low platelet counts, while RP counts (RP% × platelet count) were elevated in neonates with high platelet counts. Our findings suggest that septic neonates up-regulate Tpo production, leading to increased megakaryocytopoiesis and platelet release, although the degree of upregulation is moderate. The changes in RP% and RP count most likely reflect increased thrombopoiesis with variable degrees of platelet consumption. In addition, our findings suggest that different factors, likely including level of illness and/or specific platelet or bacterial products, can down-regulate the magnitude of the thrombopoietic response.

Closure Times Measured by the Platelet Function Analyzer PFA-100® Are Longer in Neonatal Blood Compared to Cord Blood Samples

Background: Although neonatal platelets have been shown to be hyporesponsive to most agonists in vitro, several groups have reported shorter closure times (CT) in term cord blood samples than in children and adults. It is unknown whether this is also true for preterm neonates, or for neonates of any gestational age (GA) during the 1st week of life, since limited studies have evaluated neonatal blood samples. Objectives: We designed this study to determine the effects of GA and postconceptional age on platelet function using the platelet function analyzer PFA-100®. Methods: We measured CTs in cord blood samples and in neonatal blood samples of varying GAs on days of life 1–2, and ≧7. Results: CTs were determined in 51 cord blood samples, 34 neonatal blood samples obtained on day of life 1–2, 16 neonatal blood samples from preterm neonates ≧7 days old, and 10 adults. We found a significant inverse relationship between ADP CTs and GA in both cord blood and neonatal blood day of life 1–2 samples (p = 0.02 and p = 0.01, respectively). When cord blood samples were compared with neonatal and adult blood, epinephrine and ADP CTs were significantly longer in adult blood as well as in neonatal samples obtained at either of the two time points (p ≤ 0.01 for all). Conclusions: Platelet function in response to ADP appears to improve with advancing GA. The differences between cord blood and neonatal blood CTs indicate that substantial changes in primary hemostasis occur shortly after birth. The reasons underlying these changes are unknown.

The evaluation and management of postnatal thromboses.

In the pediatric population, neonates have the highest risk for thromboembolism (TE), most likely due to the frequent use of intravascular catheters. This increased risk is attributed to multiple risk factors. Randomized clinical trials dealing with management of postnatal thromboses do not exist, thus, opinions differ regarding optimal diagnostic and therapeutic interventions. This review begins with an actual case study illustrating the complexity and severity of these types of cases, and then evaluates the neonatal hemostatic system with discussion of the common sites of postnatal thrombosis, perinatal and prothrombotic risk factors, and potential treatment options. A proposed step-wise evaluation of neonates with symptomatic postnatal thromboses will be suggested, as well as future research and registry directions. Owing to the complexity of ischemic perinatal stroke, this topic will not be reviewed.

Effects of Hypoxia on Megakaryocyte Progenitors Obtained from the Umbilical Cord Blood of Term and Preterm Neonates

Background: Placental insufficiency is associated with early-onset thrombocytopenia in preterm neonates. Prior studies demonstrated a reduction in circulating megakaryocyte (Mk) progenitors, suggesting decreased platelet production. We hypothesized that decreased Mk production is the result of a direct inhibitory effect of hypoxia on the proliferation of Mk progenitors, or a hypoxia-induced change in the fetal hematopoietic environment. Objective: To test the effects of hypoxia on the clonogenic maturation of Mk progenitors obtained from term and preterm cord blood CD34pos cells, either cultured alone or in conjunction with CD34neg light density mononuclear cells (LDMCs). Methods: CD34pos cells and CD34neg LDMCs were isolated from the cord blood of term and preterm deliveries, and mobilized peripheral blood CD34pos cells were obtained from healthy adults. CD34pos cells were then cultured alone or co-cultured with CD34neg LDMCs in a semisolid, serum-free media containing rTpo, IL-3, and IL-6. Cultures were exposed to 20%, 5%, or 1% oxygen for 10–12 days. Mk colonies were then quantified following immunohistochemical staining. Results: Pure CD34pos cells from preterm (n = 5) and term (n = 5) neonates and from adults (n = 4) generated similar numbers of Mk colonies in all three oxygen concentrations. However, the number of Mk colonies in preterm co-cultures was progressively lower with decreasing O2 concentrations. Conclusions: Hypoxia did not appear to directly inhibit colony formation of Mk progenitors from preterm and term cord blood CD34pos cells. However, co-culture studies showed a decrease in Mk colony formation with hypoxia, suggesting an indirect inhibitory effect of hypoxia on Mk clonogenic maturation mediated by non-progenitor cells in the hematopoietic microenvironment.

Benign B-Cell Precursors (Hematogones) Are the Predominant Lymphoid Population in the Bone Marrow of Preterm Infants

Bone marrow (BM) findings in 3rd-trimester stillborns and full-term living neonates have been previously described. However, there is no information regarding BM composition in living preterm infants. Specifically, it is unknown whether the BM lymphocytosis seen in full-term infants at 1–4 weeks of age also occurs in preterm infants. Furthermore, the lineage of these cells has never been investigated. We used a panel of immunohistochemical stains to characterize the BM composition in 11 neonates (8 living and 3 deceased). Unlike in the other age groups, immature B cells (hematogones) were the most common lymphoid population, accounting for 10–60% (mean 34%) of all cells. In two additional cases (both living patients), flow cytometry revealed a level of 3.8% of immature B cells in a <1-week-old neonate and 25.7% in a 19-week-old infant. Immature B cells were not identified in 6 peripheral blood samples from preterm neonates. These findings are pertinent for the interpretation of BM and peripheral blood samples in this age group as survival improves and diagnostic samples become more common.

Effects of anoxia on megakaryocyte progenitors derived from cord blood CD34pos cells

Abstract: Background: Severe hypoxic insults to the fetus and neonate are associated with the development of thrombocytopenia. The thrombocytopenia in some cases is the result of disseminated intravascular coagulation, but that mechanism fails to account for all, perhaps the majority, of cases.

Platelet Transfusion Practices Among Very-Low-Birth-Weight Infants

IMPORTANCE Thrombocytopenia and intraventricular hemorrhage (IVH) are common among very-low-birth-weight (VLBW) infants. Survey results suggest that US neonatologists frequently administer platelet transfusions to VLBW infants with mild to moderate thrombocytopenia. OBJECTIVES To characterize platelet transfusion practices in US neonatal intensive care units (NICUs), to determine whether severity of illness influences platelet transfusion decisions, and to examine the association between platelet count (PCT) and the risk for IVH in the first 7 days of life. DESIGN, SETTING, AND PARTICIPANTS This multicenter, retrospective cohort study included 972 VLBW infants treated in 6 US NICUs, with admission dates from January 1, 2006, to December 31, 2007. Data were collected from all infants until NICU discharge or death (last day of data collected, December 4, 2008). Data were entered into the central database, cleaned, and analyzed from May 1, 2009, to February 11, 2016. INTERVENTION Platelet transfusion. MAIN OUTCOMES AND MEASURES Number of platelet transfusions and incidence of IVH. RESULTS Among the 972 VLBW infants (520 [53.5%] male; mean [SD] gestational age, 28.2 [2.9] weeks), 231 received 1002 platelet transfusions (mean [SD], 4.3 [6.0] per infant; range, 1-63 per infant). The pretransfusion PCT was at least 50 000/μL for 653 of 998 transfusions (65.4%) with this information. Two hundred eighty-one transfusions (28.0%) were given during the first 7 days of life. During that period, platelet transfusions were given on 35 of 53 days (66.0%) when the patient had a PCT less than 50 000/μL and on 203 of 436 days (46.6%) when the patient had a PCT of 50 000/μL to 99 000/μL. At least 1 marker of severe illness was present on 198 of 212 patient-days (93.4%) with thrombocytopenia (PCT, <100 000/μL) when a platelet transfusion was given compared with 113 of 190 patient-days (59.5%) with thrombocytopenia when no platelet transfusion was given. Thrombocytopenia was a risk factor for intraventricular hemorrhage during the first 7 days of life (hazard ratio, 2.17; 95% CI, 1.53-3.08; P < .001). However, no correlation was found between severity of thrombocytopenia and risk for IVH. After controlling for significant clinical factors and thrombocytopenia, platelet transfusions did not have a significant effect on the incidence of IVH (hazard ratio, 0.92; 95% CI, 0.49-1.73; P = .80). CONCLUSIONS AND RELEVANCE A large proportion of platelet transfusions were given to VLBW infants with PCT greater than 50 000/μL. Severity of illness influenced transfusion decisions. However, the severity of thrombocytopenia did not correlate with the risk for IVH, and platelet transfusions did not reduce this risk.