Can Red Blood Cell and Platelet Transfusions Have a Pathogenic Role in Bronchopulmonary Dysplasia?

OBJECTIVE: To evaluate whether transfusions in infants born preterm contribute to the pathogenesis of bronchopulmonary dysplasia (BPD). STUDY DESIGN: We conducted a multihospital, retrospective study seeking associations between red blood cell or platelet transfusions and BPD. We tabulated all transfusions administered from January 2018 through December 2022 to infants born ≤29 weeks or <1000 g until 36 weeks postmenstrual age and compared those with BPD grade. We performed a sensitivity analysis to assess the possibility of a causal relationship. We then determined whether each transfusion was compliant with restrictive guidelines, and we estimated effects fewer transfusions might have on future BPD incidence. RESULTS: Eighty-four infants did not develop BPD and 595 did; 352 developed grade 1 (mild), 193 grade 2 (moderate), and 50 grade 3 (severe). Transfusions were given at <36 weeks to 7% of those who did not develop BPD, 46% who did, and 98% who developed severe BPD. For every transfusion the odds of developing BPD increased by a factor of 2.27 (95% CI, 1.59-3.68; P < .001). Sensitivity analyses suggested that transfusions might contribute to BPD. Fifty-seven percent of red blood cell transfusions and 68% of platelet transfusions were noncompliant with new restrictive guidelines. Modeling predicted that complying with restrictive guidelines could reduce the transfusion rate by 20%-30% and the moderate to severe BPD rate by ∼4%-6%. CONCLUSIONS: Transfusions were associated with BPD incidence and severity. Lowering transfusion rates to comply with current restrictive guidelines might result in a small but meaningful reduction in BPD rates.

Endocytosis of the thrombopoietin receptor Mpl regulates megakaryocyte and erythroid maturation in mice.

Dnm2fl/fl Pf4-Cre (Dnm2Plt-/- ) mice lacking the endocytic GTPase dynamin 2 (DNM2) in platelets and megakaryocytes (MKs) develop hallmarks of myelofibrosis. At the cellular level, the tyrosine kinase JAK2 is constitutively active but decreased in expression in Dnm2Plt-/- platelets. Additionally, Dnm2Plt-/- platelets cannot endocytose the thrombopoietin (TPO) receptor Mpl, leading to elevated circulating TPO levels. Here, we assessed whether the hyperproliferative phenotype of Dnm2Plt-/- mice was due to JAK2 constitutive activation or to elevated circulating TPO levels. In unstimulated Dnm2Plt-/- platelets, STAT3 and, to a lower extent, STAT5 were phosphorylated, but their phosphorylation was slowed and diminished upon TPO stimulation. We further crossed Dnm2Plt-/- mice in the Mpl-/- background to generate Mpl-/-Dnm2Plt-/- mice lacking Mpl ubiquitously and DNM2 in platelets and MKs. Mpl-/- Dnm2Plt-/- platelets had severely reduced JAK2 and STAT3 but normal STAT5 expression. Mpl-/- Dnm2Plt-/- mice had severely reduced bone marrow MK and hematopoietic stem and progenitor cell numbers. Additionally, Mpl-/- Dnm2Plt-/- mice had severe erythroblast (EB) maturation defects, decreased expression of hemoglobin and heme homeostasis genes and increased expression of ribosome biogenesis and protein translation genes in spleen EBs, and developed anemia with grossly elevated plasma erythropoietin (EPO) levels, leading to early fatality by postnatal day 25. Mpl-/- Dnm2Plt+/+ mice had impaired EB development at three weeks of age, which normalized with adulthood. Together, the data shows that DNM2-dependent Mpl-mediated endocytosis in platelets and MKs is required for steady-state hematopoiesis and provides novel insights into a developmentally controlled role for Mpl in normal erythropoiesis, regulating hemoglobin and heme production.

Transfusion practices for pediatric oncology and hematopoietic stem cell transplantation patients: Data from the National Heart Lung and Blood Institute Recipient Epidemiology and Donor Evaluation Study-III (REDS-III).

BACKGROUND: To evaluate transfusion practices in pediatric oncology and hematopoietic stem cell transplant (HSCT) patients. STUDY DESIGN AND METHODS: This is a multicenter retrospective study of children with oncologic diagnoses treated from 2013 to 2016 at hospitals participating in the National Heart Lung and Blood Institute Recipient Epidemiology and Donor Evaluation Study-III. Transfusion practices were evaluated by diagnosis codes and pre-transfusion laboratory values. RESULTS: A total of 4766 inpatient encounters of oncology and HSCT patients were evaluated, with 39.3% (95% confidence interval [CI]: 37.9%-40.7%) involving a transfusion. Red blood cells (RBCs) were the most commonly transfused component (32.4%; 95% CI: 31.1%-33.8%), followed by platelets (22.7%; 95% CI: 21.5%-23.9%). Patients in the 1 to <6 years of range were most likely to be transfused and HSCT, acute myeloid leukemia, and aplastic anemia were the diagnoses most often associated with transfusion. The median hemoglobin (Hb) prior to RBC transfusion was 7.5 g/dl (10-90th percentile: 6.4-8.8 g/dl), with 45.7% of transfusions being given at 7 to <8 g/dl. The median platelet count prior to platelet transfusion was 20 × 109 /L (10-90th percentile: 8-51 × 109 /L), and 37.9% of transfusions were given at platelet count of >20-50 × 109 /L. The median international normalized ratio (INR) prior to plasma transfusion was 1.7 (10-90th percentile: 1.3-2.7), and 36.3% of plasma transfusions were given at an INR between 1.4 and 1.7. DISCUSSION: Transfusion of blood components is common in hospitalized pediatric oncology/HSCT patients. Relatively high pre-transfusion Hb and platelet values and relatively low INR values prior to transfusion across the studied diagnoses highlight the need for additional studies in this population.

Screening With Reticulocyte Hemoglobin Increased Iron Sufficiency Among NICU Patients.

INTRODUCTION: To increase the rate of iron sufficiency among neonatal intensive care unit (NICU) patients from 16% to >35% within 12 months of implementing standardized assessment of reticulocyte hemoglobin (retHE). METHODS: We implemented a quality improvement (QI) study to improve iron sufficiency in our out-born level III/IV NICU. We screened 2,062 admissions, of which 622 were eligible based on feeding status at discharge. QI interventions included educational efforts and guideline implementation. Our primary outcome measure was the percentage of patients with their discharge retHE measure within the normal range. We also tracked the process measure of the number of retHE tests performed and a balancing measure of the incidence of elevated retHE among patients receiving iron supplementation. Statistical process control (SPC) charts assessed for special cause variation. RESULTS: The percentage of patients with a retHe within the normal range was significantly increased from a mean of 20% to 39% on SPC chart analysis. We measured significantly more retHE values after guideline implementation (11/mo to 24/mo) and found no cases of elevated retHE among patients receiving iron supplementation. CONCLUSIONS: After the implementation of a standardized guideline, a higher rate of iron sufficiency was found in NICU patients at discharge. This work is generalizable to neonatal populations with the potential for a significant impact on clinical practice.

Thrombocytopenia is associated with severe retinopathy of prematurity.

Retinopathy of prematurity (ROP) is characterized by abnormal retinal neovascularization in response to vessel loss. Platelets regulate angiogenesis and may influence ROP progression. In preterm infants, we assessed ROP and correlated with longitudinal postnatal platelet counts (n = 202). Any episode of thrombocytopenia (<100 × 109/l) at ≥30 weeks postmenstrual age (at onset of ROP) was independently associated with severe ROP, requiring treatment. Infants with severe ROP also had a lower weekly median platelet count compared with infants with less severe ROP. In a mouse oxygen-induced retinopathy model of ROP, platelet counts were lower at P17 (peak neovascularization) versus controls. Platelet transfusions at P15 and P16 suppressed neovascularization, and platelet depletion increased neovascularization. Platelet transfusion decreased retinal of vascular endothelial growth factor A (VEGFA) mRNA and protein expression; platelet depletion increased retinal VEGFA mRNA and protein expression. Resting platelets with intact granules reduced neovascularization, while thrombin-activated degranulated platelets did not. These data suggest that platelet releasate has a local antiangiogenic effect on endothelial cells to exert a downstream suppression of VEGFA in neural retina. Low platelet counts during the neovascularization phase in ROP is significantly associated with the development of severe ROP in preterm infants. In a murine model of retinopathy, platelet transfusion during the period of neovascularization suppressed retinopathy.

Developmental differences between newborn and adult mice in response to romiplostim.

Thrombocytopenia is frequent among sick neonates. While most cases are transient, some neonates experience prolonged and severe thrombocytopenia. These infants often pose diagnostic and therapeutic challenges, and may receive large numbers of platelet transfusions. Romiplostim (ROM) is a thrombopoietin (TPO)-receptor-agonist approved for treatment of adults with chronic immune thrombocytopenia (ITP). The immature platelet fraction (IPF) is a novel measure of newly produced platelets, which could aid with the diagnostic evaluation of thrombocytopenic neonates. This study had the following two objectives: (1) compare the response of newborn and adult mice to escalating doses of ROM in vivo and (2) assess the correlation between IPF and megakaryocyte (MK) mass in newborn and adult treated and untreated mice. In the first set of studies, newborn (day 1) and adult mice received a single subcutaneous (SC) dose of ROM ranging from 0 to 300 ng/g, and platelet counts were followed every other day for 14 days. Both sets of mice responded with dose-dependent platelet and IPF increases, peaking on days 5-7 post-treatment, but neonates had a blunted response (2.1-fold compared to 4.2-fold maximal increase in platelet counts, respectively). On day 5 post-treatment with 300 ng/g ROM, MKs in the bone marrow (BM) and spleen of adult mice were significantly increased in numbers and size (p < 0.0001 for both) compared to controls. MKs in the spleen and BM (but not liver) of treated neonates also increased in number, but not in size. The immature platelet count (IPC, calculated as IPF x platelet count) was highly correlated with the MK number and size in neonatal and adult BM and spleen, but not neonatal liver. The lack of response of neonatal liver MKs was not due to a cell-intrinsic reduced responsiveness to TPO, since neonatal liver progenitors were more sensitive to murine TPO (mTPO) in vitro than adult BM progenitor. In vivo treatment of newborn mice with high mTPO doses or with higher doses of ROM (900 ng/g) resulted in peak platelet counts approaching 3-fold of controls. Taken together, our data indicate that newborn mice are less responsive to ROM than adult mice in vivo, due to a combination of likely pharmacokinetic differences and developmental differences in the response of MKs to thrombopoietic stimulation, evidenced by neonatal MKs increasing in numbers but not in size. PK/PD studies in human infants treated with ROM are warranted.

A de novo T73I mutation in PTPN11 in a neonate with severe and prolonged congenital thrombocytopenia and Noonan syndrome.

We observed a neonate with severe congenital thrombocytopenia and features of Noonan syndrome where evaluations were negative for immune-mediated thrombocytopenia, congenital infections, and Fanconi anemia. The marrow findings and a significantly elevated plasma thrombopoietin (Tpo) level were consistent with congenital amegakaryocytic thrombocytopenia; we sought a genetic mutation that could explain this phenotype. No mutations were identified in c-MPL (the Tpo receptor gene). Microarray analysis of peripheral blood did not reveal an abnormality. DNA sequencing of the PTPN11 gene showed a heterozygous C>T nucleotide substitution in exon 3 (c.218C>T) predicted to result in a threonine-to-isoleucine change at residue 73 (T73I). A 6-week trial of eltrombopag (an agonist of the Tpo receptor) failed to increase the platelet count. We propose this specific PTPN11 mutation results in abnormalities of the protein product SHP-2, which, because of its role in signal transduction, results in severe congenital thrombocytopenia refractory to c-MPL agonists.

Developmental differences in megakaryocyte size in infants and children.

Developmental differences in megakaryocytes between neonates and adults have been described. However, the age at which megakaryocytes make a transition to an adult phenotype is unknown. Small megakaryocytes are often described as "dysplastic" in the pathology literature. Thus, recognizing the normal features of megakaryocytes at different ages has diagnostic implications. We identified 72 samples from 61 patients, aged 3 days to 80 years, who had negative staging based on bone marrow examination. Megakaryocyte diameters, as highlighted with anti-CD61, were measured. A scatter plot of megakaryocyte size by age revealed a normal distribution of sizes at the youngest ages, with a shift to multiple peaks starting at 24 months indicating that neonates have megakaryocytes of uniform sizes, which diverge into separate clusters of smaller and larger cells beginning at 2 years; this is followed by an overall shift toward larger megakaryocytes at age 4 years. These observations have direct implications for the evaluation of bone marrow megakaryocytes in young children.

Differences between newborn and adult mice in their response to immune thrombocytopenia.

BACKGROUND: Sick neonates frequently develop severe thrombocytopenia. OBJECTIVE AND METHODS: In order to test the ability of fetal mice to increase their megakaryocyte size and ploidy in response to thrombocytopenia, we injected an antiplatelet antibody (MWReg30) into pregnant mice daily for 7 days, and into nonpregnant adult mice to serve as controls. After that time, platelet counts were obtained and megakaryocytes in the bone marrow, liver, and spleen were stained with anti-von Willebrand factor antibody, individually measured, and quantified. RESULTS: Our study demonstrated that megakaryocytopoiesis in newborn mice shares many features of human fetal/neonatal megakaryocytopoiesis, including the small size of megakaryocytes. In response to thrombocytopenia, adult mice increased megakaryocyte volume and concentration, primarily in the spleen. Newborn mice, in contrast, increased the megakaryocyte concentration in the spleen, but exhibited no increase in megakaryocyte volume in any of the organs studied. In fact, the megakaryocyte mass was significantly lower in the bone marrow of thrombocytopenic neonates than in age-matched controls. CONCLUSIONS: We concluded that fetuses have a limited ability to increase their megakaryocyte mass in response to consumptive thrombocytopenia, compared to adult mice. These observations provide further evidence for the existence of biological differences between fetal/neonatal and adult megakaryocytopoiesis.

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% x 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.

Platelet factor 4 is a negative autocrine in vivo regulator of megakaryopoiesis: clinical and therapeutic implications.

Platelet factor 4 (PF4) is a negative regulator of megakaryopoiesis in vitro. We have now examined whether PF4 regulates megakaryopoiesis in vivo by studying PF4 knockout mice and transgenic mice that overexpress human (h) PF4. Steady-state platelet count and thrombocrit in these animals was inversely related to platelet PF4 content. Growth of megakaryocyte colonies was also inversely related to platelet PF4 content. Function-blocking anti-PF4 antibody reversed this inhibition of megakaryocyte colony growth, indicating the importance of local PF4 released from developing megakaryocytes. The effect of megakaryocyte damage and release of PF4 on 5-fluorouracil-induced marrow failure was then examined. Severity of thrombocytopenia and time to recovery of platelet counts were inversely related to initial PF4 content. Recovery was faster and more extensive, especially in PF4-overexpressing mice, after treatment with anti-PF4 blocking antibodies, suggesting a means to limit the duration of such a chemotherapy-induced thrombocytopenia, especially in individuals with high endogenous levels of PF4. We found that approximately 8% of 250 healthy adults have elevated (> 2 times average) platelet PF4 content. These individuals with high levels of platelet PF4 may be especially sensitive to developing thrombocytopenia after bone marrow injury and may benefit from approaches that block the effects of released PF4.

Megakaryocyte size and concentration in the bone marrow of thrombocytopenic and nonthrombocytopenic neonates.

Thrombocytopenia is frequent among sick neonates, but little is known about its underlying mechanisms. It is known, however, that neonatal megakaryocytes are smaller and of lower ploidy than their adult counterparts and that smaller megakaryocytes produce fewer platelets than larger, more polyploid, megakaryocytes. We hypothesized that neonatal megakaryocytes would not increase their size in response to thrombocytopenia, thus limiting the ability of neonates to mount a response. To test this, we obtained marrow specimens from thrombocytopenic and nonthrombocytopenic neonates and adults. Megakaryocytes were immunohistochemically stained, quantified using an eyepiece reticle, and measured using an image analysis system with incorporated electronic micrometer. We found that, after adjusting for differences in cellularity, neonates and adults had similar megakaryocyte concentrations. When samples from the same sources were compared (tibial clot and vertebral body sections in neonates, iliac crest biopsies in adults), there were also no differences in megakaryocyte concentration between thrombocytopenic and nonthrombocytopenic subjects. The megakaryocyte diameter, however, was greater in adults than in neonates (19.4 +/- 3.0 versus 15.3 +/- 1.7 microm, p<0.0001). Thrombocytopenic adults also had a higher proportion of large megakaryocytes than nonthrombocytopenic adults (p<0.001). This was not observed among thrombocytopenic neonates, suggesting a developmental limitation in their ability to increase megakaryocyte size.

Differential effects of recombinant thrombopoietin and bone marrow stromal-conditioned media on neonatal versus adult megakaryocytes.

Umbilical cord blood (CB) is a valuable source of stem cells for transplantation, but CB transplantations are frequently complicated by delayed platelet engraftment. The reasons underlying this are unclear. We hypothesized that CB- and peripheral-blood (PB)-derived megakaryocytes (MKs) respond differently to the adult hematopoietic microenvironment and to thrombopoietin (Tpo). To test this, we cultured CB- and PB-CD34(+) cells in adult bone marrow stromal conditioned media (CM) or unconditioned media (UCM) with increasing concentrations of recombinant Tpo and compared the effects of these conditions on CB-versus PB-MKs. PB-MKs reached highest ploidy in response to UCM + 100 ng/mL rTpo, and the addition of CM inhibited their maturation. In contrast, CB-MKs reached highest ploidy in CM without rTpo, and high rTpo concentrations (> 0.1 ng/mL) inhibited their maturation. This is the first evidence that human neonatal and adult MKs have substantially different biologic responses to Tpo and potentially to other cytokines.