Background: Thrombocytosis (>500 × 103 platelets/μL blood) occurs in infants due to infection, inflammation, and/or anemia. Thrombocytosis and extreme thrombocytosis (EXT, >1000 × 103 platelets/μL blood) can present diagnostic dilemmas, sometimes prompting invasive testing and anticoagulation therapy. We previously identified heightened thrombocytosis rates in hospitalized infants versus older children, but this population largely excluded extremely preterm infants at increased risk for infections and anemia—factors that promote thrombocytosis. Objectives: To define thrombocytosis and EXT rates, etiologies, and sequelae among infants hospitalized in tertiary neonatal intensive care units (NICUs) to assist clinical decision making and determine factors that associate with thrombocytosis risk in preterm and full-term patients. Methods: Retrospective analysis of thrombocytosis and EXT cases among 20,818 infants hospitalized in 2 tertiary NICUs from 2011 to 2023, compared to 10,323 patients hospitalized at a quaternary NICU. Results: Our results revealed thrombocytosis in 3% of all patients (8% of preterm infants). Both estimates were significantly lower than the incidence of thrombocytosis in quaternary NICU patients (20%). EXT was also reduced in our tertiary unit (0.08% vs 0.5% in quaternary NICU). Thrombocytosis was associated with leukocytosis and relative anemia, but not with thrombotic or bleeding complications. Thyroid hormone, liver-derived thrombopoietin, and vitamin D deficiency can drive thrombocytosis in adults. Vitamin D level, but not thyroid hormone or liver function, was inversely correlated with platelet count among infants with thrombocytosis. Conclusions: Inflammation, anemia, and vitamin D level correlate with infant thrombocytosis and EXT. Liver and thyroid immaturity do not appear to impact thrombocytosis risk. There were no thrombotic complications associated with EXT. These results provide important context for interpreting the origins and appropriate clinical responses to thrombocytosis in preterm infants.

Objectives:Perinatal illness and preterm birth carry lifelong multiorgan complications and are associated with hematologic derangements during neonatal intensive care unit (NICU) admission. Despite this, long term hematologic morbidities following neonatal critical illness remain undefined. Our objective was to identify associations between prematurity, perinatal critical illness, and later hematologic dysfunction. Study design:Single neonatal care network retrospective cohort study with cohorts divided by gestational age and the presence of critical illness markers. The association between hematologic dysfunction, critical illness, and prematurity was investigated using multivariate logistic regression. Results:Among 13073 infants, critical illness or prematurity was found to increase the odds of developing pancytopenia post-NICU discharge. Subsequent analyses stratified on prematurity demonstrate that a diagnosis of shock or sepsis was associated with pancytopenia. Conclusions:Our findings suggest that perinatal insults are associated with hematopoietic system dysfunction and long term morbidity. Importantly, critical illness, not prematurity itself, may drive this association in preterm infants.

Processes that direct initial colonization and maturation of the bone marrow remain elusive, despite their importance to lifelong hematopoiesis. Bone marrow mesenchymal and stromal cell (BMSC) maturation must establish supportive hematopoietic niches prior to the recruitment and colonization of hematopoietic stem cells (HSCs). Here, we define the identity of murine BMSC progenitors and temporal nature of marrow colonization through a single cell atlas spanning late gestation through 18 months of age. We define progenitor cells and developmental trajectories for Cxcl12-abundant reticular (CAR) cells and osteoblasts within the marrow, including the emergence of direct and secreted signaling modalities that impact HSC quiescence and regenerative capacity. We further identify temporal changes in Early B Cell Factor (Ebf) 1-3 transcription factor expression and activity that correlate with niche establishment. These transcriptional activities relate to change to changes in systemic physiology, including metabolic, inflammatory, and hypoxic signaling, that direct CAR cell emergence after birth in mice. These findings provide unprecedented resolution to the colonization and maturation of the murine bone marrow environment.

Liberal transfusions of adult platelets increase preterm infant morbidity and mortality. This harm may be because of functional differences between neonatal and adult platelets. Preclinical murine models remain essential for investigating the underlying mechanisms. A prerequisite for developing and using these models is a cross-species comparison of developmentally regulated molecules in platelets. The objective of this study was to define proteins and biological pathways that differ between neonatal and adult platelets in mice and ascertain developmentally regulated molecules and pathways that are consistent across murine and human platelets. By comparing proteomes from resting murine and human platelets, we identified a consistent increase in inflammatory proteins in adult platelets across species, including β2M and CXCL12. Other markers for platelet function differed between species, including P-selectin, which was increased in adult murine platelets but did not differ with development in humans. To better elucidate developmentally regulated pathways across species, we used sparse principal component and machine learning-based approaches. These analyses revealed developmentally regulated growth factors, inflammatory signaling pathways, and metabolic changes that were consistent across species as well as some discrepant molecules and signaling pathways. Our results clarify molecular differences between neonatal and adult platelets with direct relevance for altered platelet reactivity and inflammatory functions. This approach helps bridge the gap between understanding animal models and human biology to investigate the impact of developmental differences in platelet biology on neonatal transfusion. These methods can be similarly used in other biological systems to improve the translatability of preclinical research.

Genome-wide association studies (GWAS) link the Tropomyosin 1 (Tpm1) locus to quantitative blood trait variation, but related mechanisms are unclear. Tpm1 encodes an actin-binding protein that regulates actin filament diversity, cell adhesion, signaling, and actomyosin contractility. Murine Tpm1 deficiency enhances hemogenic endothelial cell (HEC) specification, but it was unclear if these effects extended to postnatal hematopoiesis. We used Cdh5Cre and VavCre models to conditionally knock out Tpm1 (Tpm1KO) in endothelial anor hematopoietic cells. Both models ablate Tpm1 in postnatal blood. Endothelial Tpm1KO increases HEC specification without altering hematopoietic progenitor cell production or adult blood counts, suggesting separate roles for Tpm1 in the embryonic and adult blood systems. Tpm1KO increases adult platelet lifespan and diminishes adhesion to fibronectin and fibrinogen. Chemical Tpm1 inhibition also reduces focal adhesion in murine and human platelets. Altered platelet morphology and reduced platelet spreading suggest perturbed actomyosin contractility underlies these findings. Platelet fibrin binding promotes blood clot contraction, which reduces occlusive thrombosis. Tpm1KO limits clot contraction and worsens vascular occlusion in ferric chloride-induced stroke models. In addition to offering a mechanistic explanation for why genetic variation at the TPM1 alters platelet traits in GWAS, our findings reveal novel roles for Tpm1 in clot contraction and thrombosis.

Extreme thrombocytosis (EXT, >1000×10 3 platelets/μl blood) occurs in infants due to infection, inflammation, and/or anemia. EXT can present diagnostic dilemmas, sometimes prompting invasive testing and anticoagulation therapy. Our prior worked identified heightened EXT rates in hospitalized infants compared with older children, but this analysis largely excluded expreterm infants at increased risk for infections and anemia – factors that promote thrombocytosis and EXT. The objectives of this study were 1) to define EXT rates, etiologies, and sequelae among infants hospitalized in tertiary neonatal intensive care units (NICUs) to assist clinical decision-making and 2) to ascertain factors that drive thrombocytosis and EXT in preterm and full-term patients. Retrospective analysis of thrombocytosis (>500×10 3 platelets/μl) and EXT cases among 20,818 infants hospitalized in tertiary NICUs from 2011-2023 revealed thrombocytosis in 3% of all patients (8% of preterm infants). Both estimates were significantly lower than the incidence of thrombocytosis in pediatric patients in a quaternary NICU (15%). EXT rates were also markedly diminished in our tertiary unit (0.08% vs 0.5% in our quaternary NICU). Thrombocytosis was associated with leukocytosis and relative anemia, but not with thrombotic or bleeding complications. Vitamin D deficiency can drive thrombocytosis in adults and Vitamin D level was inversely corelated with platelet count among infants with thrombocytosis. Our findings suggest that Vitamin D supplementation among ex-preterm infants reduces thrombocytosis and EXT incidence, as opposed to developmental differences and/or organ immaturity in these patients. These results provide important context for clinical interpretations and responses to thrombocytosis in the preterm infant population.

Trisomy 21 (T21) is associated with baseline erythrocytosis, thrombocytopenia, neutrophilia, transient abnormal myelopoiesis (TAM), and myeloid leukemia of Down syndrome (ML-DS). TAM and ML-DS blasts harbor mutations in GATA1, resulting in the exclusive expression of the truncated isoform GATA1s. Germline GATA1s mutations in individuals without T21 cause congenital cytopenias, typically without a leukemic predisposition. To dissect the developmental effects of T21 and GATA1s, we used a combination of isogenic human induced pluripotent stem cells, primary human fetal and neonatal cells, and single-cell transcriptomics to interrogate hematopoietic progenitors differing only by chromosome 21 and/or GATA1 status. Both T21 and GATA1s induced early lineage skewing, and trajectory analysis revealed that GATA1s altered the temporal regulation of lineage-specific transcriptional programs, disrupting cell proliferation and maturation irrespective of chromosomal context. These studies uncovered unexpected heterogeneity and lineage priming in early, multipotent hematopoietic progenitors and identified transcriptional and functional maturation blocks linked to GATA1s.

Mammalian blood cells originate from specialized ‘hemogenic’ endothelial (HE) cells in major arteries. During the endothelial-to-hematopoietic transition (EHT), nascent hematopoietic stem cells (HSCs) bud from the arterial endothelial wall and enter circulation, destined to colonize the fetal liver before ultimately migrating to the bone marrow. Mechanisms and processes that facilitate EHT and the release of nascent HSCs are incompletely understood, but may involve signaling from neighboring vascular endothelial cells, stromal support cells, circulating pre-formed hematopoietic cells, and/or systemic factors secreted by distal organs. We used single cell RNA sequencing analysis from human embryonic cells to identify relevant signaling pathways that support nascent HSC release. In addition to intercellular and secreted signaling modalities that have been previously functionally validated to support EHT and/or developmental hematopoiesis in model systems, we identify several novel modalities with plausible mechanisms to support EHT and HSC release. Our findings paint a portrait of the complex inter-regulated signals from the local niche, circulating hematopoietic/inflammatory cells, and distal fetal liver that support hematopoiesis.

Platelet transfusions are frequently given to preterm infants to prevent bleeding, but randomized trials demonstrated harmful effects from current practices. Many platelet transfusions were administered in 15–20 mL/kg doses. We sought to decrease platelet exposure among neonates by standardizing 10 mL/kg transfusions for non-bleeding thrombocytopenic infants in a level IV NICU. We created evidence-based platelet dosing guidelines and changed practices in 3 plan-do-study-act cycles focused on education, reinforcement and electronic clinical decision support. We reviewed 240 transfusions over 3 years. The percentage of 10 mL/kg transfusions improved from 17.6% to 100%, without increasing major bleeding and repeat transfusion rates. Monthly transfused platelet volumes decreased from 2269 ± 334 mL to 857 ± 181 mL (p < 0.001), conserving limited platelet resources and saving $2746–$4942 per month in platelets. This study improved our platelet transfusion practices and can facilitate similar transfusion guideline adoption to benefit neonates at other institutions.

Stroke remains a leading cause of death and disability worldwide. Current antiplatelet and anticoagulant treatments are prone to failure. Heritable blood and platelet traits contribute to stroke risk, but related mechanisms are not fully understood. Platelets bind to damaged endothelial walls to initiate thrombosis, but erythrocyte and leukocyte recruitment are involved in stroke pathogenesis. We aimed to identify causal blood-related cells and mechanisms that modulate stroke risk. By two sample Mendelian Randomization (MR), increased platelet count heightened stroke risk (Odds ratio [OR] 1.03 per 1 SD unit increase in platelet count, P=1×10 -2 ). However, these effects were relatively weak and complicated by similar effects from erythrocyte and leukocyte traits. To ascertain key blood traits that influence stroke, we applied Bayesian Model Averaging (MR-BMA) and identified platelet count and mean platelet volume as key positive regulators for stroke risk. We validated an epidemiologic association between increased platelet count and higher stroke risk among a large patient cohort. Taken together, these findings indicate that platelet traits are the most critical risk factors for stroke, among analyzed blood cell traits. To deconvolute multiple underlying genetic mechanisms by which platelet traits impact stroke risk, we clustered platelet count variants using noise-augmented directional clustering (NAvMix). We identified 13 clusters, two of which were highly predictive for increased stroke risk (OR 1.31 per SD unit increase in platelet count, P<1×10 -6 ). Pathway analyses on eQTLs linked to variants in these subclusters indicated enrichment for endothelial cell adhesion or platelet reactivity. Colocalization analysis of stroke and platelet count loci identified genes implicated in platelet reactivity ( RIN3 ) and peroxisome biogenesis ( PEX6/PEX29 ). These findings reflect complex mechanisms underlying platelet trait variation and reveal key pathways that influence stroke risk through multiple cell types and biological mechanisms, including platelet biology and endothelial cell adhesion. An approach combining novel MR methods with subclustering may be a viable method to ascertain causal mechanisms related to other closely related exposure traits.

The balance of hematopoietic stem cell (HSC) self-renewal versus differentiation is essential to ensure long-term repopulation capacity while allowing response to events that require increased hematopoietic output. Proliferation and differentiation of HSCs and their progeny are controlled by the JAK/STAT pathway downstream of cytokine signaling. E3 ubiquitin ligases, like Cullin 5 (CUL5), can regulate JAK/STAT signaling by degrading signaling intermediates. Here we report that mice lacking CUL5 in hematopoietic cells (Cul5Vav-Cre) have increased numbers of hematopoietic stem and progenitor cells (HSPCs), splenomegaly, and extramedullary hematopoiesis. Differentiation in Cul5Vav-Cre mice is myeloid- and megakaryocyte-biased, resulting in leukocytosis, anemia, and thrombocytosis. Cul5Vav-Cre mice had increased HSC proliferation and circulation, associated with a decrease in CXCR4 surface expression. In bone marrow cells, we identified LRRC41 coimmunoprecipitated with CUL5, and vice versa, supporting that CRL5 forms a complex with LRRC41. We identified an accumulation of LRRC41 and STAT5 in Cul5Vav-Cre HSCs during IL-3 stimulation, supporting their regulation by CUL5. Whole-cell proteome analysis of HSPCs from Cul5Vav-Cre bone marrow identified upregulation of many STAT5 target genes and associated pathways. Finally, JAK1/2 inhibition with ruxolitinib normalized hematopoiesis in Cul5Vav-Cre mice. These studies demonstrate the function of CUL5 in HSC function, stem cell fate decisions, and regulation of IL-3 signaling.

In vitro hematopoiesis systems can be used to define mechanisms for blood cell formation and function, produce cell therapeutics, and model blood cell contributions to systemic disease. Hematopoietic progenitor cell (HPC) production remains inefficient, precluded by knowledge gaps related to specification and morphogenesis of specialized hemogenic endothelial cells, which undergo an endothelial-to-hematopoietic transition (EHT) to form HPCs. We elected to define changes in gene expression and chromatin organization during HPC formation to reveal regulatory mechanisms. Using paired single cell RNA/ATAC sequencing together with Hi-C, we profiled cells before and after EHT. Pathway analysis and pseudotime inferences confirmed a continuum of stromal and endothelial cells undergoing development into HE cells and lineage-based HPCs in vitro. In these cell types, we characterize cis-regulatory elements and transcriptional regulatory activities that facilitate EHT and HPC homeostasis, including for SNAI1, SOX17, TGFβ, STAT4, as well as for GFI1b and KLF1 in megakaryocyte- and erythroid-biased progenitors, respectively. We then leveraged our insights into chromatin organization among in vitro-derived cells to assess enrichments corresponding to human trait variation reported in human genome wide association studies. HPCs revealed locus enrichment for quantitative blood traits and autoimmune disease predisposition, which were particularly enriched in myeloid- and lymphoid-biased populations. Stromal and endothelial cells from our in vitro cultures were specifically enriched for accessible chromatin at blood pressure loci. Our findings reveal genes and mechanisms governing in vitro hematopoietic development and blood cell-related disease pathology.

Down syndrome, or Trisomy 21 (T21), is uniquely associated with transient abnormal myelopoiesis (TAM) and myeloid leukemia of Down syndrome (ML-DS). Both TAM and ML-DS harbor mutations in the erythro-megakaryocytic transcription factor GATA1 that lead to exclusive expression of GATA1s, a truncated isoform lacking the amino terminus. Consistent with the megakaryocytic phenotype and gene signature of TAM and ML-DS, we found that hematopoietic differentiation of human induced pluripotent stem cells (iPSCs) with T21 and GATA1s display absent erythropoiesis but hyperproliferation of megakaryocytes and cytokine independence. Single-cell RNA sequencing of iPSC-derived hematopoietic progenitor cells (HPCs) revealed that T21 and GATA1s drive early commitment to the megakaryocyte lineage and block maturation, recapitulating a leukemic phenotype. To investigate the mechanisms underlying these diseases, we combined bulk RNA sequencing, ATAC-seq (assay for transposase-accessible chromatin with sequencing), and CUT&RUN (cleavage under targets and release using nuclease) for GATA1 in T21 HPCs expressing wtGATA1 vs. GATA1s. Multipotent CD41+235+ HPCs were differentiated and flow-purified from isogenic T21/wtGATA1 and T21/GATA1s iPSCs. RNA sequencing of these HPCs identified 1,292 differentially expressed genes (|log2FC| >0, adjusted p <0.05). Gene set enrichment analysis (GSEA) revealed enrichment of cell cycle pathways in T21/GATA1s HPCs, including G2/M checkpoint, E2F targets, and MYC targets. Parallel ATAC-seq identified 19,277 differentially accessible regions (adjusted p <0.05) in T21 HPCs expressing wtGATA1 vs. GATA1s. GSEA of differentially expressed genes with corresponding changes in chromatin accessibility again revealed positive enrichment of G2/M checkpoint genes, E2F targets, and MYC targets as well as negative enrichment of heme metabolism, consistent with the cytokine-independent hyperproliferation of megakaryocytes we observed. To determine whether GATA1s chromatin occupancy drives these transcriptional changes, we performed CUT&RUN for GATA1 in CD41+235+ HPCs and CD41+42b+ megakaryocytes differentiated from T21/wtGATA1 and T21/GATA1s iPSCs. In HPCs, GATA1s binding was globally decreased; compared to wtGATA1, 2,340 regions had decreased occupancy whereas only 62 regions had increased binding (false discovery rate ≤0.05). Genes with decreased GATA1s occupancy, chromatin accessibility, and expression in T21/GATA1s HPCs included CCND1 and CDKN1A, both of which inhibit cell cycle progression in G1 phase and play critical roles in the RB pathway. Notably, GATA1 itself plays an important role in this pathway through the GATA1-RB-E2F complex, which represses E2F to control cell proliferation, but GATA1s lacks the motif to form this complex. In stark contrast to the HPCs, GATA1s occupancy was globally increased in T21/GATA1s megakaryocytes (10,299 regions with increased occupancy compared to 279 regions with decreased occupancy; false discovery rate ≤0.05), including at both canonical megakaryocytic and erythroid loci. The essential megakaryocyte gene RUNX1 demonstrated increased chromatin accessibility and GATA1s occupancy, suggesting its role in the megakaryocyte bias and proliferation we observed with GATA1s. Taken together, our results suggest that GATA1s in T21 HPCs simultaneously promotes cell proliferation and megakaryopoiesis, yielding hyperproliferative, immature megakaryocytes due to absence of GATA1s binding in HPCs and inappropriate persistence of GATA1s at key loci in megakaryocytes. Our findings recapitulate the hallmarks of malignant transformation and offer mechanistic insights into the progression from T21 to TAM and ML-DS.

An inverse correlation between stature and risk of coronary artery disease (CAD) has been observed in several epidemiologic studies, and recent Mendelian randomization (MR) experiments have suggested causal association. However, the extent to which the effect estimated by MR can be explained by cardiovascular, anthropometric, lung function, and lifestyle-related risk factors is unclear, with a recent report suggesting that lung function traits could fully explain the height-CAD effect. To clarify this relationship, we utilized a well-powered set of genetic instruments for human stature, comprising >1,800 genetic variants for height and CAD. In univariable analysis, we confirmed that a one standard deviation decrease in height (~6.5 cm) was associated with a 12.0% increase in the risk of CAD, consistent with previous reports. In multivariable analysis accounting for effects from up to 12 established risk factors, we observed a >3-fold attenuation in the causal effect of height on CAD susceptibility (3.7%, p = 0.02). However, multivariable analyses demonstrated independent effects of height on other cardiovascular traits beyond CAD, consistent with epidemiologic associations and univariable MR experiments. In contrast with published reports, we observed minimal effects of lung function traits on CAD risk in our analyses, indicating that these traits are unlikely to explain the residual association between height and CAD risk. In sum, these results suggest the impact of height on CAD risk beyond previously established cardiovascular risk factors is minimal and not explained by lung function measures.

During development, erythroid cells are produced through at least 2 distinct hematopoietic waves (primitive and definitive), generating erythroblasts with different functional characteristics. Human induced pluripotent stem cells (iPSCs) can be used as a model platform to study the development of red blood cells (RBCs) with many of the differentiation protocols after the primitive wave of hematopoiesis. Recent advances have established that definitive hematopoietic progenitors can be generated from iPSCs, creating a unique situation for comparing primitive and definitive erythrocytes derived from cell sources of identical genetic background. We generated iPSCs from healthy fetal liver (FL) cells and produced isogenic primitive or definitive RBCs which were compared directly to the FLderived RBCs. Functional assays confirmed differences between the 2 programs, with primitive RBCs showing a reduced proliferation potential, larger cell size, lack of Duffy RBC antigen expression, and higher expression of embryonic globins. Transcriptome profiling by scRNA-seq demonstrated high similarity between FLand iPSC-derived definitive RBCs along with very different gene expression and regulatory network patterns for primitive RBCs. In addition, iPSC lines harboring a known pathogenic mutation in the erythroid master regulator KLF1 demonstrated phenotypic changes specific to definitive RBCs. Our studies provide new insights into differences between primitive and definitive erythropoiesis and highlight the importance of ontology when using iPSCs to model genetic hematologic diseases. Beyond disease modeling, the similarity between FLand iPSCderived definitive RBCs expands potential applications of definitive RBCs for diagnostic and transfusion products.

Induced pluripotent stem cell (iPSC)-based models are excellent platforms to understand blood development, and iPSC-derived blood cells have translational utility as clinical testing reagents and transfusable cell therapeutics. The advent and expansion of multiomics analysis, including but not limited to single nucleus RNA sequencing (snRNAseq) and Assay for Transposase-Accessible Chromatin sequencing (snATACseq), offers the potential to revolutionize our understanding of cell development. This includes developmental biology using in vitro hematopoietic models. However, it can be technically challenging to isolate intact nuclei from cultured or primary cells. Different cell types often require tailored nuclear preparations depending on cellular rigidity and content. These technical difficulties can limit data quality and act as a barrier to investigators interested in pursuing multiomics studies. Specimen cryopreservation is often necessary due to limitations with cell collection and/or processing, and frozen samples can present additional technical challenges for intact nuclear isolation. In this manuscript, we provide a detailed method to isolate high-quality nuclei from iPSC-derived cells at different stages of in vitro hematopoietic development for use in single-nucleus multiomics workflows. We have focused the method development on the isolation of nuclei from iPSC-derived adherent stromal/ endothelial cells and non-adherent hematopoietic progenitor cells, as these represent very different cell types with regard to structural and cellular identity. The described troubleshooting steps limited nuclear clumping and debris, allowing the recovery of nuclei in sufficient quantity and quality for downstream analyses. Similar methods may be adapted to isolate nuclei from other cryopreserved cell types.

Trisomy 21 (T21) is associated with hematopoietic abnormalities including polycythemia, thrombocytopenia, transient abnormal myelopoiesis (TAM), and myeloid leukemia of Down syndrome (ML-DS). GATA1s, the truncated isoform of GATA1, is required for TAM and ML-DS pathogenesis, but causes cytopenias without leukemia in the absence of T21. To investigate how T21 and GATA1s each impact hematopoiesis, we performed single-cell RNA sequencing (scRNA-seq) of induced pluripotent stem cell (iPSC)-derived hematopoietic progenitor cells (HPCs) differing only by chromosome 21 and/or GATA1 status.HPCs were differentiated from 4 isogenic human iPSC lines: T21/wild-type GATA1 (WT), T21/GATA1s, euploid/WT, and euploid/GATA1s. CD41+235+ multipotent HPCs on Day 7 (D7) and lineage-biased HPCs on Days 9 (D9) and 11 (D11) were collected for scRNA-seq. Cluster annotation identified HPCs, megakaryocyte (MK)- or erythroid-biased HPCs, and erythroid, MK, and myeloid cells. Although GATA1s impairs erythropoiesis, T21/GATA1s HPCs showed an early erythroid bias that diminished by D11; euploid/GATA1s cells had strong MK and myeloid skews throughout. Both T21/WT and euploid/WT cells differentiated into all 3 lineages.Trajectory analysis revealed that T21 and GATA1s each impact lineage maturation. GATA1s delayed maturation of both euploid and T21 MKs. T21/GATA1s erythroid cells were less mature than T21/WT; there were insufficient euploid/GATA1s erythroid cells for analysis. GATA1s in T21 myeloid cells delayed maturation, but in euploid cells had no consistent effect across the 3 timepoints. T21/WT MKs and myeloid cells also appeared to be less mature than their euploid counterparts. Analysis of lineage-specific driver genes showed inappropriate expression levels with T21 or GATA1s relative to euploid/WT, suggesting that the impaired maturation is due to dysregulation of transcriptional programs.

Neonates and infants hospitalized in the neonatal intensive care unit (NICU) frequently require blood product transfusions, but clinical practices vary widely. Many very low birth weight (VLBW) infants receive packed red blood cell (RBC) or platelet transfusions during their initial NICU stay, with incidence inversely proportional to gestational age at birth.1, 2 A recent study estimated that in infants less than 27 weeks gestation, 70% received RBCs, 34% received platelets, and 24% received plasma to promote coagulation during their NICU admission.3-5 These blood product transfusions are most often prophylactic, with clinical decisions made in response to numeric blood count values, as opposed to therapeutic transfusions in the context of active bleeding. Emerging evidence has suggested that some transfusion practices are harmful for certain NICU patients, such as platelet transfusions in preterm infants.6, 7 More broadly, transfusion reactions can occur with virtually all blood products.8 Although rare in the neonatal population,9 these reactions may be under-diagnosed, under-estimated, and under-reported in pediatric patients3 and some papers report that rates may be higher than in adult populations.9 Our intention was to establish optimal transfusion guidelines for our division and neonatal intensive care network, including 19 hospitals, based on a review of currently available literature. The terms "neonates" and "infants" are used throughout this manuscript to describe any patients hospitalized in the NICU. We identified blood product transfusion guidelines as a topic that could be amenable to significant quality improvement within our neonatal intensive care network. The modified Delphi method is a systematic, iterative approach geared toward developing consensus among a group of expert stakeholders.10, 11 This approach can be used to help form consensus in areas with divergent evidence base or when expert opinion is utilized. We used this method to identify and implement opportunities to align our transfusion practices with existing literature to optimize patient outcomes. We first defined the scope and specific aims of this consensus project, and then assigned neonatologists to small groups focused on specific topics. The groups reviewed and graded existing literature, and identified areas where a lack of evidence precluded clear recommendations. Each group developed literature-based guidelines and recommendations, which were shared within the broader neonatal care network. To assess agreement and consensus opportunities based on these guidelines, we posed questions to our neonatal care network providers. We inferred consensus when answers exceeded 7 on a Likert scale of 1 through 9 (1 being least likely and 9 being most likely). Our final consensus guidelines were presented to our Division of Neonatology in an open question-and-answer period. Agreement was sought, at times with live polling during the presentation. Following the larger group discussion, we edited our guidelines and distributed our finalized consensus guidelines among all network sites. We then assessed implementation of these guidelines and perspectives across our network 6 months after finalization. Platelets facilitate hemostasis and have important roles in inflammation, immunity, and vascular biology.12 Thrombocytopenia, defined as a platelet count less than 150,000 platelets/μL blood, most often occurs secondary to infection or other systemic pathology in NICU patients.13 Platelet transfusions are given most frequently to prevent major bleeding in thrombocytopenic patients, rather than in response to active bleeding.14 VLBW neonates are at increased risk for thrombocytopenia and are frequently transfused with platelets in the first 7 days of life.15 Platelet transfusion practices vary widely. Historically, clinical decision-making has been driven by concerns of increased risk of intraventricular hemorrhage (IVH) or other forms of major bleeding in thrombocytopenic infants. Platelet transfusion thresholds vary by country and institution, but have often been ~50,000/μL or higher for patients with critical illness.15 While most studies have refuted a correlation between thrombocytopenia severity and risk of IVH,15 at least one cohort study demonstrated increased IVH with lower platelet count nadirs.16 The majority of neonates with severe thrombocytopenia (platelet count <50,000 platelets/μL blood) do not have any episodes of major hemorrhage (e.g., Grade 3 or 4 IVH, pulmonary hemorrhage, or abdominal hemorrhage),14 and most neonates with significant IVH had bleeding prior to developing severe thrombocytopenia. Several trials have investigated appropriate platelet transfusion thresholds in NICU patients.6, 17, 18 Most recently, the Platelets for Neonatal Transfusion Study 2 (PlaNeT-2) was a randomized controlled trial that found a significantly higher risk of death or major bleeding in neonates transfused at a higher threshold (50,000 platelets/μL) than those transfused at a lower threshold (25,000 platelets/μL).6 The higher transfusion threshold group also had an increased risk of developing bronchopulmonary dysplasia (BPD) and a lower probability of discharge home by 38 weeks corrected gestational age.6 These effects persisted regardless of patient bleeding or mortality risk stratification.7 At 2-year follow-up, those randomized to higher transfusion thresholds were at increased risk for the composite rate of death and neurodevelopmental impairment (NDI), as defined by cerebral palsy (CP), global developmental delay (GDD), hearing impairment, or vision impairment.19 Patients in the high threshold group also had increased risk of requiring oxygen or respiratory support at 2 years of age.19 These findings support lower platelet transfusion thresholds (~25,000 platelets/μL) for NICU patients regardless of perceived bleeding risk. When a prophylactic platelet transfusion threshold of 25,000/μL was implemented in a tertiary and quaternary referral center NICU, platelet transfusions decreased overall.1 The biggest reduction in platelet transfusions was noted in non-bleeding, critically ill neonates. Importantly, there was no change in the incidence of IVH and a significant decrease in other major bleeding complications after the lower platelet transfusion threshold was introduced. Platelet transfusion dosing recommendations were also considered. Prior work has demonstrated no differences in efficacy between 10 and 15 mL/kg platelet transfusions.20-22 Several neonatal studies routinely administered 15 mL/kg platelets, including recent large clinical trials.6 However, concerns have been raised about the potential detrimental effects of this dose on neonatal physiology.12 A recent quality improvement effort demonstrated efficacy of 10 mL/kg platelet transfusions over 2 h with no change in major bleeding occurrence.1 In sum, there is no conclusive evidence of benefit for platelet transfusions at currently used thresholds in the prevention of major bleeding.23 Additionally, there are several pathophysiologic mechanisms by which platelet transfusions could harm NICU patients. Neonatal platelets are distinctly different from adult platelets in terms of reactivity to chemical agonists and in protein content.12, 24 Thus, adult platelet transfusions may disrupt normal hemostatic balance, inflammatory mediators, and/or fluid shifts in otherwise vulnerable neonatal patients.12 These potentially detrimental effects of platelets are important considerations for clinical decision-making regarding platelet transfusion indication and dosage, although these concerns may be balanced with possible benefits of transfusions given sparse evidence overall. We modeled our platelet transfusion guidelines on recent landmark studies and consensus guidelines used successfully by other institutions, distinguishing between bleeding and non-bleeding neonates and including considerations for clinical situations that may warrant higher transfusion thresholds (Table 1). For the non-bleeding neonate, we recommend platelet transfusion when the platelet count is <25,000/μL, with exceptions (Table 1). For the bleeding neonate, we recommend platelet transfusion if the platelet count is <50,000–100,000/μL. If the platelet count is >100,000/μL, the patient should be evaluated for other potential causes of bleeding. Stable—No transfusion Consider Transfusion if Transfuse Anemia of prematurity, transient erythroblastopenia of childhood (reticulocytopenia seen with the physiologic nadir), and iatrogenic blood loss are common etiologies prompting red blood cell (RBC) transfusions in NICU patients. A majority of infants born extremely prematurely are given RBC transfusions during their initial NICU stay.25 As with platelet transfusions, packed RBC transfusion practices vary widely across hospitals and even between physicians at the same institution. Indeed, we identified practice variations within our own network at the inception of this study. Several investigations have compared how liberal vs restrictive RBC transfusion thresholds impact clinical outcomes in NICU patients, which have varied among studies.26-29 Virtually, all studies found a significant decrease in RBC transfusions in the restrictive transfusion groups, in which lower hemoglobin levels were well tolerated.26, 27, 29 Despite administering fewer transfusions, there were only subtle differences in blood donor exposure since patients often received RBC aliquots from the same donor unit. One study showed no difference in donor exposures,26 while another identified fewer donor exposures in the restrictive transfusion group.29 In general, these studies have found that lower hemoglobin does not adversely impact complications of prematurity, including risks of IVH, periventricular leukomalacia (PVL), retinopathy of prematurity (ROP), bronchopulmonary dysplasia (BPD), apnea of prematurity (AOP), patent ductus arteriosus (PDA), necrotizing enterocolitis (NEC), intestinal perforation, culture-proven sepsis, clinical sepsis, pneumonia, or poor growth. When IVH, PVL, ROP, BPD, time on a ventilator, time on supplemental oxygen, time to regain birth weight, time to double birth weight, weight at 36 weeks postmenstrual age, and length of stay were evaluated separately, there was no significant difference in the liberal or restrictive transfusion groups.26-29 However, one study reported an increase in combined Grade 4 IVH and PVL outcome, and increased apneic events per day in the restrictive group.26 These differences were not observed in more recent studies, nor were there detectable differences in time requiring caffeine.27, 28 Outcomes at 18- and 24-months corrected age did not differ with regard to death and/or NDI between liberal and restrictive transfusion threshold groups.27, 28, 30 Together, these landmark studies support restrictive transfusion thresholds as being safe for NICU patients. It is possible that limitations and imprecision of using hemoglobin level as a proxy for tissue oxygen delivery can, at least in part, explain these observations. Our transfusion guidelines are based on thresholds reported in the most recent report by Kirpalani et al.,27 and includes stratification by critical vs non-critical illness as defined by being on ≥4 L high flow nasal cannula (HFNC) for respiratory support and the week of life (Table 2). There has been variation in RBC transfusion dosing among prior studies. Most trials administered 15 mL/kg RBCs per transfusion,26, 27, 29 but at least one recent trial provided 20 mL/kg RBCs.28 Based on these trials, our consensus guidelines recommend transfusion volumes of 15–20 mL/kg (Table 2). Attention to potential circulatory overload may be prudent when transfusing higher volumes. Lower transfusion volumes (10–15 mL/kg) can also be recommended at the discretion of the neonatologist. We administer all transfusions within the expiration period, which is up to 4 h. Blood supplies, additive solutions, and handling procedures vary across hospitals and hospital systems. While we cannot comment on particular additive solutions herein, we have found that most blood suppliers leukoreduce blood products pre-storage and that many pediatric institutions employ universal irradiation strategies. Our neonatal care network provides irradiated and leukoreduced products for all RBC transfusions. While RBC transfusions are necessary for many preterm neonates, numerous studies have investigated potential adverse consequences on neonatal physiology and disease pathogenesis.31-36 These concerns have largely centered on NEC, and clinical controversy continues to prompt prospective clinical studies in addition to publications spanning decades. One specific clinical concern is that RBC transfusions may lead to mesenteric ischemia, putting neonates at increased risk for NEC 48 to 72 h following a blood transfusion. While some studies have argued against changes in splanchnic oxygenation during RBC transfusions, this remains a matter of debate.31 Some clinicians have attempted to mitigate intestinal risks by withholding feeds during RBC transfusions. While studies comparing the incidence of transfusion-associated NEC before and after implementing policies to withhold feeding during and after RBC transfusions have shown a trend toward increased NEC risk, none of the studies individually reached statistical significance.32, 33 Although severe anemia may predispose to NEC,34-36 there has not been clear evidence linking RBC transfusions to an increased incidence of NEC (e.g., transfusion-associated NEC). Thus, there is insufficient evidence at this time to recommend withholding feeds during RBC transfusions, although ongoing trials aim to further evaluate this subject.37 For further information about the Wheat trial, readers are directed to NCT05213806.37 We surveyed our neonatal network providers to assess current practices (n = 102 respondents). We found that 28% of responding clinicians currently hold feeds during RBC transfusions, despite widespread opinion for a lack of strong evidence supporting this practice (88%). Of individuals who currently hold feeds during RBC transfusions, 76% reported a willingness to change practice based on updated guidelines and current literature. Of those who already continue enteral feeds during RBC transfusions, 77% offer full volume feeds or continue feeds at pre-transfusion rates. Most respondents (82%) did not consider the degree of anemia when deciding on feeding practices during RBC transfusion. Given the lack of strong evidence supporting transfusion associated NEC, our guidelines recommend continuing feeds at pre-transfusion rates during and after RBC transfusions (Table 2). Plasma contains coagulation factors and is typically indicated for bleeding with a coagulopathy. There have not been randomized controlled trials to directly evaluate the efficacy of plasma, coagulopathy, and bleeding risk in neonates.4 Nonetheless, neonates (particularly those born at <34 weeks gestation and critically ill) are given plasma more frequently than other pediatric patient groups.3 Neonatal plasma transfusions are often given prophylactically for abnormal laboratory values in patients without bleeding.3, 4 Indeed, a retrospective analysis found that approximately half of all neonatal plasma transfusions were administered empirically for abnormal coagulation studies without hemorrhage.38 It is important to note the paucity of evidence to support prophylactic plasma transfusions for coagulopathy without active bleeding. Prophylactic plasma transfusion also does not prevent future episodes of hemorrhage.3 Over the past 15 years, there has been a substantial decrease in neonatal plasma transfusions for abnormal coagulation laboratory tests without a concomitant change major bleeding.38 In the same time period, studies have shown no benefits for plasma administration in several clinical contexts. For example, infants receiving plasma in the setting of disseminated intravascular coagulation (DIC) had similar outcomes to patients not receiving any treatment.39 Similarly, there were no differences in short term outcomes, including immunologic responses to sepsis, following plasma administration in non-bleeding patients.40 Taken together, these findings offer reassurance that limited use of plasma transfusions in neonates is not associated with increased bleeding. Perhaps most concerning are studies associating plasma transfusions with harm, including increased risks of pulmonary hemorrhage and venous thrombosis,3 increased mortality,41, 42 or no benefit.43 One study reported an increase in IVH in infants who received at least one plasma transfusion (although these results did not meet statistical significance).44 Plasma transfusions in older children have been independently associated with organ dysfunction, infection, and prolonged length of hospitalization.8 Administration of 10–15 mL/kg of plasma might be expected to raise coagulation factors by 10%–15%.3, 4, 8 However, studies have shown inconsistent effects on coagulation studies following plasma transfusion, with some reporting no change in coagulation testing.3 This may be due to differences in the hemostatic systems of neonates versus older children.45 While we recommend against routine evaluation of coagulation studies in neonates, interpreting these results requires consideration of both gestational and postnatal age. Normal coagulation test values change rapidly in the neonatal period and are dependent on gestational age at delivery.46 Laboratory thresholds for coagulopathy would be PT and/or aPTT levels elevated to >2× normal references for age.46 We developed guidelines based on judicious use of 5–10 mL/kg plasma over 1 h that align with current expert opinion to transfuse patients with active bleeding, or prior to invasive procedures in coagulopathic patients likely to experience bleeding (Table 3). We defined such at-risk neonates as (1) neonates with active bleeding or uncontrolled oozing, (2) neonates with hypoxic–ischemic encephalopathy undergoing therapeutic hypothermia with bleeding or oozing, and (3) presurgical neonates with active bleeding. We recommend against empiric transfusions in response to abnormal laboratory values without bleeding or coagulopathy prior to invasive procedures. If coagulation studies are obtained, we recommend basing clinical decisions to transfuse plasma for at-risk neonates in the absence of bleeding only if studies are >2× normal values for age. Several retrospective studies also identified plasma administration in response to clinical concern for hypovolemia or hypotension in neonates, as opposed to specific concerns for coagulopathy.3 This practice is concerning, since plasma is a blood product with adverse transfusion reaction risks. Plasma is not indicated for volume expansion and we recommend against its use for this purpose (Table 3). To address volume expansion or resuscitation, we recommend isotonic crystalloid infusions. Isotonic fluids are superior to hypotonic fluids in preventing hyponatremia, with customized fluid solutions superior in preventing electrolyte disturbances in pediatric patients.47 Colloid fluids (e.g., 5% albumin—another blood derived product) have not shown superiority to crystalloids.47 Cryoprecipitate is a concentrated plasma derivative rich in factor VIII, XIII, von Willebrand Factor, fibronectin and fibrinogen. Cryoprecipitate can be used in neonates for bleeding, typically in the setting of a low fibrinogen level. Each bag of cryoprecipitate is 15 to 20 mL and contains 100 to 250 mg of fibrinogen, although this may significantly understate the fibrinogen concentration.48, 49 Pediatric dosing of cryoprecipitate is 1 to 2 bags per 10 kg of body weight to raise the fibrinogen level 60–100 mg/dL. For infants, a single unit of cryoprecipitate is a standard dose to achieve hemostasis which is equivalent to approximately 5 to 10 mL/kg.50 While there is insufficient evidence to recommend therapeutic or prophylactic cryoprecipitate transfusions in neonates and infants, our clinical guidelines outline the potential use of 5 mL/kg transfusions in actively bleeding patients with low fibrinogen or active therapeutic thrombolysis as clinically warranted4 (Table 3). While some products may be subject to supplier restrictions, these issues are often hospital-specific and outside the scope of this current recommendation. Neonates and infants are vulnerable patient populations and emerging studies have shown evidence for harm associated with some current blood product transfusion practices.3, 6, 8, 17, 18 Recent trials described herein have refined evidence-based practice for blood product transfusions in neonates. We developed transfusion guidelines for neonates and infants based on current evidence and implemented these network-wide to improve transfusion practice and safety. We surveyed providers across our 19-hospital neonatal network 6 months after finalizing the guidelines. We received 72 completed responses. We found that 89% of respondents were familiar with the new guidelines and stated they were following them, with 57% reporting a change in transfusion practices. On the survey, the most frequent changes were to transfuse at more restrictive thresholds, continuing feeds during transfusions, and transfusing lower volumes of platelets. Half of respondents (50%) stated they had a change in mindset toward transfusions, including anecdotal comments reflecting providers no longer believed transfusions to be benign, and that providers were more restrictive in transfusion administration in accordance with the guidelines. We anticipate that future trials will further clarify recommendations for specific at-risk patient populations (e.g., for extremely premature infant platelet transfusion thresholds, infants on ECMO, or those requiring massive transfusion protocols) to further refine transfusion guidelines that optimize care for neonates and infants. This project was supported through grants from the National Institutes of Health (HL156052 to CST) and the Hartwell Foundation (CST). The authors have disclosed no conflicts of interest.

Neonatal and adult megakaryocytes differ in proliferative capacity and ploidy levels, and neonatal and adult platelets differ in function, gene expression, and protein content. The mechanisms underlying these differences are incompletely understood. CDK8 and CDK19 are transcriptional kinases part of the CDK-mediator complex, which regulates gene transcription in a cell-specific manner. We discovered that cortistatin A, a potent highly selective inhibitor of CDK8/CDK19, significantly reduced cell expansion and increased ploidy in cord blood-derived megakaryocytes. These phenotypic changes were associated with gene expression changes that partially overlapped developmentally regulated genes. These findings might have relevance for the management of developmental megakaryocyte disorders.