Personalizing Preterm Neonatal Transfusions With Fetal Hemoglobin-Enriched Cord Blood

Specific AIM 1 To assess whether CB‐RBC transfusions can offer a protective advantage ofCB‐RBC transfusions in preterm newborns by preserving HbF, reducing the risk of oxidativestress‐related complications, and potentially lowering the incidence of 'free radicaldiseases of prematurity' .Biomarkers of oxidative stress will be correlated with short term outcome of the newborns(diagnosis at discharge). Moreover with the aim to evaluate the social, economic,organizational implications of this intervention the investigators will perform an HealthImpact Assessment (HIA).The goal is to provide policymakers, healthcare providers, and stakeholders withevidence‐based insights on the effectiveness, cost‐effectiveness, safety, and potentialimpact of CB‐RBC transfusions.Specific AIM 2 To evaluate pathways involved in inflammatory and hypoxic‐hyperoxicprocesses through microRNAs expression. OS cascade comprises the activation of microglia,infiltration of macrophages and release of pro‐inflammatory mediators such as cytokines,chemokines, nitric oxide, free radicals. MicroRNAs, (miRs), play an important role in theregulation of OS modulated genes, with the potentiality to induce protective proteinsynthesis such as neuroglobin and sirtuin. Specifically, miRNA 107 e 17‐5p, involved inangiogenesis and arterial remodeling are upregulated; miRNA 223‐3p associated withchronic kidney disease is downregulated.Specific AIM 3 To assess the protective role CB‐RBC transfusions in newborns, byperforming neurodevelopmental outcome of enrolled babies through clinical andneurobehavioral evaluations at 3‐6‐9‐12‐month of age.Experimental Design Aim 1 This study is designed as a multicenter, randomized,controlled, double‐blind trial.Preterm neonates born between 24+0 and 31+6 weeks of gestational age who require at leastone red blood cell transfusion during hospitalization will be randomized in a 1:1 ratioto receive either standard A‐RBCs (control group) or CB‐RBCs (intervention group).Randomization will be stratified by gestational age and study center and conducted usinga centralized computer generated system. Treating physicians, caregivers, and outcomeassessors including clinical, laboratory, and imaging personnel will be blinded totreatment allocation.‐‐Interventions: All transfusions will follow uniform clinical guidelines and thresholdsfor transfusion, aligned with current Italian neonatal transfusion standards. Theexperimental intervention involves transfusion with CB‐RBC concentrates prepared fromcord blood units donated to public cord blood banks. These units are leukodepleted usingBioR Flex filters, fractionated via Compomat G5 cell separators, suspended in SAG‐Madditive solution, and stored in DEHP‐free pediatric bags. All units will begamma‐irradiated before administration and transfused within 24 hours of irradiation.Safety screening will include infectious disease testing (HIV, HBV, HCV, syphilis,bacterial and fungal cultures), identical to the procedures for adult donor blood.‐‐Sample Size: Based on an expected 50% incidence of the composite endpoint in thecontrol group and a hypothesized reduction to 30% in the CB‐RBC group (absolute riskreduction = 20%), a one‐sided Chi‐square test with alpha = 0.05 and 80% power requires 74infants per group. To account for an interim analysis at 50% of enrollment and for up to10% attrition rate, a total of 200 infants (100 per group) will be enrolled.‐‐Data Collection and Quality Assurance: Data will be captured using the REDCapelectronic data capture system, with predefined validation rules and role‐specific accessrights. Clinical and perinatal data, transfusion details, and outcomes will be recordedprospectively.Experimental Design Aim 2‐‐Biological Sampling and Time Points:Peripheral blood samples will be collected from each enrolled neonate at three definedtime points: ‐ B1 ‐ Before the first transfusion (baseline); ‐ B2 ‐ 48 hours (±6h) after the transfusion; ‐ B3 ‐ 14 days after the transfusion. At each time point, 1 ml of whole blood will beobtained and processed for molecular and biochemical analyses. Samples will bealiquoted and stored at ‐80°C until batch analysis. Pre‐analytical procedures,storage, and shipping conditions will follow standardized SOPs across centers. Alllaboratory staff performing analyses will be blinded to treatment allocation,ensuring unbiased biomarker evaluation. ‐ Oxidative Stress and Antioxidant Biomarkers:The oxidative stress (OS) profile will be assessed using a panel of protein and lipidbiomarkers, including: ‐ Protein oxidation: Advanced Oxidation Protein Products (AOPP); ‐ Lipid peroxidation: Isoprostanes (IsoPs), malondialdehyde (MDA); ‐ Non‐enzymatic antioxidants: Vitamin E, glutathione (GSH), and ascorbic acid (AA); ‐ Enzymatic antioxidants: Superoxide dismutase (SOD), catalase (CAT), glutathioneperoxidase (GPx).Lipid mediators related to inflammation and oxidative balance, such as Resolvin D1, willalso be quantified, alongside neuroglobin and sirtuin‐1, which are involved in cellularprotection mechanisms. ‐ MicroRNA Profiling:Total RNA will be extracted from 400 μl of serum using the miRVana PARIS kit(Invitrogen). Reverse transcription will be performed using the TaqMan Advanced miRNAcDNA Synthesis Kit, followed by quantification using TaqMan miRNA assays on a 7900HT FastPCR system.MicroRNAs of interest include miR‐107, miR‐17‐5p, and miR‐223‐3p, selected for theirinvolvement in oxidative stress, angiogenesis, and vascular remodeling. Expression willbe normalized using hsa‐miR‐16‐5p as the endogenous control.The 2^deltadeltaCt method will be used to quantify expression changes, and miRNAs with Ct> 35 will be excluded from analysis. Total RNA will be isolated from 400μl of serum usingthe miRVana PARIS kit (Invitrogen, AM1556). Samples will be stored at ‐80°C. Reversetranscription will be performed using the TaqMan Advanced miRNA cDNA Synthesis Kit(Applied Biosystems, A28007). Candidate miRNAs will be quantified using TaqMan AdvancedmiRNA assays (Applied Biosystems) on a 7900HT Fast PCR System, with hsa‐miR‐16‐5p (AssayID: 477860_mir) as an endogenous control. Expression levels will be analyzed using the2^deltadeltaCt method; miRNAs with Ct > 35 will be considered not expressed and excluded.‐‐Comparative Analyses: Between‐group comparisons (CB‐RBC vs A‐RBC) will be conducted ateach time point and across time points (B1‐B3), allowing evaluation of both absolutedifferences and temporal changes in biomarker levels. Analyses will be adjusted forbaseline clinical variables, transfusion volume, and number of transfusions received.This aim will allow to link the transfusion source to molecular signatures of oxidativestress and inflammation, providing mechanistic insight into the observed clinicaloutcomesExperimental Design Aim 3 ‐‐Follow‐up Time Points‐‐ ‐ Neurodevelopmental assessments will be conducted at: ‐ 1 month corrected age; ‐ 3 months corrected age; ‐ 6 months corrected age; ‐ 12 months corrected age. These time points will allow for early detection of neurological delays and tracking of developmental trajectories over the first year of life. ‐ Neurodevelopmental Assessment Tools:Standardized and validated instruments will be used to ensure comparability andreliability across study sites: ‐ The Bayley Scales of Infant and Toddler Development ‐ Third Edition (BSID‐III) willbe used at 12 months to assess cognitive, motor, and language development; ‐ Structured neurological examinations will be performed at each follow‐up visit todetect signs of abnormal motor tone, reflexes, or neurosensory deficits; ‐ Information on growth, oxygen dependence, feeding difficulties, and the presence ofmulti‐organ sequelae will be collected longitudinally.‐‐Neuroimaging: Magnetic Resonance Imaging (MRI) of the brain will be conductedaccording to routine clinical practice and scored retrospectively using the Woodwardscoring system, which quantifies white and gray matter injury, ventricular dilation,and global structural anomalies. MRI reviewers will be blinded to treatmentallocation and clinical outcomes to ensure unbiased evaluation. ‐ Analysis Plan The primary neurodevelopmental outcome will be the proportion ofinfants with BSID‐III scores below 85 (i.e., more than one standard deviation belowthe normative mean) in at least one domain (cognitive, motor, or language) at 12months.Group comparisons will be performed using Chi‐square tests, and multivariable logisticregression models will be used to adjust for potential confounders, including gestationalage, IVH grade, BPD, and birth weight.Secondary analyses will explore continuous BSID‐III scores, composite neurodevelopmentalindices, and MRI findings.Correlations between biological markers (from Aim 2) and neurodevelopmental outcomes willbe examined to identify predictive biomarkers and potential mechanistic pathways