Study protocol of the WashT Trial: transfusion with washed versus unwashed red blood cells to reduce morbidity and mortality in infants born less than 28 weeks' gestation - a multicentre, blinded, parallel group, randomised controlled trial.

INTRODUCTION: Many extremely preterm newborns develop anaemia requiring a transfusion, with most receiving three to five transfusions during their admission. While transfusions save lives, the potential for transfusion-related adverse outcomes is an area of growing concern. Transfusion is an independent predictor of death and is associated with increased morbidity, length of hospital stay, risk of infection and immune modulation. The underlying mechanisms include adverse pro-inflammatory and immunosuppressive responses. Evidence supports an association between transfusion of washed red cells and fewer post-transfusion complications potentially through removal of chemokines, lipids, microaggregates and other biological response modifiers. However, the clinical and cost-effectiveness of washed cells have not been determined. METHODS AND ANALYSIS: This is a multicentre, randomised, double-blinded trial of washed versus unwashed red cells. Infants <28 weeks' gestation requiring a transfusion will be enrolled. Transfusion approaches will be standardised within each study centre and will occur as soon as possible with a recommended fixed transfusion volume of 15 mL/kg whenever the haemoglobin is equal to or falls below a predefined restrictive threshold, or when clinically indicated. The primary outcome is a composite of mortality and/or major morbidity to first discharge home, defined as one or more of the following: physiologically defined bronchopulmonary dysplasia; unilateral or bilateral retinopathy of prematurity grade >2, and; necrotising enterocolitis stage ≥2. To detect a 10% absolute reduction in the composite outcome from 69% with unwashed red blood cell (RBCs) to 59% with washed RBCs with 90% power, requires a sample size of 1124 infants (562 per group). Analyses will be performed on an intention-to-treat basis with a prespecified statistical analysis plan. A cost-effectiveness analysis will also be undertaken. ETHICS AND DISSEMINATION: Ethics approval has been obtained from the Women's and Children's Health Network Human Research Ethics Committee (HREC/12/WCHN/55). The study findings will be disseminated through peer-reviewed articles and conferences. TRIAL REGISTRATION NUMBER: ACTRN12613000237785 Australian New Zealand Clinical Trials Registry.

Red Blood Cell Donor Sex Associated Effects on Morbidity and Mortality in the Extremely Preterm Newborn.

Transfusion exposure increases the risk of death in critically ill patients of all ages. This was thought to relate to co-morbidities in the transfusion recipient. However, donor characteristics are increasingly recognised as critical to transfusion recipient outcome with systematic reviews suggesting blood donor sex influences transfusion recipient health. Originally focusing on plasma and platelet transfusions, retrospective studies report greater risks of adverse outcomes such as transfusion related acute lung injury in those receiving products from female donors. There is increasing awareness that exposure to red blood cells (RBCs) poses a similar risk. Recent studies focusing on transfusion related outcomes in extremely preterm newborns report conflicting data on the association between blood donor sex and outcomes. Despite a renewed focus on lower versus higher transfusion thresholds in neonatal clinical practice, this group remain a heavily transfused population, receiving on average 3-5 RBC transfusions during their primary hospital admission. Therefore, evidence supporting a role for better donor selection could have a significant impact on clinical outcomes in this high-risk population. Here, we review the emerging evidence for an association between blood donor sex and clinical outcomes in extremely preterm newborns receiving one or more transfusions.

Differential effects of ibuprofen and indomethacin on cerebral oxygen kinetics in the very preterm baby.

Background: Ibuprofen is preferred to indomethacin for treatment of a significant patent ductus arteriosus (PDA) in preterm babies despite indomethacin being associated with a lower risk of intraventricular haemorrhage. This difference is thought to relate to the discrepant effects of each medication on cerebral oxygen kinetics yet the effect of ibuprofen on cerebral perfusion is uncertain. Methods: Forty-eight babies < 30 weeks with a significant PDA, defined by echocardiography, were randomly assigned to either indomethacin or ibuprofen (n = 24 per group) and stratified by gestation and chronologic age. Cerebral blood flow [total internal carotid blood flow (TICF)] and oxygen physiology [oxygen delivery (modCerbDO2) and consumption (modCerbVO2)] were measured using cranial Doppler ultrasound and near-infrared spectroscopy, and cerebral oxygen extraction (cFTOE) calculated, immediately before and following administration. Temporal and treatment related changes were analysed. Results: A fixed effect of time was seen for TICF (p = 0.03) and therefore modCerbDO2 (p = 0.046) and cFTOE (p = 0.04) for indomethacin alone. In the indomethacin group, TICF and modCerbDO2 fell from baseline to 5 and 30 min respectively (TICF p < 0.01, cDO2 p = 0.01) before increasing from 5 min to 24 h (p < 0.01) and 30 min and 24 h (p < 0.01) timepoints. cFTOE peaked at 30 min (p = 0.02) returning to baseline at 24 h. There was a parallel increase in arterial lactate. Conclusion: Indomethacin significantly reduces cerebral blood flow soon after administration, resulting in a parallel increase in oxygen extraction and arterial lactate. This implies that the balance of oxygen kinetics at the time of treatment may be critical in very preterm babies with significant PDA.

Therapeutic hypothermia for neonatal encephalopathy with sepsis: a retrospective cohort study.

OBJECTIVE: Neonatal encephalopathy remains a major cause of infant mortality and neurodevelopmental impairment. Infection may exacerbate brain injury and mitigate the effect of therapeutic hypothermia (TH). Additionally, infants with sepsis treated with TH may be at increased risk of adverse effects. This study aimed to review the clinical characteristics and outcomes for infants with sepsis treated with TH. DESIGN AND SETTING: Retrospective cohort study of infants treated with TH within Australia and New Zealand. PATIENTS: 1522 infants treated with TH, including 38 with culture-positive sepsis from 2014 to 2018. INTERVENTION: Anonymised retrospective review of data from Australian and New Zealand Neonatal Network. Infants with culture-positive sepsis within 48 hours were compared with those without sepsis. MAIN OUTCOME MEASURES: Key outcomes include in-hospital mortality, intensive care support requirements and length of stay. RESULTS: Overall the rate of mortality was similar between the groups (13% vs 13%). Infants with sepsis received a higher rate of mechanical ventilation (89% vs 70%, p=0.01), high-frequency oscillatory ventilation (32% vs 13%, p=0.003) and inhaled nitric oxide for persistent pulmonary hypertension (38% vs 16%, p<0.001). Additionally, the sepsis group had a longer length of stay (20 vs 11 days, p<0.001). CONCLUSION: Infants with sepsis treated with TH required significantly more respiratory support and had a longer length of stay. Although this may suggest a more severe illness the rate of mortality was similar. Further research is warranted to review the neurodevelopmental outcomes for these infants.

Effect of washed versus unwashed red blood cells on transfusion-related immune responses in preterm newborns.

Objectives: Transfusion with washed packed red blood cells (PRBCs) may be associated with reduced transfusion-related pro-inflammatory cytokine production. This may be because of alterations in recipient immune responses. Methods: This randomised trial evaluated the effect of transfusion with washed compared with unwashed PRBCs on pro-inflammatory cytokines and endothelial activation in 154 preterm newborns born before 29 weeks' gestation. Changes in plasma cytokines and measures of endothelial activation in recipient blood were analysed after each of the first three transfusions. Results: By the third transfusion, infants receiving unwashed blood had an increase in IL-17A (P = 0.04) and TNF (P = 0.007), whereas infants receiving washed blood had reductions in IL-17A (P = 0.013), TNF (P = 0.048), IL-6 (P = 0.001), IL-8 (P = 0.037), IL-12 (P = 0.001) and IFN-γ (P = 0.001). The magnitude of the post-transfusion increase in cytokines did not change between the first and third transfusions in the unwashed group but decreased in the washed group for IL-12 (P = 0.001), IL-17A (P = 0.01) and TNF (P = 0.03), with the difference between the groups reaching significance by the third transfusion (P < 0.001 for each cytokine). Conclusion: The pro-inflammatory immune response to transfusion in preterm infants can be modified when PRBCs are washed prior to transfusion. Further studies are required to determine whether the use of washed PRBCs for neonatal transfusion translates into reduced morbidity and mortality.

Effect of repeat transfusion exposure on plasma cytokine and markers of endothelial activation in the extremely preterm neonate.

BACKGROUND: Very preterm newborns receive up to three to five red blood cell (RBC) transfusions, often early, after birth. Despite awareness of the association of transfusion with increases in cytokines and markers of endothelial activation, research has focused on single transfusions weeks after birth. With pathophysiologic processes contributing to the development of morbidities starting soon after delivery, we investigated the response to early, repeated transfusion exposure. STUDY DESIGN AND METHODS: Three consecutive transfusion exposures were studied in transfusion-naive infants less than 30 weeks' gestation (n = 46). Plasma cytokines and markers of endothelial activation were measured before and 2 to 4 hours after transfusion by multiplex enzyme-linked immunosorbent assay. RESULTS: The median (IQR) age was 3 (1-9) days at first transfusion, 7 (3-20) days at the second, and 18 (7-28) days at the third. Baseline concentrations did not differ between the three transfusions. Interleukin (IL)-17A and tumor necrosis factor (TNF)-α did not change after the first transfusion but increased after the second (P < .05) and third transfusions (P < .01). While IL-1ß, IL-6, and IL-8 concentrations did not differ after the first and second transfusions, all increased after the third (IL-1ß, P < .01; IL-6, P < .01; IL-8, P < .05). The magnitude of posttransfusion increase in IL-1ß, IL-17A, and TNF-α increased between the first and third transfusion exposure. CONCLUSION: Early, repeated transfusion results in alterations in proinflammatory cytokines and markers of endothelial activation in the very preterm newborn and suggests that the potential for transfusion-related immunomodulation is present in the initial days after birth rather than confined to later in the postnatal period.

Does umbilical cord blood glucose extraction discriminate the risk of early neonatal hypoglycaemia in at-risk newborns?

AIM: Despite targeting newborns at risk of hypoglycaemia based on clinical characteristics, blood glucose measured at 1 and 4 h of age is frequently normal. Identification of at-risk newborns at the greatest risk of hypoglycaemia would allow more targeted, earlier intervention. We aimed to determine the ability of calculated umbilical cord blood glucose extraction to discriminate hypoglycaemia in at-risk newborns in the first 4 h of life. METHODS: Newborns with paired arterial and venous cord blood glucose and 1 ± 4 h capillary or venous blood glucose measured using a blood gas analyser (radiometer) were retrospectively identified (n = 154). Hypoglycaemia was defined as a blood glucose ≤2.0 mmol/L. The ability of calculated umbilical cord blood glucose extraction to discriminate risk of hypoglycaemia was determined by an receiver operating characteristic (ROC) curve. RESULTS: Twenty-seven newborns (18%) had a blood glucose ≤2.0 mmol/L at either time point. Neither arterial nor venous cord blood glucose predicted early hypoglycaemia better than chance. The area under the ROC curve for umbilical cord blood glucose extraction (area under the ROC curve = 0.74, (95% confidence interval, 0.65-0.82)) was significantly better than chance and arterial or venous cord blood glucose. An umbilical cord blood glucose extraction of 16% had the best sensitivity (80%) and specificity (55%) for discriminating the risk of early hypoglycaemia. CONCLUSIONS: Umbilical cord blood glucose extraction discriminates the risk of early hypoglycaemia at 1 or 4 h of age. However, the clinical utility of this test is limited due to the low sensitivity and specificity. Its predictive value may be greater in specific subsets of at-risk newborns and warrants further investigation.

The contribution of red blood cell transfusion to neonatal morbidity and mortality.

Anaemia of prematurity will affect 90% of all very preterm infants, resulting in at least one red blood cell (RBC) transfusion. A significant proportion of preterm infants require multiple transfusions over the course of hospital admission. Growing evidence supports an association between transfusion exposure and adverse neonatal outcomes. In adults, transfusion-associated sepsis, transfusion-related acute lung injury and haemolytic reactions are the leading causes of transfusion-related morbidity and mortality; however, these are seldom recognised in newborns. The association between transfusion and adverse outcomes remains inconclusive. However, the evidence from preclinical studies demonstrates that RBC products can directly modulate immune cell function, a pathway termed transfusion-related immunomodulation (TRIM), which may provide a mechanism linking transfusion exposure with neonatal morbidities. Finally, we discuss the impact of TRIM on transfusion medicine, how we may address these issues and the emerging areas of research aimed at improving the safety of transfusions in this vulnerable population.

Antenatal steroid exposure in the late preterm period is associated with reduced cord blood neurotrophin-3.

BACKGROUND: Neurotrophins are proteins critically involved in neural growth, survival and differentiation, and therefore important for fetal brain development. Reduced cord blood neurotrophins have been observed in very preterm infants (<32weeks gestation) who subsequently develop brain injury. Antenatal steroid exposure can alter neurotrophin concentrations, yet studies to date have not examined whether this occurs in the late preterm infant (33-36weeks gestation), despite increasing recognition of subtle neurodevelopmental deficits in this population. AIM: To assess the impact of antenatal steroids on cord blood neurotrophins in late preterm infants following antenatal steroid exposure. STUDY DESIGN: Retrospective analysis. SUBJECTS: Late preterm infants (33-36weeks; n=119) and term infants (37-41weeks; n=129) born at the Women's and Children's Hospital, Adelaide. OUTCOME MEASURES: Cord blood neurotrophin-3 (NT-3), NT-4, nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) concentrations measured by ELISA. RESULTS: Cord blood NT-4 and NGF were increased at term compared to the late preterm period (p<0.001), while BDNF and NT-3 were not different. In the late preterm period, cord blood NT-3 was reduced when antenatal steroids were administered >24h prior to delivery (p<0.01). CONCLUSION: This study identified an association between reduced cord blood NT-3 and antenatal steroid exposure in the late preterm period. The reduced NT-3 may be a consequence of steroids inducing neuronal apoptosis, thereby reducing endogenous neuronal NT3 production, or be an action of steroids on other maternal or fetal NT-3 producing cells, which may then affect neuronal growth, differentiation and survival. Regardless of the specific mechanism, a reduction in NT-3 may have long term implications for child neurodevelopment, and emphasizes the ongoing vulnerability of the fetal brain across the full preterm period.

Topical thyroid hormone accelerates wound healing in mice.

Although the physiologic role of thyroid hormone in skin is not well understood, mounting evidence suggests that T3 plays an important role in epidermal proliferation. The goal of this project was to evaluate whether the topical application of supraphysiologic doses of T3 could accelerate wound healing. We evaluated mice treated with topical T3 vs. the same mice receiving vehicle alone (Novasome A). Ten-millimeter diameter (79 mm2) dorsal skin wounds were established in all animals, and wounds were remeasured 4 d after injury. All animals were evaluated twice: once with the T3 treatment and once with the vehicle alone. Daily topical application of 150 ng T3 resulted in 58% greater wound closure relative to wounds on the same animals receiving vehicle alone (P < 0.001). Furthermore, we determined that wound healing-associated keratin 6 protein expression in hair follicle keratinocytes increased in a dose-dependent manner in vivo during topical T3 treatment. The data support our previous hypothesis that T3 is necessary for optimal wound healing. Now, we further suggest that topical thyroid hormone may be an inexpensive agent to hasten healing of certain wounds.

A role for thyroid hormone in wound healing through keratin gene expression.

The importance of thyroid hormone (TH) in wound healing is not well understood. To gain insight, we evaluated the impact of TH deficiency on wound-healing genes in cultured keratinocytes. By RT-PCR, keratin 6a (K6a) and 16 (K16) gene expression in TH replete cells was 3.8- (P < 0.005) and 1.9-fold (P < 0.05) greater, respectively, than expression in TH-deficient cells. By real-time PCR, TH replete cell expression of K6a, K16, and K17 was greater than in deficient cells: 18- (P < 0.001), 10- (P < 0.001), and 4-fold (P < 0.005), respectively. To examine TH requirement for optimal wound healing, we contrasted TH-deficient vs. ip T(3)-treated mice. Four days after wounding, ip T(3)-treated mice had twice the degree of wound closure as hypothyroid mice (P < 0.001). By RT-PCR, K6a and K17 gene expression from control mouse skin was greater than from hypothyroid mouse skin: 5- (P < 0.001) and 1.7-fold (P < 0.05), respectively. T(3) is necessary for the keratinocyte proliferation required for optimal wound healing. T(3) exerts influence by stimulating expression of the wound-healing keratin genes. Thus, for hypothyroid patients undergoing surgery that cannot be delayed until euthyroidism is achieved, our data support T(3) treatment for the perioperative period.

Thyroid hormone action on skin: diverging effects of topical versus intraperitoneal administration.

Previously, we demonstrated stimulation of epidermal proliferation and hair growth in triiodothyronine (T(3)) treated mice. To distinguish skin effects of directly applied T(3) from those of systemic hyperthyroidism, we treated CD-1 mice with either intraperitoneally (IP) or topically administered T(3). Relative to controls, mice receiving T(3) IP had 10% thinner epidermis (p < 0.01) and 48% fewer hairs (p < 0.001). By contrast, mice receiving T(3) topically had 78% thicker epidermis (p < 0.01) and 160% more hairs (p < 0.01). To gain insight into factors responsible for the diverging effects, we contrasted T(3) effect on proliferation of isolated keratinocyte cultures versus keratinocytes cocultured with dermal fibroblasts. For keratinocytes grown in the absence of fibroblasts, T(3) stimulated proliferation in a dose-dependent, biphasic pattern with the peak at 0.5 nM T(3) (84 +/- 30%, p < 0.05). Paradoxically, T(3) inhibited proliferation of keratinocytes cocultured with fibroblasts, the nadir at 0.1 nM T(3) (34% +/- 4%, p < 0.001). These studies are the first describing divergent effects of IP and topically administered thyroid hormone. The data suggest that while T(3) stimulated keratinocyte proliferation, T(3) also stimulated proliferation inhibitory factor(s) from skin fibroblasts. Insight into the interplay among the competing factors will be important in understanding thyroid hormone regulation of skin physiology.