Brian H. Walsh

The use of conventional EEG for the assessment of hypoxic ischaemic encephalopathy in the newborn: A review

Neonatal hypoxic ischaemic encephalopathy continues to be one of the leading causes of morbidity and mortality among neonates around the globe. With the advent of therapeutic hypothermia, the need to accurately classify the severity of injury in the early neonatal period is of great importance. As clinical measures cannot always accurately estimate the severity early enough for treatment to be initiated, clinicians have become more dependent on conventional and amplitude integrated EEG. Despite this, there is currently no single agreed classification scheme for the neonatal EEG in hypoxic ischaemic encephalopathy. In this review we discuss classification schemes of neonatal background EEG, published over the past 35 years, highlighting the urgent need for a universal visual analysis scheme.

The Metabolomic Profile of Umbilical Cord Blood in Neonatal Hypoxic Ischaemic Encephalopathy

Background Hypoxic ischaemic encephalopathy (HIE) in newborns can cause significant long-term neurological disability. The insult is a complex injury characterised by energy failure and disruption of cellular homeostasis, leading to mitochondrial damage. The importance of individual metabolic pathways, and their interaction in the disease process is not fully understood. The aim of this study was to describe and quantify the metabolomic profile of umbilical cord blood samples in a carefully defined population of full-term infants with HIE. Methods and Findings The injury severity was defined using both the modified Sarnat score and continuous multichannel electroencephalogram. Using these classification systems, our population was divided into those with confirmed HIE (n = 31), asphyxiated infants without encephalopathy (n = 40) and matched controls (n = 71). All had umbilical cord blood drawn and biobanked at −80°C within 3 hours of delivery. A combined direct injection and LC-MS/MS assay (AbsolutIDQ p180 kit, Biocrates Life Sciences AG, Innsbruck, Austria) was used for the metabolomic analyses of the samples. Targeted metabolomic analysis showed a significant alteration between study groups in 29 metabolites from 3 distinct classes (Amino Acids, Acylcarnitines, and Glycerophospholipids). 9 of these metabolites were only significantly altered between neonates with Hypoxic ischaemic encephalopathy and matched controls, while 14 were significantly altered in both study groups. Multivariate Discriminant Analysis models developed showed clear multifactorial metabolite associations with both asphyxia and HIE. A logistic regression model using 5 metabolites clearly delineates severity of asphyxia and classifies HIE infants with AUC = 0.92. These data describe wide-spread disruption to not only energy pathways, but also nitrogen and lipid metabolism in both asphyxia and HIE. Conclusion This study shows that a multi-platform targeted approach to metabolomic analyses using accurately phenotyped and meticulously biobanked samples provides insight into the pathogenesis of perinatal asphyxia. It highlights the potential for metabolomic technology to develop a diagnostic test for HIE.

Smartphone technology enhances newborn intubation knowledge and performance amongst paediatric trainees

OBJECTIVE Smartphones are widely used by physicians, but their effectiveness in improving teaching of clinical skills is not known. The aim of this study was to determine if pre procedural use of a smartphone neonatal intubation instructional application (NeoTube) improves trainee knowledge and enhances procedural skills performance in newborn intubation. DESIGN Neonatal Resuscitation Program certified trainees in paediatrics and neonatology completed a knowledge based questionnaire on neonatal intubation, and were recorded intubating a term newborn manikin model. They then used the NeoTube iPhone application for 15 min, before completing the questionnaire and intubation again. Video recordings were later reviewed by two independent assessors, blinded to whether it was pre or post NeoTube use. RESULTS 20 paediatric trainees (12 fellows and 8 residents) participated in this study. Comparing pre and post-viewing of the application, Questionnaire Scores (median (range)) increased from 18.5 (8-28) to 31 (24-35) (P<0.001), with calculation scores increasing from 6 (0-11) to 11 (6-12) (P<0.001), Skill Scores increased from 11 (9-15) to 12.5 (9-16) (P=0.016), and the duration of intubation attempt decreased from 39 to 31 s (P=0.044) following utilisation of the application. There was a significant positive correlation with duration of specialist training for procedure performance post viewing, but not pre viewing of the application. CONCLUSIONS Bedside use of smartphones can enhance both knowledge of newborn intubation and improves procedural performance, including reducing the time to successfully intubate. Smartphones may have a useful role in bringing procedural skills training closer to the bedside.

Quantitative EEG analysis in neonatal hypoxic ischaemic encephalopathy.

OBJECTIVE To test the hypothesis that quantitative EEG (qEEG) measures are associated with a grading of HIE based on the visual interpretation of neonatal EEG (EEG/HIE). METHODS Continuous multichannel video-EEG data were recorded for up to 72 h. One-hour EEG segments from each recording were visually analysed and graded by two electroencephalographers (EEGers) blinded to clinical data. Several qEEG measures were calculated for each EEG segment. Kruskal-Wallis testing with post hoc analysis and multiple linear regression were used to assess the hypothesis. RESULTS Fifty-four full-term infants with HIE were studied. The relative delta power, skewness, kurtosis, amplitude, and discontinuity were significantly different across four EEG/HIE grades (p<0.05). A linear combination of these qEEG measures could predict the EEG/HIE grade assigned by the EEGers with an accuracy of 89%. CONCLUSION Quantitative analysis of background EEG activity has shown that measures based on the amplitude, frequency content and continuity of the EEG are associated with a visual interpretation of the EEG performed by experienced EEGers. SIGNIFICANCE Identifying qEEG measures that can separate between EEG/HIE grades is an important first step towards creating a classifier for automated detection of EEG/HIE grades.

Early continuous video electroencephalography in neonatal stroke.

Perinatal stroke is the second most common cause of neonatal seizures, and can result in long‐term neurological impairment. Diagnosis is often delayed until after seizure onset, owing to the subtle nature of associated signs. We report the early electroencephalographic (EEG) findings in a female infant with a perinatal infarction, born at 41 weeks 2 days and weighing 3.42kg. Before the onset of seizures, the EEG from 3 hours after delivery demonstrated occasional focal sharp waves over the affected region. After electroclinical seizures, focal sharp waves became more frequent, complex, and of higher amplitude, particularly in ‘quiet sleep’. In ‘active sleep’, sharp waves often disappeared. Diffusion‐weighted imaging confirmed the infarct, demonstrating left frontal and parietal diffusion restriction. At 9 months, the infant has had no further seizures, and neurological examination is normal. To our knowledge, this report is the first to describe the EEG findings in perinatal stroke before seizures, and highlights the evolution of characteristic background EEG features.

Downregulation of Umbilical Cord Blood Levels of miR-374a in Neonatal Hypoxic Ischemic Encephalopathy

OBJECTIVE To investigate the expression profile of microRNA (miRNA) in umbilical cord blood from infants with hypoxic ischemic encephalopathy (HIE). STUDY DESIGN Full-term infants with perinatal asphyxia were identified under strict enrollment criteria. Degree of encephalopathy was defined using both continuous multichannel electroencephalogram in the first 24 hours of life and modified Sarnat score. Seventy infants (18 controls, 33 with perinatal asphyxia without HIE, and 19 infants with HIE [further graded as 13 mild, 2 moderate, and 4 severe]) were included in the study. MiRNA expression profiles were determined using a microarray assay and confirmed using quantitative real-time polymerase chain reaction. RESULTS Seventy miRNAs were differentially expressed between case and control groups. Of these hsa-miR-374a was the most significantly downregulated in infants with HIE vs controls. Validation of hsa-miR-374a expression using quantitative real-time polymerase chain reaction confirmed a significant reduction in expression among infants with HIE compared with those with perinatal asphyxia and healthy controls (mean relative quantification [SD] = 0.52 [0.37] vs 1.10 [1.52] vs 1.76 [1.69], P < .02). CONCLUSIONS We have shown a significant step-wise downregulation of hsa-miR-374a expression in cord blood of infants with perinatal asphyxia and subsequent HIE.

The effect of haemolysis on the metabolomic profile of umbilical cord blood

OBJECTIVES Metabolomics is defined as the comprehensive study of all low molecular weight biochemicals, (metabolites) present in an organism. Using a systems biology approach, metabolomics in umbilical cord blood (UCB) may offer insight into many perinatal disease processes by uniquely detecting rapid biochemical pathway alterations. In vitro haemolysis is a common technical problem affecting UCB sampling in the delivery room, and can hamper metabolomic analysis. The extent of metabolomic alteration which occurs in haemolysed samples is unknown. DESIGN AND METHODS Visual haemolysis was designated by the laboratory technician using a standardised haemolysis index colour chart. The metabolomic profile of haemolysed and non-haemolysed UCB serum samples from 69 healthy term infants was compared using both (1)H-NMR and targeted DI and LC-MS/MS approach. RESULTS We identified 43 metabolites that are significantly altered in visually haemolysed UCB samples, acylcarnitines (n=2), glycerophospholipids (n=23), sphingolipids (n=7), sugars (n=3), amino acids (n=4) and Krebs cycle intermediates (n=4). CONCLUSION This information will be useful for researchers in the field of neonatal metabolomics to avoid false findings in the presence of haemolysis, to ensure reproducible and credible results.

Early Cord Metabolite Index and Outcome in Perinatal Asphyxia and Hypoxic-Ischaemic Encephalopathy

Background: A 1H-NMR-derived metabolomic index based on early umbilical cord blood alterations of succinate, glycerol, 3-hydroxybutyrate and O-phosphocholine has shown potential for the prediction of hypoxic-ischaemic encephalopathy (HIE) severity. Objective: To evaluate whether this metabolite score can predict 3-year neurodevelopmental outcome in infants with perinatal asphyxia and HIE, compared with current standard biochemical and clinical markers. Methods: From September 2009 to June 2011, infants at risk of perinatal asphyxia were recruited from a single maternity hospital. Cord blood was drawn and biobanked at delivery. Neonates were monitored for development of encephalopathy both clinically and electrographically. Neurodevelopmental outcome was assessed at 36-42 months using the Bayley Scales of Infant and Toddler Development, ed. III (BSID-III). Death and cerebral palsy were also considered as abnormal end points. Results: Thirty-one infants had both metabolomic analysis and neurodevelopmental outcome at 36-42 months. No child had a severely abnormal BSID-III result. The metabolite index significantly correlated with outcome (ρ2 = 0.30, p < 0.01), which is robust to predict both severe outcome (area under the receiver operating characteristic curve: 0.92, p < 0.01) and intact survival (0.80, p = 0.01). There was no correlation between the index score and performance in the individual BSID-III subscales (cognitive, language, motor). Conclusions: The metabolite index outperformed other standard biochemical markers at birth for prediction of outcome at 3 years, but was not superior to EEG or the Sarnat score.

165 The Metabolomic Profile of Umbilical Cord Blood in Neonatal Hypoxic Ischaemic Encephalopathy

Background Hypoxic ischaemic encephalopathy (HIE) is associated with the activation of multiple biochemical pathways. The importance of these pathways individually, and that of their interaction, in the disease process is not fully understood. The aim of this study was to describe and quantify the metabolomic profile of umbilical cord blood samples in a carefully defined population of full-term infants with HIE. Methods Full-term infants with perinatal asphyxia (with and without HIE) and healthy controls had umbilical cord blood drawn and bio-banked at –80°C, within 3 hours of birth. A combined direct injection and LC-MS/MS assay (AbsolutIDQ p180 kit, Biocrates Life Sciences AG, Innsbruck, Austria) was used for the metabolomic analyses of the samples. The degree of encephalopathy among those with asphyxia was defined using both continuous multichannel-EEG in the first 24 hours, and modified Sarnat score. Results 142 neonates were included in the analysis (HIE=31, asphyxia without encephalopathy=40, controls=71). There was a significant alteration (p<0.01) in 29 metabolites from 3 distinct metabolite classes (Amino Acids, Carnitines, and Phosphatidylcholines) between study groups. 13 of these metabolites were significantly altered between HIE and controls. Cross-validated Partial Least Square Discriminant Analysis models were developed to distinguish between the groups. The HIE model differentiated significantly between HIE, and those without HIE (AUC=0.93, R2 = 0.36, Q2 = 0.25). Conclusion The description of the metabolomic profile from umbilical cord plasma and the specific metabolite signature associated with HIE, offers insight into the disease mechanism and the possibility of an early screening test.

Cord Blood IL-16 Is Associated with 3-Year Neurodevelopmental Outcomes in Perinatal Asphyxia and Hypoxic-Ischaemic Encephalopathy

Activation of the inflammatory pathway is increasingly recognized as an important mechanism of injury following neonatal asphyxia and encephalopathy. This process may contribute to the poor prognosis seen in some cases, despite therapeutic hypothermia. Our group has previously identified raised interleukin (IL)-6 and IL-16, measured in umbilical cord blood at birth, to be predictive of grade of hypoxic-ischaemic encephalopathy (HIE). Our aim in this study was to examine the ability of these cytokines to predict the 3-year neurodevelopmental outcome in the same cohort. As part of a prospective, longitudinal cohort study set in a single tertiary maternity unit, term infants with biochemical and clinical evidence of perinatal asphyxia were recruited at birth. Umbilical cord blood was collected and analyzed for IL-6 and IL-16 using a Luminex assay. The neurodevelopmental outcome of these infants was assessed at 3 years using the Bayley Scales of Infant and Toddler Development (Edition 3). Early cord blood measurement of IL-6 and IL-16 and long-term outcome were available in 33/69 infants. Median (IQR) IL-16 differentiated infants with a severely abnormal outcome (n = 6) compared to all others (n = 27), (646 [466-1,085] vs. 383.5 [284-494] pg/mL; p = 0.012). IL-16 levels were able to predict a severe outcome with an area under the receiver-operating characteristic (ROC) curve of 0.827 (95% CI 0.628-1.000; p = 0.014). Levels ≥514 pg/mL predicted a severe outcome with a sensitivity of 83% and a specificity of 81%. IL-16 also outperformed other routine biochemical markers available at birth for the prediction of severe outcome. APGAR scores at 1 and 10 min were also predictive of a severe outcome (p = 0.022 and p = 0.036, respectively). A combination of IL-16 with these clinical markers did not improve predictive value, but IL-16 combined with electroencephalogram grading increased the area under the ROC curve. IL-6 did not show any association with 3-year outcome. This is the first report studying the association of IL-16 measured at birth with long-term outcome in a cohort of neonates with perinatal asphyxia. IL-16 may be an early biomarker of severe injury and aid in the long-term prognostication in infants with HIE.