Screening methods for neonatal hyperbilirubinemia:benefits, limitations, requirements, and novel developments

Severe neonatal hyperbilirubinemia (SNH) is a serious condition that occurs worldwide. Timely recognition with bilirubin determination is key in the management of SNH. Visual assessment of jaundice is unreliable. Fortunately, transcutaneous bilirubin measurement for screening newborn infants is routinely available in many hospitals and outpatient settings. Despite a few limitations, the use of transcutaneous devices facilitates early recognition and appropriate management of neonatal jaundice. Unfortunately, however, advanced and often costly screening modalities are not accessible to everyone, while there is an urgent need for inexpensive yet accurate instruments to assess total serum bilirubin (TSB). In the near future, novel icterometers, and in particular optical bilirubin estimates obtained with a smartphone camera and processed with a smartphone application (app), seem promising methods for screening for SNH. If proven reliable, these methods may empower outpatient health workers as well as parents at home to detect jaundice using a simple portable device. Successful implementation of ubiquitous bilirubin screening may contribute substantially to the reduction of the worldwide burden of SNH. The benefits of non-invasive bilirubin screening notwithstanding, any bilirubin determination obtained through non-invasive screening must be confirmed by a diagnostic method before treatment. ImpactKey message: Screening methods for neonatal hyperbilirubinemia facilitate early recognition and timely treatment of severe neonatal hyperbilirubinemia (SNH). Any bilirubin screening result obtained must be confirmed by a diagnostic method. What does this article add to the existing literature? Data on optical bilirubin estimation are summarized. Niche research strategies for prevention of SNH are presented. Impact: Transcutaneous screening for neonatal hyperbilirubinemia contributes to the prevention of SNH. A smartphone application with optical bilirubin estimation seems a promising low-cost screening method, especially in low-resource settings or at home.Afdeling Klinische Chemie en Laboratoriumgeneeskunde (AKCL

Diagnostic methods for neonatal hyperbilirubinemia:benefits, limitations, requirements, and novel developments

Invasive bilirubin measurements remain the gold standard for the diagnosis and treatment of infants with severe neonatal hyperbilirubinemia. The present paper describes different methods currently available to assess hyperbilirubinemia in newborn infants. Novel point-of-care bilirubin measurement methods, such as the BiliSpec and the Bilistick, would benefit many newborn infants, especially in low-income and middle-income countries where the access to costly multi-analyzer in vitro diagnostic instruments is limited. Total serum bilirubin test results should be accurate within permissible limits of measurement uncertainty to be fit for clinical purposes. This implies correct implementation of internationally endorsed reference measurement systems as well as participation in external quality assessment programs. Novel analytic methods may, apart from bilirubin, include the determination of bilirubin photoisomers and bilirubin oxidation products in blood and even in other biological matrices. ImpactKey message: Bilirubin measurements in blood remain the gold standard for diagnosis and treatment of severe neonatal hyperbilirubinemia (SNH). External quality assessment (EQA) plays an important role in revealing inaccuracies in diagnostic bilirubin measurements. What does this article add to the existing literature? We provide analytic performance data on total serum bilirubin (TSB) as measured during recent EQA surveys. We review novel diagnostic point-of-care (POC) bilirubin measurement methods and analytic methods for determining bilirubin levels in biological matrices other than blood. Impact: Manufacturers should make TSB test results traceable to the internationally endorsed total bilirubin reference measurement system and should ensure permissible limits of measurement uncertainty.Afdeling Klinische Chemie en Laboratoriumgeneeskunde (AKCL

Functional Induction of the Cystine-Glutamate Exchanger System Xc- Activity in SH-SY5Y Cells by Unconjugated Bilirubin

We have previously reported that exposure of SH-SY5Y neuroblastoma cells to unconjugated bilirubin (UCB) resulted in a marked up-regulation of the mRNA encoding for the Na+ -independent cystine∶glutamate exchanger System Xc− (SLC7A11 and SLC3A2 genes). In this study we demonstrate that SH-SY5Y cells treated with UCB showed a higher cystine uptake due to a significant and specific increase in the activity of System Xc−, without the contribution of the others two cystine transporters (XAG− and GGT) reported in neurons. The total intracellular glutathione content was 2 folds higher in the cells exposed to bilirubin as compared to controls, suggesting that the internalized cystine is used for gluthathione synthesis. Interestingly, these cells were significantly less sensitive to an oxidative insult induced by hydrogen peroxide. If System Xc− is silenced the protection is lost. In conclusion, these results suggest that bilirubin can modulate the gluthathione levels in neuroblastoma cells through the induction of the System Xc−, and this renders the cell less prone to oxidative damage

Response to H₂O₂ oxidative stress in xCT silenced SH-SY5Y cells pre-treated with bilirubin.

<p><u>Panel A</u>: Protein expression of xCT (55 kDa and 35 kDa) was analyzed by Western blot after siRNA and Bf treatment. Lane 1: MOCK and 0.6% DMSO; lane 2: NT and 0.6% DMSO; lane 3: anti-xCT and DMSO 0.6%. Lane 4: MOCK and Bf 140 nM; lane 5: NT and Bf 140 nM ; lane 6: anti-xCT and Bf 140 nM. <u>Panel B</u>: Quantification of bands shown in lanes from 1 to 6 of panel A. xCT 35 kDa and 55 kDa bands normalized by actin and expressed as relative to MOCK . <u>Panel C</u>) H₂O₂ dose-response by MTT test was evaluated in untransfected (MOCK – left), transfected with not targeting siRNA (NT – middle) and transfected with siRNA against SLC7A11 gene (anti-xCT - right) SH-SY5Y cells. After silencing, and before 1 h H₂O₂ treatment, SH-SY5Y cells were exposed for 24 h to 0.6% DMSO or Bf 140 nM as indicated in the legend of each picture.</p

Sodium –dependent and independent L-[¹⁴C] cystine transport in SH-SY5Y cells.

<p>Transport activity was measured in control (0.6% DMSO, 24 h) and treated (Bf 140 nM, 24 h) cells. L-[¹⁴C] cystine (0.8 µM) uptake was measured after 10 min incubation at 37°C in the presence and absence of sodium ions. L-quisqualate (500 µM) was used as a specific inhibitor for System X_c⁻. Each point represents the mean ± SD of three plates of cells (** p<0.001).</p

List of primer sequences designed for the quantification of specific mRNAs by qPCR.

<p>List of primer sequences designed for the quantification of specific mRNAs by qPCR.</p

Diagnostic Performance Analysis of the Point-of-Care Bilistick System in Identifying Severe Neonatal Hyperbilirubinemia by a Multi-Country ApproachResearch in context

Importance: The real prevalence and clinical burden of severe neonatal jaundice are undefined due to difficulties in measuring total serum bilirubin (TSB) outside secondary and tertiary clinical centers. Objective: To assess the diagnostic performance of the point-of care Bilistick System (BS) in identifying neonatal jaundice patients requiring treatment. Design: Between April 2015 and November 2016, 1911 neonates, were recruited to participate in the study. Blood samples were simultaneously collected for the TSB determination by BS and by hospital laboratory (Lab). Data were collected and sent to the Bilimetrix headquarter in Trieste where statistical analysis was performed. Newborns with neonatal jaundice were treated with phototherapy according to each center's guidelines. Setting: 17 hospitals from Nigeria, Egypt, Indonesia, and Viet Nam. Participants: 1911 newborns were included, of which 1458 (76·3%) fulfilled the inclusion criteria. Results: TSB level measured by BS agreed (p < .0001) with the lab result in all four countries. The diagnostic performance of BS showed a positive predictive value (PPV) of 92·5% and a negative predictive value (NPV) of 92·8%. Conclusions and Relevance: BS is a reliable system to detect neonatal jaundice over a wide range of bilirubin levels. Since Bilistick is a point-of-care test, its use may provide appropriate and timely identification of jaundiced newborns requiring treatment. Keywords: Neonatal jaundice, Severe hyperbilirubinemia, Neonatal screening, Bilirubin, Bilistick System, Point-of-care system, Diagnostic accuracy study, STARD, Low-medium income countrie