Hyperbilirubinemia is a common occurrence in early neonatal period. About 50% of term and 80% of preterm neonates have serum bilirubin levels greater than 5 mg/dl in first few days of life. The levels may go up to 15 mg/dl in 6% of term neonates . In preterm populations the levels are higher and have more serious implications. Timely phototherapy is the mainstay of treatment to avoid exchange transfusion.
Total serum bilirubin (TsB) is the gold standard to estimate serum bilirubin levels in jaundiced preterm neonates. It, however, requires a needle prick. Transcutaneous bilirubinometry (TcB), on the other hand, is quick, handy and non-invasive way of screening for hyperbilirubinemia requiring intervention. It has also been validated to assess jaundice in term neonates at the time of discharge . Once commenced, phototherapy leads to bleaching of the skin, rendering further TcB measurements unreliable. Covering a part of the skin during phototherapy has been shown to give better correlation with TsB values in some studies [3-8]. However, there has been conflicting evidence in studies [9, 10]. These studies were heterogeneous in regards to gestational age and were done in small number of babies.
Hence, we planned to do an observational study to assess the accuracy of TcB levels on covered part of the skin, as compared to corresponding TsB levels in jaundiced preterm neonates before the start and during phototherapy.
Trial design and participants:
This single center, prospective observational study was conducted in the neonatal intensive care unit of a tertiary care hospital, in northern India from January 2017 to January 2019. Preterm neonates requiring phototherapy in first two weeks of life were considered eligible for enrolment into the study. Neonates with poor peripheral perfusion, sepsis, conjugated hyperbilirubinemia, having major congenital malformations, and hydrops fetalis were excluded. Those neonates who had already received phototherapy, ones who required exchange transfusion, were also excluded. We did not include babies with hemangioma or bruise on glabella that may have interfered with TcB measurements.
Case Enrollment: Preterm neonates with clinical neonatal jaundice were accessed for eligibility. After parental consent, the TcB /TsB levels were measured. As we enrolled Indian Blacks only, hence we did not take skin pigmentation into account.
Bilirubin measurement protocol: In all study participants, TcB levels were measured simultaneously with TsB. Serum bilirubin levels were measured at three defined time points, just before starting phototherapy, at 6 hours and 24 hours of continuous phototherapy. During the pilot study, we measured TcB at two different locations: on the covered skin area (glabella) under the eye protector and on the exposed naked skin close to it. We observed higher TcB values under the covered skin than surrounding uncovered skin area in the pilot study. For TsB measurement, 1-1.5 ml of blood was collected from each neonate. We used venous blood instead of capillary blood as the previous has lesser environmental effect and more accurate values than capillary blood as mentioned in previous study . At our centre we were using venous blood more frequently than capillary blood for bilirubin measurement.
Peripheral venous blood was collected in plain vials, which was processed in the laboratory immediately. Our study centre was a dedicated pediatric hospital. The measurement of TsB was calibrated as per the reference range for neonate. Bilirubin assay was done by using diazo method. The principal of bilirubin assay was based on the Jendrassik-Grof method in which diazotized sulfanilic acid reacts with bilirubin to form azo-bilirubin, the latter of which was detected at an Optical Density of 540 nm. Blood sampling was performed within 10 min of transcutaneous measurement of bilirubin.
Protocol for starting phototherapy: The decision to start phototherapy was based on clinical observation and serum bilirubin level by a pediatrician or a neonatologist. In our unit we followed NICE (National Institute for Health and Clinical Excellence) guidelines for phototherapy in preterm neonates set by National Collaborating Centre for Women’s and Children’s Health . Preterm neonates with clinical jaundice were tested for TcB and TsB at admission. If the value of TcB/TsB was below the threshold for commencing phototherapy, the neonate was excluded from the study. Phototherapy was started if the value of TcB/TsB was higher than the threshold value. In case of different values between the TcB and TsB, we took TsB value for the point of action. Exchange transfusion was done if values of serum bilirubin were higher than reference range or signs of acute bilirubin encephalopathy were present.
FANEM Bilitron Sky 2006, Super LED Phototherapy was used in our study. The distance between baby and phototherapy unit was 30 cm. Irradiance was kept in the range of 30–50 micro W/cm2/nm and measured the same by hand held radiometer. We used uniform phototherapy machine for all study participants and the light spectrum was maintained as per standard guidelines.
BiliCare device (BiliCare REF 81000300, Israel, 2015) was used to measure TcB. As per manufacturer’s recommendation, the device was calibrated each time before use. It gave the average bilirubin value in mg/dl of three back to back applications. During the treatment with phototherapy the neonates remained naked, with diaper and eye protector. As babies eyes were always covered with eye protector during phototherapy, we chose glabella, the area between the eyes, to measure TcB levels to correlate with TsB levels at same time points. We used neonatal eye protector of size 10cm x 3 cm, opaque, non allergic and unique design manufactured by Ibis Medical, Kerala, India. It has a stretchable rim that fits in the head and minimized the chances of slip. In case of any accidental slip of the eye protector, the nursing staff on duty had placed it in same position.
Phototherapy unit was turned off only during TcB/TsB measurements and feeding. All the doctors involved in the care of these neonates were familiar with the use of BiliCare device as we have been using it for over two years in our nursery. We also performed three live demonstration of its use before the start of the study among our neonatal team.
Observations from study conducted by Lucanova et. al.  indicated that phototherapy significantly interferes with the accuracy of transcutaneous bilirubinometry. In order to have a correlation coefficient of 0.287, a sample size of 94 neonates was estimated for a study power of 80% and two tailed alpha of 0.05. This was further rounded off to 100 neonates.
Statistical analysis: Nonparametric tests were used for the outcomes on continuous scale. The mean of TcB for each measurement time point was compared with the corresponding TsB concentration. Pearson correlation coefficient (r) analysis was performed to assess correlation coefficient between TcB and TsB. In order to assess the accuracy of noninvasive bilirubin concentration measurement, difference (Δ) between TsB and TcB and their 95% confidence intervals (CI 95%) were evaluated. A P-value of 0.05 was considered statistically significant. The method of Bland and Altman was used to evaluate the agreement between bilirubin levels in blood and skin.
Ethical Approval: Written informed consent was taken from parents or caregiver of the baby before enrollment into the study. The study was approved by institutional Ethics Committee (letter no: 2823 MC/EC/2016).