Bilirubin Binding Capacity to Assess Bilirubin Load in Preterm Infants

Most preterm newborns are managed by phototherapy to reverse hyperbilirubinemia with the intent to prevent bilirubin neurotoxicity. A threshold-based relationship between a specific total bilirubin level and need for intervention has been elusive. This is most likely due to other biomarkers such as hemolysis, developmental maturation, concurrent illnesses, or even interventions, may impede bilirubin/albumin binding. The over-prescription of phototherapy has impacted clinical and family-centered care, and in the extreme preterm infants, it may have augmented their risk of mortality. Thus, the opportunity to individualize phototherapy in in order to reduce its use is unique. The investigators have assembled a transdisciplinary team to examine critical unanswered questions including the role of bilirubin binding capacity (BBC) of an individual during the first week of life in the context of clinical modifiers and antecedents for a domain of bilirubin-induced neurologic disorders, that includes neuro-anatomical, hearing, visual and developmental processing impairments. In this study, the investigator will evaluate two new innovative nanotechniques to quantify bilirubin load for the first time in the context of a clinical decision algorithm to identify those most at risk for any bilirubin-related neurotoxicity. The investigators anticipate that knowledge gained from this study will lead to ethically testable hypotheses to individualize the prescription of phototherapy.

The investigator intends to first collect simultaneous and comprehensive "acute phase" measurements of TB, BBC, ETCOc, and COHbc in MPT infants. The investigator will then seek to understand precisely the relationship between GA, TB, BBC, ETCOc, and COHbc levels and the domains of BIND. Third, The investigator will provide a comprehensive database that can be used to improve current neonatal BIND screening practices in the context of lowered and higher BBC. The investigator's working hypothesis is that exposures to modest TB levels in the presence of significantly diminished BBC in the developing neonate result in residual deficits of one or more neuroprocessing function (BIND) at TEA.

1. Patients (GA 24 to \<34 wks) will be enrolled. Subject exclusion criteria: Major life-threatening anomalies and diagnosed inborn errors of metabolic disorders; attending physician or parent refusal.

   Clinical data collection: After receiving written informed consent, the research team will complete clinical data forms for infant demographics. The data forms will be consistent with and abstracted from the medical record. No additional information will be collected for this exploratory study.

   Population: The entire cohort will compromise 60-80 patients. From this cohort, 12 at-risk infants with most impaired BBC and matched with those designated as low-risk will be re-recruited for the follow-up to identify any evidence of BIND in any or all 4 of the outcome variables.

   Laboratory data: Once inclusion criteria are met, routine neonatal laboratory tests will be as clinically ordered. Each infant will tested for BBC and ETCOc at least 2 intervals (maximum 4 over 12h-7d) during rates-of-rise and -decrease in TB. Subsequent laboratory and clinical data will be paired with research data for statistical analysis.

   The investigators will compare BIND outcomes at TEA to 3 mos-corrected age (\<54 wks PMA) using a re-consented sample size: n=12 for those at high risk with decreased BBC versus a GA-matched controls at low risk (n=12).
2. Measurements: 0.1-mL whole blood will be drawn in special heparinized tubes for COHbc determinations and anticoagulated blood set aside for the hematofluorometry.

Plasma for peroxidase UB assays will be stored and labeled without patient identifiers.

Frozen research samples will be transported to the Spectrum Child Health Research (SCHR) Lab for analyses. 1. BBC, TB, and UB will be measured directly: 1a. BBC, TB and UB in 50-μL whole blood using POC hematofluorometry; TB performed by the hospital-based clinical laboratory; and UB in plasma using the peroxidase method (Arrows device).

ETCOc will be determined for those breathing spontaneously.

c. Testing and techniques for outcome variables for select at-risk and matched control infants:

1. Screening ABR: Two or more simultaneously channels will consist of the electrode pairs of: 1) contralateral to ipsilateral mastoid prominence; 2) vertex to ipsilateral mastoid; and 3) vertex to contralateral mastoid for better identification of waves. Insert tubephone earphone will be used to introduce an acoustic delay to distinguish CM response from artifact. Rarefaction clicks at 90, (75), 60, (45), and 30 dBnHL will be delivered monaurally to the right and left ears. RE and LE, ≥2 repetitions, ≥2,000 sweeps/repetition. Separate recording to rarefaction and condensation clicks will be obtained at 90 dB. The surface electrical activity will be amplified x10,000 and filtered from 30-3,000 Hz. Latencies and peak-to-trough amplitudes of waves and CM from the outer hair cells in the inner ear of the ABRs will be scored independently by "masked" interpreters (Drs. Oghalia and Popelka).
2. Screening Visual Brainstem Responses after TEA (at 50-54 wks PMA): All infants in this subcohort will be evaluated using the sVEP technique described above.68 Electrodes are placed across the back of the visual cortex, midline and 2 cm to the left and right, with a reference lead at the occipital vertex. Thresholds and suprathreshold measurements will be compared with controls. Further, the infants in the bilirubin cohort can serve a case series with a dose response plot determined, comparing thresholds with TB levels. Bin averages for each type of vision can also be compared to the same for control infants to determine whether suprathreshold measures vary to any significant degree from controls (Fig. 3).

   Evidence from other studies of CNS damage suggests that lower signal amplitudes and thresholds correlate with CNS damage. Support for this sample size is based on practical considerations an ad hoc sample size calculation.
3. Neuroimaging of the brain will be performed by conventional MRI at TEA; this is the routine near-term neuroimaging for preterm infants in our institution. MRI is performed in unsedated infants, using a 3-Tesla platform with sequences that include Sagittal T1 FLAIR, Axial DWI, T2 FRFSE, FLAIR, GRE, and SSFSE, and Coronal SSFSE and 3D SPGR over 30 min. Drs. Barnes and Hintz, who will be masked to the acute phase biomarkers data, will interpret imaging utilizing a central reader form that includes white matter scoring according to a widely used classification system, and data regarding location, number, size, and imaging characteristics of lesions. Dr. Bhutani will correlate these data to the acute biomarkers.

Eligibility Criteria:
Inclusion Criteria:

* Patients (GA 24 to ≤34 wks)

Exclusion Criteria:

* Major life-threatening anomalies and diagnosed inborn errors of metabolic disorders
* Attending physician or parent refusal