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To assess the influence of clinical status on the association between total plasma bilirubin and unbound bilirubin on death or adverse neurodevelopmental outcomes at 18–22 months corrected age in extremely low birth weight infants.
Total plasma biirubin and unbound biirubin were measured in 1,101 extremely low birth weight infants at 5±1 day of age. Clinical criteria were used to classify infants as clinically stable or unstable. Survivors were examined at 18–22 months corrected age by certified examiners. Outcome variables were death or neurodevelopmental impairment, death or cerebral palsy, death or hearing loss, and death prior to follow-up. For all outcomes, the interaction between bilirubin variables and clinical status was assessed in logistic regression analyses adjusted for multiple risk factors.
Regardless of clinical status, an increasing level of unbound bilirubin was associated with higher rates of death or neurodevelopmental impairment, death or cerebral palsy, death or hearing loss and death before follow-up. Total plasma bilirubin values were directly associated with death or neurodevelopmental impairment, death or cerebral palsy, death or hearing loss, and death before follow-up in unstable infants, but not in stable infants. An inverse association between total plasma bilirubin and death or cerebral palsy was found in stable infants.
In extremely low birth weight infants, clinical status at 5 days of age affects the association between total plasma and unbound bilirubin and death or adverse neurodevelopmental outcomes at 18–22 months of corrected age. An increasing level of UB is associated a higher risk of death or adverse neurodevelopmental outcomes regardless of clinical status. Increasing levels of total plasma bilirubin are directly associated with increasing risk of death or adverse neurodevelopmental outcomes in unstable, but not in stable infants.
Bilirubin-related neurotoxicity is an important clinical issue in extremely low birth weight (ELBW) infants.1,2 Kernicterus at autopsy has been reported among very low birth weight (VLBW) infants at total serum/plasma bilirubin levels well below those associated with kernicterus in term infants.3–5 This is likely due, in part, to a weaker bilirubin binding to albumin in VLBW infants due to lower serum albumin concentrations and lower bilirubin/albumin binding affinities and capacities.6,7 An association between TPB measurements, the conventional clinical laboratory test used to assess the severity of jaundice, and a variety of poor neurosensory and developmental outcomes among low birth weight survivors has been demonstrated.2,8–12 Because unbound bilirubin (UB) is generally considered to be the form of bilirubin that can cross the blood brain barrier and neuronal membranes to cause neurotoxicity,13,14 it has been suggested that this bilirubin variable would have a better association with neurodevelopmental outcomes in high risk newborns.15 Furthermore, clinical conditions such as respiratory distress, acidosis, hypoxemia, and sepsis may impair bilirubin binding,6,16–19 and many pharmacologic agents administered to sick ELBW infants may also interfere with bilirubin: albumin binding resulting in higher UB levels.20–25 For these reasons, it is important to assess the relationships between intravascular bilirubin variables and adverse outcomes in these vulnerable infants with reference to their clinical status. The objective of this study was to assess the influence of clinical status of ELBW infants at 5 days of age on the association between unbound and total plasma bilirubin levels on death or adverse neurodevelopmental outcomes at 18–22 months corrected age.
From September 2002 through April 2005, the Eunice Kennedy Schriver National Institute of Child Health and Human Development’s Neonatal Research Network conducted a multicenter trial to compare the outcomes of infants with birth weight between 501–1,000 g. treated with aggressive vs conservative phototherapy.2 A subset of infants randomized into this trial whose parents consented to the sampling of an additional 0.5 mL of whole blood during routine blood sampling at 5 ±1 days of age (most likely age of peak bilirubin levels) were enrolled in this study. Blood samples were collected in lithium-heparin gel tubes (CapiJect, Terumo Medical Corporation, Somerset, NJ) immediately centrifuged for at least 1 min at 13,000×g and stored at − 4°C within 2 hours. Samples were labeled with an identifier for storage, with the code correlating to patient identification kept in a confidential logbook. The samples were sent in dry ice and in batches to two centers (Brown and Stanford Universities) for biirubin analysis. TPB and UB concentrations were measured using the UB-A1 analyzer (Arrows, Japan).26 The elapsed time between frozen storage and analysis and the presence of hemolysis and/or turbidity was recorded in the logbook. Because of concerns that hemolysis and turbidity may affect the bilirubin values, we performed an analysis of the data comparing the results of the entire cohort (n=1101) vs. a cohort that excluded 163 samples that were recorded as “hemolyzed” or “turbid” in appearance. The comparison showed similar results (data not shown). Data from the entire cohort is reported.
The clinical status of the study Infants was determined using a set of clinical criteria to label an infant as stable or unstable. Infants were classified as clinically unstable if they had one or more of the following conditions at the time of blood sampling (5±1 days of age): (a) blood pH < 7.15 at the time of blood sampling; (b) blood culture positive sepsis in the prior 24 hours; (c) apnea and bradycardia requiring cardio-respiratory resuscitation (bagging and or intubation) during the previous 24 hours; (d) hypotension requiring pressor treatment during the previous 24 hours; and (e) mechanical ventilation at the time of blood sampling. Infants not having any one of the above clinical conditions were considered as clinically stable.
Survivors were seen in each center’s follow-up clinics at 18–22 months corrected age. Interviews with parents, neurological examination, visual and hearing assessment, and developmental testing were done by trained and certified personnel who were blinded to the bilirubin values, grouping of subjects in the phototherapy trial2. and clinical status. Cerebral palsy (CP) was defined as a non-progressive central nervous system disorder characterized by abnormal muscle tone in at least one extremity and abnormal control of movement or posture with delayed attainment of motor milestones. Infants were classified as having moderate or severe CP if they were able to walk only with assistive devices or unable to walk at all. Hearing outcomes were determined by parent report and confirmed by the examiner as central hearing loss (HL) using audiologic testing including auditory brainstem evoke response. Outcomes assessed included death or neurodevelopmental impairment (NDI), death or CP, death or HL and death before follow up. NDI was defined as an infant having at least one or more of the following findings: moderate to severe CP, blindness (no functional vision in either eye), severe bilateral central HL (requiring bilateral hearing aids), or Bayley Scales of Infant Development II27 demonstrating a mental developmental index (MDI) or psychomotor developmental index (PDI) of <70. Death was included because it is a competing outcome for NDI, CP, and HL. This study was approved by each participating center’s institutional review board.
Separate multiple logistic regression analyses were performed to assess the association between TPB and UB with death or NDI, death or CP, death or hearing loss, and death before discharge. To adjust for potential confounders that might affect outcome and obscure the independent effect of bilirubin, the following were included as independent variables in all regression analyses: birth weight strata (≤750 g and >750 g), center, sex, inborn or outborn, small for gestational age, and race. The interaction between bilirubin variables and clinical status was tested in each regression analysis. If evidence of interaction existed (p-value of <0.1 was used to avoid failure to identify a true interaction), the logistic regression for stable and unstable infants was performed separately and results shown as predicted probability plots. When there was no evidence of statistical interaction for clinical status, logistic regression was done for the entire group with clinical status added as a covariate to demonstrate the adjusted association between levels of UB and TPB with each of the study outcomes. A p-value of <0.05 for the corresponding odds ratios (OR) were considered statistically significant. The statistical analysis was done using SAS software at RTI International, the data-coordinating center for the NICHD Neonatal Research Network (Research Triangle Park, NC).
Of the 1,101 infants enrolled in the study,933 survived and discharged. Of these, 25 died after discharge leaving 908 available for follow up at 18–22 months corrected age.841 of these survivors were seen in the follow up clinics (93% follow up rate). Table 1 shows the demographic, outcomes and bilirubin variables of stable and unstable infants. As expected, the unstable infants were lower in gestational age and birth weight. They were more likely to die before discharge and have higher incidence of death or abnormal neurodevelopmental outcomes as well as UB values when compared with the stable infants. Fifty-eight percent (634/1101) of the study infants were considered clinically unstable because of mechanical ventilator requirement (n=620), hypotension (n=78), acidosis (n=24), and sepsis (n=8). The total number exceeds 634 because of overlapping conditions. Forty two percent (n=467) did not have any of the clinical events and were considered stable. Significant interactions between clinical status and composite outcomes of death or neurodevelopmental abnormalities were not observed for UB (all p-values >0.1), but were observed for TPB (all p-values <0.02). Thus, we depict the probability plots showing the statistical association between UB and composite outcomes of the entire group (stable and unstable, (Fig. 1) and TPB for stable and unstable infants separately (Fig. 2). Increasing levels of UB were associated with increasing incidence of death or NDI death or CP, death or HL and death before follow up (adjusted odds ratio and 95% confidence intervals are listed in Fig. 1). Among unstable infants, increasing levels of TPB were significantly associated with an increasing rate of death or NDI, death or CP, death or HL, and death before follow-up. In stable infants, the association between TPB and death or NDI, death or hearing loss and death before discharge were not significant (all p values >0.05). However, decreasing levels of TPB were significantly associated with an increasing proportion of death or CP (Fig. 2).
A recent report suggests that presence of photoisomers resulting from phototherapy may affect the measurement of UB in vitro.28 Because our study infants were randomized to aggressive or conservative phototherapy by study design of the primary protocol,2 we examined the potential interaction between phototherapy treatment (aggressive or conservative) and bilirubin variables for all outcomes. Statistically significant treatment interaction was not observed (data not shown).
The objective of our study was to assess the influence of clinical status (stable or unstable) of ELBW infants at 5 days of age on the association between plasma total bilirubin and unbound bilirubin on death or abnormal neurodevelopmental outcomes at 18–22 months corrected age. Our observation that UB levels are directly related to poor outcomes – regardless of clinical status – is consistent with the notion that UB is the form of bilirubin responsible for neurotoxicity.13, 14 We also found that infants’ clinical status influenced the association between TPB and death or abnormal neurodevelopmental outcomes in that there was a direct relationship between TPB with study outcomes in unstable but not in stable infants. To our knowledge, the differences between stable and unstable infants in regards to the relationship between TPB and adverse outcomes have not been identified previously. This observation may in part be due to a higher UB in the clinically ill (unstable) ELBW infants (table 1) as a result of reduced bilirubin binding capacity and affinity in unstable infants.7 The data suggest that using UB levels may be useful in directing clinical decisions as to when to initiate phototherapy or perform exchange transfusion since its correlation is independent of clinical status. Unfortunately, the methodology for UB measurements is currently not available widely. The main measure for directing clinical decisions for the initiation of phototherapy is still TPB levels. The association of increasing level of TPB with adverse outcomes in unstable infants suggests that we may have to be more aggressive in lowering the plasma/serum bilirubin in this population.
An interesting and unexpected finding in our study is the inverse relationship between TPB and death or CP in stable infants. As shown in Figure 3, a decreasing level of TPB is associated with an increasing rate of death or CP. Our interpretation of this paradoxical observation is complex. Bilirubin has been shown to have an antioxidant property29–32 that may provide protection against oxidative injury in these infants. Thus, a lower level of bilirubin might have contributed to a higher rate of death or CP because of less protection against oxidative injury. An alternative and more insidious possibility is that TPB is lower in some clinically stable infants because of weaker bilirubin binding creating a circumstance reminiscent of the sulfisoxazole experience.34 if this were the case; the lower TPB would be a reflection of bilirubin movement into tissue, such as the brain, causing neurotoxicity. To verify this speculation, we calculated the ratio of UB to TPB as the surrogate for binding characteristics. The UB to TPB ratio (μg/dL/mg/dL) for stable Infants with or without death or CP was 0.053±0.03 (n=40) and 0.045±0.02 (n= 387), respectively, p=0.12. These data suggest that poor binding is probably not the explanation for the intriguing association between TPB and death or CP. In the final analysis, the data from our current study, while provocative, are insufficient for a definitive explanation of this curious association between decreasing levels of TPB and increased rates of death or CP in stable infants. While our main trial has shown that aggressive phototherapy that keeps the TPB at a lower level than that following conservative phototherapy may be beneficial for ELBW infants,2 it may be appropriate to avoid being too aggressive resulting in a TPB level that is too low in a clinically stable infant.
The National Institutes of Health and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) provided grant support for the Neonatal Research Network’s Phototherapy Study (Recruitment 2002–2005).
Data collected at participating sites of the NICHD Neonatal Research Network (NRN) were transmitted to RTI International, the data coordinating center (DCC) for the network, which stored, managed and analyzed the data for this study. On behalf of the NRN, Dr. Abhik Das (DCC Principal Investigator) and Ms. Rebecca L. Perritt (DCC Statistician) had full access to all the data in the study and take responsibility for the integrity of the data and accuracy of the data analysis.
We are indebted to our medical and nursing colleagues and the infants and their parents who agreed to take part in this study. The following investigators, in addition to those listed as authors, participated in this study:
NRN Chairs – Alan Jobe, MD PhD, University of Cincinnati (2001–2006); Michael S. Caplan, MD, Northwestern University (2006–2011).
Brown University Women & Infants Hospital of Rhode Island (U10 HD27904) – William Oh, MD; Abbot R. Laptook, MD; Betty R. Vohr, MD; Angelita Hensman, BSN RNC; Lucy Noel RN.
Case Western Reserve University Rainbow Babies & Children’s Hospital (GCRC M01 RR80, U10 HD21364) – Avroy A. Fanaroff, MD; Michele C. Walsh, MD MS; Deanne Wilson-Costello, MD; Nancy S. Newman, BA RN; Bonnie S. Siner, RN.
Duke University University Hospital, Alamance Regional Medical Center, and Durham Regional Hospital (GCRC M01 RR30, CCTS UL1 RR24128, U10 HD40492) – Ronald N. Goldberg, MD; C. Michael Cotten, MD; Ricki Goldstein, MD; Kathy Auten, BS; Melody Lohmeyer, RN.
Emory University Children’s Healthcare of Atlanta, Grady Memorial Hospital, and Emory Crawford Long Hospital (GCRC M01 RR39, U10 HD27851) – Barbara J. Stoll, MD; Ira Adams-Chapman, MD; Ellen Hale, RN BS.
Indiana University Indiana University Hospital, Methodist Hospital, Riley Hospital for Children, and Wishard Health Services (GCRC M01 RR750, U10 HD27856) – James A. Lemons, MD; Brenda B. Poindexter, MD MS; Anna M. Dusick, MD; Diana D. Appel, RN BSN; Dianne Herron, RN; Lucy Miller, RN BSN CCRC; Leslie Richard, RN.
National Institute of Child Health and Human Development – Linda L. Wright, MD; Rosemary D. Higgins, MD; Elizabeth M. McClure, MEd.
RTI International (U01 HD36790) – W. Kenneth Poole, PhD; Abhik Das, PhD; Betty K. Hastings; Elizabeth McClure, MEd; Jamie Newman; Rebecca L. Perritt, MS; Qing Yao, PhD; Carolyn Petrie Huitema, MS; Kristin Zaterka-Baxter, RN.
Stanford University Lucile Packard Children’s Hospital (GCRC M01 RR70, U10 HD27880) – David K. Stevenson, MD; Krisa P. Van Meurs, MD; Susan R. Hintz, MD MS; M. Bethany Ball, BS CCRC.
University of Alabama at Birmingham Health System and Children’s Hospital of Alabama (GCRC M01 RR32, U10 HD34216) – Waldemar A. Carlo, MD; Namasivayam Ambalavanan, MD; Myriam Peralta-Carcelen, MD; Monica V. Collins, RN BSN; Shirley S. Cosby, RN BSN; Vivien Phillips, RN BSN.
University of California – San Diego Medical Center and Sharp Mary Birch Hospital for Women (U10 HD40461) – Neil N. Finer, MD; Yvonne E. Vaucher, MD MPH; Maynard R. Rasmussen MD; David Kaegi, MD; Kathy Arnell, RN; Clarence Demetrio, RN; Martha G. Fuller, RN MSN; Chris Henderson, RCP CRTT; Wade Rich, BS RRT CCRC.
University of Cincinnati University Hospital, Cincinnati Children’s Hospital Medical Center, and Good Samaritan Hospital (GCRC M01 RR8084, U10 HD27853) – Edward F. Donovan, MD; Kurt Schibler, MD; Jean Steichen, MD; Barb Alexander, RN; Cathy Grisby, BSN CCRC; Marcia Mersmann, RN; Holly Mincey, RN; Jody Shively, RN; Teresa Gratton, PA.
University of Miami Holtz Children’s Hospital (GCRC M01 RR16587, U10 HD21397) – Charles R. Bauer, MD; Shahnaz Duara, MD; Ruth Everett-Thomas, RN BSN; Silvia Frade Eguaras, MS; Silvia Hiriart-Fajardo, MD.
University of Rochester Golisano Children’s Hospital at Strong (GCRC M01 RR44, U10 HD40521) – Ronnie Guillet, MD PhD; Dale L. Phelps, MD; Gary Myers, MD; Linda Reubens, RN; Diane Hust, RN PNP; Rosemary Jensen; Erica Burnell, RN.
University of Tennessee (U10 HD21415) – Sheldon B. Korones, MD; Kim Yolton, PhD; Marilyn Williams, LCSW.
University of Texas Southwestern Medical Center at Dallas Parkland Health & Hospital System and Children’s Medical Center Dallas (GCRC M01 RR633, U10 HD40689) – Walid A. Salhab, MD; Pablo J. Sanchez, MD; Charles R. Rosenfeld, MD; Roy J. Heyne, MD; Jackie Hickman, RN; Gay Hensley, RN; Nancy A. Miller, RN; Janet Morgan, RN.
University of Texas Health Science Center at Houston, Medical School, Children’s Memorial Hermann Hospital, and Lyndon B. Johnson General Hospital (CCTS KL2 RR24149, CCTS UL1 RR24148, U10 HD21373) – Jon E. Tyson, MD MPH; Kathleen Kennedy, MD MPH; Brenda H. Morris, MD; Pamela J. Bradt, MD MPH; Patricia W. Evans, MD; Laura L. Whiteley, MD; Esther G. Akpa, RN BSN; Patty A. Cluff, RN; Anna E. Lis, RN BSN; Georgia E. McDavid, RN; Claudia Y. Franco, RN BNS MSN NNP; Maegan Currence, RN; Nora I. Alaniz, BS; Patti L. Tate, RRT; Sharon L. Wright, MT(ASCP).
Wake Forest University Baptist Medical Center, Brenner Children’s Hospital, and Forsyth Medical Center (GCRC M01 RR7122, U10 HD40498) – T. Michael O’Shea, MD MPH; Lisa K. Washburn, MD; Nancy J. Peters, RN CCRP; Barbara G. Jackson, RN BSN.
Wayne State University Hutzel Women’s Hospital and Children’s Hospital of Michigan (U10 HD21385) – Seetha Shankaran, MD; Yvette Johnson, MD; Athina Pappas, MD; Rebecca Bara, RN BSN; Geraldine Muran, RN BSN; Deborah Kennedy, RN BSN.
Yale University Yale-New Haven Children’s Hospital (GCRC M01 RR6022, U10 HD27871) – Richard A. Ehrenkranz, MD; Patricia Gettner, RN; Harris Jacobs, MD; Christine Butler, MD; Pat Cervone, RN; Monica Konstantino, RN BSN; Elaine Romano, RN BSN.00 g.