Rethinking the Assessment of Hyperbilirubinemia in Preterm Infants

Rethinking the assessment of hyperbilirubinemia

in preterm infants

by

Thivia Jegathesan

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Institute of Medical Science University of Toronto

© Copyright by Thivia Jegathesan 2021

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Rethinking the assessment of hyperbilirubinemia in preterm infants

Thivia Jegathesan

Doctor of Philosophy

Institute of Medical Science University of Toronto

2021

Abstract

Preterm infants born at 240/7 to 356/7 weeks’ gestation are at greater risk of developing acute

bilirubin encephalopathy (ABE) and chronic bilirubin encephalopathy (CBE). However,

guidelines for the assessment of hyperbilirubinemia are consensus-based, and systematically

derived pre-treatment total serum bilirubin (TSB) percentiles are lacking for preterm infants.

Preterm infants who undergo repeat TSB testing are subjected to greater stress, pain, and risk

of anemia.

To address the aforementioned issues, two multi-site retrospective (Study 1 and 2), and one

multi-site prospective (Study 3), cohort studies were conducted at three neonatal intensive care

units (NICUs) in Ontario. Study 1 generated pre-treatment TSB percentile levels among infants

born at 290/7 to 356/7 weeks’ gestation, using quantile regression. Study 2 generated pre-

phototherapy TSB percentiles levels at 24 hours of age among infants born at 240/7 to

286/7 weeks’ gestation and contrasted those pre-phototherapy TSB percentiles with existing

consensus-based thresholds produced by Maisels. Study 3 investigated the agreement between

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transcutaneous bilirubin (TcB) and TSB measurements among preterm infants born at 240/7 to

356/7 weeks, overall, by receipt of phototherapy, by anatomical site of measurement and by

self-reported maternal ethnicity.

Study 1 produced hour-specific pre-treatment TSB levels at the 40th, 75th and 95th

percentiles, with corresponding curves by gestational week at birth. Study 2 observed

considerably higher statistically derived pre-phototherapy TSB percentile levels at 24 hours of

age at the 75th and 95th percentile than Maisels’ consensus-based TSB thresholds. Study 3

observed higher agreement between TcB and TSB values among preterm infants born at

330/7 to 356/7 weeks’ gestation, prior to phototherapy, whether measured at the forehead or

sternum. TcB-TSB agreement was not impacted by ethnicity.

The results of this thesis provide new information about bilirubin measurement in preterm

infants. Experts and health policy makers may choose to use these data to inform evidence-

based guidelines in the assessment (and management) of hyperbilirubinemia in preterm

infants.

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Acknowledgements

I would like to extend my sincere thanks to my supervisor Dr. Michael Sgro. Thank you for your

guidance, support, and patience throughout my doctoral education. Thank you for providing

me with numerous educational opportunities, for being an advocate for my career growth, and

for believing in me always, even during my moments of doubt.

I also owe a great deal of gratitude to Dr. Joel Ray, my co-supervisor. Thank you for your

ongoing words of encouragement, incredible attention to detail, constructive criticism,

assistance throughout the analytical and writing process, and invaluable feedback and revisions

to all three of my manuscripts.

I would also like to thank my thesis committee members, Dr. Howard Berger and Dr. Robin

Hayeems. Thank you both for your valuable guidance, support and suggestions throughout my

doctoral education. Dr. Vibhuti Shah, Dr. Douglas Campbell and Dr. Tony Barozzino: thank you

for checking in on me, supporting my research and providing me with valuable feedback.

I would also like to thank Dr. Vinod Bhutani and his team at Stanford University. Thank you for

providing me with valuable training and guidance to complete my graduate work and continue

working in the field of neonatal hyperbilirubinemia.

Thank you to Lori Tenuta and the Women’s and Children’s Health Program at St. Michael’s

Hospital, Unity Health Toronto, for providing me with the support and space to complete my

PhD.

Finally, I would like to thank my parents (Thirunavukkarasu and Ranymalar Jegathesan), brother

(Jegan Jegathesan), sister-in-law (Nishani Umasuthan), nephew (Jayden Jegathesan), and niece

(Maya Jegathesan) for all your emotional and moral support.

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Contributions

This thesis was prepared by Thivia Jegathesan (the doctoral candidate), who takes full

responsibility for its contents. Thivia, under the supervision and assistance of her supervisors;

Dr. Michael Sgro and Dr. Joel Ray and thesis committee (Dr. Robin Hayeems and Dr. Howard

Berger) formulated the research question and objectives, designed the study protocols, secured

research ethics approval, collected and managed relevant data, developed and executed the

analytical plan, and drafted and revised all three manuscripts according to peer reviewers’

comments.

Dr. Douglas Campbell and Dr. Vibhuti Shah assisted with study protocol designs, research ethics

approvals, data collection supervision, analyses and all three final manuscripts. Dr. Jennifer

Twiss also assisted with research ethics approvals and data collection supervision.

Dr. Vinod Bhutani assisted with the study design, analysis and final manuscript for Chapter 3.

Dr. Charles Keown-Stoneman provided guidance with the analytical plan and analyses for

Chapters 3 and 4.

Saisujani Rasiah, Gayathri Visvanathaiyer, Shangari Baleswaran, Melissa Librach, Jeffrey Antwi,

Maria Casalino, Aaditeya Jhaveri, Dishaben Prajapati, Ilham Elias and Helen Zheng assisted with

data collection and data entry for Chapters 3 and 4.

Ashvinie Sritharan, Geoffrey Travis, Aasha Gnanalingam, Mary Debono, Sureka Selvakumaran

and Heet Sheth assisted with data collection and data entry for Chapter 5.

Dr. Nithi Fernandes provided feedback for Chapters 1 and 2.

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Dr. Tony Barozzino and Dr. Douglas Campbell provided feedback for Chapters 1, 2, 6, 7 and 8

of this thesis.

Maria Ghobrial, Nazi Torabi and Alexander Davidson assisted with the review described in

Appendix 8-1.

Drager Medical System provided funding support for all studies. Drager was not involved in the

design of the thesis proposal, data collection, data analysis, data interpretation or manuscript

preparation. All data interpretation and conclusions reported in the thesis were at the sole

discretion of Thivia Jegathesan and the co-authors of the associated manuscripts.

The list of eligible infants for Chapter 3 and 4 were retrieved from the Canadian Neonatal

Network (CNN) and Better Outcomes Registry and Network (BORN). CNN and BORN were not

involved in the data collection, data interpretation or data analysis of the thesis.

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Table of Contents

Acknowledgements......................................................................................................................... iv

Contributions ................................................................................................................................... v

Table of Contents ........................................................................................................................... vii

List of Abbreviations ...................................................................................................................... xii

List of Tables.................................................................................................................................. xiii

List of Figures ................................................................................................................................. xv

List of Appendices ........................................................................................................................ xvii

Chapter 1. Introduction and Definitions ..........................................................................................1

1.1 General introduction............................................................................................................2

1.2 Thesis format .......................................................................................................................3

1.3 Bilirubin ................................................................................................................................4

1.4 Effects on conversion and excretion of bilirubin in newborns ............................................5

1.4.1 Red blood cell production and breakdown .............................................................5

1.4.2 Bilirubin-albumin binding.........................................................................................6

1.4.3 Unconjugated and conjugated bilirubin ..................................................................6

1.5 Harms of bilirubin to the neonatal brain .............................................................................7

1.5.1 Neonatal blood brain barrier ...................................................................................7

1.5.2 Bilirubin crossing the blood-brain barrier..............................................................10

1.6 Definitions of key clinical terms .........................................................................................11

1.6.1 Neonatal hyperbilirubinemia .................................................................................11

1.6.2 Acute and chronic bilirubin encephalopathy.........................................................12

1.7 Risk factors for severe hyperbilirubinemia ........................................................................13

1.8 Prevention of severe hyperbilirubinemia ..........................................................................14

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1.8.1 Phototherapy………………………………………………………………………………………… ...... ……14

1.8.2 Exchange transfusion .............................................................................................15

1.8.3 Intravenous Immunoglobulins ...............................................................................16

Chapter 2. Assessment of hyperbilirubinemia in preterm infants ...............................................17

2.1 Prematurity ........................................................................................................................18

2.2 Hyperbilirubinemia in preterm infants ..............................................................................18

2.2.1 Neurotoxic effects of significant hyperbilirubinemia in preterm infants .............19

2.2.2 ABO incompatibility in preterm infants .................................................................19

2.2.3 Feeding in preterm infants ....................................................................................20

2.2.4 Summary of hyperbilirubinemia in preterm infants ..............................................20

2.3 Overview of the assessment of hyperbilirubinemia ..........................................................21

2.3.1 Term and near-term infants...................................................................................21

2.3.2 Preterm infants ......................................................................................................22

2.4 Review of currently published pre-treatment TSB levels and phototherapy thresholds in preterm infants by prematurity ...................................................................23

2.4.1 Published pre-treatment TSB levels.......................................................................23

2.4.2 TSB thresholds used for phototherapy initiation ..................................................24

2.4.3 Review of the frequency of phototherapy in preterm infants ..............................29

2.4.4 Summary of pre-treatment TSB levels in preterm infants and relevance.............30

2.5 Total serum bilirubin and transcutaneous bilirubin measurements .................................33

2.5.1 Total serum bilirubin tests .....................................................................................33

2.5.2 Transcutaneous bilirubin tests...............................................................................35

2.5.3 Summary of TcB measurements in preterm infants and relevance ......................40

2.6 Overall summary of literature review and significance .....................................................41

2.7 Overall research aims, objectives and hypotheses............................................................42

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Chapter 3. Hour-specific total serum bilirubin percentiles for infants born at 29 to 35 weeks’

gestation……………….. .................................................................................................................46

3.1 Abstract ..............................................................................................................................47

3.2 Introduction .......................................................................................................................48

3.3 Methods .............................................................................................................................48

3.3.1 Data analyses .........................................................................................................49

3.3.2 Sample size calculation ..........................................................................................50

3.4 Results ................................................................................................................................51

3.4.1 All Infants ...............................................................................................................51

3.4.2 By subsequent receipt of phototherapy ................................................................52

3.4.3 By degree of prematurity.......................................................................................52

3.4.4 By feeding type ......................................................................................................53

3.4.5 Peak pre-treatment total serum bilirubin by hemolysis........................................53

3.5 Discussion...........................................................................................................................54

3.6 Conclusion ..........................................................................................................................56

3.7 Tables .................................................................................................................................57

3.8 Figures ................................................................................................................................61

Chapter 4. Pre-phototherapy total serum bilirubin levels in extremely preterm infants .............69

4.1 Abstract ..............................................................................................................................70

4.2 Introduction .......................................................................................................................71

4.3 Methods .............................................................................................................................72

4.3.1 Data analyses .........................................................................................................73


4.4 Results ................................................................................................................................74

4.4.1 Pre-phototherapy TSB levels..................................................................................75

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4.4.2 Pre-phototherapy TSB levels by gestational age groups .......................................75

4.4.3 Derived pre-phototherapy TSB percentiles compared with Maisels’ published thresholds ..............................................................................................................76

4.4.4 Phototherapy administration among all infants....................................................76

4.5 Discussion...........................................................................................................................77

4.6 Conclusion ..........................................................................................................................79

4.7 Tables .................................................................................................................................80

4.8 Figures ................................................................................................................................85

Chapter 5. Transcutaneous vs. total serum bilirubin measurements in preterm infants .............88

5.1 Abstract ..............................................................................................................................89

5.2 Introduction .......................................................................................................................90

5.3 Methods .............................................................................................................................91

5.3.1 Data analyses .........................................................................................................92


5.4 Results ................................................................................................................................93

5.4.1 TcB measurement before and after phototherapy, all preterm infants ..............94

5.4.2 Sites of TcB measurement .....................................................................................94

5.4.3 TcB measurement by gestational age groups........................................................95

5.4.4 Influence of ethnicity on TcB .................................................................................95

5.4.5 Performance of TcB at pre-defined TSB levels ......................................................95

5.5 Discussion...........................................................................................................................96

5.6 Conclusion ..........................................................................................................................99

5.7 Tables ...............................................................................................................................100

5.8 Figures ..............................................................................................................................103

Chapter 6. General Discussion .....................................................................................................116

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6.1 Discussion outline ............................................................................................................117

6.2 Summary of main findings ...............................................................................................117

6.2.1 Study 1: Hour-specific total serum bilirubin percentiles for infants born at 29

to 35 weeks’ gestation .........................................................................................117

6.2.2 Study 2: Pre-phototherapy total serum bilirubin levels in extremely preterm

infants…................................................................................................................119

6.2.3 Study 3: Transcutaneous vs. total serum bilirubin measurements in preterm infants…................................................................................................................120

6.3 Strengths and Limitations ................................................................................................121

6.3.1 Overall strengths ..................................................................................................121

6.3.2 Overall limitations ................................................................................................124

6.4 Overall research impact ...................................................................................................127

6.4.1 Assessment of hyperbilirubinemia ......................................................................127

6.4.2 Inform health policy efforts in professional societies .........................................130

6.4.3 Research ...............................................................................................................131

Chapter 7. Conclusions…..............................................................................................................132

7.1 Conclusions ......................................................................................................................133

Chapter 8. Future directions ........................................................................................................134

8.1 Future Directions .............................................................................................................135

8.1.1 Assessment of hyperbilirubinemia ......................................................................135

8.1.2 Management of hyperbilirubinemia ....................................................................138

8.2 Summary of future research ............................................................................................140

8.3 Appendix ..........................................................................................................................141

8.3.1 Short-term effects of phototherapy ....................................................................142

8.3.2 Long term side-effects of phototherapy..............................................................145

References....................................................................................................................................147

xii

List of Abbreviations

ABE Acute Bilirubin Encephalopathy

AAP American Academy of Pediatrics

BORN Better Outcomes Registry Ontario

BIND Bilirubin Induced Neurological Dysfunction

CNN Canadian Neonatal Network

CPS Canadian Pediatric Society

CBE Chronic Bilirubin Encephalopathy

CCC Concordance Correlation Coefficient

ET Exchange Transfusion

ELBW Extremely Low Birthweight

HDN Hemolytic Disease of the Newborn

IVIG Intravenous Immunoglobulins

LED Light Emitting Diode

MRI Magnetic Resonance Imaging

NICU Neonatal Intensive Care Unit

NADPH Nicotinamide Adenine Dinucleotide Phosphate

ROR Rate of Rise

Rh Rhesus

TcB Transcutaneous Bilirubin

TSB Total Serum Bilirubin

UDPGT UDP- glucuronyl transferase

VLBW Very Low Birthweight

xii i

List of Tables

Table 2-1. Summary of published total serum bilirubin thresholds based on gestational age at

birth by country and author.......................................................................................................... 31

Table 2-2 Total serum bilirubin thresholds used in two neonatal intensive care units in

Michigan, United States by year and gestational age at birth.87.................................................. 32

Table 3-1. Neonatal and maternal characteristics of 2549 preterm infants included in the study

by prematurity groups. All data are presented as a number (%) unless otherwise indicated. .... 57

Table 3-2. Estimated pre-treatment total serum bilirubin percentiles at birth, and the estimated

hours of age at peak total serum bilirubin at the 40th, 75th and 95th percentiles, by subsequent

receipt of phototherapy, and by gestational age. ........................................................................ 58

Table 3-3. Peak total serum bilirubin concentration within the first 72 hours after birth, as well

as the timing of that peak, by neonatal factors............................................................................ 59

Table 3-4. Neonatal characteristics by proportion of infants subsequently administered

phototherapy. ............................................................................................................................... 60

Table 4-1. Characteristics of 642 extremely preterm infants born at 240/7 to 286/7 weeks’

gestation included in the study. All data are presented as a number (%) unless otherwise

indicated........................................................................................................................................ 80

Table 4-2. Estimated pre-phototherapy total serum bilirubin concentration at 24 hours of age,

rate of rise of total serum bilirubin from birth, change in rate of rise of total serum bilirubin

from birth onward and, the hours of age of peak total serum bilirubin at the 40 th, 75th and 95th

percentiles..................................................................................................................................... 81

xiv

Table 4-3. Difference between the current study’s derived pre-phototherapy total serum

bilirubin percentile at 24 hours of age and the total serum bilirubin thresholds published by

Maisels18, presented by gestational age....................................................................................... 82

Table 4-4. Proportion of 183 infants subsequently started on phototherapy below Maisels’

published total serum bilirubin threshold18, shown in 12-hour intervals since birth. All data are

presented as number (%).............................................................................................................. 83

Table 4-5. Proportion of the 279 infants started on phototherapy at 24 to 30 hours of age in the

current study, at a total serum bilirubin level below Maisels’ published18, or below the current

study’s generated 75th and 95th percentiles at 24 hours of age................................................. 84

Table 5-1. Characteristics of the 296 preterm infants included in the study. All data are shown

as number (%) unless otherwise indicated. ................................................................................ 100

Table 5-2. Agreement between measurement of transcutaneous bilirubin measurement (TcB)

and total serum bilirubin (TSB) among all 296 preterm infants included in the study. Shown are

measures of agreement stratified by anatomical site of TcB assessment, gestational age at birth

and receipt of phototherapy....................................................................................................... 101

Table 5-3. Test characteristics of transcutaneous bilirubin measurement at recommended total

serum bilirubin cut-points........................................................................................................... 102

xv

List of Figures

Figure 1-1 Risk factors and process of unbound bilirubin crossing the blood brain barrier in

neonates published by Watcho and Tiribelli25................................................................................ 9

Figure 3-1. Flow diagram of included preterm infants born at 290/7 to 356/7 weeks’ gestation,

from January 2013 to June 2017................................................................................................... 61

Figure 3-2. Hour-specific pre-treatment total serum bilirubin percentile-based curves among

preterm infants born at 29 to 35 weeks’ gestation (A) overall (n=2549), (B) among infants not

subsequently administered phototherapy (n=696), and (C) among infants subsequently

administered phototherapy (n=1853). To convert total serum bilirubin levels to mg/dL divide by

17.1................................................................................................................................................ 62

Figure 3-3. Hour-specific pre-treatment total serum bilirubin percentile-based curves among

preterm infants born at (A) 29 to 32 weeks’ gestation (n=1120), and (B) 33 to 35 weeks’

gestation (n=1429). To convert total serum bilirubin levels to mg/dL divide by 17.1. ................ 65

Figure 3-4. Pre-treatment total serum bilirubin percentile net differences between infants born

at 33 to 35 weeks’ gestation minus those born at 29 to 32 weeks’ gestation. To convert mean

total serum bilirubin differences to mg/dL divide by 17.1. .......................................................... 67

Figure 3-5. Mean pre-treatment total serum bilirubin net differences between infants born at

33 to 35 weeks’ gestation minus those born at 29 to 32 weeks’ gestation. To convert mean total

serum bilirubin differences to mg/dL divide by 17.1. ................................................................... 68

Figure 4-1. Flow diagram of included extremely preterm infants born at 240/7 to 286/7 weeks’

gestation, from January 2013 to June 2017 ................................................................................. 85

xvi

Figure 4-2. Hour-specific pre-phototherapy total serum bilirubin percentile curves estimated

based on data from 642 extremely preterm infants born at 240/7 to 286/7 weeks’ gestation. To

convert total serum bilirubin levels to mg/dL divide by 17.1 ....................................................... 86

Figure 4-3. Proportion of the 615 extremely preterm infants born at 240/7 to 286/7 weeks’

gestation who subsequently received phototherapy after birth. ................................................ 87

Figure 5-1. Flow diagram of participant recruitment. ................................................................ 103

Figure 5-2. Bland-Altman plots of paired transcutaneous bilirubin (TcB) and total serum bilirubin

(TSB) measurements among all 296 preterm infants at 24-35 weeks’ gestation, measured

overall (A), prior to (B), and after (C), initiation of phototherapy .............................................. 104

Figure 5-3. Bland-Altman plot of paired forehead transcutaneous bilirubin (TcB) and total serum

bilirubin (TSB) measurements among neonates born at (A) 24-28 weeks’ gestation, (B) 29-32

weeks’ gestation and (C) 33-35 weeks’ gestation. ..................................................................... 107

Figure 5-4. Overall Bland-Altman plot of paired transcutaneous bilirubin (TcB) and total serum

bilirubin (TSB) measurements among preterm infants born at 24-35 weeks’ gestation whose

mother’s ethnicity is (A) African-Caribbean, (B) Caucasian, (C) Southeast Asian, and (D) South

Asian ............................................................................................................................................ 110

xvii

List of Appendices

Appendix 8-1. Review of the side effects of phototherapy...................................................... 141

1

Chapter 1 . Introduction and Definitions

2

1.1 General introduction

Neonatal hyperbilirubinemia, also known as newborn jaundice, is a common condition among

infants.1-3 Although transient physiological rise of bilirubin is self-limiting in the majority of

newborns, unmonitored and untreated hyperbilirubinemia has led to devastating, irreversible

impacts on newborns, including but not limited to, severe neurological damage, neuro-

developmental impairment and even death.4-6 Prior to 1950, severe complications from severe

neonatal hyperbilirubinemia were identified to be associated with hemolytic disease of the

newborn due to Rhesus (Rh) disease.4,7,8 After the introduction of Rh prenatal screening and Rh

prophylaxis, mortality and morbidities from severe hyperbilirubinemia associated with Rh

disease were significantly reduced.7,8 In the 1980’s and 1990’s there was a resurgence of

chronic sequelae from severe hyperbilirubinemia due to changes in management of

hyperbilirubinemia.3,9-12 This resulted in a re-review of the assessment and management of

severe hyperbilirubinemia and its associated risk factors.5,6

Subsequent research has focused on understanding the natural profile of bilirubin in newborns

and the identification of risk factors in infants experiencing complications of severe neonatal

hyperbilirubinemia.3,13,14 This reassessment has led to the introduction of treatments and

preventative strategies to reduce the incidence of severe hyperbilirubinemia and chronic

sequelae, such as kernicterus.1,3,15

Preterm infants are at higher risk of severe hyperbilirubinemia and its chronic sequelae

compared to term and near-term infants.16,17 In part, this is because the preterm brain is more

vulnerable to the toxic effects of severe hyperbilirubinemia. In addition, preterm infants

experience decreased enteral motility, higher red blood cell breakdown and have an immature

liver, putting them at higher risk of experiencing severe hyperbilirubinemia.4,16-18 Despite the

increased risk among preterm infants, the current approach to measuring bilirubin in these

newborns is highly criticized and unclear.16,17 Total serum bilirubin (TSB) thresholds

recommended for initiating phototherapy have been based on expert opinion as opposed to

studying a large cohort of preterm infants.18-20 The paucity of pre-treatment TSB levels in

3

preterm infants born at <36 weeks’ gestation has resulted in guidelines for the assessment of

hyperbilirubinemia based on concensus.18-20 In addition, repeated TSB testing in preterm

infants has been associated with these infants experiencing pain, stress and anemia.21,22 In term

and near-term infants transcutaneous bilirubin devices (TcB), a non-invasive method for

measuring bilirubin, have been used to decrease the harms of repeated TSB testing. This

method, however, has not been approved for preterm infants in Canada.2

We require a better understanding of the measurement of bilirubin in preterm infants to

optimize the assessment and management of hyperbilirubinemia in this cohort. The purpose of

this thesis is to provide new information on the current measurement of bilirubin in preterm

infants. This was achieved by: 1) defining hyperbilirubinemia in preterm infants; 2) generating

pre-treatment TSB levels in preterm infants; and 3) assessing a non-invasive method to

measuring TSB in preterm infants.

1.2 Thesis format

This thesis is presented in a “multiple paper,” format. It includes a general introduction to key

concepts in neonatal hyperbilirubinemia, a description of hyperbilirubinemia in preterm infants,

a review of the literature about the measurement of bilirubin in preterm infants and three

peer-reviewed manuscripts. There is also a final overall discussion of the thesis findings, with a

proposition for future directions and implications.

Enclosed within Chapter 1 are definitions of key terms related to hyperbilirubinemia.

Chapter 2 offers a review of the assessment of hyperbilirubinemia in preterm infants,

substantiating the purpose of the thesis. The first part of chapter 2 provides a description of

hyperbilirubinemia in preterm infants. The second part reviews two aspects of the

measurement of bilirubin in preterm infants. First, a review of currently published pre-

treatment TSB levels in preterm infants was conducted. Second, a review of the harms of

4

repeated TSB testing and an assessment of a non-invasive alternative to measuring bilirubin

was conducted. Chapter 2 forms the basis for the three subsequent manuscripts included in the

thesis.

Chapters 3 and 4 are self-contained, reformatted peer-reviewed manuscripts submitted to the

journal Neonatology and Paediatrics and Child Health, respectively. Chapter 5 is a self-

contained, published manuscript in the journal Neonatology.

In Chapter 6, the discussions from chapters 3 to 5 are further expanded, followed by a

summary of the implications of the thesis. Chapter 7 provides an overarching conclusion of the

thesis. Finally, Chapter 8, summarizes additional research that is currently underway based on

the results of the thesis and plans for future research.

1.3 Bilirubin

Bilirubin is a normal waste product of hemoglobin breakdown. Hemoglobin breaks down into

two molecules, heme and globin. Bilirubin is the byproduct of heme catabolism via two

enzymes.23 Heme oxygenase converts heme to biliverdin, then a reduced nicotinamide adenine

dinucleotide phosphate (NADPH)-dependent biliverdin reductase converts biliverdin to

bilirubin.23 Bilirubin then binds to albumin (referred to as unconjugated bilirubin) and is

transported to the liver where it is converted to conjugated bilirubin via the enzyme UDP

glucuronyl transferase (UDPGT).23 The conjugated bilirubin is then excreted by the biliary

system.23

TSB is made up of the multiple components of bilirubin but predominantly unconjugated and

conjugated bilirubin. Some other forms of bilirubin that make up TSB include: beta bilirubin

(monoconjugated bilirubin), gamma bilirubin (di-conjugated bilirubin) and delta bilirubin

(protein bound bilirubin).24 In newborn infants, these latter forms of bilirubin are not felt to be

clinically significant and are not measured separately. They are included in an overall measure

5

of both unconjugated and conjugated bilirubin. For the purpose of this thesis , TSB will be

used as an encompassing term of unconjugated and conjugated bilirubin, as this is the most

commonly used marker to manage care in newborns.25

1.4 Effects on conversion and excretion of bilirubin in newborns

1.4.1 Red blood cell production and breakdown

Among newborn infants, normal red blood cell turnover and break down accounts for 75% of

the daily production of bilirubin, while the remaining 25% of bilirubin comes from premature

red cell break down and heme breakdown from non-hemoglobin molecules (i.e. myoglobin).23

Due to the short life span of red blood cells in newborns, coupled with the increase in red blood

cell production shortly after birth, the rate of red blood cell breakdown usually increases shortly

after birth at a rate similar to that of bilirubin-albumin binding and bilirubin conjugation.23,26

Among preterm infants the life span of red blood cells is even shorter resulting in a higher rate

of physiological red blood cell turnover.27,28 These processes contribute to an overall increased

risk of hyperbilirubinemia among preterm infants.

Infants experiencing hemolytic disease of the newborn (HDN), may experience an even faster

rate of red blood cell breakdown, surpassing the rate of bilirubin binding and bilirubin

conjugation.23,26 Rh incompatibility (also known as Rh disease) is a major worldwide cause of

HDN in newborns. This occurs when mom is Rh negative and baby is Rh positive, and the

mother has anti-D (or other antibodies) primarily from exposure in a previous pregnancy,

resulting in increased red cell breakdown, also known as hemolysis.7,8,29

Another, more common, reason for hemolysis in the developed world is ABO incompatibility.

ABO incompatibility occurs in infants with blood group A or B born to mothers with blood group

O. Although common, HDN from ABO incompatibility is usually less severe than Rh disease.29,30

6

Infants with Rh disease or ABO incompatibility are at higher risk of developing severe

hyperbilirubinemia. 13,14

1.4.2 Bilirubin-albumin binding

Bilirubin-albumin binding capacity refers to serum albumin’s capacity to bind with bilirubin,

while bilirubin-albumin binding affinity refers to the strength of the bilirubin-albumin bond.23

The higher the level of serum albumin, the higher the bilirubin-albumin binding capacity.

Bilirubin that is not bound to albumin is referred to as unbound, or free, bilirubin.23,25,31 Among

healthy term and near-term infants free bilirubin levels are dictated by the bilirubin-albumin

binding capacity, which is usually not disrupted at high levels of albumin.23,25,31 In preterm

infants, however, a lower bilirubin-albumin binding affinity may result in a decrease in bilirubin-

albumin binding capacity, thus increasing levels of unbound bilirubin.23,25,31 This is largely due to

the decreased serum albumin levels in preterm infants compared to term infants.31 Some

research has demonstrated that while binding capacity increases over time among term and

near-term infants, in preterm infants it decreases after the first week of life as a potential

reason for this.31,32 The reduced binding capacity of albumin coupled with the increase in red

blood cell breakdown results in the increase of free bilirubin that can be dangerous to

newborns, especially preterm infants.31,32

1.4.3 Unconjugated and conjugated bilirubin

Unconjugated bilirubin is lipid soluble. The conversion of unconjugated bilirubin via glucuronyl

transferase results in conjugated bilirubin, which is water-soluble. In newborns, there is

frequently a delay in the up regulation of glucuronynl transferase activity leading to delays in

conjugation and a rise in the unconjugated bilirubin level. This can be even more delayed

depending on clinical characteristics of newborns including prematurity.23,25,26

The component of the bilirubin that is harmful to the infant brain is the lipid-soluble

7

unconjugated bilirubin since it can pass through the blood-brain barrier when in excessive

amounts in the bloodstream.23,25 The vast majority of TSB levels in newborn infants is

unconjugated bilirubin. However, if an infant does have elevated TSB levels and a large

percentage of it is conjugated bilirubin, these infants are managed differently. In this case, the

conjugated bilirubin does not damage the infant brain but is more likely representative of liver

disease or a condition impacting liver function unrelated to hyperbilirubinemia.26

Among newborns, there are several clinical characteristics and conditions that impact the

production (hemolysis/increased red cell degradation); conversion (decreased liver/UDPGT

function); and excretion (gastrointestinal dysfunction) of bilirubin. Disruption in any of the

above can result in excessive amounts of unconjugated bilirubin. If left unmonitored and/or

untreated, the accumulation of unconjugated bilirubin could pose a potential and dangerous

threat to newborns.23,25,26 Preterm infant brains are particularly vulnerable to the toxic effects

of unconjugated bilirubin. 16

1.5 Harms of bilirubin to the neonatal brain

1.5.1 Neonatal blood brain barrier

The blood brain barrier protects the blood vessels in the brain from toxic molecules entering

the brain through the bloodstream. The blood brain barrier is made of up of brain capillary

endothelial cells with tight intercellular junctions.23,33 Through the complex network of tight

junctions connecting endothelial cells and astrocytic end-feet surrounding the intercellular

membrane, the blood brain barrier protects the brain by preventing the diffusion of molecules

that are toxic to the brain.33-35 The blood-brain barrier is a highly selective semipermeable

membrane that only allows specific molecules to pass through either via diffusion or by binding

to transporters along the endothelial cells. For example, lipid soluble molecules like

unconjugated bilirubin can pass through the blood-brain barrier through diffusion while glucose

8

passes through the blood brain barrier via a transporter.23,33-36 The blood brain barrier also

maintains homeostasis around the extracellular fluid surrounding the brain and central nervous

system. Although the blood-brain barrier is a highly selective membrane, a change in the

equilibrium of certain molecules can result in neurotoxic molecules passing via diffusion.23,33-36

Newborn infants are at higher risk of unconjugated free bilirubin passing through the blood

brain barrier. Preterm infant brains are more vulnerable to the toxic effects of unconjugated

free bilirubin once passed through the blood brain barrier. 37 This is because the neonatal brain

is still developing and among these infants the blood brain is highly permeable compared to

adults. 37 Some research has shown that the tight junctions along the endothelial cells that

protect the brain from neurotoxins are formed early in brain development, thus providing

appropriate protection during the neonatal period.37-40 Despite this, previous research on the

neonatal brain and blood brain barrier reported that several molecules in excess can pose a

threat to the neonatal brain due to the disruption in equilibrium. These molecules include but

are not limited to bilirubin, glucose and various amino acids.23,41,42 The combination of a highly

permeable blood-brain barrier among preterm infants and increased risk of unconjugated

bilirubin levels in the bloodstream, puts the preterm brain at greater risk of unconjugated

bilirubin passing through the blood brain barrier through diffusion. 43-45,46 Figure 1-1, published

by Watcho and Tiribelli, provides a visual description of this physiological process.25 Similar to

intermittent monitoring of glucose and amino acids, shortly after birth, TSB is monitored

intermittently among newborns in the first week of life, to prevent the damage potentially

caused by excess levels of bilirubin.1,2,18,47

Clinical characteristics and/or illnesses among newborn infants , such as sepsis can increase

serum unconjugated bilirubin, and/or reduce the binding capacity of albumin to bilirubin,

increasing the risk of free bilirubin passing through the blood brain barrier and causing severe

neurological damage.23,48

9

Figure 1-1 Risk factors and process of unbound bilirubin crossing the blood brain barrier

in neonates published by Watcho and Tiribelli25

This figure was reproduced with permission from

25Watchko JF, Tiribelli C. Bil irubin-Induced Neurologic Damage —

Mechanisms and Management Approaches. New England Journal of Medicine. 2013;369(21):2021-2030, Copyright Massachusetts Medical Society

10

1.5.2 Bilirubin crossing the blood-brain barrier

Unconjugated bilirubin can pass through either an intact blood brain barrier or disrupted blood

brain barrier. Bilirubin passes through an intact blood-brain barrier through passive diffusion

when there is an increased amount of unconjugated bilirubin in the bloodstream.23 Examples

of conditions that can disrupt the blood-brain barrier include hyperosmolar load, hypercarbia

with acidosis, asphyxia and vasculitis.23 These conditions can disrupt the overall structure and

function of the blood-brain barrier making it more susceptible to neurotoxic molecules.23 For

instance, the increase of hyperosmolar load has been shown to disrupt the endothelial tight

junctions that make up the blood-barrier thus resulting in a decreased ability of the blood-brain

barrier to protect the brain from toxic molecules.23 Preterm infants are at greater risk of being

clinically unstable. This results in a disrupted blood brain barrier, increasing its vulnerability to

the toxic effects of hyperbilirubinemia. 16,23

Once passed the blood brain barrier, unconjugated bilirubin deposits into specific areas of the

brain including the globus pallidus, hippocampus, lateral ventricular walls, cerebellum and

subthalamic nuclei, resulting in a specific characterization of bilirubin neurotoxicity in magnetic

resonance (MRI) findings.48-50

Although free unconjugated bilirubin is the component of TSB that is believed to cross the

blood brain barrier, there is currently no routine measurement of free bilirubin. As such, TSB

levels, and occasionally serum albumin levels, are used as a marker of the risk of bilirubin

induced neurotoxicity.25

The increase in TSB levels and subsequent crossing of unconjugated bilirubin through the blood

brain barrier among newborns can present as several stages of illness.26 An immediate and less

harmful impact of the increase in bilirubin levels is the yellowing of an infant’s skin, referred to

as the clinical sign, jaundice. The term hyperbilirubinemia refers to the increase in TSB levels

among newborn infants.26 However, as unconjugated bilirubin passes through the blood brain

barrier, it can result in a condition called bilirubin encephalopathy (acute and chronic bilirubin

11

encephalopathy), classically referred to as kernicterus.25

1.6 Definitions of key clinical terms

1.6.1 Neonatal hyperbilirubinemia

Jaundice is identified as the yellowish discoloration of a newborn infant’s skin, conjunctivae

and sclerae, while hyperbilirubinemia is defined by the measurement of elevated TSB levels.26

Newborn infants are at increased risk of transient neonatal hyperbilirubinemia, which is a

common condition that can occur within the first three days of birth. As described earlier, this is

because newborn infants have increased red blood cell production and breakdown shortly after

birth due to the short life span of red blood cells, decreased liver function and increased

reabsorption of conjugated bilirubin through the gastrointestinal tract.23,26 This is often

referred to as physiological jaundice which is generally mild and resolves on its own.26

In healthy term and near-term infants, bilirubin levels increase after birth, continue to rise up

to 72 hours of age and then decline over the first weeks of life with no intervention.3,51 The

rate of rise (ROR) of TSB decreases after the first week of life as the liver function increases in

the newborn infant. Less is known about the specific pattern of bilirubin metabolism in preterm

infants, although it also seems to rise within days after birth and fall within weeks .16,17

However, in some instances bilirubin levels can continue to rise to extremely high levels. If not

treated, it can be toxic for newborns and result in severe hyperbilirubinemia and its chronic

clinical sequelae.51 This pathological hyperbilirubinemia generally requires treatment to prevent

the toxic effects of bilirubin in the newborn.

12

1.6.2 Acute and chronic bilirubin encephalopathy

An overproduction of bilirubin or its delayed removal through the hepatobiliary system, if left

untreated, can lead to bilirubin encephalopathy. Bilirubin encephalopathy at a gross pathology

level, is caused by deposition of unconjugated bilirubin in the brain and results in specific

yellowish discoloration in the basal ganglia and can also extend to the brain stem, auditory,

oculomotor and vestibular nuclei.52 Historically, this has been referred to as kernicterus.

Clinically, bilirubin encephalopathy can present as acute bilirubin encephalopathy (ABE) and/or

chronic bilirubin encephalopathy (CBE). Bilirubin induced neurological dysfunction (BIND) is

referred to as the subtle signs of kernicterus presenting as subtle neurological disabilities

without any classic signs of kernicterus. BIND presents as a less severe injury of bilirubin

encephalopathy including mild neurological abnormalities or isolated hearing loss. BIND can

occur following a presentation of ABE.53 BIND is included in the spectrum of neurological injury

due to bilirubin.54

ABE is the acute version of kernicterus and can present as decreased feeding, lethargy,

hypotonia, hypertonia, high pitched crying, retrocollis, opisthotonos, fever, seizures and

death.52,55,56 CBE includes clinical signs similar to kernicterus but also more permanent chronic

sequelae including developmental disabilities, hearing loss, choreoathetotic cerebral palsy,

paralysis of upward gaze and sometimes intellectual deficits.50,52,56,57 ABE and CBE have distinct

clinical and MRI findings.52,53 MRI findings for ABE and CBE usually present in specific areas of

the brain related to the clinical presentation of ABE and CBE mentioned above including , but

not limited to, the globus pallidus and subthalamic nuclei. ABE is associated with more subtle

and acute damage, while CBE is associated with more severe damage.50,55,58

13

1.7 Risk factors for severe hyperbilirubinemia

Several factors place a newborn infant at risk of developing elevated hyperbilirubinemia. These

include: hemolysis, prematurity, presence of visible jaundice within 24 hours of life,

cephalohematoma, male sex, Asian race, family history of hyperbilirubinemia and infants who

are exclusively breastfed.13,14,51 These clinical factors impact either bilirubin production and/or

an infant’s ability to excrete bilirubin. The most cited and investigated risk factors of severe

hyperbilirubinemia are hemolysis and prematurity. These conditions often impact the

overproduction and delayed removal of bilirubin in a newborn infant.16,17

Severe hyperbilirubinemia was originally recognized as most associated with hemolysis of red

blood cells, more specifically hemolysis as a result of Rh disease. With the introduction of

prenatal universal Rh screening and Rh prophylaxis, the incidence of severe hyperbilirubinemia

associated with Rh disease has significantly decreased.7,16,17

Recently, hemolysis by ABO incompatibility has started to garner attention in the incidence of

hyperbilirubinemia.59,60 Clinical presentation of ABO incompatibility commonly includes

jaundice from hyperbilirubinemia in the first 24 hours of life and high peak bilirubin requiring

treatment.61 Reports of ABO incompatibility and jaundice have been predominantly reported in

term and near-term infants with limited reports in preterm infants.61 In addition, although less

common, hemolysis in newborns can also result from the presence of other antibodies such as

Kell, Kidd and Duffy.26,29 However, as hemolysis from Rh disease and ABO incompatibility has

been predominantly discussed in hyperbilirubinemia, this thesis will restrict the discussion to

hemolysis caused by Rh disease and ABO incompatibility.29

Despite the improved management of Rh disease, and other forms of blood group

incompatibility, there continue to be rare cases of severe hyperbilirubinemia with the absence

of hemolysis, especially in preterm infants. In 1955, Crosse et al attributed a higher risk of

14

infants developing ABE and CBE to prematurity in the absence of hemolysis.4,16,17

Infants exhibiting these factors, specifically prematurity or risk of hemolysis, are monitored for

rising TSB levels and are often treated promptly with phototherapy to decrease the rise of TSB

levels to potentially unsafe levels.16

1.8 Prevention of severe hyperbilirubinemia

The standard treatments to reduce severe hyperbilirubinemia and chronic neurological

sequelae are phototherapy, exchange transfusion (ET) and, more recently, the use of

intravenous immunoglobulins (IVIG). Phototherapy is first-line therapy, can be escalated in

intensity, and followed rarely by ET if it is unable to treat severe hyperbilirubinemia.1,2,62 Over

the past few years, IVIG has garnered more interest as an alternative to ET if phototherapy has

been ineffective in reducing TSB levels among infants with hemolytic diseases or in the

presence of hemolysis from other causes.63 This review will focus on phototherapy and limit the

discussion of ET and IVIG to their definitions only.

1.8.1 Phototherapy

Phototherapy exposes an infant’s skin to a light of approximate wavelengths ranging from 420-

490nm depending on the intensity required for phototherapy.1,64 Blue florescent tubes,

compact fluorescent tubes, halogen spotlights and most recently a light-emitting diode (LED)

are the most commonly used light sources for administering phototherapy.65 Phototherapy

allows for the excretion of bilirubin without conjugation. 66,67 More specifically, phototherapy

through isomerization, converts bilirubin to a more polar (i.e. water-soluble) photoisomer.

These newly isomerized bilirubin molecules are then excreted in urine and bile.68 Phototherapy

has effectively and significantly reduced the need for ET as well as the incidence of ABE and CBE

for more than five decades. 1,64,66,69-72

15

1.8.1.1 Efficacy of phototherapy

The clinical efficacy of phototherapy is measured by the decrease in TSB levels, to levels

deemed safe for infants, as per national guidelines.1,2,66 This generally depends on the

wavelength, irradiance, amount of exposed body surface area, distance of phototherapy from

skin surface area and duration of exposure.1,65 Current guidelines for phototherapy initiation

aim to reduce TSB levels by 34-68 μmol/L within 6-12 hours.1,64,66 Types of phototherapy used

in newborns include conventional (i.e. single phototherapy) or more intense forms, often

referred to as: double phototherapy and triple phototherapy. Single, double and triple

phototherapies refer to the number of phototherapy units during the administration of

phototherapy. 73 Conventional phototherapy is used in infants without hemolytic disease or

who are experiencing a low progression of jaundice as evidenced by the ROR of their bilirubin

levels.74 If single phototherapy is ineffective, double and triple phototherapy are used based on

the increase in the ROR of bilirubin levels.1,65,66,74 Intensive phototherapy is a high level

irradiance in the 430 to 490 nm band with an irradiance level of 30 µW/cm2/nm and used in

infants with hemolytic disease or infants who demonstrate evidence of rapidly progressing

jaundice with a faster ROR.1,65,74 Bilirubin blankets are also used as part of intensive

phototherapy whereby the neonate can lie on them, providing the newborn’s back with

irradiance.1

1.8.2 Exchange transfusion

ET is administered when maximal phototherapy is ineffective in reducing TSB levels, if an infant

requires an immediate decrease in rising TSB levels or if severe hyperbilirubinemia is

accompanied with signs of ABE. Currently, it is most commonly used in infants with hemolytic

disease, when intensive phototherapy is ineffective.75,76 ET is the simultaneous removal and

replacement of an infant’s red blood cells with a donor’s appropriate compatible red blood

cells.75 Infants undergoing ET are unwell and at an increased risk of intubation and mechanical

ventilation, cerebro-vascular incidents, hemodynamic dysfunction, thrombocytopenia and

16

hypocalcemia .77-79 Compared to term and near-term infants, preterm infants are at an

additional risk necrotizing enterocolitis, arrhythmias and higher morbidity and mortality.18,80,81

As a result, every effort is made to reduce the use of ET through frequent TSB monitoring and

early administration of phototherapy.

1.8.3 Intravenous Immunoglobulins

IVIG can reduce bilirubin levels by decreasing the rate of red cell degradation caused by

antibody mediated hemolysis. IVIG is often used to prevent the use of ET among infants with

hemolytic disease.2,74 It is a preferred method of treatment over ET since it is the less invasive

of the two treatments.63 Although IVIG has been deemed safer than ET, some reported

complications include sepsis and necrotizing entercolitis.63,82,83 Despite the use of IVIG over the

past several years, its effectiveness in reducing ET is unclear due to the limited number of high

quality studies.63 Furthermore, the effect is transient, once administered, levels of

immunoglobulin decrease, underlying hemolysis can continue, resulting in rebound

hyperbilirubinemia and anemia.

17

Chapter 2 . Assessment of hyperbilirubinemia in preterm infants

18

2.1 Prematurity

Premature newborns have a significant risk of severe hyperbilirubinemia and its toxic effects.16

A greater understanding of hyperbilirubinemia and its associated risk factors in preterm infants

is required. Prematurity is defined as neonates born at < 37 weeks’ gestation.16,84 For this

thesis, preterm infants will be defined as ≤ 35 weeks’ gestation (current guidelines are based on

data from infants born at ≥ 36 weeks’ gestation with a limited number of infants born at 35

weeks’ gestation).1,2 To further clarify, in this thesis extremely preterm infants are defined as

infants born between at 240/7 to 286/7 weeks’ gestation; very preterm infants are those born at

290/7 to 326/7 weeks’ gestation; and moderate preterm infants are born at 330/7 to 356/7 weeks’

gestation. In addition to prematurity, hemolysis and mode of feeding are also important clinical

risk factors of hyperbilirubinemia among preterm infants.

2.2 Hyperbilirubinemia in preterm infants

Preterm infants experience more jaundice than full-term infants. While approximately 50%-60%

of term and near-term infants are reported to experience hyperbilirubinemia, that number is

thought to be 80% in preterm neonates.85 However, a vast majority of these infants do not

experience severe hyperbilirubinemia or clinical neurological signs of ABE and CBE. Previously,

among term and near-term infants in Canada, the minimum incidence of severe

hyperbilirubinemia with neurological consequences was estimated to be 1 in 2480 live births. 14

However, with the increase in intermittent monitoring and timely initiation of treatment among

infants at higher risk of ABE and CBE, severe neonatal hyperbilirubinemia has decreased

significantly to 1 in 8352 live births in this cohort.15 The decrease in severe hyperbilirubinemia

can be in part attributed to routine screening and closer attention to infants at higher risk of

developing ABE and CBE.15 However, this reported incidence referred to term and near-term

infants, whereas the current incidence in preterm infants is unknown.13,14

19

Preterm infants have different clinical and physiological factors that put them at more risk of

developing ABE and CBE compared to term and near-term newborns.16,17 A deeper

understanding of the risk factors and impact of hyperbilirubinemia in this population is

required.

2.2.1 Neurotoxic effects of significant hyperbilirubinemia in preterm infants

Preterm infants are at greater risk of developing ABE and CBE in the absence of classic

neurological signs. Cross et al described the acute and clinical signs of bilirubin neurotoxicity in

preterm infants to include head retraction, expressionless facies, changes in muscle tone,

cyanotic attacks, refusal to suck, vomiting and hemorrhage prior to death.4,16 Furthermore, as a

result of the complicated physiological and clinical characteristics of premature infants,

neurological deficits caused by bilirubin have been seen below the TSB thresholds for

phototherapy established in term and near-term infants.4,16,17 As such, it has been hypothesized

that preterm infants have greater susceptibility to neurologic damage at lower bilirubin levels

deemed safe in term and near-term infants.18 As a consequence of the use of lower TSB

thresholds, more than 80% of extremely preterm infants are treated with phototherapy to

prevent significant hyperbilirubinemia. 86,87

2.2.2 ABO incompatibility in preterm infants

Hemolysis from ABO incompatibility and the resultant hyperbilirubinemia has been well

reported among term and near-term infants. There is currently limited research on the

incidence of severe hyperbilirubinemia among preterm infants with ABO incompatibility.61 One

previous case-control study of 118 preterm infants with ABO incompatibility revealed no

significant difference in the incidence of hyperbilirubinemia among preterm infants less than 32

weeks’ gestation with or without ABO incompatibility. However, the same study demonstrated

20

that the incidence of hyperbilirubinemia related to ABO incompatibility increased with

increasing gestational age.61 Additional research is required in preterm infants to determine the

impact of ABO incompatibility on hyperbilirubinemia requiring treatment.

2.2.3 Feeding in preterm infants

Among term and near-term infants, guidelines for management of hyperbilirubinemia do not

consider the impact of mode of feeding (i.e. enteral vs total parenteral nutrition) since the vast

majority of these infants are started on enteral feeds by the first day of life.88 Extremely

preterm infants are started on total parenteral nutrition (TPN) potentially impacting their

gastro-intestinal tract.89 Since hyperbilirubinemia can occur due to disruptions in the excretion

of bilirubin, the impact of the mode of feeding on bilirubin levels should be explored.26

Currently, there is a paucity of knowledge on the role that feeding methods have on evolution

and the management of hyperbilirubinemia in preterm infants.

2.2.4 Summary of hyperbilirubinemia in preterm infants

Preterm infants are at higher risk of significant hyperbilirubinemia and their brains are more

vulnerable to the toxic effects of severe hyperbilirubinemia. To address the lack of

understanding of hyperbilirubinemia in preterm infants, chapter 3 attempts to define the

natural history of bilirubin in preterm infants by gestational age at birth, by receipt of

treatment, by hemolysis and by mode of feeding.

21

2.3 Overview of the assessment of hyperbilirubinemia

An integral component in the prevention of severe hyperbilirubinemia is intermittent

monitoring of bilirubin levels. As part of monitoring, routine bilirubin tests are done as early as

24 hours of age to determine if rising bilirubin levels may require treatment, namely

phototherapy.1,2,51 Among healthy term and near-term infants born at

> 35 weeks’ gestation, bilirubin tests are done either through a TSB test, or through TcB

measurements, using a TcB device to measure bilirubin levels at the skin.1,2 Among preterm

infants born at < 36 weeks’ gestation and admitted to a NICU in Ontario, TSB tests are

predominantly used to monitor bilirubin levels.18

2.3.1 Term and near-term infants

Among healthy term and near-term infants born at ≥ 35 weeks’ gestation, safe hour-specific

pre-treatment TSB levels have been established and are currently used as a reference point to

measure bilirubin in this population from birth.1-3 These values are based on gestational age

and risk factors of the infant.1-3,13,15 They were determined based on pre-treatment TSB levels

from a large prospective study of 2,840 healthy term and near-term infants born at ≥35 weeks’

gestational age with a limited number of infants born at 35 weeks’ gestation and excluding

infants admitted to NICUs. Pre-treatment TSB levels done before discharge (between 18-72

hours of age from birth) and after discharge at, a follow-up assessment (between 24-48 hours

of age after discharge) were compared. In the same study, to validate these TSB levels, the

probability, sensitivity and specificity of pre-treatment bilirubin was determined to predict

severe hyperbilirubinemia.3 As this thesis is focused on measuring bilirubin in preterm infants,

further details on the methods for assessing predictive ability of nomograms will not be

discussed.3

Using this validated nomogram, developed by Bhutani and subsequent studies thereafter, the

Canadian Pediatric Society (CPS)2 and American Academy of Pediatric (AAP)1 developed

22

guidelines for the assessment of hyperbilirubinemia in term and near-term infants. They

recommend a bilirubin test (either TSB or TcB) must be completed between 24-72 hours of age

and plotted on Bhutani’s nomogram to determine the risk of an infant developing

hyperbilirubinemia. Risk level is determined by the term and near-term infant’s gestational age

at birth, presence of hemolytic conditions and relevant previous maternal pregnancy history. If

a term infant is discharged at 24 hours of age, the guidelines recommend a follow-up plan be

made for the infant to have a bilirubin test done at least by 72 hours of age.1,2

2.3.2 Preterm infants

Among preterm infants born 240/7 to 356/7 weeks’ gestation a nomogram of pre-treatment TSB

levels does not exist and therefore current national guidelines are limited to term and near-

term infants. The current approach to measuring bilirubin in preterm infants is highly criticized

and unclear.17 Earlier guidelines recommended the use of Bhutani’s nomogram in conjunction

with considering an infant’s level of prematurity, illness and/or presence of hemolytic disease.17

However, this was proven to be problematic as there was an increase in ABE and CBE among

preterm infants.17 Among preterm infants, TSB tests are done as early as 24 hours of age and

repeated every 12 to 24 hours to determine increases in TSB levels.16-18 These readings help

determine if treatment is required based on hospital-specific guidelines, which are based on

published and unpublished expert opinions.17,20,90 Since preterm infants are at higher risk of

severe hyperbilirubinemia, TSB thresholds used to initiate phototherapy are lower for this

cohort than what is deemed safe in term and near-term infants.16

In this section, I systematically assess two aspects of bilirubin measurement in preterm infants

born at 240/7 to 356/7 weeks’ gestation. First, a review of published pre-treatment TSB levels and

phototherapy thresholds based on gestational age at birth in preterm infants was conducted.

Second, TSB and TcB measurements used in preterm infants were reviewed to determine the

impact of repeated TSB measurements and current limitations of TcB measurements in preterm

infants.

23

2.4 Review of currently published pre-treatment TSB levels and

phototherapy thresholds in preterm infants by prematurity

2.4.1 Published pre-treatment TSB levels

There is currently a paucity of published pre-treatment TSB level percentile curves in preterm

infants stratified by gestational age at birth.

One of the first studies to report TSB levels in preterm infants , by Mayer’s, was limited to a

small cohort of 150 preterm infants born at ≤ 35 weeks’ gestation stratified by birthweight

groupings. The purpose of this study was to determine the predictability of a bilirubin done at

12 hours of age from birth in determining severe hyperbilirubinemia.91 It was similar to

Bhutani’s study that assessed the predictability of the pre-discharge bilirubin in identifying

significant hyperbilirubinemia.3 Although the authors present an hour-specific nomogram of

pre-treatment TSB levels in preterm infants, it was limited to 150 subjects with a normal

bilirubin at 12 hours of age and did not stratify by gestational age at birth. Furthermore, the

focus of this study was to assess the predictive ability of a specifically timed TSB level rather

than reporting all pre-treatment TSB measurements in preterm infants.91

After Mayer’s study, a recent retrospective cohort study published pre-treatment TSB levels

from 483 preterm infants with very low birth weight (VLBW).92 This study included infants born

at <37 weeks’ gestation with a birthweight <1500 g. This study, however, did not describe the

number of infants included in the study by gestational age group of prematurity. Linear

regression was employed to plot 2,430-hour specific pre-treatment TSB levels from 483

preterm infants from birth to 72 hours of age. However only the 50th percentile curve was

generated and compared with Bhutani’s nomogram between 12 to 48 hours of age, which was

below the 40th percentile. In addition, the primary purpose of this study was to assess ROR of

TSB in preterm infants and compare it to Bhutani’s nomogram.3,92 The authors found TSB had a

slower ROR between 12 to 48 hours of age compared to term and near-term infants reported in

24

Bhutani’s nomogram. Although this study was larger than the previous study published in

2014 and presented hour-specific pre-treatment TSB levels in preterm infants up to 72 hours of

age, the study population was limited to infants <1500 g and did not differentiate TSB levels by

gestational age of prematurity. This study does highlight that TSB levels in preterm infants are

different than TSB levels in near-term and term-infants supporting the need to study preterm

infants seperately.92

2.4.2 TSB thresholds used for phototherapy initiation

Since 1985, phototherapy initiated at 12 to 24 hours of age has prevented severe

hyperbilirubinemia and its chronic sequelae in infants less than 2000g.16,93 The lack of published

pre-treatment TSB levels based on gestational age at birth in preterm infants has resulted in

limitations on guidelines and recommendations for initiating phototherapy in preterm infants.

Historically, TSB thresholds recommended for initiating phototherapy in preterm infants were

based on birthweight.94,95 Now, most recommended TSB thresholds also include gestational

age at birth.18,96,97

For this review, only guidelines for TSB thresholds in preterm infants based on gestational age,

or include gestational age, were reviewed. This is because Bhutani’s nomogram is based on

gestational age at birth and current guidelines by CPS and AAP for managing hyperbilirubinemia

are also based on gestational age at birth.1-3

As most recommended TSB thresholds for initiating phototherapy prior to 2012 were published

in textbooks and clinical handbooks, papers that reported guidelines recommending TSB

thresholds used for phototherapy were reviewed.95 If available, the methods used to develop

the guidelines and the subsequent recommended TSB thresholds were only extracted if they

were based on gestational age at birth.

Early published reports of suggested TSB thresholds to initiate phototherapy in preterm infants

25

have been based on expert opinion and small cohort studies of preterm infants .98-101 In

addition earlier reports on phototherapy treatment guidelines for preterm infants also reported

inconsistent guidelines between NICUs.19,20,90

One review of guidelines used to manage hyperbilirubinemia in newborns in the United

Kingdom (UK) revealed increased variation in practice among preterm infants.19 Among 140

NICUs included in this review, most guidelines were predominantly based on hospital

developed unpublished TSB thresholds with an unclear indication of origin. Individual site-

specific, hour-specific TSB graphs were developed based on these thresholds, however, authors

reported that these graphs were not legible, and many did not recommend TSB thresholds after

72 hours of age from birth. Some of the legible graphs were limited to term and near-term

infants or did not stratify preterm infants by gestational age week at birth. Overall, there was

limited information on how these hour-specific TSB graphs were developed.19 A small number

of guidelines were based on published TSB thresholds. Thirteen (9%) of the participating NICUs

used a published TSB graph by Finlay and Tucker based on gestational age at birth102, 9 NICUS

(6%) used a published TSB graph based on birthweight.19,94 Finally, 13 (9%) of the NICUS used a

mathematical formula to start phototherapy, among these, 11 NICUS used a gestational age

based formula .19

Upon further review of these published TSB thresholds based on gestational age at birth, the

origin of these recommendations was not explained. For instance, the original TSB graph by

Finlay and Tucker was published as a letter to the editor and the details of how the TSB graph

was developed were not provided.102 The modification of Finaly and Tucker’s TSB graph for

preterm infants was based on expert opinion with no clear methods for how the values were

derived.19,102,103

Among the 11 NICUS who used a gestational age-based formula, the mathematical formula

was: Total serum bilirubin (μmol/L) = (gestational age x 10) – 100. This paper neither provided

details on how this gestational age-based formula was developed, nor cited the origin of this

formula.19 Overall, when the authors assessed all TSB thresholds recommended by 140 NICUs,

26

they found a wide range of TSB thresholds by gestational age at birth. For example, among

infants born 24 to 28 weeks’ gestation the minimum recommended TSB threshold level was

80umol/L and maximum was 330umol/L (Table 2-1).19

The Netherlands were one of the first regions to try to address the variation in assessment and

management practices of hyperbilirubinemia by publishing nationwide, consensus-based TSB

thresholds in preterm infants.20 Using a cross-sectional survey of 10 NICUs in the Netherlands,

TSB threshold guidelines used across NICUs were amalgamated to create a large consensus-

based guideline, now adopted by the Dutch Society of Pediatrics. These amalgamated

guidelines and nomograms were based on birthweight among preterm infants and not

gestational age. Among the 10 NICUs, only one NICU used guidelines based on gestational age

at birth and all 10 NICUs used guidelines based on expert opinion. Six of the guidelines included

TSB thresholds by hours of age from birth, while four NICUS suggested fixed TSB threshold not

accounting for hours of age after birth. Furthermore, all guidelines after four days of birth

suggested fixed TSB threshold not accounting for hours of age from birth.20

In this study to develop amalgamated nationwide guidelines, TSB thresholds by hours of age

from birth were derived from recommended thresholds provided by NICUs and categorized by

birthweight groupings. These ranges were then compared with two published

recommendations for starting phototherapy and combined to develop national guidelines.69,101

The process of how TSB ranges and published recommendations were combined was not

described. Although these amalgamated guidelines have been currently adapted by the Dutch

Society of Pediatrics, these guidelines are based on consensus-based expert opinion.

Furthermore, the nomograms developed in this study were theoretically derived and not based

on preterm infant data.20

A study from Italy also used a cross-sectional survey design to identify the treatment guidelines

used to assess hyperbilirubinemia in preterm infants.90 In contrast to the study conducted in

the Netherlands, NICUs in Italy were asked specific questions around their institution’s

27

assessment of hyperbilirubinemia and currently used TSB thresholds were not directly shared.

Instead, sites were asked if they followed published TSB recommendations either based on

gestational age at birth100,101 or birthweight95 or followed other unspecified published and/or

unpublished guidelines. Among 67 NICUs who participated in the study, 45.7% of NICUS used

published guidelines. Among these 8.7% of NICUs used both guidelines based on gestational

age and birthweight100,101 while 17.4% used only the guideline based on gestational age at

birth100 and 19.6% used only the guideline based on birthweight.90,95 A total of 54.3% of NICUs

used institutionally developed specific guidelines or unspecified unpublished guidelines.90 Upon

further review of the published guidelines, the methods to develop these guidelines were not

clearly reported.

Again, in keeping with previous reports20, guidelines for assessing hyperbilirubinemia in

preterm infants lacked uniformity between NICUs in Italy and were based on predominantly

consensus-based guidelines. This study did note that the timing of bilirubin measurements

varied between sites. The published TSB thresholds based on gestational age at birth are

reported in Table 2-1. 90 TSB thresholds by unpublished and/or locally developed guidelines

were not reported in this study.

In North America, in response to reports of inconsistent guidelines and TSB thresholds used

among preterm infants between NICUs and a call by the AAP in 2007, Maisels’ et al developed

consensus-based recommendations on appropriate TSB levels to initiate phototherapy in

preterm infants born at <35 weeks’ gestation in 2012.18 These recommendations have now

been widely adopted by most sites in North America.18

The method of how these TSB levels were selected, based on gestational age at birth, was not

clearly explained in Maisels’ paper.18 He does, however, identify a range of recommended TSB

levels by gestational age groups to initiate phototherapy that he perceived may do more good

than harm, based on previous research.18-20,98 He recommends initiating phototherapy at TSB

levels between 85-103 umol/L for infants born at <28 weeks’ gestation and 103 –137 umol/L for

28

infants born 280/7 to 296/7 weeks (Table 2-1).18

There is a paucity of reports assessing Maisels’ recommended TSB thresholds18. Anecdotally,

we have heard that some sites may have modified his thresholds. A recent study reporting the

frequency of phototherapy use in preterm infants born at 23 to 34 weeks’ gestation provided

TSB thresholds used to initiate phototherapy from 2010 to 2015 as supplementary material.

The origin of these recommendations was not provided. Upon review, many of the

recommended TSB thresholds are similar to Maisels’.87 Table 2-2 includes TSB thresholds used

in this study by year.87 Experts in Canada and India recently published TSB graphs for initiating

phototherapy97 by adapting consensus-based guidelines,1,2,18,20,84,96,104 including Maisels’.18,97

They were presented as hour-specific TSB graphs as opposed to fixed TSB thresholds used in

previous reports. These recommendations were not included in Table 2-1.

After 2012, NICUs outside of North America continued to report the use of unpublished

guidelines based on expert opinion.105 For example, a national survey of 84 hospitals in Turkey

revealed that 75% of NICUs were using Turkish Neonatology Society guidelines and 25% of

these NICUs used other non-specific guidelines. The Turkish Neonatology Society guidelines

were based on birthweight rather than gestational age at birth and were developed in 2002.105

Specific details on how these guidelines were derived were inaccessible. Another institution in

Japan updated their birthweight based guidelines for treating hyperbilirubinemia to gestational

age based guidelines.96 These guidelines were updated to address the increase in CBE among

preterm infants and lack of focus on preterm infants in earlier guidelines.96

29

2.4.3 Review of the frequency of phototherapy in preterm infants

As a result of the lack of pre-treatment TSB levels and guidelines based on consensus there is

an increased rate of phototherapy use among extremely preterm infants, specifically among

those born less than 29 weeks’ gestation.18

Since most preterm infants receive phototherapy, there has been a push to evaluate the use of

phototherapy in these newborns, specifically around infants of lower birthweight and

gestational age. Infants of lower gestational age are not only at higher risk of ABE and CBE, but

they are also at higher risk of side effects from prolonged and intensive use of phototherapy. To

address these issues, NICUs have started evaluating phototherapy use in this population.86,87

Very few studies reported the frequency of phototherapy among extremely preterm infants.

Among studies that did, more than 80% of infants were started on phototherapy.86,87

Most research reporting phototherapy in extremely preterm infants is generally limited to

infants of VLBW or extremely low birthweight (ELBW) and are not specific to gestational age at

birth.106-111 Among these studies, research was limited to the impact of the amount of

phototherapy administered on neonatal outcomes and type of phototherapy device used in

infants of VLBW and ELBW.106-111In all studies conducted to date on the usage of phototherapy

among extremely preterm infants, reports of TSB levels used to initiate phototherapy were

limited.108

30

2.4.4 Summary of pre-treatment TSB levels in preterm infants and relevance

Systematically derived pre-treatment TSB levels from a large cohort of preterm infants are

lacking. Accordingly, guidelines for monitoring and measuring TSB levels to inform treatment in

preterm infants are based on consensus and expert opinion.97

Chapters 3 and 4 describe the development of an hour-specific pre-treatment TSB nomogram

derived from a multi-site retrospective cohort of preterm infants born at 290/7 to 356/7 and 240/7

to 286/7 weeks’ gestation, respectively.

31

Table 2-1. Summary of published total serum bilirubin thresholds based on gestational age at birth by country and author

First Author, Year

Region TSB

thresholds are used

Range (if provided) of published total serum bilirubin thresholds by gestational age at birth, weeks

24 25 26 27 28 29 30 31 32 33 34 35

Rennie, 200819,

United

Kingdoma

80-330

80-330

80-330

80-330

80-330

80-330

100-330

100-330

150-330

150-330

150-370

150-370

Ives, 200490,100

,101

Italyb

81

81

81

81

100

100

100

100

152

152

152

152

Maisels 201218

North

Americac

85-103

85-103

85-103

85-103

103-137

103-137

137-171

137-171

171-205

171-205

205-239

no value

indicatedd

a The TSB ranges presented are ranges authors derived from all reported TSB thresholds in this study 19 Adapted by permission from BMJ Publishing Group

Limited. Rennie JM, Sehgal A, De A, Kendall GS, Cole TJ. Range of UK practice regarding thresholds for phototherapy and exchange trans fusion in neonatal

hyperbilirubinaemia. Arch Dis Child Fetal Neonatal Ed. 2009;94(5):F323-327.

bAdapted by permission from John Wiley and Sons. Dani C, Poggi C, Barp J, Romagnoli C, Buonocore G. Current Italian practices regarding the

management of hyperbilirubinaemia in preterm infants. Acta Paediatr. 2011;100(5):666-669.90

c,d Maisels’ recommended TSB thresholds did not include infants born at 35 weeks’ gestation.18 Adapted by permission from Springer Nature. Maisels MJ,

Watchko JF, Bhutani VK, Stevenson DK. An approach to the management of hyperbilirubinemia in the preterm infant less than 35 weeks of gestation.

Journal of Perinatology. 2012;32(9):660-664.

32

Table 2-2 Total serum bilirubin thresholds used in two neonatal intensive care units in Michigan, United

States by year and gestational age at birth.87

Year total serum bilirubin threshold was recommended

Total serum bilirubin thresholds used to initiate phototherapy by gestational age at birth in Mukherjee’s study87, weeks

24 25 26 27 28 29 30 31 32 33 34

February 2010 to July 2011

85 85 85 85 85 103 103 137 137 171 205

July 2011 to June 2013

85 85 85 85

85

103 103

137

137 171 205

July 2013 to September

2015 85 85 85 85 120 120 154 154 188 188 222

aAdapted by permission from Spring Nature. Mukherjee D, Coffey M, Maisels MJ. Frequency and duration of phototherapy in preterm infants <35 weeks gestation. Journal of Perinatology.

2018;38(9):1246-1251.

33

2.5 Total serum bilirubin and transcutaneous bilirubin measurements

The paucity of published pre-treatment TSB levels in preterm infants has resulted in

intermittent monitoring of bilirubin. In this section, the potential harms of repeated TSB

sampling are reviewed followed by a review of the use TcB measurements, a non-invasive

alternative to TSB measurement in preterm infants.

2.5.1 Total serum bilirubin tests

TSB values are obtained through blood tests using spectrophotometry.112 Among preterm

infants TSB tests are done as early as 24 hours of age and repeated at least once a day to

monitor TSB levels to determine if phototherapy should be started.16,17,113 After the initiation of

phototherapy, TSB levels continue to be monitored until TSB levels are below hospital specific

guidelines for management of hyperbilirubinemia. Bilirubin tests are usually done via a heel

prick on a newborn, but can also include venipuncture.114 A minimum of 200uL of blood is

required per TSB test.112

The use of TSB screening is recommended to monitor bilirubin levels among preterm infants

born at <36 weeks’ gestation and subsequently admitted to the NICUs. However, repeated

blood sampling has been reported to be potentially harmful to newborns, time consuming

among health professions, and difficult to access in some resource poor areas.21,115,116 The

following section briefly summarizes these concerns associated with repeated TSB tests.

Repeated blood sampling has been associated with increased pain and stress among

newborns. Heel prick blood sampling has been reported as one of the more painful procedures

newborns experience while in the NICU.117,118 This is particularly a problem among preterm

infants admitted to NICUs who have blood tests daily in the first few days of life. Furthermore,

34

repeated procedural pain and stimuli among preterm infants have been associated with

impaired brain development.119,120 Consequently, repeated blood sampling for TSB monitoring

can contribute to increased pain and stress in this population.21 Some research has looked at

ways to reduce the pain and stress neonates experience during their clinical course at the

hospital.120 One way of reducing pain and stress among newborns, particularly preterm infants,

has been to reduce the frequency of blood tests.121,122 Often times, when TSB levels are being

monitored in preterm infants after the first few days of life, TSB tests are the only blood tests

done.

In addition to pain, frequent blood sampling has also been associated with increased risk of

anemia.123 Anemia in preterm infants caused by blood loss from laboratory tests is referred to

as iatrogenic anemia.124 Since a minimum 200ul of blood is required per TSB test, and a preterm

infant typically receives a routine bilirubin test every day for the first 5 days of life, a preterm

infant may lose a minimum of 2000 ul from TSB testing alone.112 As such, experts have

suggested repeated TSB tests may increase the risk of anemia especially among smaller

preterm infants.22,125 One way to reduce iatrogenic anemia is to reduce blood sampling to only

those blood tests required.

Repeated TSB tests can also be costly and time-consuming. One Canadian study reported the

cost of one TSB test to be $15.82 in hospitals.126 With approximately 500 preterm infants

admitted to a level III NICU per year with at least 5 TSB tests during their neonatal stay, TSB

tests alone contribute to $39,550 in healthcare costs. In community settings the same study

estimated one TSB test to be between $50.21 - $65.03, whereas TcB tests were estimated to be

only $3.76.126 Furthermore, the use of TSB tests in community or resource poor areas continues

to be a challenge due to the limited access to laboratories for analysis.127 The logistics and costs

around obtaining frequent TSB measurements is particularly challenging for preterm infants

who require bilirubin tests every 12-24 hours of age for the first 10 days of life. TcB tests could

address the increased cost and logistical challenges of obtaining routine TSB tests , especially in

community settings.

35

Given the increased risk of harm from repeated TSB testing in preterm infants, finding a non-

invasive alternative approach to bilirubin measurements is warranted.

2.5.2 Transcutaneous bilirubin tests

To address the concerns of repeated TSB tests, TcB devices were introduced to provide a

measure of bilirubin at the level of the skin. Several TcB devices exist including but not limited

to the JM-103, JM-105, and BiliCheck. TcB devices provide an immediate, non-invasive

approach to obtain bilirubin levels in a healthy newborn.128 To comply with current guidelines

for measuring bilirubin and managing neonatal hyperbilirubinemia in term and near-term

infants, TcB levels in term infants has been validated.1,2 Currently, among term and near-term

infants, the CPS and AAP guidelines recommend the use of TcB devices as an immediate

alternative approach to obtaining serum bilirubin measurements prior to the initiation of

phototherapy for TSB concentrations <240 mmol/L (<14 mg/dL).1,2

Although TcB levels correlate well with TSB levels, they can be affected by other factors such as

phototherapy, sunlight exposure, skin colour, prematurity and site of measurement.129 In term

infants, TcB measurements have resulted in a decrease in the number of invasive blood tests

performed to measure bilirubin. 130 The above-mentioned factors have been well studied in

term and near-term infants as a result of large prospective cohort studies looking at TcB device

use. Studies investigating the use of TcB devices have reported a high correlation and

agreement between TcB and TSB levels.131,132 This has led to clear guidelines on the use of TcB

devices in term and near-term infants.132 Among term and near-term infants, TcB devices have

been approved to be used as a screening tool for hyperbilirubinemia prior to the initiation of

phototherapy with no limitations related to skin colour or maternal ethnicity.1,2

Limited studies exist in preterm infants overall and by anatomical site of measurement,

treatment and ethnicity.128,133 Since most preterm infants require phototherapy by the first or

36

second day of life, large prospective studies of TcB levels in preterm infants are often limited

in sample size since the use of TcB devices after phototherapy is not recommended.128,133

However, given the large number of preterm infants under phototherapy, the use of TcB

devices after phototherapy in this population should be explored. While studies evaluating TcB

device use in preterm infants have increased over the past few years, these studies have been

limited to small sample sizes (<250 infants).133

An earlier systematic review looking at TcB device use in preterm infants has been limited to

neonates greater than 34 weeks’ gestation, almost completely excluding infants admitted to

NICUs.132 Prior to 2016, the only systematic review of TcB device use in preterm infants was

limited to usage prior to phototherapy.128 Only one recent review, done in 2020, included

studies using TcB devices after phototherapy. This suggests larger studies are required to

ascertain the use of TcB devices in preterm infants after phototherapy.133

Overall, studies that have looked at TcB device use in preterm infants have reached conflicting

conclusions. One study reported significant differences between TcB and TSB levels in preterm

infants134, while others have reported TcB levels to accurately predict TSB levels in preterm

infants.135-137 The conflicting results may be due to heterogeneous samples of neonates with a

small sample size per gestational age group. In this section, I systematically review data on TcB

use in preterm infants by receipt of phototherapy, by anatomical site of measurement as well

as by skin colour and/or parental ethnicity.

2.5.2.1 Phototherapy

This section will discuss phototherapy’s impact on an infant’s skin and the transcutaneous

measurement of bilirubin.

Light exposure administered through phototherapy reduces the bilirubin level measured at the

skin more than in the blood.138,139 Phototherapy converts bilirubin to a water-soluble molecule,

37

which is excreted by the liver. The direct light exposure to an infant’s skin reduces bilirubin

levels in the skin.140 TcB devices release white light into the skin of an infant and measures the

cutaneous bilirubin levels through the intensity of the wavelengths returned to the device.141

The decrease of cutaneous bilirubin may therefore impact the ability of the TcB device to

accurately measure TSB levels in infants.

There is strong evidence for the use of TcB devices prior to phototherapy in term and near-

term infants. Using TcB devices during and after phototherapy was often strongly discouraged

in large studies including term and near-term infants. 142,143 TcB device use during and after

phototherapy in preterm infants, however, still continues to be debated due to limited sample

sizes.133 This may be due to the early and frequent administration of phototherapy being a

routine part of jaundice care in preterm infants. Studies in preterm infants assessing the

performance of TcB measurements during and after phototherapy reported TcB levels to

underestimate TSB144-146, while one study of preterm infants reported TcB levels to

overestimate TSB levels.21 These studies included sample size of less than 50 preterm

infants.21,144-146 In contrast, two larger studies (i.e., 94-196 infants) found TcB measurements to

overestimate TSB levels when used after the initiation of phototherapy.125,136 More recently

and conversely, two larger studies in 2019 and 2020 reported TcB measurements to be less

accurate after the initiation of phototherapy.123,147 Overall, although the use of TcB devices

after phototherapy has been clinically discouraged, in keeping with recent studies, larger

studies in preterm infants assessing the agreement of TcB levels with TSB levels during and

after the initiation of phototherapy are required.133

As a potential solution to the impact of phototherapy on TcB devices, in term and near-term

infants, covering a small part of the skin during phototherapy has garnered interest over the

past 5 years. Over the past couple of years, some studies have started to employ and assess the

same solution in preterm infants.147,148 However, the extent of the impact of phototherapy on

TcB measurements on exposed skin remains limited due to the small sample sizes of these

studies. Given the increased frequency of phototherapy in preterm infants , a larger study using

TcB devices after the initiation of phototherapy is required before a large study using TcB

38

measures on covered skin. In fact, exploring the optimal anatomical site of measurement of

TcB levels without intervention, during and after the initiation of phototherapy may inform the

design of a larger study assessing TcB use on covered during phototherapy.

2.5.2.2 Site of measurement

TcB measurements are typically taken on the forehead and sternum in all infants regardless of

gestational age, with a few studies taking TcB measurements on the abdomen.128,133 The

appropriate site of TcB device use has continuously been under review across the different TcB

devices with varying results. Nagar et al in 2013 conducted a systematic review of 22 studies

assessing TcB device usage in preterm infants. Among these studies, only six compared the use

of TcB measurements on the forehead and sternum, while the remaining 16 studies used either

only the forehead or sternum. Among the six studies that compared the use of TcB

measurements on the forehead and sternum, overall agreement between TcB and TSB was

acceptable on both sites. These measurements were limited to prior to the initiation of

phototherapy.128 The sample sizes were also less than 70 infants.128

Since 2013, some larger studies compared TcB measurements on the forehead, sternum and

additional anatomical sites of measurement (abdomen and interscapular sites). These studies

reported conflicting results between the forehead and sternum with some reporting the

sternum to be a more accurate site.149,150 Others reported the forehead to be a more accurate

site.151

Overall, prior to the initiation of phototherapy the optimal anatomical site of measurement is

unclear as studies have shown conflicting results. In addition, these studies are based on small

sample sizes or exclude a diverse sample of neonates.128,151,152 Despite the potential change in

the accuracy of TcB by anatomical site of measurement after the initiation of phototherapy,

studies comparing anatomical sites are limited and use small sample sizes as well.133 More

research comparing anatomical sites of measurement both prior to, and after the initiation of

39

phototherapy in a diverse population of preterm infants is required.

2.5.2.3 Maternal ethnicity

The potential impact of parental ethnicity and skin colour needs to be better understood in

preterm infants. The skin in newborns has been reported to be different between preterm and

term infants. This may be due to the delay in skin maturation among preterm infants. Among

preterm infants skin maturation is delayed by approximately 2-3 weeks.153 One study also

reported a difference in skin by gestational age at birth in preterm infants.154

Initially, the accuracy of TcB devices was reported to be impacted by an infant’s skin

pigmentation.155 With the introduction of multiple TcB devices and technological

advancements, race and ethnicity seem to have less of an effect on TcB device performance.

However, studies measuring TcB levels in more diverse populations of infants are limited.128 In

recent years, additional studies have been conducted to test the accuracy of TcB devices in

specific ethnic populations.131,152,156 There is good evidence to support the hypothesis that race

and ethnicity do not impact TcB readings.131,135 However, these studies are based on small

sample sizes in premature infants and/or limited to term and near-term infants, requiring larger

scale studies to support these recommendations in preterm infants.128

Studies before 2013 looking at TcB device usage in preterm infants provided limited data with

respect to the effect of maternal ethnicity and race. A systematic review by Nagar et al in 2013

of TcB measurement studies in preterm infants reported participants’ ethnicity when available

but did not assess the performance of TcB devices by race or ethnicity.128 Among the 22 studies

reviewed, only four studies included mixed ethnicities, four studies were specific to one non-

Caucasian group (2 studies of Chinese infants, one study of Japanese infants and one study of

Persian infants), and the remaining 14 studies either included only Caucasian infants or did not

mention the ethnic background of their population.128

40

TcB device use expanded to different countries enabling a greater understanding of its use in

specific ethnicities but in these studies no comparison between ethnicities were

done.151,152,156,157 Other studies have compared and grouped non-Caucasian infants together

and limited their analysis to Caucasian vs non-Caucasian, excluding analysis between non-

Caucasian group differences. Among these studies, the impact of skin colour and ethnicity on

TcB levels is conflicting. While some studies found no impact of skin colour or ethnicity on TcB

measurements135,136,141,158 other studies reported an impact.159,160 Given the inconsistently

reported impact of maternal ethnicity in preterm infants and lack of diversity in these studies, a

larger study assessing the agreement between TcB and TSB measurements in an ethnically

diverse population of preterm infants within Canada is required.

2.5.3 Summary of TcB measurements in preterm infants and relevance

Overall, the screening of premature infants for hyperbilirubinemia with TcB devices warrants

more research. TcB devices offer a non-invasive, immediate approach to screening for

hyperbilirubinemia in preterm infants that could potentially decrease the pain and stress

infants experience from repeated blood tests. However, studies assessing the effectiveness of

TcB devices are limited to term and near-term infants or small studies including preterm infants

with a lack of diversity. A larger study of TcB use in a diverse population of preterm infants

assessing TcB measurements, overall, and by receipt of phototherapy and by anatomical site of

measurement is required.

Chapter 5 presents a multi-site prospective cohort study of preterm infants born at 240/7 to

356/7 weeks’ gestation. This study assesses the agreement between TcB and TSB levels overall,

prior to and after the initiation of phototherapy, by anatomical site of measurement and by

maternal ethnicity.

41

2.6 Overall summary of literature review and significance

In this chapter, I systematically examined two aspects of the assessment of hyperbilirubinemia

in preterm infants born at 240/7 to 356/7 weeks’ gestation. The first aspect relates to the

detection of hyperbilirubinemia. As highlighted in chapter 2 preterm infants are more prone to

significant hyperbilirubinemia and their brains may be more susceptible to the toxic effects of

hyperbilirubinemia. However, TSB cut-points or thresholds for continued monitoring and

initiating phototherapy are poorly established. Accordingly, chapters 3 and 4 attempts to derive

mathematical TSB thresholds by degree of prematurity. The second aspect relates to the

method of measurement of bilirubin levels in preterm infants, namely that repeated blood

sampling leads to both pain for the infant and risk of anemia. Accordingly, a non-invasive

approach, if accurate, is preferred. Chapter 5 assesses the agreement between TcB and TSB

measurements in preterm infants overall, by receipt of phototherapy, anatomical site of

measurement and maternal ethnicity.

42

2.7 Overall research aims, objectives and hypotheses

Even though preterm infants are at significant risk of experiencing severe hyperbilirubinemia

and its toxic effects guidelines for the assessment of hyperbilirubinemia are consensus-based,

and systematically derived pre-treatment TSB percentiles are lacking for preterm infants. In

addition preterm infants who receive repeat TSB testing are subjected to greater stress, pain,

and risk of anemia.

In order to inform the assessment of hyperbilirubinemia and optimize management of

hyperbilirubinemia in preterm infants, a better understanding of the measurement bilirubin

levels in preterm infants need to be established. In addition, a quicker and ideally non-invasive

way to conduct routine bilirubin testing in preterm infants needs to be assessed and verified to

support and achieve routine bilirubin screening universally.

Therefore, the overall aims of this thesis were to define hyperbilirubinemia in preterm infants

born 240/7 to 356/7 weeks’ gestation and to provide data on the measurement of TSB and TcB

levels in preterm infants born 240/7 to 356/7 weeks’ gestation. This was achieved by the

following three studies and research objectives:

43

Study 1: Hour-specific total serum bilirubin percentiles for infants born at 29 to 35 weeks’

gestation (chapter 3)

Primary Objective: To generate pre-treatment hour-specific TSB percentile curves among

preterm infants born at 290/7 to 356/7 weeks’ gestation, including by gestational age of

prematurity, by subsequent receipt of phototherapy and by influential factors such as enteral

feeding and hemolysis.

To address the lack of systematically derived pre-treatment TSB levels in preterm infants, pre-

treatment TSB levels directly derived from preterm infants is required. Based on previous

assumptions of risk factors associated with hyperbilirubinemia in preterm infants and TSB

nomograms developed in term and near-term infants, this study hypothesized that pre-

treatment hour specific TSB levels may be influenced by an infant’s prematurity, whether

infants’ subsequently receive phototherapy, hemolysis and feeding difficulties. Therefore, a

deeper understanding of how TSB levels may change under these important clinical conditions

needs to be established as well.

To achieve the primary research objective of study 1, a multi-site retrospective cohort study of

preterm infants born at 290/7 to 356/7 weeks’ gestation between January 2013 and June 2017

was completed at three NICUs in Ontario, Canada.

44

Study 2: Pre-phototherapy total serum bilirubin levels in extremely preterm infants (chapter

4)

Primary Objective: To generate pre-phototherapy TSB percentile levels at 24 hours of age

among extremely preterm infants born at 240/7 to 286/7 weeks’ gestation, and to contrast these

derived percentile levels with currently used consensus-based thresholds published by

Maisels.18

Secondary Objective: To determine the proportion of infants who were started on

phototherapy below Maisels’ published threshold,18 overall, and by hours of age of

phototherapy initiation.

The lack of systematically derived TSB thresholds for initiating phototherapy in preterm infants

have resulted in consensus based guidelines for the assessment of hyperbilirubinemia. These

guidelines have been further adapted by NICUs. Consequently there is an increase of

phototherapy administration among preterm infants born extremely preterm at <29 weeks’

gestation. Given the increase of phototherapy administration in this vulnerable population and

potential adaptions to Maisels’ consensus-based guidelines, study 2 hypothesized that

systemically derived pre-treatment TSB levels in extremely preterm infants may differ from

Maisels’ consensus-based TSB thresholds established for phototherapy initiation.

To achieve this study’s objectives, a multi-site retrospective cohort study of extremely preterm

infants born at 240/7 to 286/7 weeks’ gestation between January 2013 and June 2017 was

completed at three NICUs in Ontario, Canada.

45

Study 3: Transcutaneous vs. total serum bilirubin measurements in preterm infants (chapter

5)

Primary Objective: To evaluate the agreement and accuracy of TcB measurements among

preterm infants born at 240/7 to 356/7 weeks’ gestational age, including before and after

phototherapy, by the anatomical site of measurement, and the infant’s ethnicity. (Chapter 5)

Based on previous studies assessing the impact of neonatal and maternal factors on TcB device

performance, we hypothesized that TcB agreement with TSB may be influenced by prematurity,

receipt of phototherapy, anatomical site of measurement and maternal ethnicity. Given the

impact phototherapy has on TSB levels at the skin, we suspect agreement between TcB and TSB

may be different before and after the initiation of phototherapy.

To achieve the primary objective of study 3, a multi-site prospective cohort study of preterm

infants born at 240/7 to 356/7 weeks’ gestation was conducted at three NICUs in Ontario, Canada,

between September 2016 to June 2018.

46

Chapter 3 . Hour-specific total serum bilirubin percentiles for infants born at

29 to 35 weeks’ gestation

This chapter is currently under peer-review with the journal Neonatology , publisher S. Krager AG Basel.

47

3.1 Abstract

Introduction: As preterm infants are susceptible to hyperbilirubinemia; they require frequent

close monitoring. Prior to initiation of phototherapy, hour-specific total serum bilirubin (TSB)

percentile cut-points are lacking in these infants, which lead to the current study.

Methods: A multi-site retrospective cohort study of preterm infants born between January

2013 and June 2017 was completed at three NICUs in Ontario, Canada. A total of 2549 infants

born at 290/7 to 356/7 weeks’ gestation contributed 6143 pre-treatment TSB levels. Hour-specific

TSB percentiles were generated using quantile regression, further described by degree of

prematurity, and among those who subsequently received phototherapy.

Results: Among all infants, hour-specific pre-treatment TSB percentiles were 36.1 µmol/L (95%

CI 34.3-39.3) at the 40th, 52.3 µmol/L (49.4-55.1) at the 75th, and 79.5 µmol/L (72.1-89.6) at

the 95th percentiles. The corresponding percentiles were 39.3 μmol/L (35.9-43.2 ), 55.4 μmol/L

(52.1-60.2) and 87.1 μmol/L (CI 70.5-102.4) prior to initiating phototherapy, and 24.4 μmol/L

(20.4-28.8), 35.3 μmol/L (31.1-41.5) and 52.0 μmol/L (46.1-62.4) among those who did not

receive phototherapy. Among infants born at 29 to 32 weeks, pre-treatment TSB percentiles

were 53.9 µmol/L (49.4-61.0) and 95.5 µmol/L (77.5-105.0) at the 75th and 95th percentiles,

with respective values of 48.7 µmol/L (43.0-52.3), and 74.1 µmol/L (64.8-83.2) for those born at

33 to 35 weeks’ gestation.

Conclusion: Hour-specific TSB percentiles, derived from a novel nomogram, may inform how

hyperbilirubinemia is described in preterm newborns. Further validation of this nomogram is

recommended before its clinical adoption.

48

3.2 Introduction

Preterm infants are particularly vulnerable to acute (ABE) and chronic bilirubin encephalopathy

(CBE).4 However, among infants born preterm at < 36 weeks’ gestation, there is a paucity of

evidence on safe hour- specific thresholds for total serum bilirubin concentrations (TSB),

especially among those who have not yet received phototherapy.16,92 This issue is compounded

by the fact that clinical management largely relies on consensus based guidelines,1,2 leaving

care providers to rely on clinical judgement in their approach to preterm newborns.16,18

Contemporary knowledge of the natural history of pre-treatment TSB levels among preterm

infants, and the provision of hour-specific statistical cut-points to define hyperbilirubinemia,

might influence decision-making about the need for ongoing TSB testing and/or the initiation of

phototherapy in this susceptible population. Accordingly, the current study was undertaken to

generate hour-specific pre-treatment TSB percentile curves among preterm infants born at

290/7 to 356/7 weeks’ gestation, including by degree of prematurity, subsequent receipt of

phototherapy, and by influential factors such as enteral feeding and red cell hemolysis.

3.3 Methods

This multi-site retrospective cohort study included preterm infants born or transferred to St.

Michael’s Hospital (January 2013 to June 2017) and Sinai Health (January 2015 to June 2017), in

Toronto, Ontario, as well as the Hamilton Health Sciences Centre in Hamilton Ontario (January

2014 to June 2017). All healthcare in Ontario is provided under the universal Ontario Health

Insurance Plan.

From each of three hospitals, included were preterm infants born at 290/7 to 356/7 weeks’

gestation. Newborns were excluded who had Rh disease, transferred to a NICU outside of a

participating centre without a TSB level done, or who did not have an accessible TSB level in

49

their electronic medical chart. Eligible preterm infants born during the study period were

identified using the Canadian Neonatal Network (CNN) and the provincial Better Outcomes

Registry and Network (BORN) databases.161,162

Infants who met the main inclusion criteria were further assessed for completeness of their

electronic medical charts to obtain pre-treatment TSB levels -- defined as any TSB level prior to

initiation of phototherapy (among those who went on to receive such phototherapy), or any

TSB level otherwise (among those who did not go on to receive phototherapy). Also abstracted

was the postnatal age (in hours), phototherapy status, hours of age at phototherapy initiation

(if started), gestational age at birth in weeks, and relevant maternal and infant information.

Infants with at least one pre-treatment TSB within the first three days of birth were included in

the final analysis.

Infants were also included in a secondary analysis who had known hemolysis (laboratory-

confirmed ABO incompatibility), as well as mode of feeding (enteral feeds vs. total parenteral

nutrition [TPN] vs. combined enteral + TPN) within the first 10 days of birth.

3.3.1 Data analyses

The primary study analysis set out to generate an hour-specific pre-treatment TSB percentile-

based nomogram for male and female infants born at 290/7 to 355/6 weeks’ gestation.

Nomograms were developed overall (infants born 290/7 to 355/6 weeks’ gestation), by

subsequent receipt of phototherapy, and by gestational age groups (290/7 to 326/7 and 330/7 to

356/7 weeks’ gestation). All hour-specific pre-treatment TSB levels from preterm infants -- prior

to initiation of phototherapy, and regardless of whether phototherapy was ever started -- were

plotted in 6-hour increments, from birth up to 5 days thereafter. As used in previous TSB

percentile curves for term and near-term infants 3, the 40th, 75th and 95th percentiles were

generated using quantile regression, including quadratic polynomials for age (in hours). The

50

quantreg package in R version 4.0.3 was used for fitting all quantile regression models.163,164

Modelling accounting for repeated measures within an infant, and 1000 bootstrap resamples of

participants were used to generated 95% confidence intervals (CI).165 Quantile regression was

used to generate pre-treatment TSB levels, because it allows one to evaluate the relation of

independent variables across a full range of continuous dependent variables, rather than a

conditional mean.166 Missing data was labeled as “unknown”, and accounted for in relevant

sub-analyses.

To assist with the clinical interpretation of the percentile-based nomogram, the mean pre-

treatment peak TSB, and the time of an infant’s pre-treatment peak TSB from birth to 72 hours

of age, were calculated for all newborns, by receipt of phototherapy, by gestational age

groupings, by laboratory-confirmed ABO incompatibility, as well as by mode of feeding – as

described above. To determine differences in infants’, mean pre-treatment peak TSB, and hours

of age of pre-treatment peak TSB within the first 72 hours since birth, a one-way ANOVA was

conducted for these aforementioned neonatal factors, with statistical significance p-values <

0.01. Finally, to determine differences in the proportion of infants who subsequently received

phototherapy, a chi-square analysis was conducted for the above-mentioned neonatal factors,

tested at a p-value < 0.01. All statistical analyses were conducted using R version 4.0.3 and SPSS

27 for Mac OS.164,167

3.3.2 Sample size calculation

The sample size was calculated based on assumptions from previous nomograms for pre-

treatment total serum bilirubin levels in term and near-term infants.168 Using the methods for

reference limits by Bellera and Hanley, in order to obtain 2.5% and 97.5% reference limits, with

a relative margin of error of 10% for a Gaussian distribution, a minimum sample size of 448

preterm infants was required.169 Since the TSB curve-by-time was further stratified by infants

born at 29 to 32 and 33 to 35 weeks’ gestation, and while accounting for a potentially 20% loss-

to follow-up, a minimum of 537 infants was deemed necessary for the 29 to 32 and 33 to 35

51

weeks’ gestation age groups, respectively.

3.4 Results

Out of 2954 preterm infants born at 290/7 to 356/7 weeks’ gestation, 2549 infants had at least

one pre-treatment TSB level available from birth to 72 hours of age (Figure 3-1). The mean

(standard deviation [SD]) gestational age and birthweight of the study population was 32.6 (1.9)

weeks and 1915.2 (695.3) g, respectively (Table 3-1).

3.4.1 All Infants

In the creation of the main nomogram, 2549 infants contributed a total of 6143 hour-specific

pre-treatment TSB measures (Figure 3-2A). The pre-treatment estimated TSB percentiles were

36.1 µmol/L (95% CI 34.3 to 39.3) at the 40th, 52.3 µmol/L (95% CI 49.4 to 55.1) at the 75th,

and 79.5 µmol/L (95% CI 72.1 to 89.6) at the 95th percentiles (Table 3-2). The corresponding

nomogram-estimated rate of rise of TSB from birth were 2.9 μmol/L/hour (95% CI 2.7 to 3.0),

3.1 μmol/L/hour (95% CI 3.0 to 3.3), and 3.1 μmol/L/hour (95% CI 2.7 to 3.4) (Table 3- 2). The

estimated change in the rate of rise of TSB then diminished with advancing age after birth

(Table 3-2).

Overall, the estimated pre-treatment TSB level percentiles peaked at 90.6 hours of age for

the 40th percentile, 103.3 hours for the 75th percentile, and 110.7 hours at the 95th percentile

(Table 3-2 and Figure 3-2A). Upon limiting the data to the first 72 hours of age, the mean (SD)

peak TSB was 142.0 μmol/L (37.9) at a mean (SD) of 41.7 (17.3) hours of age (Table 3-3).

52

3.4.2 By subsequent receipt of phototherapy

A total of 696 infants (27.3%) did not receive phototherapy during their hospitalization, and

they provided 2306 hour-specific TSB levels (Figure 3-2B). There were 1853 infants (72.7%) who

subsequently received phototherapy at a mean (SD) of 46.5 (26.9) hours of age, and they

provided 3837 hour-specific pre-treatment TSB levels (Figure 3-2C). Their overall pre-treatment

TSB levels at birth at the 40th, 75th, and 95th percentiles were higher than the preterm infants

who did not receive phototherapy (Table 3-2). The estimated TSB rate of rise at birth was

slightly higher among infants who subsequently received phototherapy than those who did not,

except for the 95th percentile (Table 3-2). Within the first 72 hours of age, those administered

phototherapy had a higher mean peak in TSB (145.7 μmol/L vs. 132.1 μmol/L; p < 0.01), which

occurred significantly earlier (38.3 vs. 50.8 hours; p < 0.01) (Table 3-3).

3.4.3 By degree of prematurity

A total of 1120 infants were born at 290/7 to 326/7weeks’ gestation, providing 2313 pre-

treatment TSB levels (Figure 3-3A), and 1429 infants were born at 330/7 to 356/7 weeks, with a

total of 3830 pre-treatment TSB levels (Figure 3-3B). For those born at 29 to 32 weeks’

gestation, estimated pre-treatment TSB percentiles at birth were 53.9 μmol/L (95% CI 49.4 to

61.0) at the 75th percentile, and 95.5 μmol/L (95% CI 77.5 to 105.0) at the 95th percentile (Table

3-2). Among those born at 33 to 35 weeks’ gestation, the respective values were 48.7 μmol/L

(95% CI 43.0 to 52.3) and 74.1 μmol/L (95% CI 64.8 to 83.2) (Table 3-2).

The estimated TSB rate of rise from birth was generally similar between the two gestational

age groups, except at the 95th percentile, where the estimated TSB rate of rise was higher

among infants born at 33 to 35 weeks’ gestation than those born at 29 to 32 weeks (Table 3-2).

Pre-treatment TSB percentiles peaked earlier among the latter than the former (Table 3-2 and

Figure 3-3A and B). Within the first 72 hours after birth, mean pre-treatment TSB level peaked

significantly earlier in infants born at 29 to 32 weeks than those born later (36.3 vs. 46.0 hours;

53

p < 0.01), and with a significantly lower mean peak TSB level among the former vs. latter

(133.7 vs. 148.5 μmol/L; p < 0.01) (Table 3-3). Furthermore, significantly more infants born at

29 to 32 weeks’ gestation received phototherapy than those born at 33 to 35 weeks (91.1% vs

58.3%; p < 0.01) (Table 3-4).

Estimated pre-treatment TSB level percentile differences between infants born at 33 to 35

weeks’ gestation and those born at 29 to 32 weeks’ gestation widened with time since birth,

especially after 24 hours of age (Figure 3-4). Actual mean differences are shown in Figure 3-5.

3.4.4 By feeding type

When stratified by feeding type or presence of red hemolysis, there was an insufficient number

of pre-treatment TSB levels to generate hour-specific pre-treatment TSB percentile curves.

Overall, among all infants, the mean peak TSB between birth and 72 hours was significantly

higher in infants exclusively receiving enteral nutrition (146.6 μmol/L) than those receiving TPN

(134.2 μmol/L), or a combination of both (140.8 μmol/L) (p < 0.01) (Table 3-3). Significantly

more infants who were on TPN -- with or without enteral feeds -- were administered

phototherapy than those exclusively receiving enteral feeds (Table 3-4).

3.4.5 Peak pre-treatment total serum bilirubin by hemolysis

Among all 2549 infants, 327 (12.8%) had laboratory confirmed ABO incompatibility. Those with

ABO incompatibility had their TSB peak at a significantly lower concentration, and earlier, than

infants without ABO incompatibility (Table 3-3).

54

3.5 Discussion

This multi-site retrospective cohort study produced a novel hour-specific, pre-treatment TSB

percentile-based nomogram for preterm infants born at 290/7 to 356/7 weeks’ gestation. Hour-

specific pre-treatment TSB levels differed between infants born at 330/7 to 356/7 and 290/7 to

326/7 weeks’ gestation. Degree of prematurity and infant nutrition were each significantly

associated with the subsequent initiation of phototherapy.

This is one of the first and largest studies to estimate hour-specific percentiles of pre-treatment

TSB levels in preterm infants born at 290/7 to 356/7 weeks’ gestation. One previous study was

limited to fewer than 1000 very low birthweight preterm infants.92 Other studies relied on

perceived risk of hyperbilirubinemia in preterm infants, rather than pre-treatment TSB levels

directly from preterm infants.16,18 Finally, this is one of the first studies to describe the influence

of feeding type on TSB levels and phototherapy initiation in preterm infants.

This study has some limitations. First, fewer infants at more extreme prematurity were

included, and they are more likely to undergo early initiation of phototherapy; no infants born

before 29 weeks’ gestation were included. In our study, initiation of phototherapy was at the

discretion of the clinical care team, rather than by a defined study protocol. Nevertheless, a

large number of pre-treatment TSB levels were collected up to 120 hours after birth. Second,

information about infant feeding was limited to the first 10 days of life and lacked details about

the volume and type of enteral feeds or TPN. Certainly, other approaches to the management

of hyperbilirubinemia and feeding practices in preterm infants could have generated somewhat

different TSB nomograms and TSB percentiles than those based on Canadian infants from three

nearby clinical centres.

As preterm infants are known to be at a higher risk for severe hyperbilirubinemia, they tend to

receive treatment at lower TSB thresholds than term infants.16-18 In our study, within the first

72 hours of age, TSB peaked earlier in infants born more premature -- often before 48 hours –

55

which is consistent with previous research on predictors of severe hyperbilirubinemia.17,18

However, this observed early peak in TSB levels among more premature infants was likely due

to the fact that many received phototherapy soon thereafter.86,87

Delayed hyperbilirubinemia has been reported in preterm infants born at 34 to 35 weeks’

gestation.170 We observed that infants at the 95th percentile of TSB continued to experience a

TSB rise up to 110 hours of age, for example. This would suggest that TSB should continue to be

monitored beyond 72 hours of age, especially among infants whose TSB is near the 95 th

percentile.

As seen elsewhere86,87, gestational age had a significant impact on TSB levels and initiation of

phototherapy. Pre-treatment TSB percentile curves differed significantly between infants born

at 29 to 32 weeks’ or 33 to 35 weeks’ gestation, suggesting that two different nomograms

might be needed for these age groups. 16-18

Although the impact of red cell hemolysis on the risk of hyperbilirubinemia in preterm infants

has been well studied, that of infant nutrition has not.61,171 After being processed by the liver,

bilirubin is excreted into the gastrointestinal tract (GIT), and therefore, GIT abnormalities and

motility issues can further affect bilirubin clearance.172 In our study, more infants who received

TPN, either alone, or with enteral feeds, received phototherapy, and experienced an earlier

peak in TSB than those solely receiving enteral feeds. Hence, early nutrition may be an

additional factor to consider when determining the risk of hyperbilirubinemia.

For preterm infants, our study provides clinicians and policymakers with novel information

about hourly trends in pre-treatment TSB levels in preterm infants. Analogous to research

determining the optimal threshold for oxygen and carbon dioxide in preterm infants, there is

also a need to identify which hourly bilirubin threshold is safest, short-term and long-

term.173,174 A clinical trial might compare initiation of phototherapy at the lower vs. higher pre-

treatment TSB percentiles described herein.

56

3.6 Conclusion

The American Academy of Pediatrics has called for development of guidelines for the

management of jaundice in preterm infants. In response, we generated hour-specific TSB

percentiles and nomogram plots.18 While the current nomograms offer a first step in reliably

understanding bilirubin levels in preterm infants, further research is needed to determine their

predictive ability and safety in identifying infants at higher risk of jaundice.

57

3.7 Tables

Table 3-1. Neonatal and maternal characteristics of 2549 preterm infants included in the study by

prematurity groups. All data are presented as a number (%) unless otherwise indicated.

Characteristic Overall By degree of prematurity at birth

290/7 to 326/7weeks 330/7 to 356/7weeks

Mean (SD) gestational age, weeks 32.6 (1.9) 30.7 (1.1) 34.0 (0.8)

Mean (SD) birthweight, g 1915.2 (695.3) 1559.9 (381.8) 2193.6 (756.1)

Small for gestational age birthweight < 10th

percentile 471 (18.5) 152 (13.6) 319 (22.3)

Female 1139 (44.7) 484 (43.2) 655 (45.8)

Mean (SD) maternal age at birth of current infant,a y 32.2 (5.8) 31.9 (5.7) 32.5 (5.9)

Mode of delivery

Cesarean 1526 (59.9) 691 (61.7) 835 (58.4)

Vaginal 975 (38.2) 409 (36.5) 566 (39.6)

Unknown 48 (1.9) 20 (1.8) 28 (2.0)

Hemolysis

ABO incompatibility 327 (12.8) 142 (12.7) 185 (12.9)

Feeding

Enteral only 891 (34.9) 305 (27.2) 586 (41.0)

Total parenteral nutrition only 285 (11.2) 137 (12.2) 148 (10.4)

Mixed (enteral and total parenteral nutrition) 1307 (51.3) 649 (57.9) 658 (46.0)

Unknown 66 (2.6) 29 (2.7) 37 (2.6)

Sepsis with a positive blood or spinal fluid culture 141 (5.5) 73 (6.5) 68 (4.8)

Median (IQR) number of total serum bilirubin measurements per infant prior to the initiation of phtotoherapy

2.0 (1.0 to 3.0)

2.0 (1.0 to 3.0)

2.0 (1.0 to 3.0)

Mortality 24 (0.9) 12 (1.1) 12 (0.8)

a Maternal age missing in <1% of cohort overall and by degree of prematurity.

58

Table 3-2. Estimated pre-treatment total serum bilirubin percentiles at birth, and the estimated hours of age at peak total serum bilirubin at the 40th, 75th and

95th percentiles, by subsequent receipt of phototherapy, and by gestational age.

By subsequent receipt of phototherapy By gestational age at birth Measure All infants born at 29 to 35 weeks’

gestation

Did not subsequently receive phototherapy

Did subsequently receive phototherapy

29 to 32 weeks’ gestation 33 to 35 weeks’ gestation

40th 75th 95th 40th 75th 95th 40th 75th 95th 40th 75th 95th 40th 75th 95th Estimated total serum bilirubin at birth (95% CI), μmol/L

36.1

(34.3 to 39.3)

52.3

(49.4 to 55.1)

79.5

(72.1 to 89.6)

24.4

(20.4 to 28.8)

35.3

(31.1 to 41.5)

52.0

(46.1 to 62.4)

39.3

(35.9 to 43.2)

55.4

(52.1 to 60.2)

87.1

(70.5 to 102.4)

40.5

(37.0 to 44.5)

53.9

(49.4 to 61.0)

95.5

(77.5 to 105.0)

32.3

(27.6 to 35.8)

48.7

(43.0 to 52.3)

74.1

(64.8 to 83.2)

Estimated rate of rise of total serum bilirubin at birth (95% CI), μmol/L/h

2.9 (2.7 to

3.0)

3.1 (3.0 to

3.3)

3.1 (2.7 to

3.4)

2.7 (2.5 to

3.0)

2.9 (2.7 to

3.1)

3.1 (2.6 to

3.3)

3.0 (2.8 to

3.2)

3.1 (2.8 to

3.3)

2.9 (2.2 to

3.7)

2.8 (2.6 to

3.1)

3.2 (2.8 to

3.5)

2.5 (2.0 to

3.3)

2.9 (2.8 to

3.2)

3.2 (3.0 to

3.4)

3.3 (2.9 to

3.7)

Estimated change in the rate of rise from birth onward (95% CI), μmol/L/h2

-0.016 (-0.017

to -0.015)

-0.015

(-0.017 to

-0.014)

-0.014 (-0.017

to -0.009)

-0.014 (-0.016

to -0.012)

-0.013 (-0.015

to -0.011)

-0.012 (-0.014

to -0.008)

-0.014 (-0.017

to 0.012)

-0.013 (-0.016

to -0.010)

-0.009 (-0.017

to -0.003)

-0.018

(-0.021 to

-0.016)

-0.020

(-0.023 to

-0.015)

-0.012

(-0.019 to

-0.007)

-0.015

(-0.018 to

-0.014)

-0.015 (-0.018

to -0.014)

-0.014 (-0.018

to - 0.011)

Estimated hours of age at the peak of total serum bilirubin, h

90.6

103.3

110.7

96.4

111.5

> 120

107.1

119.2

> 120

77.8

80.0

104.2

96.7

106.7

117.8

59

Table 3-3. Peak total serum bilirubin concentration within the first 72 hours after birth, as well as the timing of that peak, by neon atal factors.

Measure Overall By subsequent receipt of

phototherapya

By gestational age at birth,

weeksb

By feeding type among all infants

born at 29 to 35 weeksc

By feeding type among infants born at

29 to 32 weeksd

By feeding type among infants born at 33

to 35 weekse

By ABO incompatibilityf

Yes No 29-32 33-35 Enteral TPN Mixed Enteral TPN Mixed Enteral TPN Mixed Present Absent

Mean (SD) peak total serum bilirubin concentration in the first 72 h

of life, µmol/L

142.0 (37.9)

145.7 (37.5)

132.1 (37.2)

133.7 (31.8)

148.5 (41.0)

146.6 (40.6)

134.2 (35.4)

140.8 (36.4)

138.4 (32.9)

128.9 (28.3)

132.7 (32.0)

150.8 (43.5)

139.1 (40.3)

148.8 (38.6)

135.3 (38.2)

143.0 (37.8)

Mean (SD) time since birth to peak total

serum bilirubin, h

41.7

(17.3)

38.3

(16.6)

50.8

(15.6)

36.3

(16.2)

46.0

(16.9)

44.3

(16.9)

39.1

(17.6)

40.5

(17.3)

37.8

(16.5)

35.0

(16.0)

35.7

(16.2)

47.7

(16.2)

42.9

(18.3)

45.3

(17.1)

37.6

(17.5)

42.3

(17.2)

a Mean peak total serum bilirubin and mean hours of age at the time of peak total serum bilirubin significantly differed between infants subsequently administered and not

administered phototherapy (p<0.01).

b Mean peak total serum bilirubin and mean hours of age at the time of peak total serum bilirubin significantly differed between infants born 29 to 32 weeks’ and 33 to 35

weeks’ gestation (p < 0.01).

c Mean peak total serum bilirubin and mean hours of age at the time of peak total serum bilirubin significantly differed across infants’ feeding type among all infants born

29 to 35 weeks’ gestation (p<0.01).

d Mean peak total serum bilirubin significantly differed across infants’ feeding type among infants born 29 to 32 weeks’ gestat ional age (p <0.01).

e Mean peak total serum bilirubin and mean hours of age at the time of peak total serum bilirubin significantly differed across infant’s feeding type among infants born 33

to 35 weeks’ gestational age (p<0.01).

f Mean peak total serum bilirubin and mean hours of age at the time of peak total serum bilirubin significantly differed between infants with and without ABO

incompatibility (p<0.01).

60

Table 3-4. Neonatal characteristics by proportion of infants subsequently administered phototherapy.

Overall By gestational age at birth,

weeksa

By feeding type among all infants

born at 29 to 35 weeksb

By feeding type among infants born at

29 to 32 weeks

By feeding type among infants born at 33

to 35 weeksc

By ABO incompatibility

29-32 33-35 Enteral TPN Mixed Enteral TPN Mixed Enteral TPN Mixed Present Absent

No (%) of infants subsequently administered phototherapy

1853 (72.7)

1020 (91.1)

833

(58.3)

577

(64.8)

211

(74.0)

1018 (77.9)

278

(91.1)

123

(89.8)

592

(91.2)

299

(51.0)

88

(59.5)

426

(64.7)

238

(72.8)

1615 (72.7)

a Proportion of infants subsequently administered phototherapy significantly differed between infants born 29 to 32 weeks’ and 33 to 35 weeks’ gestational age

(p < 0.01).

b Proportion of infants subsequently administered phototherapy significantly differed ac ross feeding types among all infants born 29 to 35 weeks’ gestational age

(p < 0.01).

c Proportion of infants subsequently administered phototherapy significantly differed across feeding types among infants 33 to 35 weeks’ gestational age

(p <0.01).

61

3.8 Figures

Figure 3-1. Flow diagram of included preterm infants born at 290/7 to 356/7 weeks’ gestation,

from January 2013 to June 2017

2954 electronic medical charts of preterm

infants born 290/7 to 356/7 weeks’ gestation

reviewed at participating study sites

2549 preterm infants included in the

primary analysis

405 preterm infants excluded

Receipt of phototherapy unknown (n=162)

Pre-treatment bilirubin level from birth to 72 hours unknown (n=243)

62

Figure 3-2. Hour-specific pre-treatment total serum bilirubin percentile-based curves among preterm infants born at 29 to 35

weeks’ gestation (A) overall (n=2549), (B) among infants not subsequently administered phototherapy (n=696), and (C) among

infants subsequently administered phototherapy (n=1853). To convert total serum bilirubin levels to mg/dL divide by 17.1.

No. infants contributing in each 6-h interval: 68 330 310 993 1103 308 461 447 370 273 289 258 161 143 144 135 88 92 74 73

A

95th percentile

75th percentile

40th percentile

63

Hours of age

To

tal se

rum

bili

rubin

(µ

mol/L)

0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102 108 114 120

050

100

150

20

02

50

300

35

0

95th percentile

75th percentile

40th percentile

B

64

C

Hours of age

To

tal se

rum

bili

rub

in (µ

mo

l/L

)

0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102 108 114 120

05

01

00

15

02

00

250

30

03

50

95th percentile

75th percentile

40th percentile

65

Figure 3-3. Hour-specific pre-treatment total serum bilirubin percentile-based curves among preterm infants born at (A) 29 to

32 weeks’ gestation (n=1120), and (B) 33 to 35 weeks’ gestation (n=1429). To convert total serum bilirubin levels to mg/dL divide

by 17.1.

A

Hours of age

To

tal se

rum

bili

rubin

(µ

mol/L)

0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102 108 114 120

050

100

150

20

02

50

300

35

0

95th percentile

75th percentile

40th percentile

66

B

Hours of age

To

tal se

rum

bili

rubin

(µ

mol/L)

0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102 108 114 120

050

100

150

20

02

50

300

35

0

95th percentile

75th percentile

40th percentile

67

Figure 3-4. Pre-treatment total serum bilirubin percentile net differences between infants born at 33 to 35 weeks’ gestation

minus those born at 29 to 32 weeks’ gestation. To convert mean total serum bilirubin differences to mg/dL divide by 17.1.

Hours of age

Diffe

rence

s in p

erc

entile

tota

l seru

m b

iliru

bin

(µ

mol/L

)

0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102 108 114 120

-30

-20

-10

01

020

30

40

50

60

95th percentile

75th percentile

40th percentile

68

Figure 3-5. Mean pre-treatment total serum bilirubin net differences between infants born at 33 to 35 weeks’ gestation minus

those born at 29 to 32 weeks’ gestation. To convert mean total serum bilirubin differences to mg/dL divide by 17.1.

69

Chapter 4 . Pre-phototherapy total serum bilirubin levels in extremely preterm infants

This chapter is currently under peer-review with the journal Pediatrics and Child Health.

70

4.1 Abstract

Introduction: To generate pre-phototherapy total serum bilirubin (TSB) percentile levels at 24

hours of age among extremely preterm infants, and to contrast these derived percentile levels

with currently used consensus-based thresholds published by Maisels.

Methods: A multi-site retrospective cohort study of extremely preterm infants born 240/7 to

286/7 weeks’ gestation between January 2013 and June 2017 was conducted at three NICUs in

Ontario, Canada. Pre-phototherapy TSB percentiles at 24 hours of age were generated using

quantile regression and compared with Maisels’ thresholds.

Results: Among 642 extremely preterm infants, pre-phototherapy TSB percentiles at 24 hours

of age were 103.3 µmol/L (95% CI, 101.4 to 106.2 [75th percentile]) and 130.4 µmol/L (95% CI,

125.7 to 135.2 [95th percentile]). Among infants born 240/7 to 256/7 weeks’ gestation, the

difference between our TSB percentiles vs. Maisels’ threshold of 85.0 µmol/L were 10.0 µmol/L

(95% CI, 6.0 to 16.0) at the 75th percentile and 35.3 µmol/L (95% CI, 26.1 to 42.8) at the 95th

percentile. Among infants born at 260/7 to 276/7 weeks, the respective differences were 19.4

µmol/L (95% CI, 16.8 to 23.4) and 43.3 µmol/L (95% CI, 34.7 to 46.9). For infants born at 280/7 to

286/7 weeks’ gestation, the difference between our 75th and 95th TSB percentiles and Maisels’

threshold of 103 µmol/L were 6.9 µmol/L (95% CI, 3.2 to 12.0) and 36.0 µmol/L (95% CI, 31.0 to

44.3), respectively.

Conclusion: We describe statistically derived pre-phototherapy TSB levels in extremely preterm

infants that notably differ from commonly cited consensus-based phototherapy thresholds.

71

4.2 Introduction

Infants born extremely preterm at 24 to 28 weeks’ gestation are at higher risk of bilirubin

induced neurological dysfunction (BIND). BIND can arise in preterm infants in the absence of

acute bilirubin encephalopathy (ABE) and at a total serum bilirubin (TSB) concentration lower

than in term or near-term infants.16,17,54,175

In an attempt to prevent BIND in extremely preterm infants, TSB levels are monitored more

frequently than in term infants, and at-risk infants are typically treated at lower TSB levels than

their term and moderate preterm infant counterparts.16,17,54 Although pre-phototherapy TSB

levels have been developed among term and near-term infants, less is known about the natural

history of pre-phototherapy TSB levels in extremely preterm infants. Rather, TSB treatment

thresholds that are currently in use were primarily derived from expert opinion, consensus-

guidelines, and small cohort studies.18,84,98,113

In 2012, Maisels et al. introduced recommendations to manage and treat hyperbilirubinemia in

preterm infants born at < 35 weeks’ gestation, largely based on expert opinion.18 A lack of

evidence regarding the veracity of these recommendations has led to different adaptations and

variations in their use across neonatal intensive care units (NICUs), potentially resulting in an

increase in frequency of phototherapy in this population.18,87

The primary objective of the current study was to generate percentile-based pre-phototherapy

TSB levels in a cohort of extremely preterm infants born at 240/7 to 286/7 weeks’ gestation, and

compare these TSB percentiles with Maisels’ consensus -based published thresholds.18 A

secondary objective was to determine the proportion of infants who were started on

phototherapy below Maisels’ published threshold18, overall, and by hours of age of

phototherapy initiation.

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4.3 Methods

This multi-site retrospective cohort study included extremely preterm infants born or

transferred to Sinai Health (January 2015 to June 2017) and St. Michael’s Hospital (January

2013 to June 2017) in Toronto, Ontario, as well as at Hamilton Health Sciences Centre (January

2014 to June 2017) in Hamilton, Ontario. Healthcare in Ontario is provided under the universal

Ontario Health Insurance Plan.

Extremely preterm infants born at 240/7 to 286/7 weeks gestational age were included in the

study. Excluded were infants who had Rh disease, or those who did not have a recorded TSB

level in their electronic medical chart. Infants with Rh disease are usually diagnosed and

managed prenatally by monitoring maternal antibody levels, fetal sonographic surveillance and

offering selective interventions. Eligible infants born during the study period were identified

using two databases, namely, the national Canadian Neonatal Network (CNN)162 and the

provincial Better Outcomes Registry & Network (BORN).161 Eligible infants’ medical records

were reviewed at their respective hospitals of admission to obtain pre-phototherapy TSB levels.

A pre-phototherapy TSB was defined as any TSB level prior to initiation of phototherapy (among

those who went on to receive phototherapy), or any TSB level otherwise (among those who did

not go on to receive phototherapy). Other information abstracted included postnatal age in

hours, phototherapy status, hours of age of phototherapy initiation, gestational age at birth and

relevant infant characteristic.

73

4.3.1 Data analyses

All hour specific TSB levels prior to initiation of phototherapy were plotted in 6-hour

increments, from birth to 72 hours thereafter. In keeping with previous research, hour-specific

40th, 75th and 95th TSB percentiles3 were estimated using quantile regression among all infants,

including a quadratic polynomial for age, in hours. The quantreg package in R was used for

fitting all quantile regression models. In order to account for repeated measures within

subjects, 1000 bootstrap resamples at the subject level were used to generate 95% confidence

intervals (CI).163-165

Maisels’ recommended published thresholds for the initiation of phototherapy are based on

expert opinion and do not specify the hours of age at which these thresholds should be

considered.18 Among infants born at 240/7 to 276/7 weeks’ gestation the recommended TSB for

starting phototherapy is 85 μmol/L-103 μmol/L while among infants born at 280/7 to 286/7

weeks’ gestation the recommended published threshold is 103 μmol/L - 137 μmol/L.18 Based on

previous reports suggesting that pre-phototherapy TSB at 24 hours can be used to decide on

phototherapy administration, the difference between pre-phototherapy TSB percentiles

derived at 24 hours of age and Maisels’ above mentioned lower TSB threshold at the respective

gestational age groups were calculated.16 A previous study reported NICUs using the lowest

level of Maisels’ published threshold to initiate phototherapy among infants born at < 28

weeks’ gestation.87 The differences between the study’s pre-phototherapy TSB percentiles at

24 hours of age and Maisels’ published lower threshold18 were stratified by the following

gestational age groups based on clinical guidelines; 240/7 to 256/7 weeks, 260/7 to 276/7 weeks

and 280/7 to 286/7 weeks’ gestation.

As a secondary analysis, we determined the proportion of infants in the study who were started

on phototherapy below Maisels’ published threshold.18 Infants with a TSB level done > 12 hours

before the time of phototherapy initiation were excluded from the secondary analysis. Among

infants born at 240/7 to 276/7 weeks’ gestation, those who had a TSB level < 85 μmol/L at the

74

time of phototherapy initiation, were considered as being treated below Maisels’ published

threshold.18 For infants born at 280/7 to 286/7 weeks’ gestation, those whose TSB level was < 103

μmol/L at the initiation of phototherapy were considered as being treated below Maisels’

published threshold. To determine if the proportion of infants administered phototherapy

below Maisels’ published threshold changed with time of phototherapy initiation (in hours)

since birth, a Cochran-Armitage trend test was conducted, with significance set at < 0.01.176,177

All data analyses were performed using R version 4.0.3 for macOS and SPSS 27 for macOS.164,167


The sample size was calculated based on previous pre-phototherapy curves for bilirubin levels

in term and near-term infants that demonstrated a Gaussian distribution.3,168 Using methods

for reference limits from Bellera and Hanley, in order to obtain reference limits of 2.5 and

97.5%, with a CI of 90% and relative margin of error of 10% for a Gaussian distribution, a

minimum sample size of 448 extremely preterm infants was required.169

4.4 Results

Of the 737 eligible infants born between 240/7 and 286/7 weeks’ gestation, 642 infants had at

least one available bilirubin measure prior to initiation of phototherapy within the first 72 hours

after birth (Figure 4-1). The median gestational age at birth was 26.0 weeks (Interquartile range

[IQR]) 25.0 to 28.0), and the median birthweight was 900.0 grams (IQR 760.0 to 1100.0) (Table

4-1).

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4.4.1 Pre-phototherapy TSB levels

642 extremely preterm infants born at 240/7 to 286/7 weeks’ gestation contributed a total of

1134 hour-specific pre-phototherapy TSB levels (Figure 4-2). Each infant contributed a median

of 2 (IQR 1 to 2) pre-phototherapy TSB samples. Since there was a paucity of pre-phototherapy

TSB levels after 72 hours of age, quantile regression was limited to data up to 72 hours after

birth.

At 24 hours of age, the estimated pre-phototherapy TSB levels were 84.4 µmol/L (95% CI, 82.2

to 87.0) at the 40th, 103.3 µmol/L (95% CI, 101.4 to 106.2) at the 75th, and 130.4 µmol/L (95%

CI, 125.7 to 135.2) at the 95th percentile (Table 4-2). The corresponding estimated hourly rates

of rise of TSB at birth were 2.8 μmol/L/hour (95% CI, 2.4 to 3.5), 2.8 μmol/L/hour (95% CI, 2.2 to

3.6) and 1.9 μmol/L/hour (95% CI, 0.7 to 3.4) for the 40th, 75th and 95th percentiles. The

estimated change in the rate of rise of TSB diminished with time since birth at the 40 th and 75th

percentiles; however, this change was not statistically significant at the 95th percentile (p =

0.50) (Table 4- 2). Estimated pre-phototherapy TSB percentiles peaked at 53.8 hours of age for

the 40th percentile, 63.6 hours for the 75th percentile and beyond 72 hours of age for the 95th

percentile (Table 4-2 and Figure 4-2).

4.4.2 Pre-phototherapy TSB levels by gestational age groups

Among infants born at 240/7 to 256/7 weeks’ gestation, pre-phototherapy TSB percentiles at 24

hours of age from birth were 95.0 µmol/L (95% CI, 91.0 to 101.0) at the 75th percentile and

120.3 µmol/L (95% CI, 111.1 to 127.8) at the 95th percentile (Table 4-3). Among those born at

260/7 to 276/7 weeks, the corresponding respective levels were 104.4 µmol/L (95% CI, 101.8 to

108.4) and 128.3 µmol/L (95% CI 119.7 to 131.9) (Table 4-3). For those born at 280/7 to 286/7

weeks’ gestation, the 75th percentile value was 109.9 µmol/L (95% CI, 106.2 to 115.0), and the

95th percentile value was 139.0 µmol/L (95% CI, 134.0 to 147.3) (Table 4-3).

76

4.4.3 Derived pre-phototherapy TSB percentiles compared with Maisels’ published thresholds

Among infants born at both 240/7 to 256/7, 260/7 to 276/7, and 280/6 to 286/7 weeks’ gestation, our

statistically derived pre-phototherapy TSB levels at the 75th and 95th percentiles were

significantly higher at 24 hours than Maisels’ published threshold (Table 4-3).

4.4.4 Phototherapy administration among all infants

Overall, 615 infants (95.8%) subsequently received phototherapy, of which 76.6% had started

therapy by 36 hours of age (Figure 4-3). Among these 615 infants, phototherapy was initiated at

a mean (SD) of 31.2 (16.2) hours of age, and at a mean TSB level of 104.5 (25.0) µmol/L.

Of the 615 infants who were started on phototherapy, three infants were excluded from the

analysis below as their last available TSB level was more than 12 hours from the initiation of

phototherapy. Among the 612 analyzed infants, 183 (29.9%) were started on phototherapy at a

TSB concentration below Maisels’ published threshold.18 The proportion of these 183 infants

administered phototherapy below Maisels’ threshold significantly declined with hours of age of

phototherapy initiation since birth (Cochran-Armitage trend test P < 0.01) (Table 4-4).

77

4.5 Discussion

In this retrospective cohort study of extremely preterm infants born at 240/7 to 286/7 weeks’

gestation, pre-phototherapy TSB percentiles at 24 hours of age were generated and contrasted

with Maisels’ currently published consensus-based threshold for phototherapy initiation. At

higher percentiles (i.e., 75th and 95th percentile) there was a notable difference between

Maisels’ consensus-based threshold and our study’s generated 24-hour pre-phototherapy TSB

percentiles. Furthermore, more than 95% of extremely preterm infants were administered

phototherapy, and 76% were started on phototherapy by 36 hours of age. Among those infants

who went on to have phototherapy, overall, 29.9% were started on phototherapy below

Maisels’ published threshold18, especially among infants who had phototherapy initiated at ≤ 12

hours of age.

This is the first study to report hour-specific pre-phototherapy TSB percentiles in extremely

preterm infants, and to compare them to Maisels’ consensus -based published threshold.18 This

is also one of the first studies to report the proportion of infants who started phototherapy

below Maisels’ published threshold.18 A previous study on phototherapy administration below

published thresholds (referred to therein as “subthresholds” ) was limited to term and near-

term infants, and was focused on longer hospital length of stay as a consequence of

subthreshold phototherapy.178 The only other North American study on phototherapy in

preterm infants was limited to reporting on the frequency and duration of phototherapy, and

the median TSB level used immediately preceding phototherapy. 87

Our study’s statistically derived TSB percentile levels at 24 hours of age were notably different

from Maisels’ consensus-based published threshold.18 Among the 279 infants who started on

phototherapy between 24 to 30 hours of age, 55.2% and 87.8% would hypothetically have not

received phototherapy within that time point had our study’s 75th or 95th percentile cut-points

been used instead, respectively (Table 4-5). These hypothetical estimates of the number of

infants not starting phototherapy based on our cut-points are also appreciably greater than the

78

29.7% figure based on Maisels’ threshold (Table 4-5).18 Certainly, these notable differences do

not account for the many factors that might influence the decision to initiate phototherapy in

an extremely preterm infants, or the possibility that phototherapy might otherwise be initiated

soon after the 24 to 30 hours of age interval used in this exercise – factors beyond the scope of

the current study. Nevertheless, though hypothesis generating, these finding provide novel

data on pre-treatment TSB in extremely preterm infants, for experts to consider in the

development of future guidelines for phototherapy initiation in extremely preterm infants.

Almost all infants in this study were started on phototherapy by 36 hours of age, in keeping

with previous reports of increased frequency of phototherapy use in extremely preterm

infants.86,87 The potential long-term negative consequences of early initiation or increased

frequency of phototherapy use has not been well studied in extremely preterm infants;

however, one study found that early initiation is associated with a longer duration of

phototherapy.86 Previous studies have been limited to the negative consequences of aggressive

or intensive phototherapy in preterm infants of low birth weight.98 Among extremely preterm

infants, little is known about the potential benefits and harms of initiating phototherapy below

recommended thresholds.18 Hence, more research is needed to determine the reasons for, and

impact of, administering phototherapy below percentile-based or recommended thresholds,

including long-term developmental follow-up.18

This study has some limitations. First, Maisels’ published thresholds for phototherapy initiation

were based on expert opinion, and not on data directly measured in extremely preterm

infants.18 As such in Canada, Maisels’ published thresholds18 have been further modified within

different centres and applied at the discretion of individual clinicians. Accordingly, this would

have resulted in considerable practice variation within our study. Notwithstanding, these

recommendations have been widely disseminated across North America. Second, some have

suggested that lower thresholds for initiating phototherapy are needed in very ill preterm

infants.16 Future research should focus on differences in phototherapy thresholds by

comorbidities, and then proceed to compare short- and long-term neonatal outcomes,

79

accordingly. Third, in this study, we compared our statistically derived TSB percentiles with

Maisels’ lowest TSB threshold18, typically used for infants at high risk of neurotoxicity from

severe hyperbilirubinemia. However, preterm infants born before 29 weeks’ gestation are

considered at higher risk of neurotoxicity from severe hyperbilirubinemia. Additionally, among

infants born 24 to 28 weeks’ gestation, one previous study reported using the lower TSB

threshold level recommended by Maisels.87 Finally, the current study did not describe clinical

factors that may impact the administration of phototherapy below or above/at Maisels’

threshold.18 The scope of this study was to statistically derive pre-phototherapy TSB levels for

preterm infants born extremely preterm and compare them to Maisels’ published threshold.18

Future larger studies should look at neonatal factors and administration of phototherapy above

and below Maisels’ phototherapy thresholds.18

4.6 Conclusion

There is considerable variability in the initiation of phototherapy compared to the most

commonly recommended treatment thresholds in extremely preterm infants.18 In addition, we

report statistically derived pre-phototherapy TSB percentiles from birth to 72 hours of age, and

compared 24-hour pre-phototherapy TSB percentiles with existing consensus-based

phototherapy guidelines.18 Re-assessment of the effectiveness of current phototherapy

treatment practices in these infants may be warranted.

80

4.7 Tables

Table 4-1. Characteristics of 642 extremely preterm infants born at 240/7 to 286/7 weeks’

gestation included in the study. All data are presented as a number (%) unless otherwise

indicated.

Characteristic

Median (IQR) gestational age, weeks 26.0 (25.0 to 28.0)

Median (IQR) birthweight, g 900.0 (760.0 to 1100.0)

Female 300 (46.7)

Mean (SD) maternal age at birth of current infant, ya 31.8 (6.0)

Mode of birth

Cesarean 376 (58.6)

Vaginal 257 (40.0)

Unknown 9 (1.4)

Hemolysis

ABO incompatibility 89 (13.9)

Feeding

Enteral only 139 (21.6)

Total parenteral nutrition only 91 (14.2)

Mixed (enteral and total parenteral nutrition) 408 (63.6)

Unknown 4 (0.6)

Blood or cerebral spinal fluid confirmed positive culture 133 (20.7)

Median (IQR) number of total serum bilirubin levels per infant 2.0 (1.0 to 2.0)

Death 55 (8.6)

a Maternal age missing in <1% of cohort

81

Table 4-2. Estimated pre-phototherapy total serum bilirubin concentration at 24 hours of age, rate of rise of total serum bilirubin

from birth, change in rate of rise of total serum bilirubin from birth onward and, the hours of age of peak total serum bilir ubin at the

40th, 75th and 95th percentiles.

Measure Percentile

40th 75th 95th Estimated total serum bilirubin concentration at 24 h of age since birth (95% CI), µmol/L

84.4 (82.2 to 87.0) 103.3 (101.4 to 106.2) 130.4 (125.7 to 135.2)

Estimated hourly rate of rise of total serum bilirubin concentration from birth (95% CI), µmol/L/h

2.8 (2.4 to 3.5) 2.8 (2.2 to 3.6) 1.9 (0.7 to 3.4)

Estimated change in the rate of rise of total serum bilirubin concentration from birth onward (95% CI), µmol/L/h2

-0.026 (-0.041 to -0.019) -0.022 (-0.035 to -0.011) -0.007 (-0.025 to 0.012)a

Estimated hours of age since birth to attain a peak in total serum bilirubin concentration, h

53.8 63.6 > 72

a The estimated change in the rate of rise of total serum bilirubin from birth onward was not statistically significant at the 95th percentile (p = 0.50)

82

Table 4-3. Difference between the current study’s derived pre-phototherapy total serum bilirubin percentile at 24 hours of age and the total serum bilirubin

thresholds published by Maisels18, presented by gestational age.

Gestational age at birth, weeks

Current study’s derived total serum bilirubin

percentile at 24 h of age (95% CI), µmol/L

Maisels’ published total serum bilirubin

threshold value18

, µmol/L

Difference between the current study’s derived total serum

bilirubin percentile and Maisels’ published threshold value

(95% CI), µmol/L 40th 75th 95th 40th 75th 95th

240/7

to 256/7

79.3 (75.6 to 82.0) 95.0 (91.0 to 101.0) 120.3 (111.1 to 127.8) 85.0 -5.7 (-9.4 to -3.0) 10.0 (6.0 to 16.0) 35.3 (26.1 to 42.8)

260/7 to 276/7 85.0 (81.0 to 89.4) 104.4 (101.8 to 108.4) 128.3 (119.7 to 131.9) 85.0 0.0 (-4.0 to 4.4) 19.4 (16.8 to 23.4) 43.3 (34.7 to 46.9)

280/7 to 286/7 90.9 (87.9 to 94.3) 109.9 (106.2 to 115.0) 139.0 (134.0 to 147.3) 103.0 -12.1 (-15.1 to -8.7) 6.9 (3.2 to 12.0) 36.0 (31.0 to 44.3)

83

Table 4-4. Proportion of 183 infants subsequently started on phototherapy below Maisels’

published total serum bilirubin threshold18, shown in 12-hour intervals since birth. All data

are presented as number (%).

Hours elapsed since birth, at which phototherapy was initiated a

≤ 12 (n = 17)

13 to 24 (n = 133)

25 to 36 (n = 322)

37 to 48 (n = 74)b

49 to 60 (n = 40)b

61 to 72 (n = 15)

≥ 73 (n = 11)

10 (58.8) 65 (48.9) 98 (30.4) 7 (9.5) 2 (5.0) 1 (6.7) 0 (0.0)

a The proportion of infants started on phototherapy below Maisels’ published threshold 6 for phototherapy

decreased significantly with increasing hours of age (p < 0.01)

b Excluding three infants who did not have a total serum bilirubin level measured at least 12 hours before

their initiation of phototherapy

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Table 4-5. Proportion of the 279 infants started on phototherapy at 24 to 30 hours of age in

the current study, at a total serum bilirubin level below Maisels’ published18, or below the

current study’s generated 75th and 95th percentiles at 24 hours of age.

Number (%) infants started on phototherapy below the specified total serum bilirubin level

Below Maisels’ threshold18

Below the

current study’s 75th percentile

Below the

current study’s 95th percentile

83 (29.7) 154 (55.2) 245 (87.8)

85

4.8 Figures

Figure 4-1. Flow diagram of included extremely preterm infants born at 240/7 to 286/7

weeks’ gestation, from January 2013 to June 2017

737 electronic medical charts of extremely preterm infants born at

240/7 to 286/7 weeks’ gestation reviewed at participating study sites

95 extremely preterm infants excluded

Receipt of phototherapy unknown (n=36)

Pre-phototherapy bilirubin level unknown (n=59)

642 extremely preterm infants included in the primary analysis

86

Figure 4-2. Hour-specific pre-phototherapy total serum bilirubin percentile curves estimated based on data from 642 extremely

preterm infants born at 240/7 to 286/7 weeks’ gestation. To convert total serum bilirubin levels to mg/dL divide by 17.1

95th

percentile

75th

percentile

40th

percentile

Hours of age

To

tal se

rum

bili

rubin

(µ

mol/L

)

0 6 12 18 24 30 36 42 48 54 60 66 72

05

010

015

020

0250

95th percentile

75th percentile

40th percentile

No. infants contributing within each 6-h interval: 17 191 157 234 272 62 53 51 43 17 11 18

87

Figure 4-3. Proportion of the 615 extremely preterm infants born at 240/7 to 286/7 weeks’ gestation who subsequently received

phototherapy after birth.

No. infants

not yet on phototherapy after each 6-h interval: 615 612 598 522 465 209 143 107 67 43 26 19 11

88

Chapter 5 . Transcutaneous vs. total serum bilirubin measurements in preterm infants

This chapter has been peer reviewed and published in the journal Neonatology. The

peer-reviewed, post-print version of manuscript has been reproduced in full with permission from S. Krager AG Basel as a chapter in this doctoral thesis. The final, published version of this article is available at https://www.karger.com/?doi=[10.1159/000516648.] Jegathesan T, Campbell D, M, Ray J, G, Shah V, Berger H, Hayeems R, Z, Sgro M: Transcutaneous versus Total Serum Bilirubin Measurements in Preterm Infants.

Neonatology 2021. doi: 10.1159/000516648.

89

5.1 Abstract

Introduction: Transcutaneous bilirubin (TcB) measurement offers a non-invasive approach for

bilirubin screening, however its accuracy in preterm infants is unclear. This study determined

the agreement between TcB and total serum bilirubin (TSB) among preterm infants.

Methods: A multi-site prospective cohort study was conducted at three NICUs in Ontario,

Canada, September 2016 to June 2018. Among 296 preterm infants born at 240/7 to 356/7 weeks,

856 TcB levels were taken at the forehead, sternum, and before and after the initiation of

phototherapy with TSB measurements. Bland-Altman plots and 95% limits of agreement (LOA)

expressed agreement between TcB and TSB.

Results: The overall mean TcB-TSB difference was -24.5 μmol/L (95% LOA -103.3 to 54.3), 1.6

μmol/L (95% LOA -73.4 to 76.5) before phototherapy, and -31.1 μmol/L (95% LOA-105.5 to

43.4) after the initiation of phototherapy. The overall mean TcB-TSB difference was -15.2

μmol/L (95% LOA -86.8 to 56.3) at the forehead and -24.4 μmol/L (95% LOA -112.9 to 64.0) at

the sternum. The mean TcB-TSB difference was -31.4 μmol/L (95% LOA -95.3 to 32.4) among

infants born 24-28 weeks, -25.5 μmol/L (95% LOA -102.7 to 51.8) at 29-32 weeks, and -15.9

μmol/L (95% LOA -107.4 to 75.6) at 33-35 weeks. Measures did not differ by maternal ethnicity.

Conclusion: Among preterm infants, TcB may offer a non-invasive, immediate approach to

screening for hyperbilirubinemia, with more careful use in preterm infants born at <33 weeks’

gestation, as TcB approaches treatment thresholds. It’s underestimation of TSB after the

initiation of phototherapy warrants the use of TSB for clinical decision making after the

initiation of phototherapy.

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5.2 Introduction

The decline in acute and chronic bilirubin encephalopathies in Canada and the US can be

attributed, in part, to the adoption of American Academy of Pediatrics’ and Canadian Pediatric

Society’s recommendations to perform routine total serum bilirubin (TSB) screening in infants

born at > 35 weeks’ gestation prior to hospital discharge1,2,15 and among preterm infants born

at 24-35 weeks’ gestation.16,18

Although obtaining TSB is the most common way to measure bilirubin levels in infants, blood

sampling can be painful and a time-consuming procedure.21,116 Studies have also reported

concerns about frequent TSB measurements, such as the increased risk of infection and

anemia, particularly among extremely preterm infants.22,125 Furthermore, repeated procedural

pain and stress have been documented in preterm infants.119

In term and near-term infants, the use of noninvasive transcutaneous bilirubin (TcB)

measurement has gained use in clinical practice, as it can reduce the frequency of TSB tests

when TSB concentrations are < 240 μmol/L (< 14 mg/dL).1,2 There is a conflicting body of

research on the use of TcB in preterm infants ≤ 35 weeks’ gestation. Prior studies included small

sample sizes, with conflicting data reporting the effects of phototherapy, and anatomical site of

measurement.123,133,152 There is also limited data reporting the effects of ethnicity on TcB in

preterm infants.128,133 In addition few recent studies have stratified agreement between TcB

and TSB by gestational age groups of prematurity among preterm infants, especially after the

initiation of phototherapy.123

The primary objective of this study was to evaluate the agreement (i.e., limits of agreement)

between TcB and TSB measurements among preterm infants born at 240/7 to 356/7 weeks’

gestation. We also assessed the agreement between TcB and TSB before and after the initiation

of phototherapy, by anatomical site of measurement, and the infant’s ethnicity.

91

5.3 Methods

A multi-site prospective cohort study was completed in Ontario at St. Michael’s Hospital, Sinai

Health, and Hamilton Health Sciences Centre from September 2016 to June 2018. Institutional

Research Ethics Board approval was obtained at participating sites, and informed consent was

provided by the parent(s).

Eligible participants were preterm infants born at 240/7 to 356/7 weeks’ gestation and admitted

to participating sites. Excluded were those with a condition that could interfere with TcB

measurements, such as, hydrops fetalis, congenital malformation, diffuse cutaneous conditions,

infection, or purpura.

The JM-105 TcB device (Drager Medical Systems Inc, Telford Philadelphia) was used to measure

TcB in each participating infant within 15 minutes of every TSB measurement. TSB samples

were collected by venous or capillary blood sampling as per clinical decision and was analyzed

by spectrophotometry using Beckman Coulter’s AU680 automated analyzer.112

Participating sites were provided 2-3 TcB devices which were calibrated daily as per

manufacturer’s instructions. As per hospital policy, with every medically indicated TSB

measurement a single TcB (average of 3 measurements) was concomitantly taken at both the

forehead and sternum by nurses from time of parental consent, up to 10 days, in the NICU. TcB

sampling was repeated in each infant at every subsequently required TSB. Phototherapy was

started as medically indicated based on TSB measurements. During phototherapy,

phototherapy lights were turned off when TSB and TcB measurements were taken at each site.

Nursing staff at all three sites were trained by the primary site investigator on how to perform

TcB measurements as per procedures described above. Research assistants then collected the

results directly from the meter and entered them on the data collection form.

Also recorded were infant age (in hours) at each TSB-TcB measurement and the time of

92

initiation of phototherapy. Maternal and infant demographic data and clinical information

were collected via standardized chart extraction.

5.3.1 Data analyses

The primary study outcome was the agreement between TcB and TSB. Agreement was

assessed among all infants (“overall”); before and after initiation of phototherapy; by

anatomical site (forehead vs. sternum); and by infant ethnicity, determined by maternal self-

identified ethnicity (Canadian Caucasian, Southeast Asian, South Asian, and African or

Caribbean).

To express the level of agreement between TcB and TSB measurements, Bland-Altman plots

and Lin’s concordance correlation coefficients (CCC) were generated. Adapted Bland-Altman

plots were weighted for multiple TcB and TSB measurements per infant, and the two sites of

TcB measurement (forehead and sternum), with 95% limits of agreement (LOA).179,180

Agreement was assessed, overall, in all preterm infants, and further stratified by gestational age

(24-28, 29-32 and 33-35 weeks’ gestation at birth). Bland-Altman plots were also developed for

TcB-TSB measurement differences pre- or post-phototherapy, anatomical site of measurement

(forehead vs. sternum), and infant ethnicity.

To assess the use of TcB as a potential screening test for hyperbilirubinemia, sensitivity,

specificity, positive predictive value (PPV), negative predictive value (NPV) and positive and

negative likelihood ratios were calculated at clinically-important TSB cut-points as

recommended by Maisels et al, and which is widely used in North America.18 For preterm

infants born at 24-28 weeks, the lack of TcB measurements prior to phototherapy prohibited

any similar analysis, as described below. The recommended TSB cut-points are 103 to 171

μmol/L in infants born at 29-32 weeks, and 171 to 205 μmol/L in infants born at 33-35

weeks.1,18 Accordingly, a rounded off cut-point TSB >100 μmol/L was applied to infants born at

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29-32 weeks’ gestation, and a rounded off cut-point >170 μmol/L was used for those at 33-35

weeks. Receiver operating characteristic (ROC) curves, and the respective area under the curves

(AUC), were also calculated. Finally, TcB cut-points were determined with at least 90%

sensitivity to detect a TSB of >100 μmol/L at 29-32 weeks’ gestation, and a TSB of >170 μmol/L

at 33-35 weeks.


In one previous study, the mean TSB was 146 µmol/L, with a standard deviation of the average

difference between TSB and TcB measurements of 30 µmol/L, and negligible variation by

gestational age or ethnicity.135 With our a priori sample size calculation of a minimum of 271

infants, the current study sample of 296 infants was sufficient to detect a minimum TcB-TSB

difference of 4 µmol/L, at a conventional two-sided P-value of 0.05 and a statistical power of

80%. Analyses were conducted using NCSS 12 and SPSS 26.

5.4 Results

Out of 344 preterm infants born at 240/7 and 356/7 weeks, 296 preterm infants received at least

one TcB measurement (Figure 5-1). The median gestational age at birth was 31.0 weeks (IQR

28.0-33.0) among an ethnically diverse group of mothers (Table 5-1). Each of the 296 infants

received a mean (SD) of 7.0 (3.6) TSB measurements. There were 856 paired TcB and TSB

measurements done at both the forehead and sternum, with a mean of 3.0 (1.9) paired

measurements per infant performed at a median age of 105 hours (IQR 68 -151).

Among all 296 infants, the overall mean TcB-TSB difference was -24.5 μmol/L (to convert TSB to

mg/dL divide by 17.1) (95% LOA -103.3 to 54.3) (Figure 5-2A). There were 29 (3.4%) measures

above the upper LOA, and 73 (8.5%) below the lower LOA, with no visual evidence of any

greater deviation at higher mean bilirubin concentrations (Figure 5-2A). Furthermore, TcB

94

underestimated TSB in 650 of all measures (75.9%), and overestimated TSB in 206 of all

measures (24.1%) (Figure 5-2A).

5.4.1 TcB measurement before and after phototherapy, all preterm infants

In total, 252 neonates (85.1%) received phototherapy, at a median of 29.0 hours of age (IQR

26.0 to 55.0). A total of 172 paired measurements were done prior to phototherapy, and 684

following phototherapy. Among 79 infants born at 24-28 weeks, 229 out of 241 measurements

(95.0%) were done after initiation of phototherapy, with 76 infants (96.2%) receiving

phototherapy by a median of 26.0 hours of age (IQR 15.0 to 29.0).

Among all newborns, the overall mean TcB-TSB difference at the combined forehead and

sternum was much smaller prior to the initiation of phototherapy (1.6 μmol/L, 95% LOA

-73.4 to 76.5) (Figure 5-2B) than after (-31.1 μmol/L, 95% LOA -105.5 to 43.4) (Figure 5-2C). TcB

overestimated TSB in 99 (57.6%) measures prior to the start of phototherapy, and

underestimated TSB in 577 (84.4%) measures after. The corresponding Lin’s CCC was 0.76 (95%

CI 0.69-0.81) prior to starting phototherapy, and 0.64 (95% CI, 0.60-0.67) after.

5.4.2 Sites of TcB measurement

The overall mean TcB-TSB difference at the forehead (-15.2 μmol/L, 95% LOA -86.8 to 56.3)

was less pronounced than at the sternum (-24.4 μmol/L, 95% LOA -112.9 to 64.0) (Table 5-2,

red). This was consistent after receipt of phototherapy, and upon stratifying by gestational age

at birth (Table 5-2). Lin’s CCC’s between TSB and TcB at the forehead was consistently higher

than at the sternum.

95

5.4.3 TcB measurement by gestational age groups

Regardless of phototherapy, 241 paired TcB-TSB measurements were obtained in 79 neonates

born at 24-28 weeks, 381 paired measurements in 119 infants born at 29-32 weeks, and 234

paired measurements in 98 infants born at 33-35 weeks. At the forehead and sternal sites

combined, the mean TcB-TSB difference was -31.4 μmol/L (95% LOA -95.3 to 32.4) at 24-28

weeks, -25.5 μmol/L (95% LOA -102.7 to 51.8) at 29-32 weeks, and -15.9 μmol/L (95% LOA -

107.4 to 75.6) at 33-35 weeks. However, upon limiting to TcB at the forehead (where higher

overall LOA were observed regardless of phototherapy), the mean TcB-TSB difference was less

pronounced, especially among late preterm infants (Figure 5-3C). This was consistently seen

when stratified by receipt of phototherapy and site of measurement (Table 5-2). TcB

underestimated TSB in 216 (89.6%) of the measures done in infants born at 24-28 weeks, 302

(79.3%) of those at 29-32 weeks, and 132 (56.4%) of the measures in infants born at 33-35

weeks.

5.4.4 Influence of ethnicity on TcB

The overall LOA between TSB and TcB (both prior to and after the initiation of phototherapy)

was similar across the four main ethnic groups, including infants born to a mother of African or

Caribbean ethnicity (Figure 5-4).

5.4.5 Performance of TcB at pre-defined TSB levels

Among infants born at 29 to 32 weeks‘gestation, and at the recommended TSB cut-point of >

100 μmol/L, prior to phototherapy, forehead TcB had an AUC of 0.76 (95% CI, 0.59 to 0.87)

(Figure 5-5A and Table 5-3). A forehead TcB cut-point of 85 μmol/L had a sensitivity of 92%

(95% CI 80 to 98), specificity 60% (95% CI 32 to 84), PPV 88% (95% CI 76 to 95), NPV 69% (95%

CI 38 to 91), and respective positive and negative likelihood ratios of 2.3 (95% CI 1.2 to 4.3) and

96

0.14 (95% CI 0.05 to 0.39) to detect the recommended TSB threshold of > 100 μmol/L at 29 to

32 weeks’ gestation.

In preterm infants born at 33 to 35 weeks’ gestation, and at the recommended TSB cut-point of

>170 μmol/L, prior to phototherapy, forehead TcB had an AUC of 0.86 (95% CI 0.76 to 0.92)

(Figure 5-5B and Table 5-3). A forehead TcB cut-point of 156 μmol/L had a sensitivity of 90%

(95% CI 73 to 98), specificity 69% (95% CI 57 to 80.0), PPV 55% (95% CI 40 to 70), NPV 94 %

(95% CI 83 to 99), and respective positive and negative likelihood ratios of 2.9 (95% CI 2.0 to

4.2) and 0.15 (95% CI 0.05 to 0.44) to detect the recommended TSB threshold of >170 μmol/L

among infants born at 33 to 35 weeks.

5.5 Discussion

In this multi-site, prospective cohort study of Canadian infants born preterm at 240/7 to 356/7

weeks’ gestation, TcB measured with the JM-105 at the forehead and sternum offered a

reasonably accurate assessment of TSB, especially prior to starting phototherapy in preterm

infants born between 33 and 35 weeks’ gestation. In preterm infants born at 33-35 weeks’

gestation, TcB demonstrated better agreement with TSB than among those born before 33

weeks’ gestation. Maternal ethnicity did not appreciably affect TcB.

As per our primary objective, we studied the agreement between TcB and TSB measurements

among preterm infants using multiple TcB measurements per infant. Specifically, this study

assessed the impact of phototherapy initiation, the anatomical site of measurement,

gestational age at birth, and maternal ethnicity. This study used an adapted version of the

Bland-Altman analysis, which accounts for repeated bilirubin measurements in the same

infant.179,180 Previously a regression analysis was employed to account for multiple

measurements per infant.181 The inclusion of a large number of repeated samples permitted a

comprehensive analysis of TcB measurements within a diverse sample of neonates, surpassing

the sample size from a previously completed study in India using the BiliChek.151

97

Similar to previous studies of TcB among preterm infants, our study reported a mean TcB-TSB

difference of ≤ 26 μmol/L.125,151,181 After initiation of phototherapy and at a greater degree of

prematurity, TcB-TSB agreement worsened. This is consistent with previous research that

showed greater agreement between TcB and TSB prior to initiating phototherapy using the JM-

103 and JM-105 devices.123,145,147 As in prior studies, we saw no appreciable difference whether

TcB was measured at the forehead or sternum prior to phototherapy128,152; however, after the

initiation of phototherapy the current study suggests that the forehead may be the more

preferred site for TcB measurement when using the JM-105 device. One reason for this may be

the reduced exposure to phototherapy at the forehead from the eye mask used during

treatment.182 However TSB measurements should be used to make clinical decisions, especially

after the initiation of phototherapy. As with former studies135,141 , ethnicity does not appear to

appreciably affect the agreement between TcB and TSB measurements.

As a limitation, fewer TcB measurements were available before initiation of phototherapy than

after, especially among infants born at 24-28 weeks’ gestation who started on phototherapy at

a median of 26 hours of age. The recruitment of extremely preterm infants was challenging in

terms of obtaining parental consent prior to initiation of phototherapy. This study did not

assess TcB-TSB agreement in relation to the time since phototherapy cessation and therefore

did not differentiate between during or after phototherapy. Finally, as this study used the JM-

105 device, the current findings may not be reflective of the performance of other TcB devices.

The clinical utility of TcB as a screening tool was also assessed at specific recommended

treatment TSB thresholds in two preterm infant groups. Among infants born at 29-32 weeks,

forehead TcB had an AUC of 0.76 to detect the recommended TSB threshold of >100 μmol/L. At

this gestational age, a forehead TcB cut-point of 85 μmol/L had the necessary sensitivity of 92%

to detect the recommended TSB threshold of > 100 μmol/L, even though the specificity was

only 60%. In preterm infants at 33-35 weeks’ gestation, the corresponding AUC was 0.86 to

detect the recommended TSB threshold of > 170 μmol/L. Among these infants, a forehead TcB

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cut-point of 156 μmol/L, had the requisite sensitivity of 90%, with a slightly better specificity

of 69% to detect the recommended TSB threshold of > 170 μmol/L. Accordingly, TcB may be

useful in screening for hyperbilirubinemia in low-risk preterm infants born at 33-35 weeks prior

to the initiation of phototherapy. Among preterm infants born at 29 to 32 weeks’ gestation, TcB

should be used more carefully as TSB approaches treatment thresholds. In all preterm infants

the recommended TSB thresholds of treatment need to be considered when deciding to use

TcB in this population.

Clinicians should remain cognizant of the treatment thresholds for hyperbilirubinemia, based

on gestational age at birth, and other clinical factors for developing bilirubin toxicity.18 Once an

infant is on phototherapy, blood testing is often repeated regularly, to monitor the effect of

phototherapy on bilirubin levels.113 The tendency of TcB to underestimate TSB after the

initiation of phototherapy in 84% of TcB measurements after the initiation of phototherapy

seems problematic: Relying on TcB alone might lead to improper discontinuation of

phototherapy.123 As such, TSB measurements should be used to make clinical decisions after

the initiation of phototherapy, especially since most preterm infants start phototherapy after

the first or second day of life. As a potential solution to the underperformance of TcB after

initiation of phototherapy, for example, some small studies in term and preterm infants

explored the effectiveness of measuring TcB on covered skin.147,148 A similar approach, with a

larger sample size, should be considered in preterm infants, to determine the effectiveness of

TcB after phototherapy.

In addition to procedural pain, repeated blood sampling in preterm infants can contribute to

anemia.183 Approximately 0.5 mL of blood sampling is required for TSB measurement in late

preterm infants. In the current study, each newborn received a mean of 7 TSB measurements,

amounting to 3.5 mL of cumulative blood loss. The careful use of TcB as a screening tool may

reduce blood sampling, especially in mid- and late-preterm infants.

99

5.6 Conclusion

Among preterm infants born at 33-35 weeks’ gestation, TcB with the JM-105 may offer a

noninvasive, immediate approach to screening for hyperbilirubinemia prior to phototherapy.

Careful use of TcB should be considered in infants born at < 33 weeks’ gestation when TSB

levels are approaching phototherapy thresholds. Its notable underestimation of TSB

measurements after the initiation of phototherapy warrants limited use after the initiation of

phototherapy and use of TSB measurements for clinical decision making.

100

5.7 Tables

Table 5-1. Characteristics of the 296 preterm infants included in the study. All data are shown as

number (%) unless otherwise indicated.

Characteristic Measurement

Median (IQR) gestational age, weeks 31.0 (28.0 to 33.0)

Mean (SD) birthweight, g 1558.8(612.8)

Sex (Female) 134 (45.3)

Mode of delivery

Cesarean 180 (60.8)

Vaginal 101 (34.1)

Unknown 15 (5.1)

Maternal ethnicity

Canadian Caucasian 110 (37.2)

Unknown 90 (30.4)

Southeast Asian 27 (9.1)

South Asian 26 (8.8)

African or Caribbean 20 (6.7)

Hispanic 12 (4.1)

Middle Eastern 6 (2.0)

First Nations or Inuit 5 (1.7)

Mean (SD) number of hours from birth to initiation of phototherapy 42.9 (30.9)

Total number of transcutaneous bilirubin measurements among all infants, prior to initiation of phototherapy

172

Total number of transcutaneous bilirubin measurements among all infants, after initiation of phototherapy

684

Number of total serum bilirubin measurements > 100 μmol/L prior to the initiation of phototherapy

138

Number of total serum bilirubin measurements > 170 μmol/L prior to the initiation of phototherapy

34

101

Table 5-2. Agreement between measurement of transcutaneous bilirubin measurement (TcB) and total serum bilirubin (TSB) among all 296 preterm infants included in the study. Shown are measures of agreement stratified by anatomical site of TcB assessment, gestational age at birth and receipt of phototherapy.

Group assessed Site of

measurement Stratification variable

(number of paired measurements)

Bland-Altman mean TcB-TSB difference (95%

limits of agreement), μmol/L

Minimum, maximum TcB-TSB difference,

μmol/L

Lin’s concordance coefficient (95% CI)

between TcB and TSB

All neonates, born at 24 to 35 weeks’ gestation

Forehead Regardless of phototherapy (856) -15.2 (-86.8 to 56.3) -143.3, 109.3 0.68 (0.65 to 0.71)

Prior to phototherapy (172) 5.0 (-66.0 to 76.1) -122.0, 109.3 0.74 (0.67 to 0.80)

During or after phototherapy (684) -24.2 (-91.5 to 43.2) -143.3, 86.0 0.65 (0.62 to 0.69)

Sternum

Regardless of phototherapy (856) -24.4 (-112.9 to 64.0) -176.1, 113.3 0.58 (0.54 to 0.61)

Prior to phototherapy (172) 1.1 (-80.0 to 82.2) -165.5, 113.3 0.68 (0.61 to 0.75) During or after phototherapy (684) -35.4 ( -119.7 to 48.9) -176.1, 79.0 0.54 (0.50 to 0.58)

Neonates born at 24-28 weeks’ gestationa

Forehead During or after phototherapy (229) -25.9 (-84.3 to 32.6) -133.1, 86.0 0.49 (0.42 to 0.56)

Sternum During or after phototherapy (229) -40.2 (-106.5 to 26.1) -176.1, 44.7 0.35 (0.29 to 0.42)

Neonates born at 29-32 weeks’ gestation

Forehead


Prior to phototherapy (63) -2.6 (-81.3 to 76.1) -122.0, 108.3 0.60 (0.44 to 0.72)


Sternum


Prior to phototherapy (63) -3.1 (-91.1 to 84.9) -165.5, 96.3 0.52 (0.36 to 0.65) During or after phototherapy (318) -32.2 (-111.8 to 47.4) -150.0, 71.6 0.44 (0.38 to 0.49)


Forehead Regardless of phototherapy (234) -5.0 (-88.4 to 78.4) -138.7, 109.3 0.67 (0.60 to 0.73)



Sternum




aOnly 11 neonates born at 24-28 weeks’ gestation had the opportunity to have TSB or TcB measured before initiation of phototherapy, providing 12 paired TcB

measurements. Accordingly, the related data are only presented for those infants receiving phototherapy.

102

Table 5-3. Test characteristics of transcutaneous bilirubin measurement at recommended total serum bilirubin cut-points.

Group assessed

Recommended total serum

bilirubin cut-point, μmol/L

Sensitivity (%, 95% CI)

Specificity (%, 95% CI)

Positive predictive value

(%, 95% CI)

Negative predictive value

(%, 95% CI)

Positive likelihood ratio

(95% CI)

Negative likelihood ratio

(95% CI) Neonates born at 29-32 weeks’ gestation

> 100 73

(58 to 85) 73

(45 to 92) 90

(76 to 97) 46

(25 to 67) 2.7

(1.2 to 6.4) 0.37

(0.21 to 0.64)


> 170 83

(64 to 94) 78

(66 to 87) 61

(45 to 77) 91

(81 to 97) 3.7

(2.3 to 6.0) 0.22

(0.10 to 0.50)

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5.8 Figures

Figure 5-1. Flow diagram of participant recruitment.

*Matched paired measurements of total serum bilirubin (TSB) and transcutaneous bil irubin measurements (TcB) at

the forehead and sternum.

75th

percentile

40th

percentile

Assessed and approached for

study participation (n = 417)

Analysed (n = 296)

856 matched paired

measurements

Declined to participate (n = 73)

Consented to study (n = 344)

Had matched paired

measurements* (n = 296)

Did not have matched paired measurements *

(N=48)

104

A

Mean + 1.96 SD

= 54.3

Mean

= -24.5

Mean - 1.96 SD

= -103.3

Figure 5-2. Bland-Altman plots of paired transcutaneous bilirubin (TcB) and total serum bilirubin (TSB) measurements among all 296

preterm infants at 24-35 weeks’ gestation, measured overall (A), prior to (B), and after (C), initiation of phototherapy

105

Mean + 1.96 SD

= 76.5

Mean

= 1.6

Mean - 1.96 SD

= -73.4

B

106

Mean + 1.96 SD

= 43.4

Mean

= -31.1

Mean - 1.96 SD

= -105.5

C

107

Mean + 1.96 SD

= 36.3

Mean

= -23.2

Mean - 1.96 SD

= -82.7

A

Figure 5-3. Bland-Altman plot of paired forehead transcutaneous bilirubin (TcB) and total serum bilirubin (TSB) measurements among

neonates born at (A) 24-28 weeks’ gestation, (B) 29-32 weeks’ gestation and (C) 33-35 weeks’ gestation.

108

B

Mean + 1.96 SD

= 46.0

Mean

= -18.4

Mean - 1.96 SD

= -82.8

109

C

Mean - 1.96 SD

= -88.4

Mean

= -5.0

Mean + 1.96 SD

= 78.4

110

Figure 5-4. Overall Bland-Altman plot of paired transcutaneous bilirubin (TcB) and total serum bilirubin (TSB) measurements among preterm

infants born at 24-35 weeks’ gestation whose mother’s ethnicity is (A) African-Caribbean, (B) Caucasian, (C) Southeast Asian, and (D) South Asian

Mean + 1.96 SD

= 52.0

Mean

= -12.0

Mean - 1.96 SD

= -76.0

A

111

Mean + 1.96 SD

= 45.1

Mean

= -30.6

Mean - 1.96 SD

= -106.2

B

112

C

Mean + 1.96 SD

= 49.9

Mean

= -25.2

Mean - 1.96 SD

= -100.3

C

113

Mean

= -9.8

Mean - 1.96 SD

= -94.1

Mean + 1.96 SD

= 74.5

D

114

Figure 5-5. Receiver operating curves for transcutaneous bilirubin (TcB) measured at the forehead at recommended total serum bilirubin

thresholds > 100 μmol/L at 29 to 32 weeks’ gestation (A), and > 170 μmol/L at 33 to 35 weeks’ gestation (B).

A

Forehead TcB AUC 0.76 (95% CI 0.59 - 0.87)

115

B

Forehead TcB AUC 0.86 (95% CI 0.76 - 0.92)

116

Chapter 6 . General Discussion

75th

percentile 40th

percentile

117

6.1 Discussion outline

This chapter summarizes and expands on three original research studies completed as part of

this thesis. This chapter complements the discussion sections of three original manuscripts in

chapters 3-5. First, the main findings of each study are summarized. Second, the overall

strengths and limitations of all three studies are discussed. Finally, the potential impact of this

thesis on the assessment and management of hyperbilirubinemia in preterm infants are

described.

Study 1 uses the term “pre-treatment,” to describe TSB levels measured prior to phototherapy

among those who subsequently received phototherapy or all TSB levels among those infants

that did not receive phototherapy. In study 2 we use pre-phototherapy to describe this. Both

pre-treatment and pre-phototherapy refer to the same concept.

6.2 Summary of main findings

6.2.1 Study 1: Hour-specific total serum bilirubin percentiles for infants born at 29 to 35 weeks’ gestation

Study 1 addresses the lack of systematically derived pre-treatment TSB percentiles in preterm

infants. Using a multi-site, retrospective cohort design, study 1 produced a novel hour-specific

pre-treatment TSB percentile-based nomogram among 2549 infants born at 290/7 to 356/7

weeks’ gestation overall. A total of 6143 pre-treatment TSB levels were included in the overall

nomogram (Figure 3-2A), including 2313 pre-treatment TSB levels among 1120 preterm infants

born at 290/7 to 326/7 weeks’ gestation (Figure 3-3A) and 3830 pre-treatment TSB levels among

1429 infants born at 330/7 to 356/7 weeks’ gestation (Figure 3-3B). Pre-treatment TSB curves at

the 40th, 75th and 95th percentiles were generated using quantile regression.

118

We compared pre-treatment TSB levels between infants born at 290/7 to 326/7 weeks’ gestation

and 330/7 to 356/7 weeks’ gestation. Infants born at 330/7 to 356/7 weeks’ gestation had notably

higher TSB levels than infants born at 290/7 to 326/7 weeks after 30 hours of age from birth,

highlighting the need for two nomograms (Figure 3-4).

When we looked at the behavior of TSB levels in this study, we noted two key trends. First,

within the first 72 hours of age from birth, TSB levels peaked earlier among preterm infants

born at 290/7 to 326/7 weeks’ gestation (36.3 hours) than infants born at 330/7 to 356/7 weeks’

gestation (46.0 hours), usually before 48 hours of age. This is likely because the number of

infants administered phototherapy is higher among infants of greater prematurity than infants

of less prematurity.86,87 This further supports the need for two nomograms by gestational age

group at birth. Second, at the 95th percentile, estimated TSB levels continued to rise past 72

hours of age up to 110 hours of age suggesting TSB levels among infants at the 95th percentile

may need prolonged and intermittent monitoring.

Finally, this study provides descriptive data on the potential influence that mode of feeding

(TPN vs enteral) may have on TSB levels and subsequent decisions to initiate phototherapy.

More specifically we found that more infants who received TPN, either alone (74.0%) or with

enteral feeds (77.9%) received phototherapy than infants only on enteral feeds (64.8%). In

addition, these infants on TPN either alone or with enteral feeds also experienced an earlier

peak bilirubin at 39.1 and 40.5 hours, respectively, than among those infants only on enteral

feeds (44.3 hours). (Table 3-3 and Table 3-4).

119

6.2.2 Study 2: Pre-phototherapy total serum bilirubin levels in extremely preterm infants

Given the even higher risk of significant hyperbilirubinemia and increased frequency of

phototherapy usage amongst extremely preterm infants born 240/7 to 286/7 weeks’ gestation

described in chapter 2, a separate study was conducted to address the consensus-based TSB

thresholds used in this population.18 A multi-site, retrospective cohort study at the same three

NICUs as study 1, among 642 extremely preterm infants born at 240/7 to 286/7 weeks’ gestation

was conducted. A total of 1134 pre-phototherapy TSB levels were included in the hour-specific

pre-phototherapy percentile-based TSB nomogram. TSB percentile curves at the 40th, 75th and

95th percentiles were generated using quantile regression. This study also generated pre-

phototherapy TSB percentiles at 24 hours of age from birth at the 40th, 75th and 95th percentiles

and compared them to Maisels’ published thresholds stratified by gestational age group at birth

(i.e. 24-25 weeks, 26-27 weeks , 28 weeks).18 In addition, the proportion of infants who were

administered phototherapy at TSB levels below Maisels’ treatment thresholds was also

evaluated.18

In study 2, we found the statistically derived 75th and 95th pre-phototherapy TSB percentiles for

24 hours of age (from birth) were significantly higher than consensus-based TSB phototherapy

thresholds published by Maisels (Table 4-3).18

When assessing phototherapy administration we found that almost all extremely preterm

infants (95%) were administered phototherapy and 29.9% of these infants were started on

phototherapy below Maisels’ thresholds.18 When we compared the proportion of infants

administered phototherapy below Maisels’ published threshold by hours of age of

phototherapy initiation (since birth), we found the proportion of infants administered

phototherapy below Maisels’ threshold significantly declined with hours of age of phototherapy

initiation since birth.18 This suggests that earlier initiation of phototherapy may be associated

with use of TSB thresholds below the currently recommended thresholds. Furthermore, 76.6%

of infants in this extremely preterm population were administered phototherapy by 36 hours of

120

age, further confirming the tendency to initiate phototherapy earlier in more premature

infants.87

6.2.3 Study 3: Transcutaneous vs. total serum bilirubin measurements in preterm infants

As illustrated in both studies 1 and 2, preterm infants born at 240/7 to 356/7 weeks’ gestation

are intermittently monitored for hyperbilirubinemia with frequent TSB testing being performed

as early as 24 hours of age and infants are often started on phototherapy by 48 hours of age.

TSB testing also continues after the initiation of phototherapy to monitor response and support

clinical management decisions.123 As described in chapter 2, these preterm infants are exposed

to the additional risks of pain, stress, and anemia as a result of repeat TSB testing. As a

reminder among term and near-term infants, TcB meters have been approved to monitor

bilirubin levels prior to phototherapy initiation. However, to date, TcB meters have not yet

been approved for usage in preterm infants in Canada.2

To address the increased stress, pain and risk of anemia caused by frequent TSB tests in

preterm infants, study 3 assesses the use of the JM-105, a TcB device, among preterm infants

born 240/7 to 356/7 weeks’ gestation. A multi-site, prospective, cohort study of 296 preterm

infants born at 240/7 to 356/7 weeks’ gestation was conducted. A total of 856 TcB

measurements were taken on the forehead and sternum with medically indicated TSB

measurements. To capture the optimal usage and limitations of the JM-105 in preterm infants,

the agreement between TcB and TSB measurements were assessed overall, by gestational age

at birth, receipt of phototherapy, anatomical site of measurement (i.e., forehead and sternum)

and self-reported maternal ethnicity.

In this study, consistent with previous research, agreement between TcB and TSB was better

prior to the initiation of phototherapy (1.6 μmol/L, 95% LOA -73.4 to 76.5) than after the

initiation of phototherapy (-31.1 μmol/L, 95% LOA -105.5 to 43.4).123,145,147 This was consistent

by anatomical site of measurement and gestational age at birth (Table 5-2). Prior to the

121

initiation of phototherapy, there was no appreciable difference in TcB-TSB agreement between

the forehead (5.0 μmol/L, 95% LOA, -66.0 to 76.1) and the sternum (1.1 μmol/L, 95% LOA -80.0

to 82.2). Although this was consistent with some previous studies,128,152 some studies reported

the sternum149,150 to be a more accurate site for TcB while one study reported the forehead to

be the better anatomical site.151 After the initiation of phototherapy, the forehead appeared to

be the more accurate site for TcB measurements, demonstrating better TcB-TSB agreement on

the forehead (-24.2 μmol/L, 95% LOA -91.5 to 43.2) than the sternum (-35.4 μmol/L, 95% LOA -

119.7 to 48.9). One reason for this may be because of the eye mask used to cover the eyes

during phototherapy, which reduces the exposure to phototherapy.182 These results were also

consistent when TcB and TSB agreement was stratified by gestational age at birth (Table 5-2).

When agreement was assessed by gestational age group at birth, the agreement between TcB

and TSB was better among preterm infants born at 330/7 to 356/7 weeks’ gestation than infants

of younger gestational age at birth (Figure 5-3). Finally, maternal ethnicity did not seem to

have an impact on the agreement between TcB and TSB, consistent with previous research135

(Figure 5-4).

6.3 Strengths and Limitations

6.3.1 Overall strengths

In all three studies, a multi-site cohort design was employed and carried out at three large

NICUs in Ontario. This allowed for inclusion of a diverse population of preterm infants,

sufficient statistical power to conduct analysis, and thorough representation of multiple study

sites allowing for greater generalizability of study findings.

The multi-site design of the study also resulted in large sample sizes for each research study.

Study 1 and 2 were one of the largest studies to estimate pre-treatment TSB levels in preterm

infants. Previous studies were limited to sample sizes of under 500 infants.91,92 Study 3 to date

was also one of the larger studies to assess agreement between TcB and TSB among preterm

infants.151

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All three studies also included multiple measurements per infant in the analysis, where

previous research in hyperbilirubinemia did not statistically account for repeated

measurements per infant.16,128,133 In studies 1 and 2, bootstrapping as part of quantile

regression in R allowed for the weighting of multiple measurements per infant in the

calculation of the 95% confidence intervals of pre-treatment and pre-phototherapy TSB

percentiles.165 In study 3, adapted Bland-Altman plots weighting for multiple TcB and TSB

measurements was used.179,180 A previous study used regression analysis to account for

multiple measurements per infant.181 To date these adapted Bland-Altman plots have not been

used to measure agreement between TcB and TSB measurements.128,132 The weighting of

multiple measurements per infant in both quantile regression and Bland-Altman plots allowed

for a more comprehensive and representative analysis of bilirubin levels from preterm infants

admitted to NICUs.

The studies also explored several clinical factors impacting the main outcome of each

individual study to provide a comprehensive understanding of the measurement of bilirubin in

preterm infants.

Study 1 generated pre-treatment TSB percentiles for the whole population of preterm infants

overall and stratified specifically by receipt of phototherapy and gestational age group at birth

(i.e., 29 to 32 weeks’ gestation vs 33 to 35 weeks’ gestation). Previous studies reporting pre-

treatment TSB levels did not differentiate between infants who did and did not receive

phototherapy.3,91,92 Specific to preterm infants, two previous studies reporting pre-treatment

TSB levels stratified TSB levels by birthweight and not gestational age at birth.91,92 Our study

provides novel data of how the pattern of TSB levels may differ under different clinical

conditions representative of the clinical care preterm infants receive.

In study 2, pre-phototherapy TSB percentiles at 24 hours were stratified and compared to

Maisels’ thresholds18 by gestational age at birth. By stratifying the main analysis by gestational

age at birth (i.e., 24 to 25 weeks, 26 to 27 weeks, and 28 weeks ’ gestation) experts and

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researchers are provided with a deeper understanding of how pre-phototherapy TSB levels may

change in relation to gestational age at birth, a major risk factor of hyperbilirubinemia.16,17

Finally, in study 3, agreement between TcB and TSB levels were calculated considering

gestational age at birth, receipt of phototherapy, anatomical site of measurement and maternal

ethnicity. By assessing agreement in the context of these multiple variables, a detailed report of

the strengths and limitations of TcB measurements was generated, a component that previous

studies lacked and where usually only one or two such factors were considered.128,133 This study

also provided valuable information on the change in TcB and TSB agreement after the initiation

of phototherapy by gestational age at birth and anatomical site of measurement. Based on a

systematic review of studies assessing the impact of phototherapy on TcB measurements,

previous studies were usually limited to one anatomical site of measurement, and a small

cohort of infants.128,133

By stratifying the main analyses of all three papers by key clinical factors, specifically gestational

age, this thesis provides valuable information on the measurement of TSB and TcB levels in

preterm infants.

The thesis also addresses the paucity in research around management of hyperbilirubinemia in

North America. First, studies 1 and 2 support and add to recent research reporting the high

frequency of phototherapy administration among preterm infants.86,87 Second, study 2 reports

new information related to the use of Maisels’ recommended thresholds.18 This study provides

the first direct comparison between statistically derived pre-phototherapy TSB percentiles and

Maisels’ published threshold.18 Finally study 2 was one of the first studies to report the

proportion of infants who were treated below Maisels’ recommended thresholds.18 The only

recent study to report the administration of phototherapy below recommended thresholds was

limited to term and near-term infants.178

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6.3.2 Overall limitations

All three studies had several limitations related to the availability of data and the inherent

limitations of multi-site prospective and retrospective cohort studies.

First, although multi-site studies allow for the recruitment of a diverse and large population of

infants, given the current use of consensus-based guidelines for managing hyperbilirubinemia in

preterm infants, practice variation with regards to the management hyperbilirubinemia exist. In

studies 1 and 2, each institution used adapted, unpublished guidelines developed by another

tertiary hospital to directly manage hyperbilirubinemia. However, since these guidelines were

also based on expert opinion and each site had made further adaptations to them, variation

between the individual sites and physicians may have also existed. As reported in the literature,

practice variation related to the management of hyperbilirubinemia in preterm infants is quite

common.19,20,90,105,184 Similarly in study 3, the lack of a protocol for the timing of TcB and TSB

measurements could have resulted in variation of TcB device use. The clinical staff at all three

sites received universal training by the same study personnel on the use of the TcB device and

frequent check- ins and re-training were done. Despite this, the introduction of new

technology within a clinical care team presented challenges that may have resulted in variations

in how the technology/equipment was used.185

Second, because of limited data available in the electronic medical charts reviewed for these

studies, specific details of clinical factors that may impact care in preterm infants were limited.

In study 1, specific information on the volume and duration of each mode of feeding was

difficult to obtain due to inconsistent reporting in medical charts and the reliance on physician

and nursing clinical reports to obtain this information. This presented challenges in determining

possible associations between mode of feeding and pre-treatment TSB levels and phototherapy

administration.

In study 2, the severity of illness could not accurately be assessed as a potential factor

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impacting phototherapy initiation. This was due to the limited number of extremely preterm

infants included in the study, the lack of a universal definition for severity of illness and the

difficulty obtaining key clinical factors in electronic medical charts. In addition, our study was

designed to assess the patterns of TSB levels and immediate initiation of phototherapy and

therefore did not collect follow-up information to assess long term sequelae.

Finally, within study 3, the impact of phototherapy on the agreement between TcB and TSB

levels by time from phototherapy cessation could not be reliably assessed. This was due to

several factors including incomplete information on the hours of age of phototherapy

discontinuation in medical charts and lack of timed measurements after the cessation of

phototherapy. Previous research with a set protocol on when to measure TcB and TSB levels

explored the change in agreement at specific time points after phototherapy had been initiated

and stopped.147 Since the study left the decision to conduct TSB tests to the discretion of the

physician, timed analysis was difficult to obtain. As clinical staff become more familiar with the

use of TcB devices, future research could add a timed protocol for obtaining TcB and TSB levels.

Third, the increased proportion and early administration of phototherapy (initiated at less than

48 hours of age) among infants born 240/7 to 286/7 weeks’ gestation limited the analysis for this

population. For example, in study 2, an hour-specific TSB nomogram could only be reliably

developed up to 72 hours of age, due to limited pre-phototherapy TSB levels after 36 hours of

age among infants born 240/7 to 286/7 weeks’ gestation. As well, in study 3, agreement between

TcB and TSB levels could not be reliably assessed among these more premature infants since

only 12 paired TcB-TSB measurements were obtained prior to phototherapy and infants were

started on phototherapy at a median of 26 hours of age from birth.

Fourth, all three studies were conducted in Ontario and amongst three nearby NICUs. As a

result of potential practice variation between regions on the management hyperbilirubinemia

in preterm infants, these studies may not be generalizable to other regions outside of Ontario.

Related to the generalizability of study results, since study 3 used only the JM-105, the current

findings of study 3 may not be reflective of the performance of other TcB devices.

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Finally, the results of this study do not offer clinical guidance on the assessment

hyperbilirubinemia in preterm infants.

The pre-treatment TSB levels derived from this study were statistically estimated from a large

cohort of preterm infants and were not validated as previously conducted in Bhutani’s

nomogram.3 The short and long-term outcomes of infants with these TSB levels were also not

explored and therefore we cannot confirm if these pre-treatment TSB cut-points reported in

study 1 and study 2 are safe. Also in study 2, the clinical factors that may impact the

administration of phototherapy below Maisels’ published threshold were also not explored. The

scope of this study was to statistically derive pre-phototherapy TSB levels for infants born

extremely preterm and compare them to Maisels’ published threshold. Finally in study 3, our

primary outcome to assess TcB performance was agreement between TcB and TSB. Agreement

provides a mathematical description of the bias between TcB and TSB measurements but does

not provide any guidance about the appropriate TSB levels at which to use TcB. Although study

3 reports the sensitivity and specificity of forehead TcB levels in detecting recommended TSB

thresholds18, the number of paired TcB-TSB measurements prior to the initiation of

phototherapy were limited. Additional research is required to validate our suggested TcB cut-

points.

The intention of this thesis was not to change clinical practice. The aim was to re-think current

approaches to the assessment of hyperbilirubinemia in preterm infants by first providing novel

data on pre-treatment TSB levels in preterm infants. A secondary goal was to assess the

performance of an alternative non-invasive method to measure bilirubin levels in preterm

infants. With these results we hope to conduct additional research to optimize management of


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6.4 Overall research impact

This thesis provides novel data about the measurement of bilirubin from a large cohort of

preterm infants. The information from this study could be used by experts and health policy

makers to inform evidence-based guidelines in the assessment and management of


6.4.1 Assessment of hyperbilirubinemia

First, Study 1 and Study 2 presents data on pre-treatment/pre-phototherapy TSB levels from

birth to 120 hours of age and birth to 72 hours of age, respectively. These mathematically

derived hour specific pre-treatment/pre-phototherapy TSB levels offer a first step in

understanding the behavior of bilirubin levels in preterm infants along with a description of the

potential clinical factors that can impact the behavior of these TSB levels. The thesis also

provides these hour-specific pre-treatment (Study 1) and pre-phototherapy (Study 2) TSB

percentile curves by gestational age group at time of birth (i.e., 24 to 28 weeks, 29 to 32 weeks

and 33 to 35 weeks gestational age), further enabling targeted assessment of

hyperbilirubinemia by gestational age group at birth. This fills the existing gap in the literature

around the lack of published pre-treatment/pre-phototherapy TSB levels among preterm infant

that has resulted in consensus-based guidelines for the management of hyperbilirubinemia.

Although these values cannot be used directly to assess hyperbilirubinemia, it provides experts

with data to consider, in order to inform the development future guidelines for the assessment

of hyperbilirubinemia. For example, experts may use our data to further explore the behavior of

TSB levels to inform the safety of these levels, similar to previous research used to determine

optimal thresholds for oxygen and carbon dioxide levels in preterm infants. Since a

standardized approach to the assessment of hyperbilirubinemia in preterm infants is

lacking,17,18 our pre-treatment TSB percentiles could be the first step towards gathering data to

inform the development of a standardized approach. This provides an improvement over

guidelines based on consensus and expert opinion.18

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Second, Study 3 provides more information on the use of TcB devices in preterm infants

especially after the initiation of phototherapy. We provided a detailed description of agreement

between TcB and TSB stratified by gestational age group at birth. Only one previous study

stratified TcB performance by gestational age group at birth among preterm infants. Other

studies provided an overall assessment of TcB in preterm infants.123 By providing gestational

age group specific data on TcB performance, experts can use our data to inform the

development of a more targeted approach to measuring TcB levels in preterm infants. In

addition, our data supports recommendations discouraging the use of TcB after the initiation of

phototherapy. While considering TSB thresholds in preterm infants, TSB testing could be

potentially reduced by using TcB in preterm infants at low risk of hyperbilirubinemia.

The combination of our data on pre-treatment hour specific TSB percentiles (study 1) and

agreement data between TcB and TSB in preterm infants born at 330/7 to 356/7 weeks’ gestation

(study 3), provides clinical experts and health policy makers with key information to assess and

consider in informing a more targeted and non-invasive approach to assessing

hyperbilirubinemia in this population.21,116

Third, our study also offers a Canadian specific insight into the assessment of

hyperbilirubinemia. Although hyperbilirubinemia may not change from country to country, the

current practices of measuring bilirubin and administering phototherapy may be different and

result in different nomograms. To date, only one Canadian study has proposed hour-specific

TSB curves based on gestational age group at birth in preterm infants, however, these curves

were developed by reviewing existing consensus-based guidelines and not based on data from

preterm infants.97 Our study presents hour-specific pre-treatment TSB levels directly derived

from a large cohort of Canadian preterm infants. As an alternative to relying on US-based

data,16,18,98,113 our Canadian data has the potential to inform clinical practice in Canada on the

current assessment and management practices of hyperbilirubinemia in preterm infants.

Fourth, this thesis adds to our understanding of clinical factors that could impact TSB levels.

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Study 1 introduces mode of feeding as an additional clinical factor to continue to investigate its

impact on pre-treatment TSB levels. Feeding has often been overlooked in the management of

hyperbilirubinemia in preterm infants. In term and near-term infants, exclusive breast feeding is

one of the risk factors of hyperbilirubinemia.1,2 In preterm infants, gestational age and

hemolysis have been most commonly reported as two of the key risk factors of

hyperbilirubinemia.16 Our study provides initial insight into the relationship between mode of

feeding and TSB levels in preterm infants. As mode of feeding is a significant component of

clinical care provision in preterm infants that differs from term and near-term infants, a greater

understanding of the role feeding has on the management of hyperbilirubinemia is important.

Finally, studies 2 and 3 underscore the need to review the short- and long-term effects of

phototherapy by supporting previous data noting a high rate of phototherapy administration

among extremely preterm infants. In keeping with previous reports86,87, all three studies

highlight the increased use of phototherapy among preterm infants, especially among infants

born at <29 weeks’ gestation. This is largely due to the lack of data-based guidelines for

assessing pre-phototherapy TSB levels in preterm infants born at 240/7 to 286/7 weeks’

gestation, as well as the recognition that these infants are at higher risk for long-term

neurological sequelae relative to infants at older gestational ages.16,17,97 In study 1, 91% of

preterm infants born 290/7 to 326/7 weeks’ gestation were administered phototherapy. In

studies 2 and 3, almost all infants born between 240/7 to 286/7 weeks’ gestation were

administered phototherapy by as early as 26 hours of age. With the higher rates of

phototherapy use and reports of potential harms of phototherapy, a review of phototherapy in

preterm infants is required.86,87 In addition although statistically derived, the notable variability

between our pre-phototherapy TSB percentiles and Maisels published threshold may signal to

experts to re-assess the effectiveness of current phototherapy treatment practices in their

respective regions.

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6.4.2 Inform health policy efforts in professional societies

The data from this thesis can provide valuable data for health policy makers within professional

societies (i.e., CPS) to assess and consider in three areas of neonatal health, including the

assessment of hyperbilirubinemia, feeding practices and pain management.

This thesis is the first step in re-thinking the assessment of hyperbilirubinemia in preterm

infants. This thesis provides new information on TSB levels in preterm infants and additional

information on current phototherapy use in preterm infants. This could prompt additional

NICUs to review their own local, pre-treatment TSB level thresholds for phototherapy and, in

turn, assess their current management of hyperbilirubinemia in preterm infants in general, as

has recently been conducted in the US.87 The culmination of our findings, data from additional

NICUs and data on phototherapy administration and its impact, could provide novel

information to health policy makers to consider in the assessment of hyperbilirubinemia,

specifically guidelines around when to repeat TSB testing, TcB use and the administration of

phototherapy.

As part of the policies around feeding practices in preterm infants, within the nutrition

literature, a greater push for earlier initiation of enteral feeding is currently being made.186 This

study provides initial insights into the potential relationship between mode of feeding and the

behavior of TSB levels. This study could inform the design of future studies to investigate the

impact of feeding methods on the management of hyperbilirubinemia to inform guidelines

around feeding practices in preterm infants.

With the increased awareness of the long-term effect of pain and stress among preterm

infants, results from this study could inform clinical practice on strategies to reduce

unnecessary blood tests in preterm infants. Although this study provides support for use of TcB

prior to phototherapy among preterm infants born at 330/7 to 356/7 weeks’ gestation, further

research is required to understand TcB device underestimation of TSB levels after the initiation

of phototherapy. The data from our study and additional research could both be used by health

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policy experts in pain management in preterm infants to inform guidelines around reducing

pain in preterm infants.122 Currently there is a nationwide strategy to reduce unnecessary tests,

specifically in neonatal patients, and our findings could contribute to achieving this goal.187

6.4.3 Research

This thesis is a starting point to rethinking the assessment of hyperbilirubinemia in preterm

infants. Additional research is required to support and assess the results of these studies to

shift the management of hyperbilirubinemia from consensus based to evidence based. As there

is currently a paucity of data on pre-treatment TSB levels in preterm infants, this study provides

the largest dataset of bilirubin levels in preterm infants to inform the design and the

development of additional research. Chapter 8 highlights some of the additional research that

could be conducted as next steps in this thesis.

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Chapter 7 . Conclusions

133

7.1 Conclusions

This thesis provides a breadth of novel data about measuring bilirubin levels in preterm infants.

We present hour-specific pre-treatment TSB percentile-based nomograms by gestational age

group at birth among preterm infants (study 1). We identified considerable variation between

our statistically derived pre-phototherapy TSB curves and Maisels’ published TSB thresholds

among extremely preterm infants (study 2). We offered data on a non-invasive alternative

approach, TcB measurements, to measuring bilirubin levels in preterm infants (study 3). As the

first step in re-thinking the assessment of hyperbilirubinemia, our systematically derived pre-

treatment TSB levels fills the gap in research around the lack of published pre-treatment TSB

levels in preterm infants. Our pre-treatment TSB curves will provide, experts, health policy

makers and researchers valuable information about the trends and behavior of TSB in preterm

infants to conduct future research that will inform the development of evidence-based

guidelines for the assessment of hyperbilirubinemia. In addition, to address the harms of

repeated TSB testing in preterm infants, we offer TcB as an immediate alternative method to

measuring bilirubin levels in preterm infants born at 33 to 35 weeks’ gestation prior to

phototherapy. These infants do not receive routine blood work as frequently as extreme

preterm infants and beyond the first few days of life may only receive bloodwork for bilirubin

tests. As such, TcB in this population has the potential to reduce unnecessary blood tests.

Overall, this thesis offers key information for experts, health policy makers and researchers to

consider in their efforts to move toward a more standardized evidence-based approach to

assessing and managing hyperbilirubinemia in preterm infants.

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Chapter 8 . Future directions

135

8.1 Future Directions

This thesis lays the foundation for several future areas of research which would continue to

focus on the assessment and management of hyperbilirubinemia. Below is a brief description of

current and future studies that could be completed based on the findings of this thesis,

followed by a summary of preliminary findings of one study.

8.1.1 Assessment of hyperbilirubinemia

Although the development of this novel nomogram is the initial step in achieving methods to

reliably assess TSB levels in preterm infants, further research needs to be done to determine if

TSB levels are influenced by different clinical practices and neonatal characteristics. Our large,

hour-specific, pre-treatment and pre-phototherapy TSB percentiles derived from preterm

infants provide a starting point to compare other pre-treatment TSB percentiles generated

from additional NICUs. By using our statistically derived nomogram as a basis for comparing the

trends of TSB levels from other sites, a collectively greater understanding of the behavior and

factors impacting TSB levels in preterm infants can be established. Our studies included all

preterm infants born at 240/7 to 356/ weeks’ gestation, only excluding infants with Rh disease.

Other studies may choose to apply additional exclusion criteria to generate TSB curves specific

to sub-populations of pre-term infants. These additional studies would generate additional TSB

curves for experts to consider in the development of guidelines for measuring TSB levels in

preterm infants.

The scope of Study 1 and 2 was to statistically derive pre-treatment/pre-phototherapy TSB

levels in preterm infants and therefore did not assess the short- and long-term outcomes of

neonates at these statistically derived TSB levels (Study 1 and Study 2) or those administered

phototherapy below Maisels’ published threshold (Study 2). A follow-up prospective study

could be designed to assess the safety of these TSB levels among preterm infants from birth to

one year of life, comparing neonatal outcomes between those infants who had pre-treatment

TSB levels above our percentiles and below our percentiles. This would strictly provide

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information on neonatal outcomes among infants with TSB levels like those reported herein. A

longitudinal, prospective cohort study of preterm infants followed for one year would allow us

to generate evidence on the safety of these TSB levels.

This thesis also provides additional data on measuring bilirubin with a TcB device after the

initiation of phototherapy. To address the limitations of TcB use after the initiation of

phototherapy, some small studies have used opaque skin patches to cover a portion of the skin

during phototherapy and subsequently evaluated the agreement between TcB and TSB on

exposed and unexposed areas of skin to phototherapy.147,148 In keeping with these new reports,

at our site, we are currently conducting a larger prospective cohort study among infants born at

> 35 weeks’ gestation to determine the agreement between TcB and TSB when TcB

measurements are taken on parts of the forehead exposed and unexposed to phototherapy. To

shield part of the forehead from phototherapy, an opaque skin patch is placed on a small part

of the forehead. The same study should be done in preterm infants

In addition to the direct exposure of skin to phototherapy, TcB may also be impacted by the

hours the skin is exposed to phototherapy and hours after phototherapy is completed. Our

study was unable to ascertain a difference in TcB performance by during and after the initiation

of phototherapy. However the number of hours after the cessation of phototherapy would

undoubtedly impact the use of TcB devices.22 A future study could focus specifically on TcB

device use during and after the initiation of phototherapy. Implementing a timed protocol of

measuring TcB and TSB levels during and after the cessation of phototherapy could provide a

deeper understanding on the potential use of TcB devices. Identifying the optimal time points

to use TcB after an infant has started and stopped phototherapy and could reduce the number

of repeated TSB tests in preterm infants.

Finally, TcB could also be used to address barriers to accessing timely TSB measurements in

resource poor areas and community hospitals. Of importance, these sites may lack access to

timely TSB measurements and often resort to transferring patients to other community or

tertiary hospitals for bilirubin testing.127 Our team specifically explored this issue in the Baffin

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region in Iqaluit, the capital city of the Canadian territory of Nunavut. One hospital, Qikiqtani

General Hospital, serves several communities that fall within the Baffin region which are often

plane rides away from Qikitqtani General Hospital. More specifically women often travel to

Qikiqtani Hospital to delivery their baby. Consequently, following an uncomplicated delivery at

this hospital, newborns are followed by the local general practitioner until discharge and a

mandatory TSB test is done at 24 hours of age from birth. If the TSB is high, the newborn may

stay in the hospital to monitor TSB levels and initiate treatment. However, if an infant shows

signs of jaundice after being discharged home to their communities, the infant must travel back

to Iqaluit by air for a repeat bilirubin level. The transfer of patients is not only costly for the

healthcare system but also disruptive for the family. In situations such as this, a TcB device

could offer a non-invasive and immediately accessible approach to bilirubin testing in

community clinics that do not readily have access to TSB tests. As such, we are currently

conducting a prospective study in Iqaluit to assess the clinical utility and cost effectiveness of

TcB device usage in Iqaluit. This study may offer an immediate and cost-efficient method to

measure bilirubin levels in geographically isolated communities and/or resource poor areas.

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8.1.2 Management of hyperbilirubinemia

This thesis provides additional clinical data on preterm infants that experts could consider

further exploring to inform guidelines around the management of hyperbilirubinemia. Feeding

can be used to manage hyperbilirubinemia in term and near-term infants.1,2 The association

identified between the mode of feeding, TSB levels and subsequent receipt of phototherapy in

study 1, lends itself to additional research exploring the link between mode of feeding and

hyperbilirubinemia in preterm infants. To use mode of feeding as a potential management

strategy to manage hyperbilirubinemia, additional research is required. Our study was limited

to the feeding method administered to the infant in the first 10 days of life. It did not consider

the impact of the amount of feeding on TSB levels and phototherapy administration. A

prospective study measuring the impact of volumes and modes of feeding ( i.e., nutrition via

TPN versus enteral) on TSB levels, initiation of phototherapy and short and long-term neonatal

outcomes, could explain how feeding modes impact the risk of hyperbilirubinemia in preterm

infants. Currently a larger grant application to study this is under review.

As phototherapy is the first line of treatment for jaundice future research should focus on

exploring three aspects of phototherapy administration in preterm infants. These include 1)

frequency of phototherapy use, 2) short and long-term side effects of phototherapy, 3) short

and long-term side effects by intensity of phototherapy. Since all three studies reported a high

rate of phototherapy among preterm infants born at 240/7 to 286/7 weeks’ gestation and 290/7 to

326/7 weeks’ gestation and early initiation of phototherapy in study 2, further investigation on

the implications of this are required. Currently, there is a lack of consensus and understanding

with regards to the harms and short- and long-term impacts of phototherapy, especially among

preterm infants.188,189 To address this, a systematic review of prospective and retrospective

studies evaluating the short- and long-term side effects of phototherapy in preterm infants is

currently being conducted by our team. Appendix 8-1 has a summary of the systematic review.

The preliminary results of the systematic review revealed a paucity of research ascertaining the

long-term side effects of phototherapy, specifically the potential link between malignancy and

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phototherapy. Previous research has been done in term and near-term infants to assess the risk

of malignancy from phototherapy.190,191 However, studies following preterm infants

longitudinally for more than 20 years are limited. Our team plans on using population-based

healthcare administrative data housed at the Institute for Clinical and Evaluative Sciences

(IC/ES) to determine the risk of developing malignancy and other long term health conditions

from phototherapy. Retrospectively, we aim to assess the 30-year risk of developing

pathological conditions among infants administered phototherapy at birth.

The potential harms of early and intensive phototherapy use have also been heavily debated

over the last few years. Earlier studies exploring the impact of the intensity of phototherapy

were limited to infants of VLBW and ELBW. 98,106,111 Since phototherapy is administered at a

high frequency among infants born at < 33 weeks’ gestation, a prospective cohort study

specifically collecting data related to the intensity of phototherapy and following those infants

for a year, may provide insight into the short- and long-term impacts.

Finally, based on the results of this thesis, a review of additional treatments used to reduce the

risk of severe hyperbilirubinemia needs to be conducted. Since phototherapy is used

increasingly in most preterm infants, the use of other treatments should be further

investigated. Over the past few years, the use if IVIG has garnered increasing attention as a

treatment to reduce severe hyperbilirubinemia in high risk infants, specifically those with ABO

incompatability.63 As a side project of this thesis, a population based study of the frequency of

IVIG use among term and near-term infants was conducted, highlighting the increased use of

IVIG despite limited studies on its effectiveness. Given that extremely preterm infants almost

universally receive phototherapy and are at higher risk of severe hyperbilirubinemia, a similar

study on the use of IVIG in preterm infants is warranted.

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8.2 Summary of future research

Based on the results of this thesis future research should focus on three areas of the

assessment of hyperbilirubinemia. First a better understanding of the clinical factors that

impact pre-treatment TSB levels and the safety and neonatal outcomes of our statistically

derived pre-treatment TSB levels is required. Second, solutions to address the impact of

phototherapy on TcB measurements and feasibility of introducing TcB devices in resource poor

areas should be explored. Finally, as this thesis supports recent research on the increased

frequency of phototherapy use in preterm infants the natural next step would be to explore the

impacts of the increased use of phototherapy. Where previous research focused on

mathematically deriving pre-treatment TSB levels from a large cohort of term and near-term

infants, our study did this in a large cohort of preterm infants. Additional research following our

studies is vital to contributing to the knowledge around hyperbilirubinemia in preterm infants

to shift the assessment and management hyperbilirubinemia from consensus to more evidence

based.

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8.3 Appendix

Appendix 8-1. Review of the side effects of phototherapy

Although the decrease in ABE and CBE among preterm infants has been attributed to the

intensive use of phototherapy, the potential harms of phototherapy cannot be ignored.188

There are a limited number of prospective and retrospective studies assessing the short- and

long-term effects of phototherapy in preterm infants. Although extremely preterm infants have

been noted to be at higher risk of experiencing the side effects of phototherapy, due to the

paucity of research in this area, this review will focus on prospective and retrospective studies

evaluating short- and long-term side effects of phototherapy among all newborn infants.

A review of peer-reviewed articles (from 1970-2020) was conducted to identify articles

evaluating the short- and long-term side effects of phototherapy on newborns <28 days of age.

A search was conducted in the following electronic databases: OvidMedline, PsycInfo, Embase

Classic, Embase, Ebsco, Cinhal, Scopus, Cochrane (Evidence-Based Medicine Reviews). Search

terms related to neonatal phototherapy and common side effects were used. The search terms

included were all possible synonyms related to phototherapy, neonates and side effects, as well as

the commonly found side effects mentioned by Hansen et al .188 The section below has been

divided into short- and long -term side effects of phototherapy.

142

8.3.1 Short-term effects of phototherapy

Some short-term side effects of phototherapy reported in preterm infants include188; changes

to mesenteric blood flow192, increase in body temperature193, patent ductus arteriosus194,

rashes195, oxidative stress196, DNA damage197 and increased mortality in preterm infants of

VLBW and ELBW.98,106,111

The most common side effects associated with phototherapy have been related to the

homeostatic and hemodynamic status of infants.188 Phototherapy has been reported to

decrease cardiac output, change blood flow, increase body temperature, increased renal blood

flow and reduce skin level fluid.188,198-201,202 One of the early studies to determine the impact of

phototherapy dosage on body temperature and cardiac function found body temperature to

increase at all dosages of phototherapy administered to both preterm and term infants.

Increased heart rate at all dosages of phototherapy was only identified among preterm

infants.201 In addition, LED phototherapy was reported to increase body temperature thus

prompting authors to suggest frequent monitoring of body temperature during the

administration of intensive LED phototherapy.188,193 Finally, phototherapy has also been

associated with hypocalcemia, resulting from electrolyte imbalance.189,203

Trans-epidermal water loss was often associated with an infant being thermally unstable from

phototherapy.188,204 In keeping with previous reports, Maayan-Metzger reported a 26.4%

increase in trans-epidermal water loss among preterm infants during phototherapy.199 Trans-

epidermal water loss has also been reported to be impacted by the type of phototherapy

administered in preterm infants. For example, a study comparing conventional phototherapy

with LED phototherapy found conventional phototherapy to be associated with a significantly

increased risk of trans-epidermal water-loss compared to LED phototherapy.205

Patent ductus arteriosus (PDA) was found to be an additional hemodynamic side effect of

phototherapy reported in preterm infants.188,202 The opening of the ductus arteriosus results in

increased skin blood flow.188,206 The hemodynamic adverse effects of phototherapy have been

143

associated with increase blood levels of endothelin and nitric oxide among preterm infants born

less than 32 weeks’ gestation after the initiation of phototherapy.207 As a potential solution to

reducing PDA caused by phototherapy, chest shielding has been employed and demonstrated

to reduce this issue. However, this was only replicated in two studies.208,209

Rashes have been a commonly reported side effect of phototherapy as well, especially among

preterm newborns. The skin of extremely preterm infants is fragile and immature, placing it at

high risk of injury.210,211 In addition, infants with an existing skin condition are also at greater

risk of skin injury when undergoing phototherapy.71 The type of phototherapy device used to

administer treatment in preterm infants may impact side effects related to the skin. Surmeil-

Onay et al conducted a randomized controlled trial comparing LED phototherapy and

conventional phototherapy to determine the impact on skin related side effects. In this study,

at least 30% of infants in both groups experienced some sort of skin eruption with no significant

difference between the type of phototherapy device utilized.195

Oxidative stress is another side effect of phototherapy that has garnered interest over the last

several years. Different types of phototherapy devices have been studied to determine

potential for cause of oxidative stress. Blue light phototherapy was shown to cause oxidative

stress in preterm neonates, with increased levels of thiobarbituric acid reactive substances

among preterm infants after the initiation of phototherapy.196 A recent study comparing

conventional and LED phototherapy in preterm infants reported an increase in oxidative stress

among newborns after the initiation of phototherapy especially among infants who received

conventional phototherapy.212 In summary, oxidative stress from phototherapy seems to be

largely associated with the type of phototherapy device used. Additional research is required to

obtain a better understanding of oxidative stress by type of phototherapy device.

In addition to the hemodynamic impacts of phototherapy, most recently, trends of increased

risk of death have been associated with aggressive phototherapy among infants of ELBW.106

Two randomized controlled trials compared aggressive phototherapy and conservative

phototherapy among infants of ELBW.98,111 Both randomized controlled trials found a trend of

144

increased risk of death in ELBW infants with the administration of aggressive

phototherapy.98,111 In Morris et al’s clinical trial comparing conservative and aggressive

phototherapy in ELBW infants, this study found aggressive phototherapy to significantly

decrease the rate of neurodevelopmental impairment. The authors noted a trend of increased

risk of death among infants between 501 to 750 g who were administered aggressive

phototherapy, however this difference was not significant.98 In 2012, a similar study was

conducted to determine the risk of death and impairment between infants administered

conservative versus aggressive phototherapy. Similar to Morris et al’s results, Tyson et al found

that although aggressive phototherapy significantly decreased neurological impairment, there

was an increased trend in mortality. Again, this result was not statistically significant.111 More

research needs to be conducted to determine the risk of death among infants of ELBW

administered aggressive phototherapy.

Some small clinical studies have reported an association between phototherapy and DNA

damage. For example, a very early study of phototherapy by Gala et al in 1992 reported a slight

increase in the frequency of sister chromatid exchange after the initiation of phototherapy.197

This rise in sister chromatid exchange is an indicator of DNA damage occurring before DNA

replication.213,214 However, among term infants studied, there was no increase in the frequency

of sister chromatid exchange.197 Since the increase of sister chromatid exchange among

preterm infants was not significant, the authors of this study suggested conducting a study with

a larger homogenous population of preterm infants to understand the cytogenetic effects of

phototherapy.197 A subsequent study in 2009 of 83 newborns also reported an increase in sister

chromatid exchange among infants administered both intensive and conventional

phototherapy.213 Other DNA damage associated with phototherapy include the increase of DNA

damage in the lymphocytes of infants 215 and endogenous mononuclear leukocyte DNA

damage.216 The results of these studies suggest a potential association between phototherapy

and DNA damage. Given these reports of a higher risk of DNA damage among infants

administered phototherapy, longitudinal studies of newborns administered phototherapy are

required to ascertain the potential long-term side effects of DNA damage from phototherapy.

145

8.3.2 Long term side-effects of phototherapy

Few studies have been done on the long-term side effects of phototherapy. Among those that

have reported long-term side effects of phototherapy, increased risk of melanocytic nevus

development and malignancy were reported.188,217,218

Long-term DNA changes and/or damage is a controversial and much debated side effect of

phototherapy. The potential risk of changes and/or damage of blue light to DNA has been

hypothesized to potentially lead to skin cancer.189,213,215 One method used to determine the

risk of malignancy from phototherapy has been counting melanocytic nevi as increased

melanocytic nevi has been associated with melanoma. 189,218 Two studies have found an

increase in melanocytic nevi in children who were exposed to phototherapy as newborns. One

study was done in children between 8 to 9 years of age218, and the second study was done in

school aged children 14 to 18 years of age.217 However, reports of the potential risk of

melanoma from increased melanocytic nevi among infants administered phototherapy has

been highly criticized,219 specifically since neither of these studies looked at the link between

increase melanocytic nevi and malignancy.217,218 Conversely, some studies have reported no

association between neonatal phototherapy and increase in melanocytic nevi, suggesting that

additional research is needed in this area with larger sample sizes.220

The potential long-term risk of malignancy because of phototherapy has led to some studies

investigating this association. The association between neonatal phototherapy and subsequent

malignancy in childhood and adulthood has also been highly debated. While some earlier

studies of smaller sample sizes did not find an association between infants administered

phototherapy and malignancy221-223, other later studies of larger sample sizes did find a slightly

increased risk of malignancy among infants administered phototherapy.190,191,224 For example,

in a Canadian study, phototherapy was slightly associated with the late onset of solid tumors.

Children who received neonatal phototherapy between 4-11 years of age were two times more

at risk of having a solid tumor (Hazard ratio 2.26 , 95% CI 1.3-3.81) than children unexposed to

neonatal phototherapy.190 However, this association was reported to be weak and further

146

research was required.190 Comparatively, one study that found an increased risk of malignancy

among children exposed to neonatal phototherapy did not report a significant association after

controlling for confounding factors including bilirubin levels.191 The authors of this study still

suggest the importance of reducing unnecessary phototherapy due to a potential risk of cancer

among infants administered phototherapy. 191 Both of these studies were limited to a cancer

diagnosis in childhood and did not follow these infants to adulthood. Additional longitudinal

studies following newborn infants to adulthood are required to determine the association

between neonatal phototherapy and cancer in adulthood. This review of phototherapy side

effects among newborns raises the question of whether phototherapy is truly without harm. As

the current literature is limited in the number of large-scale studies reporting the long-term

impact of phototherapy, larger prospective and retrospective cohort studies are required.

147

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Rethinking the Assessment of Hyperbilirubinemia in Preterm Infants

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