TAGEDENS E M I N A R S I N P E R I N A T O L O G Y 45 (2021) 151351

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Seminars in Perinatology
www.seminperinat.com

Neonatal hyperbilirubinemia management: Clinical

assessment of bilirubin production
Lizhong Dua,b,*, Xiaolu Maa,b, Xiaoxia Shena,b, Yinying Baoc, Lihua Chena,b, and
Vinod K. Bhutanid
a
Department of Neonatology, the Children’s Hospital, Zhejiang University School of Medicine, Hangzhou, China
b
National Clinical Research Center for Child Health, China
c
Women’s Hospital, Zhejiang University School of Medicine, China
d
Department of Pediatrics, Division of Neonatal and Developmental Medicine, Stanford University School of Medicine, Stanford, CA, USA

AB STR ACT

The predominant cause of elevated total/plasma bilirubin (TB) levels is from an increase in
bilirubin production primarily because of ongoing hemolysis. If undiagnosed or untreated,
the risk for developing extreme neonatal hyperbilirubinemia and possibly bilirubin-
induced neurological dysfunction (BIND) is increased. Since carbon monoxide (CO) and bili-
rubin are produced in equimolar amounts during the heme catabolic process, measure-
ments of end-tidal CO levels, corrected for ambient CO (ETCOc) can be used as a direct
indicator of ongoing hemolysis. A newly developed point-of-care ETCOc device has been
shown to be a useful for identifying hemolysis-associated hyperbilirubinemia in newborns.
This review summarizes the biology of bilirubin production, the clinical utility of a novel
device to identify neonates undergoing hemolysis, and a brief introduction on the use of
ETCOc measurements in a cohort of neonates in China.
Ó 2020 Elsevier Inc. All rights reserved.

and possibly bilirubin-induced neurologic dysfunction (BIND).4

Introduction
Identification of hyperbilirubinemia in the newborn can be
done noninvasively by visual inspection, using a transcutane-
ous bilirubin (TcB) device or by cell phone-based screening
applications. All have limited reliability in predicting the
degree of hyperbilirubinemia.1 3 The measurement of total
serum/plasma bilirubin (TB) level is the “gold standard” for
identifying an infant at risk for developing extreme neonatal
hyperbilirubinemia (EHB, TB levels > 25 mg/dL [428 mmol/L])
Hyperbilirubinemia depends upon the balance between an
infant’s ability to produce and eliminate bilirubin. Either
excessive bilirubin production and/or impaired hepatic conju-
gation contributes to the severity of the hyperbilirubinemia,
and thus the risk for developing BIND or even kernicterus. Dur-
ing early postnatal life, an infant’s hepatic conjugation for bili-
rubin is very limited, mainly due to a low expression of the
bilirubin-conjugating enzyme, uridine diphosphoglucurono-
syltransferase (UGT1A1), in the newborn liver (about 1% of
adult levels).5 Because all infants have a reduced capacity to

This work was supported by Zhejiang Key Laboratory for Diagnosis and Therapy of Neonatal Diseases, Key Laboratory of Reproductive
Genetics, Ministry of Education, Zhejiang University, and by grants from the National Natural Science Foundation of China (Nos.
81471480, 81630037).
*Corresponding author at: The Children’s Hospital, Zhejiang University School of Medicine, #3333 Binsheng Road, Hangzhou, China.
E-mail address: dulizhong@zju.edu.cn (L. Du).

https://doi.org/10.1016/j.semperi.2020.151351
0146-0005/Ó 2020 Elsevier Inc. All rights reserved.
2 S E M I N A R S I N P E R I N A T O L O G Y
conjugate bilirubin, increased bilirubin production rates, e.g.,
as in cases of hemolysis, primarily drives TB levels. In fact, evi-
dence have shown that hemolysis isa major causes of bilirubin
encephalopathy or kernicterus. In the US Kernicterus Registry,
hemolysis was present in 41.8% of cases.6 In a large Chinese
kernicterus case series, 44% were from infants with ABO, Rh
incompatibilities or glucose-6-phosphate dehydrogenase
(G6PD) deficiency.7 However, in many infants who had TB lev-
els at or above the phototherapy threshold, no identifiable
causes of hemolysis could befound.8 This is partly due to mul-
tiple causes of increased bilirubin production or in most
instances, these infants were not evaluated sufficiently for
possible hemolysis.
The hour-specific TB levels plotted on a nomogram can be
used to screen for infants who may develop hyperbilirubine-
mia by stratification into different risk zones based on age in
hours.9 The rate of rise of TB can also be used to track
increased bilirubin production rates.6 In this review, we focus
on the role and risks of increased bilirubin production.
Regulation of bilirubin production
Process of bilirubin production is shown in Fig. 1. Bilirubin is
derived from the heme degradation process, which is cata-
lyzed by heme oxygenase (HO). The first step of heme catabo-
lism is rate-limiting. It breaks the IX- methane bridge in
heme and releases equimolar amounts of carbon monoxide
(CO), ferrous iron (Fe2+), and biliverdin.10 In the second step,
which occurs in the cytosol, biliverdin reductase rapidly
reduces biliverdin to bilirubin. Based on this unique stoichi-
ometry, CO production can be used as a surrogate measure
for bilirubin production. However, there are other endoge-
nous sources of CO in the body, such as lipid peroxidation
and photo-oxidation which may vary in their proportional
contribution to total CO production.11,12 These sources nor-
mally only contribute only a small amount of CO. Once CO is
formed, it diffuses into circulation and becomes tightly bound
to hemoglobin as carboxyhemoglobin (COHb). Once in the
Fig. 1 – Schematic of bilirubin production.
45 (2021) 151351
lungs, it is continually excreted in breath in exchange for oxy-
gen. Thus, the CO concentration in the end-tidal breath
(ETCO) or blood (as COHb), when corrected for ambient CO
(ETCOc or COHbc, respectively)can be used as indices of bili-
rubin production rates.13,14
Because the circulating blood volume of the newborn is
small and the minute ventilation is relatively high, a short
equilibration time is only required for changes in the endoge-
nous CO production rate to be accurately reflected in COHbc
and ETCOc levels.15,16 Even in a normal term neonate, biliru-
bin production is increased 2- to 3-fold compared with an
adult on a per-body weight basis,17 mainly because of a neo-
nate’s shortened red blood cell (RBC) lifespan.18,19 Premature
infants have an even shorter RBC lifespan, and thus have rel-
atively increased bilirubin production rates compared with
healthy term neonates. Many other factors can affect biliru-
bin production in the neonate, exacerbating hyperbilirubine-
mia and increasing the risk for developing BIND.
The heme catabolic pathway can be regulated by many fac-
tors. The promoter region of the inducible form of HO gene
(HO-1) is complex, and there are many different mechanisms
by which expression can be increased.20 There are two iso-
zymes of the microsomal HO. HO-1 is the isozyme that can be
induced up to 100-fold by a wide variety of stimuli, such as
heme, stress, heavy metals, UV radiation, cytokines, and endo-
toxins.21,22 Elevated heme catabolism observed in neonates
may be associated with an increased transcription of HO.23
Dinucleotide (GT)n repeat lengths in the HO-1 promoter region
can modulate transcription with shorter alleles (
24 GT
repeats) associated with high gene expression rates and should
result in increased heme catabolism. However, clinical studies
have shown inconsistent results. In the steady state, the length
of (GT)n repeats does not appear to modulate heme catabolism
or TB values in either G6PD-normal or -deficient neonates.23 25
One Taiwanese study showed that short HO-1 promoter (GT)n
repeats was a risk factor for neonatal hyperbilirubinemia (rela-
tive risk [RR] = 2.185).26 Another Japanese study showed that
infants with short alleles [< 22 (GT)n repeats] appear to be at a
higher risk for developing hyperbilirubinemia.27
Biliverdin reductase A (BLVRA; OMIM* 10975) efficiently
reduces biliverdin to bilirubin. Both biliverdin and bilirubin
are potent antioxidants and may exert cellular protective
effects against injurious stimuli in vivo and in vitro.28,29 Only
one common non-synonymous BLVRA gene variant (rs
699512: A>G) has been reported and was found not to be asso-
ciated with Asian adult or neonatal TB levels.30,31
Hemolysis and increased bilirubin production
The common conditions associated with neonatal hemolysis
are shown in Table 1. Rh isoimmune hemolytic disease is the
most significant condition of increased bilirubin production.
Kernicterus with Rh disease ranged from 38, 28, 28, and 25
per 100,000 livebirths for Eastern Europe/Central Asian, sub-
Saharan African, South Asian, and Latin American regions,
respectively.32 With the widespread use of anti-D immuno-
globulin prophylaxis, Rh hemolysis is currently seen only in
occasionally in industrialized countries.33 Unfortunately, this
proven solution remains underused, especially in low- and
 TAGEDENS E M I N A R S I N P E R I N A T O L O G Y
Table 1 – Major conditions known to increase hemolysis and bilirubin production in the newborn.
Iso-Immune Rh isoimmunization ABO immunization
Conditions Some rare immune
conditions, anti-M,
Kell incompatibility
Non-Immune Red blood cell (RBC) Glucose-6-phosphate
Conditions enzyme deficiencies dehydrogenase
deficiency
RBC membrane Hereditary Elliptocytosis
abnormalities spherocytosis
Hemoglobinopathies Sickle cell disease Thalassemias
General Conditions Upshaw-Schulman Syn- Hemangiomatosis
drome (ADAMTS13
deficiency)
middle income countries (LMICs). Although the Rh-negative
prevalence is substantially lower in some LMICs, it is esti-
mated that in India, Pakistan, and Nigeria where the majority
of RhD-negative women do not receive post-partum anti-D
prophylaxis, thousands of women will develop anti-D anti-
bodies, and about half the infants born to these women will
develop Rh hemolytic diseaseannually.34
The most frequent cause of hemolytic disease of the new-
born is ABO heterospecificity. This is a relatively mild form of
hemolysis and can be managed by phototherapy in most
cases. However, in cases with inappropriate early discharge
without risk factor assessment for EHB, kernicterus may
occur. In the US Kernicterus Registry, 31 (25%) were from ABO
incompatibility.6 In a Chinese case series of 348 neonatal ker-
nicterus, 104(29.9%) were from ABO hemolysis.7 The direct
antiglobulin test (DAT) is an important tool for identification
of hemolytic disease of the newborn (HDN) caused by eryth-
rocyte immunization. Increasing DAT strength plays a modu-
lating role in the pathophysiology of the hemolysis and
hyperbilirubinemia. DAT++ readings were associated with a
higher incidence of hyperbilirubinemia and higher COHbc
values than their DAT+- or DAT+ counterparts.35 However, a
negative DAT may not be suggestive of HDN. When clinical
suspicion is high, an eluate test should be performed when
the DAT is negative.36 Despite the increased hemolysis docu-
mented in DAT+ ABO-heterospecific newborns, not all
affected neonates will develop severe hyper-
bilirubinemia.37 39 As stated earlier, TB levels are determined
by the balance between bilirubin production and elimination.
The UGT1A1 polymorphism or mutation also contributes to
the final TB levels in ABO-heterospecific newborns.40,41 On
the other hand, an ABO heterospecificity even if the DAT is
positive does not necessarily indicate the presence of ABO
hemolytic disease.42 Other criteria such as indirect hyperbilir-
ubinemia, microspherocytosis as observed on a peripheral
blood smear, and increased reticulocyte count are necessary
to support the diagnosis, especially during the first 24 h of
life. On this situation, a direct test for ongoing hemolysis or
bilirubin production, such as ETCOc or COHbc (see below) will
be extremely valuable.43,44
G6PD deficiency is another common cause of hemolytic dis-
ease. Its incidence varies greatly among different geographic
distribution. G6PD is critical to the redox metabolism of RBCs
45 (2021) 151351 3
Pyruvate kinase Other rare RBC enzyme
deficiency deficiencies
Ovalocytosis Pyknocytosis Stomatocytosis
Other rare
hemoglobinopathies
Sepsis Extravasated blood(e.g.,
cephalohematoma,
ecchymosis, adrenal
hemorrhage, and sub-
dural hemorrhage)
and G6PD deficiency may be associated with acute hemolysis
in newborns following exposure to oxidative stress. It is an
important cause of severe neonatal hyperbilirubinemia and
kernicterus.45,46
The presentations of hyperbilirubinemia in G6PD-deficient
neonates are characterized by an acute hemolytic event, pre-
cipitated by an environmental trigger, such as infection or
naphthalene, with a resultant rapid exponential rise in TB to
potentially hazardous levels that may not always be pre-
ventable.43 46 The role of traditional herb medicine in the
triggering of G6PD hemolysis and hyperbilirubinemia is still
controversial.47
The prevalence of G6PD deficiency has spread widely from
its population origins and geographic distributions. The high-
est G6PD deficiency prevalence rates in the US are in African-
American males (12.2%) and lowest in Caucasian(0% in
female, 0.3 in male).48 In Asian countries like China, some
regions have introduced universal G6PD screening programs.
From the data of 2017, the G6PD screening rates by province
were between 37% and 99%, with the incidence of G6PD in
each province ranging from 0.008% to 3.39%.49 There are
many genetic variations that can affect G6PD phenotypes.50
From newborn screening data and further analyses by quan-
titatively enzymatic assays and G6PD mutation analyses, the
overall incidence of G6PD deficiency was 7.3% in Guangxi
province.51Another mode of hyperbilirubinemia in G6PD-defi-
cient neonates is associated with low-grade hemolysis and
genetic polymorphisms of the UGT1A1 gene that reduce
UGT1A1 expression and thereby limiting hepatic bilirubin
conjugation.52 Collectively, variations in theG6PD gene, the
triggering factors, and hepatic UGT1A1 polymorphisms all
contribute to the observed increase in bilirubin production
and alterations in elimination, to result in hyperbilirubinemia
in newborns.
General conditions that cause increased RBC breakdown
(and consequently increase heme levels) also can increase
bilirubin production rates. The most common cause is extrav-
asated blood such as in cephalhematomas and bruises.53
There are also other conditions that increase RBC breakdown
and decrease RBC lifespan such as hemangiomatosis and
Upshaw-Schulman syndrome (USS). USS, a synonym for con-
genital thrombotic thrombocytopenic purpura (TTP), is a rare
hereditary deficiency in the activity of von Willebrand factor
4 S E M I N A R S I N P E R I N A T O L O G Y
(VWF)-cleaving protease orADAMTS13. The characteristics of
neonatal onset of USS are severe jaundice, which may require
exchange blood transfusion, and repeat episodes of thrombo-
cytopenia. USS is also reported to complicated severe pulmo-
nary hypertension of the newborn.54 TTP is diagnosed by the
deficiency of ADAMTS13 activity (< 10% of normal) due to
mutations in the ADAMTS13 gene or autoantibodies against
ADAMTS13. There is also reported case with kernicterus in
other Asian literature.55
HO inhibitors and bilirubin production
For neonates with increased TB levels that meet the photo-
therapy threshold, phototherapy has been the mainstay of
treatment. New phototherapy devices incorporate narrow
band light-emitting diodes (LEDs), which are low heat produc-
ing, highly efficient, and inexpensive.56,57 And timely inter-
mittent instead of continuous phototherapy used prudently
can deliver less intensive irradiance levels to very low birth-
weight infants and is a strategy that may decrease any possi-
ble side effects of phototherapy.58 However, the use of
phototherapy for neonates is not totally harmless. A study
conducted in extremely low birthweight infants to assess
aggressive versus conservative phototherapy.59 The results
showed that the rate neurodevelopmental impairment alone
was significantly reduced with aggressive phototherapy.
However, among the subgroup infants weighing 501 to 750 g
at birth, an increased in mortality was found. Other side
effects including erythematous, melanocytic nevi, interfer-
ence with the circadian rhythm, DNA damage, increased sei-
zure episode in childhood, and others.60 64
As such, the rationale for inhibiting bilirubin production
may be an attractive alternative strategy to phototherapy.65
The infants who could potentially benefit from such an
approach are those who have very high TB levels and do not
adequately respond to phototherapy, require exchange trans-
fusions, or whose family refuse the administration of blood
products, such as those among the Jehovah’s Witness com-
munity.66 A study registry of compassionate use of an HO
inhibitor, tin mesoporphyrin (SnMP), was listed in Clinical-
Trials.gov(NCT00076960) in 2004, but failed to meet validation
by the US Food and Drug Administration (FDA).
HO is the key rate-limiting enzyme in the catabolism of
heme to bilirubin, and therefore its inhibition can lead to a
direct reduction of bilirubin. Metalloporphyrins are potent
competitive inhibitors of HO.67,68 Earlier clinical studies and
trials in Europe and Argentina have documented the efficacy
of using SnMP for severe neonatal hyperbilirubinemia.
Recently, a masked, SnMP (4.5 mg/kg), placebo-controlled,
multicenter trial of single intramuscular injection to 213 new-
borns  35 wks’ gestational age, median postnatal age 30 h,
whose predischarge screening TcB was  75th percentile was
conducted. Infants were randomized to receiving treatment
with SnMP or ‘sham’. The authors found that the duration of
phototherapy was halved. Within 12 h of SnMP administra-
tion, the natural TB trajectory was reversed. At age 3 5 days,
TB in the SnMP-treated group was 6-fold lower than the
sham-treated group (P < 0.001). At age 7 10 days, mean TB
declined 18% (P < 0.001) compared with a 7.1% increase
45 (2021) 151351
among controls. No short-term adverse events from SnMP
treatment were noted other than few cases of photoreactivity
due to inadvertent exposure to white light phototherapy.69
SnMP (or stannsoporfin) use for treating neonatal hyperbilir-
ubinemia was tested in the US and the FDA advised that more
evidence was needed to support its effectiveness [https://
www.mdmag.com/medical-news/fda-advisory-committee-
not-in-support-of-stannsoporfin-for-neonatal-jaundice].
Noninvasive assessment of bilirubin production
Since CO and bilirubin are produced in equimolar amounts in
the heme catabolic process, measurements of ETCOc o
rCOHbc can be used as a quantitative means for assessing
ongoing hemolysis.15,70 COHbc levels can be determined by
gas chromatography (GC) or by spectrophotometry.70,71 Meas-
urements of COHb using specialized spectrophotometers (co-
oximetry) are accepted as the standard diagnostic procedure
in diagnosis of inhalation injury and CO intoxications.72 It
has also been used to identify infants undergoing hemoly-
sis.73,74 A study by Mahoney et al compared spectrophoto-
metric results with those obtained with the reference GC
method. They found that the direction of the bias relative to
GC was dependent on COHbc concentration. In general, the
co-oximeters underestimated COHbc when levels > 2.5%, but
overestimated at
2.5%. Because of the inaccuracy of co-oxi-
meters at low COHbc concentration (
2.5%), these labora-
tory-based instruments may not be suitable for some
neonatal applications.75
A point-of-care ETCOc device, CO-StatÒ (Natus Medical, Inc.
San Carlos, CA),was developed about 20 years ago. In 2004,
the American Academy of Pediatrics (AAP) guideline pro-
posed that ETCOc levels could be used to confirm the pres-
ence or absence of hemolysis.4
Recently, a newly-developed ETCOc device, CoSenseÒ (Cap-
nia, Redwood City, CA), is currently marketed in the US and
has been recently cleared for marketing in China.42,76,77 This
is an improved point-of-care analyzer in that it is battery-
powered and easily portable, no need for on-site calibration,
avoids error due to CO sensor drift, which may have occurred
with the CO-StatÒ , and reportedly requires less technical sup-
port. We evaluated the ability of the CoSenseTM device to
identify hemolysis in 51 neonates with severe hyperbilirubi-
nemia, whose TB levels met the AAP guideline for initiating
phototherapy, and having a positive DAT. We found that
ETCOc can identify severe hyperbilirubinemia in neonates
with or without isoimmune hemolysis (Table 2).
Table 2 – ETCOc and other work-up strategy for hemoly-
sis in neonatal with severe hyperbilirubinemia.
DAT+ (n = 27) DAT (n = 24) P-Value
Hematocrit (%) 47.8 § 8.7 56.5 § 9.5 0.43
Reticulocyte % 5.7 § 4.1 2.7 § 1.4 0.01
TB (mmol/L) 321 § 94 320 § 79 0.55
ETCOc (ppm) 2.9 § 2.2 1.7 § 1.0 0.03
TB, total serum/plasms bilirubin; DAT, direct anti-globulin test.
 TAGEDENS E M I N A R S
Fig. 2 – Normal trend of early postnatal ETCOc levels in Chinese neonates (n = 750 tests;dash lines show percentiles).
In the normal early postnatal life, RBC breakdown and bili-
rubin production is significantly increased. Therefore, the
rate of rise in ETCOc may be more appropriate indicator of
the severity of hemolysis, and the effectiveness of therapeu-
tics.78 We are currently establishing an ETCOc nomogram in
Chinese neonates in order to identify those at high risk for
developing neonatal hyperbilirubinemia. Our preliminary
data show that in the early postnatal period, there is a nor-
mal increase in ETCOc and a downward trend by age of
5 days (Fig. 2).
The present data from the literature support the com-
bined use of TB/TcB percentile risk assessments and
ETCOc measurements to identify infants with hemolytic
hyperbilirubinemia. 76,77 Nevertheless, many other factors
such as genetic and immunological abnormalities in bili-
rubin metabolism pathways need to be considered. ETCOc
and COHbc measurements have also been used to identify
some non-hemolytic conditions such as bronchopulmo-
nary dysplasia (BPD) and sepsis where inflammatory
mediators can lead to consequent increases in CO produc-
tion. 79,80 The present ETCOc device can detect CO with a
sensitivity of 1 ppm and ECTOc levels can serve as a bio-
marker for inflammation.
Conclusions
Hemolysis contributes significantly to the development of
EHB and subsequent BIND. While routine hemolysis work-
ups are not accurate means of identifying all actively hemo-
lyzing neonates, heme catabolic pathway and its byproducts
can be used as therapeutic or monitoring targets. Since CO
and bilirubin are produced in equimolar amounts in the
heme catabolic process, measurements of ETCOc offer a
quantitative means for assessing hemolysis.
I N P E R I N A T O L O G Y 45 (2021) 151351 5
Declaration of Competing Interest
The authors declare no conflicts of interest.
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