Download A 5-Hour-Old Male Neonate With Cyanosis

Case Studies
A 5-Hour-Old Male Neonate With Cyanosis
Melkon DomBourian, MD,1 Alok Ezhuthachan, MD,2 Amitai Kohn, MD,2
Brian Berman, MD,2 Elizabeth Sykes, MD1*
Lab Med Winter 2015;46:13-63
DOI: 10.1309/LMBQIZ5DRS4K9IMD
ABSTRACT
Clinical History
Nursing staff noted that the skin of a 5-hour-old male
Caucasian neonate born at term via forceps-assisted
vaginal delivery was dusky-blue in color. The consulting
neonatology service discovered that the patient had
perioral/central cyanosis but was breathing comfortably;
his cardiopulmonary examination results were normal. The
medical records for this patient reported that he had initial
physical examination results that pronounced him to be
Abbreviations
MetHb, methemoglobin; APGAR, appearance, pulse, grimace, activity,
respiration; ABG, arterial blood gas; pCO2, carbon dioxide pressure;
pO2, oxygen pressure; FO2Hb, fractional oxyhemoglobin; ODC, oxygen
dissociation curve; cb5R, cytochrome b5 reductase; cb5, cytochrome
b5; NAD, nicotinamide adenine dinucleotide; NADH, NAD + hydrogen;
NADPH, nicotinamide adenine dinucleotide phosphate-oxidase; HbM,
hemoglobin M disease; O2Hb, oxyhemoglobin; HHb, deoxyhemoglobin;
SHb, sulfhemoglobin; COHb, carboxyhemoglobin
Departments of 1Clinical Pathology and 2Pediatrics, William Beaumont
Hospital and Beaumont Children’s Hospital, Oakland University William
Beaumont School of Medicine, Royal Oak, Michigan
*To whom correspondence should be addressed.
[email protected]
60 Lab Medicine Winter 2015 | Volume 46, Number 1
Keywords: clinical chemistry, congenital methemoglobinemia,
cytochrome b5 reductase, pulse oximetry, CO-oximeter, arterial blood
gas
healthy. His APGAR (appearance, pulse, grimace, activity,
respiration) scores were 8 and 9 at 1 and 5 minutes,
respectively. His mother had a history of diet-controlled
gestational diabetes mellitus and was being treated for
Hashimoto thyroiditis; otherwise, the maternal prenatal
history and laboratory profile were within healthy limits.
Hemoglobin oxygen saturations (SatO2) as determined
via pulse oximetry remained between 92% and 97%
on room air with no discrepancy between upper and
lower extremity values. An arterial blood gas (ABG)
specimen was obtained from the patient and he was
transferred to the neonatal intensive care unit. Blood
cultures were obtained and the patient was treated with
broad-spectrum antibiotics. A chest x-ray showed no
acute cardiopulmonary process, and an echocardiogram
demonstrated normal cardiac anatomy and function. A
complete blood count disclosed a hemoglobin level of 19.1
g per dL (14.6-22.7 g/dL); the white cell count, differential,
and platelet count were within healthy limits for a neonate.
The ABG results of the patient, as measured by an
integrated ABG analyzer/CO-oximeter, were as follows: pH
7.40 (7.27 to 7.47), pCO2 37 mm Hg (27 to 40 mm Hg), pO2
58 mm Hg (54 to 95 mm Hg), fractional oxyhemoglobin
(FO2Hb) 71% (95% to 98%), fractional carboxyhemoglobin
2.4% (<1.5%), fractional MetHb 22.9% (0 to 2.0%), HCO3
22 mmol per L (16 to 23 mmol/L), and TCO2 23 mmol/L
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Methemoglobin (MetHb) is a form of hemoglobin in which heme iron
is oxidized and unable to bind oxygen; its normal basal production is
counteracted by an efficient MetHb-reduction pathway. The causes of
methemoglobinemia are classified as congenital or acquired. Shortly
after his birth, the 5-hour-old male Caucasian neonate, whose case
we present herein, developed central cyanosis that was unresponsive
to supplemental oxygen. Oxygen saturation as determined via pulse
oximetry was normal. In contrast, blood gas testing by multiwave
CO-oximetry indicated decreased fractional oxyhemoglobin and an
elevated MetHb fraction. The patient was subsequently diagnosed
with a congenital cytochrome b5 reductase deficiency. This case
emphasizes causes of methemoglobinemia and differences among
analytical methods used to measure oxygen status when MetHb is
present.
Discussion
MetHb represents hemoglobin in which ferrous heme iron
(Fe2+) has been oxidized to the ferric state (Fe3+), and is
unable to bind oxygen.3 In the presence of MetHb, any
remaining ferrous iron in the hemoglobin tetramer will
bind oxygen with greater affinity,4 resulting in a markedly
left-shifted oxygen dissociation curve (ODC).3 MetHb
formation occurs spontaneously in the erythrocyte due
to superoxide radical formation as oxygen dissociates
from heme iron.5 This basal production is counteracted
by a highly efficient MetHb reduction pathway involving
cytochrome b5 reductase (cb5R), also known as
MetHb reductase, a 32,000-da flavoenzyme, along with
cytochrome b5 (cb5), a 12,000-da electron-transport
protein.5 Electrons from reduced nicotinamide adenine
dinucleotide (NADH) produced during glycolysis are
transferred to cb5R, then to cb5, and finally to MetHb,
resulting in normal hemoglobin production (Figure 1).
Treatment of methemoglobinemia with reducing agents
(eg, methylene blue and ascorbic acid) uses the minor
nicotinamide adenine dinucleotide phosphate-oxidase
(NADPH oxidase)–dependent pathway for MetHb
reduction (Figure 1).6 It should be noted that methylene
blue use involves some risk because at high doses (>7
mg/kg), this substance is an oxidant and can lead to a
paradoxical rise in MetHb fraction of as high as 10%.6,7
Methemoglobinemia is classified as acquired or
congenital. Acquired forms are more common, affect
those with an intact MetHb reduction pathway, and
involve exposure to oxidative agents (eg, dapsone and
benzocaine).6 Risks for acquired methemoglobinemia
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NADH
(from Glycolysis)
NAD+
e–
Cytochrome b5
reductase
e–
Cytochrome b5
e–
Methemoglobin
(Fe3+)
e–
Hemoglobin
(Fe2+)
Methylene
blue
e–
NADPH
Methemoglobin
reductase
NADP+
e–
NADPH
(from hexose
monophosphate
shunt)
Figure 1
Summary of electron transfer in methemoglobin reduction
­pathways.
in the infant age group include exposure to oxidants
through teething gels7 or food or formula prepared with
nitrate-contaminated well water (>10 mg/L).8 Congenital
methemoglobinemia is usually caused by autosomal
recessive inheritance of a deficient cb5R gene. The
cb5R gene produces multiple isoenzymes that are
found in erythroid and nonerythroid cells, accounting
for the creation of clinical-biochemical subtypes of
enzymopenic hereditary methemoglobinemia. 5 Of 4
subtypes that have been described, 5,9 type I disease,
with loss of only erythrocyte cb5R, is most common.
Patients with this mutation often present with cyanosis
from birth but are generally asymptomatic and have a
normal life expectancy. 5 In these patients, if treatment
with reducing agents is undertaken it is often for
cosmetic purposes. 5,9
The more severe type II disease represents a deficiency
of erythroid and nonerythroid cb5R.9 Beyond cyanosis,
infants with this disease experience progressive
neurologic dysfunction, and most die in the first year
of life.5,9 The mechanism for neurological decline is
poorly understood but may include the involvement of
nonerythroid cb5R in desaturation of fatty acids.5 Type
III disease is no longer considered a distinct entity,9 and
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Neonatal cyanosis may be peripheral or central in nature.
Peripheral cyanosis involves the distal extremities and
often occurs due to sluggish blood flow in peripheral
capillaries, leading to increased oxygen extraction.1
By contrast, central cyanosis involves the entire body
and can occur due to various mechanisms, including
hypoventilation, ventilation-perfusion mismatch, rightto-left cardiac shunt, alveolar diffusion impairment, or
inadequate oxygen transport by an abnormal hemoglobin
such as MetHb.1,2
Minor Pathway
(Used in Treatment)
(17 to 24 mmol/L). Despite supplemental oxygenation,
the neonate continued to display cyanosis; his healthcare
team suspected some form of methemoglobinemia.1,2
Major Physiologic
Pathway
Case Studies
Case Studies
type IV disease (erythrocyte cb5 deficiency) has only been
reported thoroughly in 1 individial.5
Patients who are homozygous for the congenital defects
described herein are especially susceptible to acquired
methemoglobinemia.8,9 However, even patients with a
heterozygous deficiency of cb5R have a lower threshold
for acquired methemoglobinemia compared with those
with no level of deficiency.9 Also, healthy neonates are at
higher risk of acquired methemoglobinemia during the first
2 to 3 months of life because they possess 60% of healthy
adult cb5R levels.9
Laboratory Role in Diagnosis
Blood containing a MetHb fraction of 10% or greater has a
noticeable chocolate-brown appearance; this color may be
the initial clue to methemoglobinemia.3,5 Analytical methods
used to measure oxygen may not be equivalent in patients
with methemoglobinemia.7 Oxyhemoglobin (O2Hb) and
deoxyhemoglobin (HHb) each absorb light at 660 nm and
940 nm, but at varying degrees. Standard pulse oximetry
measures absorbance at both of these wavelengths and
determines a 660-nm to 940-nm absorbance ratio. This
ratio corresponds to oxygen saturation from an on-device
calibration curve and is used to calculate SatO2 as defined
in Table 1.7 The presence of MetHb poses a problem
because it also absorbs light at 660 nm and 940 nm,
thereby contributing to the measured absorbance of O2Hb
and HHb via pulse oximetry.6,7 As in our patient, this will
result in a clinically misleading calculated SatO2.
Eventually, as the MetHb fraction reaches 30% or greater,
the measured absorbance ratio will be 1.0. This value
corresponds to a SatO2, determined via pulse oximetry, of
approximately 85%.6,7
62 Lab Medicine Winter 2015 | Volume 46, Number 1
Hemoglobin oxygen saturation (SatO2) =
Fractional oxyhemoglobin (FO2Hb) =
O2Hba
O2Hb + HHbb
O2Hb x 100%
tHbc
O2Hb = oxyhemoglobin
HHb = deoxyhemoglobin
tHb = total hemoglobin
a
b
c
In contrast to pulse oximetry, modern CO-oximeters
measure absorbance at multiple wavelengths. Therefore,
CO-oximeters can estimate additional hemoglobin
fractions, including MetHb, sulfhemoglobin (SHb), and
carboxyhemoglobin (COHb), providing a total hemoglobin
value that is used to calculate FO2Hb (Table 1). COoximetry is the preferred method to assess oxygen status
in the presence of MetHb because the calculated FO2Hb is
a more accurate reflection of the oxygen-carrying capacity
of the blood, compared with SatO2 in this setting.6,7
However, there are 2 important caveats to consider
when using CO-oximeters to assess patients with
methemoglobinemia. First, HbM variants create a problem
in the assessment of MetHb concentration via COoximetry because they lack the characteristic absorbance
peak of MetHb at 630 to 635 nm. Instead, HbM variants
produce a peak at 600 nm that is poorly resolved from
O2Hb and COHb, as well as a peak at 610 to 620 nm that
contributes to the SHb fraction.7 Due to these variations
in absorption spectra, HbM variants generally require
hemoglobinopathy studies for their evaluation.7,9 Second,
CO-oximetry cannot distinguish MetHb from methylene
blue, so monitoring patients after treatment via COoximetry leads to misleading measured MetHb fractions.
In these instances, healthcare professionals can use a
method developed by Evelyn and Malloy.11
ABG analyzers assume a normal ODC and use PO2
with pH to calculate an “estimated” SatO2; this result is
largely unaffected by MetHb.7 When the estimated SatO2
level is compared to SatO2 level determined via pulse
oximetry, an abnormal saturation gap of more than 5%
may be observed, which suggests methemoglobinemia
or another dyshemoglobinemia.6 Our laboratory does not
report an estimated SatO2 level, so we did not observe
this abnormality in our case. Last, one can measure Cb5R
activity spectrophotometrically by following NADH oxidation
at 340 nm after reduction of ferricyanide or other substrates.7
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Aside from enzyme deficiencies, hemoglobin M disease
(HbM) (autosomal dominant inheritance) is another type
of congenital methemoglobinemia that results from
mutations in the globin gene.7 Most HbM cases occur
due to replacement of histidine by tyrosine in the heme
pocket region, resulting in a ferric iron-phenolate complex
that resists reduction.7,9 Because this defect is structural
in nature, using the minor NADPH-dependent pathway
via methylene blue treatment is ineffective. However, no
treatment is usually required because patients with HbM
generally experience a benign disease course.10
Table 1. Hemoglobin Oxygen Saturation and
Fractional Oxyhemoglobin Defined
Case Studies
Patient Follow-Up
11. Evelyn K, Malloy H. Microdetermination of oxyhemoglobin,
methemoglobin, and sulfhemoglobin in a single sample of blood. J
Biol Chem. 1938;126:655-662.
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No evidence of oxidant exposure was discovered in the
patient or his mother, which suggested a congenital
methemoglobinemia. The clinical team contacted our
laboratory for consultation and sent us a specimen for
a comprehensive methemoglobinemia evaluation; blood
culture results in the interim were negative. The results
obtained, after referral to an outside laboratory, confirmed
that the patient harbors a congenital methemoglobinemia,
with MetHb of 18.2% (≥1 year: 0 to 1.5%), SHb 0% (≥1
year: 0 to 0.4 %), cb5R 1.1 IU/g Hb (≥1 year: 8.2 to 19.2
IU/g Hb). HbM testing was negative.
10.Weatherall DJ, Clegg JB, Higgs DR, Wood WG. The
hemoglobinopathies. In: Scriver CR, Beaudet AL, Sly WS, Valle D,
Childs B, Vogelstein B, eds. The Metabolic & Molecular Basis of
Inherited Disease. Vol. III. 8th Ed. New York City: McGraw Hill Medical
Publishing Division. 2001;4571-4636.
Knowledge of a cb5R deficiency should result in
avoidance of oxidant exposures that place our patient
at risk for potentially life-threatening complications. In
follow-up evaluation at 7 weeks of age, the patient was
thriving, with mild compensatory polycythemia and stable
perioral cyanosis, consistent with the diagnosis of type 1
enzymopenic hereditary methemoglobinemia. LM
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