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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 www.labmedicine.com Downloaded from http://labmed.oxfordjournals.org/ by guest on October 25, 2016 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 www.labmedicine.com 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 Winter 2015 | Volume 46, Number 1 Lab Medicine 61 Downloaded from http://labmed.oxfordjournals.org/ by guest on October 25, 2016 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 www.labmedicine.com Downloaded from http://labmed.oxfordjournals.org/ by guest on October 25, 2016 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. To read this article online, scan the QR code, http://labmed. ascpjournals.org/content/46/1/60. full.pdf+html Downloaded from http://labmed.oxfordjournals.org/ by guest on October 25, 2016 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 References 1. Steinhorn RH. Evaluation and management of the cyanotic neonate. Clin Pediatr Emerg Med. 2008;9:169-175. 2. Sasidharan, P. An approach to diagnosis and management of cyanosis and tachypnea in term infants. Pediatr Clin North Am. 2004;51:999-1021. 3. Fairbanks VF, Klee GG. Biochemical aspects of hematology. In: Burtis CA, Ashwood ER, eds. Tietz Textbook of Clinical Chemistry. 3rd ed. Philadelphia: WB Saunders 1999:1642-710. 4. Darling RC, Roughton FJW. The effect of methemoglobin on the equilibrium between oxygen and hemoglobin. Am J Physiol. 1942;137:56-68. 5. Jaffe E, Hultquist D. Cytochrome b5 reductase deficiency and enzymopenic hereditary methemoglobinemia. In: Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Kinzler KW, Vogelstein B, eds. The Metabolic & Molecular Basis of Inherited Disease. Vol. III. 8th ed. New York City: McGraw Hill Medical Publishing Division. 2001:4555-4570. 6. Skold A, Cosco DL, Klein R. Methemoglobinemia: pathogenesis, diagnosis and management. South Med J. 2011;104:757-761. 7. Haymond S, Cariappa R, Eby CS, Scott MG. Laboratory assessment of oxygenation in methemoglobinemia. Clin Chem. 2005;51:434-44. 8. Greer FR, Shannon M; American Academy of Pediatrics Committee on Nutrition, American Academy of Pediatrics Committee on Environmental Health. Infant methemoglobinemia: the role of dietary nitrate in food and water. Pediatrics. 2005;116:784-786. 9. Da-Silva S, Sajan IS, Underwood J III. Congential methemoglobinemia: a rare cause of cyanosis in the newborn—a case report. Pediatrics. 2003;112:e158-e161. www.labmedicine.com Winter 2015 | Volume 46, Number 1 Lab Medicine 63