Understanding severe hyperbilirubinemia and preventing kernicterus: Adjuncts in the interpretation of neonatal serum bilirubin

Clinica Chimica Acta 356 (2005) 9 – 21 www.elsevier.com/locate/clinchim

Review

Understanding severe hyperbilirubinemia and preventing kernicterus: Adjuncts in the interpretation of neonatal serum bilirubin Michael Kaplana,b,T, Cathy Hammermana,c a

Department of Neonatology, Shaare Zedek Medical Center, P.O. Box 3235, Jerusalem 91031, Israel b Faculty of Medicine of the Hebrew University, Jerusalem, Israel c Faculty of Health Sciences, Ben Gurion University of the Negev, Be’er Sheva, Israel Received 29 October 2004; received in revised form 11 January 2005; accepted 13 January 2005

Abstract The serum total bilirubin concentration at any point in time represents the amount of bilirubin being produced minus that being excreted. Hyperbilirubinemia develops when bilirubin production exceeds the body’s capacity to excrete it, primarily by conjugation. When extreme, hyperbilirubinemia may lead to the development of free bilirubin, that form of bilirubin which may cross the blood–brain barrier and enter and damage the basal nuclei of the brain. This rare, though devastating complication, may result in irreversible bilirubin induced brain damage termed kernicterus. In this paper, adjuncts to the interpretation of the serum total bilirubin are discussed, with the purpose of singling out those few neonates in real danger of bilirubin encephalopathy. Interpretation of the serum total bilirubin should be performed in conjunction with factors unique to the particular infant being evaluated. Understanding the mechanisms and dangers of severe neonatal hyperbilirubinemia should facilitate recognition of an emergency situation and optimize the speed with which bilirubin testing is performed and blood for exchange transfusion prepared. Hyperbilirubinemia is a condition of major importance and a source of concern to all involved in the management of the newborn. Its prevention and management should be based on the recently revised American Academy of Pediatric guidelines, with special attention paid to neonates manifesting risk factors for kernicterus. Close cooperation between the clinical laboratory and the medical team managing the newborn is an essential component in the management of a hyperbilirubinemic baby. D 2005 Elsevier B.V. All rights reserved. Keywords: Serum total bilirubin; Hyperbilirubinemia; Kernicterus; Bilirubin encephalopathy; Hemolysis; Bilirubin conjugation; Newborn

Abbreviations: AAP, American Academy of Pediatrics; ABR, automated brainstem response; BIND, bilirubin induced neurologic dysfunction; CO, carbon monoxide; COHbc, carboxyhemoglobin corrected for inspired carbon monoxide; DAT, direct antiglobulin test; ETCOc, end-tidal carbon monoxide corrected for inspired carbon monoxide; G-6-PD, glucose-6-phosphate dehydrogenase; RBC, red blood cell; STB, serum total bilirubin; TcB, transcutaneous bilirubinometry. T Corresponding author. Department of Neonatology, Shaare Zedek Medical Center, P.O. Box 3235, Jerusalem 91031, Israel. Tel.: +972 2 655 5643; fax: +972 2 652 0689. E-mail address: [email protected] (M. Kaplan). 0009-8981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2005.01.008

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Contents 1. 2. 3.

Introduction: kernicterus in term and near term infants . . . . . . . . . . . . . . . . . . . . . . . bThe specter walks againQ [8] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical features of kernicterus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Chronic bilirubin encephalopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Acute bilirubin encephalopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. What does the serum total bilirubin tell us?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. The concept of equilibrium between production and elimination of bilirubin . . . . . . . . 4.2. What is hyperbilirubinemia, and when do serum total bilirubin values become dangerous? 4.3. Assessment of jaundice according to the hour-specific bilirubin nomogram. . . . . . . . . 4.4. Frequency of exceptionally high STB concentrations . . . . . . . . . . . . . . . . . . . . 4.5. Duration of hyperbilirubinemia and the role of hemolysis. . . . . . . . . . . . . . . . . . 4.6. Rate of rise of STB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7. Serum albumin and free bilirubin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Endogenous carbon monoxide production and the assessment of hemolysis . . . . . . . . . . . . 6. Blood group incompatibility and the direct Coombs’ test . . . . . . . . . . . . . . . . . . . . . . 6.1. ABO blood group incompatibility with a negative direct Coombs’ test . . . . . . . . . . . 7. Transcutaneous bilirubin measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Prevention and treatment of hyperbilirubinemia. . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction: kernicterus in term and near term infants About 60% of healthy, term neonates will develop recognizable jaundice, in which case the serum total bilirubin (STB) concentration can be expected to be at least 5 to 7 mg/dL [1]. In the majority of cases, the STB will not exceed the physiological range but may occasionally rise to very high levels and pose a threat to the neonate by entering the central nervous system tissues, thereby causing kernicterus, or bilirubin encephalopathy (usually irreversible neurologic damage). The reason for the demand for many STB determinations is to detect neonatal jaundice so as to institute treatment prior to the STB entering the danger zone. Kernicterus was a not uncommon occurrence prior to the 1970s, at which time the most frequent etiologic factor was Rh isoimmunization. Modern therapeutic techniques including exchange transfusion, phototherapy, and the use of intravenous immune globulin therapy were instrumental in almost eliminating this condition. For many years after maternal anti-D antibody (Rhogam) administration was instrumental in practically abolishing Rh disease, based on

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experience gained during that era, physicians continued to prevent the serum total bilirubin (STB) value from exceeding 20 mg/dL in term and near term neonates. As a result, during the two decades preceding 1990 there were virtually no published reports of kernicterus in healthy, full-term infants from industrialized countries [2–7].

2. bThe specter walks againQ [8] Since the 1990s, there has been some evidence of a resurgence of kernicterus. Reports have emanated from the United States and Canada [9–13], as well as Europe [14,15], Africa [16], the Middle and Far East [17,18], and New Zealand [19]. Many cases in North America have been collected in an informal Kernicterus Registry, from which 125 cases collected during this period were recently reported [20,21]. One reason leading to this change in the epidemiology of kernicterus includes the (subsequently disproved [22]) notion that kernicterus will not occur in healthy term infants in the absence of hemolysis, resulting in adoption of a less aggressive approach to such neonates [6]. In addition, newborns are being discharged earlier from hospital,

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before clinically apparent jaundice becomes manifest, and increased numbers of infants are being breast fed [23,24]. Borderline prematurity (35 to 37 weeks of gestation) is frequently ignored as an additional risk factor [25]. Public attention has recently been drawn to the changing situation by The Joint Commission for Accreditation of Hospital Organizations [26], the Centers for Disease Control [27], the National Quality Forum (Agency for Healthcare Regulation of Quality) [28] and the American Academy of Pediatrics (AAP) [25]. These bodies have emphasized that most cases of kernicterus should be preventable [25]. The AAP emphasizes paying attention to risk factors, including borderline prematurity, breast-feeding, and jaundice manifesting within the first 24 post-natal hours, and has recommended follow-up within 2 to 3 days of discharge to all neonates discharged b48 h, and those born b38 weeks of gestation [29,30]. It is not our intention in this paper to comprehensively review all aspects of bilirubin physiology, hyperbilirubinemia and kernicterus. The interested reader is referred to many recent overviews or commentaries on the subject [3,31–38]. Rather, we will highlight selected recent advances in our understanding of some biochemical aspects of severe neonatal hyperbilirubinemia so as to facilitate interpretation of STB values and assist in understanding the pathophysiology of the condition.

3. Clinical features of kernicterus Considering the rarity of the condition, it is inevitable that most clinicians will have not have encountered a neonate with classic features of kernicterus. A short clinical description of this condition follows. 3.1. Nomenclature The term bkernicterusQ originally referred to a pathologic diagnosis typified by staining of the brainstem nuclei and cerebellum with bilirubin in infants or children who had manifested signs of acute or chronic bbilirubin encephalopathyQ. However, the terms bkernicterusQ and bbilirubin encephalopathyQ have come to be used interchangeably. The Subcom-

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mittee on Hyperbilirubinemia of the AAP has suggested that the term bacute bilirubin encephalopathyQ be reserved to describe acute manifestations of bilirubin toxicity, while the term bkernicterusQ be used to depict chronic and permanent neurologic sequelae of bilirubin toxicity [30]. Another term in use is bbilirubin induced neurologic dysfunctionQ (BIND). 3.2. Chronic bilirubin encephalopathy The classical picture of chronic bilirubin encephalopathy encompasses a clinical tetrad comprising (1) extrapyramidal abnormalities with athetoid cerebral palsy and spasticity, (2) deafness or diminished hearing (auditory neuropathy), (3) impairment of upward gaze, and (4) dental emamel dysplasia [31,37,39,40]. Pathologically, these features correspond to lesions in the globus pallidus and subthalamic nucleus, auditory and oculomotor brainstem nuclei, respectively, while the cerebellum and hippocampus may also be affected. Although some children may have mild mental retardation and subtle cognitive disturbances, in many cases the intellect is normal. However, their aptitude may frequently not be apparent, as the severe, characteristic movement disorders result in the children being trapped in their own, unresponsive, uncontrollably writhing, bodies making speaking, ambulation, communication or even utilization of a computer keyboard close to impossible. Visual and auditory problems may exacerbate the situation. Other children may manifest a less severe spectrum of disease, including cognitive disturbances, mild neurologic abnormalities, isolated hearing loss and auditory neuropathy [41–43]. The latter condition is not simply sensorineural hearing loss, but rather is the result of dysfunction at the level of the auditory brainstem or nerve. These children may have normal inner ear function, and their auditory neuropathy may be missed if hearing testing is based on emission hearing screening alone [39]. 3.3. Acute bilirubin encephalopathy The acute stages of bilirubin encephalopathy may be subtle and elude the pediatrician’s identification [39]. Phase 1 includes poor sucking, lethargy, stupor, hypotonia and seizures in an obviously icteric neo-

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nate. During phase 2, starting in the middle of the first week, episodes of hypertonia including spasm of the extensor muscles, back and neck arching, opisthotonus and retrocollis may alternate with hypotonia. Fever and high-pitched cry frequently occur and limitation of upward gaze (setting sun sign) may be noted. The characteristic feature of phase 3, beginning after the first week, is hypertonia. The acute stages of bilirubin encephalopathy may be accompanied by abnormal or absent auditory evoked responses and characteristic MRI findings including hyperintense signals in the globus pallidus [15,39]. Recognition of the signs of acute bilirubin encephalopathy is vital, as prompt treatment may be successful in reversing the situation [43–45]. Shapiro [38] has emphasized that bthe faster and the more aggressive the treatment, the more reversible and better the outcome.Q

4. What does the serum total bilirubin tell us? The keystone to assessment of neonatal hyperbilirubinemia is the measurement of STB. These concentrations are used, frequently in conjunction with additional factors, to decide whether an infant is in danger of bilirubin encephalopathy, and whether treatment should be instituted, the kind of therapy, and the rapidity and intensity with which it should be administered. Knowledge of some of the factors discussed below may alert the laboratory staff to the danger of a situation and prompt them to warn the clinician of the potential jeopardy to his patient. 4.1. The concept of equilibrium between production and elimination of bilirubin Of paramount importance is the concept that the STB at any point in time represents the amount of bilirubin being produced, minus the amount of bilirubin being eliminated from the body, primarily by conjugation [47]. In most cases equilibrium between these processes should prevent the STB from exceeding the physiologic range. Should bilirubin production exceed the body’s conjugative ability, hyperbilirubinemia may ensue. Thus, moderately increased heme catabolism in the face of more severely diminished bilirubin conjugation may be

sufficient to upset the equilibrium. Some conditions associated with diminished bilirubin conjugation include prematurity [48], homozygosity for (TA)7 promoter polymorphism for the gene encoding the bilirubin conjugating enzyme, UDP-glucuronosyltransferase 1A1, associated with Gilbert’s syndrome [49,50], or the Crigler–Najjar syndromes. On the other hand, a baby with severe hemolysis may not necessarily become hyperbilirubinemic, in which cases it is likely that the specific neonate has an efficient bilirubin conjugative capacity. Kaplan et al. [51,52] have illustrated this concept mathematically, using an index comprised of blood carboxyhemoglobin corrected for ambient carbon monoxide (COHbc) (an accurate reflection of heme catabolism) divided by the serum total conjugated bilirubin, reflective of bilirubin conjugation. For the most part, values for the index correlated with STB concentrations. Highest values for the index were encountered in those homozygous for the above-mentioned (TA)7 promoter polymorphism: these neonates had both the highest carboxyhemoglobin values and lowest total conjugated bilirubin values [52]. 4.2. What is hyperbilirubinemia, and when do serum total bilirubin values become dangerous? Several approaches exist which combine STB concentrations and additional risk factors in assisting the physician in deciding when STB concentrations may potentially endanger a baby. For many years data derived from the National Collaborative Study, conducted on 30,000 newborns between 1955 and 1961, were used for this purpose [53]. In that study, infants had serum bilirubin values measured at 48 h with subsequent determinations performed, according to a preplanned protocol, until values decreased. The 95th percentile in this study was identified as a STB value of 12.9 mg/dL, and for many years this was regarded as the upper limit of physiological jaundice. These results were confirmed by Maisels et al. [54] who studied a population of over 2000 neonates admitted to a well-baby nursery between 1976 and 1980. Subsequently the 95th percentile in the first week of life was shown to be considerably higher, and in populations of predominantly breast-feeding neonates, appears to be between 15 and 17.5 mg/dL [55–57]. However, this does not imply that all neonates with

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STB concentrations within this range are safe: assessment of the jaundiced neonate must be individualized, taking into account risk factors such as direct Coombs’ positive hemolytic disease, glucose-6-phosphate dehydrogenase (G-6-PD) deficiency, borderline prematurity (35–37 weeks) or a parental history of Gilbert’s syndrome. Signs highly suggestive of hemolysis, a major risk factor, include early jaundice (within the first 24 h of life) as well as rapidly rising STB values (N0.25 mg/dL/h) [36]. In another approach to the definition of hyperbilirubinemia, Chou et al. [58] modified age-specific STB concentrations recommended by the AAP in their (now superceded [30]) 1994 Clinical Practice Parameter for the Management of Newborn Hyperbilirubinemia [29], for the consideration of institution of phototherapy. They defined hyperbilirubinemia as a STB concentration z12 mg/dL in a newborn 25–48 h of age; z15 mg/dL between 49 and 72 h, and z17.0 mg/dL after 72 h. Severe hyperbilirubinemia was defined as a STB concentration z20 mg/dL within the first 30 days of life in any infant. One limitation of these definitions may include the fact that the 1994 AAP recommendations did not encompass neonates with hemolysis, and these definitions might not be appropriate in that situation.

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Bhutani and Johnson [36] have graded the severity of hyperbiliubinemia according to the bilirubin percentile for hour-of-life [55]. According to their hour-of-life nomogram based on almost 3000 term and near term neonates who had no obvious evidence of hemolytic or other disease (Fig. 1), a predischarge STB value above the 75th percentile is considered to be clinically significant jaundice. These neonates should be vigilantly followed in order to detect further rises in STB and prevent these concentrations from entering the hyperbilirubinemia zone. Hyperbilirubinemia is defined as any STB value z95th percentile. These authors have further categorized hyperbilirubinemia into severe (z95th percentile for hour-of-life), extreme (STB values z99.9th percentile, or z25.0 mg/dL), and dangerous (STB values z99.99th percentile, or z30.0 mg/dL) [36]. 4.3. Assessment of jaundice according to the hourspecific bilirubin nomogram A modern approach to assessing the clinical implications of a specific STB value is to plot the bilirubin percentile according to hour-of-life specific bilirubin nomogram (Fig. 1) [55,59]. The nomogram was devised primarily to predict the subsequent risk of

Total Serum Bilirubin (mg/dL)

25

20 High Risk Zone

95th percentile

e 75th percentile Zon isk R e t 40th percentile dia one rme kZ Inte s i h te R Hig dia rme e t In Low

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24

36

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Fig. 1. Bilirubin nomogram showing percentiles for hour-of-life at which the baby was sampled for bilirubin. From Bhutani et al. [54], with permission.

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hyperbilirubinemia: in those whose predischarge STB values fell within the low risk or low intermediate risk zones, less than 2.5% subsequently developed a STB value z95th percentile. In contrast, 12.9% of those in the high risk intermediate zone, and 39.5% of those in the high risk zone, subsequently developed hyperbilirubinemia. Kaplan et al. confirmed usefulness of predischarge bilirubin screening by hour-of-life specific percentile determinations to detect or predict hyperbilirubinemia in G-6-PD deficient neonates, a group characterized by a high rate of hyperbilirubinemia as well as increased hemolysis [60]. The nomogram is particularly useful in detecting and monitoring neonates with early jaundice as, even if STB values are not particularly high in the first 24 post-natal hours, they may well be greater than the 95th percentile. An example of the value of the nomogram can be obtained by following a STB concentration of 11.0 mg/dL through the first days of life: This value in an infant 12 or 24 h of age will be much above the 95th percentile, while the same value at age 48 h will now have dropped to the 75th percentile, and at 72 h will pose very small risk. A similar, hour-by-hour approach, demonstrating values for the 50th and 95th percentiles, was used by Maisels [61] who drew smoothed curves from data derived from predominantly breast-feeding neonatal populations pooled from eight individual studies. An important aspect of the nomograms is the ability to plot the rate of rise of STB. By plotting two or more STB values on these graphs, neonates with STB concentrations rising at a greater than normal rate (jumping tracks) will be detected. These newborns should be regarded with special vigilance, be evaluated to determine a specific cause for the jaundice, and followed up closely to detect further increases in STB. It will also be apparent from these graphs that, upon discharge at or about 48 h, the STB will not yet have reached its peak. Fiftieth percentile values at this point approximate 6–8 mg/dL, a STB level at which clinical icterus may not always be apparent [62]. Values continue to rise over the subsequent 48 h and plateau only after completion of the 4th day of life. The pediatrician, accustomed from the past to discharging neonates on the third or fourth day, may be lulled into a sense of false security that current

predischarge STB values are benign, whereas they may in fact be approximating the 95th percentile. Chou et al. [58] have further adapted the hour-byhour approach not only to assess the risk of hyperbilirubinemia, but also to provide guidelines for the management of hyperbilirubinemia in healthy, term infants. Furthermore, in the recently published Clinical Practice Guidelines of the AAP, suggested indications for phototherapy and exchange transfusion have been graded according to smoothed curves plotted on a graph, the X axis of which reflects the infant’s age in epochs of 12 h [30]. 4.4. Frequency of exceptionally high STB concentrations The National Quality Forum at the Agency for Healthcare Research for Quality has declared kernicterus and or STB concentrations N30.0 mg/dL a bnever-eventQ [28]. Although rare, STB values in the very high range are occasionally encountered. The incidence of STB values exceeding 25.0 mg/dL within health care systems has been reported as 0.14–0.16% [63,64]. Newman et al. recently reported the bincidenceQ of STB z30.0 mg/dL in a population of 111,009 neonates born consecutively between 1995 and 1998 within the Northern California Kaiser Permanante Medical Care System as 0.01% (STB range 30.7 mg/dL to 45.5 mg/dL [56]. However, declarations of the bincidenceQ of extremely high STB values actually represent a failure of the health system in which these data were collected to prevent such values from occurring, rather than a reflection of the true or natural incidence of such values. 4.5. Duration of hyperbilirubinemia and the role of hemolysis Neonates with a hemolytic etiology for their jaundice appear to be at higher risk of bilirubin encephalopathy than those without hemolysis. While a STB concentration of 20–23 mg/dL may be associated with kernicterus in a neonate with Rh isoimmunization, a healthy, term non-hemolysing infant will rarely be endangered by STB concentrations in this range. It is also possible that the duration of hyperbilirubinemia may be of importance in relation to long-term neurological development

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[65]. Some of this evidence may be intricately interwoven with Coombs’ positivity, a condition associated with increased hemolysis resulting in both high STB concentrations as well as prolonged jaundice. Nilsen et al. [66] studied fifty-five 18year-old Norwegian males born between 1962 and 1963 and who had had neonatal hyperbilirubinemia. Their mean IQ was similar to that of the entire cohort, but 7 with a positive direct Coombs’ test and whose hyperbilirubinemia extended for longer than 5 days had significantly lower IQ values than the national average. Ozmert et al. [67], in a retrospective study of 102 children aged 8–13 years, born at term with birth weights greater than 3.0 kg, and who had been treated for indirect hyperbilirubinemia ranging from 17 to 48 mg/dL, found that Coombs’ positivity was associated with lower IQ scores and more prominent neurological abnormalities. Their observations also suggest a possible role of the duration of hyperbilirubinemia on the long-term outcome: When the indirect bilirubin level remained greater than 20.0 mg/dL for less than 6 h, prominent neurological abnormalities were detected in 2.3%. As this time period increased to 6–11 h and to 12 h or more, neurological abnormalities were noted in 18.7% and 26%, respectively. 4.6. Rate of rise of STB The rate at which the STB is rising is important in the assessment of the jaundiced neonate, and any bilirubin determination should be evaluated in light of previous STB concentrations. AAP guidelines suggest that a rate of rise of STB N0.25 mg/dL/h, equivalent to 6.0 mg/dL/day, should be regarded with concern in well babies. 4.7. Serum albumin and free bilirubin Fortunately, not all neonates with severe hyperbilirubinemia develop bilirubin encephalopathy. Many factors may interact with the high STB concentrations, either preventing or predisposing to kernicterus. One important entity to be considered is that of the serum albumin [37,68–71]. Unconjugated bilirubin binds to serum albumin and in this form is transported to the liver. Unless the blood–brain barrier is damaged, as long as the bilirubin is bound to albumin, it is unlikely to cross the blood–brain barrier and enter the brain

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basal ganglia [72]. Should the bilirubin load exceed the capacity of the serum albumin to bind it, free bilirubin may result, a form of bilirubin which can potentially cross the blood–brain barrier and enter the brain cells [73]. In animals, free bilirubin was shown to be a major determinant of bilirubin toxicity [74]. Free bilirubin, but not total bilirubin, has been shown to correlate with and induce predictable, progressive and often reversible changes in auditory brainstem response (ABR) wave latency and amplitude in neonates, and correlated with the development of kernicterus in premature newborns [75–77]. At especially high risk are newborns less than 72 h old, as even in healthy, term neonates, albumin binding to bilirubin is less effective during the first post-natal days than in older infants or adults [38]. Metabolic acidosis, infection, hyperoxia, and prematurity and drugs or preservatives including sulfisoxazole or benzyl alcohol may interfere with bilirubin–albumin binding or the integrity of the blood–brain barrier [71,78–81]. A low serum albumin concentration, frequently encountered in small premature infants, will also obviously lower the bilirubin binding capacity. Unfortunately, readily available assays for assessing serum free bilirubin are not yet available. As an alternative, the bilirubin–albumin ratio can be used to reflect the potential for free bilirubin. Albumin can bind bilirubin at a molar ratio of up to 1, which is equivalent to 8.2 mg bilirubin per gram albumin. A molar ratio of bilirubin/albumin N0.63 may be associated with ABR changes, while a ratio N0.80 may be associated with a risk of neurotoxicity [71]. In the clinical field, the ratio can be assessed by calculating the ratio of STB (mg/dL) to serum albumin (g/dL) and this ratio used as an adjunct to STB values in assessing the need for exchange transfusion or phototherapy. The Subcommittee on Hyperbilirubinemia of the AAP regards a serum albumin level of less than 3.0 gm/dL as a risk factor indicative for lowering the threshold for phototherapy. The same committee has suggested bilirubin/albumin ratios to be used in conjunction with STB concentrations in determining the need for exchange transfusion: infants z38 weeks of gestation, ratio 8.0 mg/ g; infants 35 0/7 to 36 6/7 weeks and well, or z38 weeks if higher risk, isoimmune hemolytic disease or G-6-PD deficiency, ratio 7.2 mg/g; infants 35 to 37 6/

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7 weeks if higher risk, isoimmune hemolytic disease or G-6-PD deficiency, ratio 6.8 mg/g. (30,71). To put these ratios into clinical perspective, in a neonate with a serum albumin concentration of 3.2 g/dL, for example, an accompanying STB concentration of 17 mg/dL will be equivalent to a ratio of 5.3 mg/g, while a STB of 22 mg/dL will be equivalent to a ratio of 7.0 mg/g [31].

5. Endogenous carbon monoxide production and the assessment of hemolysis Severe hemolysis may be associated with falling hemoglobin or hematocrit values and an increase in reticulocyte count. However, these indices are frequently unreliable indicators of hemolysis in neonates, as there may be overlap between hemolytic and nonhemolytic states [82]. An accurate assessment of hemolysis may be obtained by assessing the endogenous carbon monoxide (CO) production. Because CO and biliverdin (and subsequently bilirubin) are produced in equimolar quantities from the catabolism of heme by the enzyme heme oxygenase, and as the majority of heme derives from hemoglobin, measurement of CO production, with correction for ambient (inspired) CO, will allow for the rate of heme catabolism, and therefore bilirubin production, to be assessed [83–85]. Blood COHbc, accurately measured by a gas chromatographic technique [86,87] is the bgold standardQ of this method, but, because of the complexity of the assay, is not clinically available. More recently, corrected end-tidal CO (ETCOc) techniques have been developed, allowing for noninvasive sampling of end-tidal air with immediate automated analysis [88–90]. COHbc or ETCOc values usually increase in concert with increasing STB values, demonstrating the important role of hemolysis in the mechanism of neonatal jaundice [88,90, 91,93–101]. The value of COHb determinations in assessing the degree of hemolysis and the potential danger to hyperbilirubinemic babies was convincingly shown when values were correlated with bilirubin-related morbidity and mortality. In Nigerian neonates, COHb levels zthe median value of 1.40% of total hemoglobin were associated with increased need for exchange transfusion, an increased incidence of clinical findings

compatible with kernicterus, and a higher mortality rate than those with lower levels [16]. A multinational, multicenter study was recently performed to determine whether ETCOc can predict hyperbilirubinemia during the first 7 days of life [90]. Of 1370 neonates who completed the study, 120 (8.8%) developed hyperbilirubinemia, defined as a STB value z95th percentile on the hour-of-life age-specific bilirubin nomogram. As expected, ETCOc values, sampled at 30F6 h, were significantly higher in the hyperbilirubinemic babies than in those who did not become hyperbilirubinemic (1.81F0.59 vs. 1.45F0.47). However, an ETCOc value, greater than or equal to the population mean of 1.48F0.49 ppm, yielded only a 13% positive predictive value for hyperbilirubinemia, and did not improve on the predictive ability of age-specific STB determinations. On the other hand, there was a strong negative predictive value for ETCOc (95.8%), implying that in the absence of increased hemolysis, there would be little chance of developing hyperbilirubinemia. One possible reason for the failure of ETCOc to predict hyperbilirubinemia is that the technique reflects bilirubin production only, but does not take bilirubin conjugation into account.

6. Blood group incompatibility and the direct Coombs’ test ABO incompatibility is nowadays the most frequent cause of neonatal immune hemolytic disease. ABO blood group heterospecificity refers to the situation in which the mother has O blood group and the baby blood group A or B. In some instances, women with blood group O may have a high titre of naturally occurring anti-A or anti B antibodies. In contrast to Rh isoimmunization, in which the titer of anti-D antibody increases progressively following a pregnancy-related immunization, high titers of anti-A or anti-B antibodies can sometimes be found in women or girls even before their first pregnancy [102]. Because these antibodies are IgG molecules, they are able to cross the placenta and attach to the corresponding fetal red blood cell (RBC) antigens, potentially causing hemolysis. The antibodies can be detected on the newborns RBCs by the direct

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Coombs’ test, also called the direct antiglobulin test (DAT). About one-third of blood group A or B neonates born to a blood group O mother may have a positive direct Coombs’ test [103]. However, ABO heterospecificity, even in the presence of a positive direct Coombs’ test, does not necessarily indicate ABO hemolytic disease as many Coombs’ positive neonates have no evidence of ongoing hemolysis, and do not develop early jaundice or become hyperbilirubinemic. Several studies have demonstrated that ETCOc is reflective of increased hemolysis in some of these neonates, but only about 20% will develop clinically significant jaundice, and even fewer, severe hyperbilirubinemia [35,103–110]. Herschel et al. [111] determined that the positive predictive value of endtidal carbon monoxide ETCOc values z95th percentile was not significantly different than for DAT in the prediction of subsequent hyperbilirubinemia, while Madan et al. [112] demonstrated that a positive Coombs’ test was a poor predictor of readmission for treatment of hyperbilirubinemia. On the other hand, infants have been described whose mothers had identifiable antibodies in their serum, and whose neonates had clear evidence of severe hemolytic disease including hydrops fetalis and death, but whose direct antiglobulin test was negative [113]. Thus, while direct Coombs’ positivity is a risk factor for neonatal jaundice, not all ABO incompatible, direct Coombs’ positive neonates actually develop increased hemolysis or hyperbilirubinemia, suggesting that additional factors, such as diminished bilirubin conjugation, may mediate hyperbilirubinemia among neonates with increased hemolysis. 6.1. ABO blood group incompatibility with a negative direct Coombs’ test Some ABO incompatible babies with a negative Coombs’ test also develop hyperbilirubinemia, and may on occasion even exhibit evidence of ABO hemolytic disease. Although the difference did not reach statistical significance, Kaplan et al. [114] found that 12.5% of 40 direct Coombs’ negative ABO incompatible neonates developed a STB concentration z15.0 mg/dL, compared with 5.5% of 356 ABO compatible controls. However, in the subgroup of neonates who were not only ABO incompatible, but

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were also homozygous for the variant (TA)7 promoter for the gene encoding the bilirubin conjugating enzyme UDP-glucuronosyltransferase 1A1 (seen in Gilbert’s syndrome), the incidence of hyperbilirubinemia was significantly higher at 43%. This interaction is reminiscent of that demonstrated between G6-PD deficiency and the aforementioned (TA)7 gene promoter polymorphism seen in Gilbert syndrome [49,50,115]. It is possible that in some of the ABO incompatible infants the amount of antibody present was insufficient to result in a detectable direct Coombs’ test, but was sufficient to generate mild hemolysis. Suggested reasons for this apparent discrepancy include a low concentration of antibody but with a high biologic activity, or differences in the subclasses of human immune globulin, resulting in varying ability to bind to the Fc receptor of phagocytic cells [116].

7. Transcutaneous bilirubin measurement A technique which may result in decreased load on the clinical biochemistry laboratory and obviate the need for serum bilirubin testing in many instances is that of transcutaneous bilirubinometry (TcB). The amount of bilirubin in the skin tissue can be measured by quantifying the light reflected from the skin, using reflectance spectrometry of transient colorimetery, and the STB fairly accurately estimated. Modern commercially available instruments function without being affected by variations in skin pigmentation and have been used successfully, with good correlation between the TcB readings and actual serum concentrations, in mixed populations. Devices can be expected to provide a valid estimate, within 2 to 3 mg/ dL of the STB, for STB concentrations b15.0 mg/dL, but for the present, higher readings should be confirmed by serum determination [117–122].

8. Prevention and treatment of hyperbilirubinemia The American Academy of Pediatrics (AAP) has recently revised its guidelines for the management and prevention of hyperbilirubinemia in neonates of 35 weeks of gestation or more [30]. Overall, these recommendations, if adhered to, should be instrumen-

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tal in preventing kernicterus in the majority of cases. The clinical laboratory staff must realize that many neonates will have to have bilirubin testing performed and be treated with phototherapy, with the occasional exchange transfusion, in order to prevent one single case of kernicterus. Advances in non-invasive therapy of hyperbilirubinemia have rendered exchange transfusion almost obsolete, but when blood is required for an exchange transfusion, the blood bank should regard the setting up of the blood for transfusion as an emergency requiring priority. Severe neonatal hyperbilirubinemia should be regarded as an urgent situation. The clinical laboratory staff can contribute to the management of endangered neonates by following serial STB concentrations, plotting these values on the hour-of-life specific nomogram, and integrating them with results of additional laboratory tests such as G-6-PD deficiency, blood group and DAT results and a blood count. By alerting clinical caretakers to the danger of the situation laboratory workers may be instrumental in facilitating commencement of therapy as early as possible. Close cooperation between the laboratory and clinicians is of paramount importance in the prevention and management of severe neonatal hyperbilirubinemia.

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