Part D: Blood

INTRODUCTION

Neonatal jaundice, implying a yellow color of the skin and mucous membranes due to bilirubin deposition, occurs in about 60% of infants and even more often in those exclusively breast feeding. In most cases, the jaundice will be mild or moderate, and the total serum bilirubin (TSB) will not endanger the newborn infant. Occasionally, the TSB may increase to potentially dangerous levels and require treatment with phototherapy or rarely exchange transfusion to offset any further increase. Rarely, the TSB may rise to hazardous levels, at which bilirubin may cross the blood-brain barrier and enter the brain cells. The result will be the complication of acute bilirubin encephalopathy (ABE), in many cases with the devastating chronic sequela of choreoathetotic cerebral palsy known as kernicterus, or death. Despite attempts to eliminate this condition, kernicterus, although a rare condition, is still with us and accompanies us into the third millennium. Many cases should be preventable. Because the implications for affected individuals are lifelong, the public health aspects are necessarily of major importance. Understanding of the metabolism of bilirubin and the pathophysiology of hyperbilirubinemia are crucial to the prevention of this condition, as discussed in this chapter.

EPIDEMIOLOGY

Was There a Disappearance and Resurgence of Kernicterus?

Some authors refer to a disappearance and then resurgence of reported cases of kernicterus in Westernized countries during the last decades.^{1, 2, 3, and 4} Others opine that the condition never completely disappeared and is still being seen, both in countries with advanced medical systems and in developing countries.⁵ There can be no doubt that widespread use of the 2 mainstays of treatment of hyperbilirubinemia, exchange transfusion and phototherapy, along with immune prophylaxis of Rh isoimmunization, have prevented many cases of extreme hyperbilirubinemia. If the condition did indeed disappear, however, there can be no explanation for the cases of kernicterus still occurring due to conditions including glucose-6-phosphate dehydrogenase (G-6-PD) deficiency and direct antiglobulin titer (DAT) positive ABO isoimmunization, conditions that, in themselves, did not disappear.^6,7

In favor of the resurgence theory, following a period during which few cases of kernicterus were reported, several case reports were published from the United States,^{8, 9, and 10} followed by reports of the US-based Kernicterus Registry.^7,11 In Denmark, Ebbesen¹² did find cases of bilirubin encephalopathy between 1994 and 2001, whereas in a nationwide search, he uncovered no cases during the 20 years preceding 1994.

On the other hand, in the United States, Burke et al reported a 70% decrease in the number of hospitalizations between 1988 and 2005 of neonates diagnosed with kernicterus.¹³ Brooks et al found a constant incidence of kernicterus in California occurring during 2 time epochs: 1988–1993 and 1994–1997.¹⁴ On a national US basis, mortality data due to kernicterus from the Centers for Disease Control and Prevention databases revealed 31 kernicterus-related deaths between 1979 and 2006, the mortality during the first 14 years of the study period being similar to that of the second.¹³

The debate whether kernicterus did or did not disappear and then reappear is overshadowed by that fact that, well into the third millennium, cases of kernicterus are still being reported with major public health effects on neonatal and childhood morbidity and mortality.

How Frequent Is Kernicterus Currently?

National Statistics

Kernicterus is not a reportable disease, and the exact incidence may not be able to be accurately determined. From the data available, it appears that the incidence varies widely from country to country, as seen in Table 30-1.

Kernicterus in Westernized Countries

Series of affected neonates from Westernized countries have recently been published from the United States,⁷ Canada,¹⁵ the United Kingdom and Ireland,¹⁶ and Denmark.¹⁷ The US-based Kernicterus Registry has details of 125 infants who actually developed kernicterus. In addition to cases of kernicterus, the Canadian, UK and Ireland, and Danish studies also included infants with extreme hyperbilirubinemia but without evidence of ABE. The Canadian survey further included newborns who had undergone exchange transfusion. These reports shared several common epidemiologic and etiologic features, summarized in Table 30-2. It is of note that many of the infants had been discharged as healthy from birth hospitalization but were subsequently readmitted for extreme hyperbilirubinemia. Black ethnicity and minority groups were also overrepresented, relative to the home population, in the US and UK/Ireland groups.

In an Italian survey of 109 level III neonatal units, 16 cases of kernicterus (11 of these in term infants) were reported¹⁸ during the 10 years prior to 2010. A national surveillance system in Germany uncovered 11 cases of kernicterus in infants born between 2003 and 2005.¹⁹ Late prematurity and readmission of previously healthy babies were commonly encountered in this series.

Kernicterus in the Non-Western World

Kernicterus continues to occur in countries to which G-6-PD deficiency is indigenous, as well as in developing countries with underdeveloped health services, or in war zones, at a high rate. Perhaps the most devastating example emanates from Baghdad, Iraq.²⁰ Of 162 hyperbilirubinemia-related neonatal admissions, 22% had advanced ABE, 12% died within 48 hours of admission, and 21% had posticteric sequelae. Other recent reports derive from Nigeria (115 babies with ABE, of whom 42 [36.5%] died)²¹; Oman (14 cases, of whom 4 [28.5%] died)²²; and Turkey (10 G-6-PD-deficient neonates, of whom 5 [50%] developed kernicterus).²³ In Kuwait, newborns with kernicterus were reported following traditional henna applications to their skin.²⁴ Gamaleldin et al recently reported 249 newborns admitted during a 12-month period with a TSB level 25 to 76.4 mg/dL to Cairo University Children’s Hospital.²⁵ Forty-four (18%) had evidence of ABE on admission; another 55 (22%) had subtle evidence of bilirubin-induced neurologic dysfunction (BIND). Twenty-six (10.4%) died, with the deaths attributable to bilirubin neurotoxicity. The most common cause for the hyperbilirubinemia was ABO incompatibility (24%), followed by Rh isoimmunization (8.8%) and G-6-PD deficiency (8.1% of 86 tested).

Surrogates for Assessing the Incidence of Kernicterus

As kernicterus is generally rare, it is difficult to assess the incidence in any specific population group with any precision. To assess the potential for developing ABE in any population groups, surrogates have been sought, including the incidence of extreme hyperbilirubinemia (serum total bilirubin [STB] > 25 mg/dL or 30 mg/dL) or the incidence of readmission for hyperbilirubinemia.^14,26 The reported range for readmission^{27, 28, 29, 30, 31, 32, 33, 34, 35, 36, and 37} for hyperbilirubinemia lies between 0.17% and 3.2%. The main reasons for readmission include lower gestational age, early discharge, unsuccessful breast feeding, and lack of predischarge assessment of the risk for subsequent hyperbilirubinemia. Punaro et al, in Brazil, recently emphasized an important effect of late prematurity on the rate of readmission for hyperbilirubinemia.³⁸

Newman et al assessed the incidence of newborns developing extreme hyperbilirubinemia within a health care system. Despite the close surveillance and ready availability of treatment, 0.14% of newborns born at Kaiser Permanente centers in northern California developed an STB greater than 25 mg/dL; 0.01% had STB values ranging from 30.5 to 45.5 mg/dL.³⁹ Chou et al, after controlling for gestational age, sex, maternal race, feeding type, and maternal age, compared their data from the Henry Ford Health System, Detroit, Michigan, with those of Newman et al.⁴⁰ They found that the newborns under their care were less likely to have severe hyperbilirubinemia, defined as a TSB value greater than 20 mg/dL (0.6% vs 2%).⁴⁰ They attributed this difference to a rigorous bilirubin screening, follow-up, and treatment program. Under less-vigorous scrutiny, the incidence of extreme hyperbilirubinemia could be expected to be even higher.

DEFINING HYPEBILIRUBINEMIA

The Hour-Specific Bilirubin Nomogram

A major advance in our understanding of bilirubin dynamics during the first week of life has been the development of the hour-specific bilirubin nomogram.⁴¹ As there are rapid changes in STB during the first postnatal days, it is inappropriate, for the purpose of bilirubin assessment, to regard units of time in days of life, but rather in hours. The higher the bilirubin value plots for postnatal hour, the higher becomes the chance of that infant developing subsequent hyperbilirubinemia. Examination of this graph will reveal an increase in STB during the first days of life, with a plateau at about 5 days. As a result, within the normal distribution of bilirubin values, no single bilirubin value can be designated to define hyperbilirubinemia. Whereas an STB value of 10 mg/dL at 12 hours of life lies above the 95th percentile, in the high-risk zone and predictive of severe hyperbilirubinemia, the same value at age 84 or 96 hours will be of little consequence. It is clear that all bilirubin values should be plotted on the nomogram to assess these values in relation to the baby’s age.

Definition of Hyperbilirubinemia

The 95th percentile on the bilirubin nomogram has been used by some in recent years to define hyperbilirubinemia,11 and several clinical research studies have incorporated this definition.^{42, 43, and 44} This definition may not be universally practical, as the American Academy of Pediatrics (AAP, 2004) guidelines indicate the need for phototherapy below that level in cases of premature neonates or those with risk factors.⁴⁵ Use of the 75th percentile for hour of life, to designate clinically significant jaundice,⁴⁶ or STB values approaching the AAP indications for phototherapy⁴⁷ may be viable alternatives.

PATHOPHYSIOLOGY

Bilirubin Formation and Metabolism

Bilirubin is formed from the metabolism of heme (Figure 30-1). The majority of the heme derives from the continual turnover of red blood cells (RBCs), releasing hemoglobin (Hb), although some other sources, such as myoglobin, also contribute to the heme pool. Heme is catabolized by heme oxygenase to biliverdin, by which process equimolar quantities of CO are released. Biliverdin is then reduced to bilirubin via bilirubin reductase. This form of bilirubin, also known as unconjugated or indirect bilirubin, complexes reversibly with serum albumin and in this form is transported to the liver. Intrahepatocytic uridine diphosphate (UDP)–glucuronosyltranferase 1A1 controls the conjugation of bilirubin to glucuronic acid, to form the mono- and diglucuronide forms of conjugated bilirubin. This water-soluble, polarized bilirubin form is now transported into the bile by the canalicular adenosine triphosphate (ATP)–dependent transport protein MRP2⁴⁹ and thence to the intestine from which most is excreted. Some bilirubin, however, may be reconverted to the unconjugated form by intestinal bacteria, thereby allowing its reabsorption and return to the bilirubin pool (enterohepatic circulation). This process is facilitated by the presence of the enteric mucosal enzyme β-glucuronidase in the newborn.

Several of the steps noted have important clinical implications.

Release of CO

Carbon monoxide released concomitant with heme breakdown combines with Hb to form carboxyhemoglobin (COHb). As the CO is released in equimolar quantities with the biliverdin, quantification of COHb or end-tidal CO, corrected for ambient CO, represents the endogenous production of CO, which can be used as an index of the rate of heme catabolism, or hemolysis.⁵⁰ Unfortunately, there is no bedside tool currently available for clinical use.

Heme Oxygenase

Heme oxygenase 1 (HO-1) controls the first step in heme catabolism and is the rate-limiting factor in the conversion of heme to bilirubin. Polymorphisms of the HO-1 gene encoding HO-1 may have an effect on the bilirubin production rate. Although variations in the gene promoter have been associated with higher TSB levels in adults,^51,52 no effect of HO-1 polymorphisms has been reported in association with neonatal hyperbilirubinemia.⁵³ The enzyme may be important with regard to bilirubin pharmacotherapeutics. Synthetic heme analogues, metalloporphyrins, are competitive inhibitors of HO-1 and have been used clinically to inhibit the enzyme activity, thereby either preventing bilirubin formation or limiting further bilirubin production when treating established hyperbilirubinemia.⁵⁴ Some studies have documented successful use of metalloporphyrins, as reviewed,⁵⁴ but the Food and Drug Administration (FDA) has not yet approved the drug for routine clinical use.

Bilirubin Binding and Unbound Bilirubin

As long as the unconjugated bilirubin is bound to serum albumin, this complex is thought to be unable to cross the blood-brain barrier and enter the brain cells. Unbound bilirubin, on the other hand, is potentially neurotoxic and has the ability to cause neuronal damage. Indeed, the unbound bilirubin fraction concentration is a much better predictor of bilirubin neurotoxicity than the STB.^55,56 Unfortunately, there is currently no bedside tool to make unbound bilirubin measurement readily available or clinically useful, and the bilirubin-albumin ratio is sometimes used as a surrogate to determine the need for exchange transfusion.⁴⁵ Some factors that may facilitate the finding of increased serum unbound bilirubin and therefore precipitate the penetration of bilirubin into brain cells include hypoalbuminemia, asphyxia, hypothermia, metabolic acidosis, and sepsis.

Uptake Genes

Uptake of bilirubin into the liver is controlled by the solute carrier organic anion transporter protein 1B1, SLCO1B1, also known as OATP2.⁵⁷ This sinusoidal transporter facilitates the hepatic uptake of bilirubin as well as other substances, and varying expression of the gene may affect bilirubin kinetics and metabolism. SLCO1B1∗1b variant is associated with neonatal hyperbilirubinemia in Tawainese newborns, especially when coupled with UGT1A1 (uridine disphosphoglucuronosyl transferase 1A1) variants.⁵⁸ In a US-based study, coexpression of SLCO1B1∗1b with G-6-PD A- was associated with hyperbilirubinemia.⁵⁹ These studies and others⁶⁰ emphasized the importance of gene interactions and ethnic variation in the pathogenesis of hyperbilirubinemia.

Genetic Control of UGT1A1

The function of the UGT enzyme is to conjugate glucuronic acid to certain target substrates to facilitate their elimination from the body. Of major importance to the conjugation and elimination of bilirubin is the UGT1A1 gene,⁶¹ situated on chromosome 2q37. This gene encodes the UGT1A1 enzyme and is therefore of paramount importance in bilirubin conjugation. The gene consists of 4 common exons (exons 2, 3, 4, and 5) and 13 variable exons, but of the latter, only variable exon A1 is of importance regarding bilirubin conjugation (Figure 30-2). The variable exon A1 functions in conjunction with the common exons to regulate the synthesis of the individual enzyme isoform. Upstream of each variable exon is a regulatory noncoding promoter that contains a TATAA box sequence of nucleic acids. Mutations or polymorphisms of the variable exon A1, its promoter, or the common exons 2–5, may interfere in the normal process of bilirubin conjugation. While polymorphisms of the noncoding promoter area may affect bilirubin conjugation by diminishing expression of a normally structured enzyme, mutations of the coding areas may alter the structure of the enzyme molecule, thereby interfering with or abolishing enzyme function.

Imbalance Between Bilirubin Production and Elimination

The STB concentration at any point reflects 2 major processes: bilirubin production and bilirubin elimination.⁶³ Most important is the concept of lack of equilibrium between bilirubin production and conjugation. As long as these processes remain in equilibrium, the STB should remain within normal limits. In the postnatal period, increased heme catabolism in combination with diminished UGT1A1 activity results in imbalance between these processes with increasing bilirubin levels. The enterohepatic circulation may add to the bilirubin load. Should this imbalance remain mild or moderate, STB concentrations should not exceed the 95th percentile on the hour-of-life-specific bilirubin nomogram.⁴¹ However, should bilirubin production exceed its elimination, hyperbilirubinemia may occur. Some degree of increased heme catabolism, not necessarily severe, occurs in many cases of hyperbilirubinemia, even when no obvious hemolytic etiology is apparent.^42,46,64 Despite increased hemolysis and production of large amounts of bilirubin, some babies may not develop increased STB because their hepatic bilirubin-conjugating capacity is mature. On the other hand, minimally increased hemolysis in the face of immature bilirubin conjugation may well result in hyperbilirubinemia. This concept has been likened to a sink of water. Increased inflow relative to the drainage will result in a rise in the water level, whereas increased inflow along with an efficient drainage system will result in minimal rise of the water level.⁶³ Kaplan et al demonstrated this concept mathematically.⁶⁵ Using blood carboxyhemoglobin corrected for ambient CO (COHbc) to index heme catabolism as the numerator of the equation and serum-conjugated bilirubin to reflect bilirubin conjugation as the denominator, they found that the combined effect of bilirubin production and conjugation correlated better with TSB levels than any of these processes individually.

Increased Danger Associated with Hemolysis

It is generally believed that neonates with hemolytic disease are at a higher risk of bilirubin neurotoxicity than those whose hyperbilirubinemia is not the result of hemolysis.^66,67 Indeed, many original reports of kernicterus emanated from infants with Rh isoimmunization.⁶⁸ Data from a few studies are supportive of this concept. In a Turkish study, a positive DAT (or direct Coombs test), due to Rh isoimmunization or ABO incompatibility, was used as a presumed marker of hemolysis.⁶⁹ In children with indirect hyperbilirubinemia, DAT positivity was associated with lower IQ scores and a higher incidence of neurologic abnormalities than controls without a positive test but with similar bilirubin concentrations. In DAT-positive Norwegian males who had TSB levels greater than 15 mg/dL for longer than 5 days, IQ scores were significantly lower than average for that population.⁷⁰ In the Jaundice and Infant Feeding Study, 5-year IQ values of infants with TSB greater than 25 mg/dL in combination with a positive DAT were significantly lower than hyperbilirubinemic counterparts but with a negative DAT (−17.8 IQ points, 95% confidence interval [CI] −26.8 to −8.8).⁷¹ In a reanalysis of the data from the Collaborative Perinatal Project, Kuzniewicz and Newman found the presence of a positive DAT in those with a TSB of greater than 25 mg/dL was associated with a 6.7-point decrease in IQ scores.⁷² An increase in the duration of exposure to high TSB levels was also associated with an increase in neurologic abnormalities. Hemolytic conditions were among the most common etiologies for hyperbilirubinemia in the previously mentioned neonates with ABE recently reported from Egypt: ABO incompatibility (24%), Rh isoimmunization (8.8%), and G-6-PD deficiency (8.1% of 86 who were tested).²⁵

The reason for hemolysis increasing the risk for bilirubin neurotoxicity is not clear. Rapid rise in STB and earlier onset of the peak bilirubin value may contribute to this effect. Rapid saturation of the extravascular tissues with bilirubin may further increase the intravascular component. The AAP recommends institution of treatment of hyperbilirubinemia at lower levels of STB in newborns in whom hemolysis is recognized than in nonhemolyzing controls.⁴⁵

THE DREADED COMPLICATION OF EXTREME HYPERBILIRUBINEMIA: ACUTE BILIRUBIN ENCEPHALOPATHY OR KERNICTERUS

Definitions

The term kernicterus is sometimes used interchangeably for acute encephalopathy and the chronic form of choreoathetotic cerebral palsy. The clinical picture of these conditions, however, is very different.⁷³ Bilirubin encephalopathy implies the central nervous system findings that result from bilirubin toxicity to the basal ganglia and other brainstem nuclei.⁴⁵ The Subcommittee on Hyperbilirubinemia of the AAP recommends using the term acute bilirubin encephalopathy to denote the acute manifestations of bilirubin toxicity seen in the first few weeks after the hyperbilirubinemic episode. The term kernicterus, in contrast, should be used in association with the chronic and permanent clinical sequelae of bilirubin toxicity.⁴⁵ The expression bilirubin-induced neurologic dysfunction (BIND) usually refers to a form of bilirubin neurotoxicity that is subtle, including neurodevelopmental and auditory disabilities that appear to be due to bilirubin toxicity but without the classic findings of kernicterus.^74,75

Pathology of Bilirubin Toxicity

The pathologic hallmark of bilirubin neurologic damage is staining and necrosis of neurons in the basal ganglia, hippocampal cortex, subthalamic nuclei, and cerebellum followed by gliosis of these areas in survivors (Figure 30-3). The cerebral cortex is generally spared.

Clinical Picture of Kernicterus

Acute Bilirubin Encephalopathy

Early clinical features of ABE include with severe lethargy and poor feeding (phase 1). Although these signs are nonspecific, in the presence of severe hyperbilirubinemia, encephalopathy should be suspected to avoid delay in institution of therapy. During the middle of the first postnatal week (phase 2), muscle tone may fluctuate between hypo- and hypertonia, and a high-pitched cry develops. Spasm of the extensor muscles with back arching, opisthotonus, retrocollis, and impairment of upward gaze resulting in the “setting sun sign” ensue in phase 3, with fever, seizures, apnea, and death following^45,73,77 (Figure 30-4).

Chronic Kernicterus

The clinical picture of kernicterus in its chronic form has been well described.⁷³ Affected individuals may display a dystonic or athetoid movement disorder, an auditory processing disturbance that may be associated with hearing loss, motor ocular impairment of upward gaze, enamel dysplasia of the teeth, and hypotonia and ataxia due to cerebellar involvement. Many are unable to walk, require a feeding tube, or have other feeding disabilities. Mental retardation is not uncommon, but some do not have evidence of mental impairment. Hearing and visual impairments occur frequently. Motor spasticity, ataxia, and dyskinesia or hypotonia may be seen.^14,45

Subtle Bilirubin Encephalopathy and Auditory Neuropathy

Bilirubin encephalopathy may not always manifest as the classic, chronic picture of kernicterus. In some, bilirubin-induced neurological damage may result in subtle bilirubin encephalopathy, or BIND. These children have less-severe injury than those with classic kernicterus but may have cognitive disturbances, mild neurologic abnormalities, isolated hearing loss, or auditory neuropathy.^74,79 Auditory neuropathy associated with hyperbilirubinemia is not simply a sensorineural hearing loss, but rather is the result of dysfunction at the level of the auditory brainstem or nerve. Functionally, auditory neuropathy or dyssynchrony is characterized by absent or abnormal brainstem auditory evoked potentials, but with normal inner ear function. In these cases, hearing screening utilizing automated auditory brainstem responses will identify the condition. Evoked otoacoustic emission studies, reflecting inner ear function, may be normal, however, and if used alone, may result in the auditory neuropathy being missed. Awareness of bilirubin auditory neuropathy is of practical importance as cochlear implantation has been used successfully in children with this condition.⁷⁴ The mechanism by which hearing is improved is not clear, as the implant is actually proximal to the neural lesion.

DIFFERENTIAL DIAGNOSIS

A list of conditions associated with neonatal jaundice can be seen in Table 30-3. Some specific conditions, chosen because they are frequent, demonstrate a principle, or have public health implications, have been selected for further description in the following paragraphs.

Physiological Jaundice

About 60% of term newborns and even more breast-feeding infants will develop some degree of visible jaundice. During the first postnatal week, there is a natural increase in STB, reaching a mean peak of 5 to 6 mg/dL at about 5 days. Visible jaundice occurring during the first 24 hours should never be regarded as physiologic.⁴⁵ Any bilirubin concentration greater than the 95^th percentile should not be regarded as physiologic. Following the fifth day, the STB values decrease gradually.

Physiologic jaundice occurs because of increased heme catabolism in combination with diminished bilirubin conjugation, even in the absence of known risk factors for jaundice. Newborn babies have a high rate of heme catabolism, the result of shorter RBC lifespan and greater hematocrit and RBC volume than older counterparts. Also, UGT1A1 enzyme activity is immature in newborns, leading to bilirubin buildup in the immediate postnatal period.⁸⁰ This physiologic phenomenon can be exacerbated by breast feeding (below) or increased reabsorption of bilirubin from the bowel in newborns by way of the enterohepatic system, adding to the already-overloaded bilirubin pool and putting further strain on the limited conjugative system.

Jaundice in Breast-Feeding Infants

Healthy term and late-preterm infants who are breast-feeding have higher STB concentrations than formula-feeding counterparts. Some authorities recognize 2 specific entities with regard to jaundice in breast-fed infants, the first, related to inadequate feeding, leading to mild dehydration (breast feeding jaundice), and the second due to breast milk itself (breast milk jaundice).⁸¹ Breast-feeding jaundice occurs during the first postnatal week. Many of these babies have excessive weight loss, the result of delayed initiation of breast feeding, insufficient frequency of feeding, and supplementation with water. These factors may result in increased bilirubin absorption via the enterohepatic circulation. Frequent and successful breast feeding not only will enhance the milk supply, thereby increasing caloric intake and providing sufficient fluid, but also will accelerate intestinal transit time and facilitate excretion of meconium, thereby reducing the enterohepatic bilirubin circulation. Phototherapy may be necessary. In contrast, breast milk jaundice appears after the first week. The infants are healthy and vigorous and have been gaining weight appropriately. It is postulated that substances present in the breast milk may inhibit UGT1A1 activity. Suggested substances include the progesterone metabolite pregnane-3-alpha-20-beta-diol or beta-glucuronidase. It has been demonstrated that presence of the (TA)₇ UGT1A1 promoter polymorphism (UGT1A1∗28) may be responsible for prolonged jaundice in breast-feeding newborns.⁸² Most infants with breast milk jaundice will respond to phototherapy, and in most cases, it is unnecessary to discontinue breast feeding during treatment.

Hemolytic Conditions

Neonatal hemolytic conditions may be divided into immune and nonimmune pathophysiologic subgroups (Table 30-4). Although Rh isoimmunization is today largely preventable, much of our knowledge regarding the pathophysiology of kernicterus derives from the study of babies affected with this hemolytic condition. ABO immune disease is now the most common immune condition encountered. Of the nonimmune causes, G-6-PD deficiency is the most important from a public health standpoint and is associated with the development of extreme hyperbilirubinemia and, in some cases, bilirubin encephalopathy.

Immune Hemolytic Conditions

The Direct Antiglobulin Test

The DAT, otherwise known as the direct Coombs test, is the hallmark of isoimmunization⁸³ and is indicative of maternally derived immunoglobulin (Ig) G directed against fetal/neonatal RBC antigenic sites. The antiglobulin reaches the fetus via the placenta. The DAT detects antibody but does not identify the specific antibody type; for this purpose, further tests are necessary. Agglutinated clumps of RBCs may be identified visually or microscopically. Although a positive DAT is potentially associated with increased hemolysis and hyperbilirubinemia, this may not be universally so. Herschel et al found that a newborn with a positive DAT had only a 59% chance of having a 12-hour corrected end-tidal CO (ETCOc) value greater than the 95th percentile, and only 14.8% of DAT-positive neonates developed a STB equal to or greater than the 75th percentile.⁸⁴ Ozolek et al found a similar relationship between DAT and hyperbilirubinemia/jaundice in ABO-heterospecific mother-infant pairs.⁸⁵

Rh Isoimmune Hemolytic Disease

Rh disease in pregnancy may lead to intrauterine hemolysis, severe intrauterine anemia, hydrops fetalis, and intrauterine death. Following delivery, ongoing hemolysis may result in severe hemolytic disease of the newborn (HDN), anemia, and hyperbilirubinemia with the potential for bilirubin encephalopathy.

Background: The Immunization Process

Although the Rh group comprises the C, c, D, E, and e antigens, each of which may result in isoimmunization and hemolysis, RhD isommunization was formerly the most common, and when it does occur, is still the most severe. In white populations, 13% to 15% of individuals are Rh negative, but only about half that number are encountered in African Americans, and few Asian individuals are Rh negative. Because of paternal heterozygosity (D/d), an Rh-negative mother may have an Rh-negative fetus in about 50% of pregnancies. However, because of factors such as nonuniversal fetomaternal transmission of fetal blood and variable maternal immune responses, the overall incidence of Rh isoimmunization is infrequent and reported to be 6.8 cases per 1000 live births.⁸⁶

Exposure of a D-negative woman to the D antigen, occurring either antepartum or intrapartum by fetomaternal passage of fetal RBCs containing the D antigen, sets the immunization process into action. Although first-pregnancy isoimmunization has been documented, in primigravidas the process usually begins too late to allow for sufficient maternal IgG antibody to be produced and transferred across the placenta.^87,88 Transfusion of Rh-positive RBCs may also occur during abortion, blood administration, amniocentesis, chorionic villus sampling, or fetal blood sampling, thereby sensitizing the mother. In response to the antigen, the mother’s immune system produces anti-D IgG antibodies, which cross the placenta and adhere to the D-antigen sites of the fetal RBCs. The resulting antigen-antibody response culminates in hemolysis and anemia. During subsequent pregnancies, this response may become progressively more severe and rapid. The bone marrow now releases increased numbers of circulating immature RBCs (erythroblastosis), and hepatomegaly and splenomegaly may ensue due to extramedullary hematopoiesis. Fetal hydrops, including generalized tissue edema and pleural, pericardial, and peritoneal effusions, may result. Formation of this extravascular fluid results from hypoproteinemia, tissue hypoxia, and capillary leakage, along with congestive cardiac failure, itself the result of poor myocardial function and diminished cardiac output due to anemia and venous congestion.⁸⁹

Management of the Pregnancy

Hydrops fetalis is associated with a high mortality rate. Should the fetus become anemic, the option to perform intrauterine transfusion must be weighed against delivery. This decision will depend primarily on the gestational age: With increasing maturity, the potential for inherent complications involved in preterm delivery will decrease relative to the dangers involved in performing intrauterine transfusion. Aminocentesis-based regimens for detecting fetal anemia⁹⁰ are being replaced by a combination of advancing ultrasonographic techniques and developing genetic technologies, as described further in this chapter. Furthermore, modern advances in DNA technologies allow for accurate determination of paternal RhD gene status; cell-free fetal DNA determination techniques may allow for minimally invasive determination of fetal Rh type in a maternal blood sample.^{91, 92, and 93} Administration of rhesus immune globulin during pregnancy to all nonimmunized, RhD-negative women, in combination with routine postpartum administration of the globulin to those delivering an Rh-positive newborn,⁹⁴ has reduced the incidence of antenatal immunization from 14% to 0.1%. The globulin should also be administered following spontaneous or elective abortion, amniocentesis, chorionic villus sampling, or fetal blood sampling. In developing countries where Rh immune prophylaxis is not yet routine, the incidence of Rh HDN in still common.⁹⁵

Determination of the maternal anti-D titer to find the critical titer associated with a high risk of kernicterus is an important step in the monitoring of an RhD-sensitized woman. The critical titer⁸⁹ ranges from 1:8 to 1:32. Doppler assessment of the blood flow velocity in the middle cerebral artery of the fetus is replacing amniocentesis in the detection of fetal anemia, the fetal anemia resulting in increased blood flow velocity due to decreased blood viscosity and increased cardiac output.⁹⁶ Should the results suggest anemia, fetal blood is then sampled by cordocentesis. Intrauterine transfusion is an option when the hematocrit is less than 30% and the fetus less than 35 weeks’ gestation. However, if the pregnancy has reached 35 weeks’ gestation or more, delivery will be prudent. In experienced hands, the outcome of intrauterine transfusion should be good.⁹⁷

Postnatal Management of the Newborn

Management of a hydropic, severely anemic neonate, especially if complicated by respiratory and other problems of prematurity, will require intubation and ventilation, sometimes with adjunctive use of surfactant, nitric oxide, and high-frequency ventilation, with emergency drainage of pleural and pericardial effusions.⁵³ Metabolic acidosis and hypoglycemia are common.^98,99

Cord blood sample Hb values less than 10 g/dL and STB above 5 mg/dL suggest the presence of severe hemolysis. It may be preferable to correct the anemia isovolumetrically, titrating the hematocrit value until the desired concentration is obtained, using a partial exchange transfusion technique via umbilical venous or arterial catheter rather than by simple blood transfusion.

Following delivery, the placenta will no longer participate in bilirubin elimination. Extreme hyperbilirubinemia may develop, the result of continuing hemolysis and ongoing bilirubin elimination immaturity. Phototherapy and exchange transfusion, should the STB continue to rise despite intensive phototherapy, should be performed according to the 2004 AAP guidelines.⁴⁵

Intravenous immune globulin (IVIG) may be effective in preventing or limiting the number of exchange transfusions in Rh disease.^{100, 101, 102, 103, and 104} Although the 2004 AAP guideline recommends the use of IVIG to prevent exchange transfusion in cases of failing phototherapy,⁴⁵ a Dutch study recently found no benefit from the prophylactic administration of IVIG to infants with Rh hemolytic disease.¹⁰⁵ A Cochrane report concluded that more information, based on well-designed studies, was needed before IVIG could be recommended for the treatment of isoimmune hemolytic disease.¹⁰⁶ Meanwhile, even if an exchange transfusion will ultimately be necessary, delay of this procedure by IVIG can be useful in gaining time for stabilization of the patient.

Hemolytic Disease of the Newborn Caused by ABO Heterospecificity

By the term ABO setup, we refer to a blood group A or B baby born to a group O mother, a combination seen in about 15% of pregnancies. About one-third of these pregnancies will have a positive DAT due to anti-A or -B antibodies attached to RBCs.⁸⁵ Because Rh hemolytic disease has been virtually eliminated in the Western world, ABO blood group heterospecificity has become the most frequent cause of immune hemolytic disease in the neonate. The hyperbilirubinemia may at times be severe and associated with bilirubin encephalopathy. For example, 31 (25%) of 125 babies reported in the US Kernicterus Registry were ABO incompatible.⁷ A high incidence of ABO heterospecificity among infants with kernicterus or extreme hyperbilirubinemia was also reported from Canada, the United Kingdom and Ireland, Denmark, Nigeria, and Switzerland.^{15, 16, and 17,107,108}

In contrast to Rh isoimmunization, ABO disease does not follow an initial immunizing process during pregnancy. Some women with type O blood group have an inherently high titer of anti-A or anti-B antibodies even before their first pregnancy.¹⁰⁹ Furthermore, in group O individuals, anti-A or anti-B antibodies comprise IgG molecules that are able to cross the placenta. These IgG molecules may cross the placenta and attach to the corresponding A or B antigens on the fetal RBCs, thereby precipitating hemolysis in utero. Hemolysis of the IgG-coated RBCs is mediated by Fc-receptor-bearing cells within the reticuloendothelial system. The immune process is not usually sufficiently strong to result in severe hemolysis prior to delivery, but continuation of the hemolytic process following delivery can potentially result in severe hyperbilirubinemia.

It is important to realize that ABO heterospecificity, even if the DAT is positive, does not necessarily indicate the presence of ABO hemolytic disease, and many infants may not develop early jaundice or significant hyperbilirubinemia. The following criteria should be used to support the diagnosis of ABO hemolytic disease:

1. Mother blood group O, baby group A or B

2. Positive DAT

3. Indirect hyperbilirubinemia, especially during the first 24 hours of life

4. Microspherocytosis on peripheral blood smear

5. Increased reticulocyte count

Studies of Hemolysis Incorporating Endogenous Formation of Carbon Monoxide

Although not all ABO-heterospecific, DAT-positive neonates will necessarily develop hyperbilirubinemia, measurements of endogenous formation of CO, an index of heme catabolism, have demonstrated an increased rate of heme catabolism in these neonates compared with controls.^{42,110, 111, and 112} These studies confirmed the role of hemolysis in the pathogenesis of the hyperbilirubinemia and explained the high incidence of this condition in series of infants with bilirubin encephalopathy. However, not all hemolyzing babies will necessarily develop hyperbilirubinemia. In a multinational, multicenter study in which ETCOc was used to detect hemolysis, values were higher in 54 DAT-positive babies compared with the total.⁴² However, only 18.5% of the DAT-positive newborns developed a TSB greater than the 95th percentile. Despite the increased hemolysis, many, by implication, had sufficiently mature conjugating mechanisms and were able to handle the increased bilirubin load. Kaplan et al found that ABO-heterospecific, DAT-positive neonates had higher COHbc values than previously published values for a DAT-negative reference group, and that those who developed hyperbilirubinemia (STB > 95th percentile) had even higher COHbc values.⁴⁴ The percentage of newborns who developed hyperbilirubinemia increased in tandem with COHbc, reaching 100% in those with COHbc values greater than the 90th percentile.

Incidence of Hyperbilirubinemia in ABO-Heterospecific Neonates

Not all affected neonates will develop severe or clinically significant hyperbilirubinemia. For example, Kanto et al found that only 11.3% of infants developed STB values greater than 12 mg/dL¹¹³; Ozolek et al reported that only 13.7% developed STB concentrations greater than 12.8 mg/dL.⁸⁵ On the other hand, Kaplan et al found that 51.8% DAT-positive, ABO-heterospecific neonates developed STB values greater than the 95th percentile, 67% of whom had their first TSB greater than the 95th percentile documented earlier than 24 hours.⁴⁴ As early jaundice has been recognized by the AAP (2004) as a risk factor for hyperbilirubinemia, any additional stress precipitated by further hemolysis or conjugating immaturity may upset the equilibrium between the bilirubin production and elimination processes with the potential for developing severe hyperbilirubinemia.^45,114

ABO blood group incompatibility with a negative DAT, not usually predictive of hemolysis or hyperbilirubinemia, may occasionally cause early jaundice with progression to potentially dangerous bilirubin levels, reminiscent of DAT-positive hemolytic disease. Some of these infants may be displaying a manifestation of coexpression between ABO incompatibility and homozygosity for the (TA)₇ UGT1A1 polymorphism (UGT1A1∗28).¹¹⁵ This concept is discussed in the section on the genetic interactions between hemolytic conditions and UGT1A1 polymorphisms.

Treatment of ABO Heterospecificity-Associated Neonatal Jaundice

A high degree of vigilance is necessary to detect developing jaundice in newborns born to blood group O mothers. STB or TcB should be measured if jaundice is seen in the first 12–24 hours. Phototherapy and exchange transfusions should be implemented according to the 2004 AAP guideline.⁴⁵ IVIG may be helpful in modifying the rate of rise of bilirubin and is indicated if the STB is approaching the exchange transfusion threshold despite a trial of intensive phototherapy. In an analysis of 4 randomized trials involving 226 babies affected with Rh and ABO immune disease, IVIG in combination with phototherapy significantly reduced the need for exchange transfusions compared with phototherapy alone (relative risk [RR], 0.28; 95% CI, 0.17–0.47).¹⁰⁴ In newborns who responded to IVIG administration with a decrease in STB, COHbc values decreased from baseline in tandem with diminishing STB concentrations, demonstrating the effect of IVIG on limiting heme catabolism.¹¹⁶ The authors have found IVIG therapy useful in neonates who are approaching the indications for exchange transfusion despite phototherapy.

Tin mesoporphyrin (SnMP), a metalloporphyrin that inhibits HO, has been shown to reduce the need for and duration of phototherapy in DAT-positive, ABO-incompatible neonates,¹¹⁷ but this drug has not yet been approved by the FDA.

Immunization Due to Antibodies Other than RhD and ABO

Because the number of pregnancies complicated by Rh isoimmunization has decreased, uncommon RBC antigens have now become relatively more clinically important. More than 50 RBC antigens may cause HDN.¹¹⁸ Some clinically important antibodies with regard to intrauterine hemolysis include anti-c, -Kell, -Fy^a, -Jk^a, -C, -E, -C^w, -k, and -S. Anti-Kell isoimmunization warrants special mention as fetal anemia, rather than hyperbilirubinemia, often predominates the clinical picture.¹¹⁹

Fetal surveillance protocols and neonatal clinical strategies developed for RhD alloimmunization can be used for the monitoring of affected pregnancies and management of newborns affected by antibodies other than Rh.

Nonimmune Hemolytic Conditions

G-6-PD Deficiency

Deficiency of G-6-PD is a major etiologic factor in the pathogenesis of neonatal hyperbilirubinemia and a well-described cause of extreme hyperbilirubinemia and bilirubin toxicity, as reviewed in Reference 6. In the previously mentioned series of neonates with extreme hyperbilirubinemia or kernicterus from the United States, Canada, the United Kingdom, and Ireland, G-6-PD deficiency comprised a major proportion of affected infants and was overrepresented relative to the background frequency of this condition in these populations.^7,15,16 The condition therefore has major public health implications with regard to the newborn.

Deficiency of G-6-PD is estimated to affect hundreds of millions of individuals worldwide.^{120, 121, and 122} Immigration patterns and ease of travel have transformed G-6-PD deficiency into a condition that is now encountered in virtually every part of the globe and is no longer limited to its indigenous distribution, which included the Mediterranean basin, Africa, and the Middle and Far East.

Function of G-6-PD

G-6-PD^{120, 121, and 122} is a major stabilizing enzyme of the RBC membrane, protecting these cells from oxidative damage. By reducing NADP (nicotinomide adenine dinucleotide phosphate) to NADPH (reduced [hydrogenated] NADP) reduced glutathione can be regenerated from its oxidized form, a process that is essential to oxidative defenses. If there is a deficiency of G-6-PD, NADPH will not become available, regeneration of reduced glutathione will be compromised, and cells will be rendered susceptible to oxidation. The RBC is especially vulnerable because, in these cells, no alternative source of NADPH is available. Oxidative damage to the cell membrane may cause hemolysis.

As G-6-PD deficiency is an X-linked condition, males may be either normal hemizygotes or deficient hemizygotes, whereas females may be normal homozygotes, deficient homozygotes, or heterozygotes.^{120, 121, and 122} Although there should be no difficulty in differentiating between the 2 genotypes in males, in females, the heterozygotes may be difficult to classify because of the phenomenon of X chromosome inactivation. Varying ratios of active and inactivated mutated chromosomes within the cells result in differing degrees of enzyme activity. The most common G-6-PD mutations include G-6-PD A-, originally encountered in Africa; G-6-PD Mediterranean, indigenous to the Mediterranean basin and the Middle East; and G-6-PD Canton, indigenous to the Far East. Many other mutations have been described.¹²³

G-6-PD Deficiency and Hemolysis

Deficiency of G-6-PD is notoriously associated with severe hemolytic episodes with resultant jaundice and anemia, which may occur in both children and adults, following exposure to a hemolytic trigger. Classically, these episodes occur following ingestion of or contact with the fava bean (Vicia fava). Other chemical triggers, such as antimalarials, sulfonamides, and naphthalene-containing mothballs, may be equally dangerous, as are infections.¹²⁰

Extreme Neonatal Hyperbilirubinemia

The most extreme and dreaded form of hemolysis associated with G-6-PD deficiency occurs in neonates. Typically, there is acute, sudden, and unpredictable onset of jaundice. STB levels may rise exponentially, and bilirubin encephalopathy may ensue. Frequently, no trigger can be identified. Extreme hyperbilirubinemia may develop, and exchange transfusion may be the only recourse.

Studies of endogenous CO formation have demonstrated the important role of increased hemolysis in association with the extreme hyperbilirubinemia associated with this condition.¹²⁴ Often, there is an absence of hematological evidence of hemolysis, leading some to conclude, erroneously, that these events are not the result of hemolysis.^125,126

Moderate Neonatal Hyperbilirubinemia

Many G-6-PD-deficient neonates manifest a more moderate form of jaundice that usually responds to phototherapy, although exchange transfusion may sometimes be necessary.^43,127 Moderately increased COHbc concentrations do not correlate with the degree of hyperbilirubinemia, leading to the conclusion that this somewhat increased hemolysis cannot be implied as the primary icterogenic factor. Consequently, a predilection for diminished bilirubin conjugation has been demonstrated,^128,129 the result of an interaction between G-6-PD deficiency and the (TA)₇ UGT1A1 promoter polymorphism60 (Figure 30-5). These infants may be at high risk for subsequent development of severe hyperbilirubinemia as any further imbalance between bilirubin production and conjugation may result in severe hyperbilirubinemia.¹³⁰

Testing for G-6-PD Deficiency

Qualitative or quantitative screening tests should accurately determine the hemizygous state in males or the homozygous state in females. Biochemical tests are based on the oxidation of glucose-6 phosphate by G-6-PD, which results in generation of NADPH, the NADPH production reflecting G-6-PD activity. NADPH fluoresces in ultraviolet light and is the basis of the fluorescent spot test.¹³¹ Spectrophotometric measurement of absorbance at 340 nm reflects NADPH activity and is used in the quantitative measurement of G-6-PD activity. Neonatal G-6-PD enzyme values are frequently higher than adult values because neonates have many young RBCs with inherently high G-6-PD activity. Molecular screening methods will allow for heterozygote identification, but some newborns with mutations not included in any specific set of mutations tested for may be missed. The test time is substantially longer than biochemical screening tests. Neonatal G-6-PD screening programs, in combination with parental education regarding the development of jaundice and which foodstuffs or chemical substances to avoid, are in force in some countries and have been associated with a decrease in the number of cases of kernicterus, as reviewed elsewhere.^132,133

The treatment of neonatal hyperbilirubinemia associated with G-6-PD deficiency should follow the guidelines of the AAP for neonates with hemolytic risk factors.⁴⁵

Pyruvate Kinase Deficiency

Following on G-6-PD deficiency, pyruvate kinase (PK) deficiency is the second-most-common cause of nonspherocytic, Coombs-negative, hemolytic neonatal jaundice in the United States.¹³⁴ PK plays an important part in catalyzing the process resulting in formation of ATP from adenosine diphosphate in the Embden-Meyerhof pathway. It is therefore crucial to energy production. Four PK isoenzymes are encoded by 2 genes, for which 180 mutations have been identified. PK deficiency is inherited in an autosomal recessive manner.¹³⁵

In the PK-deficiency state, ATP depletion and increased cell content of 2,3-disphosphoglycerate (2,3-DPG) result in stasis, acidosis, and hypoxia, contributing to entrapment and premature destruction of poorly deformable RBCs in the microcirculation of the reticuloendothelial system. Resultant hemolysis may lead to severe neonatal anemia and early hyperbilirubinemia.¹³⁶ In patients with homozygous null mutations, no functional enzyme is formed, and newborns may be born severely anemic or even die in utero.¹³⁷ Kernicterus has been reported in association with the enzyme deficiency.¹³⁸

The condition should be suspected in cases of hemolysis and hyperbilirubinemia not associated with a positive direct Coombs test or spherocytosis. Enzyme-deficient patients have a 5% to 40% of normal enzyme activity. Molecular studies may confirm the diagnosis by identifying mutations in the coding area of the PK gene.¹³⁹ Treatment is supportive, consisting of exchange transfusion or blood transfusions when necessary. Iron overload is common, and chelation therapy may be required.

Hereditary Spherocytosis

Hereditary spherocytosis (HS) is an RBC membrane defect that can lead to acute hemolysis and hyperbilirubinemia in the newborn.^{140, 141, and 142} The condition is characterized by a deficiency in 1 or more RBC membrane proteins. Affected RBCs are abnormally shaped, have higher metabolic requirements, and are prematurely trapped and destroyed in the spleen.¹⁴³ The condition is usually inherited in an autosomal dominant fashion, but recessive or de novo mutations are also encountered. Protein deficiencies in the RBC membrane, including ankyrin, band 3, α-spectrin, β-spectrin, and protein 4.2, leave microscopic patches of the lipid bilayer inner surface bare of proteins. At these points, microvesicularization occurs, rendering the affected RBCs osmotically fragile. Affected RBCs become trapped in the spleen, the microvesicles are aspirated by macrophages, and the cell is destroyed. Hemolysis may result in jaundice, anemia, and splenomegaly. Clinically, in its most severe form, HS may result in hydrops fetalis with intrauterine death.¹⁴⁴ Hyperbilirubinemia may require exchange transfusion¹⁴⁵; kernicterus has been described.¹⁴⁶ Frequently, there is a history of hyperbilirubinemia in a sibling or a parent.

The diagnosis of HS is based on the combination of a typical clinical picture with the presence of spherocytes on a peripheral blood smear, the setting of familial hemolytic anemia, and an abnormal osmotic fragility test.¹⁴³ Christensen and Henry recently demonstrated that a mean corpuscular Hb concentration of greater than 36.0 g/dL had an 82% sensitivity and 98% specificity for identifying HS.¹⁴⁷ Flow cytometric analysis techniques may be more specific diagnostically than osmotic fragility testing.¹⁴⁸

The diagnosis of HS may be difficult in the neonatal period as splenomegaly is infrequent, and reticulocytosis may not be severe or universal. Many affected neonates may not have large numbers of spherocytes in their peripheral blood smears.^149,150 Also, the osmotic fragility test is less reliable for diagnosis of this disease in newborns than in adults. Postponement of testing until the infant is about 6 months of age may be prudent.^142,151

Treatment of hyperbilirubinemia in the neonatal period should be guided by the 2004 AAP guideline.⁴⁵ Splenectomy may become necessary later during childhood to control the anemia resulting from ongoing hemolysis.

Hereditary Elliptocytosis, Hereditary Pyropoikilocytosis, Hereditary Ovalocytosis, and Hereditary Stomatocytosis

Hereditary elliptocytosis, hereditary pyropoikilocytosis, hereditary ovalocytosis, and hereditary stomatocytosis are rare conditions affecting the erythrocyte membrane. The diagnosis is usually made by microscopic examination of the peripheral blood smear. Hemolysis may occur in the neonatal period and result in anemia and hyperbilirubinemia.^{141,152, 153, and 155}

Unstable Hemoglobinopathies

Hemoglobinopathies are the result of mutations of the globin genes. These unstable hemoglobins^156,157 may decrease RBC survival time. Heinz body inclusions may be seen in the RBCs. The γ-globin mutations Hemoglobin Poole and Hemoglobin Hasharon have been reported to cause neonatal hemolysis with jaundice and anemia. Unstable hemoglobinopathies should be sought in cases of unexplained hemolytic anemias.

Conditions Associated with Diminished Bilirubin Uptake, Conjugation, or Elimination

Gilbert Syndrome

Gilbert syndrome is a benign disorder that produces mild unconjugated bilirubinemia in about 6% of adults. Both defective hepatic uptake of bilirubin and decreased hepatic UGT activity have been demonstrated. In individuals with Gilbert syndrome, the UGT1A1-conjugating enzyme is normally structured but not fully functional because of diminished gene expression. This is because the noncoding, rather than coding, area of the gene is affected. The genetic basis of the reduced gene expression lies in the presence of additional TA repeats [(TA)₇ or occasionally (TA)₈ instead of the wild-type (TA)₆] in the TATAA box in the promoter region of the UGT1A1 gene^158,159 (Figure 30-2). In and of itself, the (TA)₇ promoter polymorphism has resulted in increased STB values compared with (TA)₆ controls, but not with hyperbilirubinemia. When in combination with additional icterogenic factors, however, the situation may be very different.

Kaplan et al described a dose-dependent genetic interaction between G-6-PD deficiency and (TA)₇ promoter polymorphism in which the incidence of any STB greater than 15 mg/dL increased dramatically when these 2 factors were in combination⁶⁰ (Figure 30-5). In Asian populations, interaction between G-6-PD deficiency and coding area UGT1A1 mutations similarly exacerbate hyperbilirubinemia.¹⁶⁰ Iolascon et al described an interaction between (TA)₇ promoter polymorphism and HS, also increasing the incidence of neonatal hyperbilirubinemia over and above that of HS, individually.¹⁶¹

Crigler-Najjar Syndrome Type I

Crigler-Najjar (CN) syndrome type I is a rare autosomal recessive disease characterized by an almost-complete absence of hepatic UGT activity. In contrast to Gilbert syndrome, the coding area of the UGT gene is mutated, so the enzyme itself is structurally abnormal with no or little bilirubin-conjugating ability. In homozygotes, severe unconjugated hyperbilirubinemia develops during the first days of life, and STB concentrations may reach 25 to 35 mg/dL. Kernicterus may occur. Stools are pale yellow, and bile bilirubin concentrations are low, with total absence of bilirubin glucuronide in bile. The diagnosis can now be obtained by sequencing the UGT1A1 gene and identifying mutations.¹⁶²

The management of these neonates involves containment of STB concentrations by phototherapy, sometimes in combination with exchange transfusion.¹⁶³ The advent of effective phototherapy systems has altered the course of this previously lethal disease, but patients remain at risk from bilirubin neurotoxicity throughout their lifetime.¹⁶³ Although liver transplant offers the only definitive treatment of the disease, in a multicenter report, 7 of 21 (33%) of transplanted children had already developed some form of brain damage by the time of their transplantation.¹⁶⁴ Hepatocyte transplantation and gene therapy may have promise for these children in the future.¹⁶⁵

Crigler-Najjar Syndrome Type II

Crigler-Najjar syndrome type II is more common than type I disease and is typically benign. Unconjugated hyperbilirubinemia occurs in the first days of life and may be exacerbated by fasting, illness, and anesthesia. The occurrence of kernicterus is rare. Unconjugated hyperbilirubinemia may persist into adulthood. Less than 50% of the daily bilirubin production is excreted in bile, and the monoglucuronide is the predominant form due to inability to convert monoglucuronide to diglucuronide.¹⁶²

Phenobarbital may be used as a simple clinical tool to differentiate between type II and type I diseases. Jaundiced neonates with type II disease respond to oral administration of phenobarbital with a sharp decline in TSB; individuals with type I disease do not respond in this way. Beyond the neonatal period, there should be no long-term risk of kernicterus.

Crigler-Najjar syndrome type II occurs as both an autosomal recessive and a dominant inheritance. The parents and other family members may appear icteric or have low-grade unconjugated hyperbilirubinemia. Testing of the neonate and the parents for the capacity to form glucuronides of bilirubin was used diagnostically in the past. Molecular diagnostic techniques with UGT1A1 gene sequencing have replaced the older methods.¹⁶²

Pyloric Stenosis

Pyloric stenosis may be associated with unconjugated hyperbilirubinemia. Hepatic UGT activity is diminished in jaundiced neonates, the result of the presence of the variant (TA)₇ UGT1A1 gene promoter.¹⁶⁶

Hypothyroidism

About 10% of congenitally hypothyroid neonates may develop prolonged jaundice due to diminished UGT activity, and testing for thyroid function should be performed in these cases. The mechanism of this association may be impairment of hepatic uptake and reduced hepatic ligandin concentrations. Absence of thyroid hormone may delay hepatic bilirubin enzyme and transport development.¹⁶⁷

Jaundice Associated with Prematurity

Jaundice in premature infants is more common and severe than in full-term neonates. STB concentrations peak around the fifth day of life. The reason for the frequency of jaundice in premature infants is immaturity of the UGT1A1 bilirubin-conjugating enzyme, over and above the level of immaturity normally encountered in term infants.⁸⁰ In premature infants, bilirubin toxicity may occur at lower concentrations of bilirubin than in term infants. Any visible jaundice in a preterm infant should be closely monitored. Recommended STB values for commencing phototherapy or performing exchange transfusions are lower for premature infants than in term newborns.¹⁶⁸

Jaundice Associated with Late Prematurity

Late-preterm gestation (newborns born who have completed between 34 weeks and 36 weeks 6 days) is becoming important as a risk factor for the development of neonatal hyperbilirubinemia. Kernicterus has been described in association with late prematurity. Immature bilirubin conjugative capacity may contribute to the greater prevalence and potential severity of jaundice in these infants. Coexpression of late prematurity with additional icterogenic factors such as G-6-PD deficiency may enhance the jaundice.¹⁶⁹ Management of late-preterm infants and discharge at 1 or 2 days, as if they were term infants, with lack of appropriate follow-up, is a major contributor to the bilirubin-related morbidity in these cases.

DIAGNOSTIC TESTS

Assessment of Level of Jaundice

Visual

Newborns should be assessed visually for the appearance of jaundice from birth and several times daily through the first days of life. Jaundice appears first on the head and progresses cephalocaudally. The point to which the jaundice has reached has been used to allow for visual assessment of the level of jaundice.¹⁷⁰ Assessment of the skin color may be facilitated by blanching the skin to reveal the skin and underlying tissue color. In dark-skinned newborns, inspection of the palms, soles, and mucous membranes may enhance the assessment. Visual assessment, however, is an inaccurate technique, and quantification of jaundice should be performed by obtaining a STB, especially in the presence of jaundice appearing during the first 24 hours. Transcutaneous bilirubinometry (TcB) technology should facilitate closer and more objective noninvasive evaluation than was available in the past (see the next sections).

Serum or Plasma Bilirubin Determinations

The STB is the mainstay of bilirubin determination for practical, clinical use. Although high-performance liquid chromatographic (HPLC) techniques are the “gold standard” of bilirubin determinations, this method of testing is expensive and labor intensive. There are still inaccuracies and variability in laboratory performance of bilirubin testing in the clinical laboratory, although efforts are being made to standardize systems and minimize inaccuracy.¹⁷¹

Transcutaneous Bilirubinometry

Transcutaneous bilirubinometry measurement techniques offer a rapid, noninvasive, point-of-care screen.¹⁷² TcB does not measure the actual bilirubin but measures the intensity of the yellow color of the skin and translates that into a value estimating the STB. TcB is useful in that it eliminates the guesswork associated with visual assessment of jaundice and can give guidance regarding which babies require a serum or plasma test. TcB readings correlate well with actual STB determinations, although TcB tends to underestimate the actual TSB determination. It may be prudent to obtain a blood test to confirm TcB readings prior to institution of therapy or should the reading be greater than the 75th percentile on the bilirubin nomogram or greater than the 95th percentile on a TcB nomogram.¹⁷³

Interpretation of the TcB or STB Value: Assessment of Risk

It is imperative to interpret the TcB or STB value by plotting the value on the hour-specific nomogram and determining the percentile for age.⁴¹ There are rapid changes in STB during the first days of life, and the identical bilirubin value may have different implications when occurring at different ages during the first week. In general, the higher the percentile is, the greater the risk of subsequently developing significant hyperbilirubinemia will be. Newborns with readings less than the 40th percentile (low-risk zone) have a low, but not negligible, risk of subsequent hyperbilirubinemia. Risk factor assessment should be incorporated in the assessment of the STB value. The presence of factors such as late prematurity, DAT positivity, or G-6-PD deficiency may have an effect on exacerbating the risk of subsequent hyperbilirubinemia.

Blood Groups and DAT

Blood grouping and DAT determinations do not need to be performed routinely, except in infants born to Rh-negative mothers to determine the need for maternal anti-D administration. Blood groups and DAT should be determined in infants of group O mothers who are developing jaundice or in cases of unexplained, severe jaundice to identify possible isoimmunization due to causes other than ABO heterospecificity.

Complete Blood Count

The CBC may sometimes be useful as a falling Hb or HCT value or increased reticulocyte count may be adjunctive in assessing whether hemolysis is present. However, the CBC is not an accurate index of hemolysis in newborns, and there may be overlap between normal and abnormal CBC values in hemolytic and nonhemolytic situations.¹⁷⁴

Examination of the blood smear may be useful in the identification of RBC morphology.

G-6-PD and Pyruvate Kinase Testing

In cases of unexplained jaundice or an unidentified cause of hemolysis, especially in families with a strong family background for G-6-PD deficiency, the G-6-PD status can be assessed by either qualitative screening tests or quantitative enzyme assays. PK can be tested by enzyme assay.

Magnetic Resonance Imaging

In many cases, ABE and kernicterus are accompanied by characteristic magnetic resonance imaging (MRI) changes. Typically noted is a bilateral, symmetric, high-intensity signal in the globus pallidus and sometimes in the hippocampus and thalamus.⁷⁴ In the acute stages of the disease process, these changes may not be universally present: Coskun et al recently reported only 8 of 13 affected neonates with this finding.¹⁷⁵ Absence of typical MRI changes does not exclude the diagnosis.⁷⁴ Gkoltsiou et al were not able to demonstrate a constant relationship between early imaging abnormality and motor outcome. Classic globus pallidus changes on T2-weighted images, however, were seen in all infants with cerebral palsy attributable to bilirubin neurotoxicity.¹⁷⁶

Brainstem Auditory Evoked Potentials

As auditory neural tissue is sensitive to the effects of bilirubin toxicity, the brainstem auditory evoked potential (BAEP) offers an early and sensitive measure of bilirubin-induced central nervous system dysfunction. Increased latency and decreased amplitude of waves III and V are early signs of bilirubin toxicity, followed by absence of these waveforms and finally complete absence of all waveforms. Automated ABR can be used at the bedside as a rapid test of auditory function in a neonate with severe hyperbilirubinemia. Absence of automated ABR, or change from “pass” to “refer,” may be an indication of bilirubin neurotoxicity.⁷³

MANAGEMENT

Successful ongoing evaluation to detect the development of jaundice, predischarge assessment of the risk for subsequent hyperbilirubinemia, and the timely management of hyperbilirubinemia when it does occur to prevent bilirubin encephalopathy are essential to ensure a safe first week of life. Several countries, including the United States, Canada, United Kingdom, Norway, Israel, and South Africa, have set up national guidelines to standardize the management of neonatal jaundice in an attempt to minimize the number of babies developing kernicterus.

Ongoing assessment during birth hospitalization includes regular assessment of babies for development of jaundice and assessment of risk factors that may exacerbate the risk of hyperbilirubinemia. Some important risk factors include exclusive breast feeding, late prematurity, a previous sibling with significant jaundice, G-6-PD deficiency, and DAT positivity. Although babies born at 37 weeks’ gestation are classified as term, their bilirubin-conjugating system is not as efficient as that of an infant at 40 weeks’ gestation. It is now recommended that each infant is assessed predischarge for the risk of hyperbilirubinemia by determining a bilirubin value, either by the STB or TcB routes, in addition to assessment of risk factors.¹⁷³ Should the risk be determined high, that baby should have a bilirubin test within 1 or 2 days of discharge. Because the bilirubin value may increase even in the absence of risk factors or a bilirubin percentile in the high-risk zone, all newborns should be seen by a health authority within 2 or 3 days of discharge to assess the success of breast feeding and to evaluate the need for bilirubin testing.^45,173

Treatment of Hyperbilirubinemia

The mainstays of treatment include phototherapy and exchange transfusion.¹⁷⁷ The AAP has published graphs indicating specific TSB values at which treatment should be instituted.⁴⁵ These graphs, akin to the bilirubin nomogram, vary according to hour of life and with the presence of risk factors or prematurity. The technical aspects of phototherapy and exchange transfusion are discussed in the Atlas section of this book.

Therapy with IVIG may be useful in cases of immune hemolysis in which the STB is reaching exchange transfusion indications. The AAP has published guidelines for its use.⁴⁵

Drug therapy has included phenobarbital administration to enhance UGT activity and facilitate bilirubin excretion. Mesoporphyrins have been demonstrated to successfully lower the STB and may in the future play a major role in the management of hyperbilirubinemia but, as discussed, have not yet been released by the FDA for routine use.

Once the chronic features of kernicterus have become established, the treatment is primarily supportive and directed toward improving the neurodevelopmental and other sequelae. Physiotherapy, occupational therapy, and speech therapy may be helpful in alleviating some of the symptoms. Additional related problems can include feeding and nutritional difficulties, sleep disturbances, and muscle hypertonicity. Hearing deficits may be alleviated by conventional hearing aid amplification. Cochlear implantation has been reported to be successful in improving hearing, even though the lesion may be proximal to the implant.⁷⁴

OUTCOME AND FOLLOW-UP

Extreme Hyperbilirubinemia

The STB is a poor predictor of bilirubin-related neurologic outcome as not all newborns with extreme hyperbilirubinemia go on to develop choreoathetotic cerebral palsy.⁵⁵ In a study of 140 newborns with STB values greater than 25 mg/dL who were treated with phototherapy or exchange transfusion, 5-year outcomes were not significantly different from those of randomly selected controls.⁷¹ Reanalyzing data from the Collaborative Perinatal Project, Kuzniewicz and Newman found no relationship between maximum STB levels and IQ scores.⁷² However, in both these studies, the presence of a positive DAT did result in poorer prognosis than the general population studied. Of 249 newborns admitted with STB values greater than 25 mg/dL to a children’s hospital in Cairo, Egypt, Gamaleldin et al found little correlation between admission STB and ABE.²⁵ However, the presence of risk factors, including Rh incompatibility, ABO incompatibility, and sepsis, decreased the threshold TSB for identifying babies with bilirubin encephalopathy relative to those without risk factors.

Acute Bilirubin Encephalopathy

Sgro et al recently documented the clinical picture of 32 Canadian newborns whose STB ranged from 24.9 to 45.2 mg/dL and who had neurological findings compatible with ABE at the time of admission.⁷⁷ The dominant clinical features included hypotonia, poor suck, lethargy, and abnormal auditory evoked responses. Opisthotonus, retrocollis, apnea, seizures, irritability, and hypertonia were also found. Infants in the highest peak bilirubin level group (>32 mg/dL), who presented within the first 2 days of life, or who had undergone exchange transfusion were at higher risk for presenting with signs of bilirubin encephalopathy. The authors suggested that the rapid increase in serum bilirubin may have potentiated the risk of ABE.

Many babies with ABE go on to develop chronic choreoathetotic cerebral palsy.

Chronic Kernicterus

The clinical picture of kernicterus in its chronic form has been well described.⁷³ A recent report of 25 California cases of strictly defined kernicterus provided a dismal picture of these severely disadvantaged children.¹⁴ Seventy-two percent were male. At a mean (SD) age of 7.8 (3.9) years, 60% did not walk at all, and only 16% were able to walk unaided. Only 52% could self-feed; a feeding tube was in place in 12%. Severe or profound mental retardation or severe disablement was found in 36%, while only 32% had no evidence of mental retardation. Epilepsy was found in 20%. Severe, profound, or untestable visual impairment was documented in one-quarter of cases. Severe, profound, or untestable hearing impairment affected 56%, with only 36% having normal hearing. Motor spasticity was seen in 32%, ataxia and dyskinesia in 12% each, and hypotonia in 8%.

Does Acute Bilirubin Encephalopathy Necessarily Predict Chronic Outcome?

Although ABE usually precedes the permanent neurological sequelae of kernicterus, some reports suggested that this may not necessarily be so. There are reports of severely hyperbilirubinemic infants with signs of ABE on admission who subsequently did not demonstrate signs of bilirubin neurotoxicity.^{16,17,178,179} These results, although most likely representing the minority of outcomes, are nevertheless encouraging. Should a baby present with features of ABE, exchange should be done as soon as feasible because of the potential for reversal of the severe outcome.

CONCLUSIONS

Despite growing awareness of the condition and the formulation of international as well as local guidelines, kernicterus continues to occur well into the third millennium. Kernicterus has been declared a never event, but because of conditions causing sudden and unpredictable rises to extreme levels of STB, such as G-6-PD deficiency, it is unlikely that kernicterus will be completely eliminated. However, if general principles and published guidelines are meticulously followed, most babies should be unaffected. It is imperative to assess each baby prior to discharge for TcB or STB as well as for risk factors and to evaluate each baby within a few days of discharge in accordance with AAP recommendations.⁴⁵ Severely hyperbilirubinemic neonates should be followed up closely clinically with auditory and MRI evaluation to detect and institute supportive therapy as indicated.

REFERENCES

INTRODUCTION

The newborn period marks a time when red blood cell (RBC) indices change significantly. Anemia can occur at various times in the neonatal period, from the perinatal and immediate postnatal period through the first months of life. Hematocrits 2 or more standard deviations below the normal range for gestation are seen frequently and should be evaluated. Conversely, true anemia, the inability to adequately deliver oxygen to tissues, is less common. Anemia can be classified into the following 3 major processes: hemolysis, hemorrhage, or hypoproliferative disease. Anemia can also result from overlapping processes. For example, sepsis can result in hemolysis, disseminated intravascular coagulation (DIC), and subsequent hemorrhage. This chapter reviews fetal and neonatal erythropoiesis, discusses the etiology and diagnosis of anemia in the neonatal period, and offers management options for anemic term and preterm infants.

Fetal Erythropoiesis

The growth factors stimulating fetal erythropoiesis during gestation are produced solely by the fetus. Erythropoietin, or Epo, is the primary growth factor responsible for erythropoiesis after birth and appears to be the principal growth factor for fetal erythropoiesis as well. Primary production of Epo occurs in the liver during fetal development. The kidney becomes the primary source of Epo following delivery; however, the etiology for the liver-to-kidney switch continues to be studied. Anephric fetuses have normal serum Epo concentrations and normal hematocrits, proving that renal Epo production is not required for erythropoiesis during fetal development. Methylation patterns in the promoter and enhancer regions of the Epo gene isolated from fetal liver and kidney suggest increased methylation in the kidney as a possible explanation for decreased Epo gene expression during fetal development.

During gestation, the site of red cell production transitions from yolk sac to liver to marrow. Primitive erythroblasts are produced in the fetal yolk sac during the first 3–4 weeks of gestation. By 6 to 8 weeks’ gestation, definitive erythropoiesis is established in the fetal liver. Although erythrocytes are noted in the fetal marrow as early as 8–9 weeks’ gestation, the liver remains the primary site of erythropoiesis until well into the second trimester. By the third trimester, erythropoiesis occurs primarily in the fetal marrow, although production can continue in extramedullary sites in the face of increased need, during times of hemolysis, or in fetal infection caused by a variety of bacteria or viruses.

Variations in hematologic indices for preterm and term infants reflect the dynamic nature of the erythron in late fetal development. The production of hemoglobin transitions from embryonic (Gower 1 [ζ₂ε₂], Gower 2 [α₂ε₂], and Portland [ζ₂γ₂]) to fetal hemoglobin (α₂γ₂) and finally to adult hemoglobin (α₂β₂), as shown in Figure 31-1.

In conjunction with changes in globin gene expression and the transition from embryonic to fetal hemoglobin production, hemoglobin concentrations gradually increase, from approximately 14.5 g/dL at 28 weeks’ gestation to 15 g/dL at 34 weeks to 16.8 g/dL at 40 weeks.¹ The relationship between cord hemoglobin concentration and duration of pregnancy is linear for infants who are appropriate for gestational age (AGA). The mean corpuscular volume (MCV) is higher in preterm infants than in term infants and is inversely proportional to gestation, averaging 5–20 fL higher than the mean MCV of 108 fL in term infants.

Reticulocyte counts are elevated at birth, averaging 6% to 10%. Nucleated red blood cells (NRBCs) are not consistently elevated, but there does appear to be an inverse relationship between number of NRBCs and gestational age. Normoblasts are cleared rapidly from the circulation during the first postnatal days, although a few may still be observed in small preterm infants into the second week of life. Variations in red cell size and shape are somewhat greater than those observed in term infants.

Neonatal Erythropoiesis

The abrupt transition from the relatively hypoxic in utero environment to the oxygen-rich postnatal environment triggers erythropoietic responses that have a profound effect on the red cell mass. Over the first 2 to 6 months of life, the infant experiences both the highest and the lowest hemoglobin concentrations occurring at any time in development. Although variable, Epo levels at birth are above the normal adult range but decrease abruptly in the immediate postnatal period, with a half-life of no more than 4 hours (2.6 ± 0.5 hours in infants with polycythemia and 3.7 ± 0.9 hours in infants born to mothers with preeclampsia.)² By 24 hours, the Epo value is nearly undetectable and remains so throughout the first month of life. The decrease in Epo is accompanied by a decrease in the number of erythroid progenitors and in the reticulocyte count.³

A combination of decreased red cell production, shortened cell survival, and growth-related expansion of the blood volume results in a progressive fall of the hemoglobin concentration to a mean of approximately 11 g/dL at 2–3 months of age.¹ The lower range of normal for infants of this age is approximately 9 g/dL. This nadir is called physiologic anemia, in that it is not associated with lack of oxygen delivery to tissues and is not abrogated by nutritional supplements. Stabilization of the hemoglobin concentration is heralded by an increase in reticulocytes in the second month of life. Thereafter, the hemoglobin concentration rises to an average of 12.5 g/dL, where it remains throughout infancy and early childhood.

ETIOLOGY

Hemolysis

Hemolysis is the premature destruction of erythrocytes and is generally categorized as immune-mediated or non-immune-mediated hemolysis (Table 31-1). Immune-mediated hemolysis occurs when immunoglobulin (Ig) G antibodies created by the maternal immune system cross the placenta and cause destruction of antigen-containing fetal red cells (isoimmunization). Hemolysis in the fetus can be severe enough to lead to hydrops fetalis. Non-immune-mediated hemolysis occurs when intrinsic abnormalities in the fetal or neonatal red cell lead to premature destruction and a shortened red cell life span.

In older children and adults with congenital hemolytic anemia, there is usually clear evidence of hemolysis, as manifested by indirect hyperbilirubinemia, reticulocytosis, and low haptoglobin levels, all markers of increased red cell destruction. In neonates, however, the recognition of congenital hemolytic anemia utilizing these laboratory measurements is more challenging. Physiologic indirect hyperbilirubinemia is common in neonates because of the increased red cell mass at birth, decreased RBC survival, and reduced hepatic glucuronyl transferase activity. Also, the normal physiologic anemia of infancy can modulate reticulocytosis in the presence of mild hemolytic anemia. Finally, serum haptoglobin is not a reliable index of neonatal hemolysis because concentrations of this protein do not increase until after 6 months of age.

Neonates with congenital hemolytic anemia may have a normal-for-age hemoglobin or there may be mild-to-severe anemia. Indirect hyperbilirubinemia presenting earlier than expected and in excess of the usual physiologic levels is the most common and occasionally only sign of hemolytic anemia. Reticulocytosis and persistence of nucleated RBCs beyond the third day of life are other reasons to evaluate a neonate for hemolytic anemia. Acquired hemolytic anemia in neonates occurs with alloimmune hemolysis (chapter 34), hemolytic anemia secondary to maternal disease, and hemolysis secondary to infection or microangiopathic anemia.

Immune-Mediated Hemolysis

The most common cause for hemolysis in the neonatal period is immune-mediated hemolysis due to ABO incompatibility or Rh incompatibility. Following the advent of RhoGAM (RhD antibody), the incidence of hydrops fetalis due to RhD sensitization decreased significantly, from approximately 1 in 200 live births to now less than 1 in 1000 live births.⁴ Immune-mediated hemolysis due to anti-A or anti-B antibodies crossing the placenta is now the most common etiology of immune-mediated hemolysis.

RhD Isoimmunization: RhD incompatibility occurs when a woman with RhD-negative blood type is exposed to Rh-positive blood cells, leading to the development of RhD antibodies (anti-D antibodies). Fifteen percent of the population lacks the D antigen (is Rh negative). RhD sensitization occurs in approximately 1 per 1000 births to women who are Rh negative.

RhD incompatibility occurs through 2 primary mechanisms. The most common route of exposure occurs when an Rh-negative pregnant mother is exposed to Rh-positive fetal RBCs secondary to fetomaternal hemorrhage (FMH), spontaneous abortion, or normal delivery. RhD incompatibility can also occur if an Rh-negative female accidentally receives Rh-positive blood. Maternal anti-D IgG antibodies freely cross the placenta into the fetal circulation, where they form antigen-antibody complexes with RhD-positive fetal RBCs. The red cells are eventually lysed, leading to isoimmune hemolytic anemia.⁵ As a result, large amounts of bilirubin are produced from the breakdown of fetal hemoglobin and are transferred via the placenta to the mother, where they are conjugated and excreted.

Once the baby is born, low levels of glucuronyl transferase can lead to significant elevations of unconjugated hyperbilirubinemia. Erythroblastosis fetalis is a severe form of the disease characterized by severe hemolytic anemia and hydrops fetalis, involving placental and fetal edema, ascites, pericardial effusion, high-output cardiac failure, and extramedullary hematopoiesis. The risk and severity of sensitization increase with each subsequent pregnancy in which the fetus is Rh positive. The risk of sensitization depends on the volume of FMH, the extent of maternal immune response, and the concurrent presence of ABO incompatibility, which actually decreases the severity of hemolysis in Rh-positive fetuses.

Administration of RhoGAM decreases the production of maternal IgG antibodies by inhibiting immune memory. RhD immune globulin coats the surface of RhD-positive fetal RBCs. These antibody-antigen complexes inhibit the stimulation of maternal B cells to produce IgG antibodies. RhoGAM is administered once at 28–32 weeks’ gestation. It has a short half-life of 23–28 days and is thus administered again within 72 hours after delivery. Rh-negative (nonsensitized) women presenting to the emergency room with antepartum bleeding or potential FMH should receive 300 μg of Rh IgG.⁶ An additional 300 μg of Rh IgG should be administered for every 30 mL of fetal blood to which the Rh-negative mother is exposed. A lower 50-μg dose (MICRhoGAM) is available and recommended for Rh-negative women who have a termination of pregnancy in the first trimester when the volume of FMH is likely minimal.⁷

ABO Isoimmunization: Hemolysis of fetal red cells caused by ABO antibodies occurs almost exclusively in infants of blood group A or B who are born to group O mothers.⁸ Anti-A and anti-B antibodies formed in group O individuals are mainly IgG antibodies and thus can cross the placenta, whereas anti-A and anti-B antibodies found in the serum of group A or group B individuals tend to be IgM antibodies. ABO incompatibility tends to be relatively mild in nature, mainly because fetal RBCs do not express adult levels of A and B antigens, resulting in few reactive sites. This allows the antibody-coated cells to remain in the circulation for a longer period of time compared to RhD disease. In addition, severity of ABO incompatibility is not dependent on birth order.⁹

The presence of microspherocytes on peripheral blood smear is a characteristic of ABO isoimmunization, with little or no increase in nucleated RBCs. In RhD isoimmunization, there are fewer spherocytes and a large number of nucleated RBCs.¹⁰ ABO isoimmunization usually presents with hyperbilirubinemia and can be managed by phototherapy alone. Exchange transfusions are occasionally required. The direct antiglobulin test (DAT) is usually weakly positive but can be negative in some cases despite evidence of immune-mediated hemolysis.

Minor Blood Group Isoimmunization: Although RhD isoimmunization is the most common cause of severe hemolytic disease of the newborn, other antibodies belonging to Kell (K and k), Duffy (Fya), Lewis, Kidd (Jka and Jkb), and MNSs (M, N, S, and s) systems can cause severe hemolytic disease of the newborn.¹¹ Kell antigen is a minor blood group antigen that often results in isoimmunization. In addition, antibodies directed against Kell antigens can bind to erythroid progenitors in the fetal marrow and result in hypoproliferative anemia.¹² Minor Rhesus blood group antigens c and E can also cause severe hemolysis, requiring exchange transfusion.

Non-Immune-Mediated Hemolysis

Enzyme Disorders

Glucose is the major metabolic substrate for RBCs, and it is metabolized via the glycolytic pathway and hexose monophosphate (HMP) shunt. The glycolytic pathway generates adenosine triphosphate (ATP), NADH (reduced nicotinamide adenine dinucleotide), and 2,3-disphosphoglycerate (2,3-DPG), all important in maintaining RBC integrity. The HMP shunt pathway is critical for protecting RBCs against oxidative insult. Glucose-6-phosphate dehydrogenase (G-6-PD) is the initial step in the HMP pathway. Among hemolytic anemias due to hereditary enzyme deficiencies, G-6-PD deficiency is the most common. The remaining glycolytic pathway enzymopathies are relatively rare, with only a few thousand cases known and 90% due to pyruvate kinase deficiency.

Glucose-6-Phosphate Dehydrogenase Deficiency: Deficiency of G-6-PD is one of the most common genetic disorders in the world, affecting over 400 million people worldwide.¹³ It is a sex-linked disorder affecting males, but females who are homozygous for G-6-PD deficiency or have skewed lyonization of the X chromosome can also have significant enzyme deficiency. This enzymopathy is found most commonly in people from Africa, Asia, and the Mediterranean region. Three clinically important types of G-6-PD deficiency are classified by the World Health Organization (WHO). Class I G-6-PD deficiency is vanishingly rare, but it is the most severe form, characterized by lifelong mild-to-moderate chronic hemolytic anemia. Class II G-6-PD variants, commonly found in Mediterranean (G-6-PD^{Mediterranean}), Middle Eastern, and Asian people, have a marked enzyme deficiency that is associated with severe anemia after exposure to oxidant drugs and chemicals (eg, primaquine, ciprofloxacin, nitrofurans, sulfonamides, dapsone, naphthalene) or fava beans. Class III G-6-PD is the mildest deficiency, found in people of African (A-variant) and Asian background who experience moderate but self-limited hemolytic anemia with oxidant exposure. In patients with class II or III G-6-PD, there is no anemia or hemolysis in the steady state when there is no oxidant exposure.

G-6-PD deficiency in neonates is associated with high risk of severe jaundice and kernicterus.^{14, 15} It is much more common in neonates with class II variants than in infants with class III G-6-PD deficiency. The possibility of G-6-PD deficiency should be considered in infants with hyperbilirubinemia beyond expected physiologic levels. The neonatal jaundice associated with G-6-PD deficiency has a late peak at day 5–6 of life; it may be especially difficult to identify in preterm infants due to their later peak bilirubin levels. Of particular importance, hyperbilirubinemia often is the only abnormality, and there may be no other signs of hemolysis. This observation has suggested that hyperbilirubinemia might not be related solely to increased bilirubin production from red cell destruction but rather may be due to a defect in liver clearance of bilirubin.¹⁶ Also, there are reports suggesting that marked hyperbilirubinemia in G-6-PD deficiency is associated with the simultaneous inheritance of the variant Gilbert polymorphism.¹⁷ In the United States, severe hyperbilirubinemia due to G-6-PD deficiency is responsible for over 30% of kernicterus cases.¹⁸ Neonates at risk for G-6-PD should have bilirubin levels measured prior to discharge. Close outpatient follow-up is particularly important given that many neonates are discharged early from the hospital. Neonatal discharge guidelines have been established by the American Academy of Pediatrics and the American College of Obstetricians and Gynecologists.¹⁹

Severe hyperbilirubinemia occurring in G-6-PD deficiency requires phototherapy and, occasionally, exchange transfusion. Packed RBC (PRBC) transfusion should be given for symptomatic anemia. Avoidance of oxidative medications and chemicals is important to prevent further hemolysis. Mothers who are breastfeeding should be counseled to avoid ingestion of fava beans and oxidant medications. Vitamin K injection is safe for neonates with class II and III variants and should be given to prevent hemorrhagic disease of the newborn.

Pyruvate Kinase Deficiency: Pyruvate kinase (PK) deficiency is the most common glycolytic pathway defect. Over 200 different mutations of the 2 different PK genes (PKM2 and PK-LR) have been reported.²⁰ It is an autosomal disorder, with most patients compound heterozygous for 2 different mutations. The prevalence of PK deficiency among Caucasians in the United States has been estimated at 1 in 20,000. The disease is common among the Amish population in Pennsylvania and other inbred communities.²¹

Pyruvate kinase catalyzes the conversion of phosphoenolpyruvate to pyruvate, at the same time generating ATP, a critical source of energy for RBCs. Impaired ATP production occurs in PK-deficient cells, leading to loss of membrane plasticity and, ultimately, splenic destruction of red cells.

The clinical features of PK deficiency are highly variable, ranging from severe anemia and jaundice at birth to an incidental finding in adults. This is likely related to the many different known mutations and the often-compound heterozygosity for different mutations causing the deficiency.

Deficiency of PK should be part of the differential diagnosis of hydrops fetalis and neonatal jaundice. Jaundice can be pronounced, and kernicterus has been reported.²² In contrast to G-6-PD deficiency, in addition to hyperbilirubinemia, there are usually obvious features of hemolysis, including anemia and reticulocytosis. PK deficiency should be suspected in a neonate with evidence of hemolytic anemia after immune-mediated hemolysis, hereditary spherocytosis (HS), and G-6-PD deficiency are ruled out.

Hyperbilirubinemia should be managed with phototherapy and exchange transfusion as needed. Severe anemia may require red cell transfusions to maintain hemoglobin or as needed to maintain normal neonatal growth and development. Many children with severe PK deficiency are transfusion dependent until they undergo splenectomy after 5 years of age.

Other RBC Enzymopathies Associated With Hemolysis: Abnormalities have been reported in all glycolytic enzymes. These conditions are rare, accounting for less than 1% of hemolytic anemia due to enzymopathies.²³ After PK deficiency, which accounts for 90% of glycolytic enzymopathies, glucose phosphate isomerase (GPI) deficiency is the next most common. All glycolytic enzymopathies are associated with chronic nonspherocytic hemolytic anemia. The degree of hemolytic anemia varies from mild to severe, and hydrops fetalis has been seen in those with the most severe hemolysis. Several glycolytic enzyme deficiencies are associated with myopathy or neurologic deficits. Most are inherited in an autosomal recessive (AR) pattern except phosphoglycerate kinase deficiency, which is X linked. This group of rare disorders should be considered when a neonate shows clear and persistent evidence of nonspherocytic hemolytic anemia and after other hemolytic causes such as immune-mediated hemolysis, membrane defects, and the more common enzymopathies discussed have been ruled out. Enzymopathies should also be considered in neonates with neurologic defects or myopathy in addition to hemolytic anemia. Specific assays of RBC enzyme activity are necessary for diagnosis of these rare conditions. In some cases, DNA-based molecular tests are available.

Membrane Disorders

Hemolytic anemias due to abnormalities of the erythrocyte membrane comprise an important group of inherited disorders. The most common disorder encountered is HS. Hemolysis due to hereditary elliptocytosis (HE), and the hereditary stomatocytosis (HSt) syndromes also occurs but is less common.

Hereditary Spherocytosis: Hereditary spherocytosis is the most common hereditary hemolytic anemia in people of northern European descent, occurring in up to 1 in 2500 individuals.²⁴ In the United States, it is estimated that 1 in 5000 people have HS. Most cases of HS are inherited in an autosomal dominant (AD) pattern. However, up to 25% of cases do not display this inheritance; some are new mutations, while others represent AR inheritance.

Hereditary spherocytosis is associated with deficiencies of membrane cytoskeleton proteins (spectrin, ankyrin, or band 3) that lead to destabilization of the RBC lipid bilayer, resulting in progressive membrane surface loss and microspherocyte formation.²⁵ Microspherocytes are vulnerable to entrapment by the spleen, which leads to further membrane loss and ultimately cell destruction by splenic macrophages. A characteristic feature of HS is the presence of spherocytes seen in the peripheral blood smear. However, even normal neonates can have a small population of spherocytes, and as noted previously, spherocytes also are found in neonates with hemolysis due to ABO incompatibility. Older children and adults with HS commonly have anemia, reticulocytosis, indirect hyperbilirubinemia, and splenomegaly.

In neonates, the most common presentation of HS is jaundice. Jaundice may present early, be more severe than usual, persist longer, and may require treatment with phototherapy. Infants with HS rarely require an exchange transfusion. Up to 50% of adults with HS have a history of jaundice during the first week of life.²⁶ Those who coinherit HS and the gene for Gilbert syndrome may develop marked jaundice requiring phototherapy.

The diagnosis of HS can be made in a neonate with a positive family history of HS who has evidence of hemolytic anemia, the presence of an increased number of spherocytes on peripheral smear, elevation of mean corpuscular hemoglobin concentration (MCHC), and a negative DAT. In these cases, further testing is not necessary. However, if the family history is in doubt or if there is coexistence of ABO incompatibility, further testing of the neonate or repeat testing of the affected family member might be necessary to confirm the diagnosis. The incubated RBC osmotic test is used to detect spherocytosis. However, it is important to recognize that this test does not differentiate the various causes of spherocytosis. If there is an ABO incompatibility setup, it is important to obtain a DAT. Occasionally, the establishment of the diagnosis of HS needs to be delayed until confounding factors (maternal antibodies, fetal RBC changes) are resolved. Since management and counseling of neonates with hemolytic anemia is not dependent on the exact cause, the complete laboratory workup can usually be deferred for 4 to 6 months.

Occasionally, severe symptomatic anemia can develop soon after birth and require a PRBC transfusion. More often, anemia develops after discharge from the nursery. It therefore is important to have close follow-up of these infants. While some neonates do not develop significant anemia at all, others may need PRBC transfusions during the first few months of life. Splenectomy is the definitive treatment of HS; however, even in the most severe cases, this surgery is often delayed until a child is at least 5 years old given the increased risk of life-threatening sepsis with encapsulated organisms in young children.

Hereditary Elliptocytosis: The HE syndromes are a heterogeneous group of disorders characterized by the presence of elliptical-shape RBCs in the peripheral blood smear.

Common HE is a dominantly inherited condition characterized by many elliptocytes in the peripheral blood smear. The clinical severity of common HE is extremely variable, ranging from an incidental asymptomatic condition, most commonly observed, to mild-to-moderate hemolytic anemia. The clinical expression of hemolytic HE ranges from a moderate hemolytic disorder to a severe, near-fatal or fatal hemolytic anemia. Hereditary pyropoikilocytosis (HPP) is a severe hemolytic anemia, with red cell fragments, poikilocytes, and microspherocytes seen on peripheral blood smear. From a clinical perspective, it is difficult to distinguish severe hemolytic HE from HPP. Once regarded as a separate condition, HPP is now recognized to be a variant of the HE disorders. Spherocytic HE is a rare condition in which both ovalocytes and spherocytes are present on the blood smear. Southeast Asian ovalocytosis (SAO), also known as stomatocytic elliptocytosis, is an HE variant prevalent in the malaria-infested belt of Southeast Asia and the South Pacific, and it is characterized by rigid spoon-shaped cells that have either a longitudinal slit or a transverse ridge. The varied clinical and hematologic manifestations of HE are an expression of the numerous molecular defects that give rise to an elliptocytic-shape erythrocyte. The most commonly encountered cases seen are nonhemolytic common HE, hemolytic HE, and HPP.

The incidence of HE is estimated at 1:2000–4000 in the United States.²⁷ It is found worldwide but is more common among people of African and Mediterranean decent. HE is inherited in an AD fashion. Heterozygous patients generally have asymptomatic elliptocytosis (common HE) that is often found incidentally. Overall, only 12%–15% of patients with HE are symptomatic at some time during their life.²⁷

Similar to HS, this membrane disorder is due to qualitative and quantitative defects of RBC membrane cytoskeletal proteins. Alternation in the amount, function, and structure of these proteins leads to elliptocyte formation, instability of the RBC membrane, and in some cases hemolysis. In the last subset of HE patients, symptoms include anemia, splenomegaly, and intermittent jaundice.

Most neonates with HE are asymptomatic. However, infants with rare HE subtypes (common HE with infantile poikilocytosis and HPP) may present with severe hemolytic anemia. These neonates present with jaundice, moderate-to-severe anemia, and peripheral blood smear findings of marked poikilocytosis. In the neonatal period, common HE with infantile poikilocytosis and HPP are clinically indistinguishable. However, the poikilocytosis and severe hemolytic anemia are transient in patients with common HE. The distorted RBCs noted at birth morph into the typical HE elliptical shape by a few months of life, and there is complete resolution of the hemolytic anemia. At birth, it is difficult to predict if an infant has common HE or HPP. Hydrops fetalis has been described in association with unusually severe hemolytic HE.

Children with common HE have anywhere from a few up to all elliptocytic RBCs on their peripheral blood smear. However, these elliptocytes usually do not appear until 4–6 months of age, so infants who present to the neonatal units generally have severe hemolytic anemia with poikilocytosis. In all cases, however, infants with hemolytic HE need to be followed to see if their elliptocytosis persists or benign common HE evolves. In the hemolytic variants seen in infants, the peripheral smear may demonstrate red cell fragments, microspherocytes, and some elliptocytes. Osmotic fragility testing in these patients is abnormal, with increased fragility. In addition to typical features of hemolytic anemia, the MCV of those with HPP can be very low (25–75 fL) due to the presence of a large number of microspherocytes.

The management of neonates with hemolytic anemia due to HE is identical to any patient with hemolytic anemia. Most neonates require no treatment, but some may have significant hyperbilirubinemia and anemia requiring phototherapy, exchange transfusion, or PRBC transfusion. Splenectomy later in childhood is helpful in minimizing or resolving the chronic hemolytic anemia in those with severe hemolytic HE or HPP.

Hereditary Stomatocytoses (HSt): HSt syndromes are a group of inherited disorders characterized by erythrocytes with a mouth-shaped (stoma) area of central pallor on peripheral blood smear. HSt is associated with abnormalities in red cell cation permeability that lead to changes in red cell volume. MCV may be increased (hydrocytosis), decreased (xerocytosis), or in some cases, near normal.²⁸ Many patients present with hemolytic anemia in the neonatal period. Pallor, jaundice, hepatosplenomegaly, and signs of gallstone disease (in older patients) are the most common physical findings. Peripheral blood smears show an increased number of stomatocytes (up to 3% is normal). The reticulocyte count is elevated during hemolysis, and the stomatocytes are osmotically fragile in the overhydrated form of stomatocytosis and are resistant to osmotic lysis in the dehydrated form. Neonates with HSt may require phototherapy and occasionally exchange transfusions. Patients with HSt do not generally require splenectomy as the results vary. There is an increased risk of life-threatening thrombosis after splenectomy.²⁹ Cholecystectomy may be considered in patients with cholelithiasis.

Erythrocyte Hemoglobin Abnormalities

Hemoglobin is a tetrameric protein made up of 4 globin chains, each associated with a heme group. The production of globin chains is directed by the alpha (α) gene cluster on chromosome 16 and the beta (β) gene cluster on chromosome 11. The α-gene cluster contains the embryonic delta (ζ) gene and 2 adult α genes. The β-gene cluster contains the embryonic epsilon (e) gene, the fetal gamma (γ) gene, and the adult β gene. During fetal development, embryonic globin production is switched to fetal or adult globin production at different times (ζ → α, e → γ, and γ → β). Various globin chains form different hemoglobin tetramers during embryonic, fetal, and postnatal life (Figure 31-1). Embryonic hemoglobins (Hb Gower 1, Hb Gower 2) are responsible for oxygen delivery during the first 8 weeks of gestation. By 10 weeks, embryonic chain production ceases, γ-chain synthesis ensues, and fetal hemoglobin (HbF: α₂γ₂) becomes the dominant hemoglobin. HbF has a high oxygen affinity, allowing the fetus to extract oxygen across the placenta. At term, 60%–90% of hemoglobin in a newborn is HbF. Following birth, γ-chain production is replaced by β-chain synthesis. By 6 months of age, 95% of hemoglobin will be adult hemoglobin (HbA), which is composed of 2 α and 2 β chains (α₂β₂). At birth, production of δ chains begins. The presence of HbA₂ (α₂δ₂), a minor hemoglobin, increases gradually to the adult level of 2%–3% during the first few months of life.

Congenital hemolytic anemias can be secondary to deficient production of normal hemoglobin (thalassemia syndromes) or production of abnormal hemoglobins (sickle cell disease). The α-globin chain disorders are manifest at birth, while β-globin chain abnormalities may not be clinically apparent until 4–6 months of age, after the switch from γ- to β-globin chain synthesis. Rare mutations affecting γ-globin chains also exist, and these can result in transient neonatal problems that resolve by 3 months of age.

Thalassemia Syndromes: Thalassemia disorders are due to decreased production of normal α- or β-globin chains, leading to a relative excess of the complementary unaffected globin chains. Thalassemias are one of the most common genetic disorders worldwide. Just like G-6-PD deficiency and sickle cell anemia, it is believed that thalassemia heterozygosity is protective against malaria. In the United States, the large influx of Southeast Asian people has led to a significant increase in the number of thalassemia syndromes seen in this country. Newborn screening programs for sickle cell disease also have led to discovery of nonsickling hemoglobinopathies, including thalassemia.

α-Thalassemia Disorders: These conditions are due to decreased or absent α-globin synthesis, a consequence of deletions or mutations of α-globin genes. More than 40 different deletions have been identified.³⁰ Most commonly, α thalassemia occurs in China and Southeast Asia and less frequently in India, Kuwait, the Middle East, Greece, Italy, and northern Europe. In African Americans, α-thalassemia trait is common, with up to 5% prevalence, but it rarely causes clinically significant problems. With increasing immigration to the United States from countries with high carrier rates of thalassemia, the prevalence of significant α thalassemia is rising. Data from the California newborn program, a state with a 13% Asian population, suggest that the prevalence rate of α-thalassemia disorder is 1/9000.³¹

Decreased α-globin chain production is associated with accumulation of the non-α-globin chains, leading to the formation of γ₄ tetramers (Hb Bart) and β₄ tetramers (HbH). The amount of Hb Bart or HbH will vary, depending on the number of α-globin genes deleted and the age of the patient.

The silent carrier state is due to deletion of one α-globin gene. There are no clinical symptoms, and the blood counts are normal.

The α-thalassemia trait is due to deletions of 2 α-globin genes. These individuals have no clinical symptoms but will have microcytosis, hypochromia, and mild anemia with a normal hemoglobin electrophoresis. At birth, the MCV is less than 100 fL, and the Hb Bart level is 2%–10% (normal is 1%). Beyond infancy, Hb Bart disappears, and the only abnormality is mild microcytosis. This is a clinically benign condition; therefore, establishing the diagnoses is not necessary in the neonatal period. In people of African extraction, α-thalassemia trait is due to 2 deletions occurring in trans (2 different chromosomes) vs cis deletions (occurring on the same chromosome), which is what is observed in Asians. If an α-thalassemia syndrome is noted in any Asian child, the parents should be referred for α-thalassemia testing and genetic counseling to determine the risk of their future pregnancies being affected by a more severe type of α thalassemia (see further discussion this chapter). The risk of more serious α-thalassemia conditions is not an issue in people of African background unless the mating partner has α-thalassemia trait and is Asian.

Hemoglobin H disease is due to deletion of 3 α-globin genes (- -/- α). Also, a combination of 2 α-globin deletions and an α-globin gene mutation, most common constant spring (- -/αα^cs), results in HbH disease. The clinical features of HbH disease beyond infancy are characterized by mild-to-moderate chronic microcytic anemia with variable degrees of intermittent hemolysis. HbH is unstable and undergoes denaturation in the presence of oxidant stress, thereby forming intracellular inclusions that damage red cells, leading to early removal from circulation. Similar to G-6-PD deficiency, exposure to oxidants can lead to increased hemolysis and worsening anemia. In most cases, HbH disease does not cause clinical problems other than microcytic anemia in neonates. Diagnosis in the neonatal period generally results from newborn screening tests, which detect increased amounts of Hb Bart (usually greater than 20%). Beyond infancy, children with HbH disease have a mild-to-moderate microcytic anemia (Hb 9–10 g/dL), and there can be evidence of hemolysis, such as indirect hyperbilirubinemia and splenomegaly. These infants and children rarely require RBC transfusions.

Hb Barts hydrops fetalis is due to deletion of all 4 α-globin genes (- -/- -). There are no functional α-globin chains produced; non-α-globin chains accumulate, forming exclusively Hb Bart (γ₄) and HbH (β4). Embryos that lack 4 α-globin genes are well until 8 weeks of age, when production of embryonic hemoglobin ceases. Subsequently, due to lack of α-globin chains, Hb Bart becomes the predominant hemoglobin. Hb Bart has a very high oxygen affinity and is unable to efficiently deliver oxygen. Consequently, these fetuses develop anemia and have severe hypoxia, resulting in hydrops with heart failure, generalized edema, and severe growth failure. Most homozygous α-thalassemia fetuses die in utero, although some survive until term due to persistent expression of embryonic globin chains. These hydropic infants often have congenital anomalies and significant neurologic defects. There are case reports of infants with 4 α-globin gene deletions supported with intrauterine transfusions until delivery. Postdelivery, these infants received PRBC transfusions until bone marrow transplantation. This management strategy is controversial as there remain concerns regarding poor in utero neurodevelopment and long-term outcome.

Besides the fetal effects, there are serious maternal complications in pregnancies of homozygous α thalassemia. Up to 30% of women develop severe preeclampsia. Antepartum hemorrhage, hypertension, renal failure, congestive heart failure, and placental abruption are other known complications. Without proper medical care, up to a 50% mortality rate has been reported.³⁰ Hemoglobin Bart hydrops fetalis can be diagnosed prenatally by DNA-based testing of amniocytes from amniocentesis or chorionic villus sampling. It is important to establish this diagnosis early to avoid the serious maternal complications seen in these pregnancies.

β-Thalassemia Major: β-Thalassemia major, due to mutations in both β-globin genes, is not a neonatal disease. Anemia from β thalassemia is not notable until 3 months of age when γ-globin production decreases. There are 2 groups of β thalassemia. β⁰ thalassemia is caused by complete absence of β-chain production; β⁺ thalassemia has significantly decreased but some β-globin production. Hemoglobin electrophoresis during newborn screening of neonates with β⁰ thalassemia reveals HbF only, consistent with absent HbA production, while β⁺-thalassemia patients will have HbF and very small traces of HbA. The diagnosis of β⁺ thalassemia is therefore only possible after the neonatal period. Infants with β-thalassemia major will become anemic and transfusion dependent by 6–9 months of age. These patients require lifelong transfusions and treatment of iron overload. Bone marrow transplantation is the only curative option.

Hemoglobin E/β thalassemia is a result of coinheritance of HbE trait and β-thalassemia trait. HbE is a β-globin variant most commonly seen in individuals in India and Southeast Asia, including Thailand, Laos, and Cambodia. There is significant clinical heterogeneity among patients with HbE/β thalassemia, ranging from a mild hemolytic process to a severe chronic transfusion-dependent anemia, identical to that seen with β⁰-thalassemia major. HbE/β thalassemia is diagnosed by newborn screening hemoglobin electrophoresis, which will demonstrate an FE or FEA pattern. Just like β-thalassemia major, clinical symptoms do not arise until after 3 months of age. However, once the newborn screening tests reveal this hemoglobin disorder, the infant and family should be referred to a hematology treatment center for advice and guidance.

Sickle Cell Disease: Hemoglobin S is one of the most common β-globin mutations encountered worldwide. The most severe form of sickle cell disease results from inheritance of 2 β^s mutations. This genetic pattern results in what is classically known as sickle cell anemia (HbSS disease). Inheritance of one β^s mutation and one β-thalassemia trait mutation causes sickle β thalassemia (HbS-β thal), which is identical to sickle cell anemia phenotypically except there is also microcytosis. Coinheritance of β^s mutations and β^c mutations results in HbS-C disease, which is a milder form of sickle cell disease. All 50 US states have newborn screening programs for sickle cell disease. Hemoglobin patterns are reported in the order of decreasing hemoglobin concentration. The normal neonatal pattern is fetal/adult (FA). Infants with sickle cell anemia and HbS-β thal will have an FS pattern on their newborn screen, while HbS-C disease will provide an FSC pattern. FSA is seen in Hb S-β thal while FAS indicates sickle cell trait.

Neonates are protected from clinical symptoms of sickle cell disease due to the presence of fetal hemoglobin. However, as HbF concentration decreases after birth, and as HbS production increases, significant complications from cells sickling arise. Newborn screening for sickle cell disease was adopted to ensure early diagnosis to prevent life-threatening complications. Two major complications during infancy are sepsis from encapsulated organisms and splenic sequestration. All neonates with sickle cell anemia should be referred to a sickle cell treatment center for family education and care. Penicillin prophylaxis should begin by 2 months of age, and it is particularly important for sickle cell patients to receive all scheduled vaccines during their first year of life. Sickle cell center staff or primary physicians should provide appropriate education to parents to help them identify splenic sequestration and other sickle cell complications. Couples with a child with sickle cell disease or trait should be referred for further testing and genetic counseling.

Hypoproliferative Disorders

Hypoproliferative anemia refers to anemia caused by impaired erythrocyte production (Table 31-2) and is the least-common etiology of anemia in the neonatal period. Lack of specific growth factors stimulating erythropoiesis can lead to hypoproliferative anemia, such as the anemia of prematurity. Decreased erythrocyte progenitors, as seen in isoimmunization with Kell antibodies, can cause decreased red cell production. Abnormalities resulting in increased Epo resistance or abnormalities in Epo receptor expression can result in a decreased red cell mass. Finally, lack of specific substrates or their carriers (such as iron, folate, vitamin B₁₂, or transcobalamin II deficiency) can also lead to deficient production of red cells.

Congenital/Genetic Hypoproliferative Disorders: Congenital Hypoplastic Anemias

Congenital hypoplastic anemias refer to a rare group of inherited disorders with impaired RBC production. The most common congenital hypoplastic anemia is Diamond-Blackfan anemia (DBA). Other very rare causes include congenital dyserythropoietic anemia (CDA), and Pearson syndrome.

Diamond-Blackfan anemia is an AD disorder, estimated at 4–7 cases per million live births, occurring equally in males and females. With the discovery of multiple ribosomal gene mutations and deletions in patients with DBA, it is now firmly established that DBA is a ribosomopathy.³² Mutations in 9 different ribosomal protein genes (RPL 5, RPL 11, RPL 35A, RPS 7, RPS 10, RPS 17, RPS 19, RPS 24, and RPS 26) have been confirmed to be associated with 50% of DBA patients. An additional 20% of patients have deletions of these same ribosomal protein genes, while 30% of patients with DBA remain genetically unclassified.

In children 6 months of age to toddler years, the main differential diagnosis of RBC aplasia is DBA vs transient erythroblastopenia of childhood (TEC). However, TEC is rarely ever seen in newborns and is unusual in the first 6 months of life. Other causes of red cell aplasia include congenital parvovirus infection, maternal drug exposure, or a marrow infiltrative process such as congenital leukemia. Anemia with reticulocytopenia can also be seen in newborns who have received in utero PRBC transfusions.

Diamond-Blackfan anemia usually presents in infancy, with approximately 90% of patients diagnosed by 12 months of age. Over 25% of affected infants have severe anemia. Newborns with DBA are otherwise healthy (aside from associated congenital defects) but have severe anemia and reticulocytopenia. DBA is usually an isolated anemia, with other cell lines normal. The anemia is generally macrocytic, but the increased MCV is only appreciated beyond the neonatal period given the large RBC size during the first few months of life. If a bone marrow aspirate is done, it typically reveals normal overall cellularity with a marked decrease in erythroid precursors.

An important clinical feature of DBA is the presence of congenital anomalies in over 50% of patients. Craniofacial anomalies are most common, but upper limb, hand (particularly thumb), genitourinary, and cardiac anomalies are common. These features can be subtle and may be hard to recognize at birth. The constellation of congenital hypoplastic anemia associated with skeletal anomalies, cleft palate, growth failure, and triphalangial thumbs was previously coined Aase syndrome.³³ It is now recognized that this is one of the variants of DBA. Diagnosis of DBA is supported by detecting elevated erythrocyte adenosine deaminase (eADA) activity, found in over 80% of DBA patients.³⁴ Genetic testing for ribosomal protein mutations is available through some commercial gene diagnostic laboratories.

Packed RBC transfusions are recommended as initial treatment if needed until 1 year of age.³⁵ After this period, steroids are started if the child remains significantly anemic. About 80% of children will respond to steroids, with reticulocytosis noted within 1–2 weeks after initiation of therapy. The natural history of DBA is variable, ranging from a need for chronic red cell transfusions, to a need for continuous administration of small doses of steroids, to spontaneous remission after years of transfusion or steroids. Treatment for those who are steroid refractory or steroid intolerant includes regular PRBC transfusions and iron chelation therapy for preventing and treating iron overload. Hematopoietic stem cell transplantation is the only curative option for DBA, and outcome of matched sibling transplants has been encouraging, with overall survival of greater than 75%. Increasing amounts of data have arisen to support the observation that DBA is a cancer predisposition syndrome. Leukemia and solid tumors have both been reported.³⁶

Congenital dyserythropoietic anemia is a rare disorder marked by ineffective erythropoiesis, macrocytic or normocytic anemia, and characteristic abnormalities of the nuclear membrane and cytoplasm seen on electron microscopy. Both AD and AR inheritance patterns have been reported. Three types of CDA have been described.³⁷ Type I CDA is characterized by AD inheritance, macrocytic anemia, nuclear chromatin bridges between marrow erythroblasts, and erythroid hyperplasia. Type I CDA has been linked to chromosome 15q15.1–15.3 in a large cohort, and the mutated gene is CDAN1 (codanin-1). Type II, the most common form of CDA, is characterized by normocytic anemia, erythroblastic multinuclearity, and a positive acidified serum test result. A specific mutation in the SEC23B gene on chromosome 20p has been demonstrated in this condition. CDA II appears to result from enzymatic defects in the cellular glycosylation pathway that affects band 3 anion transport activity. Type III CDA is marked by erythroblastic multinuclearity and macrocytosis.

Congenital dyserythropoietic anemia can present in the newborn period with macrocytic anemia, early jaundice, hepatosplenomegaly, and intrauterine growth retardation. Infants with type I CDA may have bony abnormalities and syndactyly. Fetuses with CDA can present with hydrops fetalis. Although rare, this disorder should be included in the differential diagnosis of newborns with anemia, jaundice, and hepatosplenomegaly. Treatment of this disorder consists of supportive therapy and close observation for side effects of chronic transfusions. Splenectomy may be helpful in some older patients with severe anemia.

Pearson marrow pancreas syndrome is a disorder involving the hematopoietic system, exocrine pancreas, liver, and kidneys. It presents in infancy with macrocytic anemia, sometimes associated with neutropenia and thrombocytopenia. The bone marrow has normal cellularity but with erythrocyte abnormalities, including vacuolization of erythroid and myeloid precursors and ringed sideroblasts. The disorder is associated with deletions in mitochondrial DNA and appears to involve defects in oxidative phosphorylation. There is no specific therapy. The disorder is usually considered fatal. Some of the few children who survive the hematologic cytopenias later go on to have the clinical and laboratory features associated with Kearns-Sayre syndrome.

Fanconi anemia is an AR-inherited disorder characterized by marrow failure. In many patients, there are congenital anomalies, including abnormalities in skin pigmentation, gastrointestinal anomalies, renal defects, and upper limb anomalies. In addition, FA is associated with an increased risk of myeloid leukemia and squamous cell cancers, occurring at a relatively younger age than when these tumors usually appear. This disorder is due to a defect in DNA repair, and 13 different FA genes have been identified.³⁸ Peripheral blood lymphocytes are hypersensitive to DNA cross-linking agents such as diepoxybutane (DEB) and mitomycin C, representing a sensitive and specific diagnostic test for FA. The mean age of diagnosis is 6.5 years. The hematologic features of FA are rarely ever present in the newborn period. When FA is identified in neonates, it is because early recognition of congenital abnormalities initially led to specific testing for FA. Definitive treatment of FA necessitates stem cell transplantation, which can ameliorate the hematologic problems, although the increased risk of solid tumors remains.

Anemia of Prematurity

In utero, serum Epo concentrations gradually increase through the third trimester and are highest at term. Epo measurements made on neonatal cord blood of laboring and nonlaboring mothers can reflect hypoxic stress during labor and delivery. An increase in NRBCs can be seen with chronic in utero hypoxic stress; however, acute stress (less than 24 hours) may be associated with increased Epo concentrations alone. Serum Epo concentrations at birth normally range from 5 to 100 mU/mL, while Epo concentrations in anemic, nonuremic adults range from 300 to 400 mU/mL.

In preterm infants, adaptive mechanisms to the extrauterine environment are incomplete, and the transition to term organ function often follows gestational age, regardless of premature delivery. In anemic preterm infants, Epo concentrations are significantly lower than those found in adults, given the degree of their anemia. This normocytic, normochromic anemia is termed the anemia of prematurity. It commonly affects infants 32 weeks’ gestation or less and is the most common anemia seen in the neonatal period.³⁹ The anemia of prematurity is minimally responsive to the addition of iron, folate, vitamin E, or other erythropoietic nutrients. Some infants may be asymptomatic despite hemoglobin concentrations below 8 g/dL, while others demonstrate signs of anemia that appear alleviated by transfusion. Signs associated with the anemia of prematurity include tachycardia, increased episodes of apnea and bradycardia, poor weight gain, tachypnea, an increased oxygen requirement, and elevated serum lactate concentrations that decrease following PRBC transfusion.

Multiple studies evaluating the use of recombinant Epo to prevent and treat the anemia of prematurity have been published to show that Epo is successful in preterm infants in stimulating erythropoiesis (described in the treatment section of this chapter). Epo recipients will maintain hematocrits 4%–6% greater than placebo recipients (Figure 31-2), total transfusions are decreased, and the number of nontransfused infants are increased.

Fetal/Neonatal Anemia due to Congenital Infection

Infections before and after birth can lead to anemia through a hemorrhagic, hemolytic, or hypoproliferative process or a combination of processes. Neonatal sepsis due to Escherichia coli, group B streptococcus, and other perinatal organisms may result in hemolysis, DIC, and hemorrhage. Infants are often jaundiced and have hepatosplenomegaly, although the degree of hyperbilirubinemia does not always correlate with the degree of anemia. Infants may have an elevated direct bilirubin as well, possibly due to infectious involvement with the liver. Bacteria such as E coli will produce hemolytic endotoxins, which result in increased red cell destruction, often associated with a microangiopathic process.

Fetal and neonatal parvovirus B19 infection can cause severe anemia, hydrops, and fetal demise.⁴⁰ The infant generally presents with a hypoplastic anemia, but hemolysis can occur as well. The virus replicates in erythroid progenitor cells and shuts down erythropoiesis, resulting in aplastic anemia. In utero transfusions for hydropic fetuses have been investigated but are not successful in all patients. Treatment during aplastic crises with intravenous immunoglobulin (IVIG) leads to resolution of the anemia.

Both malaria and the human immunodeficiency virus (HIV) can be associated with neonatal anemia. Congenital malaria may occur in major urban areas, where imported cases of malaria are increasing. Congenital HIV infection can be asymptomatic in newborns. Infants born to mothers on zidovudine (AZT) may have hypoplastic anemia due to transplacental side effects of AZT.

Other Erythropoietin-Responsive Anemias in the Newborn Period

Late Anemia of Rh Disease. Early anemia in infants with Rh hemolytic disease is caused by ongoing antibody-mediated hemolysis, but a late (age 1 to 3 months) anemia resulting from diminished erythrocyte production can also occur. Late anemia appears to be more common in infants who receive intrauterine transfusions and is characterized by low serum concentrations of Epo but erythroid progenitors that remain highly responsive to recombinant Epo in vitro. Infants with this late anemia will often receive PRBC transfusions until the late anemia resolves, generally by the third or fourth month of life. Following the active hemolytic period when circulating anti-D antibody levels are elevated, the administration of Epo is effective at stimulating erythropoiesis and can serve as an alternative to erythrocyte transfusion.⁴¹ Outpatient Epo administration may also diminish or eliminate the need for hospitalization in those centers in which infants are hospitalized for transfusions.

Kell isoimmunization is a unique hemolytic disorder that selectively impacts marrow erythroid precursors, resulting in both hemolytic and hypoproliferative anemia. Kell positive infants born to Kell-negative mothers can present with severe and protracted anemia.³⁷ Epo administration for neonatal anemia after active hemolysis is resolved can stimulate erythropoiesis.

Anemia of Bronchopulmonary Dysplasia. Anemia can develop in patients with bronchopulmonary dysplasia (BPD). The anemia of BPD is characterized as normocytic, normochromic, hyporegenerative anemia, with marrow normoblast iron stains that are distinct from those observed in both the anemia of chronic disorders and the anemia of prematurity. Similar to the late anemia of Rh hemolytic disease, the anemia of BPD responds to Epo administration. The explanation for reduced Epo production in patients with BPD requires further study to determine if other factors that could create a relatively Epo-resistant environment are involved, such as interleukin (IL) 1, tumor necrosis factor, and interferon γ.

Anemia in Neonates Requiring Surgery. Neonates who are born with problems that require surgical repair, such as congenital diaphragmatic hernia, gastroschisis, omphalocele, meningomyelocele, and craniofacial abnormalities often undergo surgery during the period of physiologic anemia. These hospitalized infants undergo blood loss through phlebotomy, the surgery itself, and postoperative care. As a result, the physiologic anemia is exacerbated, and transfusions are often administered to increase hemoglobin concentrations, especially around the time of surgery. These infants respond to Epo administration by increasing erythropoiesis⁴² and may also benefit from the nonhematopoietic, mitogenic effects of Epo, such as an increase in villous growth.

In addition to neonates who require surgery, other neonatal populations may benefit from Epo therapy. Epo has been used successfully to treat anemia in newborns with end-stage renal disease due to congenital nephrotic syndrome or polycystic kidney disease and in infants with congenital heart disease awaiting surgery. In addition, infants with hemolytic disease from ABO incompatibility and HS have been treated with Epo.⁴³ Further studies are required in these populations to determine whether treatment with Epo is beneficial and does not cause further harm through increased hemolysis and hyperbilirubinemia.

Hemorrhage

The process of blood loss from the intravascular space can be either acute or chronic and can occur at any time during the prenatal, perinatal, or postnatal periods (Table 31-3). Acute hemorrhage generally results in a change in hemoglobin and hematocrit only, while chronic hemorrhage can result in changes to other red cell indices, such as MCV, MCHC, reticulocyte count, and red cell distribution width (RDW).

Prenatal Hemorrhage

Twin-to-twin transfusion syndrome (TTS) is a complication of monochorionic twin gestations and occurs in 5% to 30% of these pregnancies. Depending on the timing and severity of presentation, the perinatal mortality rate can be as high as 70%–100%. In TTS, placental anastomoses allow transfer of blood from one twin to the other. Approximately 70% of monozygous twin pregnancies have monochorionic placentas. Although vascular anastomoses are present in almost all instances of monochorionic placentas, not all anastomoses result in TTS.

Acute TTS usually results in twins of similar size with hemoglobin concentrations that vary by more than 5 g/dL. Chronic TTS results in the donor twin becoming progressively anemic and growth retarded, while the recipient twin becomes polycythemic, macrosomic, and sometimes hypertensive. The donor twin can become hydropic from profound anemia, while the recipient twin can develop hydrops from congestive heart failure and hypervolemia. The donor twin exists in diminished amniotic fluid volumes, while the recipient twin has increased amniotic fluid due to significant differences in blood volume, renal blood flow, and urine output.

Chronic TTS can be diagnosed by serial prenatal ultrasounds measuring cardiomegaly, discordant amniotic fluid volume, and fetal growth discrepancy of more than 20%. After birth, the donor twin may be anemic enough to require transfusions and can also experience hypoglycemia, neutropenia, hydrops from severe anemia, growth retardation, and congestive heart failure. The recipient is often the sicker twin, experiencing congestive heart failure associated with hypertrophic cardiomyopathy, hypocalcemia, hypoglycemia, polycythemia, hyperviscosity, and respiratory difficulties. Neurologic evaluation and imaging are important as the risk of antenatally acquired neurologic cerebral lesions is 20% to 30% in both twins. Morbidities include hypoperfusion syndromes from hypotension, multiple cerebral infarctions, and periventricular leukomalacia.⁴⁴ Long-term neurologic follow-up is necessary and important for all TTS survivors.

Treatment options vary but should include close monitoring and reduction amniocenteses if needed to decrease uterine stretch and prolong the pregnancy. The average survival rates with serial reduction amniocenteses range from 40% to 70%. Selective fetocide of the hydropic twin has also resulted in the survival of the healthier twin in some studies. Laser surgery to ablate bridging vessels has the best outcomes for TTS pregnancies, resulting in improved survival rates of up to around 50%, with approximately 70% of the pregnancies having at least one survivor. The survival rate without morbidity in the surviving twin is approximately 50%. Meta-analyses have found no differences in outcome between amnioreduction, fetoscopy, septostomy, or close observation for fetuses with TTS.⁴⁵

Feto-Maternal Hemorrhage. Maternal and fetal circulating cells may cross the placental barrier at varying times during the pregnancy. The passage of fetal red cells into the maternal circulation is termed feto-maternal hemorrhage (FMH). Approximately 50%–75% of pregnancies are associated with some degree of FMH, usually occurring after the first trimester. Commonly, the volume of fetal blood transferred into the maternal circulation is relatively small, usually on the order of 0.01 to 0.1 mL. About 1 pregnancy in 400 is associated with an FMH of 30 mL or greater, and about 1 pregnancy in 2000 is associated with an FMH of 100 mL or more.⁴⁶

In an Rh-incompatible pregnancy, the overall risk of maternal blood group sensitization due to FMH is 16% if the fetus is Rh positive and ABO. The risk decreases to 1.5% if the fetus is Rh positive but ABO incompatible due to the destruction of incompatible cells early during placental transfer. Fetal transfer of cells to the mother occurs during abortions as well: there is a 2% incidence of such transfer with spontaneous abortion and a 4%–5% rate with therapeutic abortion.

The Kleihauer-Betke stain (KB stain) evaluates the acid elution of hemoglobin from red cells on a sample of maternal blood. Fetal hemoglobin resists acid elution to a greater degree than adult hemoglobin. Maternal cells (in which adult hemoglobin has been eluted from the cell) appear clear (termed ghost cells), while the contaminating fetal cells appear pink.

KB stains obtained from mothers with increased fetal hemoglobin synthesis, such as mothers with sickle cell disease, thalassemia, or hereditary persistence of fetal hemoglobin, are not reliable; in these cases, other methods should be used to detect FMH, such as flow cytometry using anti-HbF antibodies.⁴⁷ Diagnosis of a small-volume FMH may be difficult when the mother and infant are ABO incompatible as fetal cells are rapidly cleared from the maternal circulation.

One in 1000 deliveries is associated with severe (>200 mL) FMH. Severe FMH has been associated with decreased fetal movements and a fetal sinusoidal heart rate (SHR) pattern. Studies noted that decreased/absent fetal movements for a period ranging between 24 hours and 7 days were present for 10%–15% of cases evaluated.⁴⁸ In this group, two-thirds of the infants either died in utero or during the neonatal period. An SHR pattern was reported in 15% of cases and was associated with decreased fetal movement in 40% of the cases.

Infants need rapid evaluation and treatment if a significant hemorrhage is suspected. The infant with massive hemorrhage will present with pallor, tachypnea, and delayed capillary refill but may not have a significant oxygen requirement. Hemoglobin concentrations can be extremely low at birth, less than 6 g/dL. Significant metabolic acidosis is often present as a result of poor perfusion.

Other causes of pallor in the newborn period do exist and should be ruled out once the infant is stable. Infants with asphyxia and infants with chronic hemolytic anemia may also present with pallor (Table 31-4). These diagnoses can be distinguished from acute hemorrhage based on differences in clinical signs (Table 31-5). With chronic hemolytic anemia, clinical signs are mild or absent, and infants respond to conservative therapy with iron alone. Asphyxiated infants will be pale and floppy and may have poor peripheral circulation. The hemoglobin will be stable but may decrease if DIC and subsequent bleeding occur.

Other causes of prenatal hemorrhage include vaginal bleeding due to placenta previa or abruption, nonelective cesarean section, and deliveries associated with cord compression. Significant FMH has been described following trauma, and fetal hemorrhage into the placenta has been associated with placental chorioangioma.

Perinatal Hemorrhage

Obstetrical complications such as placenta previa, placental abruption, incision or tearing of the placenta during cesarean section, and cord evulsion of normal or abnormal umbilical cords can result in significant neonatal blood loss at the time of delivery. Placental anomalies such as a multilobed placenta and placental chorioangiomas may also be a source of hemorrhage during the perinatal period.

Placental abruption occurs when there is separation of the placenta from the uterus prior to delivery. Severe fetal growth restriction, prolonged rupture of membranes, chorioamnionitis, hypertension (chronic and pregnancy induced), cigarette smoking, advanced maternal age, and male gender are all risk factors for placental abruption.⁴⁹ Abruption occurs in approximately 1% of term deliveries, and the incidence of abruption increases with decreasing gestation. Mortality ranges 0.8 to 2 per 1000 births and can be as high as 15% to 20% of the deliveries in which significant abruption occurs. The risk of stillbirth increases significantly when abruption exceeds 50%.

Women with a history of a previous cesarean birth and increased parity are at increased risk of having a pregnancy complicated by placenta previa,⁵⁰ a condition in which part or all of the placenta overlies the cervical os. Cigarette smoking increases the risk of placenta previa 2.6- to 4.4-fold. Prenatal diagnosis of vasa previa (anomalous vessels overlying the internal os of the cervix) can be performed with transvaginal color Doppler and should be suspected in any case of antepartum or intrapartum hemorrhage. Although uncommon (1 in 3000 deliveries), the perinatal death rate is high, ranging from 33% to 100% when undetected before delivery.⁵¹ Infants are often stillborn.

Infants born following placental abruption or placenta previa may be anemic and may also present with signs of hypoxia. In these infants, it is important to monitor changes in hematocrit and neurologic signs. The need for postnatal transfusions in the infants is generally associated with the volume of perinatal hemorrhage. The infant’s hemoglobin should be measured at birth and again at 12 to 24 hours whenever there is evidence of placental abruption, placenta previa, or any significant vaginal bleeding. A KB stain can be performed on maternal blood to determine if FMH occurred. Monitoring mothers with a history of second- or third-trimester bleeding with Doppler flow ultrasound to detect placental abnormalities may decrease the incidence of fetal loss and anemia in newborns.

Cord rupture due to excess traction on a shortened or abnormal umbilical cord usually occurs closer to the fetus than the placenta. Cord aneurysms, varices, and cysts can all lead to a weakened cord. Cord infections, termed funicitis, can also weaken the cord and increase the risk of rupture. In addition, infants born precipitously may be at increased risk for a ruptured cord.

Hematomas of the cord occur infrequently (1 in 5000–6000 births) but can result in fetal blood loss and may be associated with significant perinatal mortality. Intrauterine fetal demise may occur due to compression of the umbilical vein and arteries by the hematoma. Cord hematomas can result from trauma due to percutaneous umbilical blood sampling (PUBS) and can also be associated with a high maternal α-fetoprotein. Cord hematomas can be accurately diagnosed in utero by ultrasound and differentiated from other lesions of the placenta and cord. The lesion can also result in poor fetal growth and FMH.

Velamentous insertion of the umbilical cord occurs when the umbilical cord enters the membranes distant from the placenta and is present in approximately 0.5% to 2% of pregnancies. Blood vessels left unprotected by Wharton’s jelly are more vulnerable to tension on the cord, and rupture of anomalous vessels in the absence of traction or trauma can occur even if the cord itself attaches centrally or paracentrally. Fetal mortality is high with velementous insertion, often because detection by routine ultrasound is rare.⁵²

Postnatal Hemorrhage

Blood loss into the placenta, termed fetoplacental hemorrhage, is one of the most common etiologies for a low hematocrit at birth. Of the 120 mL/kg of blood in the fetoplacental unit, approximately one-third remains in the placenta, and blood will continue to flow in the direction of gravity after birth. Fetoplacental hemorrhage occurs when the infant is held above the placenta after birth; for this reason, infants born by cesarean section have smaller blood volumes than those born vaginally.⁵³ In addition, infants can lose 10% to 20% of their total blood volume when born with a tight nuchal cord, which allows blood to be pumped through umbilical arteries toward the placenta while constricting flow through the umbilical vein.

Blood loss into the subgaleal space is referred to as a subgaleal hemorrhage and can occur during difficult deliveries requiring vacuum or forceps assistance, such as face presentation, occiput posterior presentation, and shoulder dystocia. It is a potentially life-threatening event and must be recognized as early as possible to prevent significant morbidity or mortality. Subgaleal hemorrhage occurs when emissary or “bridging” veins are torn, allowing blood to accumulate in the large potential space between the galea aponeurotica (the epicranial aponeurosis) and the periosteum of the skull (Figure 31-3). The subgaleal space extends from the orbital ridge to the base of the skull and can accommodate an infant’s entire blood volume, or 80 to 90 mL/kg of blood.

A subgaleal hemorrhage may occur because of preexisting risk factors, such as coagulopathy or asphyxia. The diagnosis should be considered in the presence of a ballotable fluid collection in dependent regions of the infant’s head, coupled with signs of hypovolemia such as tachycardia and metabolic acidosis. A rule of thumb for estimating the volume of blood lost is that every centimeter increase in head circumference represents 38 mL of blood loss. Treatment requires restoration of blood volume and control of bleeding. Exsanguination due to subgaleal hemorrhage has been reported, and the mortality is high if the hemorrhage goes unrecognized.

Infants undergoing vacuum extraction have an increased risk for subgaleal hemorrhage.⁵⁴ The duration of vacuum application is thought to be the best predictor of scalp injury, followed by duration of second stage of labor and paramedian cup placement. Of those infants with reported subgaleal hemorrhages, 60%–90% had some history of vacuum or instrument-assisted delivery. A cesarean delivery does not preclude the use of vacuum or forceps, and subgaleal hemorrhage can still occur via this route of delivery. Limiting the frequency and duration of vacuum assistance in high-risk infants may decrease the risk.

Infants with neonatal alloimmune thrombocytopenia (NAIT) are at increased risk for pre- and postnatal intracranial hemorrhage. NAIT results from platelet-antigen incompatibility between the mother and fetus and occurs in 1 to 2 per 1000 live births. Antibodies to human platelet antigen (HPA) 1a antigen occur most commonly. Of newborns with platelet alloimmunization, 10%–20% will have an intracranial hemorrhage, of which half occur in utero.⁵⁵ The risk for intracranial hemorrhage is increased in newborns with an affected sibling who had an antenatal intracranial hemorrhage; similar to RhD isoimmunization, the subsequent affected newborns generally have more severe disease than the first sibling. Treatment includes IVIG and platelet transfusions in severe cases.⁵⁶ Platelets obtained from the mother have a longer half-life because they lack the antigen causing consumption. Treatment of fetal thrombocytopenia includes maternal IVIG administration, maternal steroid administration, and fetal platelet transfusions.

Anemia appearing after the first 24 hours of life in an unjaundiced infant may result from postnatal hemorrhage. In addition to birth trauma causing visible hemorrhage such as a cephalohematoma, internal hemorrhage to liver, spleen, or other organs can occur. Breech deliveries may be associated with renal, adrenal, or splenic hemorrhage into the retroperitoneal space. Delivery of macrosomic infants, such as infants born to diabetic mothers, may also result in organ damage and hemorrhage.

The incidence of adrenal hemorrhage is not uncommon, occurring in 1.7 per 1000 births. In addition to causing anemia, adrenal hemorrhage may result in circulatory collapse due to the loss of organ function. Adrenal hemorrhage can also affect surrounding organs. Intestinal obstruction and kidney dysfunction have been reported in infants with adrenal hemorrhage. Diagnosis can be made using ultrasonography, which notes calcifications or cystic masses. Adrenal hemorrhage can be distinguished from renal vein thrombosis (RVT) by ultrasound, in that RVT generally results in a solid mass. Occasionally, both entities may coexist in the same patient. Infants with RVT may have gross or microscopic hematuria and may develop renal failure and hypertension.

Splenic rupture can result from birth trauma or as a result of distention caused by extramedullary hematopoiesis, such as that seen in erythroblastosis fetalis. Abdominal distension and discoloration, scrotal swelling, and pallor are clinical signs of splenic rupture.

The newborn liver is prone to iatrogenic rupture, resulting in high morbidity and mortality. Infants may appear asymptomatic until significant hemoperitoneum occurs. Hepatic rupture and hemorrhage occur in both term and preterm infants and have been associated with chest compressions during cardiopulmonary resuscitation. Surgical intervention involving vascular tamponade has been reported to save some infants; however, the mortality remains high.

Other rare causes of hemorrhage in the newborn include hemangiomas of the gastrointestinal tract, characteristic of Kassaback-Merrit syndrome; vascular malformations of the skin; and hemorrhage into soft tumors, such as giant sacrococcygeal teratomas, thoracic hamartomas, or cystic hygromas. Occult intra-abdominal hemorrhage can occur with fetal ovarian cysts, which are commonly benign and resolve spontaneously.

DIAGNOSIS

Clinical Assessment

The diagnosis of anemia in the perinatal and postnatal period often requires rapid investigation, laboratory evaluation, and treatment. An immediate assessment of the infant includes determining whether the infant is suffering from acute or chronic anemia, as treatments vary significantly (Table 31-5). Infants presenting in shock from their anemia have most likely suffered massive hemorrhage, and patient stabilization is of utmost importance to avoid organ damage and death. The “ABCs” of newborn resuscitation should be applied: stabilize the infant’s airway, administer oxygen and intubate if necessary, and determine if the infant’s cardiovascular and intravascular volume status is adequate. Immediate volume expansion using normal saline should be performed when a significant acute hemorrhage is suspected. A transfusion of type O, Rh-negative blood (“trauma” blood) may be required in the first hour if massive hemorrhage has occurred. Repeat transfusions of cross-matched PRBCs may be necessary to treat lactic acidosis if oxygen delivery to the tissues is not improved with volume expansion.

It is helpful to identify the timing of presentation of anemia. Infants with significant acute blood loss before or during delivery may be anemic and hypovolemic at birth, while infants with TTS, chronic FMH, or chronic ongoing hemolysis from isoimmunization may not be immediately symptomatic. Infants with internal trauma and hemorrhage (hepatic, splenic, adrenal, or renal) may not initially be symptomatic but may rapidly decompensate as compensation for intravascular volume loss fails. Attention to details of the infant’s transition period from fetal to postnatal life may be life-saving. As an example, an infant suffering from subgaleal hemorrhage will experience an increased heart and respiratory rate during stage 2 of transition (instead of the expected decrease in heart rate and respiratory rate) due to intravascular volume loss and worsening metabolic acidosis. Close observation during transition will allow rapid diagnosis and potentially life-saving treatment for the infant.

Maternal, Labor, and Delivery History

Once the infant is stable, information on maternal, labor, and delivery history can be gathered from both the mother’s and infant’s chart to help determine the cause of anemia. Any family history of anemia, bleeding, “low blood” counts, transfusions, jaundice, or unusual hematologic indices should be identified. Ethnicity of both parents is important to note, as some inherited disorders (eg, G-6-PD deficiency or thalassemia) are more prevalent in specific ethnic groups.

Obtaining a thorough labor and delivery history is important, including information on vaginal bleeding, trauma, infection or exposure to infected individuals, and any prescribed or nonprescribed drug use during the pregnancy. The use of cocaine prior to delivery may increase the potential for placental abruption, fetal infarction, and postinfarction hemorrhage. Important maternal laboratory information includes blood type and antibody screen, maternal hepatitis, and syphilis and rubella status.

Detailed information regarding labor and delivery can sometimes be difficult to obtain, especially if delivery was not anticipated, such as an emergent cesarean section for loss of fetal heart tones. Episodes of fetal tachycardia or decelerations or other evidence of fetal heart rate abnormalities such as loss of beat-to-beat variability should be noted. The length of labor, vaginal bleeding, evidence of placental abruption, knowledge of placenta previa or vasa previa, and route of delivery should be noted. Information regarding the placenta (cord hematoma, cord rupture, chorioangioma, velementous insertion of the cord) should be gathered and the placenta examined. The use of forceps, vacuum, or other manipulations is important to note. Finally, it is important to identify the presence of multiple gestations, especially those associated with discordant growth.

Laboratory Evaluation

Laboratory evaluation of the anemic infant includes a complete blood count including RBC indices and peripheral smear, a reticulocyte count, a direct Coombs test, and total and direct bilirubin. A KB stain of maternal blood can identify fetal cells in the maternal circulation if FMH is suspected. With a thorough history, physical examination, and minimal laboratory evaluation, most causes of anemia in the newborn period can be determined.

TREATMENT

PRBC Transfusion

Treatment of anemia in neonates should be guided by the timing and etiology of the anemia. The management of hemorrhage in the neonatal period depends in part on the etiology of the hemorrhage, its timing, and the extent of hemorrhage. Infants with chronic hemorrhage, such as those with chronic FMH or TTS, may have had time to compensate for a gradual decrease in hemoglobin and may therefore have an adequate intravascular volume despite a low circulating erythrocyte volume (Table 31-5). Those infants will require iron supplementation to replace iron stores depleted by the loss of red cells; however, they are unlikely to require an immediate PRBC transfusion or even volume expansion. Infants experiencing an acute hemorrhage will require volume expansion to improve circulation and organ perfusion.

Treatment of anemia due to hemolysis involves understanding the etiology of the hemolysis, the rate of red cell destruction, and the benefits of stimulating neonatal erythropoiesis vs administering an erythrocyte transfusion. Management of hemolytic anemia in neonates will first include managing hyperbilirubinemia. If hemolysis is significant enough and hyperbilirubinemia severe enough to require a double-volume exchange (generally with immune-mediated hemolysis), then correction of hematocrit can occur with the double-volume exchange transfusion. If a double-volume exchange transfusion is not required, acute anemia should be managed with PRBC transfusion. Epo is contraindicated when circulating antibodies directed against the infant’s blood type are still significantly elevated, as stimulating neonatal erythropoiesis may result in increased hemolysis.

Treatment of anemia due to hypoproliferative disorders has expanded with the use of erythropoiesis-stimulating agents (ESAs). Most hypoproliferative disorders are associated to a varying degree with low Epo production; thus, treatment with ESAs to increase serum Epo concentrations is often effective.

The decision to administer an immediate transfusion of O-negative “trauma” PRBCs should be made carefully, and the emergency use of whole blood prior to type and cross match is generally discouraged. Type O-negative whole blood will contain antibodies directed against A and B blood groups and leukocytes. It should only be used in a hemorrhagic emergency or if the infant’s blood type is known to be O negative.

Immediate transfusions will benefit those infants with significant metabolic acidosis and oxygen requirement, generally those with greater than 30%–40% acute blood loss. When considering a transfusion in a preterm infant with a low hematocrit (not associated with an acute drop in hematocrit), the clinician should first determine if the infant needs an immediate increase in oxygen to tissues. If the answer is yes, then treatment consists of a transfusion of PRBCs. If there is no evidence that an immediate increase in oxygen delivery is necessary, then treatment with red cell growth factors and appropriate substrates might be considered. An example of guidelines used to administer transfusions is shown in Table 31-6.

Treatment of anemic infants with modalities beyond volume expansion, PRBC transfusion, and vitamin and iron supplementation depends on the clinical care offered. The use of recombinant Epo to stimulate erythropoiesis has become more common in neonatal intensive care units; its role in the newborn nursery and in outpatient treatment continues to be refined.

Erythropoiesis-Stimulating Agents

The in vitro response of erythroid progenitors from preterm infants led investigators to begin evaluating Epo administration to preterm infants. Since the early 1990s, numerous studies evaluating the use of Epo to prevent and treat the anemia of prematurity have been performed.³⁹ The administration of Epo successfully stimulates erythropoiesis in preterm infants, and transfusion requirements are decreased. Success rates in preventing transfusions in preterm infants are dependent in part on transfusion criteria and the volume of phlebotomy losses. Side effects of Epo in published randomized controlled trials (RCTs) have not differed from placebo, although meta-analyses have suggested an association between early Epo administration and retinopathy of prematurity (ROP). Because a single relevant study was mistakenly categorized in the early Epo analysis instead of the late analysis, this association was incorrectly determined to be statistically significant. This error will be corrected in the next analysis. The development of ROP in preterm infants is likely multifactorial. Moreover, there is evidence in some adult studies of protective effects of Epo in diabetic retinopathy.

Darbepoetin alfa (Darbe), a biologically modified version of Epo, was developed by Amgen in 1998. Darbe has a longer serum half-life than Epo in adults and preterm infants, so dosing can be spread over 1 to 2 weeks. Side effects are similar to Epo, and the production of anti-Epo antibodies has not been reported. Studies are under way to evaluate short- and long-term effects of extended courses of Darbe administered to preterm infants. Recent results showed decreased donor and transfusion exposure with weekly Darbe doses of 10 μg/kg.⁵⁷

A minimal number of clinical studies evaluating Darbe administration to preterm infants have been published, and randomized trials are ongoing. In contrast, numerous RCTs evaluating Epo administration to preterm infants have consistently shown evidence of increased erythropoiesis and a decrease in transfusions.

A consistent finding in the largest RCTs has been an elevation in hematocrit in the Epo-treated infants compared to those treated with placebo/controls (Figure 31-2). For those neonatal practitioners electing to maintain the hematocrit at higher levels, the use of Epo generally results in an increase of 4%–6% hematocrit points and a decrease in the number of transfusions administered. The increased hematocrit may benefit the infant by increasing available oxygen to tissues and may also provide indirect benefit by avoiding transfusions, which have recently been associated with both necrotizing enterocolitis (NEC) and intraventricular hemorrhage (IVH).^{58, 59, and 60} Guidelines for Epo and vitamin/iron administration to preterm infants as part of clinical care are presented in Table 31-7.

Given the risks of transfusion, such as transmission of infectious agents like hepatitis, Trypanosoma cruzi, West Nile virus, and HIV⁶⁴; the possible development of graft-vs-host disease; and possible associated risks, such as the development of NEC or the worsening of IVH, treatment of anemic neonates using recombinant Epo is an important alternative. Regardless of treatment strategy, a critical understanding of the physiologic influences affecting oxygen availability, delivery, and extraction at the tissue level will best serve clinicians in developing the best evidence-based approach to managing the red cell mass in term and preterm infants.

ACKNOWLEDGMENTS

We wish to thank Erin Adair for figure production and Mary Merchant and Cyndie Suniga for their assistance editing the manuscript.

REFERENCES

INTRODUCTION

Thrombocytopenia is a well-recognized phenomenon in the neonatal intensive care unit (NICU); it can lead to morbidity and mortality from severe and life-threatening bleeding. The epidemiology, pathophysiology, causes, management, and outcome of neonatal thrombo­cytopenia are reviewed in this chapter.

Thrombocytopenia is traditionally defined as a platelet count below 150,000/μL. This definition is based on several population studies, which have demonstrated that in mothers with normal platelet counts, over 98% of healthy term neonates have platelet counts greater than 150,000/μL at birth.^{1, 2, and 3} Thrombocytopenia is rare in healthy newborns; however, it affects up to one-third of patients in the NICU.^{4, 5} The increased incidence of thrombocytopenia in NICU patients is reflective of the inverse relationship of thrombocytopenia risk to birth weight and gestational age.^{4, 6} Christensen et al reported that up to 70% of infants with birth weight less than 1000 g are thrombocytopenic.⁶ Preterm infants, particularly those at less than 24 weeks’ gestation, have platelet counts as low as 100,000/μL in the first few days of life.^{4, 7}

Severe thrombocytopenia is defined as a platelet count of less than 50,000/μL. Severe thrombocytopenia occurs in less than 1% of healthy newborns^{2, 3}, but affects 5%–10% of hospitalized neonates.^{4, 8, 9}

CAUSES OF NEONATAL THROMBOCYTOPENIA

The causes of neonatal thrombocytopenia can be classified into 2 major pathophysiologic categories: increased destruction (including consumption) and decreased production. In some neonates, especially sick preterm infants, both processes can occur concomitantly. The time of thrombocytopenia onset has often been used to help with diagnosis. Early-onset thrombocytopenia occurs within 72 hours of birth, while late-onset thrombocytopenia occurs after 72 hours of life (Table 32-1). Diagnostic algorithms and general management of neonatal thrombocytopenia are highlighted in Chapter 92.

The diagnoses of thrombocytopenia based on underlying pathophysiologic classifications are summarized in Table 32-2. The most common causes include infection, necrotizing enterocolitis (NEC), neonatal alloimune thrombocytopenia, maternal platelet autoantibodies, and chronic fetal hypoxia. The major causes of neonatal thrombocytopenia are reviewed next.

Congenital Infection

Congenital infections are a major cause of thrombocytopenia in the neonatal population. Cytomegalovirus and herpes simplex virus are the most likely of the congenital TORCH (toxoplasmosis, rubella, cytomegalovirus, herpes simplex virus) infections to cause thrombocytopenia. Cytomegalovirus is the most common of the congenital infections, affecting 0.5%–1.5% of live births. Most of these neonates are asymptomatic, but up to 35% develop thrombocytopenia.¹⁰ Other congenital infections that have been implicated include Coxsackievirus, syphilis, varicella zoster virus, human immunodeficiency virus (HIV), and parvovirus B19.

Mechanisms of thrombocytopenia with congenital infection include increased platelet aggregation, hypersplenism secondary to splenomegaly and platelet pooling, and platelet membrane damage secondary to viral neuraminidase cleavage of sialic acid. Infants with congenital infections are typically well appearing but can present with clinical manifestations such as a petechial rash, microcephaly, intracerebral calcifications, and hepatosplenomegaly. Thrombocytopenia is generally mild to moderate (>50,000/μL), and affected infants usually do not have bleeding apart from cutaneous manifestations. Positive results for a specific congenital infection typically confirm the diagnosis. Most patients do not require therapy for thrombocytopenia, and management generally is close observation. Platelet transfusions may be given for rare cases of severe thrombocytopenia or bleeding.

Bacterial and Fungal Infections

Perinatal bacterial infections particularly implicated in neonatal thrombocytopenia include group B Streptococcus (GBS) and Escherichia coli (E. coli). Other late-onset (>72 hours postdelivery) bacterial and fungal infections may also occur, particularly in vulnerable preterm infants. Mechanisms of thrombocytopenia include endothelial damage and increased platelet aggregation caused by adherence of bacterial products to platelet membranes. Neonates can present with vital sign instability, leukocytosis, and mild-to-severe thrombocytopenia. Sepsis and disseminated intravascular coagulation (DIC) may occur and are associated with rapid onset of severe thrombocytopenia, consumptive coagulopathy, and clinical bleeding or thrombosis. Management includes platelet transfusions for severe thrombocytopenia and bleeding, and most important, aggressive treatment of the underlying infection.

Necrotizing Enterocolitis

Necrotizing enterocolitis occurs in up to 10% of neonates, particularly in those with a birth weight less than 1500 g.¹¹ Platelets are destroyed at sites of gut ischemia, injury, and necrosis, resulting in thrombocytopenia. The degree of thrombocytopenia correlates with gut necrosis severity, particularly within the first 3 days of NEC onset.¹² The presence of severe thrombocytopenia may also be predictive of overall morbidity and mortality.¹² DIC can also complicate NEC, thereby increasing morbidity and mortality risk from significant bleeding or thrombosis. Platelet transfusions are given for severe thrombocytopenia and clinical bleeding. Thrombocytopenia typically improves with improvement and resolution of the NEC.

Immune-Mediated Thrombocytopenia

There are 2 major immune-mediated causes of neonatal thrombocytopenia: neonatal alloimmune thrombocytopenia (NAIT) and maternal platelet autoantibodies. Immune-mediated neonatal thrombocytopenia most commonly occurs in otherwise-well, term neonates without other reasons for thrombocytopenia. Patients typically have decreased platelet counts at birth (early-onset thrombocytopenia).

Neonatal Alloimmune Thrombocytopenia

Epidemiology

Neonatal alloimmune thrombocytopenia is estimated to occur in 1:1000 live births.¹³ NAIT is the platelet equivalent of hemolytic disease of the newborn, or Rhesus (Rh) antigen incompatibility. It occurs in the first pregnancy in nearly half of cases, unlike Rh incompatibility, in which neonates are affected only after the index pregnancy.¹³ Differences between Rh incompatibility and NAIT are outlined in Table 32-3. The majority of patients have severe thrombocytopenia, and often the thrombocytopenia is severe (platelets <20,000/μL).¹⁴ Neonates are at high risk of major bleeding, including intracranial hemorrhage (ICH). Large retrospective studies have reported ICH in 10%–20% of untreated NAIT cases.^{15, 16} The bleeding may occur in utero, as thrombocytopenia develops after 20 weeks’ gestation in untreated cases.^{15, 16}

Pathophysiology

Neonatal alloimmune thrombocytopenia results from transplacental passage of maternal alloantibodies against fetal platelets expressing paternal human platelet antigens (HPAs) that the mother lacks. Sixteen HPA antigens have been identified, and 3 of them cause 95% of NAIT in Caucasians^{16, 17}: HPA-1a, HPA-5b, and HPA-15b. HPA-4 has been implicated in NAIT in Asians.^{16, 17} HPA-1a in particular causes nearly 75% of all NAIT cases.^{16, 17} HPA-1a incompatibility occurs in 1:350 pregnancies, but NAIT occurs in only 1:1500 cases.¹⁶ The reason for this disparity is due to differences in human leukocyte antigen (HLA) alleles. Specifically, HLA DRB3*0101-positive women are up to 140 times more likely to form HPA-1a antibodies than are HLA DRB3*0101-negative women, thereby indicating a strong association of HLA with NAIT.^{18, 19}

Differential Diagnosis

The major and most important differential diagnoses are bacterial and congenital infections. Evaluation for bacteremia with blood counts and cultures is particularly important in the vulnerable neonatal population, and empiric broad-spectrum antibiotic therapy is warranted until bacteremia is excluded. Patients with congenital infections typically have other physical stigmata (see the section on congenital infections), which may aid in diagnosis.

Thrombocytopenia secondary to maternal autoantibodies (see the section on this topic) is an alternative diagnosis that can be excluded by the absence of maternal thrombocytopenia or maternal autoimmune disease (ie, systemic lupus erythmatosus). Thrombocytopenia secondary to hypoxia or acidosis may occur in neonates with complicated pregnancies or deliveries and thus can be excluded in cases of uncomplicated term pregnancies and deliveries. Inherited thrombocytopenia syndromes (see the section on this topic) may also present similarly but are much rarer. The diagnosis of inherited syndromes typically includes evaluation for specific physical examination or peripheral smear findings and genetic testing.

Diagnostic Tests

Thrombocytopenia is evident at birth. Generally, a diagnosis of NAIT requires both evidence of maternal-paternal platelet antigen incompatibility and maternal antiplatelet antibodies. Occasionally, maternal antibodies are found without evidence of platelet antigen incompatibility. In these cases, the reference laboratory should distinguish between “unimportant” antigens (ie, HLA) vs incompatibilities of a previously undefined platelet antigen.¹⁴ Evidence of a platelet antigen incompatibility without maternal antibodies is insufficient for an NAIT diagnosis.

Management

Treatment involves platelet transfusions and intravenous immunoglobulin (IVIG; 1 g/kg administered over 1–2 days). Historically, recommendations were for transfusion of maternal platelet antigen-matched platelets or HPA-1a-negative platelets if maternal platelet antigen type was unavailable. However, recent studies showed no difference in outcome between antigen-specific and random donor platelets.^{20, 21} Therefore, given the often-lengthy delay in platelet typing or obtaining antigen-specific platelets, recommended initial therapy for NAIT is the administration of IVIG and random donor platelets. The duration of effect of a platelet transfusion needs to be closely monitored to evaluate the need for further transfusion. A head ultrasound should always be performed to evaluate for ICH.

Antenatal management of NAIT has been controversial. Previous recommendations were for fetal platelet monitoring and intrauterine transfusions. However, the invasiveness and complications associated with these procedures have since decreased their appeal. In cases of NAIT in index pregnancies, guidelines for subsequent pregnancies include maternal infusion of IVIG (1 g/kg) weekly, starting at 16 weeks if the previous sibling had ICH and at 32 weeks if not.²² In some protocols, steroids also have been used antenatally in particularly difficult NAIT cases. Because of the intensity of antenatal management, it is important to be certain of NAIT diagnosis.

Outcome and Follow-up

The majority of neonates with thrombocytopenia do well, with the platelets resolving within 7 days after delivery, without relapses or long-term sequelae.^{15, 16} However, a small subset of infants has thrombocytopenia that lasts several weeks and may require additional platelet transfusions. Those neonates who develop ICH have poorer neurodevelopmental outcomes, including cerebral palsy and sensory impairment.¹⁶

Maternal Platelet Autoantibodies

Epidemiology

Neonatal thrombocytopenia can also occur in infants born to mothers with platelet autoantibodies. Immune thrombocytopenia (ITP) is the most common underlying disease in mothers, and 10%–15% of maternal ITP cases result in neonatal thrombocytopenia.²³ Platelet autoantibodies can also occur in mothers affected with systemic lupus erythematosis (SLE) and hyperthyroidism, among other less-common autoimmune disorders.

Fortunately, neonatal thrombocytopenia associated with maternal autoantibodies is much less of a clinical problem than NAIT. The thrombocytopenia is generally transient, though infants should be closely monitored in the first several days of life, as platelet counts can rapidly drop during this time. The majority of neonates have mild-to-moderate thrombocytopenia (50,000 to 100,000/μL), and most are clinically asymptomatic except for minimal bruising or petechiae.²⁴ However, up to 10% of affected neonates develop more severe thrombocytopenia. ICH is rare and has been reported in less than 1% of infants with severe thrombocytopenia. Predictive factors for clinical bleeding and severe neonatal thrombocytopenia include history of older sibling(s) with severe neonatal thrombocytopenia, maternal platelet count at delivery less than 100,000/μL, and maternal ITP refractory to splenectomy.²³

Pathophysiology

Neonatal thrombocytopenia occurs secondary to maternal antiplatelet autoantibodies that cross the placenta to also affect the fetus and newborn. Autoantibodies are typically directed against platelet glycoprotein (Gp) IIbIIIa, although there are also cases of anti-Gp1b autoantibodies.

Differential Diagnosis

Maternal ITP should not be confused with gestational thrombocytopenia, a benign transient entity that occurs in women only during pregnancy and resolves spontaneously after delivery. Affected women are typically healthy, have no history of autoimmune disease, and have an uncomplicated pregnancy. Maternal platelet counts are mildly low in the majority of cases. Furthermore, neonatal thrombocytopenia rarely occurs. Both mothers and fetuses typically are asymptomatic and do not require any medical treatment.

Other major differential diagnoses include bacterial and congenital infections. Bacterial infections should be strongly considered and empirically treated until cultures are proven negative, particularly in high-risk neonates. NAIT may be excluded by the presence of maternal thrombocytopenia or maternal autoimmune disease. Inherited thrombocytopenia syndromes are alternative diagnoses that are typically associated with distinct physical examination or peripheral smear findings and are often diagnosed with specific genetic testing.

Diagnostic Tests

There is no specific test for thrombocytopenia secondary to maternal autoantibodies. Tests for anti-GpIIbIIIa or anti-Gp1b antibodies have low sensitivity. Instead, diagnosis is typically based on maternal and neonatal history and other laboratory findings. The greatest diagnostic clue is a maternal history of ITP or other autoimmune disorder. Affected mothers and neonates typically have isolated thrombocytopenia and variably large platelets on peripheral smear, typical of what is seen with ITP. Thrombocytopenia in the neonate is evident at birth. Mothers may also have a previous history of ITP treatment, including steroids, IVIG, and splenectomy. Previously splenectomized mothers actually may have a normal platelet count but still have circulating autoantibodies that can cross the placenta to destroy fetal platelets. Therefore, if maternal platelet counts are normal but maternal autoantibodies are suspected, it is important to query mothers regarding whether they previously had their spleen removed or if they have any other history of autoimmune disease.

Management

Treatment of thrombocytopenia is typically supportive. In the presence of severe thrombocytopenia or life-threatening bleeding, first-line therapy includes IVIG (1 g/kg administered over 1–2 days). Platelet transfusions also may be indicated, although since maternal antibodies are directed against common antigens, the transfusion may not be effective.

Previously, the antenatal management of known maternal ITP cases included removing the mother’s spleen and fetal blood sampling to determine the safety of vaginal delivery. However, current recommendations are less invasive, with IVIG or steroids for the mother to delay splenectomy until at least after delivery. Fetal blood sampling is no longer done. Furthermore, cesarean sections now are recommended only for obstetric indications.

Outcome and Follow-up

Platelet counts typically nadir between day 2 and day 5 of life and recover 1 week after birth. There are a relatively small number of patients with persistent thrombocytopenia beyond 7 days, in which case antiplatelet antibodies persist.²⁵ With documented platelet normalization to greater than 150,000/μL, there is no risk of recurrent thrombocytopenia since the maternal autoantibodies will have been cleared from the neonate.

Chronic Fetal Hypoxia and Acidosis

Chronic fetal hypoxia typically occurs in neonates born to mothers with pregnancy-associated hypertension, intrauterine growth restriction (IUGR), and gestational diabetes mellitus. All result in placental insufficiency and therefore decreased fetal oxygenation. The underlying mechanism for the thrombocytopenia is thought to be related to decreased megakaryopoiesis.⁵ Low platelet counts in these cases are evident at birth, are typically mild to moderate (eg, platelet counts of 50,000–100,000/μL), and commonly self-resolve within 1 to 2 weeks after birth. Most patients do not require treatment of their thrombocytopenia, but platelet transfusions can be given if indicated for bleeding or if there is risk of increased bleeding (eg, invasive procedures).

Kasabach-Merritt Syndrome

Kasabach-Merritt syndrome, associated with capillary hemangiomas, can present with neonatal thrombocytopenia, along with microangiopathic anemia and DIC. Affected neonates have one or more enlarging vascular lesions. Diagnosis is typically straightforward in cases of cutaneous lesions; however, approximately 20% of patients have visceral involvement (eg, of the liver) without cutaneous lesions.^{26, 27, and 28} Thrombocytopenia occurs secondary to sequestration of platelets within the vascular endothelium and is compounded in cases complicated by DIC. Supportive therapy entails replacement of platelets and clotting factors, along with definitive treatment of the vascular lesions. Definitive therapies used for Kasabach-Merritt syndrome include steroids, vincristine, interferon, and sirolimus.²⁶

Inherited Thrombocytopenia Syndromes

Neonatal thrombocytopenia that persists more than 2 weeks after birth is unusual and warrants further investigation. As most other forms of thrombocytopenia resolve by this time, the likely cause of prolonged, otherwise-unexplained, thrombocytopenia is an inherited syndrome. Inherited thrombocytopenias can be classified into 3 major groups, based on platelet size: increased platelet size, normal platelet size, and decreased platelet size (Table 32-4).