Red Blood Cells
The great questions of the day are not decided by speeches and majority votes but by blood and iron. Otto von Bismarck, September 30, 1862
Fetal Erythropoiesis and Changes in Erythropoiesis after Birth
Rapid growth during fetal development demands a brisk pace for red blood cell production, and this capacity must expand with the increase in blood volume, which is proportionate to the weight of the fetus. Blood volumes average 80 mL/kg of fetal body weight at term, but the ratio is larger in the preterm fetus (~90 mL/kg). The rapid pace of erythropoiesis is reflected by a rise in hematocrit throughout gestation (from a mean of 40% at 28 weeks of gestation to 50% at term) and by high reticulocyte counts and the presence of circulating nucleated red blood cells at birth.
Hematopoiesis during mammalian embryonic development proceeds from the yolk sac blood island to the aorta-gonad-mesonephros region, the fetal liver, and subsequently the fetal bone marrow, and is tightly regulated by the stromal cells in each of these unique areas that make up the hematopoietic niche ( Fig. 17-1 ). Moreover, there are likely distinct myeloid-erythroid progenitors in the early yolk sac niche that may exist transiently and contribute to the unique regulation of the β-globin locus in the mammalian embryo. The control of red blood cell production and progression of fetal erythropoiesis from yolk sac to liver (in utero) to bone marrow at birth is also in part orchestrated by Kruppel-like factors (KLFs) that control cell differentiation and embryonic development. KLF1 (erythroid Kruppel-like factor) is essential during both embryonic and adult erythropoiesis. KLF2 is a positive regulator of the mouse and human embryonic β-globin genes. KLF1 and KLF2 have highly homologous zinc finger DNA-binding domains and have overlapping roles in embryonic erythropoiesis. The ontogeny of fetal erythropoiesis has been reviewed elsewhere.
Fetal erythropoiesis also occurs during chronic bone marrow failure and recovery from marrow suppression. Fetal erythrocytes have hemoglobin F, with more G-γ than A-γ chains, i antigen, large mean corpuscular volume, characteristic enzyme levels, low carbonic anhydrase, low hemoglobin A 2 , and short life span. Many of these fetal characteristics are present in the red blood cells of patients with temporary or chronic hematopoietic stress. Chronic fetal erythropoiesis is seen in patients with constitutional aplastic anemia, such as Fanconi anemia or Diamond-Blackfan anemia. Thus fetal erythropoiesis occurs during hematopoietic stress, whether chronic or transient, if there is some marrow activity and may be due to expansion of fetal clones.
Several endogenous proteins contribute to the changes in regulation of erythropoiesis after birth, with erythropoietin being the most recognized. Fetal erythropoiesis is regulated by endogenous (fetal) erythropoietin produced in the liver, but in infancy the main site of production converts to the kidneys. Although the rate of erythropoiesis in the fetus is quite high, serum erythropoietin levels are low, and the erythropoietin response to hypoxia in the fetus and neonate is reduced compared with that in adults. After delivery erythropoietin levels vary among species, which is probably related to the oxygen transport capacity of the hemoglobin mass. In all mammals, hemoglobin level declines following birth and erythropoiesis nearly ceases, which gives rise to “early anemia.” Except in humans, erythropoietin levels increase proportionally with the fall in hemoglobin, but there is a discrepancy between the curves for serum immunoreactive erythropoietin and for erythropoiesis-stimulating factors. The latter include other stimulatory factors in addition to erythropoietin. These other factors work in concert with erythropoietin to control erythropoiesis and probably contribute to enhanced erythropoiesis during periods of rapid growth, which is unlikely to be attributable to the same molecular controls that enhance erythropoiesis during periods of stress or hypoxia. For example, it is known that erythropoietin acts in concert with general growth-promoting factors, particularly growth hormone (GH) and the insulin-like growth factors (IGF-I and IGF-II). The erythropoietin and GH/IGF systems are both activated by hypoxia and share similar receptors and pathways. Recent studies indicate that human fetal and infant growth is stimulated by GH, IGF-I, and IGF-II. Erythropoietin, GH, and IGFs are expressed early in fetal life. IGF-I levels are low in the fetus and increase slowly following birth except in preterm infants, in whom the levels decline. The physiology of erythropoietin during mammalian development has been reviewed elsewhere.
The low level of erythroid production noted earlier persists for over a month following birth, during which time the hematocrit gradually declines. Late in the second or third month of life, the hematocrit approaches 30%. This is commensurate with a rise in serum erythropoietin levels, which prompts a resumption of erythropoiesis and leads to a rise in red blood cell mass. This rise in red blood cell mass keeps pace with rapid overall growth and blood volume, and the hematocrit rises relatively little as a consequence.
Erythropoietin and Neuroprotection in the Neonate
One other aspect of the erythropoietin system that is important to the fetus and newborn deserves mention here. Erythropoietin is a pleiotropic neuroprotective cytokine, and recent studies have shed light on the biological basis of its efficacy in the damaged developing brain. Coordinated expression of erythropoietin ligand and receptor expression occur during central nervous system development to promote neural cell survival. Studies of fetal hypoxia-ischemia in rat models have demonstrated that prenatal third-trimester global hypoxia-ischemia disrupts the developmentally regulated expression of neural cell erythropoietin signaling and predisposes neural cells to death. Furthermore, exposure of the neonate to exogenous sources of recombinant erythropoietin can restore the mismatch of erythropoietin ligand and receptor levels and enhance neural cell survival. The data generated by these studies suggest the potential utility of neonatal recombinant erythropoietin when administered in the days immediately after a global prenatal hypoxic-ischemic insult as a means to rescue neural cells and present a novel clinically relevant paradigm in which the benefits of erythropoietin in the context of a stress are linked to the induction of signaling pathways in both erythroid and nonerythroid lineages.
Placental Transfusion and Distribution of Blood at Birth
The effect of early and late umbilical cord clamping on neonatal hematocrit has been well studied. Delayed cord clamping has been shown to be associated with a higher hematocrit in very low-birth-weight infants, which suggests effective placental transfusion. Several analyses have confirmed that delaying cord clamping (by at least 30 seconds) increases average blood volume across the full range of gestational ages studied. On average, the infant will gain roughly 14 mL/kg of blood during this first 30 seconds, which leads to a blood volume of 89 mL/kg. This process has been termed placental transfusion. It occurs due to the continued circulation of blood through the umbilical arteries and veins, and leads to a net shift of blood from the placenta to the newborn infant. At birth, the partition of blood volume between the infant and the fetal placental vasculature is nearly 2:1 (75 mL/kg body weight in the infant and 40 mL/kg in the placenta). If the umbilical cord is not clamped quickly, a major shift in blood can lead to significant effects on blood volume, hematocrit, and hemoglobin concentration during the first days of life. Infants exposed to a significant delay in umbilical cord clamping may experience excessive placental transfusion, with attendant decreases in plasma volume, increased hematocrit, and elevated blood viscosity. Regardless of the extent of placental transfusion, postnatal adjustments of blood volume and hematocrit begin within 15 minutes after birth and continue for several hours. A controlled trial has suggested that delayed cord clamping in very preterm infants may reduce the incidence of intraventricular hemorrhage and late-onset sepsis.
Delayed cord clamping has continued to show benefits and little, if any, risk in preterm infants. The benefits of delayed cord clamping in preterm infants include increased blood volume, improved circulatory and respiratory function, reduced need for blood transfusion, improved cerebral oxygenation, and reduced intraventricular hemorrhage and sepsis. The cord blood of extremely preterm infants is a rich source of hematopoietic progenitor cells such as hematopoietic stem cells, endothelial cell precursors, mesenchymal progenitors, and stem cells of multipotent-pluripotent lineage; hence the merit of delayed cord clamping has been magnified. Tolosa et al referred to this aspect of delayed cord clamping as “realizing mankind’s first stem cell transfer” and proposed that “it should be encouraged in normal births.” The extra endowment of progenitor cells resulting from delayed cord clamping has the potential to both increase red blood cell production and boost host immune defenses through production of leukocytes.
There has been a shift in thinking to explore milking of the umbilical cord as an alternative to delayed cord clamping, which may provide the same benefits without the need to delay resuscitation.
Properties of Fetal Hemoglobin and the Switch to Adult Hemoglobin
The different types of human hemoglobin consist of various combinations of the embryonic, fetal, and adult hemoglobin subunits that are present at distinct times during development. This orderly transition from one form of hemoglobin to another represents a major paradigm of developmental biology but remains poorly understood. Studies have pointed to a competition between subunits for more favorable partners with stronger subunit interactions, so that the protein products of gene expression can themselves play a role in the developmental process due to their intrinsic properties. Fetal hemoglobin, or Hb F (two α-globin chains and two γ-globin chains, [α 2 γ 2 ]), is the main hemoglobin synthesized up to birth, at which point it makes up more than 80% of the hemoglobin in circulating red blood cells. However, Hb F subsequently declines, and adult hemoglobin, Hb A (α 2 β 2 ), becomes predominant.
The main reason for this shift is the transition from synthesis of mainly γ chains during fetal development to mainly β chains during late gestation, with a concomitant gradual shift from Hb F to Hb A beginning at 34 weeks’ gestation. Several studies have indicated that expression of the Hb F subunit γ-globin might also be regulated posttranscriptionally. One recently identified mechanism for posttranscription regulation of gene expression is through the production of micro-RNAs. These micro-RNAs are approximately 22 nucleotides in length and can specifically target messenger RNAs (mRNAs) for selected genes, thus acting as disease modifiers as well as molecules that control gene expression during development and in response to environmental stimuli. A study comparing micro-RNA expression in reticulocytes from cord blood and adult blood revealed several micro-RNAs that were preferentially expressed in adults, among them micro-RNA-96, which appears to directly suppress γ-globin expression and thus contributes to control of Hb F production and its suppression during the switch to postnatal erythropoiesis.
Although new hemoglobin produced during postnatal life is essentially all Hb A, there are exceptions to this rule. Perhaps the most well known is the persistence of Hb F in patients with sickle cell disease and the contribution of Hb F to amelioration of disease severity in these individuals (see later discussion). Hb F expression can be increased during periods of stress erythropoiesis, and in the infant recovering from anemia of prematurity, there is a transient phase in the recovery of erythropoiesis during which Hb F is the predominant hemoglobin synthesized.
Functional Differences of Specific Hemoglobins
The major physiologic function of hemoglobin is to bind oxygen in the lungs and deliver it to the tissues. This function is regulated and/or made efficient by endogenous heterotropic effectors.
Hb A is the major oxygen-binding tetrameric protein found in the blood. It is one of the best-recognized proteins in the human body because of its uniquely bright red color, and its color changes in diseases such as anemia, hypoxia, and cyanide and carbon monoxide poisoning. Hemoglobin has drawn the attention of physicians and physiologists since ancient times. Modern quantitative analysis of the structure and function of hemoglobin started in the late 1800s and early 1900s. Important observations of the hemoglobin allostery have been attributed to Christian Bohr (who reported in 1903 that its oxygen-binding process was sigmoidal or cooperative) and to Bohr, Hasselbalch, and Krogh, who reported in 1904 that the position of the oxygen-binding curve of the blood was sensitive to changes in P co 2 (and H + or pH), known as the Bohr effect. These observations regarding the allosteric behaviors of hemoglobin are reviewed elsewhere.
It is widely recognized that the most important functional difference between Hb F and Hb A is their different oxygen-binding properties. The higher oxygen affinity of Hb F is an advantage to the fetus because of the site of oxygen uptake, the placenta, where the umbilical venous P o 2 is just 35 to 40 mm Hg, and represents the highest P o 2 in all the fetal circulation. Because the oxygen dissociation curve is “shifted” to the left (because of higher affinity), there is a capacity to maintain a higher O 2 content, but this capacity is no longer needed after birth because the lungs provide an environment with a significantly higher oxygen tension (typically above 75 mm Hg) in the pulmonary capillaries. More importantly, the persistence of Hb F is a disadvantage to the newborn because the release of oxygen in the capillary bed depends on a much lower P o 2 for efficient oxygen delivery and maintenance of tissue metabolism, which is in contrast to the dynamics of O 2 release by Hb A. This difference has been shown in clinical investigations to influence morbidity in newborns with cardiopulmonary disease. Studies evaluating the impact of exchange transfusion in extremely premature infants demonstrated a link between improved survival and substantial replacement of Hb F by Hb A, despite the absence of a significant change in hematocrit. This effect is often achieved as a consequence of frequent phlebotomies and multiple small transfusions of packed red blood cells in very low-birth-weight infants.
Globin gene mutations are a rare but important cause of cyanosis. Crowley et al identified a missense mutation in the fetal G γ-globin gene ( HBG2 ) in a father and daughter with transient neonatal cyanosis and anemia. This newly recognized mutation modifies the ligand-binding pocket of fetal hemoglobin. The mechanisms described include a diminutive effect of the relatively large side chain of methionine on both the affinity of oxygen for binding to the mutant hemoglobin subunit and the rate at which it does so. In addition, the mutant methionine is converted to aspartic acid posttranslationally, probably through oxidative mechanisms. The presence of this polar amino acid in the heme pocket is predicted to enhance hemoglobin denaturation, causing anemia.
Methemoglobinemia arises from the production of nonfunctional hemoglobin containing oxidized Fe 3+ , which results in reduced oxygen supply to the tissues and manifests as cyanosis in the patient. It can develop by three distinct mechanisms: genetic mutation resulting in the presence of abnormal hemoglobin, a deficiency of the methemoglobin reductase enzyme, and toxin-induced oxidation of hemoglobin. The normal hemoglobin fold forms a pocket to bind heme and stabilize the complex of heme with molecular oxygen. This process prevents spontaneous oxidation of the Fe 2+ ion chelated by the heme pyrroles and the globin histidines. In the abnormal M forms of hemoglobin (Hb M) amino acid substitution in or near the heme pocket creates a propensity to form methemoglobin instead of oxyhemoglobin in the presence of molecular oxygen. Under normal conditions, hemoglobin is continually oxidized, but significant accumulation of methemoglobin is prevented by the action of a group of methemoglobin reductase enzymes. In the autosomal recessive form of methemoglobinemia, there is a deficiency of one of these reductase enzymes, which allows accumulation of oxidized Fe 3+ in methemoglobin. Oxidizing drugs and other toxic chemicals may greatly enhance the normal spontaneous rate of methemoglobin production. If levels of methemoglobin exceed 70% of total hemoglobin, vascular collapse occurs resulting in coma and death. Under these conditions, if the source of toxicity can be eliminated, methemoglobin levels will return to normal. Disorders of oxidized hemoglobin are relatively easily diagnosed and in most cases, except when congenitally defective Hb M is present, can be treated successfully.
Neonatal anemia is a condition with a diverse etiologic spectrum. To reach an accurate diagnosis, the pediatrician must have some knowledge of the more common causes of low hemoglobin concentrations and hematocrit in the neonate. Proper history taking, physical examination, and interpretation of diagnostic test results can narrow the focus and aid in establishing an accurate diagnosis and in directing the appropriate therapeutic interventions.
Hemorrhagic anemia in a newborn is often heralded by some features of the history or clinical findings that allow time to anticipate and prepare for treatment of the infant. The fetus may lose blood through a variety of routes. Hemorrhage commonly occurs through the placenta into the mother’s circulation and may be detected most readily through a Kleihauer-Betke test performed on the mother’s blood. For monozygotic twins, there is an additional risk that one fetus may hemorrhage through the placental vascular anastomosis into the other twin (see the section on twin-to-twin transfusion syndrome later in this chapter). The fetus may also bleed through the placenta into the birth canal. In many cases of placental abruption, the vaginal blood contains a mixture of fetal and maternal blood. The fetus may lose a large volume of blood into the fetal placental circulation at the time of birth (see also Chapter 2 ). All of the latter circumstances have the same effect as hemorrhage. Some of these mechanisms, such as placental abruption and trapping of blood in the placenta by cord compression, also produce asphyxia, and the coexistence of asphyxia with hypovolemia complicates both the assessment and the management of the infant. Even though most newborn babies with asphyxia are not hypovolemic, there is a subset of infants who have lost blood volume around the time of delivery and most also experienced asphyxia.
Before delivery, internal hemorrhage may occur, with the most common type being intraventricular hemorrhage. The true incidence of intracranial hemorrhage is not certain, but in cases of alloimmune thrombocytopenia in which there is severe thrombocytopenia, intracranial hemorrhage may be seen in as many as 20% of cases. Administration of intravenous gammaglobulin (IVIG) and/or corticosteroids to the mother during a subsequent pregnancy with an affected fetus is widely practiced to increase the fetal platelet count and thus avoid intracranial hemorrhage (see later discussion). Internal hemorrhage can also result from trauma around the time of delivery. Important common types of hemorrhage secondary to birth-related trauma are subgaleal hematomas, hepatic subcapsular and mediastinal hematomas, intracerebral and cerebellar hemorrhage, and hematomas in fractured limbs. Adrenal hemorrhage is more common in neonates than in children or adults. The incidence of detected cases ranges from 1.7 to 2.1 per 1000 births. Because adrenal bleeding may remain asymptomatic, the real incidence is probably higher. In published series, the most common clinical feature in infants with adrenal hemorrhage was jaundice, which was observed in 67.6% of cases in at least one series. Thus, it has been suggested that in cases of hyperbilirubinemia of unknown cause, adrenal hemorrhage must be kept in mind.
For hemorrhages that are associated with birth trauma, the occurrence is highest in difficult term deliveries, particularly in infants who are large for gestational age and require multiple applications of vacuum to assist delivery. Splenic rupture is uncommon but may lead to catastrophic intraabdominal hemorrhage in infants with hemophilia and in babies in whom intrauterine splenomegaly develops as a result of erythroblastosis or other causes. Extensive trauma to the perineum in babies born through breech deliveries can lead to hypovolemia and anemia, and these symptoms are typically exaggerated in the preterm infant.
In the newborn the clinical presentation and symptoms associated with fetal hemorrhage are directly related to the interval between hemorrhage and delivery, as well as to the extent of hemorrhage. When the bleed occurs only shortly before birth, there is little time for hemodilution; thus these babies will not be anemic initially and will show few if any signs that would indicate hypovolemia or anemia. The hematocrit will fall during the first hour after delivery, but a rise in reticulocyte count will not typically be seen until the anemia has been present for several days.
When approaching the treatment of hemorrhagic anemia presenting in the newborn period, one needs first to consider any attendant cardiorespiratory effects of blood loss that are present. Because of the capacity for rapid transport of fluid across the placenta and a limitless reservoir for volume replacement, a hemorrhage that occurs sufficiently far in advance of delivery will likely manifest only the consequences of decreased oxygen-carrying capacity from the anemia. This capacity for rapid replacement of volume loss via the placenta is an important safety net for the fetus, who might not otherwise tolerate intermittent hypoxemia during the contractions associated with labor. For any infant known to have chronic in utero anemia, volume expansion must be approached carefully. The infant in this situation is often anemic but has normal intravascular volume. Consequently, additional volume may lead to heart failure secondary to volume overload. For symptomatic infants with chronic anemia, partial exchange transfusion with packed red blood cells can be performed to achieve a desired hematocrit and avoid volume fluctuation and severe volume shifts. The amount to be exchanged depends on both the baby’s blood, the severity of the anemia, and the hematocrit of the packed red blood cells, but an exchange of between 30 and 50 mL/kg body weight may be required.
When hemorrhage occurs acutely during delivery, the hematocrit will not fully reflect the degree of blood loss because there has been little hemodilution. In this situation, one will need to aggressively manage shock, paying close attention to the hemodynamic and cardiorespiratory parameters. Measures of metabolic acidosis, capillary filling time, and both arterial and/or central venous pressures are important to monitor, because they will guide the approach and extent of fluid resuscitation. Almost invariably, newborn babies with hypovolemic shock will have experienced some degree of asphyxia, the manifestations of which will influence the assessment of shock. Thus, one must consider this to be an important variable, and it needs rapid attention, so that the treatment of shock must not delay correction of asphyxia. Acute situations may include placenta previa, vasa previa, abruption, or blood loss from the cut umbilical cord. In such cases, immediate volume replacement with blood is preferable because this rapidly enhances oxygen delivery to tissues, which is not the case when crystalloid solutions are used. Anticipation of the need for resuscitation is a key factor, so recognition of maternal vaginal bleeding should be a signal to anticipate for the need for transfusion. Most labor and delivery units have type O Rh-negative uncrossed red blood cells available. The classic approach to shock in the newborn is to transfuse 10 mL/kg of blood over 5 to 10 minutes and to repeat infusions until there are signs of adequate circulation.
Twin-to-Twin Transfusion Syndrome
Twin-to-twin transfusion syndrome (TTTS) is a complication that may occur in monochorionic twins which may originate in either imbalance or abnormality of the single placenta serving two twins. It is a serious complication in 10% to 20% of monozygous twin gestations with an overall incidence (i.e., including those in which it is not a serious complication) of 4% to 35% in the United States. The diagnosis is well established in overt clinical forms through the association of polyuric polyhydramnios and oliguric oligohydramnios. TTTS is a progressive disease in which sudden deterioration in clinical status can occur, leading to the death of a twin. Up to 30% of survivors have abnormal neurologic development as a result of the combination of profound antenatal insult and complications of severe prematurity. Newer treatment options have improved the outcomes.
TTTS results from an unbalanced blood supply through placental anastomoses in monochorionic twins. These anastomoses may be arterial to arterial, but arterial to venous are believed to be responsible for a majority of the cases presenting clinically. TTTS induces growth restriction, renal tubular dysgenesis, and oliguria in the donor and visceromegaly and polyuria in the recipient. Studies have shown a potentially important role of the renin-angiotensin system with upregulation in the donor twin, whereas in recipients, renin expression was virtually absent, possibly because it was downregulated by hypervolemia. In the donor, congestion and hemorrhagic infarction were accompanied by severe glomerular and arterial lesions resembling those observed in polycythemia- or hypertension-induced microangiopathy. Thus, fetal hypertension in the recipient twin in TTTS might be partly mediated by the transfer through the placental vascular shunts of circulating renin produced by the donor.
The degree of transfusion from one twin to the other and the time course of the transfusion may be highly variable. It may begin as early as the second trimester and therefore be long-standing at the time of delivery. In the most severe cases in which the transfusion is of long duration, the donor is substantially anemic and exhibits significantly increased erythropoiesis that can even be present in the dermis (“blueberry muffin baby”); the donor also becomes progressively small for gestational age and develops oligohydramnios. Simultaneously, the recipient twin continues to grow normally and becomes polycythemic; in extreme cases, this progresses to polyhydramnios and potentially to hydrops fetalis. If the growth-restricted twin dies in utero, the risk exists for embolization through vascular anastomoses to the surviving twin as a consequence of intravascular coagulation in the dying twin. Embolization in the surviving twin will have major consequences, often affecting vital organs including the brain, gastrointestinal tract, and kidneys. Postpartum management of liveborn twins affected by this syndrome will be quite complicated, even when the pediatrician is prepared well in advance of delivery. The polycythemic twin will need reduction of the hematocrit, whereas the treatment of the anemic donor is more straightforward. If either of the newborn twins demonstrates evidence of cardiomyopathy, the infant would be intolerant of blood volume shifts and may be particularly sensitive to blood volume expansion, in which instance a partial exchange transfusion is again the preferred approach.
In twin-to-twin transfusion syndrome (TTTS) the likelihood of perinatal survival of at least one twin was not found to vary with severity as classified by Quintero stage (stage I, 92%; stage II, 93%; stage III, 88%; stage IV, 92%). However, dual twin survival did vary by stage (stage I, 79%; stage II, 76%; stage III, 59%; stage IV, 68%; P < .01), primarily because stage III TTTS was associated with decreased donor twin survival. Sequential selective laser photocoagulation of communicating vessels in pregnancies with TTTS was associated with higher dual survival and donor twin survival rates compared with a nonsequential technique. Overall survival of one or both twins was 91% and dual twin survival was 72%.
Hyperbilirubinemia is far more common and more severe in neonates with hemolytic anemia than in older children. The bilirubin level may increase rapidly in the first hours after birth, so identification of the underlying cause is extremely important. To diagnose the cause of hemolysis, it is essential to obtain good information about any family history of anemia or neonatal jaundice, and without exception, both the baby and a blood smear should be examined. Clues to the appropriate diagnostic tests are often present in these smears, suggested by morphologic abnormalities of the red blood cells. It is standard to perform a direct antiglobulin test (DAT, or direct Coombs test), because the majority of hemolytic episodes are a consequence of maternal antibodies that cross the placenta in late gestation and then react with paternal antigen expressed on the infant’s erythrocytes. These maternal immunoglobulin G (IgG) alloantibodies against paternal antigen are responsible for most cases of hemolytic disease of the newborn. If the DAT result is negative but hyperbilirubinemia persists and the hematocrit is declining, a more thorough evaluation is required. This typically does not include a bone marrow examination, which is reserved for cases in which anemia is not associated with hemolysis and in which there is evidence suggesting a primary disorder of erythropoiesis.
Fetal-neonatal alloimmune disease is the most common cause of severe hemolytic anemia in an otherwise healthy newborn. Of these disorders, Rh disease remains the most common cause of severe anemia. Anemia varies from mild to severe, the DAT result is strongly positive, and the reticulocyte count is almost uniformly elevated. Antenatal assessment is an important aspect of good management and should include maternal screening and frequent surveillance of fetal well-being. In sensitized pregnancies, vigilance in pursuing these evaluations is essential if one is to define the appropriate time for intervention with premature delivery or intrauterine transfusions.
Hyperbilirubinemia is a problem in the majority of cases of Rh disease, and in patients with the most severe degree of hemolysis, the elevated bilirubin level cannot be managed by phototherapy alone and ultimately requires exchange transfusion. For patients with Rh sensitization and intrapartum asphyxia, correction of anemia is essential to minimize cardiorespiratory distress and is thus an important part of resuscitation in this group of patients. The most extreme cases of Rh sensitization are associated with hydrops fetalis in utero. Antenatal therapies have been implemented to prevent this severe manifestation of alloimmunization, including intrauterine transfusions. Treated infants are born with only mild to moderate anemia, with all of their red blood cells derived from the intrauterine transfusions. In these infants, the DAT finding may often be negative, but the result of the indirect antiglobulin test is strongly positive. Quite frequently, the consequence of intrauterine transfusion is that the newborn will have no reticulocytosis despite moderate anemia, and with the majority of the infant’s red blood cells derived from intrauterine transfusions with Rh-negative blood, there is no hemolysis. It is imperative that these infants be watched closely over the first several months, because a late episode of hemolysis and anemia may arise when the donor erythrocytes eventually decline in number. As erythropoiesis begins to accelerate, these infants produce their own Rh-positive blood cells, which are susceptible to attack by residual maternal antibodies. In such infants, the DAT result may remain strongly positive for months and they will require supplemental folic acid to keep pace with the demands of increased erythropoiesis.
It is truly remarkable that after the discovery of the blood groups in the 1940s, the virtual elimination of erythroblastosis with anti-D globulin took a mere 30 years, and this is one of the more notable accomplishments in modern medicine. Anti-D, a polyclonal immunoglobulin G, is purified from the plasma of D-alloimmunized individuals. It is routinely and effectively used to prevent hemolytic disease of the fetus and newborn caused by the antibody response to the D antigen on fetal red blood cells. This therapy has effectively reduced the number of cases of Rh isoimmunization from 13% to less than 1% and the mortality rate from one in four to fewer than 5%. The residual cases are few and far between and have been attributed mainly to failed maternal prophylaxis caused by improper timing or dosage of immunoglobulin anti-D therapy and by immunization during pregnancy resulting from an early occult fetomaternal hemorrhage (at <28 weeks’ gestation). With erythroblastosis, the late-onset anemia may be either hemolytic or hyporegenerative.
Alloimmune disease may also occur as a consequence of other blood group incompatibilities (anti-c, anti-e, and anti-C in the Rh system and anti-Kell). In alloimmune anemia of the newborn, the level of hemolysis caused by the presence of antibodies to antigens of the Kell blood group system is less than that caused by antibodies to the D antigen of the Rh blood group system, and the numbers of reticulocytes and normoblasts in the baby’s circulation are inappropriately low for the degree of anemia. These findings suggest that sensitization to Kell antigens results in suppression of fetal erythropoiesis as well as hemolysis. Vaughan et al compared the ex vivo growth of Kell-positive and Kell-negative hematopoietic progenitor cells from cord blood in the presence of human monoclonal anti-Kell and anti-D antibodies and serum from women with anti-Kell antibodies. The growth of Kell-positive erythroid progenitor cells (erythroid burst-forming units and colony-forming units) from cord blood was markedly inhibited by monoclonal IgG and IgM anti-Kell antibodies in a dose-dependent fashion (range of concentrations: 0.2% to 20%), but monoclonal anti-D antibodies had no effect. The growth of these types of cells from Kell-negative cord blood was not affected by either type of antibody. Neither monoclonal anti-Kell antibodies nor monoclonal anti-D antibodies inhibited the growth of granulocyte or megakaryocyte progenitor cells from cord blood. Serum from 22 women with anti-Kell antibodies inhibited the growth of Kell-positive erythroid burst-forming units and colony-forming units but not of Kell-negative erythroid burst-forming units and colony-forming units ( P <.001 for the difference between groups). The maternal anti-Kell antibodies had no inhibitory effects on granulocyte-macrophage or megakaryocyte progenitor cells from cord blood. These data indicate that anti-Kell antibodies specifically inhibit the growth of Kell-positive erythroid burst-forming units and colony-forming units, a finding that supports the hypothesis that these antibodies cause fetal anemia by suppressing erythropoiesis at the progenitor cell level.
A third form of alloimmune disease is caused by ABO incompatibility. This is perhaps one of the most frequent causes of hyperbilirubinemia in the newborn but is rarely responsible for a significant hemolytic anemia. The peripheral blood smear of patients with ABO incompatibility shows microspherocytes, and in most cases, the mother is type O, whereas the baby is either type A or type B. The elevation in serum bilirubin concentration typically resolves within 1 to 2 weeks, and it is rare for this form of alloimmune hemolytic disease to result in a drop in hemoglobin level or hematocrit sufficient to require transfusion in the absence of other complicating factors such as infection. Clinical disease rarely occurs in group A mothers with group B babies or in group B mothers with group A babies.
Congenital infections may be associated with hemolytic anemias and have most often been observed in the setting of TORCH infections ( to xoplasmosis, r ubella, c ytomegalovirus infections, h erpes simplex) as well as syphilis. The association of hemolytic anemia with cytomegalovirus infection is well described, and cytomegalovirus infection has been documented as a cause of autoimmune hemolytic anemia in the setting of vertically acquired neonatal infection with human immunodeficiency virus (HIV). Parvovirus B19 has an affinity for erythroid progenitors and produces severe erythroid hypoplasia, with severe infection during fetal development resulting in hydrops fetalis or congenital anemia. Diagnosis is based on examination of bone marrow and virologic studies. Much is known of the pathophysiology of the virus, and studies are in progress to develop a vaccine to prevent this widespread infection. Bacterial infections can precipitate a hemolytic episode, particularly in individuals with glucose-6-phosphate dehydrogenase deficiency, and thus this diagnosis should be considered in the setting of sepsis and severe hemolysis in the neonate.
Specific erythrocyte glycolytic enzyme defects can be the cause of hemolytic syndromes in the neonate. Two of the most commonly observed enzymopathies are described in the following sections.
Glucose-6-Phosphate Dehydrogenase Deficiency
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect and is present in more than 400 million people worldwide. As with sickle cell disease, the global distribution of G6PD is remarkably similar to that of malaria, which lends support to the hypothesis that these red blood cell disorders confer protection against malaria. G6PD deficiency is an X-linked genetic defect caused by mutations in the G6PD gene, which lead to functional variants with many biochemical and clinical phenotypes. Significant deficiency occurs almost exclusively in males. About 140 mutations have been described; most are single-base changes leading to amino acid substitutions. The most common G6PD mutation in North America is the G6PD-A variant, present in approximately 10% of African Americans. Term infants are rarely symptomatic. The most frequent clinical manifestations of G6PD deficiency are neonatal jaundice and acute hemolytic anemia, which are usually triggered by an exogenous agent. Jaundice in the neonate with G6PD deficiency may occur without any known oxidant exposure. In contrast to G6PD-A, G6PD-Canton, a variant common in South China, is commonly associated with significant neonatal jaundice. Some G6PD variants cause chronic hemolysis, which leads to congenital nonspherocytic hemolytic anemia. The most effective management of G6PD deficiency is to prevent hemolysis by avoiding oxidative stress.
Glucose-6-phosphate dehydrogenase is the rate-limiting enzyme in the hexose monophosphate shunt pathway. This pathway is principally important for the production of reduced glutathione, and this antioxidant has a vital role in protecting the red blood cell membrane from oxidant damage. G6PD deficiency is common worldwide, with certain molecular variants associated with neonatal hemolysis and hyperbilirubinemia. A case recently reported in the literature described a novel missense mutation in a white neonate with chronic nonspherocytic hemolytic anemia caused by a class I G6PD deficiency. The missense mutation in exon eight of the G6PD gene (c.827C>T p.Pro276Leu) was associated with severe elevation in serum bilirubin level, which peaked on day 5 at 24 mg/dL with a conjugated bilirubin level of 17 mg/dL. Jaundice resolved within 4 weeks. A detailed work-up failed to reveal other specific factors contributing to cholestasis. Severe hemolytic disease of the newborn may cause cholestasis, even in the absence of associated primary hepatobiliary disease. The diagnosis of G6PD-deficient hemolytic anemia should be suspected in male infants with evidence of acute hemolytic anemia and a negative result on Coombs test/DAT. Because the reticulocyte has higher levels of G6PD, screening tests for G6PD activity performed on the heels of a hemolytic episode are less reliable and should be repeated 2 to 3 months after an acute hemolytic episode in conjunction with family studies.
Pyruvate Kinase Deficiency
Pyruvate kinase deficiency is a rare cause of neonatal hemolytic jaundice, with a prevalence estimated at 1 case per 20,000 live births in the United States, but with a higher prevalence in the Amish communities in Pennsylvania and Ohio. One report described four neonates with pyruvate kinase deficiency born in a small community of individuals practicing polygamy. All four had early, severe hemolytic jaundice. Pyruvate kinase deficiency should be considered in neonates with early hemolytic, Coombs test–negative, nonspherocytic jaundice, particularly in communities with considerable consanguinity. Such cases should be recognized early and managed aggressively to prevent kernicterus. (See also Chapter 13 .)
Defects of the Red Blood Cell Membrane
Inherited abnormalities of one of the proteins of the red blood cell membrane may be associated with neonatal hemolysis and jaundice. Hereditary spherocytosis is an autosomal dominant condition and the most common of this class of disorders. Most cases of spherocytosis result from decreased production of spectrin. Hereditary spherocytosis, including the very mild or subclinical forms, is the most common cause of nonimmune hemolytic anemia among people of Northern European ancestry, with a prevalence of approximately 1 in 2000. However, very mild forms of the disease may be much more common. Hereditary spherocytosis is inherited in a dominant fashion in 75% of cases; the remaining are truly recessive cases and de novo mutations. A negative family history does not rule out the diagnosis, because new mutations are quite common. Diagnosis may be aided by the evaluation of a peripheral blood smear in infants suspected of one of these disorders of the red blood cell membrane.
Other Congenital Anemias
Congenital Dyserythropoietic Anemias
Congenital dyserythropoietic anemias (CDAs) are rare hereditary disorders characterized by ineffective erythropoiesis and by distinct morphologic abnormalities of erythroblasts in the bone marrow. Although historically these disorders have been largely diagnosed through identification of characteristic morphologic aberrations, the recent discovery of underlying etiologic genetic abnormalities has established the usefulness of molecular diagnostic approaches that might serve as rapid tools for the identification of these conditions. The first CDA partly accounted for genetically has been CDA I, for which the responsible gene CDAN1, encoding codanin-1, was discovered in 2002. Genetic defects linked to CDA II ( SEC23B ) and a previously unrecognized CDA ( KLF1 ) have been identified. SEC23B encodes SEC23B, which is a component of the coated vesicles transiting from the endoplasmic reticulum to the cis compartment of the Golgi apparatus. KLF1 encodes the erythroid transcription factor KLF1 (Kruppel-like factor 1), and the recently identified mutation leads to major ultrastructural abnormalities, the persistence of embryonic and fetal hemoglobins, and the absence of some red blood cell membrane proteins. The current understanding of the various CDAs, including genotype-phenotype relationships, has recently been reviewed elsewhere.
Deficiencies of Red Blood Cell Production
Among the anemias that present in the newborn period, those resulting from inadequate production are rare but, when present, may point to one of the rare congenital disorders affecting red blood cell production. These congenital defects of erythropoiesis exhibit a very low prevalence ranging from 4 to 7 per million live births and include Blackfan-Diamond anemia and Fanconi anemia, which are described in the following sections.
Blackfan-Diamond syndrome (also called congenital hypoplastic anemia ) is the most common congenital disorder of red blood cell production in the neonate. Infants with Blackfan-Diamond syndrome are often small for gestational age and may have other anomalies (including renal abnormalities) that must be considered when pursuing this diagnosis. Blackfan-Diamond anemia may result in severe fetal anemia requiring transfusion. Although autosomal dominant inheritance of Blackfan-Diamond syndrome is considered uncommon, it has been described ; the onset of anemia characteristically occurs within the first year of life, with 10% of cases presenting at birth. Affected infants exhibit variable degrees of anemia, with normal circulating white blood cell and platelet counts. Hydrops fetalis has been reported in rare cases. Among women with this disorder, a percentage are at risk for having an infant with substantial anemia in both the fetal and perinatal periods. Because the penetrance of the disorder is variable, pregnant women with a history of Blackfan-Diamond anemia should be considered at risk. Recommendations for the prenatal management of Blackfan-Diamond syndrome include prepregnancy counseling for parents with Blackfan-Diamond syndrome, detailed and serial fetal ultrasound and echocardiographic studies, cordocentesis if there are signs of anemia, consideration of in utero transfusion, and planned early delivery if the fetus is affected.
Fanconi anemia is a rare chromosomal instability disorder associated with a variety of developmental abnormalities, bone marrow failure, and predisposition to leukemia and other cancers. The Fanconi anemia gene family is a recently identified addition to the group of genes coding for the complex network of proteins that respond to and repair certain types of DNA damage in the human genome, but little is known about the regulation of this novel group of genes at the DNA level. A homozygous missense mutation in the RAD51C gene has been described in a consanguineous family with multiple severe congenital abnormalities characteristic of Fanconi anemia. RAD51C is a member of the RAD51-like gene family involved in homologous recombination-mediated DNA repair. The mutation results in loss of RAD51 focus formation in response to DNA damage and in increased cellular sensitivity to the DNA interstrand cross-linking agent mitomycin C and the topoisomerase-I inhibitor camptothecin. Fanconi anemia generally affects children and results in bone marrow failure requiring blood or marrow transplantation for survival. A unique feature of the condition is the long waiting period, often many years, between genetic diagnosis and treatment, which presents a significant challenge to the family and requires a strong, supportive multidisciplinary approach to care.
Anemia Secondary to Hemoglobinopathies
Hemoglobinopathies arise from mutations in the globin genes, with the most common hemoglobinopathies resulting from mutations in the β-globin gene. These are typically clinically silent at birth due to the persistence of Hb F but manifest as the expression switches from γ- to β- chain production.
Mutations in the β-globin gene that lead to a decrease in production are referred to as β -thalassemias. The β-thalassemias resulting from large structural deletions of the β-globin gene cluster are a rare familial cause of microcytic anemia and hyperbilirubinemia. Although blood cell counts are normal at birth, this disorder can be detected by demonstrating the absence of Hb A on electrophoresis. In most states, umbilical cord blood is routinely screened to identify infants with thalassemia and other hemoglobin disorders (including sickle cell disease; see later discussion) before they become symptomatic.
α-Thalassemia is one of the most common human genetic disorders and is found extremely frequently in populations in Southeast Asia and southern China, and the expanding populations of Southeast Asian immigrants in the United States, Canada, the United Kingdom, and Europe mean that this disorder is no longer rare in these countries. Couples in which both partners carry α 0 -thalassemia traits have a 25% risk of producing a fetus affected by homozygous α-thalassemia or hemoglobin Bart’s (Hb Bart’s) disease, with severe fetal anemia in utero, hydrops fetalis, and stillbirth or early neonatal death, as well as various maternal morbidities.
The α-thalassemias present a different, greater challenge than β-thalassemia to the neonatologist and pediatrician. The α-thalassemias are characterized by the decrease or complete suppression of α-globin polypeptide chains, with reduced or absent synthesis of one to all four α-globin genes. In the fetus, a complete deficiency of chain synthesis results in an absence of Hb F and the production of Hb Bart’s. Hb Bart’s is composed of tetrads of the γ-globin chain (γ 4 ) and exhibits a profoundly abnormal oxygen dissociation curve reflecting the reduced capacity to off-load oxygen at the tissue capillary bed. The gene cluster, which codes for and controls the production of these polypeptides, maps near the telomere of the short arm of chromosome 16 within a G+C-rich and early-replicating DNA region. The genes expressed during the embryonic stage (ζ) or fetal and adult stage (α-2 and α-1) can be modified by point mutations that affect either the processing-translation of mRNA or make the polypeptide chains extremely unstable. Much more frequent are the deletions of variable size (from approximately 3 kilobases to more than 100 kilobases) that remove one or both α genes in cis or even the whole gene cluster. Deletions of a single gene are the result of unequal pairing during meiosis, followed by reciprocal recombination. These unequal crossovers, which produce also α-gene triplications and quadruplications, are made possible by the high degree of homology of the two α genes and of their flanking sequences.
The interaction of the different α-thalassemia determinants results in three phenotypes: α-thalassemic trait, clinically silent and presenting with only limited alterations of hematologic parameters; Hb H disease, characterized by the development of a hemolytic anemia of variable degree; and Hb Bart’s hydrops fetalis syndrome (lethal), a consequence of compromised oxygen delivery to tissues. The diagnosis of α-thalassemia caused by deletions is based on electrophoretic analysis of genomic DNA digested with restriction enzymes and hybridized with specific molecular probes. Recently, polymerase chain reaction (PCR)–based strategies have replaced Southern blot analysis. Hemoglobin H disease, a mutation of three α-globin genes, is more severe than previously recognized. Anemia, hypersplenism, hemosiderosis, growth failure, and osteoporosis are commonly noted as the patient ages. Infants with one or two functional α-globin genes have microcytosis at birth (mean corpuscular volume is <95) and an elevated percentage of Hb Bart’s on electrophoresis. α-Thalassemia major is usually fatal in utero. Surviving newborns who did not undergo intrauterine transfusion often have congenital anomalies and neurocognitive injury. Serious maternal complications often accompany pregnancy. Doppler ultrasonography with intrauterine transfusion ameliorates these complications. The high incidence in selected populations mandates population screening and prenatal diagnosis of couples at risk. Universal newborn screening has been adopted in several regions with DNA confirmatory testing using the methods noted earlier.
Sickle Cell Anemia
Sickle cell disease (SCD) is caused by a single point mutation in the β-globin gene that causes the hydrophilic amino acid glutamic acid to be replaced with the hydrophobic amino acid valine at the sixth position. SCD is an autosomal recessive genetic blood disorder with incomplete dominance, characterized by red blood cells that assume an abnormal, rigid, sickle shape. Sickling decreases the cells’ flexibility and carries a risk of various complications. The introduction of newborn screening in the United States has had a significant impact on morbidity and mortality from SCD. Historically, the failure to achieve early identification of SCD resulted in a high rate of mortality because of the susceptibility of these patients to overwhelming infection, particularly with encapsulated organisms. Penicillin prophylaxis and the introduction of the pneumococcal vaccine have had an additional impact on the risk of sepsis and mortality in this population. Inheritance of the sickle gene with a thalassemia variant, such as β-thalassemia, can alter the presentation, in part by increasing the relative concentration of Hb S.
Anemia of Prematurity
Anemia of prematurity is thought to be principally a direct consequence of delivery before placental iron transport and fetal erythropoiesis are complete and is exaggerated by various factors, including blood losses associated with phlebotomy to obtain samples for laboratory testing, low plasma levels of erythropoietin due to both diminished production and accelerated catabolism, rapid body growth and the need for commensurate increases in red blood cell volume and mass, and disorders causing red blood cell losses due to bleeding and/or hemolysis. Blood losses resulting from the phlebotomy required for frequent laboratory studies can be a frequent cause of anemia of prematurity, despite advances in blood conservation with microsampling methods. The sick preterm infant receiving ventilatory assistance can often have more than 5 mL of blood per day withdrawn for laboratory studies. At this rate, an 800-g infant would lose his or her entire blood volume for laboratory studies in approximately 13 days. Large infants are less affected because of their greater blood volumes.
Rapid somatic growth of the preterm and very low-birth-weight infant also contributes substantially to anemia of prematurity. Very low-birth-weight infants will typically more than double their body weight and blood volume by the time they are ready for discharge from the nursery. In addition to the factors mentioned earlier, possibly the most significant contributing factor to this process is the prolonged cessation of erythropoietin production. As noted previously, reactivation of erythropoietin production in the infant kidney appears to be determined more by a biologic clock than by a response to stress. Indeed, there is no erythropoietin response to even severe anemia until the infant reaches a corrected gestational age of about 34 to 36 weeks. After this time, the erythropoietin system will respond when the hematocrit declines into the range of 25% to 30%. The reticulocyte count will typically rise within 1 week after the increase in erythropoietin production. Because transfusion during this critical period suppresses the release of endogenous erythropoietin, it can delay the recovery from anemia of prematurity, particularly in the seriously ill preterm requiring multiple transfusions, in whom recovery may not be observed until an even later corrected gestational age. Ultimately, it is the tissue oxygen tension that stimulates erythropoietin release, and recipients of multiple transfusions in whom Hb F has largely been replaced by Hb A will be less likely to achieve a low enough tissue oxygenation to stimulate timely or early erythropoietin release.
The treatment for anemia of prematurity has evolved substantially. Because placental iron transport is incomplete in the preterm infant, these babies require supplemental iron to mount an effective erythroid response. Iron stores are largely acquired during the last month of intrauterine life, thus term infants are born with large iron stores. The combination of a lack of these iron stores and a rapid rate of growth (and concomitant increase in blood volume) during the first 6 months of life place the preterm infant at significant risk of anemia of prematurity. Most infants with a birth weight of less than 1000 g are given multiple red blood cell transfusions within the first few weeks of life. Red blood cell transfusions have typically been the mainstay of therapy for anemia of prematurity; recombinant human erythropoietin (rHuEPO) is largely unused because of the view that it fails to substantially diminish red blood cell transfusion needs despite exerting substantial erythropoietic effects on neonatal marrow.
Multiple randomized, controlled trials have shown that treatment of extremely preterm infants with rHuEPO during the period when their endogenous erythropoietin system is inactive stimulates erythropoiesis, maintains a higher hematocrit, and reduces the need for transfusions. Reticulocytosis appears about 1 week after the start of treatment. The main population thought to benefit are those infants born before 30 weeks of gestation, with the smallest, least mature in this group exhibiting the greatest benefit.
Treatment is usually started after the infant has tolerated the introduction of enteral feedings. Large multicenter trials have demonstrated that administration of rHuEPO plus iron supplementation cannot prevent early transfusions, particularly in very low-birth-weight newborns and in infants with severe neonatal diseases. However, this approach may be effective in preventing late transfusions. Doses of 100 U/kg body weight given 5 days per week or 250 U/kg given three times per week are equally effective, and there is no evidence that larger doses are more effective. Current treatment of anemia of prematurity should focus on efforts to minimize factors that reduce erythrocyte mass (phlebotomies, noninvasive procedures) and promote factors that increase it (placental transfusion, adequate nutritional support).
Extremely low-birth-weight preterm infants often develop anemia of prematurity from frequent and excessive blood draws, a process referred to by Ed Bell as “gradual exsanguination.” The hypoproliferative anemia is marked by inadequate production of erythropoietin. Recombinant human erythropoietin (rHuEPO) has been available since 1990, but trials looking at reduction of red blood cell transfusions with rHuEPO achieved limited success. There has been a focus recently on autologous transfusion, blood-sparing technologies, and limitation in the number of donors. Treatment of anemia of prematurity includes red blood cell transfusions, which are given to preterm infants based on indications and guidelines (hematocrit and hemoglobin levels, ventilation and oxygen needs, apneas and bradycardias, poor weight gain) that are relatively nonspecific.
The need for transfusions can be reduced by limiting phlebotomy losses, providing good nutrition, and using standard guidelines for transfusion based on hemoglobin level or hematocrit. What those guidelines should be is not clear. Analysis of data for the Premature Infants in Need of Transfusion (PINT) trial, which compared management according to restrictive and liberal transfusion guidelines in infants weighing less than 1000 g and used a composite primary outcome of death before home discharge or survival with either severe retinopathy, bronchopulmonary dysplasia, or brain injury on cranial ultrasonography, revealed no statistically significant differences between groups in any secondary outcome. The investigators concluded that in extremely low-birth-weight infants, maintaining a higher hemoglobin level results in more infants receiving transfusions but gives little evidence of benefit. Data on the impact of transfusion practices on long-term outcome are very limited and inconclusive. Until further evidence surfaces, the tendency will probably be to adopt more liberal indications for transfusion. Many centers continue to use the Shannon criteria which call for transfusion in infants if any of the following conditions are met: (1) a requirement for more than 35% inspired oxygen on continuous positive airway pressure (CPAP) or positive pressure ventilation with a mean airway pressure of more than 6 cm H 2 O; (2) a requirement for less than 35% inspired oxygen on CPAP or positive pressure ventilation with a mean airway pressure of less than 6 cm H 2 O, significant apnea and bradycardia, tachycardia (>180 beats per minute) or a respiratory rate of more than 80 breaths per minute, weight gain of less than 10 g/day over 4 days, or sepsis; or (3) a hematocrit of less than 20%. Valieva et al modified these criteria to be more restrictive because of concerns about complications in the transfused group. Their criteria include the following:
Hematocrit of less than 35% in the first week of life and in unstable condition ∗
∗ Instability is defined as an increased risk for poor oxygen delivery (e.g., prolonged oxygen desaturation episodes or hypotension requiring treatment).
Hematocrit of less than 28% in the first week of life or in unstable condition ∗
Hematocrit of less than 20% if older than 1 week of age and in stable condition
Several conditions are associated with polycythemia in utero. These include chronic hypoxia due to maternal toxemia and placental insufficiency, placental insufficiency with postmaturity syndrome, pregnancy at high altitudes, pregnancy in a diabetic woman, and trisomy 21. In most instances, newborns who have clinically significant polycythemia have a preexisting high hematocrit in utero due to one of the causes listed previously, which is then exaggerated by excessive placental transfusion at delivery. Conversely, early cord clamping and reduced placental transfusion can lead to a normal hematocrit in an infant who developed polycythemia in utero.
The complications of polycythemia are a consequence of the rise in blood viscosity that occurs as the hematocrit rises, which compromises circulation to a variety of tissues and organs. The clinical manifestations are distinct in each organ system. Skin manifestations include plethora and delayed capillary filling. Renal symptoms include proteinuria and hematuria, and in extreme conditions, renal disease can be indistinguishable from renal vein thrombosis. If the severity of polycythemia is poorly appreciated and early feeding is instituted, infants can develop necrotizing enterocolitis (NEC). The central nervous system manifestations of polycythemia may be mild, including poor feeding, irritability, and an abnormal cry; more concerning cases are those manifesting apnea, seizures, and cerebral infarction.
The diagnosis of polycythemia is not based solely on hematocrit, because there is no precise hematocrit at which symptoms appear in all infants. This is partly due to the fact that other factors affect viscosity in addition to hematocrit. Although symptoms are common when the venous hematocrit exceeds 66%, serious signs of organ dysfunction develop in some infants with lower hematocrits. It is essential that polycythemia be confirmed by measuring the venous blood hematocrit, because capillary values correlate poorly with the central venous hematocrit (capillary hematocrits are generally higher). The treatment for the neonate with polycythemia is partial exchange transfusion in which blood is replaced with a plasma substitute. Isotonic saline, plasma, and a mixture of saline and albumin have all been used with equal efficacy. The goal for the hematocrit is 50%. To achieve this through exchange, the following formula is typically used: V = [(HCT 1 − HCT D ) × body weight (kg) × 90 mL]/HCT 1 , where V = the exchange volume, HCT 1 is the baby’s hematocrit, and HCT D is the desired hematocrit. The hematocrit must be monitored carefully after this procedure, because it will ultimately rise and if the HCT D is not reached with the initial volume exchange, it may rise again to a dangerous level.
The greatest dilemma is that posed by the asymptomatic newborn with polycythemia. Although one might advocate observation, the reality that the first manifestations are neurologic argues for early intervention and extremely close observation. Supporting early prophylactic exchange is the observation that the incidence of neurologic handicaps is increased in children who had untreated neonatal polycythemia. However, the benefit of this approach for preventing neurologic complications remains controversial.
Erythrocyte Transfusion in the Fetus and Newborn
Packed red blood cell transfusions are often administered to patients in the neonatal intensive care unit (NICU). Infants who have significant cardiopulmonary disease are transfused when they become anemic, because it is thought that a higher oxygen-carrying capacity improves their tolerance of cardiorespiratory distress. Current blood transfusion guidelines are useful in establishing parameters for transfusion, but it is essential that physicians also modify the application of these guidelines based on their own perceptions and assessments in identifying patients in need of a packed red blood cell transfusion. In an evaluation of the influence of caregiver perception and assessment on transfusion practices, neonates who underwent transfusion based on caregivers’ perceptions rather than adherence to strict guidelines were more likely to be receiving noninvasive ventilatory support and were more symptomatic. Neonates who improved after transfusion had a lower pretransfusion hematocrit and were more symptomatic compared with the group that did not show clinical improvement. In this study, tachycardia was the most sensitive predictor of benefit from packed red blood cell transfusion.
Extremely low-birth-weight infants are the most heavily transfused, yet the indications for transfusion do continue to represent an area of controversy. A very important concept to which one should always adhere is that there is no single critical hematocrit that always requires transfusion. In reality, there will be a range of critical hematocrits at which transfusion may be required even for an individual patient, and these different thresholds are values that are influenced by the severity of illness. Several studies have suggested an association between red blood cell transfusion and NEC in premature neonates. Withholding feeds during transfusion has never been clearly demonstrated to be beneficial but may have a protective effect against the development of NEC. In a retrospective case-control study of premature low-birth-weight infants (<32 weeks’ gestation and <2500 g) who developed NEC over a 6-year period (25 infants with NEC and 25 controls who never developed NEC), more infants in the NEC group received transfusions in the 48 to 72 hours preceding diagnosis (56% versus 20% within 48 hours [ P = .019] and 64% versus 24% within 72 hours [ P = .01]). The total number of transfusions and age of red blood cells were not different in the two groups. The same investigators implemented a policy of withholding feeds during transfusion, and this practice was associated with a decrease in the incidence of NEC from 5.3% to 1.3% ( P = .047). These data support the recognized association of NEC with the administration of red blood cell transfusions in the 48 to 72 hours preceding presentation of NEC and provide a rationale for exercising caution in feeding around the time of packed red blood cell transfusions in the neonate.
The risk-to-benefit ratio of blood transfusions for preterm infants will continue to be defined by ongoing experience. Although use of a more restrictive transfusion threshold for hemoglobin level or hematocrit, or both, may decrease the number of blood transfusions in preterm infants, the impact of such an approach on long-term outcomes must be defined.
White Blood Cells
But so long as you stimulate the phagocytes, what does it matter which particular sort of serum you use for the purpose? George Bernard Shaw, The Doctor’s Dilemma
Mature white blood cells are derived from pluripotent hematopoietic stem cells. In early development, hematopoietic stem cells emerge separately from the yolk sac, chorioallantoic placenta, and aorta-gonad-mesonephros region. Following the initial erythropoietic stage, myeloid progenitor cells can be found in the yolk sac during the third to fourth week of gestation. From the yolk sac, these progenitor cells sequentially migrate to the liver, thymus, and spleen and eventually take up permanent residence in the bone marrow at the eleventh to twelfth week of gestation. Hematopoietic stem cells with self-renewal capacity give rise to pluripotent progenitors that progress to common lymphoid or common myeloid progenitors. Common lymphoid progenitors differentiate into natural killer (NK) cells, B lymphocytes, T lymphocytes, and immature lymphoid dendritic cells. Common myeloid progenitors differentiate into granulocytes (neutrophils, eosinophils, and basophils), monocytes, and immature myeloid dendritic cells. Monocytes give rise to tissue macrophages.
The systems mediating innate immunity have qualitative and quantitative deficiencies that affect the newborn’s response to infections. For example, neonatal neutrophils ingest and kill bacteria as efficiently as their adult counterparts, but adhesion and subsequent migration of these cells to sites of infection are impaired. The migratory defect of neonatal neutrophils is exacerbated by limited production of the chemoattractant C5a and low generation of C3b, which is necessary for opsonization and phagocytosis. Neutrophil storage pools are rapidly exhausted in the face of serious infection, and the capacity to replenish those stores is limited in the neonate. Acquired immunity in the newborn is affected by qualitative and quantitative deficiencies in lymphoid lineage as well. Cell-mediated killing by NK and cytotoxic T cells is diminished, which leaves the newborn vulnerable to certain viral and intracellular pathogens. The newborn infant produces primarily IgM, and little IgG and IgA, in response to antigenic challenge. Neonatal T and B cells are predominately of a naive phenotype. Since the lymphocyte maturation process is directed largely by cytokines and the capacity of neonatal cells to produce key cytokines such as interleukin-4 and IFN-T interferon-gamma is limited, the acquisition of adult-type functional capabilities is delayed in vivo.
Despite encountering a pathogen-rich environment at the time of birth, most newborn infants, do not become ill. The relative immunodeficiency of the neonate has been viewed by some as an adaptive mechanism to optimize survival by balancing the conflicting immunologic requirements of life in utero with those of the external environment.
The neutrophil counts in an infant vary by birth weight and postpartum age. For term and near-term infants, values published by Manroe et al are considered appropriate. An absolute neutrophil count (ANC) of 1800 to 5500/µL is seen at birth, and ANC increases by threefold to fivefold over the next 12 to 18 hours of life. By 24 hours of life, the ANC begins to fall, decreasing steadily to 1800 to 7200/µL at 5 days, from then it falls to and remains at 1800 to 5400/µL through 28 days of age. Studies by Mouzinho et al show that normal preterm very low-birth-weight neonates have leukocyte reference ranges that differ significantly from those of older neonates (neutrophil counts have lower minimum values in the former group). Publications recommend using the Mouzinho et al chart for infants of less than 1500 g birth weight and the Manroe et al chart for larger infants (see also Appendix C ). Studies suggest that neonates with neutrophil counts above 1000/µL are not likely to be at high risk of acquiring a nosocomial infection. Counts below 500/µL (particularly when they remain below 500/µL for many days) are associated with an increased risk for developing a nosocomial infection. Persistent counts between 500 and 1000/µL may pose some intermediate risk.
Causes of Neonatal Neutropenia
Box 17-1 lists the most common causes of neutropenia in newborns. In general, neutropenia can be caused by either decreased neutrophil production or increased destruction.
Increased neutrophil destruction or utilization
Autoimmune neutropenia in the mother
Infection: bacterial, viral
Reduced neutrophil production
Infants of hypertensive mothers
Donors of twin-to-twin transfusion
Kostmann disease (severe congenital agranulocytosis)
Pure white cell aplasia
Hyperimmunoglobulin M syndrome
TORCH infections ( to xoplasmosis, r ubella, c ytomegalovirus, h erpes simplex)
Excessive neutrophil margination
Neutropenia Secondary to Increased Neutrophil Destruction
Alloimmune Neonatal Neutropenia
In alloimmune neonatal neutropenia (ANN) the mother becomes immunized to a father’s neutrophil antigen that is expressed on the fetal neutrophils. Subsequently, IgG antibodies directed against fetal neutrophil antigen crosses the placenta and destroys the fetal granulocytes. The severity of neutropenia is influenced by the titer and subclass of the maternal IgG neutrophil antibodies, the phagocytic activity of the infant’s reticuloendothelial system, and the capacity of the infant’s marrow to compensate for the shortened survival of antibody-coated neutrophils. Antineutrophil antibodies have been found in as many as 20% of surveyed pregnant and postpartum women, but studies have documented ANN in 0.2% to 2% of consecutively sampled newborns. A wide variety of antigenic targets have been identified, including human neutrophil alloantigen (HNA) groups HNA-1, HNA-2, and HNA-3 as well as NC1, SH, SAR, LAN, LEA, and CN1. The role of human leukocyte antigen (HLA) is controversial. Despite all the available data, in nearly half of cases the antigens cannot be recognized. Symptomatic infants can manifest delayed separation of the umbilical cord, skin infections, otitis media, or pneumonia within the first 2 weeks of life. Although most infections are mild, overwhelming sepsis is known to occur and is associated with a mortality rate as high as 5% in infants with ANN. When neutropenia is prolonged (>7 days), severe (ANC of <500/µL), or associated with serious infections, ANN can be treated with subcutaneous recombinant human granulocyte colony-stimulating factor (rG-CSF). The use of growth factor in this setting is discussed later in the chapter. Fortunately, in the majority of cases the disorder is self-limiting and resolves over a period of weeks to a few months as levels of the transplacentally acquired maternal antibody diminish.
Autoimmune Neutropenia of Infancy
Autoimmune neutropenia of infancy (AIN) is a disorder caused by increased peripheral destruction of neutrophils as a result of antibodies in the infant’s blood that are directed against the infant’s own neutrophils. It is analogous to immune thrombocytopenic purpura or autoimmune hemolytic anemia. Primary AIN, which is not associated with other diseases such as systemic lupus erythematosus, is often observed in infants and has an incidence of 1 in 100,000. A large number of children with primary AIN show the presence of antibodies specific to HNA-1a or HNA-1b. Less frequently, the antineutrophil autoantibodies recognize adhesion glycoproteins of the CD11/CD18 (HNA-4a, HNA-4b) complex, the CD35 molecule (CR1), and FcγRIIb. The origin of these autoantibodies is not known. The mechanism proposed include molecular mimicry of microbial antigens, modification of endogenous antigens as a result of drug exposure, increased or otherwise abnormal expression of HLA antigens, or loss of suppressor activity against self-reactive lymphocyte clones. There have been reported associations with parvovirus B19 infection and exposure to β-lactam antibiotics. AIN is usually diagnosed during the first few months of life (3 to 8 months).
Diagnosis of AIN in premature twins has been reported, which suggests that sensitization can occur even in utero. Although there is significant neutropenia at presentation (500 to 1000/µL), the clinical course is usually benign with mild infections. Severe infectious complications (pneumonia, sepsis, meningitis) are seen in about 12% of these patients. AIN resolves spontaneously by the age of 2 or 3 years in 95% of cases. Therefore most cases require no specific therapy. The usefulness of antibiotic prophylaxis must be assessed on a case-by-case basis. Administration of rG-CSF is currently the first-line therapy to achieve remission of the neutropenia. Treatment with IVIG is effective in less than 50% of cases and the benefit lasting less than 2 weeks. Steroids have limited effect in immune-mediated neutropenia.
Neonatal Autoimmune Neutropenia
Neonatal autoimmune neutropenia is seen when mothers with autoimmune disease transfer their neutrophil autoantibodies passively to the fetus. Most often, the mother and the infant are neutropenic. The infant’s neutropenia is transient and asymptomatic. The recovery process takes a few weeks to a few months and depends on the time it takes to clear IgG antibodies.
Neutropenia in Neonates with Sepsis
Neonates have immature granulopoiesis. This frequently results in neutropenia after sepsis, which is likely secondary to exhaustion of the storage and proliferative pools of the bone marrow. Neutropenic septic neonates have a higher mortality rate than nonneutropenic septic neonates. Whether growth factor or granulocyte infusions should be used in such a setting remains controversial (see later discussion). Neutropenia commonly occurs in neonates who have NEC as well. In this instance, neutropenia results from increased use and/or destruction in tissues, margination due to endotoxinemia, and increased mobilization of neutrophils into the peritoneum.
Neutropenia Secondary to Decreased Neutrophil Production
Severe Congenital Neutropenia
Severe congenital neutropenia is a genetically heterogeneous bone marrow failure syndrome characterized by maturation arrest of myelopoiesis at the promyelocyte-myelocyte stage. Estimated frequency is approximately 1 to 2 cases per million with equal male-female distribution. Severe congenital neutropenia follows an autosomal dominant or autosomal recessive pattern of inheritance. About 60% of cases are attributable to mutations in the gene for neutrophil elastase ( ELA2 ). Less commonly, mutations in HAX1, G6PC3, and other genes cause this disorder. From early infancy, patients who have severe congenital neutropenia experience bacterial infections. Omphalitis, beginning directly after birth, may be the first symptom; however, otitis media, pneumonitis and infections of the upper respiratory tract, and abscesses of the skin or liver are also common and can lead to the diagnosis of severe congenital neutropenia. Patients with the disorder have severe chronic neutropenia with ANCs continuously below 200/µL; in many cases, peripheral blood neutrophils are completely absent. Peripheral monocytosis or eosinophilia may be present. The bone marrow usually shows a maturation arrest of neutrophil precursors at an early stage (promyelocyte-myelocyte level) with few cells of the neutrophilic series beyond the promyelocyte stage. The use of rG-CSF remains first-line treatment for most patients with severe congenital neutropenia. Transplantation of hematopoietic cells from an HLA-identical sibling is beneficial for patients who are refractory to rG-CSF therapy. Patients who have severe congenital neutropenia are at risk of leukemic transformation, and those who develop myelodysplasia or leukemia should proceed urgently to hematopoietic stem cell transplantation.
Shwachman-Diamond syndrome is an autosomal recessive marrow failure syndrome associated with exocrine pancreatic insufficiency and predisposition to leukemia. Approximately 90% of patients meeting clinical criteria for the diagnosis of Shwachman-Diamond syndrome harbor mutations in the SBDS gene (Shwachman-Bodian-Diamond syndrome) that maps to the 7q11 centromeric region of chromosome 7. The initial symptoms typically are diarrhea and failure to thrive beginning in early infancy, and it is truly rare for the disease to present in the neonatal period. Growth failure and metaphyseal chondrodysplasia associated with dwarfism are seen in some patients. The most common hematologic abnormality, affecting 88% to 100% of patients with Shwachman-Diamond syndrome, is neutropenia. Patients with Shwachman-Diamond syndrome are susceptible to recurrent bacterial, viral, and fungal infections; in particular, otitis media, sinusitis, mouth sores, bronchopneumonia, septicemia, osteomyelitis, and skin infections. The illness may progress to bone marrow hypoplasia or dysplasia, leading to moderate thrombocytopenia and anemia. For treatment, rG-CSF has been used. The only definitive therapy for marrow failure, myelodysplasia, or leukemia is hematopoietic cell transplantation.
Neutropenia in Neonates with Hypertensive Mothers
Infants born to mothers who have pregnancy-induced hypertension (PIH) or HELLP syndrome ( h emolysis, e levated l iver enzymes, and l ow p latelet count) are observed to have neutropenia in 40% to 50% of cases, with the most severe neutropenia in the very low-birth-weight infants. This type of neutropenia is the result of placental production of an inhibitor of myelopoiesis. It can be severe, with blood neutrophil counts below 500/µL. With no specific treatment, this variety of neutropenia generally resolves in about 72 hours and almost always resolves by the fifth day after birth. Whether a risk of sepsis is associated with neutropenia in infants born to mothers with preeclampsia remains a topic of discussions.
Neutropenia in Donor Twins
Neutropenia occurs in the donor twin (the twin who becomes anemic) in twin-to-twin transfusion. It is usually transient. Since the myelopoiesis shifts toward erythropoiesis, neutrophil production decreases, which results in neutropenia. No left shift is present.
Neutropenia in Neonates with Rh Hemolytic Disease
The neutropenia in neonates with Rh hemolytic disease is likely caused by a shift of myelopoiesis toward erythropoiesis, which diminishes neutrophil production. It is usually transient.
Neutropenia Secondary to Mixed Causes
Drugs can cause neutropenia through suppressive effects on progenitors, changes in marrow extracellular matrix, development of autoantibodies, and other mechanisms. Ganciclovir has been strongly associated with neutropenia, and cessation of therapy may be necessary. Other drugs used in the NICU that have been implicated in causes of neutropenia include β-lactam antibiotics, thiazide diuretics, and ranitidine.
Intrauterine cytomegalovirus and rubella virus infections can be associated with neutropenia or pancytopenia. Neutropenia in this instance is likely secondary to splenomegaly; however, there might be an element of decreased production as well.
Artifactual neutropenia has been described that is caused by ethylenediaminetetraacetic acid (EDTA)–induced neutrophil agglutination in vitro. The condition can be diagnosed by the presence of neutrophil clumps on peripheral smears.
Evaluation of the Neonate with Neutropenia
Neutropenia in the NICU requires little diagnostic evaluation if the cause is clear (e.g., NEC, sepsis, maternal PIH). However, if neutropenia persists more than 3 to 5 days, particularly if the count is less than 500/µL, additional evaluation is needed. Helpful findings on physical examination include characteristic dysmorphic features such as skeletal dysplasia, radial or thumb hypoplasia (congenital bone marrow failure syndromes), hepatosplenomegaly (TORCH syndrome, storage disorders), and skin or hair pigmentary abnormalities (Chédiak-Higashi syndrome). A complete blood count, including microscopic examination of the peripheral blood smear to determine neutrophil morphology, can be useful in identifying congenital neutropenia syndromes. The immature-to-total (I/T) neutrophil ratio can be helpful in differentiating defects in production from destruction of neutrophils. The I/T ratio can be calculated as follows:
( Bands + Metamyelocytes + Myelocytes ) / ( Segmented neutrophils + Bands + Metamyelocytes + Myelocytes )