Hematologic Problems

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.

Hematopoiesis during mammalian embryonic development proceeds from the yolk sac blood island to the aorta-gonad-mesonephros (AGM) 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. 16.1 ). 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 beta-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 KLF) is essential during both embryonic and adult erythropoiesis. KLF2 is a positive regulator of the mouse and human embryonic beta-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-gamma than A-gamma 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 because of 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.

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 because of 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.

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 because of their intrinsic properties. Fetal hemoglobin, or Hb F (two alpha-globin chains and two gamma-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 gamma chains during fetal development to mainly beta 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 gamma-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 gamma-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 (SCD) 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.

Hemorrhagic Anemias

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.

Before delivery, internal hemorrhage may occur, with the most common type being intraventricular hemorrhage. The true incidence of intracranial hemorrhage (ICH) is not certain, but in cases of alloimmune thrombocytopenia in which there is severe thrombocytopenia, ICH may be seen in as many as 20% of cases. Administration of intravenous gamma-globulin (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 decrease the risk of ICH (see later discussion). Internal hemorrhage can also result from forces incurred around the time of delivery. Important common types of hemorrhage secondary to birth-related injuries 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 should be kept in mind.

For hemorrhages that are associated with the birth process, 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 may not 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 vital signs are important to monitor because they will guide the approach and extent of fluid resuscitation. Acute situations may include placenta previa, vasa previa, abruption, or blood loss from the cut umbilical cord. In such cases, timely 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 can help decrease the time required to initiate 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.

Nonhemorrhagic Anemias

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 important. To diagnose the cause of hemolysis, obtain information about any family history of anemia or neonatal jaundice, and examine both the baby and the blood smear. Clues to the appropriate diagnostic tests are often present in these smears, suggested by morphologic abnormalities of the red blood cells. It is recommended 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.

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

Anemia Secondary to Hemoglobinopathies

Hemoglobinopathies arise from mutations in the globin genes, with the most common hemoglobinopathies resulting from mutations in the beta-globin gene. These are typically clinically silent at birth because of the persistence of Hb F but manifest as the expression switches from gamma- to beta-chain production.

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 as a result of 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 as a result of 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 may 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).

Neutropenia

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.