Regulation of Hematopoiesis

Few hematopoietic stem cells enter the cell cycle at any given time. Most are in a resting state. Proliferation and differentiation occur within a suitable microenvironment of stroma and humoral factors. The WNT/β-catenin and Notch-δ signaling pathways drive stem cell development. Transcription factors involved in stem cell development include GATA2, RUNX1, TEL/ETV6, SCL/TAL1, and LM02. Other transcription factors such as PU.1, GFIX, C/EBPα, and GATA1 are considered to be more lineage-specific, but most also participate in lineage priming, where stem cells differentiate along a pathway depending upon cellular and environmental stimuli. Multiprotein complexes assemble and bind DNA regulatory elements to modulate transcription. Epigenetic regulatory mechanisms of transcription include DNA methylation on CpG residues and histone modification.³⁴

Factors promoting hematopoiesis include BMP4, VEGF, WNT, and FGF. Hematopoietic cytokines such as stem cell factor, fms-like tyrosine kinase receptor-3 ligand, interleukin-6, thrombopoietin, erythropoietin, and granulocyte colony-stimulating factor (G-CSF) play critical roles in the maintenance and differentiation of human hematopoietic cells. Some hematopoietic growth factors are produced in the vicinity of hematopoietic progenitors, and others are synthesized remotely (Table 88-2). Few of the glycoprotein growth factors are available for clinical use, but that number is expected to increase.

Some of the earliest hematopoietic growth factors discovered were referred to as colony-stimulating factors (CSFs), because in culture they stimulate progenitor cells to form colonies of recognizable maturing blood cells. The prefixes refer to the maturing cell produced. GM-CSF is granulocyte-macrophage CSF. The interleukins (ILs) were named for the fact that they are derived from, or act upon, leukocytes. Other factors are named for the cell surface receptor to which they bind, such as thrombopoietin receptor agonists that stimulate megakaryocyte differentiation and platelet production. Growth factors such as IL-3 and GM-CSF stimulate proliferation, differentiation, and survival of a broad range of precursors, including stem cells. Others such as erythropoietin and granulocyte CSF (G-CSF) are lineage restricted.

Hemoglobin Switching

The hemoglobin tetramer is composed of two heterodimers, consisting of an α- and β-type globin. Different globin genes are sequentially expressed in RBC precursors, a process known as hemoglobin switching (see Figure 88-2 and Table 88-1). During development, α- and β-type globin gene clusters are activated sequentially from the 5′ (embryonic) end to the 3′ (adult) end. The α-type globin genes, ζ-globin, α₁-globin, and α₂-globin, are located on chromosome 16 with the ζ gene 5′ to a pair of duplicated α-globin genes. The β-type genes on chromosome 11 are oriented 5′ to 3′ as ε-, ^Gγ, ^Aγ, δ, and β (see Figure 88-2). The protein products of the ^Aγ- and ^Gγ-globin genes are functionally similar and differ by a single amino acid residue. Globin gene expression is controlled by cis-elements of individual globin gene promoters, proximal and distal enhancer regions, and positively acting transcription factors, such as GATA1, GATA2, NFE2, MYB, EKLF, RBTN2, and SCL. Other mechanisms that also regulate globin switching are silencers, DNA conformational changes, and DNA methylation.^34,76 In fact, discovery of the ability to chemically demethylate CpG residues in the silenced γ-globin gene promoter launched translational research efforts to enhance fetal hemoglobin production in patients with β-globin defects such as β thalassemia and sickle cell anemia.

During yolk sac hematopoiesis, RBCs produce the embryonic hemoglobins (see Table 88-1). Hb F is the predominant hemoglobin in the fetus and neonate. Production of the major adult hemoglobin (Hb A) increases significantly between birth and 6 months of age, as Hb F production declines. Synthesis of HbA₂, a minor adult globin, also increases gradually over the first months of life (see Figure 88-2). After 6 months of age, Hb F usually constitutes less than 1% of the total hemoglobin and is unevenly distributed among red blood cells. Each of the different types of globin exhibits distinctive functional properties. Fetal erythrocytes, which contain mostly Hb F, have a higher oxygen affinity than adult red blood cells. This allows the transport of oxygen from maternal Hb A–containing erythrocytes across the placenta to fetal red blood cells. The increased O₂ affinity of fetal hemoglobin has been ascribed to the diminished interactions of Hb F with red blood cell 2,3-BPG. Embryonic erythrocytes also display a greater affinity for oxygen than adult cells.

Erythropoietin, Erythropoiesis, and the Physiologic Anemia of Infancy

Erythropoietin (EPO), the essential glycoprotein growth factor for erythropoiesis, binds to erythropoietin receptors on early erythroid progenitor cells and via the JAK2 signaling pathway regulates RBC production by protecting them from apoptosis. Erythropoietin is produced primarily in the fetal liver and later in the cortical peritubular cells of the kidney, so that in adults renal production of EPO is the most important. Erythropoiesis is highly responsive to blood oxygenation. Hypoxia inducible factors (HIFs), constitutively expressed EPO transcription factors, are destroyed in the presence of oxygen. Under hypoxic conditions, EPO production increases. Levels of EPO in cord blood are higher than in adult blood samples (Table 88-3), but there is a dramatic decrease after birth in response to higher levels of tissue oxygenation. By 1 month of age, serum levels in healthy term infants reach their nadir. This is followed by a rise to maximal levels at 2 months of age and then a slow drift down to adult values.

The postnatal changes in tissue oxygenation and erythropoietin production result in a physiologic anemia of infancy with a mean minimal hemoglobin concentration in healthy term infants of about 11 g/dL at 8 to 12 weeks of life (Figure 88-4; Table 88-4). Because of the shorter life span of RBCs in preterm infants with low EPO levels, the nadir is noted by 6 weeks of age and ranges from 7 to 10 g/dL. In VLBW and ELBW infants, the nadir is more than 20% below the value of the Hb at birth. In ELBW infants whose nadir falls below 7 g/dL, this so-called physiologic anemia of prematurity can be associated with pallor, tachypnea, tachycardia, poor feeding, and poor weight gain.² Other causes of blood loss and suppression of erythropoiesis in the ill neonate can contribute to more severe and earlier anemia. Although preterm infants will respond to hypoxia with a rise in EPO levels, the increase is lower than that expected for term infants. The suboptimal EPO response may be due to developmental changes in transcription factors or to the site of fetal EPO production. The use of recombinant EPO in premature and sick newborn infants is discussed later.

Red Blood Cell Indices During Prenatal and Postnatal Development

The RBC count, Hb concentration, and hematocrit (Hct) increase throughout gestation, as shown in Table 88-5. In term infants, the mean capillary hemoglobin at birth is 19.3 g/dL (see Table 88-4). The Hct has a mean of 61 g/dL. Premature infants have lower Hb levels than do full-term infants. In addition to gestational age, Hb levels are influenced by a variety of factors that must be kept in mind when analyzing the neonate with anemia or polycythemia. One important determinant is the site of sampling: Capillary Hb values are higher than peripheral venous samples, and umbilical venous Hb results are the lowest. The interval between delivery and clamping of the umbilical cord and the height of the baby relative to the placenta can significantly affect a newborn’s blood volume and total RBC mass. The placenta contains about 100 mL of blood. The mean blood volume of a full-term infant is about 85 mL/kg. Early or delayed clamping of the umbilical cord alters this mean blood volume by about 10% lower or higher, respectively. The average Hb at birth is relatively unchanged; however, 48 hours later, after redistribution of plasma volume, Hb values will reflect the lower or higher red cell mass. Racial differences also occur. One study reported significantly higher Hb, Hct, and MCV in white infants compared with black infants of similar gestational ages.³ Reticulocyte counts in the cord blood of infants average 4% to 5%, and nucleated RBCs are evident in most cord blood samples (40,000/µL). These findings are presumed to reflect high EPO production secondary to low oxygen retention in utero. Infants who experience placental insufficiency and intrauterine growth restriction have higher than normal EPO production and an even greater degree of erythrocytosis. The mean MCV of RBCs in the newborn is increased. The RBCs of the neonate have an increased Hb content, but the mean corpuscular hemoglobin concentration (MCHC) is comparable to that of adults.

Delayed (30-90 seconds) cord clamping continues to generate considerable interest because it has been shown to prevent hypotension, raise hematocrit, and decrease the need for transfusions in preterm infants. In term infants who have had delayed cord clamping, there has been a reduction in iron deficiency anemia in the first year of life, but an increased risk of early jaundice.^40a

Red Blood Cell Survival

The normal life span of adult RBCs is about 120 days. The life span of RBCs in newborns at term is 60 to 80 days and 30 to 50 days in ELBW infants. In general, red blood cell survival is affected by changes related to aging (senescence) and by random hemolysis of red blood cells, or portions of red blood cells, in the spleen and the rest of the reticuloendothelial system. Some of the changes in neonatal RBCs compared with adult RBCs listed in Table 88-6 affect survival. Aging erythrocytes with declining RBC enzyme activity become progressively less tolerant of oxidative challenges during the transportation of oxygen molecules and exposure to circulating oxidants. Any additional deficiencies in the enzymatic pathways of the RBC may affect the ability of the erythrocyte to tolerate oxidative challenges and further reduce red blood cell survival. With transit through the kidneys and lungs, the RBCs experience cycles of osmotic swelling and shrinkage. Shear forces in high-pressure areas of the circulation buffet the erythrocytes. Each passage through the cords of Billroth within the spleen requires the RBCs to deform and squeeze through tiny slits in the walls of the cords or face destruction if they cannot. Congenital or acquired defects in membrane stability or decreases in the ratio of surface area to red blood cell volume will also decrease erythrocyte survival. Alterations in the deformability of neonatal erythrocytes and relative intolerance to oxidative challenges result in shorter survival for neonatal red blood cells. Random hemolysis can be increased with splenic enlargement or activation of the phagocytic system. Infants with hemolysis may have exaggerated anemia because of decreased erythropoiesis, enhanced splenic filtration, and activation of phagocytes.