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GMP: Granulocyte monocyte progenitor matopoiesis. Nature. 2019;574(7778):365–371.
GPCR: G protein-coupled receptors 23. Höfer T, Rodewald HR. Differentiation-based model of hematopoietic stem cell
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HSC: Hematopoietic stem cell 51. Bjerregaard MD, Jurlander J, Klausen P, Borregaard N, Cowland JB. The in vivo
HSPA1A: Heat shock protein 1 profile of transcription factors during neutrophil differentiation in human
KC: Keratinocyte chemoattractant bone marrow. Blood. 2003;101(11):4322–4332.
LAD: Leukocyte adhesive deficiency 52. Cowland JB, Borregaard N. Granulopoiesis and granules of human neutrophils.
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LMPP: Lymphoid-primed multipotent progenitor 65. Hoogendijk AJ, Pourfarzad F, Aarts CEM, et al. Dynamic transcriptome-pro-
LPS: Lipopolysaccharide teome correlation networks reveal human myeloid differentiation and neutro-
LXA4: Lipoxin A4 phil-specific programming. Cell Rep. 2019;29(8):2505–2519.
MEP: Megakaryocyte erythroid progenitor 71. Grassi L, Pourfarzad F, Ullrich S, et al. Dynamics of transcription regulation in
human bone marrow myeloid differentiation to mature blood neutrophils. Cell
MIP: Macrophage inflammation protein Rep. 2018;24(10):2784–2794.
MPO: Myeloperoxidase 75. Mistry JJ, Marlein CR, Moore JA, et al. ROS-mediated PI3K activation drives
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139. Rørvig S, Østergaard O, Heegaard NH, Borregaard N. Proteome profiling of
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PAMP: Pathogen-associated molecular pattern ule subsets is determined by timing and not by sorting: the specific granule
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109 Developmental Erythropoiesis

Timothy M. Bahr | Robin K. Ohls

present in the mesenchymal and endodermal layers of the yolk

ERYTHROCYTE KINETICS sac.1 These red blood cells, or hematocytoblasts, are the product
of primitive megaloblastic erythropoiesis,2 and they differ
SITES AND STAGES OF FETAL RED BLOOD CELL from erythrocytes formed later in gestation when definitive
PRODUCTION normoblastic erythropoiesis occurs. Primitive erythroblasts
EXTRAEMBRYONIC ERYTHROPOIESIS are nucleated and macrocytic (20 to 25 μm in diameter). They
Extraembryonic erythropoiesis begins in the fetal yolk sac have a mean corpuscular volume (MCV) of more than 180
by 14 days’ gestation. Small nests of nucleated blood cells are fL and have a characteristic fine nuclear chromatin pattern

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and a polychromatophilic cytoplasm containing abundant
hemoglobin.2,3 Red blood cells enter the embryonic circulation
at 3 to 4 weeks’ gestation, coincident with joining of the vitelline
and umbilical circulations.4
INTRAEMBRYONIC ERYTHROPOIESIS
Definitive normoblastic erythropoiesis of the fetus begins
in the liver during the early first trimester.5 By 6 to 8 weeks’
gestation, the liver replaces the yolk sac as the primary site of
red blood cell production, and by 10 to 12 weeks’ gestation,
extraembryonic erythropoiesis has essentially ceased. Red blood
cell production occurs in the liver throughout the remainder
of gestation, although production begins to diminish during
the second trimester as bone marrow erythropoiesis increases
(Fig. 109.1). Erythroblasts are first noted in the marrow at 8 to
9 weeks’ gestation.5 By the end of the third trimester, almost all
erythropoiesis is occurring in the bone marrow, although resi­
dual erythropoiesis may continue in the liver and may be found
in other sites. In rodents, the spleen is also a site of erythropoiesis
before the onset of marrow red blood cell production. As with
the liver, this site ceases production shortly after birth.6 It is
unclear whether the spleen contributes to erythropoiesis in the
human fetus. Although erythroid precursors have been identi­
fied as early as 6 to 7 weeks’ gestation in human splenic tissue,7
it is not clear whether such cells are actually developing within
splenic tissue or whether they are simply part of the circulation.8
ONTOGENY OF STEM CELLS
Red blood cell precursors in the yolk sac are extremely primitive
cells, and they may either disappear or seed other areas where
erythropoiesis later becomes prominent. The unicentric theory
proposes that all hematopoietic stem cells originate from the
yolk sac, then migrate from one hematopoietic site to another.
Moore and Metcalf9 demonstrated circulating pluripotent stem
cells in the peripheral blood just before the development of
liver erythropoiesis. They suggested that the development of
intraembryonic hematopoiesis requires an intact yolk sac, and
migration of stem cells from the yolk sac to hematopoietic tissue is
necessary for the development of intraembryonic hematopoiesis.
The development of other organ systems via migration of cells
(such as neural crest tissue) gives credence to this theory.
The multicentric theory suggests that a new clonal formation of
hematopoietic stem cells occurs at different sites of hematopoiesis
Primitive Definitive
erythropoiesis erythropoiesis
Yolk sac
100 Liver
80
% Production
60
40
20
0
0 4 8 12
16 20 24 28 32 36 40
Gestational age (wk) Birth
Fig. 109.1 Sites and stages of fetal erythropoiesis. Primitive erythro-
poiesis begins in the yolk sac at 2 to 3 weeks after conception. By the
end of the first trimester the liver has become the primary erythroid
organ. The liver is the primary source of red blood cells during the
second trimester, whereas the bone marrow is the primary source of
red blood cells during the last trimester.
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CHAPTER 109 — Developmental Erythropoiesis 1105
during fetal development.Yolk sac cells are capable of producing
granulocytic, megakaryocytic, and erythroid colonies when
transplanted into adult irradiated recipients and in conditioned
newborn recipients,9,10 a finding that supports the view that
fetal stem cells possess the capacity to be pluripotent and their
differentiation is controlled by microenvironmental factors.
MARROW, LIVER, AND BLOOD DIFFERENTIALS
Table 109.1 describes the differential counts in fetal liver, marrow,
and circulating blood according to gestational age.11–14 The liver
of the second-trimester human fetus is the primary organ for
erythropoiesis,with a myeloid-to-erythroid ratio ranging from 0.07
at 14 to 17 weeks’ gestation to 0.2 at 21 to 24 weeks’ gestation.11
By 24 weeks the composition of the fetal bone marrow begins
to resemble that of adult bone marrow, differing by the presence
of a large number of stromal elements, the absence of plasma
cells and lymph follicles, and an overall increased cellularity in
the fetal bone marrow. Unlike adult bone marrow, large fat cells
are not present in fetal bone marrow. Between 18 and 22 weeks’
gestation, the mitotic index of the fetal bone marrow becomes
virtually identical to that of adult bone marrow. The fetal bone
marrow myeloid-to-erythroid ratio starts to exceed the normal
adult bone marrow ratio (1.5 ± 0.4) early in midgestation and
remains elevated even at the time of birth.
PROGENITOR CELL CONCENTRATIONS
Studies of bone marrow cells in tissue culture have identified
specific committed red blood cell precursors, termed erythroid
progenitors,15–18 on the basis of their characteristic growth
in vitro. When bone marrow cells are placed in semisolid media
culture systems for 5 to 7 days, an erythropoietin (EPO)-sensitive
erythroid progenitor cell, termed colony-forming unit–erythroid
(CFU-E), clonally matures into a single cluster containing 30 to
100 normoblasts (Fig. 109.2). An erythroid-specific progenitor
that is less differentiated than a CFU-E (and therefore a more
primitive cell) is termed a burst-forming unit–erythroid (BFU-
E). Twelve to 14 days after cultures of bone marrow cells are
initiated, a BFU-E develops into a large, multicentered colony
of normoblasts, in which each center contains 200 to 10,000
normoblasts. Finally, the most primitive erythroid progenitor cell
identifiable through in vitro culture is termed a colony-forming
unit–granulocyte, erythrocyte, macrophage, megakaryocyte
(CFU-GEMM, or CFU-MIX). Twelve to 14 days after marrow cells
are placed in culture, this multipotent progenitor develops into
a mixed colony of both normoblast clusters and granulocyte-
macrophage clusters.
The ability of an organ to produce red blood cells is based on
the number of progenitor cells it contains, as well as the growth
Marrow
factors stimulating those cells to proliferate. Determination of
erythroid progenitor numbers obtained from cell suspensions
of liver, marrow, spleen, and blood of second-trimester human
fetuses (Table 109.2) showed twice the number of multipotent
progenitors and erythroid progenitors in the fetal liver compared
with marrow.8,11,12 Erythroid progenitors from fetal liver also
appeared to be more sensitive to EPO (the primary erythroid
growth factor) than were progenitors from fetal marrow.
Progenitor cell concentrations in the spleen were nearly
identical to concentrations in the circulation.8–12 Whether the
progenitors isolated from liver and marrow represent different
subpopulations of progenitors (e.g., populations that express
different erythroid growth factor receptors, different receptor
numbers, different receptor affinity, or different cycling rates) is
not known.
REGULATION OF ERYTHROPOIESIS
Erythropoietic regulation in the human fetus differs markedly
from that in the adult. In the adult, erythropoietic regulation
primarily involves maintaining the red blood cell mass. In contrast,
1106 SECTION XVIII — Developmental Hematopoiesis

Table 109.1 Differential Counts in Liver, Marrow, and Blood During Gestation.

Differential Counts (Percentage ± SD) of Liver Cell Suspensions From Fetuses at a Gestational Age of 14–24 wk
Gestational Age (wk)
Cells 14–17 (n = 5) 18–20 (n = 7) 21–24 (n = 8)
Normoblast
Pronormoblast 3.1 ± 1.4 3.4 ± 1.0 2.9 ± 1.8
Basophilic 18.4 ± 7.6 13.7 ± 1.6 13.7 ± 4.7
Polychromatophilic 57.5 ± 12.6 55.3 ± 7.5 51.1 ± 5.5
Orthochromic 13.9 ± 5.2 15.9 ± 3.7 14.2 ± 5.0
Total erythroid cells 9.3 ± 6.3 87.8 ± 4.8 81.9 ± 5.0
Neutrophil
Promyelocyte 0±0 0.2 ± 0.2a 1.2 ± 0.4a,b
Myelocyte 0±0 0±0 0.2 ± 0.2a,b
Metamyelocyte 0±0 0±0 0±0
Band 0±0 0±0 0±0
Segmented 0±0 0±0 0±0
Total neutrophils 0±0 0.2 ± 0.2 1.4 ± 0.5a,b
Undifferentiated blast 0.5 ± 0.6 3.1 ± 1.8a 2.2 ± 0.7a
Macrophage 0.5 ± 0.6 1.2 ± 4.0a 1.3 ± 5.0
Lymphocyte 5.4 ± 2.6 3.9 ± 3.6 11.3 ± 4.2a,b
Eosinophil 0±0 0±0 0±0
Otherc 0.8 ± 0.8 3.4 ± 2.4 1.9 ± 2.4
Differential Counts (Percentage ± SD) of Marrow Cell Suspensions From Fetuses at a Gestational Age of
14–24 wk
Gestational Age (wk)
Cells 14–17 (n = 6) 18–20 (n = 6) 21–24 (n = 8)
Normoblast
Pronormoblast 0.3 ± 0.2d 1.1 ± 0.4d 0.5 ± 0.2d
Basophilic 1.4 ± 0.6d 3.1 ± 0.8d 1.7 ± 1.0d
Polychromatophilic 9.2 ± 5.0d 12.9 ± 6.2d 12.3 ± 5.2d
Orthochromic 12.1 ± 11.1 20.7 ± 12.2 8.1 ± 4.4
Total erythroid cells 23.0 ± 13.3d 37.8 ± 16.8d 22.6 ± 9.0d
Neutrophil
Promyelocyte 5.9 ± 2.9d 5.8 ± 2.6d 5.0 ± 1.7d
Myelocyte 2.7 ± 1.4d 3.4 ± 1.6d 2.1 ± 1.0d
Metamyelocyte 2.1 ± 1.0d 2.5 ± 1.8d 1.7 ± 1.0d
Band 3.1 ± 1.8d 3.2 ± 2.7d 1.6 ± 1.2d
Segmented 1.2 ± 1.3d 1.1 ± 1.3d 0.4 ± 0.2d
Total neutrophils 13.8 ± 6.1d 16.1 ± 8.0d 10.7 ± 5.0d
Undifferentiated blast 8.9 ± 12.4d 11.4 ± 5.4d 11.6 ± 3.6d
Macrophage 5.4 ± 3.8d 4.2 ± 2.4d 3.1 ± 1.6d
Lymphocyte 35.3 ± 15.8d 28.8 ± 8.0d 50.3 ± 6.8d
Eosinophil 0.5 ± 0.6 1.1 ± 1.2 1.2 ± 0.8d
Othere 0.8 ± 0.4 0.5 ± 0.4 0.4 ± 0.2
Differential Counts (Percentage ± SD) of Umbilical Cord Blood Samples From Fetuses at a Gestational Age of 18–29 wk
Gestational Age (wk)
Cells 18–21 (n = 186) 22–25 (n = 230) 26–29 (n = 144)
Normoblast (%WBCs) 45.0 ± 86.0 21.0 ± 23.0 21.0 ± 67.0
Basophilic 0.5 ± 1.0 0.5 ± 1.0 0.5 ± 1.0
Neutrophil 6.0 ± 4.0 6.5 ± 3.5 8.5 ± 4.0
Lymphocyte 88.0 ± 7.0 87.0 ± 6.0 85.0 ± 6.0
Eosinophil 2.0 ± 3.0 3.0 ± 3.0 4.0 ± 3.0
Monocyte 3.5 ± 2.0 3.0 ± 2.5 3.0 ± 2.5

aP < .05 versus 14 to 17 weeks.

bP < .05 versus 18 to 20 weeks.
cHepatocyte, megakaryocyte, or cell of undetermined origin.
dP < .05 versus fetal liver.
eMegakaryocyte or cell of undetermined origin.

WBCs, White blood cells.

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Pluripotent stem CFU-GEMM
cell
Stem cell factor
IL-6
IL-9
IL-3
GM-CSF
Fig. 109.2 Erythropoietic progenitors and the growth factors influencing erythropoiesis. BFU-E, Burst-forming unit–erythroid; CFU-E, colony-
forming unit–erythroid; CFU-GEMM, colony-forming unit–granulocyte, erythrocyte, macrophage, and megakaryocyte; EPO, erythropoietin; GM-
CSF, granulocyte-macrophage colony-stimulating factor; IL-3, interleukin-3; IL-6, interleukin-6; IL-9, interleukin-9.
Table 109.2 Progenitor Cell Concentrations in
Fetal Liver, Marrow, Spleen, and Blood.
CFU-MIX BFU-E
Liver 12.7 ± 2.1 20.7 ± 3.1
Marrow 6.7 ± 1.4a 9.3 ± 2.7a
Spleen 4.2 ± 2.7a 5.9 ± 3.7a
Blood – 8.0 ± 5.4a
aP
< .05 versus liver.
Numbers represent cell concentrations per 5 × 103 cells plated.
BFU-E, Burst-forming unit–erythroid; CFU-MIX, colony-forming unit–
granulocyte, erythrocyte, macrophage, and megakaryocyte.
constant and dramatic changes characterize erythropoiesis in the
embryo and fetus. The incredible rate of somatic growth and the
resultant need to constantly increase the fetal red blood cell mass
necessitate an extraordinary erythropoietic effort. Moreover,
the relatively low oxygen tensions but high metabolic rates
of fetal tissues require a system of oxygen delivery that differs
significantly from the adult system.
ERYTHROID GROWTH FACTORS
The production of erythrocytes, from pluripotent stem cell to
mature red blood cell, is governed by various growth factors.
These erythropoietic growth factors are produced by accessory
cells such as liver macrophages and marrow stromal cells, and
they stimulate maturation, growth, and differentiation at various
stages of red blood cell production. The progenitors involved in
red blood cell production and the factors that stimulate their
cellular maturation are depicted in Fig. 109.2. Although these
growth factors all facilitate production of red blood cells, none
plays a more important regulatory role than EPO. EPO is a 30-
to 39-kDa glycoprotein that binds to specific receptors on the
surface of erythroid precursors and stimulates their differentiation
and clonal maturation into mature erythrocytes.19,20 The EPO
gene (EPO) is located on chromosome band 7q21-22.21 During
fetal erythropoiesis, EPO is produced principally by cells of
monocyte/macrophage origin residing in the liver. Postnatally, EPO
is produced almost exclusively by peritubular cells of the kidney.
Other growth factors besides EPO play a role in the
differentiation and clonal expansion of erythroid progenitors.
These include granulocyte-macrophage colony-stimulating factor
(GM-CSF),15 stem cell factor (also known as c-kit ligand),22 and
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CHAPTER 109 — Developmental Erythropoiesis 1107
BFU-E CFU-E Normoblast Reticulocyte Mature
erythrocyte
EPO
interleukin (IL)-3, IL-6, IL-9, and IL-12.23–26 Thrombopoietin, a
growth factor involved in platelet production whose receptor is
similar in structure to the EPO receptor, also stimulates erythroid
colony formation.27 IL-3 and GM-CSF were the first growth
factors noted to have erythroid-stimulating properties, initially
described as “burst-promoting activity.” In combination with
EPO, these factors synergistically stimulate differentiation and
proliferation of BFUs-E and CFUs-GEMM.
Stem cell factor is a multipotent growth factor that, in
combination with other factors, supports clonal maturation of
hematopoietic progenitors. Murine studies indicate that stem cell
factor may be expressed during embryogenesis, thereby affecting
embryonic and fetal erythropoiesis. In vitro studies show that
stem cell factor alone stimulates clonal maturation of fetal
(but not adult) multipotent progenitors. Erythroid progenitors
isolated from term umbilical cord blood are more responsive to
stem cell factor, alone and in synergism with GM-CSF and IL-3,
than are adult marrow progenitors.22
Term circulating erythroid progenitors are also more sensitive to
IL-6 and IL-9 than are adult progenitors.24 IL-6 is a multifunctional, 22-
to 26-kDa glycoprotein cytokine involved in B-cell stimulation and
immunoglobulin production, acute-phase reactions, and induction
of hematopoietic progenitors from a noncycling (G0) phase into an
active cycling (S) phase. IL-6 alone supports clonogenic maturation of
newborn umbilical cord blood BFUs-E and CFUs-GEMM and induces
progenitor cell cycling.24 IL-9 is also a multipotent cytokine and is
similar to IL-6 in its ability to stimulate primitive erythroid progenitors
and multipotent progenitors isolated from umbilical cord blood.25
Insulin-like growth factor 1 has been shown to cause clonal expansion
of erythroid progenitors isolated from adult marrow28,29 and on
erythroid and mesenchymal-like cells in the fetal spleen. A recently
identified growth factor termed regulator of human erythroid cell
expansion is an erythroid growth factor that promotes formation
of hemoglobinizing erythroblasts.30 It comprises a new EPO/EPO
receptor target and regulator of human erythroid cell expansion that
additionally acts to support late-stage erythroblast development.
UNIQUE FEATURES OF FETAL PROGENITORS
Fetal erythroid progenitors respond in a slightly different fashion
than do adult erythroid progenitors. In addition to the features
noted earlier, fetal progenitors appear more sensitive to EPO than
adult erythroid progenitors.31 Specifically, BFUs-E of fetal origin
develop more rapidly into erythroid colonies, and the colonies
generally contain significantly more normoblasts. In addition,
BFUs-E from adult bone marrow require a combination of EPO
plus another factor, such as IL-3 or GM-CSF, to mature clonally;
1108 SECTION XVIII — Developmental Hematopoiesis
Table 109.3 Blood Cell Indices During Gestation.
Gestational Age White Blood Total White Blood
(wk) Cellsa (×109/L) Cells (×109/L)
18–21 (n = 760) 4.68 ± 2.96 2.57 ± 0.42
22–25 (n = 1200) 4.72 ± 2.82 3.73 ± 2.17
26–29 (n = 460) 5.16 ± 2.53 4.08 ± 0.84
>30 (n = 440) 7.71 ± 4.99 6.40 ± 2.99
aIncluding
normoblasts.
From Forestier F, Daffos F, Catherine N. Developmental hematopoiesis in normal human fetal blood. Blood. 1991;77:2360.
however, many fetal BFUs-E mature in the presence of EPO
alone.32 Studies have shown that fetal clones produce GM-CSF
and IL-3, which may explain their unique capability for growth
factor independence and auto-stimulation.33
CONTROL OF ERYTHROPOIETIN PRODUCTION
Erythropoiesis in utero is controlled by erythroid growth factors
produced by the fetus, not the mother. EPO is the primary
regulator of erythropoiesis in adults and appears to be the
controlling factor for fetal erythropoiesis, especially during
late gestation. EPO does not cross the placenta in humans,34,35
monkeys, or sheep,36 although it has been reported to do so in
mice.37 In the mouse, suppression of maternal erythropoiesis
by hypertransfusion does not suppress fetal erythropoiesis.38 In
humans, stimulation of maternal EPO production does not result
in stimulation of fetal red blood cell production.39
The expression of EPO is thought to be controlled by an oxygen-
sensing mechanism in the liver and kidney.40 Both hypoxia and
anemia stimulate erythropoiesis by stimulating messenger RNA
(mRNA) transcription and EPO production.41 Two factors, hepatic
nuclear factor 4 and hypoxia-inducible factor 1 (HIF-1), serve as
transcription factors for EPO as well as other hypoxia-inducible genes.
Hepatic nuclear factor 4 binds to the EPO promoter and enhancer
regions of the gene. HIF-1 is a basic helix-loop-helix transcription
factor composed of HIF-1α and HIF-1β subunits that bind to cis-
acting hypoxia-response elements and induce EPO transcription.
HIF-1 is expressed in many cells and is involved in up-regulating a
variety of oxygen-regulated proteins, including vascular endothelial
growth factor. HIF-1α appears to be constitutively expressed and
rapidly degraded under normoxic conditions. RNA stability depends
on the ubiquitin proteasome degradation system; inhibition of
this system leads to increased levels of HIF-1 and EPO, even under
normoxic conditions. Promoter and enhancer elements within the
EPO gene are responsive to hypoxia, as well as to cobalt exposure
in vitro.42 In animal models, the liver-sensing mechanism has a
decreased sensitivity to hypoxia, producing one tenth the amount
of EPO in response to comparable stimuli in the kidney.43,44 The
liver also appears to require more prolonged hypoxia to achieve
an EPO response.45,46 Other substances, including testosterone,47
estrogen,48 thyroid hormone,49 prostaglandins,50 vitamin E,51 and
lipoproteins,52 have been shown to enhance EPO production or its
effects, both in vivo and in vitro.53,54
It is not known what factors regulate the switch of EPO
production from the liver to the kidney. Renal production of EPO
is not necessary for normal fetal erythropoiesis.55 The lack of a
renal contribution to EPO production is illustrated by the normal
serum EPO concentrations and normal hematocrits of anephric
fetuses.56 In the sheep, EPO production in both the liver and the
kidney is highest at 60 days’ gestation (term being 140 days).57
EPO production in the fetal liver decreases by 90 days’ gestation;
however, increased renal production of EPO continues until 130
days’ gestation, when production falls to levels seen in adult sheep.58
Studies in human fetal and neonatal kidney obtained from
postmortem specimens also report measurable quantities
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2023. Para uso personal exclusivamente. No se permiten otros usos sin autorización. Copyright ©2023. Elsevier Inc. Todos los derechos reservados.
Platelets Red Blood Cells Hemoglobin Hematocrit Mean Corpuscular
(×109/L) (×1012/L) (g/dL) (%) Volume (fL)
234 ± 57 2.85 ± 0.36 11.69 ± 1.27 37.3 ± 4.3 131.1 ± 11.0
247 ± 59 3.09 ± 0.34 12.2 ± 1.6 38.6 ± 3.9 125.1 ± 7.8
242 ± 69 3.46 ± 0.41 12.91 ± 1.38 40.9 ± 4.4 118.5 ± 8.0
232 ± 87 3.82 ± 0.64 13.64 ± 2.21 43.6 ± 7.2 114.4 ± 9.3
of EPO mRNA,59 and quantitative mRNA studies in second-
trimester human fetuses reveal that the fetal kidney produces
approximately 5% of the amount of EPO mRNA that the fetal
liver produces during the second trimester.60 Thus it appears that
regulation of EPO gene transcription differs between the liver
and the kidney in utero.
One mechanism by which gene expression can be
differentially regulated is through methylation of promoter and
enhancer regions of a gene, and another is through deacetylation
of histones.61–65 Increased methylation generally results in
decreased gene expression, whereas histone deacetylation causes
relaxation of DNA structures and allows gene expression to
occur.These two mechanisms may be involved in developmental
EPO gene expression. For example, methylation of the enhancer
region (leading to decreased expression) of the EPO gene in
developing human kidney during the second trimester was
much greater than in the liver.66
EPO levels have been measured in umbilical cord blood
during the third trimester, and these levels gradually increase
throughout later development. 67–69 From EPO measurements
made in umbilical cord blood from infants of laboring and
nonlaboring mothers70 and from infants undergoing labor
stress,71–75 it seems likely that individual umbilical cord
EPO levels primarily reflect hypoxic stress during labor
and delivery. Serum EPO concentrations at birth normally
range from 5 to 100 mU/mL. For comparison, serum EPO
concentrations in anemic, nonuremic adults may be as high
as 300 to 400 mU/mL.76,77
RED BLOOD CELL INDICES IN THE FETUS AND
NEONATE
RED BLOOD CELL CONCENTRATIONS AND
HEMATOCRIT
Red blood cell indices change during gestation and continue to
change through the first year of life. Circulating red blood cell
concentrations gradually increase during the second trimester,
from (2.85 ± 0.36) × 106/μL at 18 to 21 weeks to (3.82 ±
0.64) × 106/μL at 30 weeks (Table 109.3).12 At term gestation,
circulating red blood cell concentrations range from 5.0 × 106
to 5.5 × 106/μL.78 In parallel with increasing red blood cell
concentrations, hematocrits increase from 30% to 40% during the
second trimester and continue to increase to term values over
the latter part of the third trimester.78 Changes in hematocrit
from 22 to 42 weeks’ gestation can be seen in Fig. 109.3.79 Term
hematocrits range from 50% to 63%, with some variability noted
because of delayed clamping of the umbilical cord.80 Values
are also dependent on the sampling site. Capillary hemoglobin
concentrations are as much as 3.5 g/dL higher than venous
hemoglobin concentrations.81
HEMOGLOBIN
The hemoglobin concentration gradually rises during gestation.82
At 10 weeks gestation the average hemoglobin concentration
is approximately 9 g/dL.83 By 22 to 24 weeks’ gestation, fetal

70
60
50
HCT (%)
40
30
20
10
0 concentrations (by 0.4 g/dL) by 6 months of age than infants
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
Gestational age (wk)
20
15
HGB (g/dL)
10
5 Infants of diabetic mothers, infants of mothers who smoke,
0
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
Gestational age (wk)
Fig. 109.3 Reference ranges for hematocrit and hemoglobin from 22
to 42 weeks’ gestation. Reference ranges are shown for hematocrit
(HCT; upper panel) and hemoglobin concentration (HGB; lower panel)
at 22 to 42 weeks’ gestation. Tests were done during a 6.5-year pe-
riod on more than 20,000 neonates. Values were excluded when the
diagnosis included abruption, placenta previa, or known cases of fetal
anemia or when a blood transfusion was given before the first hema-
tocrit was measured. The solid line represents the mean value, and
the dashed lines represent the 5% and 95% reference range. (From
Christensen RD, Jopling J, Henry E, et al. The erythrocyte indices of
neonates, defined using data from over 12,000 patients in a multihos-
pital system. J Perinatal. 2008;28:24–28.)
hemoglobin values reach 11 to 12 g/dL, and by 30 weeks the
hemoglobin concentrations are 13 to 14 g/dL.12 Premature
male infants reach term umbilical cord hemoglobin values
earlier than premature female infants,84 possibly because
of the erythropoietic effects of testosterone.47 Hemoglobin
concentrations are relatively constant during the last 6 to
8 weeks of gestation (see Fig. 109.3); at term the average
hemoglobin concentration is approximately 16 to 17 g/dL.85–87
An increase in hemoglobin concentration by 2 hours of
postnatal life occurs in most infants, resulting from a decrease
in plasma volume. By 8 to 12 hours after birth, the hemoglobin
concentration achieves a relatively constant level. Red blood cell
production decreases significantly at birth, so that hemoglobin
concentrations gradually decline by the end of the first postnatal
week.80 The decrease in red blood cell production after birth
is predominantly the result of increased availability of oxygen
in the extrauterine environment, which greatly reduces EPO
production and endogenous erythropoiesis.The continued fall in
hemoglobin concentration over the next several weeks results
from (1) decreased red blood cell production, (2) a shortened
red blood cell life span of the fetal/neonatal erythrocyte, and
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CHAPTER 109 — Developmental Erythropoiesis 1109
(3) plasma dilution and an increase in blood volume related to
growth. Multiple groups have also hypothesized that a process
termed neocytolysis (the selective lysis of young erythrocytes)
contributes to excessive hemolysis after birth, and thus a rapid
decrease in hematocrit as the neonate adapts to its new, relatively
hyperoxic environment.88–90
The nadir of hemoglobin concentration in term infants
occurs at approximately 8 weeks, with an average hemoglobin
concentration of 11.2 g/dL.78,83 Hemoglobin values gradually
rise such that, by 6 months, the average term infant has a
hemoglobin concentration of 12.1 g/dL. Altitude may have
a modest effect on the postnatal changes in hemoglobin
concentration; infants living at 1600 m had higher hemoglobin
living at sea level.91,92
The average decline in the hemoglobin concentration of
preterm very low-birth-weight infants (<1500 g) is remarkably
different from that of term infants, in part due to phlebotomy
losses that invariably occur in preterm infants, as well as the
effects of transfusions on endogenous erythropoiesis. Very low-
birth-weight infants reach a nadir of hemoglobin concentration
of 8 g/dL at 4 to 8 weeks of age.93 Fig. 109.4 and Table 109.4
demonstrate relationships among birth weight, chronologic age,
and red blood cell indices in term and preterm infants.78,79,83,92–96
Red blood cell indices may differ from normal ranges in
infants born small for their gestational age, in whom placental
insufficiency and secondary polycythemia are common.91–98
and infants born at higher altitudes also tend to have higher
hemoglobin concentrations at birth.91–96,98–100 In growth-
restricted infants born to hypertensive mothers, the blood
supply to the placenta and the capacity to deliver oxygen to
the fetus are diminished. Accelerated erythropoiesis is likely
part of a compensating mechanism that raises oxygen-carrying
capacity, maintaining an adequate supply to the fetus. In infants
of diabetic mothers, increased metabolic demands of the fetus
(resulting from increased glucose availability) may account
for higher fetal oxygen needs and compensatory increase in
hemoglobin concentration by the fetus. The increased red
blood cell mass in infants of diabetic mothers does not result
from higher maternal levels of hemoglobin A1C (a high-affinity
hemoglobin capable of decreasing the oxygen transferred to
the fetus). In mothers who smoke, the increase in fetal carbon
monoxide and the subsequent decrease in available oxygen are
the likely causes of the compensatory increase in hemoglobin
levels in the fetus.
MEAN CORPUSCULAR VOLUME
The size of the red blood cell gradually decreases during
development. The MCV is more than 180 fL in the embryo, falls
to 130 fL by midgestation, and decreases to 115 fL by the end
of pregnancy (Fig. 109.5). By 1 year of age, the MCV reaches
an average of 82 fL.95 Similar to cells of other organs in infants
born prematurely, the red blood cell MCV declines quickly
after birth, and the postpartum changes in MCV appear to be
related to chronologic age rather than postmenstrual age.94 The
mean corpuscular hemoglobin concentrations remain relatively
constant, and the mean corpuscular hemoglobin decreases
slightly.100
BLOOD VOLUME
The placenta and umbilical cord contain 75 to 125 mL of blood
at term, or approximately one fourth to one third of the fetal
blood volume.78 Umbilical arteries constrict shortly after birth,
but the umbilical vein remains dilated, and blood flows in the
direction of gravity. Infants held at or below the level of the
placenta can receive half of the placental blood volume (30 to
50 mL) in 1 minute. Blood can also travel from the neonate into
1110 SECTION XVIII — Developmental Hematopoiesis

65 25

60

55
20
50

45
40 15

HGB (g/dL)
HCT (%)

35
30

25 10

20

15
5
10

5
0 0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
A Day of life B Day of life

65 25
60
55
20
50

45
40 15
35 HGB (g/dL)
HCT (%)

30

25 10

20

15
5
10

5
0 0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
C Day of life D Day of life
Fig. 109.4 Hematocrit and hemoglobin reference ranges for term and preterm infants in the first month of life. (A) Hematocrit (HCT) over the
first 28 days of life for neonates born at 35 to 42 weeks’ gestation. (B) Blood hemoglobin (HGB) concentration over the first 28 days of life for
neonates born at 35 to 42 weeks’ gestation. (C) HCT over the first 28 days of life for neonates born at 29 to 34 weeks’ gestation. (D) Blood HGB
concentration over the first 28 days of life for neonates born at 29 to 34 weeks’ gestation. (Reprinted with permission from Henry E, Christensen
RD. Reference intervals in neonatal hematology. Clin Perinatol. 2015;42:483–497.)

Table 109.4 Mean Hemoglobin Values (± 1 SD; g/dL) in Low-Birth-Weight Infants.

Age (wk)
Birth Weight (g) 2 4 6 8 10
800–1000 16.0 (14.8–17.2) 10.0 (6.8–13.2) 8.7 (7.0–10.2) 8.0 (7.1–9.8) 8.0 (6.9–10.2)
1001–1200 16.4 (14.1–18.7) 12.8 (7.8–15.3) 10.5 (7.2–12.3) 9.1 (7.8–10.4) 8.5 (7.0–10.0)
1201–1400 16.2 (13.6–18.8) 13.4 (8.8–16.2) 10.9 (8.5–13.3) 9.9 (8.0–11.8) 9.8 (8.4–11.3)
1401–1500 15.6 (13.4–17.8) 11.7 (9.7–13.7) 10.5 (9.1–11.9) 9.8 (8.4–12.0) 9.9 (8.4–11.4)
1501–2000 15.6 (13.5–17.7) 11.0 (9.6–14.0) 9.6 (8.8–11.5) 9.8 (8.4–12.1) 10.1 (8.6–11.8)

Data from Williams ML, Shoot RJ, O’Neal PL, et al. Role of dietary iron and fat in vitamin E deficiency anemia of infancy. N Engl J Med. 1975;292:887.

the placenta if there is a significant gradient, resulting in 20 to 30
mL of blood loss per minute.101 Studies evaluating the benefits of
delayed cord clamping or cord milking showed improvements
in blood volume, hematocrit, decreased transfusions, decreased
intraventricular hemorrhage (IVH), decreased late onset sepsis,
and improved iron stores.102
The blood volume of infants with early umbilical cord
clamping averages 72 mL/kg, while the blood volume of
infants with delayed umbilical cord clamping averages 93
mL/kg. Preterm infants have slightly larger blood volumes
(89 to 105 mL/kg), owing to an increased plasma volume. By
1 month of age, blood volumes in term infants average 73 to
77 mL/kg.
NUCLEATED RED BLOOD CELLS
Though nucleated red blood cells (NRBCs) are rarely observed
in healthy adults and children, they are often observed in term
and preterm neonates.103–105 Multiple studies have hypothesized
that high concentrations of NRBCs at birth (or within the first
hours to days of life) are a marker of in utero hypoxia. Indeed,
a high concentration of NRBCs at birth has been associated
with an increased risk of developing IVH and/or retinopathy of

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140
MCV (fL)
120
100
80
60
MCH (pg)
40
20
0
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
Gestational age (wk)
Fig. 109.5 Mean corpuscular volume (MCV) and mean corpuscular
hemoglobin reference ranges from 22 to 42 weeks. Reference ranges
are shown for the (MCV; upper set of lines, measured in femtoliters)
and the mean corpuscular hemoglobin (MCH) concentration (lower
set of lines, measured in picograms) at 22 to 42 weeks’ gestation.
Tests were done during a 6.5-year period on more than 20,000 neo-
nates.
prematurity.106–108 Placental abruption, intraamniotic infection,
and intrauterine growth restriction have also been associated
with significantly higher NRBCs at birth.108 NRBCs are measured
as either NRBC/μL or NRBC per 100 white blood cells (WBC;
NRBC/100 WBC). The normative patterns of NRBCs are similar,
regardless of units used. The number of NRBCs on the day of
birth decreases from a median of ∼25 NBRC/100 WBC for
a neonate born at 23 to 28 weeks gestation to a median of 7
NRBC/100 WBC for a term neonate (Fig. 109.6).108
RETICULOCYTE INDICES
Erythrocytes are released from the marrow into the blood
as reticulocytes. The cytoplasmic organelles (e.g., ribosomes,
mitochondria, Golgi apparatus) persist for 1 to 2 days after
release before involution. Special staining detects elements of the
residual organelles and allows for a manual reticulocyte count.
Automated hematology counters that utilize flow cytometry have
made more accurate reticulocyte enumeration possible because
much larger samples of cells are evaluated than in a manual count.
These analyzers also make it possible to identify subpopulations
of reticulocytes that appear to be clinically helpful.
The reticulocyte count reflects the rate of effective
erythropoiesis. Because EPO production and endogenous
erythropoiesis falls dramatically after birth, the number of
reticulocytes in circulation also falls. In an otherwise well
neonate, the low number of reticulocytes in circulation persists
through the first 90 days of life until the physiologic nadir of
hemoglobin concentration is reached.109
The immature reticulocyte fraction (IRF) quantifies the
proportion of reticulocytes that are particularly high in RNA as a
ratio to total reticulocytes, and is an early and sensitive index of
marrow erythropoietic activity. Because flow cytometric gating
on automated hematology analyzers is not standardized, the IRF
may vary among analyzer manufacturers for any given sample.
RED BLOOD CELL INDICES IN NEONATES WITH
ANEUPLOIDY
Neonates with aneuploidy have consistent differences in their
RBC indices. This phenomenon has been best described in
neonates with trisomy 21.110,111 Polycythemia at birth is common
among this population. Henry and colleagues estimated that 33%
of neonates with trisomy 21 have hematocrits over 65% during
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CHAPTER 109 — Developmental Erythropoiesis 1111
120
100
NRBC/100 WBC
80
60
40
20
A 0
10,000
8,000
6,000
NRBC/µL
4,000
2,000
0
23 31 33 35 37 39 41
25 27 29
B Gestation (wk)
Fig. 109.6 Reference ranges for blood concentrations of nucleated
red blood cell (NRBC) on the day of birth by gestational age. The
lower and upper lines represent the 5th and 95th percentile limits.
The middle line represents the mean value. (A) Data expressed as
NRBC/100 white blood cell (WBC). (B) Data expressed as NRBC/μL.
(From Christensen RD, Henry E, Andres RL, et al. Reference ranges
for blood concentrations of nucleated red blood cells in neonates.
Neonatology. 2011;99[4]:289–294.)
the first 3 days of life, and 14% have hematocrits over 70%.112
Widness and colleagues reported elevated EPO concentrations
in umbilical cord blood samples of neonates with trisomy 21,
speculating that hypoxemia in utero resulted in elevated EPO
and consequent polycythemia. In addition, neonates with trisomy
21 have an elevated MCV in the first 3 days of life.112
Less data is available on the hematologic abnormalities in
neonates with trisomy 18 and trisomy 13. Like neonates with
trisomy 21, platelet and leukocyte abnormalities are more
pronounced. Wiedmeier and colleagues observed anemia in 40%
and polycythemia in 17% of neonates with trisomy 18. Only 1
of 22 neonates in their cohort with trisomy 13 had abnormal
erythrocyte findings.113
FETOMATERNAL RED BLOOD CELL TRANSFER
Maternal and fetal circulating cells may, at different times,
cross the placental barrier. Fetal contamination of the maternal
circulation can occur before delivery, as evidenced by studies
of maternal blood group immunization. About 50% to 75% of
pregnancies are associated with some degree of fetomaternal
transfer of blood. This event is uncommon in the first trimester
(3%). The volumes of fetal transplacental transfer are relatively
small, usually on the order of 0.01 to 0.1 mL, but on occasion
they may be much greater. About one pregnancy in 400 is
associated with fetal transplacental bleeding of 30 mL or greater,
and about one pregnancy in 2000 is associated with a potential
1112 SECTION XVIII — Developmental Hematopoiesis
fetal transplacental hemorrhage of 100 mL or more.114 The
overall risk of Rh immunization occurring in an Rh-incompatible
pregnancy is 16% if the fetus is Rh positive and ABO compatible
with its mother. This risk decreases to 1.5% if the fetus is Rh
positive and ABO incompatible. Fetal transfer of cells to the
mother occurs during abortions as well (about a 2% incidence
of such a transfer with spontaneous abortion and a 4% to 5%
rate if abortion is induced).115 Because fetal hemoglobin is
resistant to acid elution, cells containing fetal hemoglobin
can be distinguished from cells containing hemoglobin A. The
Kleihauer-Betke stain of peripheral maternal blood uses this
characteristic of fetal hemoglobin to detect fetal cells in the
maternal circulation,116 although results from mothers with
increased fetal hemoglobin synthesis (i.e., sickle cell disease,
thalassemia, and hereditary persistence of fetal hemoglobin) are
not reliable. Diagnosis of fetomaternal hemorrhage may also be
missed when the mother and infant are ABO incompatible. In
these cases, the fetal cells are rapidly cleared from the maternal
circulation by maternal anti-A or anti-B antibodies.
ERYTHROCYTE BIOCHEMISTRY
During fetal erythropoiesis, an orderly evolution of the
production of different hemoglobins occurs. Eight globin
genes direct the synthesis of six different polypeptide chains,
designated α, β, γ, δ, ε, and ζ. These globin chains combine in
the developing erythroblast to form seven different hemoglobin
tetramers: hemoglobin Gower 1 (ζ2-ε2), hemoglobin Gower 2 (α2-
ε2), hemoglobin Portland I (ζ2-γ2), hemoglobin Portland II (ζ2-β2),
fetal hemoglobin (α2-γ2), hemoglobin A (α2-β2), and hemoglobin
A2 (α2-δ2).
GLOBIN GENES
The globin genes are organized into two clusters (Fig. 109.7).The
α-like genes are located along a 20-kb distal segment of the short
arm of chromosome 16. The cluster contains three functional
genes (α1, α2, and ζ2), three pseudogenes (evolutionary remnants
of genes that are not expressed because of inactivating mutations
that prevent production of a functional globin protein), and
one gene of undetermined function (a globin-like gene without
inactivating mutations). The β-like gene cluster is located along a
60-kb segment of the short arm of chromosome 11 and contains
five functional genes (β, δ, Gγ, Aγ, and ε) and one pseudogene.
Within each complex, the genes are all in the same 5′ to 3′
orientation and are arranged in the order in which they are
expressed during development.117
The α genes are duplicated in humans. Each α gene
is approximately 4 kb long, interrupted by two small
nonhomologous regions. The exons and first introns of the two
α-globin genes have identical sequences, but the second intron
of α1 is nine bases longer and differs by three bases from that
of the α2 gene. Despite the high degree of homology between
these two genes, the sequences diverge in the 3′ untranslated
regions, 13 bases beyond the TAA stop codon. Because of these
sequence differences, the relative output of the two genes can be
measured: α2 mRNA is produced 2 to 3 times more abundantly
than α1 mRNA in fetal liver and marrow and in fetal and adult
erythrocytes.117
The ζ gene appears critical to normal fetal development.
In a murine model, both α-gene and ζ-gene expression can be
measured from the onset of erythropoiesis in the yolk sac.118 In
addition, expression of both genes occurs concomitantly within
the same cells. Expression of the ζ gene occurs predominantly
through the first 6 to 8 weeks of gestation, although minute
quantities of ζ-globin can be measured in fetal and neonatal red
blood cells.119
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2023. Para uso personal exclusivamente. No se permiten otros usos sin autorización. Copyright ©2023. Elsevier Inc. Todos los derechos reservados.
5' 3'
Chromosome 11
LCR ε Gγ Aγ ψβ1 δ β
ψα2 α2 θ1
Chromosome 16
ζ2 ψζ1 ψα1 α1
0 10 20 30 40 50 60 70
kb ladder
Embryonic: Hemoglobin Gower 1 ζ2ε2
Hemoglobin Gower 2 α2ε2
Hemoglobin Portland I ζ2γ2
Fetal: Hemoglobin F α2γ2
Adult: Hemoglobin A α2β2
Hemoglobin A2 α2δ2
Fig. 109.7 Organization of the globin genes. Transcription of mes-
senger RNA occurs from the 5′ to the 3′ end, and for both chromo-
somes the genes are arranged in order of their developmental ac-
tivation. The upper segment represents the β-like globin genes on
the short arm of chromosome 11, and the lower segment represents
the α-like genes on the distal part of the short arm of chromosome
16. Regions of the gene that code for primary globin proteins are
shown as shaded ovals, and regions that code for pseudogenes (ψ-
nonexpressed remnants, which have a number of inactivating muta-
tions that prevent transcription and translation into functional globin
protein) are shown as open ovals. θ1 is a globin-like gene without
inactivating mutations. The locus control region (LCR) is shown as
a hatched segment and contains five DNase I hypersensitivity sites,
represented by rectangles. A downstream hypersensitivity site known
as 3′ hypersensitivity site 1 is shown at the 3′ end. The composition of
embryonic, fetal, and adult hemoglobins is listed.
Like the α1 and α2 genes, the Gγ and Aγ genes appear to be
virtually identical over a span of 1.5 kb, suggesting a mechanism
for gene matching during evolution. Amino acid sequencing of
normal umbilical cord blood has shown that either glycine (Gγ)
or alanine (Aγ) is present at the 136 position in the γ chain.120
Short-chain organic acids, such as butyrate and acetate, increase
γ-gene promoter activity. This activity can be enhanced even
further by incorporation of sections of the β-gene locus control
region.121,122
The β-globin gene cluster occupies a region of approximately
17 kb on the short arm of chromosome 11.117 Each of its
constituent genes, their flanking regions, and large stretches of
the regions between them have been sequenced. Studies suggest
that the transcription factor NF-E1 regulates increased β-globin
gene expression during erythroid maturation. Deletion of the
NF-E1 binding site in the upstream segment of the β-globin
promoter blocks induction of transcription in murine leukemia
cells.123
δ-Globin gene expression occurs early at the erythroid
progenitor stage. By the reticulocyte stage, no δ-globin mRNA
synthesis can be detected.The δ-globin gene promoter functions
at a much lower level than the β-globin gene promoter, resulting
in significantly lower levels of δ-globin mRNA production. In
addition, decreased expression occurs because of a globin
promoter–specific silencer element located upstream of the
δ-globin gene.124 Moreover, δ-globin mRNA is less stable than
β-globin mRNA, which likely accounts for the early cessation of
 CHAPTER 109 — Developmental Erythropoiesis 1113

60
Globin chain production (% of total) 100
HGB A: α2β2
50 α HGB F: α2γ2 97%

40 80
Gγ β 75%

% HGB
30 60
HGB Gower 1: ζ2ε2
20 40 HGB Portland I: ζ2γ2
ζ Aγ
HGB Gower 2: α2ε2 25% HGB A2: α2δ2
ε
10 20
δ 2%
1%
10 20 30 Birth 10 20 30 40 10 20 30 Birth 10 20 30 40

A Gestational age (wk) Weeks after birth B Gestational age (wk) Weeks after birth
Fig. 109.8 Changes in globin subunits (A) and hemoglobin tetramers (B) during human development from the embryo to early infancy. HGB,
Hemoglobin. (Data collated from Bunn HF, Forget BG. Hemoglobin: Molecular, Genetic and Clinical Aspects. Philadelphia: WB Saunders;
1986:68.)

δ-globin synthesis and significantly lower levels of hemoglobin A2 in normal adults. The relative rates of Gγ-chain and Aγ-chain
compared with hemoglobin A. production are also constant throughout fetal life at a Gγ/Aγ ratio
The ε gene is located 5′ of the other β-like genes, and it is of approximately 3:1.120 It is not known whether these pairs of
expressed during embryonic development. The ε gene is similar genes are initially activated at this ratio; however, this production
in sequence to the γ genes. A silencing element of the promoter ratio is reached early in development.
region may be responsible for inactivating gene expression after Somewhere between the 32nd week and the 36th week of
the embryonic stage.125 The only known mutation that results in gestation, the relative rate of β-chain synthesis increases and that
persistent expression of this globin gene occurs in trisomy 13, of γ-chain production declines such that at birth β-chain synthesis
in which there is a delay in the switch from ε-globin to γ-globin makes up 30% to 50% of non–α-chain synthesis. It is generally
production.126 held that the transition from fetal to adult erythropoiesis starts
at 30 to 36 weeks after conception, but the rate of the transition
GLOBIN CHAIN SYNTHESIS has been controversial. A gradual transition from fetal to adult
It has been possible to analyze the patterns of globin chain hemoglobin synthesis occurs, starting in the first trimester.128,129
production at early ages of embryonic development during Considerable variation among infants occurs, however, because
the transition from yolk sac (primitive) to hepatic (definitive) many infants show prolonged dependence on fetal hemoglobin.
erythropoiesis. It is unknown why primitive erythroid After birth the level of γ-chain production steadily declines and
progenitors programmed to produce one type of hemoglobin, that of β-chain production increases, so by the end of the first
such as hemoglobin Gower 1, give way to definitive progenitors year, γ-chain synthesis reaches the low level characteristic of adult
programmed to produce a different type of hemoglobin, such life. The normal range of postnatal fetal hemoglobin production
as fetal hemoglobin. Quantification of globin gene synthesis can be seen in Fig. 109.8. During the first few months of life, the
reflects production by numerous red blood cells, and production Gγ/Aγ ratio changes from 3:1 to 2:3, although this ratio is variable

of a specific hemoglobin is usually reported as a percentage of in adults.130,131

the total hemoglobin measured. Studies evaluating hemoglobin Production of the δ chain has been observed as early as 32
production by erythroid colonies in culture, however, show that weeks. Activation of the δ gene lags β-gene activation, so the
individual cells in a colony produce predominantly one type of adult β/δ synthesis ratio is not reached until 4 to 6 months after
hemoglobin.127 birth.
During the fourth to fifth week, ζ, δ, and ε chains are the
main globin chains synthesized (Fig. 109.8). During the sixth HEMOGLOBIN PRODUCTION
to seventh week, α, δ, ε, Gγ, and Aγ chains are produced in the Developmental changes in the production of the various
remaining primitive erythroblasts, and α, ε, Gγ, and Aγ chains are hemoglobins are noted in Fig. 109.8. Before the onset of formation
produced in definitive erythrocytes. By the seventh to eighth of other chains, unpaired globin chains may form tetramers,
week, ε- and ζ-chain synthesis is no longer detectable, and the resulting in the presence of ε4.132 Almost immediately thereafter,
main globin chains produced are α, Gγ, and Aγ. Production of α-chain and ζ-chain production begins, and hemoglobins Gower
the β chain is just barely detectable at this time and gradually 1 (ζ2-ε2), Gower 2 (α2-ε2), and Portland I (ζ2-γ2) are formed.133
increases, such that by 10 weeks, it makes up 10% of total By 5 to 6 weeks’ gestation, hemoglobins Gower 1 and Gower
non–α-chain production. As soon as β-chain production occurs, 2 constitute 42% and 24% of the total hemoglobin, respectively,
genetic disorders associated with β-chain synthetic or structural with fetal hemoglobin (α2-γ2) making up the remainder. By 14 to
abnormalities may be detected in utero, a finding suggesting an 16 weeks, hemoglobin F constitutes 50% of the total hemoglobin,
asynchronous transition from ζ-chain to α-chain production as and by 20 weeks, it forms more than 90% of the hemoglobin.131,134
compared with ε-chain to γ-chain production, with the ζ-α switch Small quantities of hemoglobin A (α2-β2) are found, beginning
occurring slightly earlier.128 at 6 to 8 weeks’ gestation. The increase in β-chain production
From the 10th week to about the 33rd week of gestation, the occurring between 12 and 20 weeks’ gestation accounts for the
main globin chains synthesized are α, Gγ, Aγ, and β. Assessment of sudden rise in the amount of hemoglobin A found at the end
the output of the two linked α-globin genes by mRNA analysis of the first trimester of pregnancy. Tetramers of γ chains (γ4, or
suggests that they are expressed in a ratio ranging from 1.5:1 hemoglobin Barts) and β-chains (β4, or hemoglobin H) can be
to 3.0:1 (α2/α1) throughout fetal life. This does not appear to found in conditions in which α-chain synthesis is impaired or
change during development and is the same as that observed absent, such as the α-thalassemia syndromes.

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1114 SECTION XVIII — Developmental Hematopoiesis
Fetal hemoglobin is easily distinguished immunologically
and biochemically from adult hemoglobin. The most significant
physiologic characteristic of fetal hemoglobin is the decreased
interaction with 2,3-diphosphoglycerate (2,3-DPG). 2,3-DPG
binds to deoxyhemoglobin in a cavity between the β chains and
stabilizes the deoxy form of hemoglobin, resulting in a reduced
hemoglobin-oxygen affinity. 2,3-DPG binds less effectively
to the γ-globin chains, because of the differing amino acid
sequence in the non-α chain. Consequently, 2,3-DPG does not
reduce the oxygen affinity of hemoglobin F as much as that of
hemoglobin A.
Other differences in physical properties exist between fetal
and adult hemoglobin. Hemoglobin F is more soluble in strong
phosphate buffers than hemoglobin A.116 Hemoglobin F is
oxidized to methemoglobin more easily than is hemoglobin A,
and it has a considerably greater affinity for oxygen than does
adult hemoglobin as a result of differences in binding to 2,3-
DPG. Fetal hemoglobin is resistant to acid elution, which allows
differentiation of cells containing fetal hemoglobin from cells
containing hemoglobin A.116
The total γ chains in the blood of the fetus and newborn
comprise 70% to 80% of Gγ chains. This fraction falls to about
40% by 5 months of age. This unique difference in Gγ chain
production found in the fetus helps to distinguish fetal
hematopoiesis from that found in later life. Under stress, the
older infant and adult revert to this intrauterine form of fetal
hemoglobin structure. This often occurs in leukemic states
in children and adults, and in other conditions as well.135,136
The delay in the switch of hemoglobin F to hemoglobin A has
been noted in conditions of maternal hypoxia,137 in infants
small for their gestational age,138 and in infants of diabetic
mothers.139,140 Elevated levels of fetal hemoglobin may have
protective effects in some disease states, and much research
has gone into identifying fetal to adult hemoglobin transition,
in order to “switch on” γ-globin gene expression and increase
fetal hemoglobin production.141 The regulators implicated in
hemoglobin F production include B-cell lymphoma/leukemia
11A, myeloblastosis protooncogene protein, and Krüppel-like
factor 1. In addition, microRNAs 15a and 16-1 play a role in
gene regulation.142
The postpartum decline of fetal hemoglobin production and
of the intercellular distribution of fetal and adult hemoglobins
has been extensively examined during the first few months of
life. Immediately after birth, there is a brief rise in hemoglobin F
concentration, followed by a steady decline (Fig. 109.9). Studies of
the intercellular distribution of hemoglobin F, using the relatively
insensitive acid-elution technique, have shown that during the
first few months of life the distribution of hemoglobin F is quite
heterogeneous. At 3 months the distribution of hemoglobin F
becomes bimodal, with populations of cells that contain acid-
resistant hemoglobin F and populations of adult “ghost” cells.
These observations have suggested that fetal hemoglobin-
containing cells are replaced by a population of cells containing
adult hemoglobin.
Profound changes occur in the rates of red blood cell
production immediately before birth and during the first few
months after birth. On a body-weight basis, red blood cell
production during the latter months of gestation is significantly
greater compared with that in adult life. Immediately after
birth, erythropoiesis is considerably reduced, presumably as an
adaptation to the extrauterine environment, and red blood cell
production occurs at a low level for the first few weeks of life.
It is clear from globin-chain synthetic studies that there is a
steady and linear decline in γ-chain synthesis during the period
of reduced neonatal erythropoiesis. Newly synthesized red
blood cells appearing in the circulation when erythropoiesis
resumes contain predominantly adult hemoglobin. These
observations may explain the short plateau in the proportion
of fetal hemoglobin (but not absolute levels) after birth and
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140
120
100
HbF (g/L)
80
60
40
20
0
35 40 45 50 55 60 65 70
Postconceptional age (wk)
Fig. 109.9 Correlation between postconceptional age and absolute
levels of fetal hemoglobin. Postnatal fetal hemoglobin concentrations
(HbF) are shown for 280 infants, examined at postnatal ages of 6
weeks (circles), 12 weeks (triangles), and 6 months (diamonds). (From
Berglund SK, Lindberg J, Westrup B, et al. Effects of iron supplements
and perinatal factors on fetal hemoglobin disappearance in LBW in-
fants. Pediatr Res. 2014;76:477–482.)
the appearance of predominantly adult hemoglobin–containing
cells during the second and third months of life. These findings,
together with the results of analyses of the intercellular
distribution of fetal and adult hemoglobin by sensitive imm­
unologic methods, suggest, although they do not prove, that the
transition from fetal to adult hemoglobin production occurs
in the same erythrocyte population. This conclusion is also
consistent with the patterns of fetal and β-chain production in
red blood cell colonies grown from neonatal blood.143
Studies show that the type of globin chains produced at
different stages of development are not closely related to the site
of erythropoiesis. It appears that ζ and ε chains are synthesized in
both primitive and definitive cell lines. Moreover, the switch from
γ-chain to β-chain production occurs synchronously throughout
the liver and bone marrow during the later stages of fetal
development. The transition from γ-chain to β-chain synthesis is
most closely related to postconceptional age and not chronologic
age.135 Thus premature infants continue to synthesize relatively
large quantities of γ chains (and fetal hemoglobin) until 40
weeks’ gestation.
ERYTHROCYTE METABOLISM
The mature red blood cell differs from other cells in the body in
that it has no nucleus; it consequently lacks the ability to engage
in de novo protein synthesis. It has no mitochondria or ribosomes,
no nucleic acid or deoxyribonucleic acid synthesis, no Krebs
cycle of intermediary metabolism, and no electron transport
system for oxidative phosphorylation. Thus cellular metabolism
is dependent on a limited supply of preexisting enzymes. These
enzymes, coenzymes, and the substrates of glucose metabolism
interact with hemoglobin and the red blood cell membrane to
perform all the primary functions of the red blood cell, the most
important being oxygen transport.
Other important functions of red blood cell metabolism
include maintaining adequate amounts of energy in the form of
adenosine triphosphate (ATP), producing reducing substances to
act as antioxidants, and maintaining appropriate amounts of red

Box 109.1 Metabolic Characteristics of Neonatal Erythrocytes
Carbohydrate Metabolism
Glucose consumption increased
Galactose more completely utilized as a substrate both under
normal circumstances and for methemoglobin reductiona
Decreased activity of sorbitol pathwaya
Decreased triokinase activitya
Glycolytic Enzymes
Increased activity of hexokinase, phosphoglucose isomerase,a
aldolase, glyceraldehyde 3-phosphate dehydrogenase,a phos-
phoglycerate kinase,a phosphoglycerate mutase, enolase,a
pyruvate kinase, lactate dehydrogenase, glucose 6-phosphate
dehydrogenase, 6-phosphogluconic dehydrogenase, galacto-
kinase, and galactose 1-phosphate uridyltransferase
Decreased activity of phosphofructokinasea
Distribution of hexokinase isoenzymes differs from that of adultsa
Nonglycolytic Enzymes
Increased activity of glutamic oxaloacetic transaminase and
glutathione reductase
Decreased activity of NADP-dependent methemoglobin
reductase,a catalase,a glutathione peroxidase, carbonic
anhydrase,a adenylate kinase,a and glutathione synthetasea
Presence of α-glycerol-3-phosphate dehydrogenasea
Adenosine Triphosphate and Phosphate Metabolism
Decreased phosphate uptake,a slower incorporation into ATP
and 2,3-diphosphoglyceratea
aAppears to be a unique characteristic of the newborn’s erythrocytes and not merely a function of the presence of young red blood cells.
From Oski FA, Naiman JL. Hematologic Problems in the Newborn. 3rd ed. Philadelphia: WB Saunders; 1982:107.
ATP, Adenosine triphosphate; NADP, nicotinamide adenine dinucleotide phosphate.
blood cell 2,3-DPG to assist in the modulation of hemoglobin’s
oxygen affinity. Energy metabolism is largely a function of the
Embden-Meyerhof pathway. This pathway also regulates the
quantity of red blood cell 2,3-DPG within the cell. The pentose
phosphate pathway, among other functions, has a vital role in the
production of reducing substances such as reduced nicotinamide
adenine dinucleotide phosphate (NADPH). Box 109.1 lists the
metabolic characteristics of neonatal erythrocytes. A few of the
variations seen in comparison with adult red blood cells are
clinically significant in that they affect the life span of neonatal
red blood cells.
GLYCOLYSIS
Red blood cells need a constant supply of carbohydrate to
maintain adequate levels of ATP. Although glucose is the pre­
fer­red carbohydrate, the red blood cell metabolizes fructose or
mannose almost as readily. Galactose is metabolized more slowly.
Intracellular glucose concentrations equilibrate immediately
with changes in plasma glucose concentrations. Glucose enters
the human erythrocyte by facilitated transfer, and it is either
converted to glucose 6-phosphate or reduced to its polyol
derivative, sorbitol, which is then converted to fructose.144 Once
formed, glucose 6-phosphate is metabolized by one of three
pathways. The Embden-Meyerhof pathway converts glucose
6-phosphate to lactate or pyruvate and in the process generates
ATP. Metabolism by way of the pentose phosphate pathway
produces reduced intermediates and a phosphorylated pentose
sugar (ribulose 5-phosphate).This sugar ultimately returns to the
Embden-Meyerhof pathway. Finally, glucose 6-phosphate may be
converted to glucose 1-phosphate and then to glycogen, although
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CHAPTER 109 — Developmental Erythropoiesis 1115
Accelerated decline of 2,3-diphosphoglycerate into red blood
cell incubationa
Increased ATP levels
Accelerated decline of ATP levels during brief incubation
Storage Characteristics
Increased potassium efflux and greater degrees of hemolysis
during short periods of storage
More rapid assumption of altered morphologic forms on stor-
age or incubationa
Membrane
Decreased ouabain-sensitive ATPasea
Decreased potassium influxa
Decreased permeability to glycerol and thioureaa
Decreased membrane filterabilitya
Increased sphingomyelin content, decreased lecithin content
of stromal phospholipids
Decreased content of linoleic acida
Increase in content of lipid phosphorus and cholesterol per cell
Greater affinity for glucosea
Other
Increased methemoglobin contenta
Increased affinity of hemoglobin for oxygena
Glutathione instabilitya
Increased tendency for Heinz body formation in the presence
of oxidant compoundsa
less than 1% of glucose is metabolized to glycogen within the red
blood cell.145
EMBDEN-MEYERHOF PATHWAY
At least 90% of glucose is metabolized via the Embden-Meyerhof
pathway.146,147 Two moles of ATP are produced for every mole
of glucose catabolized, yielding two moles of lactic acid. This
potential for the production of ATP is not fully achieved because
approximately 20% of metabolized glucose traverses the 2,3-DPG
cycle, thus bypassing one of the kinase steps, which is a site of
ATP generation.148
The Embden-Meyerhof pathway has several unique
characteristics with respect to fetal and newborn cells. These
cells consume greater quantities of glucose than do the red
blood cells of adults.149 Galactose metabolism in the newborn
red blood cell also differs.150 Galactokinase activity is three
times greater in the erythrocytes of newborns, and these cells
consume galactose more rapidly than do those of the adult.The
glycolytic enzymes phosphoglycerate kinase and enolase are
much more active in the cells of the fetus and newborn infant
than would be anticipated from their young cell age.151,152 In
contrast, the activity of phosphofructokinase (a rate-controlling
enzyme in glycolysis) is lower than normal in the erythrocytes
from newborn infants.151,153,154 Developmental changes in
the activities of these three enzymes towards normal adult
values during the first year of life appear to be independent
of red blood cell age and reflect a transition from fetal to adult
erythropoiesis. The decreased phosphofructokinase activity of
fetal cells may be a consequence of accelerated decay of an
unstable enzyme.The relative deficiency of this enzyme appears
1116 SECTION XVIII — Developmental Hematopoiesis
to result in alterations in glucose metabolism, and it could be
functionally significant. Several of the other enzymes of the
Embden-Meyerhof pathway have shown differences in the
staining intensity of certain isoenzyme zones as compared with
adult controls, although the significance of this observation is
not known.
The ATP generated via the Embden-Meyerhof pathway
is necessary for the maintenance of the normal biconcave
shape of the erythrocyte.155 It is also necessary for pyrimidine
nucleotide synthesis, completion of purine nucleotide synthesis
and glutathione synthesis,156 incorporation of fatty acids into
membrane phospholipids,157 active cation transport,154 and the
initial step in the phosphorylation of glucose by the enzyme
hexokinase. Not all these synthesizing functions are important
in the mature red blood cell, which lacks a nucleus. However,
loss of red blood cell ATP produces a marked decrease in red
blood cell deformability. As the cell ages, ATP levels fall, and
older cells have lower glucose utilization, greater osmotic
fragility on incubation, lower membrane lipid content, lower
potassium concentration, and greater sodium concentration.
They are less deformable and have a shorter life span. The red
blood cells of term and preterm infants, when studied in the first
several days of life, contain higher levels of ATP than do cells
from adults.158,159 The small premature infant has even higher
ATP levels.158
Newborn red blood cells seem to demonstrate a transient
immaturity in their metabolism. This results in a slower uptake
of phosphorus, a delayed incorporation into 2,3-DPG, and a
marked decline in 2,3-DPG levels, as well as ATP levels, during
short periods of incubation in vitro. The precise reason for
this relatively transient immaturity is not known, but it may
explain why the newborn red blood cell loses potassium at an
accelerated rate and undergoes marked morphologic alteration
during short incubations in vitro.
Pyruvate kinase deficiency is a well-known enzymopathy that
affects the normal function of the Embden-Meyerhof pathway
and results in clinically significant erythrocyte pathology.
Deleterious mutations in the PKLR (pyruvate kinase L/R) gene
result in low levels of pyruvate kinase. This results in abnormal
erythrocyte morphology (echinocytes, anisocytes, poikilocytes)
and a lifelong hemolytic anemia. The exact mechanisms of
hemolysis are not known. Though other Embden-Meyerhof
pathway enzymopathies have been described in the literature,
they are even more rare than pyruvate kinase deficiency.
PENTOSE PHOSPHATE PATHWAY
Glucose 6-phosphate undergoes oxidative decarboxylation
through the pentose phosphate pathway, consuming oxygen
and producing carbon dioxide. The pentose pathway requires
NADPH as a cofactor. In the first step, oxidation of glucose
6-phosphate to 6-phosphogluconolactone is catabolized
by glucose 6-phosphate dehydrogenase (G6PD), generating
NADPH. This step is followed by enzymatic hydrolysis of
6-phosphogluconolactone to 6-phosphogluconate, which is
then oxidized (in the presence of 6-phosphogluconic dehy­
drogenase) to ribulose 5-phosphate, with the production
of carbon dioxide. Approximately 3% to 10% of all glucose
metabolized by the cell is cycled through the pentose phosphate
pathway. Hypoxia and acidosis increase the proportion of
glucose metabolism shunted through this pathway.
The pentose phosphate shunt results in the production of
two important products, ribose 5-phosphate and NADPH. Ribose
5-phosphate is a vital constituent of the pyridine nucleotides
nicotinamide adenine dinucleotide phosphate and NADPH
and the purine nucleotides adenosine diphosphate and ATP.
There is no pathway for de novo purine formation inside the
red blood cell; however, the mature red blood cell does retain
the ability for pyridine nucleotide formation. NADPH is critical
for preservation of cell integrity because it is necessary for
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methemoglobin reduction, the reduction of glutathione, and the
stabilization of certain enzymes.160 NADPH serves as a hydrogen
donor in the presence of the enzyme glutathione reductase,
resulting in reduction of glutathione. Reduced glutathione
ultimately serves as a substrate for the enzyme glutathione
peroxidase, which is responsible for the detoxification of
hydrogen peroxide. Hydrogen peroxide is a byproduct of the
conversion of oxyhemoglobin to methemoglobin, which is
a naturally occurring reaction inside the red blood cell in the
presence of oxidative stress. The absence of NADPH (or anyth­
ing that interferes with the production of reduced glutathione),
the synthesis of glutathione, or the inability to detoxify hydrogen
peroxide severely impairs the viability of the red blood cell.
This is the case in G6PD deficiency. Patients with this condition
can experience hemolytic crises when they experience oxidative
stress from illness, certain foods, and certain drugs.
The pentose phosphate pathway in the newborn red blood
cell differs from that in the adult red blood cell. Two enzymes
of the pentose pathway, G6PD and 6-phosphogluconic acid
dehydrogenase, are active at levels higher than those seen in adult
red blood cells.161 Carbon dioxide production by erythrocytes of
term and preterm infants is equal to or greater than that seen in
red blood cells of adults.
Although there are suggestions that the pentose phosphate
pathway activity in the newborn is normal, there is also
evidence that newborn infants are more susceptible to oxidant-
induced injury, leading to glutathione instability, Heinz body
formation, and the development of methemoglobinemia.162,163
This oxidant vulnerability may be caused by factors unrelated
to the pentose phosphate pathway. For example, the red blood
cell membrane in the fetus and newborn may have a decreased
number of membrane sulfhydryl groups, making these cells
more susceptible than mature red blood cells to Heinz body
formation.163 Additionally, NADPH-methemoglobin reductase
activity is decreased in neonatal red blood cells, and the plasma
of newborn infants appears to have a diminished antioxidant
capacity.164 The precise mechanisms surrounding the newborn
red blood cell vulnerability to oxidant injury are not known.
Fetal and newborn red blood cells have diminished levels
of glutathione peroxidase, which may render the cells more
vulnerable to hydrogen peroxide–induced oxidant injury. In
addition, the newborn red blood cell may have a diminished
capacity for handling other activated oxygen radicals, such
as singlet oxygen and the superoxide radical.165 The latter
is converted to hydrogen peroxide in the presence of the
enzyme superoxide dismutase. Superoxide dismutase levels
differ widely among infants. Diminished activity of superoxide
dismutase could result in accumulation of superoxide radicals.
Free radicals are generally detoxified by antioxidants such as
α-tocopherol (vitamin E). However, if superoxide dismutase
levels are increased (as has been described in some infants), the
hydrogen peroxide presented to reduced glutathione may not
be adequately detoxified.Therefore a delicate balance appears to
exist between enzymes involved in production and detoxification
of free radicals and oxidative intermediates. The use of inhaled
nitric oxide as treatment for pulmonary hypertension in sick
neonates may affect this balance, and further studies are required
to determine its impact on oxidant injury beyond an increase in
methemoglobin formation.
2,3-DIPHOSPHOGLYCERATE METABOLISM
The affinity of a hemoglobin solution for oxygen can be
decreased by interaction of hemoglobin with certain organic
phosphates.166 Among the organic phosphates tested, 2,3-DPG
and ATP are the most effective in lowering oxygen affinity. The
highly charged anion 2,3-DPG binds to deoxyhemoglobin but
not to oxyhemoglobin. Various conditions increase the amount
of 2,3-DPG present within the red blood cell, as regulated in
the Embden-Meyerhof pathway. Once formed, each molecule

of 2,3-DPG binds reversibly to one deoxyhemoglobin tetramer
under physiologic conditions of solute concentration and pH.
Fetal deoxyhemoglobin does not possess as great an affinity
for 2,3-DPG as does adult deoxyhemoglobin, and therefore it
cannot bind 2,3-DPG to the same degree as adult hemoglobin.
Thus the fetal leftward-shifted hemoglobin-oxygen dissociation
curve is not easily modulated by the presence of 2,3-DPG. The
half-saturation pressure (P50) of fetal blood is 19 to 21 mm
Hg, some 6 to 8 mm Hg lower than that of adult blood. As the
fetal hemoglobin concentration declines (see Fig. 109.9), the
increasing percentage of adult hemoglobin and increasing red
blood cell 2,3-DPG content produce a marked rightward shift in
the hemoglobin-oxygen equilibrium curve. Infants with a greater
proportion of adult hemoglobin but less 2,3-DPG may have the
same P50 as those with increased quantities of fetal hemoglobin
but a high red blood cell 2,3-DPG content.
ERYTHROCYTE PHYSIOLOGY
PHYSICAL PROPERTIES OF NEONATAL
ERYTHROCYTES
The fetal and neonatal red blood cell differs from the mature
red blood cell of the older infant, child, and adult in various
ways. Specific characteristics of fetal red blood cells include a
shortened life span, macrocytosis, high fetal hemoglobin content
with a Gγ/Aγ ratio of 3:1, the presence of i antigen,167 and low
carbonic anhydrase enzyme activity.49
LIFE SPAN
The differences in physical properties of red blood cells
derived from term and preterm infants may in part account for
the decreased life span of neonatal red blood cells within the
circulation. The average life span for a neonatal red blood cell is
60 to 90 days,168 approximately half to two thirds that of an adult
red blood cell. When neonatal red blood cells are transfused into
adults, they exhibit a shortened life span, owing to alterations
intrinsic to the neonatal red blood cell.169 In contrast, cells
transfused from adult donors appear to survive normally in
newborns.169 With increasing degrees of prematurity, remarkably
shorter red blood cell life spans (35 to 50 days) are found. Fetal
studies using [14C] cyanate-labeled red blood cells in sheep
revealed an average red blood cell life span of 63.6 ± 5.8 days.170
The mean red blood cell life span increased linearly from 35 to
107 days as the fetal age increased from 97 days (midgestation)
to 136 days (term).
The shortened red blood cell life span of the preterm and
term neonate may be explained by some of the characteristics
specific to newborn cells—namely, a rapid decline in intracellular
enzyme activity and ATP levels,171 loss of membrane surface
area by internalization of membrane lipids, decreased levels of
intracellular carnitine,172 increased susceptibility of membrane
lipids and protein to peroxidation,173 and increased mechanical
fragility resulting from increased membrane deformability.174
SIZE
Red blood cell dimensions change markedly during fetal and
neonatal development. Early in embryogenesis, cell diameters
range from 20 to 25 μm, and the MCV averages 150 to 180 fL.3
During fetal development red blood cells gradually decrease in
size and volume (see Fig. 109.5); at term the average cell is 8 to 10
μm in diameter, with an MCV between 108 and 118 fL. During the
first year of life red blood cells continue to diminish in size, and
at 1 year of life they resemble adult red blood cells (Table 109.5).
SHAPE AND DEFORMABILITY
Just as there is variation in the size of newborn red blood cells,
there is variation in shape. Irregularly shaped cells are present
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CHAPTER 109 — Developmental Erythropoiesis 1117
in much greater numbers in the peripheral blood of newborn
infants than in that of adults.175 Target cells, acanthocytes,
puckered immature erythrocytes, and other irregular projections
may normally be found. For example, a greater percentage of
neonatal red blood cells have membrane surface pits, which are
most likely the sites of formation of endocytic vacuoles. In normal
adults, 2.6% of erythrocytes appear to have surface pits or craters
ranging in size from 0.2 to 0.5 μm in diameter, demonstrated
by interference-contrast microscopy.176 In contrast, pits can be
found in almost half of the erythrocytes of preterm infants and in
one fourth of the erythrocytes of term infants.
Red blood cell deformability is principally governed by three
factors: the surface area to volume relationship of the red blood
cell, the viscosity of the cytoplasm of the cell, and intrinsic red
blood cell membrane rigidity.174 The deformability of erythrocytes
is important for several reasons. First, red blood cell deformability
appears to be an important determinant of red blood cell life span
in vivo. The removal of a red blood cell from the circulation is
thought to be a consequence of declining deformability, making
the red blood cell susceptible to sequestration in the spleen and
other organs, where it must negotiate extraordinarily narrow
passages. Second, red blood cell deformability directly influences
blood flow in the peripheral circulation. Third, red blood cell
deformability affects whole blood viscosity, which, in turn, affects
peripheral vascular resistance and cardiac workload.177
Neonatal red blood cells with the greatest density (representing
the oldest cells in the circulation) lose more volume than adult
red blood cells, have a higher mean corpuscular hemoglobin
concentration than adult red blood cells, and are less deformable
than the oldest red blood cells seen in adults. This suggests an
accelerated decrease in deformability of aging red blood cells
related to a more pronounced increase in the mean corpuscular
hemoglobin concentration, the principal determinant of the
internal viscosity of the red blood cell. Neonatal red blood cell
membranes deform more readily to a given shear force than do
adult red blood cell membranes, resulting in greater susceptibility
of neonatal cell membranes to yield and fragment.177 These
mechanical properties may lead to accelerated membrane loss
and a decreased life span. Recent studies evaluating the red blood
cell deformability of preterm infants receiving recombinant EPO
reported that erythrocyte deformability was significantly related
to the reticulocyte count, indicating that the improvement of
erythrocyte deformability with recombinant EPO therapy was
due to the formation of well-deformable young erythrocytes.178
SURFACE CHARGE
The surface charge of newborn red blood cells is more negative
than that of adult red blood cells. The negative charge at the red
blood cell surface is largely responsible for the electrophoretic
mobility of the cell, and it appears to reflect the sialic acid
content of the red blood cell membrane.179,180 Proteases expose
more negative sites on neonatal red blood cells than on adult
red blood cells and increase the electrophoretic mobility to a
greater degree.181 The sialic acid content of the newborn infant’s
erythrocyte membrane shows a gradual but significant decrease
in the first several weeks of life.182 Most studies, however, have
shown that the electrophoretic mobility of the neonatal cell is
similar to that of the adult cell.180 The more negative surface
charge of the neonatal red blood cell membrane is one of the
many characteristics that results in a decreased sedimentation
rate in newborns.183
OSMOTIC FRAGILITY
The osmotic fragility of red blood cells is a composite index of
their shape, hydration, and, within certain limitations, proneness
to in vivo destruction.174,184 Preterm and term infants have an
increased osmotic resistance.185The osmotic fragility of neonatal
cells begins to revert towards adult values shortly after birth, and
1118 SECTION XVIII — Developmental Hematopoiesis

Table 109.5 Postnatal Changes in Red Blood Cell Indices in Term Infants.

Days Weeks Months

Birth
(Umbilical
RBC Indices Cord Blood) 1 3 1 2 4 2 3 4 6 9 12
Hemoglobin 16.5 18.5 18.6 17.5 16.6 13.9 11.2 (9.4) 11.5 12.2 12.6 12.7 12.7
(g/dL) (13.0) (14.5) (16.5) (13.5) (13.4) (10.7) (9.5) (10.3) (11.1) (11.4) (11.3)
Hematocrit 51 (42) 56 (45) 55 (42) 54 (41) 53 (33) 44 (28) 35 (29) 35 (32) 38 (31) 36 (32) 36 (33) 37
(%)
Red blood 4.7 (3.9) 5.3 (4.0) 5.6 (3.9) 5.1 (3.9) 4.9 (3.3) 4.3 (3.1) 3.7 (3.1) 3.8 (3.5) 4.3 (3.9) 4.7 (4.0) 4.7 (4.1) 4.7
cells
(×1012/L)
Mean 108 (98) 108 (95) 110 107 (88) 105 (88) 101 (91) 95 (84) 91 (74) 87 (76) 76 (68) 78 (70) 78 (71)
corpuscular (104)
volume (fL)
MCH (pg) 34 (31) 34 (31) 36.7 34 (28) 33.6 32.5 (29) 30.4 (27) 30 (25) 28.6 (25) 26.8 (24) 27.3 (25) 26.8 (24)
(30.0)
MCHC (g/dL) 33 (30) 33 (29) 33.1 33 (28) 31.4 31.8 31.8 33 (30) 32.7 35 (32.7) 34.9 34.3
(28.1) (28.1) (28.3) (28.8) (32.4) (32.1)

The values are the means (values in parentheses are −2 standard deviations).
MCH, Mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RBC, red blood cell.
Data from Saarinen UM, Siimes MA. Developmental changes in red blood cell counts and indices of infants after exclusion of iron deficiency by laboratory criteria and
continuous iron supplementation. J Pediatr. 1978;92(3):412–416; and Dallman PR. In: Rudolph A, ed. Pediatrics. 16th ed. New York: Appleton-Century-Crofts;
1977:1111.

osmotic resistance reaches adult values by 4 to 6 weeks of age.
There is no known advantage to the increased osmotic
resistance seen in the neonatal red blood cell. However, studies
of osmotic fragility in neonates have practical implications. It
has been suggested that the diagnostic criteria for hereditary
spherocytosis used in adults and older children are unreliable
in newborn infants. For example, spherocytes are not regularly
seen in all infants with hereditary spherocytosis. Indeed,
spherocytes may be frequently found in ABO incompatibility.
When one is performing an osmotic fragility test, a neonatal
osmotic fragility curve must be used rather than an adult
curve.186 A relatively new diagnostic test for hereditary
spherocytosis, eosin-5-maleimide (EMA)-flow cytometry,
quantifies the erythrocyte membrane band 3 complex using
an eosin-5-maleimide dye flow cytometric analysis.187,188 A
reduction in band 3 complex is consistent with hereditary
spherocytosis. This test appears to perform very well in
neonates even in the first days of life.189,190
SURFACE RECEPTORS AND ANTIGENS
The neonatal red blood cell differs from the adult red blood cell
in its ability to bind various substances. For example, the neonatal
red blood cell binds more insulin than the adult red blood cell
because of the presence of greater numbers of insulin receptors
per cell.191,192 Newborn red blood cells have approximately 2.5
times the number of digoxin receptors in comparison with adult
red blood cells,193 and as a consequence they have erythrocyte-
to-plasma digoxin ratios three times those of adults. This may
explain the greater tolerance of newborns who are receiving
maintenance digoxin therapy.
Another unique characteristic of the fetal red blood cell is the
manifestation of the i antigen on the cell surface (adult red blood
cells express i antigen). Membrane i antigen is a carbohydrate
moiety located on protein membrane band 3, which, during
development, is converted from a linear polylactosamine to a
branched carbohydrate chain of N -acetyllactosamine units.167
Red blood cells bearing the i antigen are usually not detectable
by the first year of age. It has been suggested that the switch from
fetal to adult hemoglobin and the transformation of i antigen
expression that occur during the first year of life are governed
by a common control mechanism; therefore the presence of i
antigen can serve as a marker of fetal hematopoiesis.
Red blood cell antigens in the ABO, MN, Rh, Kell, Duffy, and
Vel systems are well developed in early intrauterine life.194 They
are easily demonstrated in the fifth to seventh gestational weeks
and remain constant through the remainder of intrauterine
development. Other antigens, such as the Lutheran and Xg
systems, develop more slowly but are present at birth. Lewis
antigens are lacking in the newborn. By approximately 2 years of
age, the child’s red blood cell and plasma antigens have developed
a pattern that is seen throughout the remainder of life.78
Although A and B antigens are present early in utero, A and B
isoagglutinin production occurs much later.195 By 30 to 34 weeks’
gestation, however, about 50% of infants have some measurable
anti-A or anti-B antibodies.The fetal production of such antibodies
is not related to maternal ABO blood type. Intrauterine exposure
to gram-negative organisms, whose antigens are chemically related
to those of blood groups A and B, is a potent stimulus for the
development of these antibodies. Isohemagglutinin antibodies
ultimately are demonstrable in normal infants by 6 months of age
and approach adult values at 2 years of age. Low to absent titers
after this time are suggestive of immune deficiency.196
OXYGEN TRANSPORT
At no other time of life are the mechanisms controlling oxygen
transport more complicated than in utero and during the immediate
postpartum period. During prenatal life the fetal arterial oxygen
tension (Po2) is approximately 30 mm Hg, and the venous Po2 is
approximately 15 mm Hg (Fig. 109.10). This low Po2 contributes
to the development of relative polycythemia in the fetus. After
birth, numerous factors affect oxygenation, including the inspired
gas mixture, pulmonary function, the arterial oxygen dissociation
curve, and the ability to extract oxygen at the tissue level.197,198
It has been speculated that the actual amount of oxygen released
to tissues may be greater in utero, given the characteristics of the
hemoglobin-oxygen dissociation curve (Fig. 109.11).
If pulmonary function is normal, there will be a rise in the Po2
of pulmonary blood in adults and neonates, from the 40 mm Hg of
pulmonary arterial blood to the 100 mm Hg of pulmonary venous
blood. Because of the shape of the hemoglobin-oxygen dissociation

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 CHAPTER 109 — Developmental Erythropoiesis 1119

curve, these Po2 values permit 95% saturation of hemoglobin by
oxygen. Further increases in Po2 produce little additional rise in
saturation. In the normal adult (see Fig. 109.10), approximately
50% of hemoglobin will be saturated with oxygen when the Po2
has fallen to 27 mm Hg (P50 = 27). In situations in which the
hemoglobin-oxygen dissociation curve has shifted to the right, the
affinity of hemoglobin for oxygen is reduced. Thus at any given
Po2, more oxygen is released to tissues. Conversely, if the curve is
NORMAL TERM INFANTS
90
1 2
80
70
HbO2 saturation (%)
shifted to the left, the affinity of hemoglobin for oxygen is increased.
Thus at any given Po2, less oxygen is released to the tissues.
Certain factors are known to alter hemoglobin’s affinity for
oxygen (Box 109.2). The most important of these are the fetal
hemoglobin concentration and the red blood cell 2,3-DPG
content. The level of red blood cell 2,3-DPG gradually increases
with gestation. By the end of the first postnatal week, the 2,3-DPG
levels are considerably higher than they are at birth (Table 109.6).
After the first week, red blood cell 2,3-DPG levels remain relatively
unchanged for the next 6 months. In term infants the hemoglobin-
oxygen dissociation curve gradually shifts to the right, and by 4
to 6 months of age, the P50 values approximate those of the adult.
3 4 5 6 7
The situation is somewhat different in preterm infants. Fetal
hemoglobin synthesis is still quite active; therefore, increases
in P50 seen in term infants as a result of the switch from fetal to
adult hemoglobin do not occur. The red blood cell 2,3-DPG
concentrations are slightly lower in preterm infants as well.96 These
concentrations can be increased with the use of EPO, thereby
shifting the hemoglobin-oxygen dissociation curve to the right.199

60 A precise relationship between the decrease in oxygen affinity of

a neonate’s blood and the progressive decline in the concentration
(1) Day 1 of fetal hemoglobin does not exist.198 Rather, changes in P50 reflect
50 (2) Day 5 the interplay between the levels of red blood cell 2,3-DPG, the
(3) 3 wk decline in fetal hemoglobin levels, and the subsequent increase in
(4) 6–9 wk hemoglobin A levels. A unifying concept has emerged consisting
40
(5) 3–4 mo of a “functioning 2,3-DPG fraction,”96 which determines how well
hemoglobin is able to release oxygen to tissues. The functioning
(6) 6 mo
30 2,3-DPG fraction (expressed as nanomoles per milliliter of red
(7) 8–11 mo blood cells) is calculated by multiplying the red blood cell 2,3-DPG
content by the percentage of adult hemoglobin93:
20
[2,3‐DPG ]functional = [2,3‐DPG ]observed × [100− % hemoglobin F]
10 
[109.1]
If P50 cannot be measured directly, it may be calculated from
the following equation86 after determining the functioning 2,3-
10 20 30 40 50 60 DPG fraction:
PO2 (mm Hg, pH 7.4) P50 = 18.4 + 0.0016[2,3‐DPG]functional [109.2]
Fig. 109.10 Oxygen equilibrium curve of blood from term infants
at different postnatal ages. The oxygen half-saturation pressure of Table 109.6 illustrates the expected changes in hemoglobin
hemoglobin (P50) on day 1 is 19.4 ± 1.8 mm Hg and has shifted to concentration, 2,3-DPG concentration, functional 2,3-DPG
30.3 ± 0.7 at age 11 months (normal adult 27.0 ± 1.1 mm Hg). (From fraction, and P50 during the first 3 months of life in low-birth-
Oski FA, Naiman JL. Hematologic Problems in the Newborn. 3rd ed. weight infants.96 In low-birth-weight infants, the amount of
Philadelphia: WB Saunders; 1982:250.) oxygen released by hemoglobin can be determined (on the basis

mm Hg
20 40 60 80 100
100 10.0
31 mmol/L

16 mmol/L

0.80 Fetal 8.0

45 mmol/L

0.60 Adult 6.0

mmol/L
ctO2
SO2

0.40 4.0
40 mmol/L

0.20 Adult 2.0

Adult
venous arterial
Fetal Fetal
venous arterial
0 0
4 8 12
PO2/kPa
Fig. 109.11 Differences in the hemoglobin-oxygen dissociation curve between the newborn and the adult. This figure demonstrates the effects
of these differences on oxygen tension (ctO2) and oxygen saturation (SO2). (From Stockman JA. Fetal Hematology. In: Eden RD, Boehm J, eds.
Assessment and Care of the Fetus. Norwalk, CT: Appleton & Lange; 1989:124.)

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1120 SECTION XVIII — Developmental Hematopoiesis
Box 109.2 Factors Affecting
Hemoglobin-Oxygen Affinity
Increased Amount of Red Blood Cell 2,3-Diphospho-
glycerate, Increased P50
Adaptation to high altitude
Hypoxemia associated with chronic pulmonary disease
Hypoxemia associated with cyanotic heart disease
Anemia
Secondary to iron deficiency
Secondary to chronic renal disease
Caused by sickle cell anemia
Decreased red blood cell mass
Chronic liver disease
Hyperthyroidism
Red blood cell pyruvate kinase deficiency
Decreased Amount of Red Blood Cell
2,3-Diphosphoglycerate, Decreased P50
Septic shock
Severe acidosis
Following massive transfusions of stored blood
Neonatal respiratory distress syndrome
Increased P50, No Consistent Alteration in Amount
of Red Blood Cell 2,3-Diphosphoglycerate
Abnormal hemoglobins (Kansas, Seattle, Hammersmith,
Tacoma, E)
Vigorous exercise
Decreased P50, No Consistent Alteration in Amount
of Red Blood Cell 2,3-Diphosphoglycerate
Abnormal hemoglobins (Kempsey, Chesapeake, J Cape
Town, Yakima, Rainier)
of unloading from a normal arterial Po2 adjusted for age to an
arbitrary central venous Po2 of 40 mm Hg).93 Although oxygen-
carrying capacity (hemoglobin concentration × percentage of
oxygen saturation × 1.36 mL oxygen per gram of hemoglobin)
decreases during the first few months of life as a consequence
of a decline in hemoglobin concentration, the amount of
oxygen capable of being delivered to tissues actually increases.
For example, a newborn weighing 1000 g with a hemoglobin
concentration of 15 g/dL, a P50 of 19, and a central venous Po2 of
40 mm Hg will unload 1 mL of oxygen to tissues for every 100
mL of blood that passes through the capillary bed. At 10 weeks
of age, the P50 has shifted to the right and is now 24 mm Hg.This
same infant will now deliver 2.1 mL of oxygen per 100 mL of
blood, even though the hemoglobin concentration has declined
to 8 g/dL (see Fig. 109.11).
These calculations emphasize the importance of understanding
an infant’s ability to deliver oxygen to tissues when one is
determining whether to administer an erythrocyte transfusion.
The decision to transfuse erythrocytes should not be based
on hemoglobin concentration alone. Transfusions significantly
affect an infant’s endogenous erythropoiesis. For infants who
undergo exchange transfusion or multiple transfusions, both EPO
concentrations and reticulocyte counts are lower at any given
hemoglobin concentration (Fig. 109.12).93,200
It is often assumed that oxygen delivery is decreased in
newborns because of the presence of high-affinity hemoglobin.
In fact, a leftward shift in the hemoglobin-oxygen dissociation
curve resulting from high levels of fetal hemoglobin may better
maintain oxygen delivery during episodes of severe hypoxemia.
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Fig. 109.13 illustrates the arterial Po2 below which shifts to the
right in the neonatal hemoglobin-oxygen dissociation curve are
no longer advantageous. The gas tension at which this occurs is
known as the crossover Po2.201 The crossover Po2 is dependent
on how low the venous Po2 falls before oxygen delivery ceases.
Wimberley202 calculated that if the arterial Po2 fell to less than 32
mm Hg, and if the venous Po2 fell to 10 mm Hg (a value found
in the cerebral venous blood of some sick newborns), the infant
would achieve better oxygen delivery with a fetal hemoglobin-
oxygen dissociation curve than with an adult curve. Conversely,
if arterial Po2 can be maintained at a higher value, better oxygen
delivery would exist if the hemoglobin-oxygen dissociation
curve were shifted to the right.
In neonates with hypoxemia (e.g., infants with cyanotic
congenital heart disease), a shift to the right (a higher P50) in
the hemoglobin-oxygen dissociation curve tends to lower the
arterial and venous oxygen saturation at any given Po2. It has
been documented that if arterial hypoxemia results from right-
to-left shunting, shifts to the right in the hemoglobin-oxygen
dissociation curve always tend to increase the arterial-venous
oxygen difference and improve oxygen delivery.201
After intrauterine transfusion, infants have oxygen-unloading
properties characteristic of those of adult blood. Despite the
decrease in oxygen affinity that accompanies intrauterine
transfusion, no deleterious effects of this procedure with respect
to oxygen uptake by the fetus have been documented.203 The
physiologic significance of manipulating the hemoglobin-oxygen
affinity of fetuses and extremely preterm infants remains to be
fully studied.
PHYSIOLOGIC ANEMIA IN NEONATES
AND THE ANEMIA OF PREMATURITY
A gradual decrease in hemoglobin concentration occurs during
the first 2 to 3 months of life in term and preterm infants. The
hemoglobin concentration remains stable during the next several
weeks and then slowly rises. In term infants the fall in red blood
cell production after birth is the result of improved oxygenation
and occurs as a natural adaptation to extrauterine life. Because
of this, the decrease in hemoglobin concentration has been
termed a physiologic nadir rather than true anemia. However,
in preterm infants, adaptive mechanisms may not be complete.
Preterm infants generally decrease hemoglobin concentration
to values lower than those seen in term infants, and the nadir
varies with the degree of prematurity. Fig. 109.4 illustrates the
decline in hemoglobin concentration in the first month of life on
the basis of birth weight and gestational age. Hemoglobin values
as low as 7 g/dL are common in preterm infants who have not
undergone phlebotomies.94,204
Assessment of the factors that characterize oxygen supply and
oxygen demand has provided insight into the adaptive changes
occurring in preterm infants in response to low hemoglobin
concentrations. In preterm infants, a low central venous Po2
correlates with a mild increase in EPO production (Fig. 109.14).205
The decline in central venous Po2 may indicate the presence of
anemia, representing the integration of variables that determine
oxygen supply and demand: hemoglobin concentration, red
blood cell and oxygen affinity, intravascular volume, oxygen
consumption, heart rate, cardiac stroke volume, and arterial
oxygenation.The hemoglobin concentration is only one of many
important variables ensuring adequate oxygen delivery in both
term and preterm infants.186
ANEMIA OF PREMATURITY
CHARACTERISTICS
Despite the slight increase in EPO production that occurs
in response to declining central venous Po2 values, EPO
 CHAPTER 109 — Developmental Erythropoiesis 1121

Table 109.6 Changes in Hemoglobin Concentration, Hematocrit, and Other Markers of Oxygen Delivery During
the First 3 Months of Life in Low-Birth-Weight Infants.

Total Fetal
Hemoglobin O2 Capacity P50 at pH 7.40 2,3-Diphosphoglycerate Hemoglobin FFDPG
Age Blood (g/dL) Hematocrit (%) MCHC (%) Blood (mL/dL) (mm Hg) (nmol/mL) (% of Total) (nmol/mL)
Group I (<1000 g)a
2 wk 17.2 47.0 36.6 23.9 18.0 6255 83.0 1002
4 wk 8.5 26.0 32.7 11.8 15.0 3923 81.0 761
9 wk 7.2 22.0 32.7 10.0 15.0 4636 87.1 974
11 wk 7.7 22.5 34.2 10.7 17.0 5867 78.0 1290
Group II (1001–1500 g)b
1–2 days 15.1 ± 1.3 45.7 ± 3.7 33.0 ± 0.7 21.0 ± 1.8 18.0 ± 1.7 4124 ± 1562 86.6 ± 3.1 580 ± 287
5–8 days 13.4 ± 1.1 41.4 ± 3.2 33.5 ± 2.9 18.7 ± 1.5 18.9 ± 3.0 4501 ± 1919 84.4 ± 3.8 903 ± 689
2–3 wk 12.6 ± 3.1 33.6 ± 6.0 34.2 ± 1.1 15.9 ± 3.1 21.2 ± 1.9 5721 ± 1375 83.3 ± 5.1 1119 ± 557
4–5 wk 8.8 ± 0.9 25.3 ± 1.8 34.9 ± 1.7 12.3 ± 1.3 20.5 ± 1.7 6095 ± 2081 85.2 ± 2.3 931 ± 456
6–9 wk 9.1 ± 1.7 24.5 ± 5.8 35.1 ± 2.2 11.8 ± 2.4 23.4 ± 1.1 8734 ± 1834 77.2 ± 1.9 1995 ± 480
9–10 wkc 8.2 24.0 34.0 11.1 24.0 9000 77.0 2070
Group III (1501–2000 g)b
1–2 days 16.1 ± 0.9 47.8 ± 1.9 33.7 ± 1.9 22.4 ± 1.2 19.3 ± 0.9 4475 ± 1174 87.2 ± 3.6 703 ± 331
5–8 days 16.8 ± 3.3 48.5 ± 10.0 34.7 ± 0.5 25.3 ± 4.7 19.8 ± 1.3 5489 ± 1428 79.4 ± 5.0 1056 ± 590
2–3 wk 13.6 ± 3.0 40.4 ± 9.8 34.4 ± 1.5 18.8 ± 4.0 21.3 ± 1.8 6002 ± 998 80.6 ± 5.8 1184 ± 329
4–5 wk 11.2 ± 2.8 31.9 ± 9.9 35.5 ± 2.2 15.5 ± 3.8 20.8 ± 1.6 5841 ± 839 75.8 ± 7.8 1569 ± 577
6–9 wk 8.0 ± 0.7 22.1 ± 1.7 35.9 ± 0.7 11.1 ± 1.0 24.0 ± 0.9 7290 ± 634 67.5 ± 6.2 2457 ± 575
Group IV (2001–2500 g)b
1–2 days 15.9 ± 0.9 46.2 ± 5.8 35.8 ± 1.9 21.9 ± 1.5 20.2 ± 1.6 5306 ± 1075 76.8 ± 5.43 1258 ± 392
5–8 days 15.6 ± 1.7 47.0 ± 5.0 34.2 ± 1.1 21.5 ± 2.4 21.3 ± 3.3 6417 ± 1527 77.7 ± 6.3 1457 ± 603
2–3 wk 12.3 ± 1.1 35.1 ± 3.2 34.9 ± 0.5 17.1 ± 1.5 22.0 ± 1.3 7145 ± 1737 76.9 ± 4.7 1666 ± 472
6–9 wkc 14.0 44.0 34.0 19.5 25.5 7100 43.0 3212

aOnly one patient.

bValues are given as the mean ± the standard deviation.
cFewer than five infants.
FFDPG, Functioning fraction of 2,3-diphosphoglycerate; MCHC, mean corpuscular hemoglobin concentration; RBC, red blood cell.
From Delivoria-Papadopoulos M, Roncevic N, Oski FA. Postnatal changes in oxygen transport of term, preterm and sick infants: the role of red cell 2,3
diphosphoglycerate in adult hemoglobin. Pediatr Res. 1971;5:235.

concentrations in preterm infants are still significantly low, given
the degree of anemia (Fig. 109.15).76,111,205,206 This anemia, termed
the anemia of prematurity, affects infants born at less than
approximately 32 weeks gestation, and it is the most common
anemia encountered in the neonatal period. It is a normocytic,
normochromic anemia, generally associated with hemoglobin
concentrations less than 10 g/dL and low reticulocyte counts.
Some infants may be asymptomatic, whereas others demonstrate
signs of anemia that are alleviated by transfusion. These signs
traditionally include tachycardia, increased episodes of apnea and
bradycardia, poor weight gain, an increased oxygen requirement,
and elevated serum lactate concentrations that decrease after
transfusion.207–210
Anemia of prematurity was first described by Schulman,211
who divided the anemia into three phases. Early anemia was
marked by an initial fall in hemoglobin concentration. The second,
or intermediate, phase was characterized by maintenance of
low hemoglobin concentrations. The third phase, late anemia of
prematurity, resulted in hemoglobin concentrations that continued
to fall, despite symptoms of anemia. The anemia of prematurity
usually resolved spontaneously by 3 to 6 months of life.
Although much information has accumulated since the 1970s,
detailed mechanisms responsible for the anemia of prematurity
remain to be determined. Shortened erythrocyte survival,212
hemodilution associated with a rapidly increasing body mass,213
and the transition from fetal to adult hemoglobin96 have all been
implicated. Despite diminished levels of oxygen available to
tissues93 and the appearance of signs of anemia,206 serum EPO
concentrations remain low.206 However, erythroid progenitors
are highly sensitive to EPO,214,215 and the concentrations of other
erythropoietic growth factors, including IL-3 and GM-CSF, appear
to be normal.216
Infants born prematurely lack a normal response to anemia
and fail to increase EPO production despite an apparent need
for improved tissue oxygenation.217 This occurs regardless of the
cause of anemia, whether it is the early anemia of phlebotomy
loss or the later anemia of prematurity. Possible molecular and
cellular mechanisms responsible for this lack of responsiveness
include defects in transferring the hypoxic signal to the nucleus,
diminished or defective binding of transcription factors to the
promoter or enhancer regions of the gene, decreased production
or stability of the transcriptional factors, decreased production
or stability of EPO mRNA, diminished or unstable protein
production, or increased production of counterregulatory
proteins such as IL-1 and tumor necrosis factor. The inability of
the kidney or liver in the premature infant to produce EPO mRNA
with magnitude equal to that seen in the fetus may also involve
a developmental delay in expression of transcriptional factors
responsible for increasing EPO production, or in persistence of
fetal EPO gene methylation patterns. Because EPO produced by
the liver is indistinguishable from that produced by the kidney,
it is unknown whether preterm infants rely on EPO produced
by the liver, the kidney, or a combination of the two. However,
macrophages from preterm infants generate EPO mRNA and
protein as effectively as do those from term infants and adults.218
The anemia of prematurity likely involves some sort of delay
in activation of EPO gene expression, from prenatal (liver) to
postnatal (kidney) sites of production.

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1122 SECTION XVIII — Developmental Hematopoiesis

PREMATURE BABIES (1000–1500 g)

Theoretical arteriovenous oxygen saturation difference (%)

100
20 P50 = 22 torr
1.0 P50 = 27 torr
1–2 days P50 = 32 torr
80
Oxygen content (m/100 mL blood)

1.2

15
1.6 60
5–8 days

6–9 wk 1.8
40
10 2–3 wk 2.1

20

5
0
0 20 40 60 80 100
9–10 wk Arterial PO2
Fig. 109.14 The effect of arterial partial pressure of oxygen (Po2)
on theoretical arteriovenous oxygen content difference when venous
10 20 30 40 50 60 70 150 partial pressure of oxygen is 40 mm Hg (bottom set of curves), 20
Oxygen tension (PO2 mm Hg) mm Hg (middle set of curves), or 10 mm Hg (top set of curves) at
various oxygen half-saturation pressures of hemoglobin (P50). (From
Fig. 109.12 Oxygen equilibrium curves of blood from premature in- Woodson RD. Physiological significance of oxygen dissociation curve
fants (1000 to 1500 g) at different postnatal ages. Double-headed ar- shifts. Crit Care Med. 1979;7:368.)
rows represent the oxygen unloading capacity between a given arterial
and venous Po2. Points corresponding to 150 mm Hg on the abscissa
are the oxygen capacities. Each curve represents the mean value of CLINICAL TRIALS
the infants studied in each group. (From Delivoria-Papadopoulos M, Multiple clinical studies have been performed that have
Roncevic N, Oski FA. Postnatal changes in oxygen transport of term, evaluate EPO administered to preterm infants.219 Randomized
preterm and sick infants: the role of red cell 2,3 diphosphoglycerate in controlled studies using EPO dosages greater than 500 U/kg/
adult hemoglobin. Pediatr Res. 1971;5:235.) week for at least 2 weeks220–233 have been reviewed elsewhere.
A randomized placebo-controlled study of darbepoetin and
EPO evaluated infants with a birth weight of 500 to 1250 g and
48 hours of age or younger who were randomized to receive
35
darbepoetin (10 μg/kg, once per week subcutaneously), EPO
(400 U/kg, three times per week subcutaneously), or placebo
30 (sham dosing) through 35 weeks’ gestation.234 Infants in the
darbepoetin and EPO groups received significantly fewer
25 transfusions (P = 0.015) and, importantly, were exposed to fewer
donors (P = 0.044) than the placebo group (darbepoetin, 1.2 ±
HbF >60%
2.4 transfusions and 0.7 ± 1.2 donors per infant; EPO, 1.2 ± 1.6
EP (mU/mL)

20 transfusions and 0.8 ± 1.0 donors per infant; placebo, 2.4 ± 2.9
transfusions and 1.2 ± 1.3 donors per infant). Morbidities were
15 similar among groups, including the incidence of retinopathy of
∆2.8 prematurity. In this population of preterm infants, darbepoetin
and EPO successfully serve as adjuncts to transfusions in
10 ∆3.0
maintaining red blood cell mass.
Recently published trials evaluating higher doses of EPO
HbF <30% ∆3.4
5 administered to extremely preterm infants evaluated potential
neuroprotective effects, with varied outcomes.235,236 It is clear
from the most recent trials that EPO administration to preterm
0
infants significantly decreases transfusions and improves
0 4 6 8 10 12 14 16 18
hematocrit during the initial neonatal intensive care unit
Hemoglobin (g/dL) (NICU) hospitalization. Further evaluation regarding potential
Fig. 109.13 Relationship between erythropoietin concentrations and neuroprotection is ongoing.
postnatal hemoglobin. In infants born prematurely who have under-
gone exchange transfusion or multiple transfusion (fetal hemoglobin PHARMACOKINETICS
[HbF] <30%), the hemoglobin concentration may fall several grams Pharmacokinetic studies in newborn monkeys and sheep
per deciliter before resumption of erythropoietin production (EP). indicate that neonates have a larger volume of distribution and
(From Stockman JA, Garcia JF, Oski FA. The anemia of prematuri- a faster elimination of EPO,237,238 necessitating the use of doses
ty: factors governing the erythropoietin response. New Engl J Med. higher than those required for adults.239 These pharmacokinetic
1977;296:647.) differences apply to preterm infants as well.240,241 Although

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42
20
15
EP mU/mL
10
5 seen in recently completed multicenter trials evaluating EPO
0 priority in clinical trials in preterm infants. Although there
25 30 35
PVO2 (torr)
Fig. 109.15 Changes in plasma erythropoietin concentrations in re-
sponse to declines in central venous oxygen tension (PVO2) in pre-
term neonates. The arrow represents the position of 38 mm Hg, which
for the purpose of this figure is the lower limit of normal for venous
oxygen tension. The horizontal dashed line is the upper limit of normal
for erythropoietin when venous oxygen tension of 38 Torr or greater is
taken as normal. EP, Erythropoietin production. (From Stockman JA,
Graeber JE, Clark DA, et al. Anemia of prematurity: determinants of
the erythropoietin response. Pediatr. 1984;105:786.)
various EPO doses and dosing schedules have been evaluated
in preterm infants, the erythrokinetics of EPO (the interaction
between erythroid progenitors and their growth factors) in
preterm infants are still being explored.
The route of administration also influences the effectiveness
of EPO. Rapid intravenous administration of EPO generates
peak serum concentrations that far exceed physiologic
concentrations.242 This may result in wasted drug via increased
renal excretion. Dosing strategies that achieve lower peak
serum concentrations over a more prolonged period may be
more effective. Dosages in the range of 500 to 1400 U/kg/week,
with administration subcutaneously every day or every other
day, result in an adequate erythropoietic response. Newer, long-
acting erythropoiesis-stimulating agents such as darbepoetin
are currently being studied.243,244 Data from preterm infants
(considering half-life, volume of distribution, and clearance)
suggest that higher doses will be required, but dosing intervals
may be as great as once every 2 to 3 weeks.
SIDE EFFECTS OF ERYTHROPOIESIS-STIMULATING
AGENTS
Side effects in neonates receiving erythropoiesis-stimulating
agents have been rare. A side effect unique to neonates, transient
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2023. Para uso personal exclusivamente. No se permiten otros usos sin autorización. Copyright ©2023. Elsevier Inc. Todos los derechos reservados.
CHAPTER 109 — Developmental Erythropoiesis 1123
neutropenia, was noted in early studies but has not been reported
in the last decade.245–247 The neutropenia was not associated
with depletion of either neutrophil reserves or colony-forming
unit–granulocyte-macrophage concentrations, but it appeared
to involve reduced production of neutrophils from granulocytic
progenitors248,249; it resolves after discontinuation of EPO
administration. In newborn mice, neutropenia occurs after
administration of very high doses of recombinant EPO as a result
of decreased production of neutrophils from progenitors.248
Overall, the side effects of EPO administration in preterm
infants have been minimal compared with those reported in
adults (hypertension, bone pain, rash, and, rarely, seizures). None
of these effects were reported in preterm infants involved in
clinical trials. Moreover, no studies have reported differences
between placebo and EPO recipients in the incidence of neonatal
morbidities such as chronic lung disease, IVH, necrotizing
enterocolitis, or late-onset sepsis. Meta-analyses first published in
2006 reported that, compared with late EPO administration, early
(first week of life) EPO administration increased the incidence
of retinopathy of prematurity (ROP) greater than stage 2.250,251
Revised meta-analyses corrected a previous misclassification of a
single-center EPO study into the late EPO administration group.
The association between retinopathy of prematurity and EPO
is no longer statistically significant.252,253 No differences were
administered to over 2000 extremely preterm infants.235,236,254 In
fact, when those infants are included in updated meta-analyses,
the trend in greater than stage 2 ROP is greater in those infants
randomized to placebo or control groups. Close ophthalmologic
evaluation for retinopathy of prematurity continues to be a
40 have been few long-term follow-up studies, it appears that the
administration of EPO has no adverse effect on developmental
outcome or growth, measured at 18 to 22 months.255
NUTRITIONAL SUPPLEMENTATION
Functional iron deficiency has frequently been reported in
pediatric and adult patients receiving EPO, and it likely limited
the success of some of the early EPO trials in preterm infants.
Studies evaluating iron stores in EPO recipients220,221 noted
increased iron requirements in infants receiving EPO, as
evidenced by diminished ferritin concentrations and elevated
numbers of hypochromic red blood cells, despite iron doses of 6
mg/kg/day. Infants receiving EPO are likely at greater risk for iron
deficiency than for iron overload and increased oxidant stress.
However, further evaluation is still required to determine the
optimal dose and most effective route of administration of iron
in preterm infants receiving EPO.
Vitamin E is an antioxidant, inhibiting peroxidation of
polyunsaturated fatty acids in the lipid bilayers of all cell
membranes.Vitamin E requirements may be increased secondary
to increased iron supplementation, because iron promotes
oxidation of polyunsaturated fatty acids. Pathak and colleagues228
evaluated high-dose vitamin E in preterm infants receiving EPO
and found it showed no benefit compared with doses of 50
IU/kg/day. Further study is needed to determine the optimal dose
of vitamin E in preterm infants receiving EPO.
Folate and vitamin B12 have recently been evaluated in
combination with EPO and iron, and have shown significant
promise.232,256 In an evaluation of preterm infants weighing
800 g or less, Haiden and colleagues232 achieved significantly
greater success (38% of infants did not receive transfusion) when
vitamin B12 at a dosage of 21 mg/kg/week subcutaneously was
added to a regimen of EPO, iron, vitamin E, and folate.239 When
combined with limited phlebotomy losses, this therapy shows
great promise in extremely low-birth-weight infants.
Regardless of the treatment strategy, a critical understanding
of the developmental, physiologic, and pathologic influences
1124 SECTION XVIII — Developmental Hematopoiesis
affecting oxygen delivery in term and preterm infants is important
in determining the optimal strategy in maintaining or altering
the hematocrit through the administration of an erythrocyte
transfusion or the administration of an erythropoiesis-stimulating
agent.
CONCLUSION
The fetal and neonatal periods mark a time when erythrocyte
indices and characteristics differ remarkably from indices of
children and adults. The stages of erythropoiesis move from
yolk sack to liver to marrow, whereas production of erythrocyte
growth factors migrates from liver to marrow and kidney. Infants
born prematurely have altered production of erythrocytes; the
ability to deliver oxygen to tissues based on fetal and adult
hemoglobin concentrations varies greatly during the early
months of life in extremely low-birth-weight infants. A detailed
understanding of erythrocyte physiology and the nutritional and
growth factor needs of preterm and term infants will aid the
clinician in caring for neonates in the first weeks of life.
ACKNOWLEDGMENTS
Parts of this chapter were adapted from sections of Chapter 134
by authors James A. Stockman III and Pedro A. DeAlarcon in the
first edition of this book. We thank Erin Adair for assistance with
illustrations.
A complete reference list is available at www.ExpertConsult.com.
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