Red blood cells (RBC) indices

such as mean corpuscular volume,

mean
corpuscular
hemoglobin
concentration, and red cell distribution
width are laboratory parameters that
are frequently overlooked in clinical
practice but they can provide
assistance in establishing a diagnosis
in anemic patients.
The first few weeks to months
after birth are marked by dramatic
physiologic and anatomic changes in
every organ system as the neonate
adapts to extrauterine life independent
of the placenta. Clinical presentation of
illness and laboratory data must be
interpreted against a backdrop of
major developmental alterations. This
is especially true when assessing a
neonate
with
a
hematologic
disturbance.
Fetal and neonatal erythrocytes
differ significantly from those produced
by older infants, children, and adults.
They are larger in size, have a shorter
life
span,
altered
shape
and
deformability,
and
high
fetal
hemoglobin concentrations. While
these differences do not impact their
ability to deliver oxygen to the tissues
to meet their metabolic needs, these
characteristics may confound the
interpretation of hematologic data and
may complicate the diagnosis of
neonates with certain disorders such
as anemia.
Embryonic
and
fetal
hematopoiesis occurs in three phases:
megaloblastic, hepatic, and myeloid.
At each phase of RBC development
both the sites of production and the
cell
composition
change.[4]
Erythrocytes,
lymphocytes,
granulocytes, and megakaryocytes
(platelets) all originate from the same

common
progenitors
called
[1,4,5]
pleuripotent stem cells,
however,
only RBC development is presented in
this article. Figure 1 illustrates the sites
and stages of erythropoiesis in utero.
Extraembryonic erythropoiesis
is detected in the yolk sac at
approximately
14
days
after
[1,3]
conception.
RBCs
enter
the
circulation at 3 to 4 weeks when the
umbilical and vitelline circulations are
joined. The yolk sac is the principle
site of RBC production until 6 to 8
weeks' gestation and by 10 to 12
weeks extraembryonic erythropoiesis
has virtually ceased.[3]
Small groups of erythroblasts,
hematocytoblasts, are detected in the
mesenchyme and endoderm of the
yolk sac. These primordial cells give
rise to two distinctive types of RBCs:
megaloblasts
and
normoblasts
(erythroblasts). Megaloblasts are the
most primitive RBCs. They differ
morphologically from more definitive
erythroblasts
produced
later
in
gestation in that they are large
(macrocytic),
remain
nucleated
throughout their life span, and have a
greater mean cell volume (MCV) (see
Table 1 ). As gestation progresses
megaloblasts are gradually replaced
by normoblastic erythrocytes.[13]
The hepatic phase begins by
the 5th to 6th gestational week. The
exact source of hepatic stem cells
continues to be a subject of
investigation. Either stem cells from
the yolk sac migrate to the liver and
other hematopoietic organs or there is
independent production of stem cells
at
these
sites
during
fetal
development.[1,3,4,6]
Normoblasts

are

the

predominant form of circulating RBCs

by 10 weeks of gestation. They are
smaller than yolk sac cells and they
lose their nuclei in the course of
maturation.[1][4]
The production of RBCs in the
fetal liver increases until the 2nd
trimester when the rate of synthesis
begins to decline as erythropoiesis
increases in the bone marrow. RBCs
are still produced in the liver for
approximately 1 week after birth.[1,3]
Bone marrow RBCs are first
noted at 8 to 9 weeks' gestation and
output from this site becomes more
significant as gestation progresses.
The myeloid period begins at the 4th to
5th month and the bone marrow
becomes the principle site of
erythropoiesis during the 3rd trimester
of gestation.[1,3]
Megaloblasts remain primitive
cells and do not mature into other
forms. However normocytes undergo a
process of development, which is
identical whether it occurs in the liver
or the bone marrow. The production of
a mature red blood cell is complex,
beginning with initial differentiation of
the pleuripotent hematopoietic stem
cell into the colony-forming unit
granulocyte, erythrocyte, macrophage,
megakaryocyte (CFU-GEMM). The
CFU-GEMM differentiates sequentially
into the burst-forming unit erythroid,
(BFU-E), colony-forming unit erythroid
(CFU-E), normoblast, reticulocyte, and
finally, the mature erythrocyte.[3]
Immature cells contain nuclei; the
nuclei are normally extruded before
release of the cells from the bone
marrow. The process of RBC
maturation and the various growth
factors that influence it are illustrated
in Figure 2.

Mature RBCs are nonnucleated

biconcave disks surrounded by a
flexible lipid membrane. Their function
is to transport oxygen from the
pulmonary vasculature to the cells to
meet metabolic needs. This role is
facilitated by hemoglobin, a protein
composed of four globin chains.
Different globin chains, thus different
types of hemoglobin, are expressed
during development and also in certain
diseases
(sickle
cell
anemia,
[1,3,7,8]
thalassemias).
Embryonic
hemoglobin (Grover, Portland) and
fetal hemoglobin (Hgb F) have higher
affinity for oxygen than adult
hemoglobin
(Hgb
A).[2,5,9]
The
production of Hgb F predominates
during gestation; at the time of birth
the
majority
of
hemoglobin
synthesized is Hgb F. However, toward
the end of the second trimester, there
is a gradual shift from the manufacture
of fetal hemoglobin to the production
of adult hemoglobin.[13]
Erythropoietin (Epo) regulates
the production of RBCs in utero as
well as after birth. Its secretion is
stimulated by low tissue oxygen. [4,5,7,10]
The fetal liver initially produces Epo
but during the 3rd trimester the kidneys
become the major source of this
growth factor.[3,10,11] At the end of
gestation, when the Epo concentration
is at its peak, erythropoiesis is
approximately three to five times that
of normal adults. Subsequent to the
onset of respiration and the increase in
PaO2, the stimulus for Epo release is
eliminated. There is a decline in both
erythropoiesis
and
hemoglobin
[12]
synthesis.
This is reflected in
changes in hematologic values and
RBC indices in the first weeks and
months of life as illustrated in Table 2 ,
Table 3 , Table 4 , and Table 5 .

Normal levels of RBCs at birth

range from 5.1 to 5.3 million/mm3 for
term newborns and 4.6 to 5.3
million/mm3 for premature neonates.[4]
Nucleated red blood cells (NRBC) are
immature erythrocytes rarely found in
the peripheral circulation of adults in
the absence of illness. It is normal to
see NRBCs in newborns shortly after
the stress of delivery. This is likely
reflective of elevated Epo levels in the
presence of the normally low oxygen
tensions in utero.[13] NRBCs generally
disappear by 4 days and 7 days of life
in term and premature neonates,
respectively, although they may persist
longer than 1 week in very immature
neonates.[5] The continued presence of
NRBCs for a longer than expected
time may indicate a pathologic
condition.

Because of active in utero

erythropoiesis, the reticulocyte count
at birth is 3 to 7% in full-term babies
and 8 to 10% in premature babies.
This declines to 0 to 1% by the first
week of age, reflecting diminished
erythropoiesis.[4] Reticulocyte counts
are frequently increased in anemia
and are useful in differentiating acute
from chronic anemia. "Increased
numbers of immature RBCs reflect the
degree of hematopoietic activity in
response to anemia."[4] There is a
delay between the onset of anemia
and the stimulation of erythropoiesis.
Therefore, in acute anemia the
reticulocyte count may be normal
whereas in chronic anemia the
reticulocyte count would be elevated.
[2,3,5,11]
A neonate delivered after a
placental abruption would be expected
to have anemia and a normal
reticulocyte count. In the case of in
utero hemolysis due to ABO
incompatibility, the neonate might be
anemic with an elevated reticulocyte
count.
The life span of adult
erythrocytes is 120 days. RBCs in
term neonate will survive between 60
and 90 days. Erythrocytes from
premature
neonates
have
considerably shorter life spans,
ranging from 35 to 50 days. [3,7,10,11] A
hemolytic process causes RBCs to be
destroyed before the end of their
expected life spans. Reticulocytosis
should be seen with hemolysis unless
there is coexisting bone marrow
suppression. In this case the
reticulocyte count might be low
compared with the degree of anemia.[2]
Hemoglobin and hematocrit are
dependent on both gestational and
chronologic ages. Further variations in
these parameters are due to timing of

cord clamping (early versus late) and

sampling sites (capillary versus
venous).[2,3,11]
In most term neonates there is
a rise in hemoglobin by 2 hours of life
due to postnatal fluid shifts with levels
reaching a plateau by 8 to 12 hours of
age.
Hemoglobin
concentrations
decline from this point on and continue
to fall over the next several weeks.
This
is
due
to
decreased
erythropoiesis, a shorter life span of
neonatal RBCs, and the increased
plasma volume seen with growth. By
approximately 8 weeks of age
hemoglobin levels achieve their lowest
point,
approximately 11.2 g/dL.
Erythropoiesis
and
hemoglobin
synthesis resume with the production
of Hb A.[1,2,3,11]
The predominance of Hb F in
early postnatal life postpones the
clinical
presentation
of
certain
hemoglobinopathies
(B-thalassemia
minor, sickle cell anemia); however
these congenital anemias may be
detected
by
hemoglobin
[2,8]
electrophoresis.
Postnatal
changes
in
hemoglobin
concentration
in
premature infants with birth weights
under 1,500 grams differ markedly
from that of term infants. This is due to
significant phlebotomy losses and the
effects of blood transfusions that
depress erythropoiesis. The nadir of
hemoglobin concentration is reached
at 4 to 8 weeks of life in this population
with an average level of 8 g/dL.[11] See
Table 2 , Table 3 , Table 4 , and Table
5 for the relationship between birth
weight,
chronologic
age,
and
hemoglobin levels in premature
babies.

Early embryonic RBCs are

large; diameters range from 20 to 25
m with a mean cell volume (MCV) of
180 femtoliters (fl) or m3. Cell size
decreases
gradually
during
development reaching 130 fl at
midgestation and 115 fl at term. MCV
at 1 year of age is 82 fl.[3,11]
When the MCV is normal for
age
and
gestation,
cells
are
normocytic. Microcytic cells have
smaller than normal MCV while
macrocytosis refers to large cell size.
[9,14,15]
The MCV is larger than normal in
megaloblastic or macrocytic anemia
and smaller than normal in microcytic
anemia.[15][17] An increased MCV is
seen in hyperviscosity/polycythemia.[5]
The
mean
corpuscular
hemoglobin concentration (MCHC) is
fairly constant from birth through
adulthood.[9] It averages 34 pg in fullterm cord blood, 35 pg on the first day
of life, and 33 picograms (pg) at 1
week of age. Premature neonates,
however, have higher MCHCs; values
range from 40 pg at 28 weeks to 38 pg
at 34 weeks.[18]
When the MCHC is normal,
erythrocytes
are
described
as
normochromic; that is, they are normal
in color due to normal amount of
hemoglobin.[9,14,15] "Increases in MCHC
reflect distortions of RBC volume that
cause compression of hemoglobin into
a smaller space."[11] In certain
disorders,
such
as
hereditary
spherocytosis or ABO incompatibility,
the MCHC is elevated since the
surface area of the erythrocytes is
decreased, although the hemoglobin
concentration in the cells remains
stable.[11]
Red

cell

distribution

width

(RDW) indicates variation in RBC size

and is used to detect anisocytosis.[14,15]
RDW can also be a sensitive and
specific early indicator of iron
deficiency anemia especially in infants
with cyanotic congenital heart disease.
[19,20]
Greater heterogeneity of cell size
yields a larger RDW. "Because
immature cells are larger than older
red
cells,
infants
with
active
erythropoiesis
have
elevated
RDWs."[11] Infants who have received
blood transfusions have lower RDWs
since
transfusions
suppress
[11]
erythropoiesis.
Values in normal individuals
vary from 11.5 to 14.5% while RDWs
in infants and children range from 1.5
to 15%.[12,21] Mean RDWs in premature
neonates on the first day of life as
described by Alur and coworkers[22] are
shown in Table 6 .
Mature
erythrocytes
are
biconcave disks. RBCs with abnormal
morphology may be removed from the
circulation
leading
to
anemia.
Therefore, certain types of anemia
may
be
characterized
by
poikilocytosis.
It is not uncommon to see
variation in shape in neonatal RBCs.
Irregularly shaped cells are more
frequent in newborns than in adults,
making it difficult to discriminate
between
normal
variants
and
pathological changes in the newborn
period.[3,11]
Anemia can result from one or
more of the following processes:
hemorrhage,
hemolysis,
and/or
inefficient production of erythrocytes.
Regardless of the etiology anemia can
be further classified according to 1) the
amount of hemoglobin contained in

RBCs (normochromic, hypochromic);

2) the size of the RBCs (microcytic,
normocytic, macrocytic); and 3) the
pathologic process causing the
anemia (hemolytic, hypoplastic, RBC
membrane defect).[7,12,23]
A combination of the above is
helpful in the approach to the young
infant with anemia.[12] However, until
the transition from the production of
fetal to adult RBCs is completed, the
definitive diagnosis of certain types of
anemia might be delayed. Laboratory
tests in the neonatal period may be
inconclusive
following
blood
transfusions required to treat anemic
patients. Also, abnormalities in RBCs
might escape detection because
defective
cells
are
frequently
destroyed during hemolytic episodes
occurring early in life. Often a definitive
diagnosis is confirmed only after
repeat testing of the patient later in
infancy since an older infant would
have increased numbers of adult
erythrocytes. Performing blood tests
on the parents might facilitate
diagnosis.[2]
Figure 3 outlines an algorithm,
which illustrates an approach to the
infant with anemia. Table 7 shows how
RBC indices and characteristics are
used to assist in establishing a
diagnosis.
Laboratory evaluation of the
anemic patient involves more than
consideration of hematocrit and
hemoglobin
concentrations.
As
hematologic parameters continue to
change and evolve over the first few
months of life, data must be assessed
in light of developmental differences
between newborns and older infants
and adults. RBC characteristics and
indices are frequently overlooked in

clinical practice but they can provide

assistance in establishing a diagnosis

in anemic patients.