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Developmental Changes in Hemoglobin F

Levels During the First Two Years of Life in
Normal and Heterozygous fJ-Thalassemia
Infants

Anna D. Metaxotou-Mavromati, MD, Helene K. Antonopoulou, MD,

Sophie S. Laskari, MD, Helene K. Tsiarta, BSc, Vasilis A. Ladis, MD,
and Christos A. Kattamis, MD

From the First Department of Pediatrics, Tha/assemia Unit, Athens University,

Athens, Greece

ABSTRACT.To studythe developmental

patternof he has been postulated that the decreasing level of
moglobin F (HbF) during the first two years of life, levels HbF is related to the life span of the red blood cells,
of HbF were estimated in two groups of infants: 117
the relative rates of their generation and destruc
normal infants and 98 heterozygotes for f.@-thalassemia,
all aged between 1 and 24 months. The results may be tion, and the switch in synthesis from HbF (a2-y2)to
summarized as follows: (1) Levels of HbF in $-thalassemia HbA (a2$2) in the red cells. The factors that control
heterozygotes were significantly higher than those of the switching at the molecular or cellular level are
normal infants of the same age (P < .01). (2) A reference as yet unknown. Of all factors studied, only fetal
curve for the decline of HbF in infants with /3-thalassemia
anoxia due to placental insufficiency (small-for
trait was established to facilitate the diagnosis of hetero
zygotes during this period of life. (3) Hemoglobin A2 dates infants) and some specific chromosomal ab
(HbA2) was also higher in /3-thalassemia heterozygotes normalities causing developmental retardation of
than in normal infants of the same age. HbA2 increases the fetus have been reported to have high HbF
with increasing age, reaching normal adult values at age levels.35
5 to 6 months. It is postulated that the higher level of
High levels of HbF in adults are known to be
HbF in heterozygous infants during the first two years of
life is associated with the presence of the /3-thalassemia associated with several genetic disorders of globin
gene, which influences the increased synthesis of HbF in chain synthesis, particularly hereditary persistence
red cells. Pediatrics 69:734738,1982; hemoglobin F, of fetal hemoglobin (HPFH), 6fl(high F) thalasse
hemoglobin A2, heterozygous f3-thalassemia infants. mias, homozygous /3-thalassemia, sickle cell//3-thal
assemia, sickle cell anemia, and several combina
tions of thalassemias to other abnormal hemoglo
bins.@9
In the perinatal period, the synthesis of hemoglo Information on the developmental changes of
bin switches from fetal (hemoglobin F [HbF]) to HbF levels in the above conditions during the neo
that of adult (hemoglobin A [HbA]). Studies of natal period is limited.'In particular, the possible
normal fetuses and neonates have shown that the influence of heterozygous /3-thalassemia on the de
sudden decrease in hemoglobin F and the relative velopmental changes of HbF in the neonatal period
increase in hemoglobin A is represented by a sig and early infancy has not been examined. The lack
moid curve that starts between the 32nd to 36th of such data and the existing difficulties in estab
week of gestation (HbF-.-90%) and is almost com lishing the diagnosis of$-thalassemia heterozygotes
plete by the third month of life (HbF.-10%).2 It at this age led us to study the levels of HbF in
heterozygous /3-thalassemia and compare them
with the levels of HbF in normal infants. The main
Received for publication May 18, 1981; accepted July 17, 1981. objectives were to ascertain whether a difference
Reprint requests to (C.K.) First Department of Pediatrics, St.
Sophie's Children's Hospital, Athens 608, Greece.
exists in the levels of HbF between the two groups,
PEDIATRICS (ISSN 0031 4005). Copyright 1982 by the and if so what degree of difference, and for how
American Academy of Pediatrics. long is the difference maintained.

734 PEDIATRICS Vol. 69 No. 6 June 1982

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 MATERIALS AND METHODS red cell morphology and screening of osmotic fra
gility in 0.36% NaCl, search for inclusion bodies,
Two groups of infants aged 1 to 24 months were
hemoglobin electrophoresis (on starch gel and cel
examined. The first group comprised 117 normal
lulose acetate), and quantitation of HbF and HbA2.
infants, who were characterized as normal either
Routine laboratory methods were used for the
because they were the offspring of normal parents
above determinations. Levels of HbA2 were meas
(95 infants) or because they had normal results on
ured by elution following cellulose acetate electro
hematologic screening and normal levels of HbA2
phoresis. Fetal hemoglobin levels were determined
after the sixth month (22 infants).
by alkali denaturation.'2 The reproducibility of the
The second group comprised 98 infants with /3-
method at levels <20% in the same sample is
thalassemia trait with high levels of HbA2. One or
0.2%. Normal values for these estimations in our
both parents had /3-thalassemia trait; namely, the
laboratory have been published previously.
classic variant characterized by increased HbA2 and
normal or slightly increased HbF. The final diag
nosis in infants less than 6 months of age was RESULTS
established after reestimation of HbA2 levels at an The levels of HbF in the group of normal infants
older age (>6 months). are shown in Fig 1 and are similar to the data in the
Hematologic screening of the two groups and literature.'4 HbF levels in 98 heterozygous /3-thal
their parents consisted of: hemoglobin and hema assemia infants,aged 1 to 24 months compared with
tocrit estimation, red cell and reticulocyte counts, the range of IThF in normal infants is shown in Fig
2. After the third month of age the results were
grouped into two-month and three-month intervals,
until the 12th month, and into four-month intervals,
until the second year. Levels of HbF in heterozy
@
@

._E
: d m mw@fot @o
rm 0 e@,e Is ( t@e@ns, @en.gi@)

gotes were, in general, higher than in normal infants

of the same age. In the first three months of life the
levels of HbF in heterozygotes were found to be
within the normal limits but, after the fourth
month, the majority of heterozygotes had signif
icantly higher levels of HbF. These differences were
generally maintained until the age of 24 months.
For example, in the 5- to 6-months age group, 11 of
5 6 7 8 9 O@ 11 12
AGE IN MONTHS 14 heterozygotes had higher than normal HbF
Fig 1. Hemoglobin F (HbF) levels in 117normal infants levels whereas in the 16- to 20-months age group 12
and pattern of decline of HbF levels during first two years of 15 heterozygotes had higher than normal HbF
oflife. levels.
P<.2 .02 .001 .001 .001 .001 .002 .001 .005
N. het.rozygotes
000 2 11 9 9 9 12 7 with high HbF
1 3 3 2 14 12 14
.@o T@ @
N.studled

@ Normal range

a? r@-i
Individual
levels
heterozygotes p-thaI

1 2 3 4 5 6 7 8 9 10 11 12 13 14

AGE IN MONTHS

Fig 2. Hemoglobin F (HbF) levels in 98 heterozygous fl-thalassemia infants and relation

to pattern of decline of HbF of normal infants.

ARTICLES 735
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 The significant differences in the levels of HbF whom the levels of HbF decreased within the stand
between heterozygotes and normal infants is clearly ard deviation of the mean for the respective age,
indicated in Fig 3, which presents the mean and SD but in infants 2 and 3 the decline in HbF was further
for each age group. delayed, whereas in infant 4, the level of HbF was
The developmental changes in the level of HbF high (15%) at 22 months. One of this child's parents
in 14 heterozygotes was determined by serial esti was normal and the other was a /3-thalassemia
mations. In most of the infants the decrease in HbF heterozygote with increased HbA2 (4.5%) and HbF
followed the same pattern as infant 1, (Fig 3), in of 2.7%.
Considering the absolute values of HbF instead
of the percent levels, similar differences were found
in the developmental changes of HbF between nor
mal and heterozygous /3-thalassemia infants (Fig 4).
a..- heterozygotes @@(hc@i
Differences in the changes of HbA2 in the two
.-.-e.
ser oi determ,noto@s groups of infants are shown in the Table. Hetero
rn @-thOI fleterozygotes
zygous infants had considerably higher HbA, than
normal infants of the same age. HbA2 increases
gradually, until the age of 5 to 6 months when it
reaches the adult level. It is of interest that infants
with /3-thalassemia had higher levels even at the
1 2 3 4 56 7891211 213i4151517et92021222324 53
age of 3 months.
AGE IN MONTHS

Fig3. Pattern of declineof hemoglobinF (HbF) in

normal and heterozygous /3-thalassemia infants during DISCUSSION
first two years of life, and means SD of HbF in relation
to agegroups. Serial determinations and individual values The changes in the levels of HbF following birth
for infants 1 to 4. in normal infants have been studied previously.'4'6
Our data in the group of normal infants are
similar to those of the literature, confirming the
sharp decline in levels of HbF during the first
months of life, reaching approximately 10% at 4 to
5 months and the subsequent gradual decline until
. heterozygotes @thoI.
the age of approximately 1 year when HbF is ap
proximately 1%, the level found in adults.
In cord blood, a wide range of HbF (55% to 90%)
Iz. was reported.' In this study a wide variability in the
I level of HbF was also observed during the first three
months of life. This could be due to the fact that
the alkali denaturation procedure is not very accu
rate when measuring such high levels of HbF and
A G E I N M 0 N T H S usually gives lower values when compared with
Fig4. Absolutevaluesof hemoglobin
F (HbF) in nor values attained by chromatography.'7
mal and heterozygous /3-thalassemia infants, means The influence of genetic disorders of globin chain
SD, in relationto agegroups. synthesis on the levels of HbF during this period of

TABLE. Hemoglobin
InfantsAge A2Levels(%)in andHeterozygous /3-Thalassemia
During First Two Years of LifeNormal
(mo) NormalHeterozygotesn

SDRange1 Mean SDRangenMean

5 0.8 0.4
2 9 1.3 0.5 0.41.9 1.8 0.3 1.62.1
3 8 2.20.6 1.03.0 3 3.8 0.9 3.35.0
4 3 2.4 0.4 2.02.8 2 4.0 4.04.0
56 15 2.5 0.3 2.13.1 14 5.2 0.7 4.26.0
79 22 2.7 0.4 1.93.5 12 5.3 0.7 3.86.3
1012 14 2.7 0.4 2.03.3 14 5.0 0.6 3.95.8
1316 13 2.6 0.5 1.63.3 20 5.4 0.8 3.86.7
1720 13 2.90.4 2.13.6 15 5.5 0.6 4.46.3
2124 15 2.80.40.41.3
2.03.63 14... 5.6 0.9... 3.86.7

736 HEMOGLOBIN F LEVELS

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 life is not well defined. The conditions of HPFH synthesis of -y-chains and a simultaneous retarded
and 8$-thalassemia clearly influence the synthesis increase in the synthesis of /3-chains. A second
of -y-chains, maintaining high levels of HbF.6'9 High possibility is that there is selective survival of /3-
levels of HbF have also been found in infants and thalassemia erythrocytes that contain fetal hemo
children with homozygous sickle cell anemia.8 It globin. This mechanism seems to operate in homo
has been postulated that in these conditions age zygous /3-thalassemia, in which red cells with a
contributes significantly in determining the levels higher HbF content have a longer life span because
of HbF in most of the patients, although other of a more balanced chain synthesis.@Also in homo
factors such as hemolysis and clinical heterogeneity zygous sickle cell anemia, the increased HbF con
may also be involved.'0 tent of the red cells protects them from sickling and
Previously, there was no information on the pos hemolysis.8'21'22 In heterozygous fI-thalassemia, he
sible effect of /3-thalassemia trait on the levels of molysis, if it exists, is mild and its participation in
HbF during the first years of life. From our detailed maintaining elevated levels of HbF in the first two
hematologic study of 98 infants with heterozygous years of life is questionable. On the other hand, in
/3-thalassemia, we have found that their levels of the first three months of life the levels of HbF in
HbF were often significantly higher than those ob the heterozygotes were within the upper range of
served in 117 normal infants. normal. Immediately after birth, erythropoiesis de
It is of interest that after the fourth month of life creases significantly while red cell destruction con
the standard deviation and range of HbF in heter tinues, resulting in a rapid decline in hemoglobin
ozygotes is much higher than that in normal infants levels in the neonate. The production of new red
(Figs 2 and 3). The upper level of HbF may be as cells starts to increase between the first and second
high as 27% in the second trimester and 15% in the month of life whereas red cell destruction continues
second year of life. In these cases, it is difficult to for two months. After the third month the eryth
exclude certain mild forms of thalassemia interme rocyte mass consists entirely of new postnatally
dia characterized by a rather low HbF (15% to 40%), produced red cells.3'23 Thus it could be argued that
such as double heterozygotes for /3-thalassemia the observed differences in the levels of HbF, start
(high A2) and /3-thalassemia silent I (normal A2 and ing after the first three months of life, are not
@ F) and homozygous thalassemia, unless family genetically determined but are a consequence of
studies are undertaken.'89 other factors, eg, a selective survival advantage of
The graphic presentation of the decline in HbF erythrocytes with a higher HbF content. However,
in /3-thalassemia heterozygotes during the first two it is more likely that the presence of the genes for
years of life may be used as a reference curve (Fig /3-thalassemia continuously influences the produc
3) to help in the diagnosis of thalassemia trait at tion of higher levels of HbF; the linear but slower
this period of life. decline in the levels of HbF is consistent with this
The difference in levels of HbF remains relatively hypothesis. In support of this contention are the
constant after the first six months until approxi considerably higher levels of HbF found in certain
mately the 24th month of life. There is evidence types of /3 and &fJ-thalassemia heterozygotes, such
that in heterozygotes the fetal hemoglobin remains as /3
Dutch (high A2 high F), and &/3(high F), which
at elevated levels for much longer than the second are characterized by high HbF (>5%) in adult life.
year of life. Heterozygous children for these two genotypes
Serial determinations of the levels of HbF in 14 maintain much higher HbF levels than the classic
heterozygotes showed that in some cases the rate high HbA2 type of /3-thalassemia, and the level of
of decline of HbF may be considerably slower (Fig HbF is greatly influenced by age (unpublished ob
3, infants 2, 3, and 4). It is possible that in some of servations).
these infants high levels of HbF are maintained
until adult life. This is substantiated by the slightly
elevated levels of HbF (2% to 5%), which are found ACKNOWLEDGMENTS
in a proportion of adult heterozygotes for the classic This investigation was supported by grants from the
/3-thalassemia trait (increased HbA2). The high National Research Institute and the Ministry of Social
levels found in infant 4 may thus be explained by Services.

the fact that one of the parents had a slight increase

ofHbF (2.7%).
REFERENCES
Several assumptions may be postulated to ex
plain the higher HbF levels in /3-thalassemia het 1. Huehns ER, Shooter EM: Human hemoglobins. J Med
Genet 2:48, 1965
erozygotes. According to one, the slower decline of 2. Wood WG: Haemoglobin synthesis during fetal develop
HbF in heterozygotes is possibly due to prolonged ment. Br Med Bull 32:282, 1976

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 3. Oski FA, Naiman JL: Hematologic Problems in the New 14. Huehns ER, Beaven GH: Development changes in human
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147 ders Co, 1974, pp 5674

A HIGHER DUTY

We are but transient passengers on this planet Earth. It does not belong to
us. We are not free to doom generations yet unborn. We are not at liberty to
erase humanity's past or dim its future. Social systems do not endure for an
eternity. Only life can lay claim to uninterrupted continuity. This continuity is
sacred. We physicians, who shepherd human life from birth to death, are aware
of the resiliency, courage, and creativeness that human beings possess. We have
an abiding faith in the concept that humanity can control what humanity
creates. This perception provides optimistic purpose in reversing the direction
of humankind's potential tragic destiny.
Submitted by John T. McCarthy, MD

From Lown B: JAMA 246:2333, 1981.

738 HEMOGLOBIN F LEVELS

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Developmental Changes in Hemoglobin F Levels During the First Two Years of Life in
Normal and Heterozygous -Thalassemia Infants
Anna D. Metaxotou-Mavromati, Helene K. Antonopoulou, Sophie S. Laskari, Helene K.
Tsiarta, Vasilis A. Ladis and Christos A. Kattamis
Pediatrics 1982;69;734
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PEDIATRICS is the official journal of the American Academy of Pediatrics. A monthly publication, it
has been published continuously since 1948. PEDIATRICS is owned, published, and trademarked by the
American Academy of Pediatrics, 141 Northwest Point Boulevard, Elk Grove Village, Illinois, 60007.
Copyright 1982 by the American Academy of Pediatrics. All rights reserved. Print ISSN: 0031-4005.
Online ISSN: 1098-4275.

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Developmental Changes in Hemoglobin F Levels During the First Two Years of Life in
Normal and Heterozygous -Thalassemia Infants
Anna D. Metaxotou-Mavromati, Helene K. Antonopoulou, Sophie S. Laskari, Helene K.
Tsiarta, Vasilis A. Ladis and Christos A. Kattamis
Pediatrics 1982;69;734

The online version of this article, along with updated information and services, is located on
the World Wide Web at:
/content/69/6/734

PEDIATRICS is the official journal of the American Academy of Pediatrics. A monthly publication,
it has been published continuously since 1948. PEDIATRICS is owned, published, and trademarked
by the American Academy of Pediatrics, 141 Northwest Point Boulevard, Elk Grove Village,
Illinois, 60007. Copyright 1982 by the American Academy of Pediatrics. All rights reserved. Print
ISSN: 0031-4005. Online ISSN: 1098-4275.

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