From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
Sex, Gestational Age, and Weight Dependancy of Adult Hemoglobin
Concentration in Normal Newborns
By F. Galacteros, M. Guilloud-Bataille, and J. Feingold
We have measured by cation exchange high pressure liquid
chromatography adult hemoglobin (HbA) concentration at
birth in 6,123 unselected single newborn individuals. Probably because of the high precision of the analytical method
used, we could demonstrate clear a relationship between
HbA concentration and, respectively, gestational age and
birth weight. We also demonstrated a significant difference
in the Hb switching process between male and female
newborns. Reference percentile distribution curves are given
that could be used to define more precisely those children
having slow or fast Hb switching.
o 1991 by The American Society of Hematology.
T
Laboratory investigations included thin-layer isoelectric focusing,z'
citrate agar electrophoresisz2when an abnormality was detected by
isoelectric focusing or cation exchange HPLC. This latter method
was applied according to referenced method^.^.'^ The equipment
used was a VARIAN 5000 liquid chromatograph. The column was
a Brownlee 3CM cartridge (Acquapore CX-300, 0 4.6 mm X 30
mm; Brownlee Laboratories, Inc, Santa Clara, CA). The gradient
used was similar to that referenced with a slight modification
permitting Hb Bart's detection and quantitation. We have not
included in this study results obtained when an abnormal Hb
fraction was detected either by isoelectric focusing or by HPLC.
The only excluded infants either carried an abnormal Hb (4%) or
had more than 0.5% Hb Bart's (5%). This policy was applied
because we had no information about the possible influence of
these traits on adult Hb A synthesis, gestational age, or birth
weight. Ethnic distribution in the studied cohort was 60% white
Caucasians, 15% from North Africa, 13% from French West
Indies, 9% from different regions of Sub-Saharian Africa, and 3%
from Asia. According to reported data on P-thalassemia gene
frequencies in these ethnic subgroups, the overall incidence of this
trait must be lower than 1%.
All HbA values were expressed as the mean ? standard
deviation. Comparisons of HbA means between males and females
were made using the Student's t-test. A P value below .05 was
considered significant. In gestational age (in weeks) groups and in
birth weight groups, the percentiles were computed. The correlations between either gestational age or birth weight and HbA
concentration were also computed.
HE ADULT HEMOGLOBIN (HbA) to fetal hemoglobin (HbF) switching process during the perinatal
period had been the subject of many in vivo and in vitro
studies. This topic has been deeply investigated, and up to
date reviews are regularly published.'
Numerous reports have been focused on the relative
rates of HbF and HbA synthesis during the first 6 months of
life. However, available data on physiologic HbA concentration range in normal newborns are contained in a small
number of r e p o r t ~ .These
~ . ~ studies did not give satisfactory
reference data that would allow clear identification of the
small subgroup of newborns having HbA concentration out
of the 2 2 SD limits of normal range. These methodologic
inaccuracies came mainly from imprecise laboratory methods, uncertainties in regard to gestional age, which in many
case was not taken into account, and insufficiently population-studied newborn groups.
Many pathologic circumstances have been shown to
modify Hb phenotype (mostly by slowing the switching
process) in this period of life. Among them are maternal
anoxia,' placental insufficien4 prematurity,' intrauterine
growth retardation: some chromosomal abn~rmalities,'~''~
hyperinsulinemia secondary to maternal diabetes mellitus,1s~'6
and the sudden infant death syndrome," although
with some controversy.'8 @-thalassemiaand other related
genetic conditions at the heterozygous state were reported
to decrease HbA concentration at birth.'920
During an experimental systematic survey for Hb abnormalities, we have measured HbA concentration by cation
exchange high pressure liquid chromatography (HPLC) in a
large group of unselected newborns. A clear relationship
between HbA concentration, birth weight, and gestational
age was found and normal ranges between the 5% and 95%
percentiles were defined. Furthermore, significant differences between male and female newborn mean HbA
concentration were demonstrated.
RESULTS
Hb studies were performed on 6,123 blood samples from
unselected single newborns without detectable Hb abnormality. The studied group was composed of 3,168 males and
2,955 females; the sex ratio was 51.74%. Their respective
mean gestational ages were 39.07 5 1.67 and 39.06 2 1.70
weeks of amenorrhea. Corresponding mean birth weights
were 3,306.2 2 521.4 g and 3,166.6 2 505.1 g. These data
were very close to corresponding, published reference
~~~~~~~~
MATERIALS AND METHODS
An experimental testing program that started in 1988 was
designed to define appropriate methods for providing satisfactory
newborn screening for hemoglobinopathies in the Val de Marne
district. After the mother gave informed consent, 100 pL of EDTA
(K,)-anticoagulated capillary blood from the newborns was obtained, and at the same time the filter paper-dried blood specimen
for PKU and hypothyroidism testing was sampled. Each newborn
was identified by a sequential number. Sex, weight, and gestational
age (measured in weeks of amenorrhea) were recorded by maternity staffs for all tested babies. The prematurity rates (gestational
age 1 3 7 weeks) were 13.9% in males and 13.5% in females.
Samples stayed no more than 3 days at 4°C before analysis.
B/uuc/, VOI 78, N04(AUgust 15). 1991: pp 1121-1124
From the INSERM U91 and CNRS UA 607, Henri Mondor
Hospital, Criteil; and the INSERM U 155, Chriteau de Longchamp,
Paris, France.
SubmittedJuly 19,1990; acceptedApril 16, 1991.
Supported by grants from Conseil General du Val de Mame,
CNAMTS, AFDPHE, and CNAM-INSERM.
Address reprint requests to Fridiric Galacteros, MD, Laboratoire de
Biochimie, H6pital Henri Mondor, 94000 Crgteil, France.
The publication costs of this article were defiayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 I991 by The American Society of Hematology.
0006-4971l91l7804-0025$3. OOlO
1121
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
1122
GALACTEROS, GUILLOUD-BATAILLE, AND FEINGOLD
% HbA
% HbA
35 T
A
40 1
1
3
A
*5
-
0 1 0
-
t
I
~
33
34
% HW
T
45
A
40
I
\
=!
20
35
1
33
34
35
37
36
38
39
40
41
37
38
39
G e ~ l a l i ~ n aAge
l
20
15
(U]
10
42
5
B
Weight (e)
0
1500
-
2000
2500
3000
3500
4500
4000
B
35
30
25
-*-25
20
15
Gestational Age(U)
-
36
25
-* 2 5
40
10
42
41
5
1
Fig 1. Relationship and percentile distribution curves of HbA
concentrationvalues accordingto gestational age (in week of amenorrhea) in male (A) and female (B) newborns.
values obtained for newborns in France?' The classical
relationship between sex, birth weight, and gestational age,
observed in French newborns,2' was found again in the
studied group of newborns.
HbA concentrations were obtained by cation exchange
HPLC. The relationship between HbA concentration and
gestational age was studied in newborns having 33 to 42
weeks of intrauterine life. However, infants under 36 and
over 41 weeks of gestational age were insufficiently numerous to permit accurate statistical calculations. Figure 1A
(males) and B (females) illustrates the relationship between those two parameters, as well as the percentile
distribution curves. The correlation coefficients --rere,respectively, 0.39 for males (P < .0001) and 0.36 I- females
(P < .0001). However, the HbA concentration cannot be
used to define gestational age, because the part of the
variance explained by the HbA concentration is small. The
2000
1500
2500
3000
3500
4000
Weight (g)
4500
Fig 2. Relationship and percentile distribution curves of HbA
concentration values according to birth weight (in grams) in male (A)
and female (E)newborns.
corresponding percentile data for HbA concentration are
given in Table 1.A significant difference between males and
females was evidenced. Mean HbA concentrations were,
respectively, 18.0% 2 6.72% in males and 19.2% & 7.2% in
females. The sexual difference in HbA concentration was
significantly present from 37 to 41 weeks of amenorrhea.
By the same time, the relationship between HbA concentration and birth weight according to sex was studied.
Results are given in a similar way in Fig 2A and B and Table
2. The correlation coefficients were, respectively, 0.30 for
males (P < .0001) and 0.27 for females (P < .0001). A
clear relationship was observed between birth weight and
HbA concentration, and at birth weight ranging from 2,000
to 4,500 g, the HbA concentration differed significantly
between the sexual groups. Although the male newborns
were heavier, they had synthetized less HbA than females
Table 1. HbA Concentrations in Male and Female Newborns
According to GestationalAge
Males
Gestational
nun \
5 33
34-35
36
37
38
39
40
41
2 42
Total
31
54
99
256
517
807
782
509
70
3,125
Table 2. HbA Concentration in Male and Female Newborns
According to Birth Weight
Females
10,
11.2 2 10.0
11.7 f 6.1
12.5 4.1
14.1 f 6.0
15.6 f 5.4
17.8 f 5.9
19.72 6.4
21.5 2 6.5
22.5 2 8.1
18.0 ? 6.7
*
""" \
*
10,
33 14.1 12.8
59 11.5 f 4.3
87 13.4f 4.1
232 15.2 2 5.1
454 16.6 2 5.0
776 18.9 f 6.8
749 21 .O 6.8
466 22.6& 7.1
60 22.9 7.9
2,916 19.22 7.2
*
NS
NS
NS
~~~
NB
HbA (%)
Females
NB
HbA ( O h )
*
<.01
NS
< .0001
P
~
7.1 f 1.3
14 13.2 f 11.3
9
s 1,500
44 13.1 2 10.4
36 12.2 f 8.2
1,501t o 2,000
206 14.9 6.5
139 12.6& 5.2
2,001 to 2,500
776 17.8 f 6.2
635 15.82 6.4
2,501 to 3,000
3,001t o 3,500 1,289 18.2 f 6.5 1,204 19.9 -t 6.8
590 20.9 -t 7.2
803 20.0 f 6.6
3,501t o 4,000
200 19.7 f 6.3
94 22.6 f 8.6
4,001t o 4,500
45 21.7 2 7.0
20 20.9 f 7.2
t 4,500
< .05
< .01
< ,001
< ,0001
~~
Abbreviations: NB, numbers; NS, not significant.
Males
Birth
Weight (9)
~
Abbreviations: NB. numbers; NS, not significant.
NS
NS
< .0001
< .0001
< ,0001
< .01
<.01
NS
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
HE SWITCHING IN MALE AND FEMALE NEWBORNS
having identical mean gestational age. The partial correlations between HbA concentration and gestational age, birth
weight being the fixed variable, are 0.29 in males and 0.27 in
females (P < .0001). On the contrary, the partial correlations between HbA concentration and birth weight, gestational age being the fixed variable, are 0.12 in males and
0.10 in females (P < .0001). The comparison of these
values indicates that the important correlation concerns
gestational age and HbA concentration.
DISCUSSION
This work was initiated during the course of an experimental program for hemoglobinopathy detection at birth. Because it had been reported that HbA concentration is
significantly lower when measured in P-thalassemia carrier
n e w b o m ~ , our
' ~ ~ aim
~ ~ was to define the fifth percentile
threshold of HbA concentration allowing to recognize the
subgroup of newborns at risk for having P-thalassemia or
hereditary persistance of HbF synthesis traits.
In fact, it became evident that it was necessary to take
into account gestational age, birth weight, and sex simultaneously to provide correct interpretation of HbA concentration at birth.
Although electrophoretical analysis of newborn Hb is the
method of choice in most of the clinical laboratories, HPLC
is a reliable and reproductible alternative procedure that
can be used as a mass screening assay. It also allows for the
precise quantitation of Hb fractions, which was probably
necessary to obtain the present results. Differences were
more significant when HbA concentration was matched to
birth weight and sex probably because newborn weight
measure is more precise than gestational age determination.
Results were not demonstrative for extreme gestational
age and birth weight groups probably because they were not
sufficiently populated, but it could also result from pathologic or therapeutic interferences (therapeutic transfusion,
maternal to fetal transfusion, maternal diabetes). In fact, in
the most premature infants, transfusion therapy was often
used very soon after birth. This therapy produced a paradox-
1123
ical elevation of the 95th percentile value in these subgroups of newborns (Figs 1A and B and 2B).
The results reported here could be used as reference
values to define slow and fast Hb switching in newborn
individuals. These results will be useful in properly characterizing or further studying factors that may slow the Hb
switching process like maternal diabetes, maternal chronic
hypoxemia, or intrauterine growth disturbances. They may
also help to define the subgroup of babies with an accelerated Hb switch that has been reported to occur, for
instance, in some chromosomal abn~rmalities'~~'~
and in
y-thalassemia traits.26
Hb studies have been proposed to precisely determine
fetal maturity and correct estimation of gestational
Previously described methods were difficult to use routinely. The present report shows that HbA concentration
reference values could be useful in that way, all the more so
because it could appear as a routine secondary result of
newborn screening for hemoglobinopathies by HPLC procedure.
We were able to find only one previous report that
mentioned a sexual difference in Hb switching in the
neonatal period.z9The authors comparing HbF concentration between 47 male and 74 female newborns with no Hb
abnormality found significantly less HbF (67.6%) in females than in males (72.6%). The present results suggest
that a sex-associated factor affects Hb switching. This
observation could be brought together with some recent
reports. One report described X-linked dominant control
of F cells in normal adult life3' aad the second located the
major human erythroid-specific DNA-binding protein (GF,)
on the X chromo~ome.~'
It is tempting to speculate on a
relationship between these sets of results after authors have
suggested that GF, is implicated in human Hb
ACKNOWLEDGMENT
The authors greatly acknowledge the maternity staffs that
participated in the study, the technical staff of our laboratory, and
G. Dubus for typing the manuscript.
REFERENCES
1. Huens ER, Beaven GH: Developmental changes in human
M, Beuzard Y: Hematological values of 163 normal fetuses
haemoglobins, in Benson P F (ed): The Biochemistry of Developbetween 18 and 30 weeks of gestation. Pediatr Res 20:342,1986
ment. Portsmouth, NH,Heineman, 1971, p 175
7. Bromberg YM, Abrahamov A, Salzberger M: The effect of
2. Wood WG, Clegg JB, Weatherall DJ: Developmental biology
maternal anoxaemia on the foetal haemoglobin of the newborn. J
of human haemoglobins, in Brown EB (ed): Progress in HaematolObstet Gynaecol Br Commonw 63:875,1956
ogy (~0110).Philadelphia, PA, Grune & Stratton, 1977, p 43
8. Bard H: The effect of placental insufficiency on fetal and
3. Stamatoyannopoulos G, Nienhuis A W Hemoglobin switchadult hemoglobin synthesis. Am J Obstet Gynecol 120:67,1974
ing, in Stamatoyannopoulos G, Nienhuis AW, Leder Ph, Majerus
9. Bard H, Makowski EL, Meschia G, Battaglia F The relative
PhW (eds): The Molecular Basis of Blood Diseases. Philadelphia,
rates of synthesis of hemoglobin A and F in immature red cells of
PA, Saunders, 1987, p 66
the newborn infants. Pediatrics 45:766,1970
4. Bard H: The postnatal decline of hemoglobin F synthesis in
10. Huens ER, Hecht F, Keil JV,Motulsky AG: Developmental
normal full term infants. J Clin Invest 55395,1975
hemoglobin anomalies in a chromosomal triplications: D, trisomy
5. Colombo B, Kim B, Perez Atencio R, Molina C, Terrenato L
syndrome. Proc Natl Acad Sci USA51:89,1964
The pattern of fetal haemoglobin disappearance after birth. Br J
11. Wilson MG, Schroedder WA, Graves DA, Kach VD:HemoHaematol32:79,1976
globin variations in D trisomy syndrome. N Engl J Med 277:953,
6. Forestier F, Daffos F, Galacteros F, Bardakdjian J, Rainaut
1967
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
1124
12. Bard H: Postnatal fetal and adult hemoglobin synthesis in D,
trisomy syndrome. Blood 40523,1972
13. Wilson MG, Schroedder WA, Graves D A Postnatal change
of hemoglobins F and A* in infants with Down’s syndrome (G
trisomy). Pediatrics 42:349, 1968
14. Weller SDV, Apley J, Raper AB: Malformations associated
with precocious synthesis of adult hemoglobin. Lancet 1:777, 1966
15. Perrine SP, Greene MF, Faller DV: Delay in the fetal globin
switch in infants of diabetic mothers. N Engl J Med 312234,1985
16. Bard H, Prosmanne J: Relative rates of fetal hemoglobin
and adult hemoglobin synthesis in cord blood of infants of
insulin-dependent diabetic mothers. Pediatrics 75:1143, 1985
17. Giulian GG, Gilbert EF, Moss R L Elevated fetal hemoglobin levels in sudden enfant death syndrome. N Engl J Med
316:1122,1987
18. Zielke HR, Meny RG, O’Brien MJ, Smialek JE, Kutlar F,
Huisman THJ,Dover GJ: Normal fetal hemoglobin levels in the
sudden infant death syndrome. N Engl J Med 321:1359,1989
19. Galanello R, Melis MA, Rugerri R, Cao A Prospective
study or red blood cell indices, hemoglobin A, and hemoglobin F in
infants heterozygous for p thalassemia. J Pediatr 98:105, 1981
20. Cosso G, Manca M, Pirastru MG, Bulitta R, Bosisio AB,
Ganazza E, Righetti PG: Neonatal screening for beta thalassemia
by thin layer isoelectric focusing. Am J Hematol13:149,1982
21. Galacteros F, Kleman K, Caburi-Martin J, Beuzard Y, Rosa
J, Lubin B: Cord blood screening for hemoglobin abnormalities by
thin layer isoelectric focusing. Blood 56:1068,1980
22. International Committee for Standardization in Haematology: Recommandations for neonatal screening for haemoglobinopathies. Clin Lab Haematol10:335,1988
23. Huisman THJ,Jonxis JHP (eds): The Hemoglobinopathies;
Techniques of Identification. Clinical and Biochemical Analysis
(vol6). New York, NY,Marcel Dekker, 1977
24. Wilson JB, Wrightstone RN, Huisman THJ: Rapid cationexchange high-performance liquid chromatographic procedure for
the separation and quantitation of hemoglobins S, C and 0 Arab in
cord blood samples. J Lab Clin Med 108:138,1986
GALACTEROS, GUILLOUD-BATAILLE,AND FEINGOLD
25. Cornuau P, Breart G: Caractiristiques de I’enfant a la
naissance. Naitre en France, 10 ans d’kvolution, in RumeauRouquette C, Du Mazaubrun C, Rabarison Y (eds): INSERM
Editions. Doin, Paris, France, 1984
26. Huisman THJ,Reese MB, Gardinier MB, Wilson JB, Lam
H, Reynolds A, Nagle S, Trowell P, Yi-Taoz, Shu-Zheng H,
Sukumaran PK, Miwa S, Efremov GD, Petkov G, Sciaratta GV,
Sansone G: The occurence of different levels of Gy chain and of
the AyT variant of fetal hemoglobin in newborn babies from
several countries. Am J Hematol 14:133,1983
27. Peterson CM, Kalan G, Jovanovic L, Jovanovic R: Use of the
minor hemoglobin ratio for the determination of gestational age.
Am J Hematol 135:85,1979
28. Felicetti L, Noveletto A, Benincasa A, Terrenato L, Colombo B: The HbA/HbA, ratio in newborns and its correlation with
fetal maturity. Br J Haematol56:465, 1984
29. Mason KP, Grandison Y, Hayes RJ, Serjeant BE, Serjeant
GR, Vaidya S, Wood WG: Post-natal decline of fetal haemoglobin
in homozygous sickle cell disease: Relationship to parental HbF
levels. Br J Haematol52455,1982
30. Miyoshi K, Kaneto Y, Kawai H, Ohcni H, Niki S, Hasegawa
K, Shirakami A, Yamano T X linked dominant control of F cells in
normal adult life: Characterization of the Swiss type as hereditary
persistence of fetal hemoglobin regulated dominantly by gene(s)
on X chromosome. Blood 72:1854,1988
31. Zon LI, Shih-Feng T, Burgess S, Matsudaira P, Bruns GAP,
Orkins SH: The major human erythroid DNA-binding protein
(G-F,): Primary sequence and localization of the gene to the X
chromosome. Proc Natl Acad Sci USA 87:668,1990
32. Superti-Furga G, Barberis A, Schaffner G, Busslinger M:
The -117 mutation in Greek HPFH affects the binding of three
nuclear factors to the CCAAT region of the y globin gene. EMBO
J 7:3099, 1988
33. Tsai SF, Martin DIK, Zon LI, D’Andrea AD, Wong GG,
Orkin SH: Cloning of cDNA for the major DNA-binding protein of
the erythroid lineage through expression in mammalian cells.
Nature 339:446,1989
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
1991 78: 1121-1124
Sex, gestational age, and weight dependancy of adult hemoglobin
concentration in normal newborns
F Galacteros, M Guilloud-Bataille and J Feingold
Updated information and services can be found at:
http://www.bloodjournal.org/content/78/4/1121.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.