The Effect of Blood Transfusion on the

Hemoglobin Oxygen Dissociation Curve of

Very Early Preterm Infants During the First
Week of Life
Virginie De HaUeux, Anita Truttmann, Carmen Gagnon, and Harry Bard

A study was c o n d u c t e d during the first w e e k o f life to determine the changes in Ps0 (PO2 required to
achieve a saturation o f 50% at p H 7.4 and 37~ and the proportions o f fetal h e m o g l o b i n (I-IbF) and
adult h e m o g l o b i n (HbA) prior to and after transfusion in very early preterm infants. Eleven infants
with a gestational age <--27 w e e k s have b e e n included in study. The h e m o g l o b i n dissociation curve and
the Ps0 was determined by Hemox-analyser. Liquid chromatography was also p e r f o r m e d to determine
the proportions o f HbF and HbA. The m e a n gestational age o f the 11 infants was 25.1 weeks (-+1
weeks) and their m e a n birth weight was 736 g (-+125 g). They received 26.9 m L / k g o f packed red
cells. T h e m e a n Ps0 prior and after transfusion was 18.5 +- 0.8 and 21.0 + 1 m m Hg (P = .0003) while
the m e a n percentage o f HbF was 92.9 -+ 1.1 and 42.6 -+ 5.7%, respectively. T h e data o f this study
show a decrease o f h e m o g l o b i n oxygen affinity as a result o f b l o o d transfusion in very early preterm
infants p r o n e to O 2 toxicity. T h e shift in H b O 2 curve after transfusion should be taken into
consideration w h e n oxygen therapy is being regulated for these infants.
Copyright 2002, Elsevier Science (USA). All rights reserved.

here are frequently reported associations
T between early blood transfusions (first
week of life) in p r e t e r m infants and retinopa-
thy 1-3 as well as b r o n c h o p u l m o n a r y dysplasia. 4
This p h e n o m e n a could be explained in part by a
decrease in h e m o g l o b i n oxygen affinity (HbO2)
as the result of a transfusion. T h e decrease in
H b O 2 could expose the tissues of very early pre-
term infants (-<27 weeks of gestation) to in-
creased levels o f oxygen and therefore m o r e
oxidative stress before their i m m a t u r e antioxi-
d a n t defenses 5 can a d a p t to the higher tissue
levels of oxygen.
Very early p r e t e r m infants are b o r n at a time
when m o r e than 90% of their red blood cells
contain fetal h e m o g l o b i n (HbF) 6 and their
blood has a high affinity for o x y g e n . 7 Because o f
frequent blood sampling, early p r e t e r m new-
borns frequently require transfusions for blood
volume r e p l a c e m e n t during their first week o f
life. These transfusions are p e r f o r m e d with adult
blood red cells containing adult h e m o g l o b i n
(HbA) and therefore decrease the H b O 2 affin-
ity. T h e shift in HbO2 affinity could expose the
i m m a t u r e tissues of these infants to higher levels
of oxygen.
A study was conducted during the first week
o f life in very early p r e t e r m infants to d e t e r m i n e
the changes in H b O 2 affinity based on the P~0's
(PO 2 required to achieve a saturation of 50% at
p H 7.4 and 37~ prior to and after a transfu-
sion. Also to be analyzed was the relationship of
Pso a n d the proportions of H b F a n d HbA.
Materials and Methods
Eleven very early p r e t e r m infants of <-27 weeks
o f gestation, free o f any congenital anomalies,
and transfused during the first week of life were
included in the study. Gestational age was deter-
m i n e d by menstrual history, confirmed by pre-
natal ultrasound examination and physical ex-
amination on the day of birth. T h e s e infants
were all intubated at birth, were normotensive,
and considered to having mild to m o d e r a t e re-
From the Division of Neonatotogy, Department of Pediatrics, Uni-
versity of Montreal, St Justine Hospital and Research Center, Que-
bec, Canada.
Supported to, Canadian Institutes of Health Researchgrant # MOP
49464.
Address reprint requests to Harry Bard, MD, Research Center,
Hospital Sainte-Justine, 3175, (~te-Sainte-Catherine, Montreal,
Quebec, H3T 1(5, Canada; e-mail."harry.bard@umontreaLca.
Co]o'right 2002, FLwvierScience (USA). All rights reserved.
O146-0005/ 02/2606-0005 535. 00/0
doi: l O.1053/sper.2002.37313

Seminars in Perinatology, Vol 26, No 6 (December), 2002: pp 411-415 411

412 De Halleux et al

spiratory distress syndrome requiring 30% to
40% 0 2 when sampled.
T h e decision regarding the n e e d of transfu-
sion was based on b l o o d volume r e p l a c e m e n t
and left to the discretion of the attending neo-
natologist. A b l o o d sample o f 0.1 m L was ob-
tained before and after transfusion and was used
for the determination of the p r o p o r t i o n of H b A
a n d H b F and the h e m o g l o b i n oxygen dissocia-
tion curve. T h e p r o p o r t i o n of H b F and H b A
levels before a n d after transfusion were deter-
m i n e d by high-performance liquid chromatog-
raphy (HPLC). 8 T h e red blood cells were
washed a n d lysed and the hemolysate was sub-
j e c t e d to globin chain separation and quantifi-
cation by reverse phase H P L C e q u i p p e d with an
integrator by using a m e t h o d described previ-
ously. 9 This m e t h o d uses a gradient between
aqueous trifluoroacetic acid and trifluoroacetic
acid in acetonitrile and gives an excellent reso-
lution of h u m a n globin chains.
T h e oxygen dissociation curve (ODC) a n d
the P50 were d e t e r m i n e d by the Hemox-Analyser
(TCS Scientific Corp, New H o p e , PA). 1~ Fifty
microliters of whole blood was a d d e d to 4 m L o f
buffer (135 m m o l / L NaC1, 30 m m o l / L TES, 5
m m o l / L KC1, a n d N a O H adjusted to p H 7.4 _+
0.02) (TCS buffer; TCS Scientific Corp.), 10 ~L
of antifoam solution, and 20 /xL of 20% BSA.
Samples were analyzed immediately u p o n collec-
tion f r o m the patient. Nitrogen (100%) was bub-
bled t h r o u g h the sample at a constant rate that
resulted in complete deoxygenation within 20
min, followed by reoxygenation with air for 15
min. T h e analyzer m e a s u r e d the oxygen tension
with a standard Clark Oe electrode (model 5331
Data were expressed as m e a n a n d standard de-
viation. A two-sample t-test was used to c o m p a r e
the data before a n d after transfusion. A correla-
tion between P50 a n d H b F was also carried out. A
P value less than .05 was considered significant.
T h e s e studies were p e r f o r m e d in accordance
with the principles established by the Sainte-
Justine's Hospital Ethics C o m m i t t e e on Clinical
Investigation.
Results
Figure 1 illustrates examples of the O D C ' s ob-
tained by Hemox-Analyser prior to a n d after
transfusion along with an adult control. T h e
m e a n P50 increased from 18.5 prior to 21.0 m m
H g after the transfusion. T h e adult control was
27.1 m m Hg. Table 1 indicates the gestational
age, birth weights, the postnatal age, a n d the
volume of red b l o o d cells transfused. Also in-
cluded are the Ps0'S and the p e r c e n t a g e of H b F
before a n d after transfusion. T h e m e a n volume
of red blood cells transfused (Hct 55%) was 20.1
m L (26.9 m L / k g ) . H e m o g l o b i n levels before
and after transfusion were 108 + 30 and 129 _+
19 g / L , respectively. T h e p e r c e n t a g e of H b F was
92.9 + 1.1 before a n d 42.6 -+ 5.7%, 2.8 +- 1 days
after a transfusion, while the value for P50 in-
creased f r o m 18.5 _+ 0.8 to 21.0 -+ 1.0 m m H g
( P -- .0003). Figure 2 shows the relationship
between the p e r c e n t a g e of H b F and the P50
before a n d after a transfusion. T h e p e r c e n t a g e
100-

Oxygen Probe; Yellow Springs I n s t r u m e n t Co., 90-

80- A C
Yellow Springs, O H ) .
Eleven infants were included in the study be- ~ 70
cause of a transfusion during their first week of =
.2
60-

life. T h e total volume of the transfusion was "~ 50

divided in 2 parts with a 12-h interval between i i i

each portion a n d was considered as a single 3a- 'I ',

i i i
20-
transfusion. T h e patients did not receive any , , ,

10 ~ 'i
b l o o d at all before the first sample. Eight of i
,
L

n . , ,
these infants had their Ps0 d e t e r m i n e d prior to 0 '1'0 2'0 ab ~o ~'0 60 7'0 8b
the transfusion, seven had a P50 d e t e r m i n e d af- POz (mmHg)
ter the transfusion. T h e h e m o g l o b i n type analy-
sis using H P L C was carried out on seven infants Figure 1. An illustration of the oxygen dissociation
curve's obtained by Hemox-Analyser. (A) before, (B)
before the transfusion and seven after the trans- after transfusion in very early preterm infant, and (C)
fusion. Four of these infants had all the analysis in an adult. The Pso before is 18 and after 21 mm Hg.
carried out before and after the transfusion. The normal adult Pso is 27 mm Hg.
 Decrease on 02 Saturation After Transfusion 413

Table 1. The Effects of a Transfusion on P~o, HbA, and HbF of Very Low Birth Weight Infants

Prior to Transfusion After Transfusion

Estimated Pso Transfusion Pso

GA Wt Blood HbF mm (packed cell) HbF mm
wk g Vol. mL % Hg mL % Hg
1 23 645 52 -- -- 15 -- 19.6
2 25 695 56 -- -- 20 36 21.7
3 24 770 62 92.2 18.7 -- -- --
4 27 785 63 -- -- 20 46.1 21.7
5 24 605 48 -- 19 -- -- --
6 26 900 72 92 17.0 22 42.4 19.5
7 24 670 54 92.9 18.0 13 50.8 22.0
8 26 915 73 92.1 19.5 26 43.5 21.5
9 25 535 43 94.6 18.0 25 36.6 21.0
10 25 695 56 94.2 18.6 -- -- --
11 27 875 70 92.1 19 -- -- --
Mean 25.1 736 59 92.9 18.5" 20.1 42.6 21.0"
SD 1.3 125 10 1.1 0.8 4.8 5.7 1.0
* P = .0003

o f H b F was inversely p r o p o r t i o n a l to the Ps0 (r -- g e n t e n s i o n P v O 2 w a s significantly i n c r e a s e d

.8512, P = . 0 0 0 2 ) . with low affinity r e d cells ( f r o m 23 to 33 m m
Hg). I n a r e c e n t r e p o r t it was also s h o w n t h a t
w h e n a n e m i c p r e t e r m i n f a n t s were transfused,
Discussion
Ps0 i n c r e a s e d w i t h o u t any c h a n g e s i n O 2 con-
It has b e e n s h o w n i n previous studies u s i n g new- s u m p t i o n o r in m y o c a r d i a l f u n c t i o n . 14 Since the
b o r n l a m b s t h a t the Ps0 d u r i n g the first day o f levels o f m i x e d v e n o u s P O 2 can b e c o n s i d e r e d as
life is a r o u n d 18 m m H g / 1 w h i c h is similar to the a close a p p r o x i m a t i o n o f tissue PO215 a n in-
early p r e t e r m i n f a n t . 6 I n a d d i t i o n a l e x p e r i m e n - crease in P~O2 in the very early p r e t e r m i n f a n t s
tal studies p e r f o r m e d o n n e w b o r n l a m b s ~z,13 w h e n t r a n s f u s e d w o u l d m e a n that the i m m a t u r e
w h e n h i g h o x y g e n affinity fetal b l o o d was ex- tissues o f these i n f a n t s are e x p o s e d to h i g h e r
c h a n g e d for low affinity a d u l t b l o o d , the P.~0 level o f O,~ b e c a u s e o f the rightward shift o f the
i n c r e a s e d f r o m 18 to 29 m m H g w i t h o u t a n y o x y g e n dissociation curve after a transfusion.
c h a n g e s in t h e i r m y o c a r d i a l f u n c t i o n , arterio- I n the data o b t a i n e d by Nicolaides e t a116 i n
v e n o u s 0 2 c o n t e n t difference, 0 2 c o n s u m p t i o n , utero, the m e a n P O 2 i n the u m b i l i c a l artery at 24
o r cardiac o u t p u t . However, m i x e d v e n o u s oxy- to 26 weeks o f gestation, which c a n b e consid-
e r e d as m i x e d v e n o u s , was 28 m m Hg. This
100- w o u l d c o r r e s p o n d to a m i x e d v e n o u s s a t u r a t i o n
(S'~O2) o f 66% with a H b O 2 curve that corre-
s p o n d s to a n early p r e t e r m i n f a n t .
~ 9 IQ9 9 9 9a V a n d e r H o e v e n w d e s c r i b e d the r a n g e o f
Sf~D2 as a r e f l e c t i o n o f residual o x y g e n after
O o x y g e n e x t r a c t i o n in stable p r e t e r m n e w b o r n
40-
infants, the m e a n a n d SD was 73.56 _+ 5.25%.
W h e n the m e a n value o f S~O2 r e p o r t e d by V a n
20-
d e r H o v e n et aP v is p l o t t e d o n the O D C ' s ob-
t a i n e d in this study p r i o r to a n d after t r a n s f u s i o n
a l o n g with the O D C o b t a i n e d f r o m a n adult, as
16 17 18 19 20 21 22 23 s h o w n in F i g u r e 3 (top), the S~O 2 o f 74% cor-
Pso (mmHg)
r e s p o n d s to P ~ ) 2 o f 28 m m H g p r i o r to a n d 34
Figure 2. The relationship between HbF as a percent m m H g after t r a n s f u s i o n a n d 39 in the adult,
of total hemoglobin and Paw (r = .851, P = .0002). which w o u l d i n d i c a t e a n i n c r e a s e in residual
4014 De Halleux et al

ical damage. This a p p r o a c h can also allow for a

Arterio-venous
saturation difference
m o r e rapid weaning of the high risk infant f r o m
oxygen therapy and mechanical ventilation di-
minishing the risk of b a r o t r a u m a and could re-
A pre-b'ansfusion =18mmHg duce the incidence of chronic lung disease. Re-
Pso B post-lransfusion =21mrnHg
C adult =27rnmHg
cently Tin et a119 r e p o r t e d that maintaining 0 2
A 28mrnHg
saturation at 70-90% in infants less then 28 wks
20-
P9o2 B 34mmHg
C 39mmHg
of gestation decreased the incidence of retinop-
athy without increasing neurological sequellae.
10 20 30 40 50 60 70 80 90 100
PO 2 (mmHg)
T h e P50 after a transfusion, if known, could be
100 useful to predict the range of adequate satura-
90 ~ tion. While 85% saturation in very early p r e t e r m

60-
70-

50

40
,
saturation difference
infants after an early transfusion should be ade-
quate the o p t i m u m and safe range of oxygen
saturation during oxygen therapy of very early
p r e t e r m infants remains to be clearly estab-
30 lished.
20

PO2
10
2
. , . , I , . , , , , ,
10 20 30 40 50 60
(mmHg)
F i g u r e 3. U p p e r panel, oxygen dissociation curve's
(A) b e f o r e , (B) after transfusion, a n d (C) in a n o r m a l
a d u l t with SvO 2 74% 17 a n d t h e arterio-venous satura-
tion d i f f e r e n c e f r o m 95% to 74%. T h e 74% S~O 2
c o r r e s p o n d s to a P~O 2 o f 28 m m H g b e f o r e , 1~O 2 o f
34 m m H g after transfusion, a n d a P~O 2 o f 39 m m H g
in a n adult. Lower panel, T h e oxygen dissociation
curve (B) after transfusion u s i n g P~O 2 o f 28 m m H g
(S~O 2 66%) a n d a similar arterio-venous saturation
d i f f e r e n c e as in u p p e r panel. T h e arterial O.~ satura-
tion w o u l d b e 85%.
oxygen as the ODC shifts to the right. If there
was a reversion to the pretransfusion PvO2,
which was 28 m m Hg, using the post-transfusion
ODC (B) with a Ps0 of 21 m m Hg, the S~r
would decrease to 66% (shown in Figure 3 bot-
tom). T h e r e f o r e with the same arterio-venous
difference that existed prior to the transfusion,
an arterial 0 2 saturation of 85% is all that would
be required after transfusion. As a general
guideline, a S~O 2 greater then 65% can be con-
sidered to represents an adequate reserve of
oxygen, is
T h e decreased oxygen affinity after transfu-
sion with adult red cells increases the a m o u n t o f
oxygen available to the i m m a t u r e tissues o f pre-
t e r m infants, and should be taken into consid-
eration when oxygen therapy is being regulated
in these infants. Lowering oxygen saturation af-
ter a transfusion may be an excellent protective
measure against hyperoxygenation and free rad-
. , , , , , ,
References
70 80 90 100
1. Hesse L, Eberl W, Schlaud M, Poets CF: Blood transfu-
sion. Iron load and retinopathy of prematurity. EurJ Pe-
diatr 156:465-470, 1997
2. Inder E, Clemett RS, Austin NC, et al: High iron status in
very low birth weight infants is associated with an in-
creased risk of retinopathy of prematurity. J Pediatr
131:541-544, 1997
3. Dani C, Reali MF, Bertini G, et al: The role of blood
transfusions and iron intake on retinopathy of prematu-
rity. Early Hum Dev 62:57-63, 2001
4. Silvers KM, Gibson AT, Russell JM, Powers HJ: Antioxi-
dant activity, packed cell transfusions, and outcome in
premature infants. Arch Dis Child Fetal Neonatal Ed
78:F214-F219, 1998
5. Sullivan JL, Newton RB: Serum antioxidant activity in
neonates. Arch Dis Child 63:748-757, 1988
6. Bard H, Makowski EL, Meschia G, et al: The relative
rates of synthesis of hemoglobins A and F in immature
red cells of newborn infants. Pediatrics 45:766-772, 1970
7. Bard H, Teasdale F: Red cell oxygen affinity, hemoglo-
bin type, 2,3-diphosphoglycerate, and pH as a function
of fetal development. Pediatrics 64:483-487, 1979
8. Bard H, WidnessJA, Ziegler EE, et al: The proportions of
cy. and AT-globins in the fetal hemoglobin synthesized
in preterm and term infants. Pediatr Res 37:361-364,
1995
9. Shelton JB, Shelton JR, Schroeder WA: High perfor-
mance liquid chromatographic separation of globin
chains on a large-pore C4 column. J Liq Chromatogr
7:1969-1977, 1984
10. Bard H, Peri KG, Gagnon C: The biologic implications
of a rare hemoglobin mutant that decreases oxygen
affinity. Pediatr Res 49:69-73, 2001
11. Bard H, FouronJC, Grothe AM, et al: The adaptation of
the fetal red cells of newborn lambs to extrauterine life:
The role of 2,3-diphosphoglycerate and adult hemoglo-
bin. Pediatr Res 10:823-825, 1976
12. Fouron JC, Bard H, Le GuennecJC, et al: Effect of fetal
 Decrease on 02 Saturation After Transfusion
or adult red cells on tissue oxygenation a n d myocardial
function in n o r m o x e m i c newborn lambs. Pediatr Res
15:967-970, 1981
13. Van A m e r i g e n MR, Fouron JC, Bard H, et al: Oxygen-
ation in anemic newborn lambs with high or low oxygen
affinity red cells. Pediatr Res 15:1500-1503, 1981
14. Lachance C, Chessex P, F o u r o n JC, et al: Myocardial,
erythropoietic, a n d metabolic adaptations to a n e m i a of
prematurity. J Pediatr 125:278-282, 1994
15. T e n n e y SM: A theoretical analysis of the relationship
between venous blood a n d m e a n dssue oxygen pres-
sures. Respir Physiol 20:283-296, 1974
16. Nicolaides KH, E c o n o m i d e s DL, Soothill PW: Blood
4 15
gases, pH, a n d lactate in appropriate- a n d small-for-
gestational-age fetuses. Am J Obstet Gynecol 161:996-
1001, 1989
17. Van d e r Hoeven MAHBM, Maertzdorf wJ, Blanco CE:
C o n t i n u o u s central venous oxygen saturation (ScvO2)
m e a s u r e m e n t u s i n g a fibre optic catheter in newborn
infants. Arch Dis Child 74:F177-F181, 1996
18. Shapiro BA: Assessment o f oxygenation: Today a n d to-
morrow. S c a n d J Lab Invest 50:197-202, 1990
19. Tin W, Milligan DWA, P e n n e f a t h e r P, Hey E: Pulse
oximetry, severe retinopathy, a n d o u t c o m e at o n e year
in babies of less than 28 weeks gestation. Arch Dis Child
Fetal Neonatal Ed 84:FI06-F110, 2001