HEMOGLOBIN, 1 ( 6 ) , 547-560 (1977)
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AN IMPROVED Cl1ROMATOGRAPHIC PROCEDURE
FOR
QUANTITATION OF HUMAN FETAL HEMOGLOBIN.
E. C. Abraham, A. Reese, M. S t a l l i n g s , F. A. Carver and T.H.J.
Huisman
Laboratory of P r o t e i n Chemistry, Department of C e l l and Molecular Biology and Comprehensive S i c k l e Cell Center, Medical College of Georgia, and Veterans Administration Hospital, Augusta, Georgia 30901, U.S.A. Abstract The D W - c e l l u l o s e chromatographic procedure which uses glycine-KCN-NaC1 s o l u t i o n s as Developers and a NaCl g r a d i e n t t o e l u t e t h e hemoglobin zones was found t o be u s e f u l f o r the q u a n t i t a t i o n of Hb F I n samples containing Hb A, provided t h e l e v e l of Hb F exceeds 2%. The method has been evaluated through a study of a r t i f i c i a l mixtures and of blood samples from p a t i e n t s with various d i s o r d e r s . The d a t a have been compared with r e s u l t s obtained by f o u r o t h e r procedures t h a t are used f o r the q u a n t i t a t i o n of Hb F. Introduction Several types of a l k a l i d e n a t u r a t i o n procedures (1-3) have been widely used for t h e determination of t h e percentage of human f e t a l hemoglobin (Hb F) i n blood, but t h e r e l i a b i l i t y of t h e s e procedures has been t h e s u b j e c t of controversy (4). Chrmatographic procedures using e i t h e r an anion exchanger o r
8
cation
exchanger are a l s o o f t e n less s a t i s f a c t o r y because of incomplete
547 Copyright 6 1977 hy Marcel Dekker. Inc. All Rights Rrserved. Neither this work nor any part niay lie reproduced o r transmitted in any fcirtii or hy any means. electronic or nwchaniral. including photocopying. microfilming, and recordieg. or hy any information storage and retrieval system. without permission in writing lroni the puhlisher.
AURAIlAE1 ET AL.
548
s e p a r a t i o n o f lib F and the m i n o r Hb component Hb A 1 a n d / o r of lib F and lib A,
(5-15).
A cliemical p r o c e d u r e which is b a s e d on t h e
r e s u l t s oE amino a c i d a n a l y s i s of t h e llb F
+
llb h i z o n e , i s o l a t e d
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by DEAE-Sephadex chromatograptiy, a p p e a r s t o p r o v i d e s a t i s f a c t o r y d a t a (16) b u t t h e t e c h n i q u e is cumbersome and t i m e consuming. R e c e n t l y w e h a v e i n t r o d u c e d a new c h r o m a t o g r a p h i c method which u t i l i z e s DEAE-cellulose and glycine-KCN-NaC1
developers; r e s u l t s
o f some c h r o m a t o g r a p h i c e x p e r i m e n t s h a v e i n d i c a t e d t h a t Hb F c a n b e s e p a r a t e d from Ilb A and tlb A 1 ( 1 7 ) .
I n t h i s communication w e
r e p o r t on t h e u s e f u l n e s s o f t h e method f o r t h e q u a n t i t a t i o n oE Hb F i n t h e p r e s e n c e o f t h e s e n o r m a l l y o c c u r r i n g a d u l t hemoglobin types
Materials a n d Methods Blood Samples.
These were o b t a i n e d from n o r m a l newborns,
normal a d u l t s , and a few p a t i e n t s w i t h l e u k e m i a , from persons who
are h e t e r o z y g o u s f o r 6 t h a l a s s e e l a , 66 ttialassemia, or t h e h e r e d i t a r y p e r s i s t e n c e o f Hb F (HPFH), from homozygotes f o r lib S o r Hb C or f o r HPFH ( w i t h 100%Hb F ) , a n d from p a t i e n t s w i t h SC d i s -
ease, S-6'
t h a l a s s e m f a , o r t h e S-HPFH c o n d i t l o n .
w a s 110; 70 p e r s o n s had t h e AF phenotype.
The t o t a l number
U s u a l l y 5 t o 10 m l blood
was c o l l e c t e d i n a v a c u t a i n e r w i t h EDTA as a n t i c o a g u l a n t .
Red c e l l
h e m l y s a t e s were p r e p a r e d as d e s c r i b e d b e f o r e ( 1 7 ) . Chromatographic Procedure.
The p r o c e d u r e u t i l i z e s D W E -
c e l l u l o s e as a n i o n e x c h a n g e r (DE-52, m i c r o g r a n u l a r and p r e s w o l l e n ,
IPII'IIOVED I'ROCEDUKE FOR Hb F QUANTITATION
549
from Wtiatman, I n c . ) and glycine-KCN (0.2 M glycine-0.01% KCN) w i t h v a r i o u s m o l a r i L i e s of N a C l a s Developers.
The p r e p a r a t i o n of t h e
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ion exchanger, t h e developers and t h e c o n d i t i o n of t h e chromatog r a p h i c procedure were d e s c r i b e d in d e t a i l elsewhere (17).
About
30 mg henloglobin ( i n 0.5 m l , and d i a l y z e d o v e r n i g h t a t 4'C
against
0.1 M glycine-0.005% KCN) is a p p l i e d t o a 1 x 25 cm column.
A
c o n t i n u o u s l y i n c r e a s i n g s a l t g r a d i e n t is o b t a i n e d by i n t r o d u c i n g t h e 0.03 M developer ( 0. 2 M glycine-0.01% KCN-0.03 M NaCl) from a s e p a r a t o r y f u n n e l i n t o a 500 m l mixing f l a s k c o n t a i n i n g t h e 0.005 M developer (0.2 M glycine-0.01Z
KCN-0.005 M N a C l ) .
15-18 ml/lir (5-6 ml/tube) is maintained.
A flow rate of
About 24 hours l a t e r t h e
0.03 PI developer is r e p l a c e d by t h e 0.06 M developer (0.2 H glycine-
0.019: KCN-0.06 M NaC1). p l e t e d in two days.
The chromatograms shown i n Fig. 1 were com-
Ca c u l a t i o n of t h e p e r c e n t a g e s of t h e hemoglo-
b i n components is based on t h e absorbance a t 415 run of t h e e f f l u e n t fractions. The percentage FDE is t h e sum of t h e p e r c e n t a g e s of t h e Fo and t h e F1 zones, b u t such a c a l c u l a t i o n w a s o n l y p o s s i b l e when a dist i n c t lib F1 zone was observed ( s e e Fig. 7 of r e f . 1 7 ) .
A t moderate
l e v e l s of Hb F (below 30%) t h e Hb F1 and t h e A 1 zones are overl a p p i n g (Fig. 1) and then t h e percentage FDE is t h e same as t h e p e r c e n t a g e o f Hb Fo.
--
Other Methods.
The a l k a l i d e n a t u r a t i o n procedure of Betke
e t a2 (3) was used t o determine t h e l e v e l of a l k a l l r e s i s t a n t hemo-
g l o b i n (FAD).
DWIE-Sephadex cliromatography (15) allowed t h e
s e p a r a t i o n of fib A,
and lfb F
+
fib A 1 , t h u s Hb A1 cochrornatograplied
A
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5S0
B
W ET AL.
E 0.2
C
? !
1
< k
a
0.2 v)
a
20
40
60
80
100
120
140
TUBE NUMBER FIGURE 1
DUE-cellulose chromatograms of r e d c e l l hemolysates with d i f f e r e n t q u a n t i t i e s of Hb F. A: Normal a d u l t . B and C: Mixtures of h e w l y s a t e s of a normal a d u l t and a cord blood. The numbers g i v e t h e p e r c e n t a g e s of t h e hemoglobins.
v i t h Hb F.
The Hb A, Hb F, Hb A 1 and Hb F1 zones from v a r i o u s
chronatograas were analyzed for t h e r a t i o of i s o l e u c i n e t o leucine and to phenylalanine t o assess t h e Hb F c o n t e n t of each f r a c t i o n , thua e n a b l i n g t h e d e t e r m i n a t i o n of t h e t o t a l Hb F c o n t e n t (File)
of t h e hemolysate (16).
The percentage of lib F h a s also been
determined by a newly developed radioimnunoassay (FRIA) which uses s p e c i f i c antisera €or Hb F (18).
IMPROVED PROCEDURE FOR Ilb F QUANTITATION
551
Results The quantitation of lib F by DEAE-cellulose Chromatography has
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been assessed through analysis of artificial mixtures containing tlb A2, Hb A, Hb Al, Hb F and Hb F1.
The samples used were freshly
prepared red cell hemolysates of a normal adult and either a newborn or a HPFH homozygote.
Table I illustrates the reproducibi-
lity; the values listed are the percentages of Hb Fo i n two mixtures of normal adult and cord blood hemolysates which were analyzed repeatedly over a period of 10 days. Fig. 2 illustrates the results of analyses of similar mixtures in which the quantity of cord blood hemolysate varied from 2.5 to 1OoX. Hb F was quantitated by alkali denaturation (Fm) and by DEAEcellulose chromatography (FDE) , and occasionally by amino acid analysis of the &, Fo, A1 and F1 hemoglobin zones
(File).
Based
on the latter data, the Hb F, zone contained 90 to 100% Hb F; however, when the Hb F i n the mixture was below 5% the Hb Fo zone contained only 70 to 90% Hb F.
The Hb A1 and Hb F1 zones were cross-
contaminated to a great extent, and Hb A1 overlapped Hb F1 com-
TABLE I
Repeated analyses of Ilb F in hemolysates. Mixture #
X Cord
% Adult
1
20
80
2
40
60
Fchrom (%) 11.2, 12.5, 24.5, 26.5,
12.7, 12.6, 25.0, 27.3,
11.6, 11.7 27.4, 26.4
Mean f S.D. 12.1 f 0.6 26.2 f 1.2
ABRAIIAM ET AL.
552
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80 70
60
at 5 0 I . i
n I 40
30
20 10
20
40
60
80
100
CORD BLOOD HEMOLYSATE % FIGURE 2 The percentages of Hb P in mixtures of hemolysates of a normal
adult and a cord X FDE File = X X FAD
-
blood sample. Hb F by DEAE-cellulose chromatography Hb F by a chemical procedure Hb P by alkali denaturation.
pletely when the tocal Hb F content was low (Fig. 1). lation between the FDE ( X Hb Fo
+
The corre-
X Hb F1) and the FAD values was
relatively poor with the F m percentages being coneistantly lower.
553
IMYiWJED PROCEDURE FOR lib F QUAHTITATION Figure 3 presents similar data for mixtures of a normal hemolysate and the hemolysate from a HPFIi homozygote containing 100% ftb F.
The mixtures were analyzed by four procedures, namely
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alkali dsnaturation (FAD), DEAE-cellulose chromatography (FDE), the chemical procedure (File) and the immunochemlcal procedure (F~IA). The percentages FDE, F i l e , and FRIA correlated w l l with the theoretical values for IIb F but the FAD values were consistently lower and deviated from the expected values by a proportionality
r/; /
Ip 0
0
L)
I
10
20
30
40
50
60
70
@O
nb’Ffh.or~lkol
FIGURE 3 The percentages of Hb F i n mixtures of hemolysates of a normal adult and a IiPFH homozygote (with 100% Hb F). FDE % Ub F by DEAE-cellulose chromatography File % Hb F by a chemical procedure FAD % Hb F by alkali denaturation FRIA = X Hb F by radioinmunoassay.
--
ABRAHAM ET AL.
554 factor of about 0 . 8 (determined visually).
The correlation co-
efficients (R) of FDE. FRIA, and PAD with the theoretical F value were 0.9920, 0.9975 and 0.9200, respectively.
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Results of Hb F quantitation in hemolysates of 109 patients with various hematologic conditions by alkali denaturation and by DEhe-cellulose chromatography are given in Figure 4.
The line
has been drawn on the basis of a least-squares treatment of the data.
The proportionality factor of 0.86 and the correlation co-
efficient of 0.94 are closely similar to the values obtained in the recovery experiments (Fig. 3).
Red cell hemolysates from a
R * 094
SLOPE ( m l 0 8 6
Y
INTERCEPT ( y l - 0 . 1 4
/ *
10
20
.
30
SO
40
60
70
80
*'
FIGURE 4 The percentages of llb F (a0 X FDE and X FAD) in blood samples of 109 persona with elevated levels of Hb F.
IMPROVED PROCEDURE FOR Ilb F QUANTITATION
555
feu of these p a t i e n t s and one a r t i f i c a l l y prepared mixture were analyzed f o r Hb F by four d i f f e r e n t techniques, and t h e r e s u l t s
As expected, the X FDE (DEAE-cellulose)
a r e given i n Table 11.
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and the X
File
are i n good agreement but t h e X FDB (DEAE-Sephadex)
is c o n s i s t e n t l y high because of the presence of v a r i a b l e amounts of Hb A 1 in t h e Hb F zone.
The d a t a presented here show t h a t DW-cellulo8e chromatography w i t h glycine-KCN-NaC1
developers provide a new procedure f o r an
accurate q u a n t i t a t i o n of Hb F in Hb A containing samples.
Even
though t h i s procedure is s u i t a b l e f o r t h i s purpose, its a p p l i c a t i o n
is nore general and can be extended t o samples without Hb A but containing various o t h e r hemoglobin v a r i a n t s (17).
The method has
TABLE 11 Comparison of Hb P values in blood from p a t i e n t s w i t h various hemat o l o g i c d i s o r d e r s , as determined by DEAE-cellulose chromatography (FDE), by DEAE-Sephadex chromatography (FDEs), by a chemical procedure (File), and by an a l k a l i denaturation procedure (FAD).
X F D ~
X FDE 29.1
35.3
20.1
C.W. A. C.
Hom-B-Thal. A-HPFH
4.9 35 .O 64.0
11.1
R.E.
A-WFH A-HPPH c-HPPH
H.S.
cJML**
4.2 34.0 64.0 15.5 54.0 25.9
L.B. J.S.
***
Mixture
* ** ***
--
15.0
54.0 23.4
--
68.4
30.6
-
X
File
Condition*
Subject
X FAD
-
3.3 29.4
50.4 13.6 45.3 16.5
WFH h e r e d i t a r y p e r s i s t e n c e of Hb F; C Hb C. CJML chronic j u v e n i l e Payelogenous leukemia. Prepared from hemolysates of a normal a d u l t and a HPM homozygote.
556
ABRAlMM ET AL.
d i s t i n c t a d v a n t a g e s o v e r t h a t which u s e s D M E - c e l l u l o s e and Tris-
HC1 d e v e l o p e r s (19); i t is s i m p l e r , f a s t e r , and i t g i v e s an i m proved s e p a r a t i o n o f Ilb A, tlb F and llb A1.
When t h e l e v e l of Hb F
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in a sample is 2 t o 3% or lower, q u a n t i t a t i o n of t h e Hb F becomes less a c c u r a t e ; however, i t s p r e s e n c e i n t h e chromatogran can r e a d i l y b e demonstrated (Fig. 1 ) .
Moreover, t h e small amount o f tib F t h a t
is p r e s e n t in a normal hemolysate (average v a l u e 0.22% w i t h a r a n g e o f 0.01 t o 1.30%. determined by radioinnnunoassay ( 2 0 ) ) . is not sep a r a t e d from lib A a t a l l (Fig. 1 ) . and t h u s t h e q u a n t i t a t i o n of
Hb F by DEAE-cellulose chromatography in samples c o n t a i p t n g less t h a n 2% Hb F is n o t p o s s i b l e .
Another s o u r c e o f e r r o r is t h e
( o f t e n ) i n c o m p l e t e s e p a r a t i o n o f t h e minor hemoglobins A 1 and F1, p a r t i c u l a r l y in samples w i t h a modest amount (10 t o 30%) of Ilb F, which makes i t r a t h e r d i f f i c u l t t o c a l c u l a t e t h e t o t a l Hb F (9
Hb Fo + Hb F1) p e r c e n t a g e . Aging o f a r e d cell hemolysate w i l l c a u s e a n error in t h e
FDE d e t e r m i n a t i o n b e c a u s e t h e in
uitm formed F1 d e r i v a t i v e
(i.e. lib P w i t h one g l u t a t h i o n e r e s i d u e a t t a c h e d t o e a c h y c h a i n ) h a s d i f f e r e n t chromatographic p r o p e r t i e s .
Fig. 5 shows t h e pre-
s e n c e o f s e v e r a l c h r o m a t o g r a p h i c a l l y d i s t i n c t minor F1 components
in an u n d i a l y z e d r e d c e l l hemolysate from a HPFH homozygote t h a t w a s k e p t a t 4.C Fld).
for s e v e r a l d a y s ( d e s i g n a t e d as F l a ,
Fib.
F l c , and
F r a c t i o n s F l c and Fl& seem t o be t h e p r o d u c t s of i n u i t r o
a g i n g , and are presumably formed by t h e d i s u l f i d e i n t e r c h a n g e rea c t i o n o f o x i d i z e d g l u t a t h i o n e w i t h Hb F.
Zone Fib a p p e a r s t o be
t h e n o r m a l l y o c c u r r i n g minor hemoglobin in which t h e amino-terminus
IMPROVED PROCEDURE FOR Hb F QUANTITATION
557
0 DAYS 0.4
b (10.6)
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0.2
IIr
E
C
:
' w
0.4
u
5 0.2
a3
a
:m: a
0.4
0.2
60
80
100
120
140
160
TUBE NUMBER FIGURE 5 The e f f e c t of a g i n g of a n u n d i a l y z e d hemolysate from a HPM homozygote on t h e formation of minor f e t a l hemoglobin components. The numbers g i v e t h e p e r c e n t a g e s of t h e hemoglobins. The nomenclature a. b , c. and d is d i s c u s s e d i n t h e text. o f t h e y c h a i n is a c e t y l a t e d ( 8 ) whereas zone F l a may c o n t a i n Hb F t h a t h a s r e a c t e d w i t h g l u c o s e i n a similar f a s h i o n as h a s Hb A t o form Hb A l c (21,221.
I n t e r e s t i n g l y , t h e q u a n t i t i e s o f b o t h P l a and
Fib a r e n o t changed d u r i n g t h e in vitro a g i n g p r o c e s s , s u p p o r t i n g t h e s u g g e s t i o n t h a t b o t h a r e formed i n t h e r e d c e l l s in
V~VO.
The
chromatographic m o b i l i t i e s of t h e s e minor components i n d i c a t e t h a t
558
ABRAHAM
ET AL.
t h e i n dtro formation of Hb Flc and Hb Fld w i l l not d i r e c t l y i n t e r f e r e w i t h t h e q u a n t i t a t i o n of Hb F provided t h e i r percentages a r e included i n t h e t o t a l X FDE.
Hb T i a , however, has t h e same
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chromatographic mobility as Hb A 1 and t h e s e two minor hemoglobins will
cochromatograph when an AF hemolysate is analyzed.
In
a d d i t i o n , minor a d u l t hemoglobins, formed d u r i n g t h e in v i t r o aging process, o f t e n o v e r l a p with t h e minor Hb F zone, thus i n t r o ducing e r r o r s i n t h e f i n a l X FDE c a l c u l a t i o n .
Most of t h e s e e r r o r s
can be eliminated or minimized by analyzing f r e s h l y prepared red c e l l hemolysate.
Such a hemolysate can be preserved a t 4'C f o r
s e v e r a l days and subsequently analyzed a t a l a t e r d a t e provided i t has been dialyzed a t 4'C f o r a t least 24 hours a g a i n s t 0.1 M glycine-0.005% KCN t o remove t h e g l u t a t h i o n e .
The a u t h o r s are indebted t o Dr. W. A. Schroeder f o r u s e f u l discussion.
This r e s e a r c h w a s supported i n p a r t by U. S. Public
Health Service Research Grants HLB-05168 and HLB-15158. References 1.
Joaxis, 3. P. and H u i s m a n , T.H.J., A Laboratory Manual on Abnormal Hemoglobins, 2nd Ed., Blackwell S c i e n t i f i c , Oxford, Eng1.t 1969.
2.
Singer, K., Chernoff, 1951.
3.
Betke, K., 1959.
4.
I., and S i a g e r , L., Blood,
H a r t i , H., and S c h l i c h t , I.. Nature,
2: 413,
184: 1877,
Hemoglobin Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/29/14 For personal use only.
IHPROVED PROCEDURE FOR Ilb F QUANTITATION
559
4.
Chernoff, A. I., I n t e r n a t i o n a l Symposium on Comparative llemoglobin S t r u c t u r e , e d i t e d by D. J. Polychronakos, p. 45, M. T r i a n t a f y l o u Sons, T h e s s a l o n i k i , Greece, 1966.
5.
Clegg, M. D. and Schroeder, W. A., 6065, 1959.
6.
Schnek, A. G. and Schroeder, W. A., 1472, 1961.
7.
J o n e s , R. T. and Schroeder, W. A., 1963.
8.
S c h r o e d e r , W. A., Cue, J . T., Matsuda, G. and Fenninger, W. D., Biochim. Biophys. Acta, 63: 532, 1962.
9.
Huisman, T.H.J. 1960.
and Meyering, C. A.,
10.
Huisman, T.H.J.,
Clin. Chim. Acta,
11. l)ozy, A. M. and Huisman, T.H.J.,
J . h e r . Chem. SOC.,
J . h e r . Chem. SOC., 8J:
J. Chromatogr.,
g:421,
C l i n . Chim. Acta,
9: 159,
5:
103,
1972.
9: 62,
J. Chromatogr.,
12.
Abraham, E. C., Biochem. Med.,
13.
Dozy, A. M., K l e i h a u e r , E. F. , and Huisman, T.H.J. J. Chromatogr,, 2:723, 1968.
14.
Huisman, T.H.J.
and Dozy, A. H., J. Chromatogr.,
15.
Huisman, T.H.J.
and Dozy, A. M., J . Chromatogr.,
16.
S h e l t o n , J . R., Schroeder, W. A., Huisman, T.H.J., J. B., A n a l y t i c a l Biochem., 35: 235, 1970.
17.
Abraham, E. C., HEMOGLOBIN,
&:
1969.
Walker, D., Gravely, M. and Huisman, T.H.J., 1975.
2:56,
Reese, A., S t a l l i n g s , M., 1976-77.
1: 27,
1: 180, 1962. 2:160, 1965. and Wilson,
and Huisman, T.H.J.,
18.
Garver, F. A., J o n e s , C. S., Baker, M. M., A l t a y , G., Barton, B. P., Gravely, M., and Huisman, T.H.J., h e r . J. Hemat., 1: 459, 1976.
19.
Efremov, G. D. and Huisman, T.H.J., 1974.
20.
Abraham, E. C., Ozawa, T., N i a z i , G. A., Hudson, J . B., Garver, F., and Huisman, T.H.J., i n preparation.
J. Chromatogr.,
B: 191,
ABRAHAM ET AL.
560
21.
Bolmquist, W. R. and Schrocder, W. A.,
Biochemistry,
5:
2489,
1966.
Hemoglobin Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/29/14 For personal use only.
22.
Buon, B. P., Raney, D. N., Gabbay, K. H., and Gallop, P. X., Biocha. Biophps. Rar. am.,67: 103. 1975.
Received: A p r i l 29, 1977;
Accepted: EfIy 15, 1977.
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AN IMPROVED Cl1ROMATOGRAPHIC PROCEDURE
FOR
QUANTITATION OF HUMAN FETAL HEMOGLOBIN.
E. C. Abraham, A. Reese, M. S t a l l i n g s , F. A. Carver and T.H.J.
Huisman
Laboratory of P r o t e i n Chemistry, Department of C e l l and Molecular Biology and Comprehensive S i c k l e Cell Center, Medical College of Georgia, and Veterans Administration Hospital, Augusta, Georgia 30901, U.S.A. Abstract The D W - c e l l u l o s e chromatographic procedure which uses glycine-KCN-NaC1 s o l u t i o n s as Developers and a NaCl g r a d i e n t t o e l u t e t h e hemoglobin zones was found t o be u s e f u l f o r the q u a n t i t a t i o n of Hb F I n samples containing Hb A, provided t h e l e v e l of Hb F exceeds 2%. The method has been evaluated through a study of a r t i f i c i a l mixtures and of blood samples from p a t i e n t s with various d i s o r d e r s . The d a t a have been compared with r e s u l t s obtained by f o u r o t h e r procedures t h a t are used f o r the q u a n t i t a t i o n of Hb F. Introduction Several types of a l k a l i d e n a t u r a t i o n procedures (1-3) have been widely used for t h e determination of t h e percentage of human f e t a l hemoglobin (Hb F) i n blood, but t h e r e l i a b i l i t y of t h e s e procedures has been t h e s u b j e c t of controversy (4). Chrmatographic procedures using e i t h e r an anion exchanger o r
8
cation
exchanger are a l s o o f t e n less s a t i s f a c t o r y because of incomplete
547 Copyright 6 1977 hy Marcel Dekker. Inc. All Rights Rrserved. Neither this work nor any part niay lie reproduced o r transmitted in any fcirtii or hy any means. electronic or nwchaniral. including photocopying. microfilming, and recordieg. or hy any information storage and retrieval system. without permission in writing lroni the puhlisher.
AURAIlAE1 ET AL.
548
s e p a r a t i o n o f lib F and the m i n o r Hb component Hb A 1 a n d / o r of lib F and lib A,
(5-15).
A cliemical p r o c e d u r e which is b a s e d on t h e
r e s u l t s oE amino a c i d a n a l y s i s of t h e llb F
+
llb h i z o n e , i s o l a t e d
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by DEAE-Sephadex chromatograptiy, a p p e a r s t o p r o v i d e s a t i s f a c t o r y d a t a (16) b u t t h e t e c h n i q u e is cumbersome and t i m e consuming. R e c e n t l y w e h a v e i n t r o d u c e d a new c h r o m a t o g r a p h i c method which u t i l i z e s DEAE-cellulose and glycine-KCN-NaC1
developers; r e s u l t s
o f some c h r o m a t o g r a p h i c e x p e r i m e n t s h a v e i n d i c a t e d t h a t Hb F c a n b e s e p a r a t e d from Ilb A and tlb A 1 ( 1 7 ) .
I n t h i s communication w e
r e p o r t on t h e u s e f u l n e s s o f t h e method f o r t h e q u a n t i t a t i o n oE Hb F i n t h e p r e s e n c e o f t h e s e n o r m a l l y o c c u r r i n g a d u l t hemoglobin types
Materials a n d Methods Blood Samples.
These were o b t a i n e d from n o r m a l newborns,
normal a d u l t s , and a few p a t i e n t s w i t h l e u k e m i a , from persons who
are h e t e r o z y g o u s f o r 6 t h a l a s s e e l a , 66 ttialassemia, or t h e h e r e d i t a r y p e r s i s t e n c e o f Hb F (HPFH), from homozygotes f o r lib S o r Hb C or f o r HPFH ( w i t h 100%Hb F ) , a n d from p a t i e n t s w i t h SC d i s -
ease, S-6'
t h a l a s s e m f a , o r t h e S-HPFH c o n d i t l o n .
w a s 110; 70 p e r s o n s had t h e AF phenotype.
The t o t a l number
U s u a l l y 5 t o 10 m l blood
was c o l l e c t e d i n a v a c u t a i n e r w i t h EDTA as a n t i c o a g u l a n t .
Red c e l l
h e m l y s a t e s were p r e p a r e d as d e s c r i b e d b e f o r e ( 1 7 ) . Chromatographic Procedure.
The p r o c e d u r e u t i l i z e s D W E -
c e l l u l o s e as a n i o n e x c h a n g e r (DE-52, m i c r o g r a n u l a r and p r e s w o l l e n ,
IPII'IIOVED I'ROCEDUKE FOR Hb F QUANTITATION
549
from Wtiatman, I n c . ) and glycine-KCN (0.2 M glycine-0.01% KCN) w i t h v a r i o u s m o l a r i L i e s of N a C l a s Developers.
The p r e p a r a t i o n of t h e
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ion exchanger, t h e developers and t h e c o n d i t i o n of t h e chromatog r a p h i c procedure were d e s c r i b e d in d e t a i l elsewhere (17).
About
30 mg henloglobin ( i n 0.5 m l , and d i a l y z e d o v e r n i g h t a t 4'C
against
0.1 M glycine-0.005% KCN) is a p p l i e d t o a 1 x 25 cm column.
A
c o n t i n u o u s l y i n c r e a s i n g s a l t g r a d i e n t is o b t a i n e d by i n t r o d u c i n g t h e 0.03 M developer ( 0. 2 M glycine-0.01% KCN-0.03 M NaCl) from a s e p a r a t o r y f u n n e l i n t o a 500 m l mixing f l a s k c o n t a i n i n g t h e 0.005 M developer (0.2 M glycine-0.01Z
KCN-0.005 M N a C l ) .
15-18 ml/lir (5-6 ml/tube) is maintained.
A flow rate of
About 24 hours l a t e r t h e
0.03 PI developer is r e p l a c e d by t h e 0.06 M developer (0.2 H glycine-
0.019: KCN-0.06 M NaC1). p l e t e d in two days.
The chromatograms shown i n Fig. 1 were com-
Ca c u l a t i o n of t h e p e r c e n t a g e s of t h e hemoglo-
b i n components is based on t h e absorbance a t 415 run of t h e e f f l u e n t fractions. The percentage FDE is t h e sum of t h e p e r c e n t a g e s of t h e Fo and t h e F1 zones, b u t such a c a l c u l a t i o n w a s o n l y p o s s i b l e when a dist i n c t lib F1 zone was observed ( s e e Fig. 7 of r e f . 1 7 ) .
A t moderate
l e v e l s of Hb F (below 30%) t h e Hb F1 and t h e A 1 zones are overl a p p i n g (Fig. 1) and then t h e percentage FDE is t h e same as t h e p e r c e n t a g e o f Hb Fo.
--
Other Methods.
The a l k a l i d e n a t u r a t i o n procedure of Betke
e t a2 (3) was used t o determine t h e l e v e l of a l k a l l r e s i s t a n t hemo-
g l o b i n (FAD).
DWIE-Sephadex cliromatography (15) allowed t h e
s e p a r a t i o n of fib A,
and lfb F
+
fib A 1 , t h u s Hb A1 cochrornatograplied
A
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5S0
B
W ET AL.
E 0.2
C
? !
1
< k
a
0.2 v)
a
20
40
60
80
100
120
140
TUBE NUMBER FIGURE 1
DUE-cellulose chromatograms of r e d c e l l hemolysates with d i f f e r e n t q u a n t i t i e s of Hb F. A: Normal a d u l t . B and C: Mixtures of h e w l y s a t e s of a normal a d u l t and a cord blood. The numbers g i v e t h e p e r c e n t a g e s of t h e hemoglobins.
v i t h Hb F.
The Hb A, Hb F, Hb A 1 and Hb F1 zones from v a r i o u s
chronatograas were analyzed for t h e r a t i o of i s o l e u c i n e t o leucine and to phenylalanine t o assess t h e Hb F c o n t e n t of each f r a c t i o n , thua e n a b l i n g t h e d e t e r m i n a t i o n of t h e t o t a l Hb F c o n t e n t (File)
of t h e hemolysate (16).
The percentage of lib F h a s also been
determined by a newly developed radioimnunoassay (FRIA) which uses s p e c i f i c antisera €or Hb F (18).
IMPROVED PROCEDURE FOR Ilb F QUANTITATION
551
Results The quantitation of lib F by DEAE-cellulose Chromatography has
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been assessed through analysis of artificial mixtures containing tlb A2, Hb A, Hb Al, Hb F and Hb F1.
The samples used were freshly
prepared red cell hemolysates of a normal adult and either a newborn or a HPFH homozygote.
Table I illustrates the reproducibi-
lity; the values listed are the percentages of Hb Fo i n two mixtures of normal adult and cord blood hemolysates which were analyzed repeatedly over a period of 10 days. Fig. 2 illustrates the results of analyses of similar mixtures in which the quantity of cord blood hemolysate varied from 2.5 to 1OoX. Hb F was quantitated by alkali denaturation (Fm) and by DEAEcellulose chromatography (FDE) , and occasionally by amino acid analysis of the &, Fo, A1 and F1 hemoglobin zones
(File).
Based
on the latter data, the Hb F, zone contained 90 to 100% Hb F; however, when the Hb F i n the mixture was below 5% the Hb Fo zone contained only 70 to 90% Hb F.
The Hb A1 and Hb F1 zones were cross-
contaminated to a great extent, and Hb A1 overlapped Hb F1 com-
TABLE I
Repeated analyses of Ilb F in hemolysates. Mixture #
X Cord
% Adult
1
20
80
2
40
60
Fchrom (%) 11.2, 12.5, 24.5, 26.5,
12.7, 12.6, 25.0, 27.3,
11.6, 11.7 27.4, 26.4
Mean f S.D. 12.1 f 0.6 26.2 f 1.2
ABRAIIAM ET AL.
552
Hemoglobin Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/29/14 For personal use only.
80 70
60
at 5 0 I . i
n I 40
30
20 10
20
40
60
80
100
CORD BLOOD HEMOLYSATE % FIGURE 2 The percentages of Hb P in mixtures of hemolysates of a normal
adult and a cord X FDE File = X X FAD
-
blood sample. Hb F by DEAE-cellulose chromatography Hb F by a chemical procedure Hb P by alkali denaturation.
pletely when the tocal Hb F content was low (Fig. 1). lation between the FDE ( X Hb Fo
+
The corre-
X Hb F1) and the FAD values was
relatively poor with the F m percentages being coneistantly lower.
553
IMYiWJED PROCEDURE FOR lib F QUAHTITATION Figure 3 presents similar data for mixtures of a normal hemolysate and the hemolysate from a HPFIi homozygote containing 100% ftb F.
The mixtures were analyzed by four procedures, namely
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alkali dsnaturation (FAD), DEAE-cellulose chromatography (FDE), the chemical procedure (File) and the immunochemlcal procedure (F~IA). The percentages FDE, F i l e , and FRIA correlated w l l with the theoretical values for IIb F but the FAD values were consistently lower and deviated from the expected values by a proportionality
r/; /
Ip 0
0
L)
I
10
20
30
40
50
60
70
@O
nb’Ffh.or~lkol
FIGURE 3 The percentages of Hb F i n mixtures of hemolysates of a normal adult and a IiPFH homozygote (with 100% Hb F). FDE % Ub F by DEAE-cellulose chromatography File % Hb F by a chemical procedure FAD % Hb F by alkali denaturation FRIA = X Hb F by radioinmunoassay.
--
ABRAHAM ET AL.
554 factor of about 0 . 8 (determined visually).
The correlation co-
efficients (R) of FDE. FRIA, and PAD with the theoretical F value were 0.9920, 0.9975 and 0.9200, respectively.
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Results of Hb F quantitation in hemolysates of 109 patients with various hematologic conditions by alkali denaturation and by DEhe-cellulose chromatography are given in Figure 4.
The line
has been drawn on the basis of a least-squares treatment of the data.
The proportionality factor of 0.86 and the correlation co-
efficient of 0.94 are closely similar to the values obtained in the recovery experiments (Fig. 3).
Red cell hemolysates from a
R * 094
SLOPE ( m l 0 8 6
Y
INTERCEPT ( y l - 0 . 1 4
/ *
10
20
.
30
SO
40
60
70
80
*'
FIGURE 4 The percentages of llb F (a0 X FDE and X FAD) in blood samples of 109 persona with elevated levels of Hb F.
IMPROVED PROCEDURE FOR Ilb F QUANTITATION
555
feu of these p a t i e n t s and one a r t i f i c a l l y prepared mixture were analyzed f o r Hb F by four d i f f e r e n t techniques, and t h e r e s u l t s
As expected, the X FDE (DEAE-cellulose)
a r e given i n Table 11.
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and the X
File
are i n good agreement but t h e X FDB (DEAE-Sephadex)
is c o n s i s t e n t l y high because of the presence of v a r i a b l e amounts of Hb A 1 in t h e Hb F zone.
The d a t a presented here show t h a t DW-cellulo8e chromatography w i t h glycine-KCN-NaC1
developers provide a new procedure f o r an
accurate q u a n t i t a t i o n of Hb F in Hb A containing samples.
Even
though t h i s procedure is s u i t a b l e f o r t h i s purpose, its a p p l i c a t i o n
is nore general and can be extended t o samples without Hb A but containing various o t h e r hemoglobin v a r i a n t s (17).
The method has
TABLE 11 Comparison of Hb P values in blood from p a t i e n t s w i t h various hemat o l o g i c d i s o r d e r s , as determined by DEAE-cellulose chromatography (FDE), by DEAE-Sephadex chromatography (FDEs), by a chemical procedure (File), and by an a l k a l i denaturation procedure (FAD).
X F D ~
X FDE 29.1
35.3
20.1
C.W. A. C.
Hom-B-Thal. A-HPFH
4.9 35 .O 64.0
11.1
R.E.
A-WFH A-HPPH c-HPPH
H.S.
cJML**
4.2 34.0 64.0 15.5 54.0 25.9
L.B. J.S.
***
Mixture
* ** ***
--
15.0
54.0 23.4
--
68.4
30.6
-
X
File
Condition*
Subject
X FAD
-
3.3 29.4
50.4 13.6 45.3 16.5
WFH h e r e d i t a r y p e r s i s t e n c e of Hb F; C Hb C. CJML chronic j u v e n i l e Payelogenous leukemia. Prepared from hemolysates of a normal a d u l t and a HPM homozygote.
556
ABRAlMM ET AL.
d i s t i n c t a d v a n t a g e s o v e r t h a t which u s e s D M E - c e l l u l o s e and Tris-
HC1 d e v e l o p e r s (19); i t is s i m p l e r , f a s t e r , and i t g i v e s an i m proved s e p a r a t i o n o f Ilb A, tlb F and llb A1.
When t h e l e v e l of Hb F
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in a sample is 2 t o 3% or lower, q u a n t i t a t i o n of t h e Hb F becomes less a c c u r a t e ; however, i t s p r e s e n c e i n t h e chromatogran can r e a d i l y b e demonstrated (Fig. 1 ) .
Moreover, t h e small amount o f tib F t h a t
is p r e s e n t in a normal hemolysate (average v a l u e 0.22% w i t h a r a n g e o f 0.01 t o 1.30%. determined by radioinnnunoassay ( 2 0 ) ) . is not sep a r a t e d from lib A a t a l l (Fig. 1 ) . and t h u s t h e q u a n t i t a t i o n of
Hb F by DEAE-cellulose chromatography in samples c o n t a i p t n g less t h a n 2% Hb F is n o t p o s s i b l e .
Another s o u r c e o f e r r o r is t h e
( o f t e n ) i n c o m p l e t e s e p a r a t i o n o f t h e minor hemoglobins A 1 and F1, p a r t i c u l a r l y in samples w i t h a modest amount (10 t o 30%) of Ilb F, which makes i t r a t h e r d i f f i c u l t t o c a l c u l a t e t h e t o t a l Hb F (9
Hb Fo + Hb F1) p e r c e n t a g e . Aging o f a r e d cell hemolysate w i l l c a u s e a n error in t h e
FDE d e t e r m i n a t i o n b e c a u s e t h e in
uitm formed F1 d e r i v a t i v e
(i.e. lib P w i t h one g l u t a t h i o n e r e s i d u e a t t a c h e d t o e a c h y c h a i n ) h a s d i f f e r e n t chromatographic p r o p e r t i e s .
Fig. 5 shows t h e pre-
s e n c e o f s e v e r a l c h r o m a t o g r a p h i c a l l y d i s t i n c t minor F1 components
in an u n d i a l y z e d r e d c e l l hemolysate from a HPFH homozygote t h a t w a s k e p t a t 4.C Fld).
for s e v e r a l d a y s ( d e s i g n a t e d as F l a ,
Fib.
F l c , and
F r a c t i o n s F l c and Fl& seem t o be t h e p r o d u c t s of i n u i t r o
a g i n g , and are presumably formed by t h e d i s u l f i d e i n t e r c h a n g e rea c t i o n o f o x i d i z e d g l u t a t h i o n e w i t h Hb F.
Zone Fib a p p e a r s t o be
t h e n o r m a l l y o c c u r r i n g minor hemoglobin in which t h e amino-terminus
IMPROVED PROCEDURE FOR Hb F QUANTITATION
557
0 DAYS 0.4
b (10.6)
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0.2
IIr
E
C
:
' w
0.4
u
5 0.2
a3
a
:m: a
0.4
0.2
60
80
100
120
140
160
TUBE NUMBER FIGURE 5 The e f f e c t of a g i n g of a n u n d i a l y z e d hemolysate from a HPM homozygote on t h e formation of minor f e t a l hemoglobin components. The numbers g i v e t h e p e r c e n t a g e s of t h e hemoglobins. The nomenclature a. b , c. and d is d i s c u s s e d i n t h e text. o f t h e y c h a i n is a c e t y l a t e d ( 8 ) whereas zone F l a may c o n t a i n Hb F t h a t h a s r e a c t e d w i t h g l u c o s e i n a similar f a s h i o n as h a s Hb A t o form Hb A l c (21,221.
I n t e r e s t i n g l y , t h e q u a n t i t i e s o f b o t h P l a and
Fib a r e n o t changed d u r i n g t h e in vitro a g i n g p r o c e s s , s u p p o r t i n g t h e s u g g e s t i o n t h a t b o t h a r e formed i n t h e r e d c e l l s in
V~VO.
The
chromatographic m o b i l i t i e s of t h e s e minor components i n d i c a t e t h a t
558
ABRAHAM
ET AL.
t h e i n dtro formation of Hb Flc and Hb Fld w i l l not d i r e c t l y i n t e r f e r e w i t h t h e q u a n t i t a t i o n of Hb F provided t h e i r percentages a r e included i n t h e t o t a l X FDE.
Hb T i a , however, has t h e same
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chromatographic mobility as Hb A 1 and t h e s e two minor hemoglobins will
cochromatograph when an AF hemolysate is analyzed.
In
a d d i t i o n , minor a d u l t hemoglobins, formed d u r i n g t h e in v i t r o aging process, o f t e n o v e r l a p with t h e minor Hb F zone, thus i n t r o ducing e r r o r s i n t h e f i n a l X FDE c a l c u l a t i o n .
Most of t h e s e e r r o r s
can be eliminated or minimized by analyzing f r e s h l y prepared red c e l l hemolysate.
Such a hemolysate can be preserved a t 4'C f o r
s e v e r a l days and subsequently analyzed a t a l a t e r d a t e provided i t has been dialyzed a t 4'C f o r a t least 24 hours a g a i n s t 0.1 M glycine-0.005% KCN t o remove t h e g l u t a t h i o n e .
The a u t h o r s are indebted t o Dr. W. A. Schroeder f o r u s e f u l discussion.
This r e s e a r c h w a s supported i n p a r t by U. S. Public
Health Service Research Grants HLB-05168 and HLB-15158. References 1.
Joaxis, 3. P. and H u i s m a n , T.H.J., A Laboratory Manual on Abnormal Hemoglobins, 2nd Ed., Blackwell S c i e n t i f i c , Oxford, Eng1.t 1969.
2.
Singer, K., Chernoff, 1951.
3.
Betke, K., 1959.
4.
I., and S i a g e r , L., Blood,
H a r t i , H., and S c h l i c h t , I.. Nature,
2: 413,
184: 1877,
Hemoglobin Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/29/14 For personal use only.
IHPROVED PROCEDURE FOR Ilb F QUANTITATION
559
4.
Chernoff, A. I., I n t e r n a t i o n a l Symposium on Comparative llemoglobin S t r u c t u r e , e d i t e d by D. J. Polychronakos, p. 45, M. T r i a n t a f y l o u Sons, T h e s s a l o n i k i , Greece, 1966.
5.
Clegg, M. D. and Schroeder, W. A., 6065, 1959.
6.
Schnek, A. G. and Schroeder, W. A., 1472, 1961.
7.
J o n e s , R. T. and Schroeder, W. A., 1963.
8.
S c h r o e d e r , W. A., Cue, J . T., Matsuda, G. and Fenninger, W. D., Biochim. Biophys. Acta, 63: 532, 1962.
9.
Huisman, T.H.J. 1960.
and Meyering, C. A.,
10.
Huisman, T.H.J.,
Clin. Chim. Acta,
11. l)ozy, A. M. and Huisman, T.H.J.,
J . h e r . Chem. SOC.,
J . h e r . Chem. SOC., 8J:
J. Chromatogr.,
g:421,
C l i n . Chim. Acta,
9: 159,
5:
103,
1972.
9: 62,
J. Chromatogr.,
12.
Abraham, E. C., Biochem. Med.,
13.
Dozy, A. M., K l e i h a u e r , E. F. , and Huisman, T.H.J. J. Chromatogr,, 2:723, 1968.
14.
Huisman, T.H.J.
and Dozy, A. H., J. Chromatogr.,
15.
Huisman, T.H.J.
and Dozy, A. M., J . Chromatogr.,
16.
S h e l t o n , J . R., Schroeder, W. A., Huisman, T.H.J., J. B., A n a l y t i c a l Biochem., 35: 235, 1970.
17.
Abraham, E. C., HEMOGLOBIN,
&:
1969.
Walker, D., Gravely, M. and Huisman, T.H.J., 1975.
2:56,
Reese, A., S t a l l i n g s , M., 1976-77.
1: 27,
1: 180, 1962. 2:160, 1965. and Wilson,
and Huisman, T.H.J.,
18.
Garver, F. A., J o n e s , C. S., Baker, M. M., A l t a y , G., Barton, B. P., Gravely, M., and Huisman, T.H.J., h e r . J. Hemat., 1: 459, 1976.
19.
Efremov, G. D. and Huisman, T.H.J., 1974.
20.
Abraham, E. C., Ozawa, T., N i a z i , G. A., Hudson, J . B., Garver, F., and Huisman, T.H.J., i n preparation.
J. Chromatogr.,
B: 191,
ABRAHAM ET AL.
560
21.
Bolmquist, W. R. and Schrocder, W. A.,
Biochemistry,
5:
2489,
1966.
Hemoglobin Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/29/14 For personal use only.
22.
Buon, B. P., Raney, D. N., Gabbay, K. H., and Gallop, P. X., Biocha. Biophps. Rar. am.,67: 103. 1975.
Received: A p r i l 29, 1977;
Accepted: EfIy 15, 1977.
