1. health and fitness
2. disease
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HEMOGLOBIN ELECTROPHORESIS IN THE DIFFERENTIAL
DIAGNOSIS OF HEMOGLOBINOPATHIES
Lena
Scherba-Krugliak,
M.D.
June,
1987
Introduction
Hemoglobin i^a complex protein with a molecular weight of
645958 and consisting of two separate pairs of globin chains,
each chain being associated with one molecule of heme.
During early fetal life, hemoglobin Gower I 7 2^2'
hemoglobin Portland J7 2 J/7 and hemoglobin Gower II &/ 2G2 are
found in humans. Later during fetal life, hemoglobin
F (HbF)tf rj2#V
oyo,
HbA-rv/0>Do
and
KbA--^-!- are found.
HbA~0(28
2 and
HbA2^2(}2
The major hemoglobin in the erythrocytes of the normal adult
is HbA. In addition, over 400 mutant hemoglobins are now
known, some of which may cause serious clinical conditions,
especially in the homozygous state or in combination with
another abnormal hemoglobin. (1,2)
All inherited abnormal hemoglobinopathies seem to fall into
one of three categories: (a) inherited abnormalities of the
structure of one or more globin chains, such as HbS, HbC;
(b) inherited abnormalities related to the rate of synthesis
of one or more of the globin chains such as thalassemia
syndromes; (c) failure of the normal switch from fetal HbF to
adult HbA synthesis.
Hemoglobin electrophoresis is generally considered the best
method for separating and identifying the hemoglobins.
Practical approach to the diagnosis of hemoglobinopathies
consists of the following tests: (1) electrophoresis in
cellulose acetate - pH 8.6; (2) quantitative estimation of
jgfpfes
-2-
HbA2; (3) quantitative estimation of HbF; (4) test for
deoxyhemoglobin solubility; (5) citrate agar gel
electrophoresis; (6) isoelectric focusing; and (7) globin
chain electrophoresis. (3,4) In cases of thalassemia where
d i a g n o s i s i s d i f fi c u l t , d e t e r m i n a t i o n o f g l o b i n c h a i n
synthesis rates, chromatography, finger printing, amino acid
sequencing is indicated. (5,6,7)
Cellulose Acetate Electrophoresis (pH 8,6) (8,9)
Principle
Hemoglobin molecules in an alkaline solution have a net
negative charge which causes them to move towards the anode
at a rate proportional to their negative charge.
i|pSfes
D i ff e r e n t h e m o g l o b i n s , h o w e v e r, h a v e d i ff e r e n t c h a r g e s .
Those with an electrophoretic mobility greater than that of
HbA at pH 8.8 are known as the "fast hemoglobins:11 these
include Hb Barts, HbH, Hbl, HbN and HbJ. Substitution of
valine for glutamic acid in the beta chain HbS causes slower
migration by reducing the negative charge. Substitution of
lysine with its positive charge for glutamic acid, HbC,
causes the slowest migration.
I n t e r p r e t a t i o n ( 1 0 , 11 , 1 2 )
The relative order of increasing electrophoretic mobility of
the common hemoglobin bands from cathode to anode is as
follows: HbA2; E=0=C; G=D=S; F; A; Bart's, N and H. (Fig.
1) Usually, HbA is preceded by a fainter band that merges
-3-
w i th H b A . Th i s h a s b e e n c a l l e d A ~ a n d i s b e l i e v e d to
represent glycosylated hemoglobins.
It must be noted
differentiated on
this medium. Nor
distinguished from
that hemoglobins A-, C, O and E cannot be
the basis of electrophoretic mobility in
can hemoglobins D, G and Lepore be
HbS.
HbF is not seen unless it comprises 2% or more of the total
hemoglobin.
A very slowly moving band that constitutes more than 15% of
the total hemoglobin may be presumed to be HbC, O or E, since
HbA2 is rarely more than 10% of the total hemoglobin. HbC
and HbO are usually about 40 to 45% of the total hemoglobin
and virtually limited to patients of African descent, whereas
HbE is usually about 30% of the total hemoglobin and limited
to those of Southeast Asian ancestry.
When present, HbH constitutes approximately 5 to 15% of the
total hemoglobin and most commonly seen in persons from
Southeast Asia.
When present, hemoglobin Lepore constitutes only 10 to 15% of
the total hemoglobin, whereas HbS, D or G constitutes 25 to
45% of the total hemoglobin.
Thus, by measuring densitometrically the proportion of these
hemoglobin bands and by knowing the ethnic origin of a
patient, a preliminary assessment of hemoglobinopathies can
be made.
Combinations of hemoglobinopathies and thalassemias can also
o c c u r. E l e c t r o p h o r e s i s p a t t e r n s , q u a n t i t a t i v e H b A 2 a n d
quantitative HbF values are usually required to assess the
type of hemoglobin abnormality and its severity.
-4-
r
Additional confirmatory tests are needed:
1. Solubility test if S band present
2. Hemoglobin stability tests for unstable hemoglobins,
e.g., HbKfiln, HbH.
3. Citrate agar electrophoresis at pH 6.2 is necessary to
distinguish: (a) HbE, 0 and A2 from HbC, (b) HbS from
HbD and G.
Acid Citrate Agar Electrophoresis (pH 6,2) (13)
Electrophoresis of hemoglobin in an acid citrate agar gel
aids in the identification of hemoglobins D, E, G and 0 on
the basis of differences in charge and molecular
configuration. This procedure complements cellulose acetate
electrophoresis at an alkaline pH and permits ready
separation of hemoglobins that migrate together on cellulose
acetate.
Interpretation
The relative order of hemoglobin migration in citrate agar at
an acid pH is as follows: HbF, A=D=E=G, 0, S, C. (Fig. 2)
HbD
HbE
from
HbA
HbA,
and G migrates with HbA and thus separate well from HbS.
migrate with HbA and thus also is easily differentiated
HbC. HbO has a slightly lower cathodal migration than
and thus can be distinguished from HbC as well as from
D, G, E.
HbA2 mobility is the same as that of HbA.
-5-
Isoelectric Focusing (15,16)
Principle
Thin-layer isoelectric focusing is based on the migration of
proteins to their isoelectric points (pi's) when placed on a
stable pH gradient of an electric field. The pH gradient is
maintained by a mixture of ampholytes incorporated in a
polyacrylamide gel. Hemoglobins migrate according to their
pi's and are separated as sharp bands with high resolution
a n d s e n s i t i v i t y.
The greatest advantages of isoelectric focusing over
conventional electrophoresis are: much better resolution
between proteins having the same or similar migration by
electrophoresis and the ability to detect small amounts of
minor components. In addition, an abnormal hemoglobin can
o f t e n b e i d e n t i fi e d s o l e l y b y i s o e l e c t r i c f o c u s i n g r a t h e r
than using several electrophoretic procedures, different pH's
and complementary tests. Isoelectric focusing is a cost
effective procedure considering the fact that up to 50
s a m p l e s c a n b e r u n o n o n e p l a t e w i t h , u s u a l l y, n o
accompanying tests necessary.
Disadvantages of isoelectric focusing in comparison to
conventional electrophoresis are: the test is technically
more complicated and densitometric quantitative analysis of
hemoglobins is not an easy task due to small size of the
bands and background opacity.
Interpretation
The relative
anodal end
S, E, A2,
established
positions of common hemoglobins from the acidic
to the basic cathodal end are: H, Bart's, Af F,
C. (Fig. 3) The identity of each band is
b y t h e r e f e r e n c e t o A , F, S , C a n d B a r t ' s
-6-
r
control, measurement of the distance of the band from HbA and
reference to the chart of known hemoglobin positions.
Conclusion
Hemoglobin electrophoresis is the most useful standard
l a b o r a t o r y p r o c e d u r e f o r t h e d e t e c t i o n a n d i d e n t i fi c a t i o n o f
abnormal hemoglobins. Different media and different buffers
v a r y i n e f fi c i e n c y a n d a d e q u a c y f o r a l l s e p a r a t i o n s a n d
screening purposes. The simplest and most popular routine
methods employ cellulose acetate membranes at alkaline pH.
Cellulose acetate electrophoresis has come into general use
because it is easier and quicker to perform, provides sharp
resolution of hemoglobin bands and permits densitometric
quantitation and permanent storage of a transparent film.
Electrophoresis in citrate agar gel at acid pH is a useful
procedure for further fractionation and separation of the
hemoglobins that migrate together on cellulose acetate
electrophoresis.
in recent years, thin-layer isoelectric focusing in
polyacrylamide gels has been increasingly used in identifying
variant hemoglobins. isoelectric focusing may replace
conventional electrophoresis in the routine laboratory
investigation of hemoglobinopathies since this method is cost
e ff e c t i v e a n d o ff e r s s h a r p b a n d r e s o l u t i o n a n d s e n s i t i v e
detection of minor components.
Hemoglobin electrophoresis separates proteins according to
charge. Thus, its principal limitation is its inability to
detect amino acid substitutions that do not alter charge.
Hence, structurally different hemoglobins such as unstable
hemoglobins and hemoglobins with increased oxygen affinity
w i l l b e e l e c t r o p h o r e t i c a l l y i n d i s t i n g u i s h a b l e . H o w e v e r, m o s t
-7-
/^* presently known hemoglobins are
and, despite the limitations,
remains the method of choice
identification of the abnormal
electrophoretically abnormal,
hemoglobin electrophoresis
for the screening, detection and
hemoglobins.
REFERENCES
1. Huehns ER, Shooter EM: Hemoglobin. Sci Prog 52:353,
1964.
2. Fishleder AJ et al.: A practical approach to the
detection of hemoglobinopathies. Part I: The
introduction and thalassemia syndromes. Lab Medicine
18:368-372, 1987.
3. Fairbanks VF: Hemoglobinopathies and thalassemias.
Laboratory methods and case studies. Brian C. Decker, a
D i v i s i o n o f T h i e m e - S t r a t t o n I n c . , N e w Yo r k . G e o r g
Thieme Verlag Stuttgart, New York, 1980.
4. Schneider RG: Differentiation of electrophoretically
similar hemoglobins — such as S, D, G and P; or A2# C,
E and O — by electrophoresis of the globin chains.
Clin Chem 20:1111, 1974.
5. Chernoff Al, Horton BF: Laboratory approach to
diagnosis of hemoglobin abnormalities. Clin Obstet#^
Gynecol 12:76, 1969.
6. Huisman THJ et al.: Chromatography of hemoglobin types
on carboxymethyl cellulose. J Lab Clin Med 53:312,
1958.
7. Huisman THJ, Dozy AM: Chromatography behavior of
different human hemoglobins on anion-exchange cellulose
(DEAE cellulose). J Chromatogr 7:180, 1962.
8. Bar^lett RC: Rapid cellulose acetate electrophoresis,
part II. Qualitative and quantitative hemoglobin
fractionation. Clin Chem 9:325-329, 1963.
9. Briere RU, Golias T and Batsakis JG: Rapid qualitative
and quantitative hemoglobin fractionation: cellulose
acetate electrophoresis. Am J Clin Pathol 44:695-701,
1965.
1 0 . We a th e r a l l , D J : Th e Th a l a s s e m i a s . Wi l l i a m s , WJ ( e d ) :
Hematology. McGraw-Hill Book Co., New York, 1983.
11 . W i n t r o b e , M M : C l i n i c a l H e m a t o l o g y. L e a & F e b i g e r,
Philadelphia, 1981.
12. Henry JB: Clinical Diagnosis and Management by
L a b o r a t o r y M e t h o d s , 6 7 3 - 6 8 7 . W. B . S a u n d e r s C o . ,
Philadelphia, 1984.
13. Robinson AR, Robson M, Harrison AP et al.: A new
technique for differentiation of hemoglobin. J Lab Clin
Med 50:745-752, 1957.
14. Schneider RG, Barwick RC: Hemoglobin mobility in
citrate agar electrophoresis - its relationship to anion
binding. Hemoglobin 6:199-208, 1982.
15. Drysdale JW, Righetti P and Bunn HF: The segregation of
human and animal hemoglobins by isoelectric focusing in
polyacrylamide gels. Biochim Biophys Acta 229:42-50,
1971.
16. Basset P, Beuzard Y, Garel MC and Rosa J: Isoelectric
focusing of human hemoglobin. Blood 51:971, 1978.
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