Laboratory Evaluation of
Hemoglobin Disorders
The differential diagnosis of hemoglobinopathies and
some thalassemias is made in the laboratory. Some of the
laboratory methods are described below.
Electrophoretic Procedures
Most of the
hemoglobin mutants can be identi-
fied by electrophoretically separating the hemoglobins on
cellulose acetate at pH 8.4-8.6 and on citrate agar at
pH 6.0-6.2. At pH 8.4, the hemoglobins are negatively
charged and migrate toward the anode (positive electrode)
in an electric field at a rate dependent on their net charge
(see Figure VII-1 and Table VII-1). Separation of the
hemoglobins with citrate agar as the support medium is
based on net charge and adsorption of the hemoglobin to
the supporting gel. The degree of adsorption may depend
on hemoglobin solubility and may be due to an impurity
in the agar rather than to the agar itself. Patterns obtained
on citrate agar with various hemoglobins are shown in
Figures VII-2 through VII-4.
The value of using these two electrophoretic systems
can be seen in the following examples.
(1) Hbs S, D, and G migrate together about halfway
between Hbs A and A
on cellulose acetate
electrophoresis. With citrate agar gels, Hbs D and G
migrate cathodally with HbA, while HbS continues
to migrate anodally separating it from the other
(2) Similarly, HbC comigrates with Hbs A
O, and
C-Harlem on cellulose acetate but is readily
separated from them on citrate agar.
(3) On cellulose acetate at pH 8.4, HbC-Harlem (two
mutations, each changing the net charge on the
tetramer by +2) migrates with HbC (one mutation,
change of +4). These two hemoglobins are well
separated from each other on citrate agar.
of classical
trophoresis are also useful for studying these proteins: iso-
electric focusing and electrophoresis of individual globin
chains, with or without the heme. These techniques can
be used to identify some hemoglobins that are not sep-
arable by electrophoresis of the complete hemoglobin
Nonelectrophoretic Procedures
It is apparent from the above discussion that many
hemoglobins with different amino acid substitutions
demonstrate identical electrophoretic mobilities. Methods
that rely on differences other than net charge are needed
to establish the identity of these hemoglobins. Definitive
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