High-performance liquid chromatography (HPLC)

High-performance liquid chromatography (HPLC)
In most cases, the diagnosis of a hemoglobin disorder can be made using
complementary protein chemistry methods such as isoelectric focusing and
HPLC. Additional testing using electrophoresis at both alkaline and acid pH may
be used to confirm a presumptive diagnosis.
Cation exchange HPLC is a complementary method used to identify and quantify
relative fractions of Hb F, Hb A2, Hb S, Hb C, Hb Barts and other Hb variants.
High-resolution separation of Hb variants permits unambiguous characterization
of Hb patterns and may also signal the presence of other electrophoretically
silent hemoglobins.
The HPLC assay utilizes the principles of cation-exchange for the separation and
determination of the relative percent of normal and abnormal hemoglobins. HPLC
is performed using a PolyCat A column run in a Bis-Tris/ KCN buffer with
increasing concentrations of NaCl until all globin proteins are eluted.
See EDTA-RT sample collection requirements.
Normal reference range:
Adult: AA, HbA2 2.4-3.5%, HbF <2.0%
Newborn: FA, Hb A2 <3.3%, HbF 60-90%