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Routine Hemoglobinopathy



Isoelectric focusing (IEF)
Cation exchange HPLC
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Routine Hemoglobinopathy

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.

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Normal reference range: 
Adult:  AA, HbA2 2.4-3.5%, HbF <2.0%
Newborn:  FA, Hb A2 <3.3%, HbF 60-90%

 

HPLC sample report:

HPLC