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Routine Hemoglobinopathy


Isoelectric focusing (IEF)
Cation exchange HPLC
Cellulose acetate electrophoresis
Citrate agar electrophoresis
Erythrocyte Protoporphyrin (FEP)
Pitted red blood cells (Pitcount)
Sickle Solubility Test (SST)

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Clinical Diagnostics and Research tools

Routine Hemoglobinopathy Testing

In most cases, the diagnosis of a hemoglobin disorder can be made using complementary protein chemistry methods such as IEF and HPLC.

Cellulose acetate electrophoresis (CAE) at alkaline pH (8.2 to 8.6) permits separation of the major hemoglobins (Hb A, Hb F, Hb S/D, Hb C/E/O-Arab, Hb G-Philadelphia) based on the relative mobilities of variant hemoglobins.

Citrate agar acid electrophoresis at acid pH (6.0 to 6.2) permits separation of the major hemoglobins based on the relative mobilities of Hb C from Hb E and Hb O, as well as Hb S from Hb D and Hb G.

Isoelectric focusing (IEF) on agarose gels separates hemoglobin (Hb) fractions and variants based on their isoelectric points.  Hb A and Hb F are clearly resolved by this method.  Hb C can also be distinguished from Hb E and Hb O, and Hb S can be distinghuished from Hb D and Hb G.

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.

Additional testing using electrophoresis at both alkaline and acid pH may be used to confirm a presumptive diagnosis.

The Free Erythrocyte Protoporphyrin (FEP) test measures the non-complexed, non-heme protoporphyrin concentration in blood.  This test alone is not sensitive enough to diagnose iron deficiency, but an elevated FEP in conjunction with a low MCV is suggestive.

Counts of  pitted red blood cells (Pit count) can be used to assess splenic reticuloendothelial function in patients with sickle cell disease.  There is an inverse relationship between the pit count and the degree of splenic dysfunction in SCD.

The Sickle Solubility Test (SST) is used to screen for the presence of sickling hemoglobins. A positive result must be confirmed by another method (HPLC or electrophoresis) for the presence of Hb S and to distinguish Hb AS (carrier state) from Hb SS (sickle cell disease).   Other insoluble hemoglobins, such as Hb C-Harlem, will also give a positive result.

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Clinical Diagnostics and Research tools