Capillary Electrophoresis Resolves Inconclusive HPLC Analysis for Hemoglobin Variants: a Study of Two Cases

Hb I and Hb Shaare Zedek are rare hemoglobin variants in the Chinese population. High-performance liquid chromatography (HPLC) is a widely used technique for screening of thalassemias and hemoglobin variants in Chinese primary hospitals. However, some rare hemoglobin variants cannot be effectively separated by HPLC. Here, we report one case of Hb I and one case of Hb Shaare Zedek which could not be detected by HPLC but required capillary electrophoresis (CE) in our hospital.

Two blood samples with high Hb F level were analyzed by HPLC as part of routine screening, and then globin genes were analyzed using Gap-PCR, PCR-Reverse dot-blot (RDB), and DNA sequencing. Subsequently, samples were analyzed by CE and results compared to HPLC.

In case 1, results were as follows: Hb F 16.9%, Hb A0 72.5% (failed to show the value of Hb A in HPLC) and Hb A2 2.2% for HPLC. No mutations were detected using Gap-PCR and PCR-RDB, but there was a mutation of codon (CD) 16 in the α2 globin gene (AAG>GAG, corresponds to Hb I) by DNA sequencing. CE showed Hb A2 1.9%, Hb A 74%, Hb I 24.1%. In case 2, results were as follows: Hb F 11.0%, Hb A0 76.6%, and Hb A2 4.7% for HPLC. It showed a CD 41 - 42 mutation of the β-globin gene (-TTCT) using PCR-RDB and the CD 56 mutation in α2/α1 globin gene (AAG>GAG, corresponding to Hb Shaare Zedek) with DNA sequencing. CE displayed Hb A2 4.5%, Hb A 84.6%, Hb F 0.4%, Hb Shaare Zedek 10.5%.

Hb I and Hb Shaare Zedek cannot be separated by HPLC because of co-elution with Hb F. However, they were identified and quantified using CE. As a general precaution, therefore, and owing to the existence of rare variants that may co-elute with normal hemoglobin fractions, it is recommended to use at least two complementary methods for the diagnostic detection of hemoglobin species.