Comparison of the characteristics of two hemoglobin variants, Hb D-Iran and Hb E, eluting in the Hb A2 window

Abstract

Background: Cation exchange-high performance liquid chromatography (CE-HPLC) is most commonly used to evaluate hemoglobin (Hb) variants, which elute in the Hb A2 window. This study aimed to assess prevalence of an uncommon Hb variant, Hb D-Iran, and compare its red cell parameters and peak characteristics with those of Hb E that commonly elutes in the Hb A2 window.

Methods: Generally, we assess abnormal Hb using CE-HPLC as the primary technique along with alkaline and acid electrophoresis. All cases with Hb A2 window >9%, as assessed by CE-HPLCs during 2009-2013, were selected.

Results: Twenty-nine cases with Hb D-Iran variant were identified-25 heterozygous, 2 homozygous, 1 compound heterozygous Hb D-Iran/β-thalassemia, and 1 Hb D-Iran/Hb D-Punjab. Overall prevalence of Hb D-Iran was 0.23%. Compared to patients with Hb E, those with Hb D-Iran had significantly higher Hb (12.1 vs. 11.3 g/dL, P=0.03), MCV (82.4 vs. 76.4 fL, P=0.0044), MCH (27.9 vs. 25.45 pg, P =0.0006), and MCHC (33.9 vs. 33.3 g/dL, P=0.0005). Amount of abnormal Hb (40.7 vs. 26.4%, P=0.0001) was significantly higher while retention time (3.56 vs. 3.70 min, P=0.0001) was significantly lower in Hb D-Iran than in Hb E.

Conclusion: Hb D-Iran peak can be easily missed if area and retention time of the Hb A2 window are not carefully analyzed. To distinguish between variants, careful analysis of peak area and retention time is sufficient in most cases and may be further confirmed by the second technique-alkaline electrophoresis.

Keywords: CE-HPLC; Hb A2 window; Hb D-Iran; Hb E.

Fig. 1. (A) CE-HPLC of a case of Hb D-Iran/β-thalassemia showing a predominant peak in the Hb A2 window with area of 66.0% and retention time of 3.61 min with elevated Hb F with area of 21.3%. (B) CE-HPLC of a case of Hb D-Iran/Hb D-Punjab with two predominant peaks, the first in A2-window with area of 47.3%, and the second in the Hb D window comprising 41.4% with retention times 3.58 and 4.14 min, respectively.

Fig. 2. (A) CE-HPLC of a case of heterozygous Hb D-Iran showing a peak in the Hb A2 window with area of 39.4% and retention time of 3.55 min. (B) CE-HPLC of case of heterozygous Hb E showing a peak in the Hb A2 window with area of 25.4% and retention time of 3.71 min. (C) Alkaline electrophoresis at pH 6.8 shows a case of heterozygous Hb E and heterozygous Hb D-Iran in lane 1 and 10 marked with dashed arrow and uninterrupted arrow respectively. (D) Acid electrophoresis at pH 6.0 shows a case of heterozygous Hb E and heterozygous Hb D-Iran in lane 1 and 10, marked with dashed arrow and uninterrupted arrow respectively. In both (C) and (D), lane 4 represents a case of Hb E/β-thalassemia, while lanes 5 and 6 are homozygous Hb E cases.