Interference of Hemoglobin D in Hemoglobin A2 Measurement by Cation-Exchange HPLC

We read the letter from Dr. Dash with interest (1), but we do not agree with her demonstration. We use the same cation-exchange HPLC (Bio-Rad Variant) as Dr. Dash, and we have concluded that hemoglobin (Hb) A2 cannot be quantified in the presence of Hb D by this method. As shown on Fig. 1 , an incomplete return to baseline between the Hb A2 and Hb D peaks does not allow accurate integration of Hb A2. To confirm this observation, we compared the Hb A2 values obtained by HPLC and anion-exchange microchromatography (Quik-Sep; Isolab) in nine samples from Hb D heterozygotes. The latter method separates hemoglobins on the basis of their pI, and the Hb A2 measurement is not influenced by Hb S or Hb D but is influenced by Hb C, Hb E, or Hb O (2). When anion-exchange chromatography was used, all results (mean, 2.6%; range, 2.4–2.9%) were within the reference interval (2.0–3.5%), whereas those obtained with HPLC were somewhat lower (mean, 1.8%; range, 1.2–2.0%). It has been shown that these methods give similar results in patients without hemoglobin variants (3)(4). In our opinion, the low Hb A2 values measured by HPLC are most probably attributable to integration problems only. The Hb A2 values measured by a specific method seem similar in Hb D heterozygotes and Hb A homozygotes. Separation of Hb A2 by cation-exchange HPLC. (top), Hb A homozygote; (bottom), Hb D heterozygote. To detect β-thalassemia carriers, a precise and specific method for Hb A2 measurement is needed. This is not the case with HPLC in Hb D heterozygotes or in Hb S heterozygotes (3)(4). In these cases, another method is required, such as anion-exchange chromatography or capillary electrophoresis (4), which in turn are not suitable in the presence of Hb C, Hb E, or Hb O. This illustrates that each laboratory should have an alternative method adapted to each case and keep in mind the performances of each method. Sumitra Dash Salmaniya Medical Complex, P.O. Box-12, Bahrain, Fax 973-279649 I am glad to note that Cotton et al. have corroborated our observation of low hemoglobin (Hb) A2 values in Hb D cases when measured by HPLC (Bio-Rad Variant). Our documentation was to emphasize the unreliability of Hb A2 measurements by this method in the presence of abnormal hemoglobins because the value not only can be spuriously increased, as in Hb S cases, but may also be decreased, as in Hb D cases. Thus, additional methods are required to quantify Hb A2 in the presence of abnormal hemoglobins.