Identification of Hemoglobin Variants by HPLC

We read with interest the recent article by Joutovsky et al. (1) in which the authors evaluated ion-exchange HPLC for identification of hemoglobin (Hb) variants. The authors concluded that “confirmatory testing by electrophoresis can be eliminated in the majority of cases by use of retention time, proportion of total hemoglobin, and peak characteristics of HPLC.” We disagree with their conclusion, which was based on the evaluation of only 34 Hb variants. A recent search of the hemoglobin variant database (http://globin.cse.psu.edu) showed that there are currently 482 known β-chain variants and 297 variants of either the α1- or α2-globin chain. Our laboratory has extensive experience in the evaluation of Hb variants by HPLC (Table 11 ). All 268 variants included in Table 11 were evaluated by multiple methods and were confirmed by DNA sequencing methods; δ- and γ-chain variants are not included in this table. A minor difference is that we used the Bio-Rad Variant™ rather than the Variant-II™ system used by Joutovsky et al. (1). However, the retention times for these 2 systems are only slightly different, and the procedures are identical. The total number of variants listed in Table 11 still represent only a minority of the total number of Hb variants known. It is important to note that the retention time of an Hb variant can change with different lots of reagents (including the ion-exchange column). For example, in >10 years of experience with the Variant system, we have seen the following ranges in retention time (in min): Hb D-Los Angeles, 4.06–4.20; Hb Korle-Bu, 3.80–3.96; and Hb D-Iran, 3.39–3.58. It can be expected that the range in retention times of Hb variants attributable to manufacturing changes would be larger over a 10-year period than the 32-month period in the study by Joutovsky et al. (1). Although it is not possible to show our data in great detail, a few examples should suffice: Hb E elutes in the A2 window (range, 3.61–3.70 min). We have identified 18 other Hb variants, including 13 β-chain variants, that have a retention time that overlaps with the retention time range of Hb E. Hb S elutes in the S window (range, 4.38–4.50 min). We have observed 28 variants that have a retention time that overlaps with the retention time range of Hb S, including 10 other β-chain variants. In Fig. 2F of their article (1), the authors present an example of Hb G-Philadelphia that elutes in the D window and can be identified by a small additional peak that indicates the G2 variant. We have observed 9 other α-chain variants that elute in the D window that would show a similar pattern, including 4 within 0.1 min of Hb G-Philadelphia. In Fig. 2C, the authors present the HPLC chromatogram of Hb Twin Peaks, which shows a hump off the downward slope of the A0 peak. We have observed 5 other Hb variants that show a very similar hump on the downward slope. As the authors readily admit in their discussion (1), Hb New York (the second most common β-chain variant seen in Chinese) is not seen on HPLC but is easily identified on alkaline electrophoresis as a band slightly anodal to Hb A. Other examples of such variants include Hb P-Nilotic and Hb P-Congo. It should be mentioned that many of the variants included by Joutovsky et al. (1) were not identified by HPLC alone and required DNA sequencing performed in our laboratory for definitive identification. These data, which are part of our research efforts, were published in their article without our knowledge or consent. In summary, we agree with the authors that the retention time obtained by HPLC, as well the percentage of the variant and the appearance of the chromatogram, are very useful pieces of information that can help in the identification of many Hb variants. However, the overlap of retention times of variants precludes definitive identification of Hb variants by HPLC alone. HPLC should not be used as the sole means of identification of Hb variants, particularly for laboratories that analyze only small numbers of samples. Summary of Hb variant retention times by HPLC. Summary of Hb variant retention times by HPLC.