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Letters to the Editor |
Department of Laboratory Medicine, and Pathology, Mayo Clinic, Rochester, MN
aAddress correspondence to this author at: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905.
To the Editor:
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 ß-chain variants and 297 variants of either the 
1- or 2-globin chain. Our laboratory has extensive experience in the evaluation of Hb variants by HPLC (Table 1
). All 268 variants included in Table 1
were evaluated by multiple methods and were confirmed by DNA sequencing methods; 
- and 
-chain variants are not included in this table. A minor difference is that we used the Bio-Rad VariantTM rather than the Variant-IITM 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 1
still represent only a minority of the total number of Hb variants known.
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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:
-chain variants that elute in the D window that would show a similar pattern, including 4 within 0.1 min of Hb G-Philadelphia. 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.
References
The following articles in journals at HighWire Press have cited this article:
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  L. B. Thomas, S. J. Agosti, M. A. Man, and S. M. Mastorides Screening for Hemoglobinopathies During Routine Hemoglobin A1c Testing Using the Tosoh G7 Glycohemoglobin Analyzer Ann. Clin. Lab. Sci., January 1, 2007; 37(3): 251 - 255. [Abstract] [Full Text] [PDF]
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