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Clinical Chemistry 45: 1317-1318, 1999;
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(Clinical Chemistry. 1999;45:1317-1318.)
© 1999 American Association for Clinical Chemistry, Inc.

Letters

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

Frédéric Cottona

a Author for correspondence. Fax 32 2 555 66 55; e-mail fcotton{at}ulb.ac.be

Béatrice Gulbis, Valérie Hansen and Françoise Vertongen

Department of Clinical Chemistry, Hôpital Erasme, Université Libre de Bruxelles, 808 Route de Lennik, B1070 Brussels, Belgium


To the Editor:

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.



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  		Figure 1. Separation of Hb A2 by cation-exchange HPLC.

(top), Hb A homozygote; (bottom), Hb D heterozygote.

To detect ß-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.


References

  1. Dash S. Hb A2 in subjects with Hb D [Letter]. Clin Chem 1998;44:2381-2382. [Free Full Text]
  2. Huisman THJ, Schroeder WA, Brodie AN, Mayson SM, Jakway J. Microchromatography of hemoglobins. III. A simplified procedure for the determinations of hemoglobin A2. J Lab Clin Med 1975;86:700-702. [ISI][Medline] [Order article via Infotrieve]
  3. Suh DD, Krauss JS, Bures K. Influence of hemoglobin S adducts on hemoglobin A2 quantification by HPLC. Clin Chem 1996;42:113-114. [Free Full Text]
  4. Cotton F, Lin C, Fontaine B, Gulbis B, Janssens J, Vertongen F. Evaluation of a capillary electrophoresis method for routine determination of hemoglobins A2 and F. Clin Chem 1999;45:237-243. [Abstract/Free Full Text]

Dr. Dash responds:

Sumitra Dash

Salmaniya Medical Complex, P.O. Box-12, Bahrain, Fax 973-279649
a Author for correspondence. Fax 32 2 555 66 55; e-mail fcotton{at}ulb.ac.be


To the Editor:

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.




The following articles in journals at HighWire Press have cited this article:


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K. Zurbriggen, M. Schmugge, M. Schmid, S. Durka, P. Kleinert, T. Kuster, C. W. Heizmann, and H. Troxler
Analysis of Minor Hemoglobins by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
Clin. Chem., June 1, 2005; 51(6): 989 - 996.
[Abstract] [Full Text] [PDF]

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 Clin. Chem.Home page
G. M. Clarke and T. N. Higgins
Laboratory Investigation of Hemoglobinopathies and Thalassemias: Review and Update
Clin. Chem., August 1, 2000; 46(8): 1284 - 1290.
[Abstract] [Full Text] [PDF]

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