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Mass Spectrometry: A Tool for Enhanced Detection of Hemoglobin Variants

Divisions of¹ Clinical Chemistry and Biochemistry and ² Hematology, Department of Pediatrics, University of Zurich, Zurich, Switzerland; ³ Functional Genomics Center Zurich, Zurich, Switzerland; and ⁴ IMD Institute for Medical and Molecular Diagnostics Ltd., Zurich, Switzerland.

^aAddress correspondence to this author at: Department of Pediatrics, University of Zurich, Division of Clinical Chemistry and Biochemistry, Steinwiesstrasse 75, 8032 Zurich, Switzerland. Fax 41-44-266-71-69; e-mail heinz.troxler{at}kispi.unizh.ch.

Background: More than 900 hemoglobin (Hb) variants are currently known. Common techniques used in Hb analysis are electrophoretic and chromatographic assays. In our laboratory, we routinely apply chromatographic methods. To ascertain whether Hb variants are missed with our procedures, we additionally analyzed all samples with mass spectrometry (MS).

Methods: Database evaluation was performed using all entries made in the Hb variant database HbVar, and possible Hb variants were calculated based on DNA variations. During a 5-year period, we analyzed 2105 lysates with cation-exchange HPLC (PolyCAT A column) and reversed-phase HPLC and additionally with electrospray ionization or MALDI-TOF MS. Globin chains were identified by their molecular masses.

Results: Database evaluation revealed that 43.2% of all possible Hb- and β-chain variants were found to date (considering only single-point mutations). Currently, 68.2% of the possible charge difference variants and only 28.7% of the neutral variants are found. Among 2105 Hb samples we identified 4 samples with Hb variants that were detected only with the MS method; 2 were new Hb variants (Hb Zurich-Hottingen and Hb Zurich-Langstrasse). With cation-exchange HPLC, 1 sample was found to be a β-thalassemia and was identified by MS to be a β-variant (Hb Malay). More common variants, such as Hb C, Hb D, and Hb E, and thalassemias could not be detected with the MS method.

Conclusions: Application of MS improves the sensitivity of Hb analysis. The combination of MS with electrophoretic and chromatographic methods is optimal for the detection of Hb variants.

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