Hemoglobin, 33 (2):137–142, (2009)Copyright © Informa Healthcare USA, Inc.ISSN: 0363-0269 print/1532-432X onlineDOI: 10.1080/03630260902861956

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LHEM0363-02691532-432XHemoglobin, Vol. 33, No. 2, March 2009: pp. 1–5Hemoglobin

SHORT COMMUNICATION

A NEW b-CHAIN VARIANT: Hb STOCKHOLM [b7(A4)Glu®Asp]

CAUSES FALSELY LOW Hb A1C

A New b-Chain Variant: Hb Stockholm [b7(A)Glu→Asp]A.-C. Bergman et al.

Ann-Charlotte Bergman, Soheir Beshara, Iréne Byman,

Raja Karim, and Britta Landin

Department of Clinical Chemistry, Karolinska University Hospital, Stockholm, Sweden

� A new b-hemoglobin (Hb) variant, Hb Stockholm [b7(A4)Glu®Asp], is described. The variantwas characterized by mass spectrometry and DNA sequencing. The new variant is clinically silentbut interferes with Hb A1c quantification using ion exchange chromatography, causing a falselylow Hb A1c level when using the Bio-Rad VARIANT II™ System.

Keywords Hemoglobin variant, Hb A1c, Electrospray ionization mass spectrometry

To date, more than 900 hemoglobin (Hb) variants have been reportedin the HbVar database (1). A substantial amount of hitherto unknown variantshave been detected during routine quantification of glycated Hb (Hb A1c)using chromatographic methods (2,3). The degree of interference for agiven variant might vary significantly between different Hb A1c methodsused (4). Great care should be used to detect the cases where the Hb variantcoelutes with Hb A, while the glycated Hb variant is resolved from Hb A1c,since Hb A1c will in these cases be significantly underestimated. We heredescribe a new b-chain variant, which interfered with Hb A1c measurementusing the VARIANT II™ system (Bio-Rad Laboratories, Hercules, CA, USA).

The new variant was discovered during Hb A1c analysis of a sample froma Swedish patient. Although the software did not give any indication of thepresence of a variant fraction, the operator noted the presence of a smallunknown fraction (2%) between the Hb A1c and the Hb A0 peaks. In addition,

Received 10 October 2008; Accepted 17 December 2008.Address correspondence to Dr. Ann-Charlotte Bergman, Department of Clinical Chemistry L7:04,

Karolinska University Laboratory, Karolinska University Hospital, SE-171 76 Stockholm, Sweden; Tel:+46-8-51773251; Fax: +46-8-310376; E-mail: ann-charlotte.r.bergman@karolinska.se

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138 A.-C. Bergman et al.

a slight shoulder was noted on the downward slope of the Hb A0 peak. Thesample was reanalyzed using Hb A1c immunoassay (Cobas Integra 400;Roche Diagnostics Scandinavia AB, Bromma, Sweden) by which methodthe Hb A1c result was about 45% higher compared to that determined bythe VARIANT II™ chromatography (6.1 and 4.2%, respectively). The ensuinginvestigation supported the view that the higher value obtained by the non-chromatographic method was more reliable.

Routine hematological investigation, including complete blood countand reticulocyte count, revealed normal results. Quantifications of plasmairon, plasma transferrin, plasma ferritin, and calculation of transferrin satu-ration were also normal.

The sample was further analyzed by the method we use for quantifyingHb A2 and Hb F (VARIANT II™ b-Thalassemia Short Program; Bio-RadLaboratories), and an abnormal pattern was obtained. A Hb variantcomprising approximately 35% of total Hb was seen as a shoulder on thedownward slope of the Hb A0 peak (Figure 1). The findings suggested het-erozygosity for a b-globin variant.

FIGURE 1 Chromatograms of (a) normal and (b) patient sample using the VARIANT II™ b-ThalassemiaShort Program (Bio-Rad Laboratories).

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A New b-Chain Variant: Hb Stockholm [b7(A4)Glu®Asp] 139

The EDTA blood sample was further analyzed using electrospray ionizationmass spectrometry (ESI/MS) on a Q-TOF 2 mass spectrometer (Waters MSTechnologies, Manchester, Lancashire, UK). All parameters were set aspreviously described by Rai et al. (5). MS of intact globin chains revealed ab-variant with a mass approximately 14 Da lower than the normal b-chain(Figure 2). This mass difference is compatible with several different aminoacid substitutions, which will not cause a dramatic change of isoelectricpoint. After tryptic cleavage, performed essentially as in Rai et al. (6) withthe exception that the reduction and alkylation steps were omitted, avariant bT-1 peptide could be demonstrated (Figure 3). Sequencing of thevariant bT-1 peptide using tandem MS (5) demonstrated a Glu→Asp substi-tution at b7(A4) (Figure 4). This variant, which we have named Hb Stock-holm, was also confirmed by DNA sequencing of amplified genomic DNAusing standard conditions (7). The nucleotide substitution was demon-strated to be GAG ;>GAT (data not shown).

Two other b-variants due to single nucleotide substitutions at b7(A4)have been described before, i.e., Hb G-Siriraj (Glu→Lys) and Hb G-San José(Glu→Gly). As expected from the b7(A4)Glu position, none of the substitu-tions described today seem to affect the clinically relevant properties of theHb tetramer.

FIGURE 2 Mass spectra of intact globin chains from a sample containing the variant bX, which isapproximately 14 Da lower in mass than the normal b-chain.

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140 A.-C. Bergman et al.

The observation of small peaks between Hb A1c and Hb A0 when usingthe VARIANT II™ (Bio-Rad Laboratories) system is usually attributed toacetylated adducts of Hb. Reanalysis of theses samples using immunoassayusually indicates a slight Hb A1c overestimation using chromatography.However, in this particular case, the Hb A1c result obtained by immunoassaywas significantly higher than the result from chromatography, since themajor part of the glycated variant was not included in the Hb A1c duringintegration.

This finding demonstrates that great care must be paid to the evaluationof the chromatograms when using chromatographic methods for thequantification of Hb A1c. Although the widespread use of such methods hasled to an immense increase in the number of Hb variants identified, therealso needs to be an increasing awareness of the different interference pat-terns. Keen interest must be given to the task of evaluating the chromato-graphic profile, thereby avoiding false results for the clinically importantHb A1c level.

Declaration of Interest: The authors report no conflicts of interest. Theauthors alone are responsible for the content and writing of this article.

FIGURE 3 Part of the mass spectra of peptides following digestion with trypsin. The doubly protonatedvariant form of the bT-1 peptide is indicated as bXT-12+.

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A New b-Chain Variant: Hb Stockholm [b7(A4)Glu®Asp] 141

FIGURE 4 Normal tandem mass spectra (a) and patient sample (b), demonstrating the variant bT-1peptide carrying the Glu→Asp substitution at b7(A4).

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142 A.-C. Bergman et al.

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