Unexpected Hemoglobin A1c Results

Summary

DOI: 10.1373/clinchem.2010.155804

Student Discussion

Alina-Gabriela Sofronescu, Laurie M. Williams, Dorinda M. Andrews, and Yusheng Zhu*

Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC.
*Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 171 Ashley Ave., MSC 908, Suite 309, Charleston, SC 29425. Fax 843-792-0424; e-mail [email protected]

Nonstandard abbreviations: Hb, hemoglobin; CE-HPLC, cation-exchange high performance liquid chromatography; Hb S, sickle cell Hb.

Case Description

A 52-year-old woman with a medical history of hepatitis B, hyperlipidemia, hypertension, anemia, and depression presented to the internal medicine clinic for a routine visit. Laboratory tests 3 months previously had revealed an impaired fasting glucose concentration of 5.9 mmol/L (106 mg/dL) [reference interval, 3.9 –5.6 mmol/L (70–100 mg/dL)]. Therefore, a hemoglobin (Hb)2 A1c analysis was performed. The initial Hb A1c evaluation by cation-exchange HPLC (CE-HPLC) (Hb A1c Program on the VARIANT IITURBOLink System; Bio-Rad Laboratories) showed an Hb A1c value of 115.8% (reference interval, 4.0%–6.0%) (Fig. 1). In an effort to determine if the unusual Hb A1c result was due to potential hemoglobinopathies, we performed an Hb variant analysis with the Bio-Rad VARIANT CE-HPLC β-Thalassemia Short Program. The analysis revealed the absence of Hb A and the presence of sickle cell Hb (Hb S) (37.4%), along with normal Hb A2 (3.2%) and Hb F (<1.0%) (Fig. 2). Also evident was another large peak (53.0%) that eluted earlier than Hb A, which we called P2. This study suggested the presence of an Hb variant with a chromatographic retention time virtually identical to that of Hb A1c, in addition to Hb S (Figs. 1 and 2). A subsequent Hb electrophoretic analysis at pH 6.0 (QuickGel Acid; Helena Laboratories) identified Hb S and another abnormal band with a mobility similar to Hb F (not shown).

Patient Follow-up
To identify the Hb variants, we investigated DNA sequences corresponding to the patient’s_β-globin genes. This analysis identified a substitution at codon 6 [GAG to GTG (Glu to Val)] on one allele, corresponding to Hb S, and a substitution at codon 1 [GTG to GCG (Val to Ala)] on the other allele, corresponding to Hb Raleigh. The presence of these hemoglobinopathies suggested that the spuriousHbA1c result obtained with the CE-HPLC method was due to the elution of Hb Raleigh, which has a retention time similar to that of Hb A1c.We evaluated the Hb A1c result with a turbidimetric inhibition immunoassay (Dimension® Clinical Chemistry System; Siemens) and obtained an Hb A1c value of 4.1%, which was not consistent with the impaired fasting glucose concentration of 5.9 mmol/L (106 mg/dL).

CE-HPLC chromatogram for Hb A1c analysis

Fig 2 Chromatogram of Hb variants analysis with the CE-HPLC beta-thalassemia short program

Questions to Consider

What are the various types of methods used for measuring HbA1c?
How do Hb variants interfere with each of these Hb A1c methods?
What actions should be taken when a spurious Hb A1c result is present?

Final Publication and Comments

The final published version with discussion and comments from the experts appears in the February 2011 issue of Clinical Chemistry, approximately 3-4 weeks after the Student Discussion is posted.

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