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Letters to the Editor |
1 Laboratory, Helios Hospital Wuppertal, Wuppertal, Germany
2 Department of Gastroenterology, Endocrinology and Diabetelogy, Helios Hospital Wuppertal, University of Witten/Herdecke, Witten/Herdecke, Germany
3 Laboratory-Institute Dortmund, Dortmund, Germany
aAddress correspondence to this author at: Helios Hospital Wuppertal, Laboratory, Heusnerstrasse 40, D-42283 Wuppertal, Germany. Fax 49-202-8962044; e-mail nostendorf{at}wuppertal.helios-kliniken.de.
To the Editor:
Hemoglobin (Hb) A1c is widely used for retrospective monitoring of blood glucose concentrations for the past 2–3 months. Repeated measurements of Hb A1c are recommended in the management of patients with diabetes. Hemoglobinopathies are known to interfere with measurement of Hb A1c in various ways, depending on the test method used (1)(2). Either falsely increased or decreased values may occur. HPLC, usually performed on analyzers specifically designed for this purpose, is a common method to determine Hb A1c values relative to total Hb A. Alternatively, immunoassays or boronate affinity chromatography are used. The influence of Hb variants on HPLC assays is well recognized, and the interaction of numerous Hb variants have been described previously (3). We report here the findings in a case of Hb Stanleyville II that caused gross overestimation of Hb A1c in our routine HPLC assay.
Case report.
A 54-year-old man was transferred from the department of neurology to our department of internal medicine (gastroenterology, endocrinology, and diabetology) with the diagnosis of a diabetic polyneuropathy. The patient suffered from anesthesia and dysesthesia in both legs. He had been treated with 300 mg of alphaliponic acid twice a day. Within a year he had lost 30 kg of weight through diet and sports to improve his blood glucose concentrations. However, in the last few months his general condition had deteriorated with the symptoms mentioned above. We saw a 54-year-old man (height, 186 cm; weight, 75 kg) in moderately good general condition. Apart from the neurologic findings there were no other pathologies evident in the physical examination. The blood glucose ranged from 1 to 1.80 g/L under an intensive insulin therapy. The Hb A1c value, expected to be <7% under insulin treatment, was 37.9% in one sample and 39.4% in another, both assayed by HPLC.
HPLC was performed on a Variant hemoglobin testing system with a standardized Hemoglobin A1c Program (Variant; Bio-Rad). Briefly, whole blood is hemolyzed by addition of an acidic hemolysis solution. The hemoglobin solution is automatically injected on a cation-exchange column at 3-min intervals and eluted by a buffer gradient of increasing ionic strength. The hemoglobin content is measured photometrically in a flow cell at 415 nm. The resulting chromatograph (absorbance against time) is printed, and the different peaks are identified according to their elution times. Under these conditions, the patient sample gave the following results: Hb A0 = 55.2%; Hb A1c = 39.4%. Additionally, there was a peak labeled "unknown", accounting for another 4.1% (Fig. 1
). These results were considered improbable.
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For further investigation, hemoglobin electrophoresis and genotyping were performed. Genomic DNA was extracted from peripheral blood by the NucleoSpin® Blood QuickPure Kit (Macherey-Nagel). The hemoglobin 
1 gene (HBA1) was amplified by use of the Expand High Fidelity PCR System (Roche) with 0.4 µM each of primers C10 (5'-GATGCACCCACTGGCACTCCT-3') and BHB1 (5'-CCATTGTTGGCACATTCCGGGAYA-3', where Y is C or T). The amplified PCR product was purified by use of the High Pure PCR Product Purification Kit (Roche). Cycle sequencing of the purified PCR product was performed with the Big Dye Terminator (Ver. 1.1) Cycle Sequencing Kit (Applied Biosystems). The sequencing reactions were purified from residual dye terminators and run on an ABI Prism 3100 genetic analyzer. Analysis was performed with the Sequencing Analysis and Sequence Navigator Software (Applied Biosystems).
Hemoglobin genotyping revealed a point mutation of the HBA1 gene at codon 78 (lysine for asparagine; N78K), which is characteristic for Hb Stanleyville II (4). Bearers of Hb Stanleyville II are clinically normal. Hb Stanleyville II is not uncommon in some regions of Africa, but has only once been reported in a family of European descent (5). The index patient is of German origin.
To obtain a reliable value for the patient, we sent the blood to a neighboring laboratory where Hb A1c is measured by immunoassay (Roche Tinaquant; Roche). In this test, Hb A1c was 6.31%, 6.25%, and 6.37% (triplicate measurement from one blood sample).
We conclude that clinicians and laboratory staff should be made aware of the limitations of Hb A1c measurement arising from hemoglobin variants. The Hb A1c value in this case was clearly improbable, but a less pronounced false increase might have been overlooked. For these cases, an alternative test should be available.
References
asparagine replaced by lysine). Br Med J 1968;12:92-93.
75 [EF 7] Asn yields Lys) found in France. Acta Haematol 1975;53:56-59.[Medline]
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