A spurious haemoglobin A1c result associated with double heterozygote for haemoglobin Raleigh (β1[NA1]Val → Ala) and α+-thalassaemia

Abstract

Background: Accurate measurement of haemoglobin A1c (HbA1c) is useful for long-term glycaemic control in patients with diabetes. Many Hb variants can interfere with HbA1c measurement and cause inaccurate results. Methods: The subject was a 31-year-old Thai man who was discovered because of an unexpected HbA1c result; other diabetic parameters were within the normal range. Abnormal Hb was investigated using automated high-pressure liquid chromatography (HPLC) and a capillary electrophoresis system. Mutation analysis was done by cDNA sequencing, polymer- ase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and multiplex allele-specific PCR assays. Results: Evaluation of HbA1c by cation-exchange HPLC showed a value of 34.9% (reference interval, 4.0-6.0%), but a value of only 4.0% (reference value, 4.8-5.9%) was found with a turbidimetric immunoassay. Haematological analysis revealed a mild anaemia but other parameters were within the normal range. Hb-HPLC analysis demonstrated an unknown Hb variant (47.0%) separating from HbA (46.7%), but capillary electrophoresis identified no abnormal peaks. Mutation analysis identified the Hb Raleigh (β1[NA1]Val → Ala → Ala [GTG → GCG]) mutation in combination with an α+-thalassaemia, a hitherto undescribed associ- ation. The Hb Raleigh mutation could be detected by PCR-RFLP or a multiplex allele-specific PCR assay. Conclusions: Hb Raleigh can cause falsely increased HbA1c values on cation-exchange HPLC. Definitive diagnosis of this variant using combined Hb and DNA analyses is therefore essential.