Heterozygote Hemoglobin G-Coushatta as the Cause of a Falsely Decreased Hemoglobin A1C in an Ion-Exchange HPLC Method

Abstract

SummaryGlycated hemoglobin (HbA1c) is used for the assessment of glycemic control in patients with diabetes. The presence of genetic variants of hemoglobin can profoundly affect the accuracy of HbA1c measurement. Here, we report two cases of Hemoglobin G-Coushatta (HBB:c.68A>C) variant that interferes in the measurement of HbA1c by a cationexchange HPLC (CE-HPLC) method. HbA1c was measured by a CE-HPLC method in a Tosoh HLC-723 G7 instrument. The HbA1c levels were 2.9% and 4%. These results alerted us to a possible presence of hemoglobinopathy. In the hemoglobin variant analysis, HbA2 levels were detected as 78.3% and 40.7% by HPLC using the short program for the Biorad Variant II. HbA1c levels were measured by an immunoturbidimetric assay in a Siemens Dimen sion instrument. HbA1c levels were reported as 5.5% and 5.3%. DNA mutation analysis was performed to detect the abnormal hemoglobin variant. Presence of Hemoglobin G-Coushatta variant was detected in the patients. The Hb G-Coushatta variants have an impact on the determination of glycated hemoglobin levels using CE-HPLC resulting in a false low value. Therefore, it is necessary to use another measurement method.Keywords: Hb G-Coushatta (HBB:c.68A>C), HbA1c, cation-exchange HPLC Kratak sadr`ajGlikolizirani hemoglobin (HbA1c) koristi se za procenu glikemijske kontrole kod obolelih od dijabetesa. Prisustvo genetskih varijanti hemoglobina mo`e sna`no uticati na ta~nost merenja HbA1c. Ovde je prikazan izve{taj o dva slu ~aja gde je varijanta hemoglobina G-Coushatta (HBB:c.68A>C) uticala na merenje HbA1c metodom HPLC sa katjonskom izmenom (CE-HPLC). HbA1c meren je metodom CE-HPLC na instrumentu Tosoh HLC-723 G7. Nivoi HbA1c bili su 2,9% i 4%. Ovi rezultati ukazali su nam na potencijalno prisustvo hemoglobinopatije. Prilikom analiziranja varijante hemoglobina, otkriveni su nivoi HbA2 od 78,3% i 40,7% metodom HPLC uz kori{}enje kratkog programa za Biorad Variant II. Nivoi HbA1c mereni su pomo}u imunoturbidimetrijskog eseja na instrumentu Siemens Dimension. Dobijeni su nivoi HbA1c od 5,5% i 5,3%. Analiza mutacije DNK izvedena je kako bi se otkrile abnormalne varijante hemoglobina. Kod pacijenata je otkriveno prisustvo varijante hemoglobina G-Coushatta. Va rijante Hb G-Coushatta uti~u na odre|ivanje nivoa glikoliziranog hemoglobina pomo}u metode CE-HPLC, {to ima za posledicu la`no niske vrednosti. Dakle, neophodno je primeniti druge metode merenja.

HbA mutation causing false‑normal HbA1c results determined by HPLC in a patient with type 2 diabetes mellitus: A case report

“…The interindividual variation in HbA1c, defined as the glycation gap, is not attributable to mean blood glucose values and has been indicated to be associated with diabetic nephropathy ( 3 , 11 ). Several studies have described low HbA1c levels in patients with Hb variants ( 9 , 12 , 13 ). According to the laboratory test data obtained for the patient of the present study, numerous factors that may lead to low HbA1c levels were excluded, except for Hb variants.…”

“…Previous studies have indicated that this variant usually leads to underestimation of HbA1c and HbA variants constitute a well-known cause of analytical interference in the measurement of HbA1c levels ( 7-10 ). One of these studies suggested that the Hb G-Coushatta variant affected the determination of HbA1c levels, with the ion-exchange HPLC method measuring an HbA1c level that was 45% lower than that obtained by the immunoturbidimetric assay method ( 9 ). Thus, it may be concluded that the Hb G-Coushatta variant is the most likely cause of the HbA1c measured by HPLC appearing normal, which was clearly inconsistent with the clinical manifestations.…”

“…The HBB:c.9T>C variant is a silent mutation, resulting in no change in the charge of the patient's HbA β-chain; thus, the HBB:c.9T>C variant has no effect on the HbA1c level when detected by ion-exchange HPLC. However, previous studies have indicated that the Hb G-Coushatta variant usually leads to underestimation of HbA1c (7)(8)(9). HbA variants constitute a well-known cause of analytical interference in HbA1c measurements (10).…”