Effects of 49 Different Rare Hb Variants on HbA1c Measurement in Eight Methods

Abstract

Background: Previous studies have shown interference with HbA1c measurement from the 4 most common heterozygous Hb variants (HbAS, HbAE, HbAC, and HbAD) with some assay methods. Here we examine analytical interference from 49 different less common variants with 7 different HbA1c methods using various method principles. Methods: Hb variants were screened using the Bio-Rad Variant or Variant II beta thal short program, confirmed by alkaline and acid electrophoresis, and identified by sequence analysis. The Trinity ultra2 boronate affinity high-performance liquid chromatography (HPLC) method and Roche Tinaquant immunoassay were used as primary and secondary comparative methods, respectively, since these methods are least likely to show interference from Hb variants. Other methods included were the Tosoh G7 and G8, Bio-Rad D-10 and Variant II Turbo, Diazyme Enzymatic, and Sebia Capillarys 2 Flex Piercing. To eliminate any inherent calibration bias, results for each method were adjusted using regression verses the ultra2 with nonvariant samples. Each method’s calibration-adjusted results were compared and judged to be acceptable if within the 99% prediction interval of the regression line for nonvariant samples. Results: Almost all variant samples were recognized as such by the ion-exchange HPLC methods by the presence of abnormal peaks or results outside the reportable range. For most variants, interference was seen with 1 or more of the ion-exchange methods. Following manufacturer instructions for interpretation of chromatograms usually, but not always, prevented reporting of inaccurate results. Results: Laboratories must be cautious about reporting results when the presence of a variant is suspected.

New trends in the long and puzzling history of HbA1c

“…For example, Ji et al demonstrate here that β-thalassemia can still lead to misinterpreting HbA 1c results, and highlight the importance of the optimal characterization and knowledge of every method with respect to interferences [17]. Moreover, less common variants may induce unexpected interferences with different impacts on the methods, as recently demonstrated elsewhere by Little et al [18]. This is also illustrated here by the studies of Camacho Benitez et al [19] and Bots et al [20] in this issue.…”

Haemoglobin J-Baltimore can be detected by HbA1c electropherogram but with underestimated HbA1c value

“…The analytical impact of this rare variant is well known on HbA 1c measurement by HPLC ( 5 , 6 ). Only a few recent publications have been published on capillary electrophoresis (CE) methods ( 7 , 8 ). Barakat and Roberts explained that the very light load change due to the presence of a glycine in position 16 of the haemoglobin β-chain does not allow clear separation of HbJ or HbJ 1c from respectively HbA or HbA 1c with HPLC methods and subsequent risk of underestimating HbA 1c value ( 5 , 6 ).…”

“…Rhea reported the same results between HPLC method, known with a negative bias, and CE ( 7 ). Little considered that the HbA 1c results of the two samples tested were accurate with CE method ( 8 ). Here, we clearly observe that we are able to identify the presence of all the fractions of interest of HbA, HbA 1c , HbJ and HbJ 1c on electropherogram obtained by C2FP analyser.…”

HbA1C as a marker of retrograde glycaemic control in diabetes patient with co‐existed beta‐thalassaemia: A case report and a literature review

“…20 Thus, determining appropriate measures that can deal with the validation of results suspected of the presence of Hb variants is of interest. 24 Developing more appropriate measures requires an understanding of the HbA1C measurement methods, as well as the information on the presence of Hb variants. 20,21,25 Even more factors that could interfere with HbA1C measurement are being identified.…”