Validation, reference intervals and overlap performance of a new commercially available automated capillary electrophoresis assay for the determination of the major fraction of glycated haemoglobin (HbA1c) in dogs

Abstract

Haemoglobin A1c (HbA1c), the major fraction of glycated haemoglobin, is widely used for the diagnosis and monitoring of diabetes mellitus in human beings. However, there is a paucity of literature on the most reliable methods available for measurement of canine HbA1c. The aim of this study was to validate a new automated capillary electrophoresis assay for canine HbA1c, to generate a reference interval and to assess the overlap performance of the assay for the diagnosis of diabetes mellitus. Eighty-three blood samples treated with EDTA were included in the study, comprising 63 from healthy dogs and 20 from diabetic dogs. Linearity was assessed by mixing canine samples of known HbA1c percentage in different proportions, precision was assessed by repeated (n=8) measurement of five canine samples, and stability was assessed by measuring canine samples stored at 4°C for 96h and −20°C for 4 weeks. The robust method was used to determine the reference interval. The assay was demonstrated to be linear (R2=0.943). Intra-assay and inter-assay coefficients of variation (CVs) were 4.8% and 7.0%, respectively. CVs for blood samples stored at 4°C and −20°C were 7.2% and 11.2%, respectively. The reference interval was 0.6–2.7%. Dogs with diabetes mellitus had significantly (P<0.001) higher mean HbA1c (5.24±0.88%) compared to the reference population (1.64±0.55%), with no overlap between results. A HbA1c cut-off of 3.3% clearly differentiated diabetic from healthy dogs. The capillary electrophoresis assay was properly validated for canine HbA1c and the reference interval was determined, while the overlap performance of the assay was excellent.