P3 fraction: Effect on HbA1c values by HPLC

Introduction: The availability of the haemoglobin A1c test has been a major advance in diabetic care and its measurement has become an integral part for the management of diabetes. When glycated haemoglobin (HbA1c) is estimated by High Performance Liquid Chromatography (HPLC), one of the fractions that are eluted is known as “P3” fraction which is labelled as “degenerated haemoglobin”. The P3 fraction is not taken into account while estimating HbA1c or HbA values. Aim: To study the effect of P3 on the final reported value of HbA1c by HPLC and the significance of this fraction in the final chromatogram. The possibility that HbA1c fraction is under reported due to increase in P3 fraction is also examined. Materials and Methods: HbA1c, various fractions of HbA1 and P3 were estimated by HPLC method in 430 subjects. Patient data was divided into 3 groups on the basis of HbA1c% (group I - <6%, group II - 6 – 12%, group III - >12%). Result: P3% as well as P3 area increases as the HbA1c increases (p-value - <0.001). Statistical significant increase was seen as HbA1c% increases, P3% also increases and correlation (r = 0.6) became stronger with increasing HbA1c levels. HbA1a%, HbA1b% fraction increases with increase in HbA1c% and HbA1a area, HbA1b area also shows the same increase with increasing HbA1c%. Conclusion: From this study we conclude that sub fractions of glycated haemoglobin and P3 fraction influence the final reported value of HbA1c by HPLC. P3 fraction might indicate the possible presence of variant haemoglobin in the sample.