Affinity chromatography method for the measurement of glycosylated haemoglobin: comparison with two methods in routine use

Abstract

Blood glucose reacts with haemoglobin to form an unstable Schiffs base which is then converted by Amadori rearrangement to a stable keotamine [l]. This glycosylated haemoglobin (G Hb), formed over the life span of the erythrocyte, can be used as an indicator of long-term control in diabetic patients [2]. The glycosylation of haemoglobin and its clinical usefulness have been the subject of a number of reviews [3,4]. The methods available for the estimation of G Hb either measure the total HbAl (i.e. HbAla + b + c), or measure the main stable glycosylated fraction HbAlc after isolation from the other fractions. The former methods (including electrophoresis and mini-column ion-exchange chromatography) are more appropriate for use in routine clinical laboratories handling large work loads from diabetic clinics. However, agar electrophoresis [5] using commercially prepared agar plates and scanning densitometers is expensive although technically simple, and separation of G Hb by mini-column ion-exchange chromatography [6] is sensitive to pH and ionic strength of buffer and operating temperature. Affinity chromatography methods are claimed to be less sensitive to these factors [7]. We have investigated the affinity chromatographic medium Glycogel B (Pierce and Warriner, Chester, Cheshire, UK) and compared the results with those from commercially available kits based on agar electrophoresis and mini-column ion-exchange chromatography being used routinely in two other hospital laboratories.