Effects of Nine Hemoglobin Variants on Five Glycohemoglobin Methods

Abstract

Most studies of the effects of variant hemoglobins (Hbs) on specific glycohemoglobin (gHb) methods have been case reports of a single variant Hb and one or two analytical methods (1)(2)(3)(4). Few studies have systematically examined multiple Hb variants with several widely used analytical methods. In this report, we describe the effects of nine heterozygous Hb variants on five gHb methods. A boronate affinity method was chosen as the comparative method because it has high specificity for glycated Hb and negligible interference by variant Hbs (5). Over 10 months, we studied 40 samples with nine variant Hbs; 38 were detected during routine gHb analysis by a cation-exchange method. Two samples (Hb E trait) were identified during routine Hb phenotype analysis. Samples were stored at 2–8 °C until analysis within 10 days of collection. Cation-exchange chromatography was performed on a Variant system with the Hb A1c program (Bio-Rad Laboratories) and on an A1c 2.2 Plus analyzer using a 3-min protocol (Tosoh Medics). Immunoassays were performed on a DCA 2000 (Bayer Corporation, Elkhart, IN) and on a Hitachi 717 with Tina-quant HbA1c II reagents (Roche Diagnostics, Indianapolis, IN). Boronate affinity analysis on a CLC 385 analyzer (Primus Corporation) served as the comparative method. All methods used the manufacturers’ reagents as recommended, had imprecision (CV) <5%, are certified traceable to the Diabetes Control and Complications Trial by the National Glycohemoglobin Standardization Program, and reported results as percentage of Hb A1c. Hb phenotype analysis was performed on all samples using a PVS99 system (Primus). This HPLC system uses a poly-aspartic acid cation-exchange column and a complex nonlinear salt and pH gradient at 40 °C. Rare phenotypes, including Grady, Hope, and Raleigh were identified using a poly-aspartic acid column with a nonlinear salt and pH gradient …