Automated Measurement of Carbohydrate-Deficient Transferrin Using the Bio-Rad %CDT by the HPLC Test on a Variant™ HPLC System: Evaluation and Comparison with Other Routine Procedures

Alcohol & Alcoholism Vol. 43, No. 5, pp. 569–576, 2008 Advance Access publication 30 July 2008 doi: 10.1093/alcalc/agn058 ASSESSMENT AND DETECTION Automated Measurement of Carbohydrate-Deficient Transferrin Using the Bio-Rad %CDT by the HPLC Test on a VariantTM HPLC System: Evaluation and Comparison with Other Routine Procedures François Schellenberg1,∗ , Louise Mennetrey2 , Catherine Girre3 , Bertrand Nalpas4 and Jean Christophe Pagès1 1 Laboratoire de Biochimie et Biologie Moléculaire, Pôle de Biologie, Hôpital Trousseau, CHRU de Tours, France, 2 CCAA, CHRU, Tours, France, 3 Addictology Unit, Hôpital Fernand Widal, Paris, France and 4 INSERM U567, Hôpital Necker, Paris, France ∗ Corresponding author: Laboratoire de Biochimie et Biologie Moléculaire, Pôle de Biologie, Hôpital Trouseau, 37044 Tours, France. Tel: +33-2-47474684; Fax: +33-2-47474688; E-mail: (Received 9 November 2007; first review notified 3 January 2008; in revised form 22 May 2008; accepted 24 June 2008; advance access publication 30 July 2008) Abstract — Aims: In this study, we evaluated the new %CDT by the HPLC method (Bio-Rad, Germany) on a VariantTM HPLC system (Bio-Rad), checked the correlation with well-known methods and calculated the diagnostic value of the test. Methods: Intra-run and day-to-day precision values were calculated for samples with extreme serum transferrin concentrations, high trisialotransferrin and interfering conditions (haemolysed, lactescent and icteric samples). The method was compared with two routine procedures, the %CDT TIA (Bio-Rad, Hercules, CA, USA) and the CapillarysTM CDT (Sebia, France). A total of 350 clinical sera samples were used for a case-control study. Results: Precision values were better in high CDT and medium CDT pools than in low CDT pools. The serum transferrin concentration had no effect on CDT measurement, except in samples with serum transferrin <1 g/L. Haemolysis was the only interfering situation. The method showed high correlation (r2 > 0.95) with the two other methods (%CDT TIA and CZE %CDT). The global predictive value of the test was >0.90 at 1.9% cut-off. Conclusions: These results demonstrate that the %CDT by the HPLC test is suitable for CDT routine measurement; the results from the high-throughput VariantTM system are well correlated with other methods and are of high diagnostic value. INTRODUCTION Among the biological markers of alcohol abuse investigated over the past decades, CDT (carbohydrate-deficient transferrin) (Stibler, 1991) is considered the most accurate marker for the detection of alcohol abuse and possible relapse (Stibler et al., 1991; Helander 2003; Bortolotti et al., 2006). The main transferrin (Tf) glycoform comprises one polypeptide chain bearing two bi-antennary N-linked oligosaccharide chains, and each antenna is terminated with a sialic acid residue (i.e. four sialic acid residues). The overall number of sialic acid residues (Hatton et al., 1982) is theoretically between 0 (no oligosaccharide chain) and 8 (two tetra-antennae oligosaccharide chains terminated with four sialic acid residues on each). The Tf structure is modified following an alcohol-induced mechanism, resulting in the synthesis of Tf molecules lacking one (disialotransferrin) or both (asialotransferrin) glycans. As disialotransferrin (DiST) is the main modified glycoform (Stibler et al., 1979) and the increase of asialotransferrin (AST) can be observed only in samples with considerable amount of DiST, it was proposed as the target molecule for CDT standardization and calibration (Jeppsson et al., 2007). Over recent years, the use of CDT as a routine test for alcohol abuse has been hampered by laborious and time-consuming methods (Bortolotti et al., 2006). The methods based on microcolumn ion-exchange chromatography allow the global quantification of asialo-, monosialo-, disialo- and a variable amount of trisialotransferrin. The final result is expressed as the ratio between CDT and total Tf. More recently, new methods based on capillary zone electrophoresis (CZE) (Bortolotti et al., 2007; Schellenberg et al., 2007), high-performance liquid chromatography (HPLC) (Jeppsson et al., 1993, Helander and Bergström, 2006) and direct immunonephelometric measurement (Delanghe et al., 2007) have been developed that allow reliable and automated measurement of CDT. HPLC methodology allows the quantitative spectrometric determination of each Tf glycoform and has been proposed as a candidate reference method (Helander et al., 2003). The aim of this study was to evaluate the analytical features and diagnostic value of the commercial kit %CDT by HPLC on the routine high-throughput VariantTM HPLC system (Bio-Rad, Munich, Germany). MATERIALS AND METHODS Analytical procedures %CDT by the HPLC technique. The Bio-Rad %CDT by HPLC was run on a VariantTM HPLC system. This system comprises a two dual-piston pump system, a thermostated (20◦ C) 100-sample auto sampler and a column compartment (35◦ C) and a dual wavelength (460 nm and 690 nm) detector. The system is connected to the CDM software (Bio Rad). Before analysis, the samples are pre-treated by 30-min incubation with a ferrous and dextran sulphate solution, followed by 10-min (10,000 g) centrifugation, for both iron saturation and lipid precipitation. The supernatant is then ready for chromatographic analysis. The Tf fractions are separated using an ion-exchange column (600 tests) protected by a guard column (100 tests). The absorbance of the ferrous iron–Tf complex is measured at 460 nm; the secondary 690 nm wavelength is used for background noise reduction. The system calculates the area of each Tf glycoform peak following baseline integration from disialo- to pentasialotransferrin. The hexasialotransferrin peak is not integrated; asialotransferrin, when present, is integrated in a separate baseline mode (Fig. 1A and C). The area of each identified peak is expressed as the ratio of its  C The Author 2008. Published by Oxford University Press on behalf of the Medical Council on Alcohol. All rights reserved 570 Schellenberg et al. Fig. 1. Transferrin glycoform pattern obtained using the %CDT by the HPLC test on a VariantTM system. The Y-axis in arbitrary absorbance units. (A) Chromatogram of a homozygote transferrin-C control with a low CDT concentration (1.12%). (B) Chromatogram of a patient with suspected liver cirrhosis, low serum transferrin (1.23 g/L) and DST as a shoulder on the TriST peak, leading to erroneous integration (5.40%). (C) Chromatogram of an alcohol-abusing patient with elevated DST (15.60%) and AST (4.77%) levels. (D) Chromatogram of a serum with high (2 g/L) haemoglobin content. PST: pentasialotransferrin, TeST: tetrasialotransferrin, TriST: trisialotransferrin, DST: disialotransferrin, AST: asialotransferrin. surface to the total under-peak area. The total analysis time is ∼10 min. %CDT TIA technique. This assay is based on separation of the Tf glycoforms on micro anion ex (...truncated)