CDTect-RIA AND CDTect-EIA FOR DETERMINATION OF SERUM CARBOHYDRATE-DEFICIENT TRANSFERRIN COMPARED

TORSTEN ARNDT 2 JUERGEN KROPF 2 RAGNHILD BRANDT 2 AXEL M. GRESSNER 2 ROLF HACKLER 2 MANFRED HEROLD 1 2 JOHANNES VAN PELT 0 2 OLA MARTENSSON 2 KARIN SALZMANN 1 2 MATHffiU H. VELMANS 0 2 0 St Maartens Gasthuis, Klinisch Chemisch en Hematologisch Laboratorium, PO Box 1926, NL-5900 BX Venlo. The Netherlands 1 Universitats Klinik fiir Innere Medizm. Hauptlabor , AnichstraBe 35, A-6020 Innsbruck, Austria 2 Klinikum der Philipps-Universitat Marburg , Abteilung fiir Klinische Cherrue und Zentrallaboratorium, BaldingerstraBe, D-35033 Marburg. Germany , 'Pharmacia & Upjohn Diagnostics AB, S-75182 Uppsaia. Sweden CDTect-RIA and CDTect-EIA for determination of serum carbohydrate-deficient transferrin (CDT) by radioimmunoassay and enzyme immunoassay respectively were tested for equality and precision in four European laboratories. For correlational studies, serum samples with CDT concentrations up to 130 U/l were analysed in accordance with a uniform trial schedule. The regression of CDT values obtained by the two procedures was computed for each laboratory using the method of Passing and Bablok. Slopes and intercepts of the regression functions did not differ significantly from the values 1 or 0, as proved by the corresponding 95% confidence intervals. Precision studies were computed using analysis of variance. For CDT concentrations at the upper reference limit for men, the within-day coefficients of variation (CVs) ranged between 0.7 and 6.4% (median 5.2%) for CDTect-RIA and from 4.3 to 9.2% (median 6.2%) for CDTect-EIA. The corresponding pure between-day CVs were 5.0-18.5% (median 9.8%) and 3.5-14.5% (median 10.9%). The study demonstrates the equality of CDT values obtained by CDTect-RIA and CDTect-EIA. According to this study, the two methods can be used interchangeably without getting fluctuating CDT values, e.g. in longitudinal studies. - INTRODUCTION Human serum transferrin shows a distinct microheterogeneity owing to: (a) altered protein moieties (genetic variants, e.g. transferrin-C-, transferrin-D-, and transferrin-B); (b) differing iron load (iron-free transferrin, Feo-transferrins; iron bound to the C-terminal or N-terminal binding site, Feic- o r Fe|N-transferrins; both binding sites loaded with iron, Fe2-transferrins); and (c) different carbohydrate chains with 0 to 8 sialic acid residues (asialo-, monosialo-, . . . , octasialo-transferrins) (de Jong and van Eijk, 1988; de Jong et al., 1990; van Noort et al., 1994) Using isoelectric focusing, Stibler et al. (reviewed in Stibler, 1991) found elevated concentrations of sialic acid-deficient transferrins (a-, *Author to whom correspondence should be addressed at: Woscientia, Institut fur Laboruntersuchungen Ingelheim GmbH, Hamburger Str. 1, D-55218 Ingelheim. Germany. mono-, and mainly disialo-transferrin) in the serum of alcoholics. The serum concentration of these isotransferrins, summarized as carbohydratedeficient transferrin (CDT) (Stibler, 1991), is used for detection and follow-up of chronic alcohol abuse (e.g. Allen et al., 1994; Anton and Moak, 1994; Sillanaukee et al., 1994; Conigrave et al., 1995; Gr0nbaek et al., 1995) as well as for the detection of the carbohydrate-deficient glycoprotein syndrome, a hereditary disorder of serum glycoprotein metabolism (e.g. Jaeken and Carchon, 1993, Stibler and Cederberg, 1993; van Pelt etal., 1996). Specific chemical reactions or antibodies for analysis of CDT are not yet available. Therefore, determination of CDT is usually done after elimination of Fe0- and Fei-transferrins by iron saturation in vitro, followed by separation of CDT-isotransferrins from higher sialylated non-CDT-isotransferrins by chromatographic (Jeppsson et al, 1993; Simonsson et al., 1996; Renner and Kanitz, 1997) or electrophoretic (e.g. Bean and Peter, 1994; Hackler et al, 1995; Arndt et al., 1997) methods. These procedures are usually sophisticated and not particularly suitable for large analysis series. In 1992 the first set of reagents for determination of CDT, CDTect-RIA (Pharmacia & Upjohn, Sweden), became available commercially. Later, %CDT (AXIS, Norway), CDTect-RIA (Pharmacia & Upjohn, Sweden) and %CDT-TIA (also called CDTri-TIA) (AXIS, Norway) were launched commercially. These four sets of reagents use (after in vitro transferrin iron saturation for elimination of F e r and Feo-transferrins) anionexchange microcolumns for separation of CDT and the other isotransferrins. Subsequent quantification of CDT in the column effluxes or eluates is carried out by radioimmunoassays (CDTect-RIA, %CDT), an enzyme immunoassay (CDTect-EIA) or turbidimetrically (%CDT-TIA), using antitransferrin-antibodies. The availability of sets of reagents for determination of CDT has accelerated the acceptance of CDT as the most specific marker of alcohol abuse available so far. However, these methods summarize different isotransferrins as CDT and report the results in different units, e.g. U/l for CDTectRIA and CDTect-EIA vs CDT/transferrin ratios for %CDT and %CDT-TIA. This complicates comparison of CDT values and diagnostic specificities and sensitivities obtained in different studies. The majority of clinical studies published so far have used the CDTect-RIA for quantification of serum CDT. However, working with radioactive material requires special laboratory equipment and expensive disposal of the contaminated material. An alternative method to CDTect-RIA could be the CDTect-EIA, since these two assays differ only in the final CDT quantification step (radioimmunoassay vs enzyme immunoassay), but not in the isotransferrin fractionation procedure (the same isotransferrins are summarized as CDT) or the reported units (U/l). The aim of our study was to investigate the equality and precision of CDT values obtained by CDTect-RIA and CDTect-EIA, since appropriate data on these aspects are not available. With the information presented here, we hope to contribute to a better comparability of CDT values obtained using both methods. MATERIALS AND METHODS All procedures were in accordance with the Helsinki Declaration of 1975, as revised in 1983. Materials All materials were delivered with the CDTectRIA and CDTect-EIA test kits (Pharmacia & Upjohn, Uppsala, Sweden). Blood was drawn after overnight fasting into tubes containing a gel separator (Gel-Monovette Sarstedt, Nlimbrecht, Germany). After clotting at room temperature for 30 min, serum was obtained by centrifugation (2000 g for 10 min at 4C). Serum aliquots were stored at -70C and sent to the participating laboratories on dry ice. Methods Assay of serum CDT concentration. Serum concentration of CDT was determined by CDTect-RIA and CDTect-EIA in accordance with the instructions of the manufacturer. In short, 50 u.1 of serum sample were mixed with ferric citrate solution (200 ul) and elution buffer (1 ml) for in vitro transferrin iron saturation (elimination of Fe0- and Fertransferrins). An aliquot (500 ul) of this mixture was applied to the top of the ani (...truncated)