Department of Clinical Neuroscience, Karolinska Institutet, and Division of Clinical Chemistry, Karolinska Hospital, Stockholm, Sweden
Received 23 November 2001; in revised form 7 February 2002; accepted 11 February 2002
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ABSTRACT |
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INTRODUCTION |
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Immunological assays for CDT are very convenient and time-efficient in routine laboratories with a high specimen throughput. The immunoassays utilise an initial separation of the CDT isoforms on anion-exchange chromatography minicolumns. Most studies published to date have used the CDTect® radioimmunoassay (Axis-Shield ASA), which measures the sum of asialo, monosialo and a minor part of disialo transferrin in units, or mg, per litre of serum. However, having an abnormally high or low serum total transferrin concentration might render falsely high and falsely low CDT results, respectively, in identification of heavy drinking when expressing the CDT content as an absolute amount, but rarely in percentage of total transferrin (Helander, 1999). A new version of the AxisShield %CDT immunoassay for quantification of CDT normalized to the total transferrin concentration was recently introduced (Helander et al., 2001b
). The new assay measures primarily the asialo, monosialo and disialo transferrin isoforms (i.e. the classical' CDT isoforms), and was found to perform favourably compared with the CDTect and previous %CDT immunoassays (Anton et al., 2001
; Helander et al., 2001b
).
This multicentre trial was designed to document the inter-laboratory transferability and agreement of results for five instrument applications of the AxisShield %CDT new version turbidimetric immunoassay.
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MATERIALS AND METHODS |
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The study was carried out with two different production lots (A and B) of the AxisShield %CDT assay manufactured by AxisShield ASA (Oslo, Norway). Quantification was performed following the manufacturer's instructions in the package insert. The samples compared were two lyophilized %CDT kit controls (low and high %CDT level; reconstituted in deionized water) and three pools of authentic sera. Serum sample 1 was selected to have a low %CDT value, serum 2 to have a value close to the cut-off of the method, and serum 3 to have an elevated %CDT value. Tentative target %CDT values for the controls and serum samples set by a high-performance liquid chromatography (HPLC) method (Bean et al., 1998) were as follows (data from AxisShield ASA): low control = 2.2%, high control = 5.2%, serum 1 = 1.7%, serum 2 = 2.5%, and serum 3 = 4.0%. The reconstituted controls were stored at 28°C and the serum samples at 18°C or below until testing.
Calibration of the instruments was done for each run using the %CDT Calibrator set. Prior to the main assay precision study, the laboratory personnel carrying out the assays underwent a familiarization training where the kit controls of lots A and B were tested in six replicates per day on 3 days within a 10-day period. The acceptable range for %CDT results was defined as the target value ± 20%, and the total coefficient of variation (CV) should be <10%. Based on these results, individual laboratory reference ranges for the low and high kit controls were established as the mean values ± 3 SD.
Once the familiarization training was successfully completed, the assay precision study was started in which controls and serum samples were run with both kit lots in four replicates per day on 4 days (separate aliquots of the sera were used) within a 10-day period. Finally, the reproducibility of the instruments was determined by measuring one low and one high kit control in nine replicates in one run. Statistical calculations were carried out using MedCalc software.
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RESULTS AND DISCUSSION |
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The %CDT mean values obtained with the different instrument applications of the immunoassay differed from the tentative target values set by an HPLC method. For example, the %CDT mean values for all low and high controls (lots A and B combined) were 2.1 and 4.9%, respectively, compared with target values of 1.7 and 5.2%, and for the serum samples the immunoassay consistently produced higher values (Table 1). However, that %CDT results obtained by HPLC may deviate from the corresponding immunoassay values is well known (Bean et al., 1998
; Helander et al., 2001a
,b
). This may result from differences in the separation of transferrin isoforms between the HPLC analytical columns and the immunoassay minicolumns, and also the integration mode (baseline or valleyvalley) used in the HPLC chromatograms.
The relative imprecision (CV) of the %CDT results between all study sites and instrument applications ranged from 4.7 to 15.1%. As expected, the highest CV values were obtained at low %CDT values (i.e. low kit control and serum sample 1) (Table 1). When the outliers were omitted from these calculations, the CV values were typically <10% at all %CDT levels. In absolute terms, %CDT values of 2.43.3% (outliers omitted) were obtained for serum sample 2 which was selected to have a value close to the cut-off limit of the method. Accordingly, irrespective of using a cut-off of <2.6% (AxisShield %CDT Instruction manual; Anton et al., 2001
) or <3.0% (Helander et al., 2001b
), %CDT values at, below, as well as above, the threshold limit were obtained for this sample. In doubtful cases of a high %CDT result with the immunoassays, it is therefore recommended that the result be verified by HPLC, or possibly capillary electrophoresis or isoelectric focusing, to rule out any interference by genetic variants and isoform types of transferrin (Helander et al., 2001a
). This is especially important whenever a positive test result could lead to serious consequences for the individual.
In summary, this standardized multicentre investigation evaluated the transferability and agreement of results for five instrument applications (Immage, BN A, BN II, Cobas Mira, and Microtiter) of the AxisShield %CDT new version immunoassay. The test results showed an overall good agreement both within and between different instrument models, although it was also demonstrated that the analysis might be biased due to site performance. Ongoing activities for the use of certified reference materials and proficiency testing, and development of an HPLC reference method, will help to improve further the quality of intra- and inter-laboratory test results and, hence, the value of CDT as an alcohol marker. To conclude, the evaluated %CDT applications for some of the most widespread chemistry analyser systems showed adequate performance, and may thus be used interchangeably in routine quantification of %CDT in serum.
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ACKNOWLEDGEMENTS |
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This study was supported in part by Axis-Shield ASA. The author has no other financial involvement with Axis-Shield related to this work, and have complete independence in the interpretation of data and writing of the report.
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FOOTNOTES |
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* Alcohol Laboratory, L7:03, Karolinska Hospital, SE-171 76 Stockholm, Sweden.
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REFERENCES |
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