1 Department of Clinical Neuroscience, Karolinska Institutet and
2 Division of Clinical Chemistry, Karolinska Hospital, Stockholm, Sweden
Received 19 December 2000; in revised form 1 March 2001; accepted 23 March 2001
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ABSTRACT |
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INTRODUCTION |
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The major advantage of CDT compared with traditional laboratory tests used to indicate prolonged harmful alcohol consumption and associated liver damage, such as the mean corpuscular volume of erythrocytes (MCV), and -glutamyltransferase (GGT), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in plasma or serum, is the higher specificity of CDT for alcohol exposure (Stibler, 1993
; Meerkerk et al., 1998
). However, a disadvantage is that, whereas measurement of the traditional alcohol markers has long been standardized, this is not yet the case for CDT. A multitude of analytical techniques, including various immunoassays, isoelectric focusing (IEF), high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE), and also different definitions of CDT (the transferrin isoforms covered vary between different methods, and the CDT value may be expressed as an absolute or relative amount) have been and are still in use. This is sometimes confusing and often hampers direct comparison of data between studies.
Immunological CDT assays are very convenient and time-saving when large numbers of samples are to be analysed on a routine basis. Most alcohol studies published to date have used the original CDTect radioimmunoassay which measures the sum of asialo, monosialo and part of disialo transferrin as an absolute amount (in units/l, with 1 unit of CDT equivalent to ~1 mg of transferrin). Having a very high or low total transferrin concentration might, however, lead to falsely high and falsely low CDT results when expressing the content as an absolute amount, but rarely when expressed as the ratio to total transferrin (Helander, 1999). Axis %CDT-TIA (variants of the %CDT-TIA and new %CDT immunoassays are distributed by Bio-Rad and Roche) turbidimetric immunoassay measures the sum of asialo, monosialo, disialo, and a portion (~50%) of trisialo transferrin as the relative amount to total transferrin. A specific drawback with this test is that having a very high or low relative amount of the trisialo transferrin isoform, which is found in a few per cent of the population (Helander et al., 2001
), might yield falsely high and falsely low %CDT-TIA results, respectively.
The main objective in this study was to demonstrate the performance of AxisShield new %CDT turbidimetric immunoassay. The new %CDT assay measures primarily the asialo, monosialo and disialo transferrin isoforms, and the result is expressed as the relative amount to total transferrin. Comparison was made with the previous %CDT-TIA and %CDT by the HPLC technique (%CDT-HPLC) (Jeppsson et al., 1993).
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MATERIALS AND METHODS |
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Methods
Measurements of serum %CDT with the new %CDT assay (all measurements were carried out with the microtitre application, but the analytical precision was also determined for the Cobas Mira application) and %CDT-TIA were carried out according to the manufacturer's instructions. Measurement of %CDT-HPLC was carried out according to Jeppsson et al. (1993), using valley-to-valley integration of the peaks representing asialo, monosialo, disialo and trisialo transferrins and baseline integration of isoforms with higher sialic acid content (mainly tetrasialo and pentasialo transferrin). Except for the performance study, single determinations were used with all methods. All CDT measurements were blinded in relation to the test results obtained with the other methods.
Statistics
The 2-test was used to test for normal distribution. Comparison between groups was made with t-test (parametric) or Wilcoxon test (non-parametric). Correlation tests were carried out with Pearson correlation coefficient (parametric) or Spearman rank correlation coefficient (non-parametric). Comparison of the overall sensitivity and specificity (threshold 60 g alcohol/day) of the CDT assays was made by receiver operating characteristics (ROC) curve analysis (Zweig and Campbell, 1993
). The statistical calculations were carried out using MedCalc software.
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RESULTS |
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The new %CDT values obtained with serum samples from healthy social drinkers are given in Table 2 and Figure 1
. The corresponding mean and median %CDT values in serum from healthy non-drinkers (Table 2
and Fig. 2
) were lower compared with healthy social drinkers, but the differences did not reach statistical significance (t = 1.802, P = 0.074). The %CDT values in abstinent alcohol patients were significantly higher, compared with the values in social drinkers (t = -2.225, P = 0.028). In the chronic heavy drinkers, the %CDT mean and median values were both markedly increased (Table 2
and Fig. 2
).
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Using ROC curve analysis, a cut-off of <2.8% was obtained for AxisShield new %CDT assay. However, based on the distribution of %CDT values in the healthy non-drinking (97.5th percentile = 2.9%) and socially drinking (97.5th percentile = 2.8%) populations (Fig. 2 and Table 2
), the reference value to be used in clinical practice was tentatively set at <3.0%. This threshold secured a very high specificity of the test results in the present material.
The %CDT values in healthy social drinkers obtained with the %CDT-TIA and %CDT-HPLC methods are given in Table 3. It should be noted that the volume of some serum samples was not sufficient to be used with all three %CDT methods. The %CDT-TIA reference value originally proposed by the manufacturer was <6.0%, but the present results, like some previous publications (Bean et al., 1997
; Viitala et al., 1998
), indicate that this cut-off may be reduced to <5.5% or even lower (97.5th percentile in this study = 5.2%) with retained high specificity. The routine reference value for the %CDT-HPLC method is <1.2% which is in accordance with the present results (97.5th percentile = 1.11%). When all samples were included, the new %CDT assay showed good overall correlation with %CDT-TIA (r = 0.986, P < 0.0001, n = 248) (Fig. 4
) and %CDT-HPLC values (r = 0.978, P < 0.0001, n = 222) (Fig. 5
).
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DISCUSSION |
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The proposed reference value for the new %CDT assay of <3.0% was slightly higher than that obtained by ROC curve analysis (<2.8%), and also higher than that proposed by the manufacturer in the Instruction Manual (<2.6%), which was based on a US population. By lowering the cut-off limit to <2.7% in our study, the sensitivity for recent heavy drinking among the chronic heavy drinkers was increased from 87 to 91%, but this gain in sensitivity was balanced by a reduction in specificity for the healthy social drinkers from 98 to 94%. It should be noted that the mean and median %CDT values obtained with the new %CDT assay (microtitre application) in our study were constantly 0.10.2% higher than those obtained in the US population (US data obtained from AxisShield ASA), both for healthy female and male social drinkers separately as for both groups combined. Whether this small difference resulted from methodological causes, or was due to differences between the Swedish and US control populations, remains to be elucidated.
The new %CDT immunoassay showed good overall correlation with the %CDT-TIA immunoassay and the %CDT-HPLC reference method. A major advantage of the new %CDT assay, compared with %CDT-TIA, is that it focuses principally on the asialo, monosialo and disialo transferrin isoforms, whereas %CDT-TIA also measures part (~50%) of trisialo transferrin. Hence, because trisialo transferrin normally makes up ~46% of total transferrin (Mårtensson et al., 1997; Dibbelt, 2000
), the %CDT values obtained with the new method were typically 23% lower than the corresponding %CDT-TIA values. Although including part of trisialo transferrin in the CDT measurement was originally suggested to improve diagnosis of elevated alcohol intake (Heggli et al., 1996
), other studies have found trisialo transferrin not to be correlated to chronic alcohol consumption (Mårtensson et al., 1997
), nor to the amount of disialo transferrin (Dibbelt, 2000
) which is considered the main CDT isoform. On the contrary, because the amount of trisialo transferrin may vary considerably from <2% to >10%, having a very high or low relative amount of trisialo transferrin might lead to falsely high and falsely low %CDT results, respectively, with the %CDT-TIA method in identification of alcohol abuse (Helander et al., 2001
). However, this risk will be markedly reduced with the new %CDT assay.
A common weakness with the immunoassays for CDT is that, unlike most methods based on HPLC, IEF and CE, they do not distinguish single transferrin isoforms. It is therefore difficult to establish whether the test result really indicates the true amount of CDT isoforms, or may be influenced by genetic B and D variants of transferrin, even though these are rare in most populations (Kamboh and Ferrell, 1987), or other chromatographic interference such as a very high or low amount of trisialo transferrin. As also demonstrated in this study, genetic transferrin variants may be an underlying cause of incorrect determination of CDT in detection of alcohol abuse with the immunoassays (Helander et al., 2001
). For this reason, although the immunoassays mostly yield accurate determination of CDT in clinical practice (Helander, 1999
), it is recommended to verify a positive analytical result to exclude the risk for inaccurate determination due to genetic variants. This should be done at least when a positive test result might lead to serious consequences for the individual, such as in regranting of driving licenses or workplace testing (Helander and Jones, 2000
; Bjerre et al., 2001
). In accordance with the decision of a recent meeting on CDT standardization (held in Berlin, Germany, in May 2000, chaired by Dr J.-O. Jeppsson and including clinical chemists and alcohol researchers from seven European countries), an ion-exchange HPLC method may be recommended for verification because, unlike IEF, the separation method is more similar to that used in the microcolumns in the CDT immunoassay.
In summary, AxisShield new %CDT turbidimetric immunoassay (the microtitre and Cobas Mira applications evaluated in this study) showed good overall analytical precision. The new assay also showed good correlation with the previous %CDT-TIA immunoassay and the %CDT-HPLC reference method, with the added advantage over %CDT-TIA that it measures primarily the asialo, monosialo and disialo transferrin isoforms. The reference value to be used in clinical practice was tentatively set at <3.0%, which gave a very high specificity (98%) in healthy social drinkers. The new %CDT assay can be recommended for routine use.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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