Alcohol and Alcoholism Vol. 38, No. 3, pp. 270-275, 2003
© 2003 Medical Council on Alcohol
MULTICENTRE EVALUATION OF A NEW ASSAY FOR DETERMINATION OF CARBOHYDRATE-DEFICIENT TRANSFERRIN
Psychiatrische Klinik der Ludwig-Maximilian-Universität, München, Germany,
1 Zentrallabor, Otto-Wagner-Spital, Wien, Austria,
2 Roche Diagnostics GmbH, Mannheim, Germany,
3 Department of Clinical Neuroscience and Clinical Chemistry, Karolinska Institute, Stockholm, Sweden,
4 Department of Clinical Chemistry, Ziekenhuis Rijnstate, Arnhem and
5 Department of Clinical Chemistry, Ziekenhuizen Noord-Limburg, Venlo, The Netherlands
Received 30 July 2002; in revised form 12 December 2002; accepted 6 January 2003
 |
ABSTRACT |
|---|
 TOPFOOTNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
Aims: The analytical performance of the new Tina-quant
% carbohydrate-deficient transferrin (%CDT) was assessed in a multicentre study on Roche/Hitachi analysers. Methods: Intra-assay/total precision studies revealed median coefficients of variation (CVs) of 4.7/7.4% within the sites. Precision between the sites was proven using a serum panel. Results: Inter-laboratory CVs from 6.3 to 10.7% were obtained. The results of the participating laboratories compared well with high-performance liquid chromatography-UV technique fulfilling the criteria of a reference method for %CDT determination (slope 1.03, intercept -0.09% CDT, correlation 0.984). Good agreement was also found with the Axis-Shield %CDT microtitre test. Conclusions: Data from this study indicate that reliable, well standardized %CDT results are obtained using the new assay. |
INTRODUCTION |
|---|
|
TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
There is broad consensus that carbohydrate-deficient transferrin (CDT) is an excellent biochemical marker for excessive alcohol consumption (Burke et al., 1998
; Lieber, 1999; Salaspuro, 1999; Allen et al., 2000, 2001; Mundle et al., 2000; Reynaud et al., 2000; Scouller et al., 2000; Anton, 2001; Sillanaukee et al., 2001; Walter et al., 2001; Forsberg et al., 2002). Besides 
-glutamyl transferase (GGT), CDT is reported to be the most specific marker of chronic alcohol misuse and to be suitable for monitoring alcohol abstinence (for review see Allen et al., 2001; Anton, 2001; Arndt, 2001; Himmerich et al., 2001; Sillanaukee and Olsson, 2001). Recent data from the WHO/ISBRA collaborative project suggest CDT to be slightly better than GGT in detecting high-risk alcohol consumption in men (Conigrave et al., 2002).
CDT can be measured by various commercial immunoassays (Hackler et al., 2000; Anton et al., 2001; Arndt, 2001; Myrick et al., 2001; Schellenberg et al., 2001; Turpeinen et al., 2001; Helander, 2002). International consensus has been reached that these tests should be calibrated against a sensitive high-performance liquid chromatography (HPLC) method separating the a-, mono-, and di-sialo transferrin isoforms (Helander et al., 2001a; Arndt et al., 2002). The results should be expressed as a percentage of total transferrin. The recently developed Tina-quant %CDT 2nd generation assay (Roche Diagnostics GmbH) fulfils these criteria—in contrast with the previous Tina-quant %CDT assay which additionally detects about 50% of tri-sialotransferrin. Aside from adaptation of the new test to an internationally acknowledged standardization procedure, the Tina-quant %CDT 2nd generation test assay is adapted for runs on high-throughput Roche/Hitachi systems in combination with clinical chemistry and drug testing.
It was the goal of the present multicentre study to test the technical performance characteristics of this new assay under routine conditions. The study was performed in five laboratories (Table 1) according to a standardized protocol.
|
|
MATERIALS AND METHODS |
|---|
|
TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
In the Tina-quant
%CDT 2nd generation assay, CDT isoforms are separated on ion-exchange microcolumns after iron saturation. The eluate collected from the columns containing CDT as well as total transferrin (fraction before column separation) are determined turbidimetrically. Two different instrument applications are used for measuring either CDT or total transferrin, which use the same six-point calibration curve generated automatically on Roche/Hitachi systems. The percentage of CDT is calculated from these results. The measuring range extends from 1.0 to 24 mg/l transferrin. %CDT Control Set (Roche Diagnostics GmbH) is recommended for quality control. The method is standardized against an HPLC technique with UV detection (Axis-Shield in-house method), which fulfills the requirements of a reference method for %CDT in serum (Helander et al., 2002).
The main difference between the Tina-quant %CDT 2nd generation assay and the previous Tina-quant %CDT test is that tri-sialotransferrin is not included in the CDT fraction. This is achieved by modification of the column separation step. Acceleration of column separation is an additional benefit of this modification. In order to overcome the frequently discussed lack of standardization for CDT immunoassays, the Tina-quant %CDT 2nd generation assay uses the same standardization procedure as the new Axis-Shield %CDT test (microtitre version) and its different instrument applications (Helander, 2002). In agreement with this method, the upper limit of the reference range is claimed to be 2.6% by Roche Diagnostics GmbH. The recommended cut-off of the previous Tina-quant %CDT test is 6.0%.
The Axis-Shield %CDT test (microtitre version) was used as immunochemical comparison method in four laboratories. CDT detection has to be performed manually in this assay. In contrast, the Tina-quant %CDT 2nd generation assay is applied to fully automated Roche/Hitachi systems, which allow a high throughput of CDT measurements in combination with routine clinical chemistry.
In the method comparison studies described here, two HPLC methods (Clin-Rep HPLC, RECIPE plc.; modified Jeppson’s HPLC) were included, each of which was employed in one laboratory. Statistical validation of the method comparison studies was performed by Passing–Bablok regression analysis (Passing and Bablok, 1983).
Studies on the analytical range and on interferences of the new test were performed in the laboratory of the manufacturer. The limit of detection was estimated by 21-fold measurements of 0.9% saline using the CDT application and reported as the mean + 3 SD of these measurements. Linearity was verified for the CDT and the total transferrin application by serial dilutions of samples with approximately 24 mg/l transferrin with 0.9% saline according to a published procedure (Bablok, 1993).
Endogenous interferences according to the Glick model (Glick et al., 1986), as well as drug interferences, were tested on Roche/Hitachi 911. Potential interfering drugs were spiked into a human serum pool at 5–10-fold concentrations of the maximum daily dosage.
|
RESULTS AND DISCUSSION |
|---|
|
TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
Precision
Intra-assay precision (10 replicates) and total precision (three replicates on 10–21 days) were determined in controls and human pool sera. The results are shown in Fig. 1
. The median intra-assay/total coefficients of variation (CVs) at low %CDT values (1.8–2.4% CDT) are 6.2/9.7%, and 4.0/5.3% at higher %CDT values (3.5–5.0% CDT), respectively.
|
Inter-laboratory survey
Nine frozen serum samples with target values assigned by the HPLC-UV method (Helander et al., 2002
) were distributed to all participants and measured in singlets in three different runs. Two additional Roche/Modular P analysers at Roche Diagnostics GmbH were included in this experiment. The median recovery of the assigned values using the Tina-quant %CDT 2nd generation test is in the range from 93 to 110% (Table 2). Considering all measurements in the five laboratories, inter-laboratory CVs of 9.4–10.7% were obtained at lower %CDT values from 1.7 to 2.8%, whereas CVs of 6.3 to 7.4% were found at higher %CDT values from 3.0 to 6.6%, respectively. The comparability between the HPLC method (x) and the Tina-quant %CDT 2nd generation test (y) was judged by Passing–Bablok regression analysis (Fig. 2). Very good agreement, with a slope of 1.03, a negligible intercept of -0.1% CDT and a correlation coefficient (r) of 0.984, was obtained.
|
|
Method comparison studies using routine samples
The method comparison studies between Tina-quant
%CDT 2nd generation and Axis-Shield %CDT on microtitre plates carried out in four laboratories are summarized in Table 3. A good comparability between the methods was found with slopes from 0.98 to 1.11, intercepts from 0.0 to 0.2% CDT, and correlation coefficients from 0.960 to 0.985. One representative example is shown in Fig. 3a. Acceptable correlation coefficients, but systematic deviations were obtained in method comparison studies between the Tina-quant %CDT test and two HPLC procedures. It has been stated that the Clin-Rep HPLC method lacks the detection of mono-sialo transferrin (Arndt, 2001). This is a reasonable explanation for the poor resolution in the low concentration range by this method, as well as for the high intercept in comparison with the Tina-quant %CDT test (Fig. 3b). The modified Jeppson’s HPLC method uses valley-to-valley integration for a-, di-, and tri-sialotransferrin, in contrast to the Axis-Shield HPLC method, which uses baseline integration and which is the reference method for the Tina-quant test. Therefore, differences in this method comparison should also be expected (Fig. 3c).
|
|
Analytical range
The limit of detection was determined as 0.64 mg/l CDT on Roche/Hitachi 917. Linearity was verified throughout the claimed measuring range from 1.0 to 24 mg/l. The recovery of the theoretical concentrations was 93–107% using the CDT application and 98–101% for the total transferrin application (data not shown).
Interferences
No effect of endogenous interferences was found up to approximately 1000 mg/dl of haemoglobin (H index of 1000), 60 mg/dl conjugated and unconjugated bilirubin (I index of 60), highly lipaemic samples (L index of 750, which corresponds to turbidity), and rheumatoid factors up to 1000 IU/ml (data not shown).
Seventeen frequently used drugs were spiked at high concentrations into a human serum pool. As shown in Table 4, the recovery of the CDT target values in the spiked samples was in the range from 92 to 108%. This demonstrates a high resistance of the test against endogenous and drug interferences. However, as with other immunochemical CDT assays, the Tina-quant %CDT 2nd generation assay bears the risk of erroneous results in cases of rare genetic transferrin variants, which can be identified by HPLC or isoelectric focusing (Helander et al., 2001b).
|
General conclusions and comments
To date, CDT is one of the most important markers for recent excessive alcohol consumption in men (Conigrave et al., 2002
). The exact mechanisms that underlie production of CDT are not yet fully understood. An inhibition of protein synthesis and a general effect of alcohol on protein glycosylation are most likely (Sillanaukee et al., 2001). Data from this study indicate a good validity of results obtained with the Tina-quant %CDT 2nd generation test. The assay compares very well with a HPLC method, which is in agreement with the requirements of a reference method for determination of %CDT, and it is highly resistant against interferents. It can be considered to be valuable and practicable for routine CDT determination on Roche/Hitachi analysis systems, which allow the parallel determination of clinical chemistry parameters and a variety of other drugs. Future clinical studies using this assay will address the issues of the upper limit of the reference range and its validity and reliability in clinical samples of subjects with recent heavy alcohol consumption when compared to patients with unspecific GGT elevations and severe liver dysfunction, one of the few reasons for false positive results with CDT (DiMartini et al., 2001).
|
ACKNOWLEDGEMENTS |
|---|
|
TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
The skillful technical assistance of Monika Wuertemberger is gratefully acknowledged.
|
FOOTNOTES |
|---|
|
TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
* Author to whom correspondence should be addressed at: Psychiatric Hospital, University of Munich, Nubbaumstrasse 7, 80336 Munich, Germany.
Tina-quant is a trademark of a member of the Roche group.
|
REFERENCES |
|---|
|
TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
|---|
Allen, J. P., Litten, R. Z., Fertig, J. B. and Sillanaukee, P. (2000) Carbohydrate-deficient transferrin, gamma-glutamyltransferase, and macrocytic volume as biomarkers of alcohol problems in women. Alcoholism: Clinical and Experimental Research 24, 492–496.[CrossRef][ISI][Medline]
Allen, J. P., Litten, R. Z., Fertig, J. B. and Sillanaukee, P. (2001) Carbohydrate-deficient transferrin: an aid to early recognition of alcohol relapse. American Journal of Addictions 10, 24–28.
Anton, R. F. (2001) Carbohydrate-deficient transferrin for detection and monitoring of sustained heavy drinking. What have we learned? Where do we go from here? Alcohol 25, 185–188.[CrossRef][ISI][Medline]
Anton, R. F., Dominick, C., Bigelow, M. and Westby, C. (2001) CDTect Research Group: Comparison of Bio-Rad %CDT TIA and CDTect as laboratory markers of heavy alcohol use and their relationships with gamma-glutamyltransferase. Clinical Chemistry 47, 1769–1775.
Arndt, T. (2001) Carbohydrate-deficient transferrin as a marker of chronic alcohol abuse: a critical review of preanalysis, analysis, and interpretation. Clinical Chemistry 47, 13–27.
Arndt, T., Korzec, A., Bar, M. and Kropf, J. (2002) Further arguments against including trisialo-Fe2-transferrin in carbohydrate-deficient transferrin (CDT): a study on male alcoholics and hazardous drinkers. Medical Science Monitor 8, CR411–CR418.
Bablok, W. (1993) Range of linearity. In Evaluation Methods in Laboratory Medicine, Haeckel, R. ed., pp. 251–258. UCH, Weinheim, Germany.
Burke, V., Puddey, I. B., Rakic, V., Swanson, N. R., Dimmitt, S. B., Beilin, L. J., Ching, S. and Beilby, J. P. (1998) Carbohydrate-deficient transferrin as a marker of change in alcohol intake in men drinking 20 to 60 g of alcohol per day. Alcoholism: Clinical and Experimental Research 22, 1973–1980.[ISI][Medline]
Conigrave, K. M., Degenhardt, L. J., Whitfield, J. B., Saunders, J. B., Helander, A., Tabakoff, B. and WHO/ISBRA Study Group (2002) CDT, GGT, and AST as markers of alcohol use: the WHO/ISBRA collaborative project. Alcoholism: Clinical and Experimental Research 26, 332–339.[CrossRef][ISI][Medline]
DiMartini, A., Day, N., Lane, T., Beisler, A. T., Dew, M. A. and Anton, R. (2001) Carbohydrate deficient transferrin in abstaining patients with end-stage liver disease. Alcoholism: Clinical and Experimental Research 25, 1729–1733.[CrossRef][ISI][Medline]
Forsberg, L., Halldin, J., Ekman, S. and Ronnberg, S. (2002) Screening of binge drinking among patients on an emergency surgical ward. Alcohol 27, 77–82.[CrossRef][ISI][Medline]
Glick, M. R., Ryder, K. W. and Jackson, S. A. (1986) Graphical comparisons of interferences in clinical chemistry instrumentation. Clinical Chemistry 32, 470–475.
Hackler, R., Arndt, T., Helwig-Rolig, A., Kropf, J., Steinmetz, A. and Schaefer, J. R. (2000) Investigation by isoelectric focusing of the initial carbohydrate-deficient transferrin (CDT) and non-CDT transferrin isoform fractionation step involved in determination of CDT by the ChronAlcoI.D.assay. Clinical Chemistry 46, 483–492.
Helander, A. (2002) Multicentre validation study of instrument applications for %CDT, an immunoassay for quantification of carbohydrate-deficient transferrin in serum. Alcohol and Alcoholism 37, 209–212.
Helander, A., Eriksson, G., Stibler, H. and Jeppsson, J. O. (2001a) Interference of transferrin isoform types with carbohydrate-deficient transferrin quantification in the identification of alcohol abuse. Clinical Chemistry 47, 1225–1233.
Helander, A., Fors, M. and Zakrisson, B. (2001b) Study of Axis-Shield new %CDT immunoassay for quantification of carbohydrate-deficient transferrin (CDT) in serum. Alcohol and Alcoholism 36, 406–412.
Helander, A., Husa, A., Westerlund, M., Arnetorp, A., Bergström, J. and Jeppsson, J.-O. (2002) Evaluation of an HPLC reference method for carbohydrate deficient transferrin (%CDT) in serum. Alcoholism: Clinical and Experimental Research 26, 5.
Himmerich, H., Müller, M. J., Anghelescu, I., Klawe, C., Wetzel, H. and Szegedi, A. (2001) Beziehungen von biologischen Markern exzesiven Alkoholkonsums. Suchtmed 3, 67–72.
Lieber, C. S. (1999) Carbohydrate deficient transferrin in alcoholic liver disease: mechanisms and clinical implications. Alcohol 19, 249–254.[CrossRef][ISI][Medline]
Mundle, G., Munkes, J., Ackermann, K. and Mann, K. (2000) Sex differences of carbohydrate-deficient transferrin, gamma-glutamyltransferase, and mean corpuscular volume in alcohol-dependent patients. Alcoholism: Clinical and Experimental Research 24, 1400–1405.[CrossRef][ISI][Medline]
Myrick, H., Henderson, S. and Anton, R. F. (2001) Utility of a new assay for carbohydrate-deficient transferrin (Biorad %CDT TIA) to monitor abstinence during a treatment outcome study. Alcoholism: Clinical and Experimental Research 25, 1330–1334.[CrossRef][ISI][Medline]
Passing, H. and Bablok, W. (1983) Comparison of several regression procedures for method comparison studies and determination of sample sizes. Application of linear regression procedures for method comparison studies in clinical chemistry. Journal of Clinical Chemistry and Clinical Biochemistry 21, 709–720.[ISI][Medline]
Reynaud, M., Schellenberg, F., Loisequx-Meunier, M. N., Schwan, R., Maradeix, B., Planche, F. and Gillet, C. (2000) Objective diagnosis of alcohol abuse: compared values of carbohydrate-deficient transferrin (CDT), gamma-glutamyl transferase (GGT), and mean corpuscular volume (MCV). Alcoholism: Clinical and Experimental Research 24, 1414–1419.[CrossRef][ISI][Medline]
Salaspuro, M. (1999) Carbohydrate-deficient transferrin as compared to other markers of alcoholism: a systematic review. Alcohol 19, 261–271.[CrossRef][ISI][Medline]
Schellenberg, F., Mennetrey, L., Bacq, Y. and Pages, J. C. (2001) Carbohydrate-deficient transferrin (CDT) determination by nephelometry using a commercial kit. Analytical and diagnostic aspects. Clinical Chemistry and Laboratory Medicine 39, 866–871.[CrossRef][ISI][Medline]
Scouller, K., Conigrave, K. M., Macaskill, P., Irwig, L. and Whitfield, J. B. (2000) Should we use carbohydrate-deficient transferrin instead of gamma-glutamyltransferase for detecting problem drinkers? A systematic review and metaanalysis. Clinical Chemistry 46, 1894–1902.
Sillanaukee, P. and Olsson, U. (2001) Improved diagnostic classification of alcohol abusers by combining carbohydrate-deficient transferrin and gamma-glutamyltransferase. Clinical Chemistry 47, 681–685.
Sillanaukee, P., Strid, N., Allen, J. P. and Litten, R. Z. (2001) Possible reasons why heavy drinking increases carbohydrate-deficient transferrin. Alcoholism: Clinical and Experimental Research 25, 34–40.[CrossRef][ISI][Medline]
Turpeinen, U., Methuen, T., Alfthan, H., Laitinen, K., Salaspuro, M. and Stenman, U. H. (2001) Comparison of HPLC and small column (CDTect) methods for disialotransferrin. Clinical Chemistry 47, 1782–1787.
Walter, H., Hertling, I., Benda, N., Konig, B., Ramskogler, K., Riegler, A., Semler, B., Zoghlami, A. and Lesch, O. M. (2001) Sensitivity and specificity of carbohydrate-deficient transferrin in drinking experiments and different patients. Alcohol 25, 189–194.[CrossRef][ISI][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  F. Schellenberg, L. Mennetrey, C. Girre, B. Nalpas, and J. C. Pages 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 Alcohol Alcohol., July 30, 2008; (2008) agn058v1. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. SCHELLENBERG, R. SCHWAN, L. MENNETREY, M.-N. LOISEAUX, J. C. PAGES, and M. REYNAUD DOSE-EFFECT RELATION BETWEEN DAILY ETHANOL INTAKE IN THE RANGE 0-70 GRAMS AND %CDT VALUE: VALIDATION OF A CUT-OFF VALUE Alcohol Alcohol., November 1, 2005; 40(6): 531 - 534. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||