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Letters |
1
Institute of Forensic Medicine, Catholic University of the Sacred Heart, Largo Francesco Vito 1, 00168 Rome, Italy
2
Department of Public Medicine and Health, Unit of Forensic Medicine, University of Verona, 37134 Verona, Italy
3
Clinical Chemistry and Hematology Laboratory, Hospital of Verona, 37126 Verona, Italy
aAuthor for correspondence. Fax 39-045-505259; e-mail franco.tagliaro{at}univr.it.s.
To the Editor:
Carbohydrate-deficient transferrin (CDT) refers to a group of minor isoforms of transferrin that, according to most authors, includes asialo-, monosialo-, and disialo-Fe2-transferrin (1). CDT is a widely used marker of chronic alcohol abuse in Europe and, after its recent approval by the Food and Drug Administration, a candidate to become a popular diagnostic tool in the US (2). Arndt (1) recently concluded that "CDT is the most specific marker of chronic alcohol abuse to date". Its specificity, however, is affected by the choice of analytical method, which must selectively determine only the CDT components, without interference from the other transferrin glycoforms (trisialo-, tetrasialo-, pentasialo-, and hexasialo-Fe2-transferrin), which are present in large excess.
Interference from trisialo-Fe2-transferrin has been reported for most commercial CDT tests, which are based on anion-exchange microchromatographic separations followed by immunoassays. Unfortunately, much of the literature does not report any correlation between trisialo-transferrin and alcohol intake (3). Moreover, Dibbelt (4) recently demonstrated by HPLC that increased relative concentrations of disialo- and asialo-transferrin associated with alcohol abuse are not correlated with increased trisialo-transferrin concentrations and consequently stated that "trisialo-transferrin is obviously of no diagnostic value, I strongly recommend not including this isotransferrin in the CDT fraction measured for laboratory diagnosis of alcoholism".
To avoid generation of false-positive results in the presence of isolated increases of trisialo-transferrin, data from immunoassays are currently confirmed by separative methods such as HPLC and isoelectric focusing (1). Recently, capillary electrophoresis methods have been developed and validated to provide selective determination of the individual CDT isoforms (3).
It appears quite unfortunate that a recent paper by Wuyts et al. (5) describes a capillary zone electrophoretic method for CDT determination that is clearly unable to separate disialo-transferrin, the major component of CDT, and trisialo-transferrin. By contrast, several capillary electrophoresis methods provide baseline separation and individual quantification of the two isoforms (6)(7)(8)(9)(10).
The claimed advantage of direct serum injection offered by the method reported by Wuyts et al. (5) is clearly of minor relevance if compared with the lack of selectivity, which may lead to false-positive results. On the other hand, most of the recently published capillary electrophoretic methods (7)(8)(9) require only minimal sample pretreatment, limited to iron saturation and dilution of the serum sample.
We suggest that assays for CDT be based on only specific methods and that analytical selectivity and diagnostic effectiveness should not be sacrificed in favor of operative simplicity, throughput, and automation.
References
4 Department of Clinical Chemistry, University Hospital Gent, De Pintelaan 185, 9000 Gent, Belgium
bAuthor for correspondence. Fax 32-9-240-49-85; e-mail joris.delanghe{at}rug.ac.be.
Tagliaro et al. (1) state, in essence, that carbohydrate-deficient transferrin (CDT) should be measured by only highly selective methods and that any loss of analytical selectivity in favor of operative simplicity, throughput, and automation is unacceptable.
In the early definition of CDT, the sum of the asialo-, monosialo-, and disialotransferrin isoforms was called "carbohydrate-deficient transferrin" because in alcoholics, some of the serum transferrin (Tf) molecules apparently lacked two to four of their terminal trisaccharides (2). Subsequent CDT methods differ in the Tf fractions measured and the portion of trisialo-Tf included in the final results.
Clinical studies have demonstrated high diagnostic efficiencies in CDT assays that include trisialo-Tfs (3)(4)(5). Furthermore, both Axis procedures performed better diagnostically than isoelectric focusing with immunoblotting and laser densitometry, which measures asialo- and disialo-Tf but not the trisialo-Tf isoforms (6). Similarly, CDTect outperformed isoelectric focusing, a finding that supports the reliability of the former as a comparison method for the validation of new CDT tests (6). Tagliaro et al. (7) reported a marked difference not only in disialo- but also in trisialo-Tf between sera from selected controls who showed no clinical evidence of chronic alcohol abuse (social drinkers were included) and those with clear anamnestic, clinical, and laboratory evidence of chronic alcohol abuse (>50–80 g of ethanol per day). The relative disialo-Tf fraction (as area percentage of tetrasialo-Tf) was fourfold higher in alcoholics than the controls, and the relative trisialo-Tf fraction was increased by a factor of five in alcoholics compared with controls. They found no significant correlation between either disialo- or trisialo-Tf and the values obtained from a minicolumn ion-exchange chromatographic method (CDTect), but a good correlation (r = 0.775; n = 14; P = 0.001) was found between CDTect and the sum of the disialo- and trisialo-Tf. Others (8)(9) observed no association between an increase in disialo- and asialo-Tf percentages attributable to increased consumption of alcohol and increased trisialo-Tf percentages. Therefore, the significance of trisialo-Tf remains unclear, and case-control studies correlating the individual Tf fractions to alcohol consumption in a variety of CDT methods are required. CDT methods capable of separating the disialo- from the trisialo-Tf may have advantages, but no published clinical studies demonstrate (e.g., by ROC analysis) a significant improvement of diagnostic performance when disialo- and trisialo-Tf can be separated.
Unlike some methods (10)(11)(12), our method (13) does not separate disialo- from trisialo-Tf, but it is simple and rapid and exhibits excellent reproducibility. We obtained CVs for the di-trisialo-Tf peak area percentage of 3.2%, whereas Crivellente et al. (11) found a CV of 6.9% for the disialo- and 10.9% for the trisialo-Tf peak areas. Our method (13) can also detect Tf variants, reducing the risk of false-positive results. Some other methods risk false-positive CDT results in genetic Tf variants because the fractions are not visualized (9).
High sample throughput, automation, and operative simplicity are needed in the clinical laboratory. The need is reflected by the FDA approval of a test that measures part of the trisialoforms (No. K992502; December 21, 1999). In view of the high prevalence of alcohol abuse, only fully automated, time-saving, and simple methods can introduce capillary electrophoresis determination of CDT in clinical laboratories.
Dr. Tagliaro’s letter confirms the urgent need for further clinical studies on the significance of the various Tf fractions, which should ultimately lead to an international standardization of CDT.
References
-glutamyltransferase for detecting problem drinkers? A systematic review and metaanalysis. Clin Chem 2000;46:1894-1902.The following articles in journals at HighWire Press have cited this article:
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  P. Anttila, K. Jarvi, J. Latvala, and O. Niemela METHOD-DEPENDENT CHARACTERISTICS OF CARBOHYDRATE-DEFICIENT TRANSFERRIN MEASUREMENTS IN THE FOLLOW-UP OF ALCOHOLICS Alcohol Alcohol., January 1, 2004; 39(1): 59 - 63. [Abstract] [Full Text] [PDF]
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P. Anttila, K. Jarvi, J. Latvala, J. E. Blake, and O. Niemela DIAGNOSTIC CHARACTERISTICS OF DIFFERENT CARBOHYDRATE-DEFICIENT TRANSFERRIN METHODS IN THE DETECTION OF PROBLEM DRINKING: EFFECTS OF LIVER DISEASE AND ALCOHOL CONSUMPTION Alcohol Alcohol., September 1, 2003; 38(5): 415 - 420. [Abstract] [Full Text] [PDF]
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 F. J. Legros, V. Nuyens, M. Baudoux, K. Zouaoui Boudjeltia, J.-L. Ruelle, J. Colicis, F. Cantraine, and J.-P. Henry Use of Capillary Zone Electrophoresis for Differentiating Excessive from Moderate Alcohol Consumption Clin. Chem., March 1, 2003; 49(3): 440 - 449. [Abstract] [Full Text] [PDF]
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F. J. Legros, V. Nuyens, E. Minet, P. Emonts, K. Z. Boudjeltia, A. Courbe, J.-L. Ruelle, J. Colicis, F. de L'Escaille, and J.-P. Henry Carbohydrate-deficient Transferrin Isoforms Measured by Capillary Zone Electrophoresis for Detection of Alcohol Abuse Clin. Chem., December 1, 2002; 48(12): 2177 - 2186. [Abstract] [Full Text] [PDF]
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