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Clinical Chemistry 45: 131-135, 1999;
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(Clinical Chemistry. 1999;45:131-135.)
© 1999 American Association for Clinical Chemistry, Inc.

Technical Briefs

Absolute or Relative Measurement of Carbohydrate-deficient Transferrin in Serum? Experiences with Three Immunological Assays

Anders Helander

Karolinska Institutet, Departments of Clinical Neuroscience and Clinical Chemistry, Alcohol and Drug Dependence Unit at Karolinska Hospital, SE-17176 Stockholm, Sweden, fax 46-8-6721904, e-mail anders.helander{at}bekl.csso.sll.se

Individuals who have consumed at least 50–80 g of alcohol per day during the previous week(s) often show an abnormal microheterogeneity of the iron-transporting glycoprotein transferrin in serum (1). After chronic exposure to alcohol, the concentrations of transferrin molecules that lack 2–4 of the four normal terminal sialic acid residues (di-, mono-, and asialo transferrin, respectively) (1) or that lack the entire biantennary carbohydrate chain(s) (2)(3) increase. The presence of increased concentrations of this "carbohydrate-deficient" transferrin (CDT) is a specific and sensitive biochemical indicator of recent excessive drinking (1)(4).

Whether the CDT result should be expressed as the absolute amount or as the amount normalized to the total transferrin concentration has been a matter of debate (4). The present study compared three commercial immunological test kits for quantification of the abnormal microheterogeneity of serum transferrin observed after excessive drinking: one kit that measures CDT as an absolute amount (CDTectTM RIA; Pharmacia & Upjohn Diagnostics),1 and two kits that measure the result relative to total transferrin [an RIA (%CDT RIA) and a turbidimetric immunoassay (%CDT TIA), both from Axis Biochemicals].

With the CDTect test, the CDT content is expressed as an absolute amount (in units/L, with 1 unit of CDT in the CDTect assay equivalent to ~1 mg of transferrin) of the transferrin isoforms with a pI >=5.7. According to a recent report, the CDTect assay measures part of the asialo, monosialo, and disialo isoforms as well as traces of trisialo transferrin (5). In brief, transferrin in the serum sample is saturated with Fe3+, and the isoforms are separated on an anion-exchange chromatography microcolumn. Quantification of CDT is carried out by a double antibody RIA. Because of a gender-based difference in the baseline concentrations of asialo and monosialo transferrin (6), different upper reference limits must be applied for males (20 units/L) and females (27 units/L).

The %CDT RIA test2 also utilizes an initial ion-exchange microcolumn separation; however, the CDT result is expressed as the percentage of asialo, monosialo, and disialo transferrin relative to total transferrin. This assay apparently measures only a portion of the asialo, monosialo, and disialo transferrin, however, because the upper reference limit of this assay has been defined as 2.5% for both males and females, whereas these isoforms make up ~3% of the total transferrin concentration (6).

The %CDT TIA3 also measures only a portion (50%) of the trisialo isoform in addition to asialo, monosialo, and disialo transferrin. The CDT result is expressed relative to the total transferrin concentration. The desialylated isoforms eluted from an ion-exchange microcolumn form immune complexes with anti-transferrin antibodies, which are quantified turbidimetrically. The concentration of total transferrin is measured separately, using the same antibodies. An upper reference limit of 6% for both males and females has been recommended by the manufacturer. The intraassay imprecision (CV) of this and the other two methods is <10%.

In this study, serum {gamma}-glutamyltransferase (GGT) activity and total transferrin concentration (traceable to CRM 470) were determined at the local clinical chemistry laboratory, using accredited routine methods.

The same serum sample was used for all analyses, and all tests were done blind to each other and the reference calibrator. Correlations were evaluated through the Spearman coefficient of rank correlation. P <=0.05 was considered significant. Areas under the ROC (7) curves and the significance of differences between areas were calculated using MedCalc software.

Serum samples were collected within 24 h of admission from 239 consecutive patients, ages 17–82 years (94 females, ages 44.6 ± 11.4 years, mean ± SD; and 145 males, ages 47.4 ± 11.2 years) admitted to the addiction treatment center at St. Görans Hospital for detoxification or treatment of alcohol or drug-related problems. Serum was stored at -80 °C until analysis. Information regarding quantity and frequency of alcohol consumption was obtained by use of a confidential self-report questionnaire, wherein the patients were asked to specify their intake of alcohol (volume and type of beverage) in the previous week, the time of last intake, and also the number of drinking days in the last month. Informed consent was obtained from all participants, and the procedures followed were in accordance with the Helsinki Declaration.

Twenty-three females and 31 males claimed not to have consumed any alcoholic beverages in the month before sample collection. In those who admitted drinking, the average daily intake was 195 ± 155 g of ethanol (mean ± SD; median, 150 g/day; range, 10–600 g/day) for the females (n = 56) and 241 ± 182 g (median, 250 g/day; range, 7–1000 g/day) for the males (n = 111). The majority of females (66%) and males (72%) reported a regular daily intake of alcohol exceeding 100 g. The number of patients consuming between 30–60 g/day on average was limited (6 females and 3 males) in the present material. Data on daily alcohol consumption were not obtained from 15 females and 3 males.

No overall correlation between CDTect and serum total transferrin was found in this study (r = -0.08; P = 0.22), which concurs with previous observations (8)(9), whereas a weak positive correlation has been demonstrated by others (10)(11)(12)(13). Apparently, the relationship between CDT (CDTect) and total transferrin is largely dependent on the population studied. A moderate correlation was demonstrated for the nondrinkers in this study (r = 0.36; P <0.05), and a higher correlation (r = 0.45; P <0.001; unpublished data) was recently found for teetotalers (14). In a drinking population, however, this correlation will become obscured, partly because of the different amounts of alcohol consumed but also because of the highly variable interindividual response in CDT to a given alcohol dose. Consequently, for the patients who admitted consuming >60 g of alcohol daily, no correlation between CDTect and total transferrin was detected (r = -0.12; P = 0.23). These results are in good agreement with a recent report that showed a significant correlation for hospitalized nondrinking patients but no correlation for alcohol abusers (15).

The CDTect, %CDT RIA, and %CDT TIA test results correlated weakly with reported average daily intake of alcohol (range, 0–1000 g/day) during the 1-week period preceding the test (r = 0.36–0.43). A relatively high correlation was obtained for the subpopulation consuming 0–250 g of alcohol per day (r = 0.70; P <0.001; n = 164), whereas no significant correlation was seen for those who admitted consuming >=250 g of alcohol daily (r = 0.06; P = 0.67; n = 80). A similar lack of correlation between CDT and reported intake at very high daily alcohol consumption has been observed in a previous study (16).

The CDT assays were evaluated by ROC analysis (Fig. 1 ). The difference in areas under the ROC curves for %CDT RIA (0.86) and CDTect (0.81) just reached statistical significance (P = 0.05) for males and females combined (Fig. 1C ). A similar test accuracy for %CDT RIA and CDTect had been found previously (17)(18). This seemed to contrast with recently published data showing a considerably higher sensitivity, albeit a lower specificity, for CDTect compared with %CDT RIA and %CDT TIA when the cutoffs recommended by the manufacturers were used (15). Nevertheless, when patients with the most severe alcohol dependence (>80 g daily) were analyzed separately (15), the sensitivities of CDTect and %CDT TIA were reported to be more equal (73% and 64%, respectively). This agrees well with the results of the present study, where the sensitivity of the three CDT tests at the manufacturers' suggested cutoffs was 65–66% for females and 74–81% for males consuming >=60 g of alcohol daily according to self-reports. Considering the high alcohol consumption reported by these patients (median, 150 g daily for females and 250 g daily for males), the sensitivity values may seem low; however, it is known that some individuals (~10–30%) do not respond to excessive drinking with a marked increase in the serum concentration of CDT (non- or low-responders) (19)(20)(21). The higher sensitivity of CDT for males compared with females is in accordance with previous observations (22)(23)(24)(25). For each method, the specificity at the manufacturer's cutoffs was 93–95% among males and females reporting a mean daily intake of <30 g of alcohol in the previous month. In four subjects ingesting <30 g/day, the results from all three tests were consistently increased, thereby suggesting a much higher alcohol intake than that reported (26)(27) or extreme sensitivity to the effects of alcohol on CDT.



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  		Figure 1. ROC curves for the three immunological tests for CDT quantification, comparing patients consuming >=60 g alcohol/day according to self-reports vs those consuming <60 g/day.

(A) females (n = 79); (B) males (n = 142); (C) females and males combined. Areas under the ROC curves for %CDT RIA (——–), %CDT TIA (- - -), and CDTect (· · · · ·), respectively, were 0.84, 0.81, and 0.78 for females; 0.87, 0.88, and 0.84 for males; and 0.86, 0.86, and 0.81 for males and females combined. Proposed cutoff points are indicated by {square}; the corresponding values are: for females (A), >1.9% for %CDT RIA, >5.7% for %CDT TIA, and >28 units/L for CDTect; for males (B), >1.8% for %CDT RIA, >5.9% for %CDT TIA, and >18 units/L for CDTect; and for females and males combined (C), >1.8% for %CDT RIA, >5.9% for %CDT TIA, and >24 units/L for CDTect.

Among the patients who admitted to recent regular heavy drinking (>=60 g/day), the CDTect test provided apparently false-negative results in 4 of 23 cases (~17%) when the serum total transferrin concentration was below the reference interval (Table 1 ). However, four false-negative results were also obtained with the %CDT RIA test, despite the fact that this test reports the CDT value as the relative amount, which should compensate for variations in transferrin concentration. In two subjects admitting to ethanol consumption of >=60 g daily, the results from all three tests fell within the reference limits (possibly CDT non- or low-responders). Conversely, CDTect apparently gave a false-positive result in one of six (~17%) abstinent patients with a total transferrin concentration well above the upper reference limit. Although transferrin synthesis and glycosylation are two distinct processes, a total transferrin concentration outside the reference limits can evidently give rise to false CDTect results (11)(12)(13)(14)(28). Nonetheless, in the vast majority of patients with a serum total transferrin concentration outside the reference interval of 1.9–3.3 g/L, the CDTect result was concordant with the self-reported drinking history (Table 1 ). Among the 239 subjects examined, 14 (7 females and 7 males; 5.9%) showed a total transferrin concentration >3.3 g/L, whereas 30 (9 females and 21 males; 12.6%) showed a concentration <1.9 g/L.


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  		Table 1. CDT values in patients with a serum transferrin concentration outside the reference interval1 and either no alcohol consumption or an alcohol consumption of >=60 g daily in the previous week according to self-report.

In a female patient with an estimated average intake of ~75 g of alcohol daily in the last month, both the CDTect and %CDT TIA results were well above the respective cutoff limits, whereas %CDT RIA gave 0%. Interestingly, when a second sample was collected from the same subject a few weeks later, the result of %CDT RIA was again 0%. Finally, in a male subject who reported drinking only ~10 g alcohol/day, the CDTect and %CDT RIA results fell within the reference limits, but %CDT TIA gave 6.3%, thus suggesting a higher alcohol intake.

The results of the present study suggest that the newer %CDT TIA test offers no striking advantage, but also no disadvantage, over the other commercial kits in terms of overall diagnostic performance. However, although this study was based on a limited number of patients, it suggests that the risk of erroneous results arising from total transferrin concentrations outside the reference interval might be lower with %CDT TIA (Table 1Up ). CDT and GGT appeared to be independently associated with alcohol consumption. Thus, increased GGT was present in samples from 10 of 14 male patients (71%) who reported drinking >=60 g/day but still showed %CDT TIA values within the reference interval, and GGT was within reference limits in 17 of 59 patients (29%) with increased %CDT TIA values. CDT and GGT should, therefore, be considered as complementary (21), rather than alternative (29), markers.

Some studies have reported the relative amount of CDT to be more accurate (28)(30)(31), whereas others have recommended use of an absolute amount of CDT (9)(10)(11)(12); however, most prior studies, like this one, found no marked difference (6)(13)(17)(18)(25)(32)(33)(34). In some cases, absolute and relative measurements disagreed, although discrepancies were also evident between the two tests expressing the CDT value as a relative amount. In the majority of patients with a serum transferrin concentration outside the reference interval, the absolute and relative CDT results agreed with the self-reported history of drinking, whereas a relative amount appeared to be more reliable among those few individuals with low or very high total transferrin concentrations.


Acknowledgments

This study was supported in part by Axis Biochemicals, Oslo, Norway. I thank Marie Olsson for skillful technical assistance.


Footnotes

1 Axis Biochemicals recently acquired the alcohol-related business from Pharmacia & Upjohn Diagnostics, including the CDTect kit (effective October 1, 1998).

2 The %CDT RIA kit is no longer commercially available.

3 Variants of the %CDT TIA kit are also distributed by Roche and Bio-Rad.


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