Carbohydrate deficient transferrin and forensic medicine

Clinica Chimica Acta 406 (2009) 1–7

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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m

Invited critical review

Carbohydrate deficient transferrin and forensic medicine Joris R. Delanghe ⁎, Marc L. De Buyzere Department of Clinical Chemistry, Ghent University Hospital, De Pintelaan 185, B 9000 Gent, Belgium

a r t i c l e

i n f o

Article history: Received 15 January 2009 Received in revised form 19 May 2009 Accepted 20 May 2009 Available online 30 May 2009 Keywords: Alcoholism Driver's license Liver disease Transferrin

a b s t r a c t In forensic medicine, diagnosing chronic alcohol abuse is a difficult task. Carbohydrate deficient transferrin (CDT) is one of the most common diagnostic markers for detecting chronic alcohol abuse. However, its diagnostic value is not 100% sensitive and specific. A number of caveats are known (e.g. pre-analytical issues, mutant transferrins, some metabolic diseases, body composition, arterial hypertension, drug treatment, exposure to organic solvents). In view of the numerous caveats, a careful interpretation of CDT analysis is needed. For forensic and occupational medicine applications, such as delegation to working sites where alcoholism may have fatal consequences or to obtain a driver's license, a well-balanced interpretation is needed. For these purposes, high performance liquid chromatography or capillary zone electrophoresis are to be preferred for CDT analysis as their main advantage is the separation of different CDT isoforms. In problem cases, the use of additional alternative tests (e.g. ethyl glucuronide and fatty acid ethyl esters in hair) can be considered. © 2009 Elsevier B.V. All rights reserved.

Contents 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alcoholic drivers . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pre-analytical aspects. . . . . . . . . . . . . . . . . . . . . . . . . . Analytical aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Standardisation . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. CDT methods . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Analytical problems . . . . . . . . . . . . . . . . . . . . . . . 4.3.1. Plasma proteins. . . . . . . . . . . . . . . . . . . . . 4.3.2. Transferrin variants . . . . . . . . . . . . . . . . . . . 4.3.3. Carbohydrate deficient glycoprotein and related syndromes 4.4. Reference values and cut-off limits . . . . . . . . . . . . . . . . 5. Adjustments of reference intervals . . . . . . . . . . . . . . . . . . . 6. Dose–response relation of CDT . . . . . . . . . . . . . . . . . . . . . 7. Confounding factors . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Biological and social confounders . . . . . . . . . . . . . . . . . 7.2. Hypertension, metabolic syndrome, anorexia nervosa . . . . . . . 7.3. Iron metabolism . . . . . . . . . . . . . . . . . . . . . . . . . 7.4. Interpretational problems in liver and metabolic disease . . . . . . 7.5. CDT determination and occupational exposure to solvents . . . . . 7.6. Drug interactions . . . . . . . . . . . . . . . . . . . . . . . . 8. Alternative tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: BMI, body mass index; CDG, congenital disorders of glycosylation; CDT, carbohydrate deficient transferrin; CZE, capillary zone electrophoresis; DST, disialotransferrin; EtG, ethyl glucuronide; FAEE, fatty acid ethyl esters; HPLC, high performance liquid chromatography; PBC, primary biliary cirrhosis. ⁎ Corresponding author. Tel.: +32 9 3322956. E-mail address: [email protected] (J.R. Delanghe). 0009-8981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2009.05.020

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1. Introduction Diagnosing chronic alcohol abuse is difficult because excessively many subjects deny or minimise alcohol abuse and because diagnostic parameters with both high sensitivity and specificity are lacking. From both a medical and from a social perspective there is a need for early detection and correct judgement of excessive alcohol drinking. Indications in the field of forensic medicine are, for instance, drunken drivers [1–3], license reapplication [4], especially of professional drivers, or postmortem diagnosis of alcoholism from vitreous humor [5,6]. In occupational medicine, compelling indications are delegation to working sites where the impact of chronic alcohol consumption may have fatal consequences, such as aviation, working at high altitudes, handling of dangerous materials, and driving of potentially endangering machines [7]. Among the currently used laboratory tests, carbohydrate deficient transferrin (CDT) is regarded as the parameter with the highest diagnostic efficiency [8,9]. At least eight countries in Europe use biomarkers routinely as part of the clinical evaluation for drunk drivers and to assess if the person is abstaining. In Switzerland, Italy, and Austria, repeat offenders are sent to therapy and biological markers, including CDT, are measured quarterly for an entire year to monitor alcohol abstinence. After 1 year, if treatment was successful and biomarkers were kept in check, the driver's license is reinstated. The mean half-life of CDT is approximately 14–17 days [10,11]. Due to the rapid decline of CDT values compared with indicative liver enzymes and mean corpuscular volume [12], CDT is a valuable parameter in the early stage of alcohol withdrawal. If liver enzymes are still elevated, a decreasing CDT value supports the presumption of abstinence. In the present review, the possibilities, limitations and pitfalls regarding the use of CDT for evaluating driving ability, workplace testing and related applications in forensic medicine are discussed. 2. Alcoholic drivers Morgan [4] investigated the potential impact of CDT values on the decision of license reapplication in “high-risk offenders”. CDT values were useful in 70% of the cases. In 8% the CDT values would have produced a change in the reapplication decision and in another 22% the CDT values might have produced confusion. Iffland [2] investigated the significance of CDT values in 534 drivers suspected of having driven impaired. Approximately 20–25% of these drivers had serious alcohol problems. In the entire group, 55% of the subjects showed CDT values exceeding 20 U/L, and approximately 30% of the group exceeding 30 U/L (for units: see below). CDT was proposed as a valuable parameter in combination with the commonly used parameters for detecting drivers with chronic alcohol abuse. 3. Pre-analytical aspects Bacterial or viral contamination may produce false-positive CDT values due to cleavage of the transferrin molecule by neuraminidase [13]. CDT values may be increased due to prolonged storage or

shipment of uncentrifuged whole-blood samples [14]. The diurnal variability of CDT is low [15]. Thus, it is not necessary to restrict sample collection to a specific time of the day. Noncontaminated serum samples, stored at −20 °C, have been stable up to 8 years [14]. For frozen samples (− 20 °C),repeated thawing and freezing had no apparent effect on CDT [16]. Serum may be stored for several weeks at 4 °C before CDT analysis. Storage at 25 °C is acceptable for 5 days but not more than 7 days. After prolonged exposure to room temperature, transferrin degradation may be assessedby measuring the hexasialo-transferrin concentration and the hexasialo-transferrin/pentasialo-transferrin ratio [16].

4. Analytical aspects 4.1. Standardisation At present, an international IFCC (International Federation of Clinical Chemistry) working group has proposed a candidate reference method [17,18]. A candidate standard reference material [19] for assaying CDT is already available. Disialotransferrin has been proposed as the isoform for CDT calibration.

4.2. CDT methods CDT values obtained depend on the method and the standardisation used [20]. Table 1 summarizes some basic characteristics of historically important ion exchange or current CDT assays. Generally, results obtained by high performance liquid chromatography (HPLC) [17,21] and capillary zone electrophoresis (CZE) methods (Fig. 1) [22– 25] are reported as % DST (disialotransferrin; fraction DST to total transferrin) or as the sum of low-sialylated isoforms (asialo- and disialotransferrin). Results obtained by the direct N-Latex method are expressed as %CDT (ratio CDT to total transferrin) [26]. The older ion exchange methods initially reported as U/L: 1 U corresponded to approximately 1 mg transferrin [27]. CDTect (Pharmacia/Axis-Shield ASA and Bio-Rad) considered the asialo-, monosialo-, and disialoisoforms as CDT [8]. Later on, %CDT considered the amounts of asialo-, monosialo-, and disialotransferrin isoforms relative to total transferrin. This test is no longer commercially available [8]. The first %CDT turbidimetric immunoassay (TIA) (Axis-Shield, Bio-Rad) considered asialo-, monosialo-, disialo-, and about 50% of the trisialoisoform as CDT [28]. Between 1999 and 2009, Axis-Shield produced a new version of the %CDT TIA, in which only asialo-, monosialo-, and disialoisoforms were considered as CDT [29]. The Dutch CDT working group (NVKC) [30] concluded that next to the reference method [17], also the N-Latex® method (Siemens), the ClinRep® CDT HPLC method (Recipe), and the CEofix® CDT CZE method (Analis) were acceptable as routine methods for forensic purposes. In Sweden, for regranting of a driver's license after drunk driving, the HPLC candidate reference method [13] is the standard (confirmatory) method for any medico-legal application of the CDT test with focus on the relative amount of disialotransferrin.

Table 1 Available commercial CDT assays on the European market. CDT kit

Reference limit (manufacturer)

Reference limit (literature)

CV (between-assay), %

Axis-Shield (no longer available), Roche Tina Quant (in exchange chromatography) N-Latex CDT Dade Behring (direct immunoassay) Sebia-Capillarys (CZE) Analis CEofix CDT (CZE) Bio-Rad (HPLC)

2.6%

3.0 [25]

3.1–8.5 [25]

2.59% 1.3% 1.8% 2.3 (CDT index 1.9% (Variant system) 1.8% (Agilent system) 1.75%

2.35 [22] 1.4 [20] 1.57 [21]

6.8 [22] 3.2–7.2 [26] 3.5 [18]

1.9 [17]

4.3–8.5 [17]

2 [26]

3.1–4.6 [26]

Recipe (HPLC)

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Fig. 1. Some typical and atypical patterns encountered in CDT pherograms obtained by capillary zone electrophoresis: A: normal pattern; B: heterozygous transferrin CD pattern; C: interference caused by a complement factor C3 variant; D: interference originating from an IgG paraprotein.

In subjects applying to re-obtain their driving license, previously withdrawn for “drunk driving”, there is a need to confirm all the results from CDT immunoassays by alternative techniques. CZE, because of its inherent characteristics of high selectivity, easy operation, high productivity and low operational costs looks wellsuited for becoming the method of choice [31]. 4.3. Analytical problems 4.3.1. Plasma proteins CZE provides high-resolution separations of serum transferrin isoforms, a prerequisite for the monitoring of unusual and complex transferrin patterns, including those with genetic variants and disorders of glycosylation. Except for selected samples of patients with severe liver diseases and sera with high levels of paraproteins [19], interference-free transferrin patterns are detected. In progressed liver cirrhosis, interferences in the beta region can be observed. Efforts to identify, reduce or even eliminate these interferences have been undertaken. The interferences are caused by increased levels of IgA and IgM and can be eliminated by immunopurification using avian IgY

antibody spin column immunocapture of serum transferrin followed by CZE analysis of the stripped and concentrated fraction [32]. Interference by complement C3 and its β-globulin split products in the CZE analysis of CDT can be counteracted by adding inulin to the fresh serum in order to activate the alternate complement pathway and convert native C3 into various degradation products whose electrophoretic mobility no longer coincides with the transferrin glycoforms. This inulin treatment permits rapid CZE evaluation of CDT glycoforms [33]. For HPLC based methods, the wavelength used for detection (460– 470 nm) is not specific for transferrin isoforms. Analytical interferences may occur due to presence of hemoglobin–haptoglobin complexes in case of hemolytic samples [34]. In case of pronounced hemolysis, high-resolution CZE may represent an alternative to HPLC [20] since its detection is based on the peptide bonds (detection wavelength: 200 nm). 4.3.2. Transferrin variants In Caucasians at least 20 variants of transferrin, different from the most common transferrin C1, can be observed. The most frequent

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variants that can be separated by electrophoresis are the B variant (anodal to C1) and the D variant (cathodal to C1) [35]. Only the rare transferrin D3 variant but not the variants D1, D2, or Dx migrate at the same isoelectric point as the bands relevant for CDT determination [35]. The transferrin subtype D may produce false-positive results in nonelectrophoretic or chromatographic analytical methods [35], and the transferrin subtype B may produce false-negative results when these methods are used [36]. The allele frequency of the transferrin subtypes varies between different ethnic origins [37,38]. Outside Europe, the prevalence of transferrin mutants is higher. In particular, in Africans and native populations in America and Australia, the frequency of transferrin mutants is high. Thus, in non-Caucasian populations, the risk of falsepositive and false-negative results increases. In case of transferrin CD heterozygotes, the alcohol consumption can still be estimated using a chromatographic or electrophoretic method when provided an adaptation of the reference values [37]. 4.3.3. Carbohydrate deficient glycoprotein and related syndromes Congenital disorders of glycosylation (CDG), formerly named carbohydrate deficient glycoprotein syndrome, are rare hereditary disorders caused by mutations in the genes coding for enzymes involved in the biosynthesis of glycoproteins and other glycoconjugates. The clinical characteristics are variable, but often include psychomotor, growth, and mental retardation from early childhood. CDG are the result of defects in the assembly and transfer (type I) or processing (type II) of the glycan moieties, and as a result, the carbohydrate chains are either completely missing (type I) or structurally abnormal (type II). CDG-Ia is the most frequent subtype, whereas less frequently patients have been diagnosed with types Ib– Ik and with type II (IIa–IId) [39]. Stibler [40] investigated patients with a CDG type I. All patients showed elevated CDT values. In patients older than 15 years, a decline of the CDT values was observed. Patients also showed alterations in other glycoproteins with sialic acid moieties (e.g. antithrombin, alpha 1-antitrypsin, and thyroxinebinding globulin). When CDT is assayed using an HPLC or a CZE method, asymptomatic CDG carriership can be detected [41]. 4.4. Reference values and cut-off limits When analysing CDT values one should not confuse the terms “reference values” and “cut-off values”. The reference range is based on 95% of the “normal” population. This implies that 5% of the normal population falls outside the reference range, which creates problems when reference values are used in legal applications of CDT testing. In order to interpret the result with certainty outside the reference range, the Dutch guideline (NVKC) adds a method-dependent critical difference (μ1 −μ0) to the upper reference limit. This addition leads to their cut-off limits. Both inter-laboratory variation (CVA,B = analytical coefficient of variation between labs) and the biological variation (CVB,W = biological intraindividual coefficient of variation) contribute to the uncertainty and are therefore included in the method-dependent critical difference. The cut-off value (decision limit) is the value which allows one to conclude with 95% certainty that the result does not belong to the normal distribution. When comparing a result to a chosen border line value (with an unknown uncertainty) the critical difference therefore can be calculated [42]: μ1 −μ0 =z × √2 × √ [CV2A,B + CV2B,W] × RL/100 in which: μ1 −μ0 = critical difference, CVA,B = analytical coefficient of variation (B = between labs), CVB,W = biological intraindividual coefficient of variation (W = within person), RL = reference limit, and z = number of standard deviations, corresponding with the chosen certainty (for 95% certainty: 1.65, for 99% certainty: 2.33). As only changes in one direction (higher) are considered, unidirectional z-scores are used. The Dutch CDT working group [30] suggests the use of a single cut-off point of 2.1% DST (HPLC and CZE) and a value of 2.8% CDT for the N-Latex® method. The HPLC upper reference limit applied in Sweden is 1.9% DST (i.e., cut-off b2.0% if using one digit), corresponding to the mean value + 3

SD for control populations [21]. Other CDT methods may also be used but HPLC is recommended for confirmation. Bortolotti [3] proposed a cut-off set at 2.0% (CDT index) in the fitness evaluation for obtaining a driving license, based on a CZE method. Generally, the diagnostic information obtained by CDT analysis is combined with results of “conventional” markers such as gammaglutamyltranspeptidase and mean cellular volume. Increasing the number of parameters and tests raises an important issue: an apparently “abnormal” test result may not be abnormal at all when viewed from the perspective of multiple tests. This is well understood by statisticians, who routinely adjust for multiple testing. Test results much more unusual than the 99th percentile among non-alcoholics should be required before they can be labelled ‘unfit’ [43]. So, decision criteria with respect to driver's license reapplication should be supported by adequate statistical evidence, in particular when multiple parameters are considered.

5. Adjustments of reference intervals Over the years, differences have been reported in basal and alcohol-related CDT levels in relation to factors such as age, gender and body mass index (BMI). A study, using an HPLC candidate reference method for measurement of individual transferrin glycoforms, instead of a CDT fraction, indicated that the previous findings were largely method-dependent, and did not reflect true baseline differences or alcohol-induced changes in the transferrin glycoprotein complex. Therefore, adjustment of reference intervals for DST, the main CDT glycoform, in relation to either of ethnic origin, age, gender, BMI and smoking is not required [44].

6. Dose–response relation of CDT The dose–response relation between daily ethanol intake in the range 0–70 g and the CDT value is characterized by a rather broad variation [45]. Total body water significantly influences the alcohol consumption/CDT dose–response relationship [46]. BMI, components of the metabolic syndrome, and smoking had notable effects on the probability of an abnormal CDT result with excessive alcohol use. CDT is a poor marker of excessive alcohol intake in both women and men who are overweight or obese. CDT is also less useful in nonsmokers. The effects of obesity and smoking do not appear to be method-dependent [27,47].

7. Confounding factors 7.1. Biological and social confounders Sillanaukee [48] reported a comparable sensitivity for CDT in female and male heavy drinkers. However, other authors using similar ion chromatography immunoassays [49–51] described gender differences. CDT elevations are less significant in females with high long-term alcohol consumption, compared to males [49–51]. Several mechanisms may contribute to this finding. Differing drinking patterns [52] may induce desialylation differences of transferrin. The liver in women appears more susceptible to alcohol than that in men. Women in general have a lower body weight and a lower distribution volume for alcohol [53]. Thus, alcohol dose should be reported as g/kg body weight. Another factor that may contribute to a higher degree of toxicity is the lower alcohol dehydrogenase activity in the stomach of women compared to men [54]. At amounts that do not affect CDT values in men, the desialylation step is possibly influenced in women by low-dose chronic alcohol consumption. CDT is more reliable in those drinking beer than in those drinking other alcoholic beverages [49].

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7.2. Hypertension, metabolic syndrome, anorexia nervosa High blood pressure (or some characteristic associated with it) suppresses the CDT response to high alcohol consumption. Hypertension (particularly diastolic hypertension) [27] is often by itself associated with metabolic syndrome and alcoholism [55]. The associations between CDT response and alcohol and smoking, plasma lipids and lipoproteins, obesity, and hypertension indicate a link with insulin resistance or “syndrome X” [27]. Hypertensive subjects with insulin resistance (established by hyperinsulinemic euglycemic clamp studies) were less likely to have high CDT concentrations [56]. Patients with both kidney and pancreas transplants (who have hyperinsulinemia because of the connection of their pancreatic venous drainage to the systemic circulation) tended to have high CDT concentrations [57]. These findings do implicate insulin or insulin resistance in the setting of circulating CDT concentrations. Although high blood pressure has some effect on CDT and its response to alcohol, it was less than the effect of BMI or obesityrelated characteristics such as triglycerides or HDL-cholesterol. Blood pressure per se did not show significant correlation with CDT [27]. Anorexia nervosa does not cause by itself increased CDT results [58]. Reports dealing with false-positive CDT values from the past are most likely due to an incomplete separation of trisialotransferrin from CDT and thus overestimation of CDT. 7.3. Iron metabolism Iron overload may reduce the sensitivity of CDT, whereas an iron deficiency may reduce the specificity of CDT [59]. Iron depletion in patients with hemochromatosis was associated with increased CDT values [60]. Alcohol intake in iron-depleted patients might even produce higher CDT values than in untreated patients [60]. The Fe3+ binding capacity was important for the determination of CDT using the older ion exchange chromatography methods. In a recent study using HPLC, the false positivity of CDT in hemochromatosis was questioned [61]. 7.4. Interpretational problems in liver and metabolic disease CDT was not suitable to differentiate between subjects with alcoholic or nonalcoholic liver diseases [62]. Higher CDT values were observed in patients with nonalcoholic liver diseases, comprising primary biliary cirrhosis (PBC), chronic active hepatitis, cryptogenic cirrhosis, chronic persistent hepatitis, and hemochromatosis. These patients with nonalcoholic liver diseases denied alcohol consumption within the last 12 months. PBC was for a long time considered as an important cause for increased CDT. The assumed pathomechanism was difficult to explain by the pathogenesis and/or consequences of PBC. In contrast to earlier findings, PBC should no longer be overestimated as an important cause for false-positive CDT results regarding chronic alcohol abuse. Although CDT might be at odds in PBC, PBC by itself is not an important cause for increased CDT values [63]. An incomplete separation of DST and trisialotransferrin may cause false-positive CDT results in alcohol abuse testing. A disialotransferrin– trisialotransferrin-bridging phenomenon (di–tri-bridge) appears with high prevalence in serum from liver cirrhosis patients. The frequently seen di–tri-bridging phenomenon in transferrin HPLC analysis for patients with liver cirrhosis is not explained by genetic transferrin variants or by an increased trisialotransferrin fraction [64]. Increased CDT values (determined using the Axis-Shield assay) have also been observed in chronic hepatitis C patients [65]. In untreated galactosemia, increased CDT values have been reported [66]. During a galactose-free diet the CDT values were normal in most cases. Similarly, in hereditary fructose intolerance, increased CDT values have been observed [67]. In celiac disease, elevated anti-transglutaminase titers were associated with increased CDT levels. A significant

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correlation was found between CDT and anti-transglutaminase values [68]. It has been suggested that the CDT test should not be used in celiac patients to authorize the return of the driving license after its withdrawal due to alcohol abuse [68]. 7.5. CDT determination and occupational exposure to solvents Van Thriel (using ion exchange chromatography followed by immunological detection) reported on CDT values in rotogravure printers in printing plants [69]. Despite the absence of chronic liver diseases, CDT values were elevated in 6.6% of the study group. CDT values, as well as subjective ratings of alcohol consumption, were not correlated with any cognitive function in this study. In workers exposed to the organic solvent N,N-dimethylformamide, CDT values in exposed persons and controls from the same factory were similar [70]. 7.6. Drug interactions Based on the older immunoassay literature, the effect of the intake of contraceptives on CDT values is controversial [71,72]. Nevertheless, CDT values in postmenopausal women were lower [71]. Postmenopausal women on estrogen replacement therapy had higher CDT values than postmenopausal women without estrogen replacement. In men, no age-related effects were observed. Increased CDT values were observed in pregnant women [73]. Lower CDT values were observed in women using oral contraceptives and in postmenopausal women [73]. Other commonly prescribed medications for hypertension, asthma/ bronchitis, diabetes mellitus, lipid metabolism disorders, angina pectoris, depression, or disorders of the gastrointestinal tract did not influence the specificity of the CDT assay in patients from general practices [74]. Factors associated with increased CDT levels were alcohol consumption, female sex, and bupropion use [75]. Two additional drug classes, the angiotensin II receptor blockers and the tricyclic antidepressants, were associated with lower CDT levels. The effects of bupropion and tricyclic antidepressants on CDT levels, however, are confounded by alcohol intake. Only one of 20 drug classes–the angiotensin II receptor blockers–affected CDT levels in the primary care sample [75]. The alcohol-deterrent drug disulfiram does not affect CDT values. Therefore, declining CDT values during treatment with disulfiram are suitable for relapse control [76]. 8. Alternative tests Alternatively, direct ethanol metabolites can be used as measures of abstinence in case CDT results cannot be interpreted. Phosphatidylethanol in blood can be regarded as a promising alternative [77]. Also IgAs against acetaldehyde-modified red cell protein have been suggested as promising alternatives [78]. Ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEE) in hair, phosphatidylethanol in whole blood and EtG and ethyl sulphate in urine can be measured. Distribution of EtG and FAEE over the hair length can be analyzed (distal and proximal parts of hair). In addition to traditional markers, a combination of direct ethanol metabolites can be useful in the expert assessment of judging driving ability [79]. 9. Conclusion Detection of CDT values is just one means in elucidating suspected alcohol abuse [8,80,81]. The psychological pressure on the subjects being told that a long-term alcohol consumption biomarker will be applied should not be underestimated. The high validity of self-reported alcohol consumption in alcoholics may be due to the announcement of CDT determination [82]. This might be of interest if patients are focused on low-dose exposures to other substances as the cause of their

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complaints. In such situations, patients might conceal the actual alcohol consumption to not forfeit a compensation. In some cases, patients might even erroneously suggest environmental exposures to low levels of potentially hepatotoxic substances, like chlorinated hydrocarbons, or by far less hepatotoxic nonchlorinated solvents, other low-level indoor pollutions, or intake of food possibly contaminated with hepatotoxins at a very low level. These elements hamper the use of CDT in a forensic context. Confounding factors have to be taken into account when interpreting CDT results. It should be noted that older methods for CDT determination were less accurate and more prone to caveats than modern CDT assays. For forensic purposes, attention has to be paid to the method-dependent cut-off limits for CDT. CZE and HPLC methods are to be preferred, since they allow visualisation of all transferrin isoforms [3,79]. Analytical errors can further be reduced by using a confirmatory CDT method since analytical interferences are partly assay dependent. In doubtful cases, also alternative tests may become very useful. References [1] Gjerde H, Morland J. Concentrations of carbohydrate-deficient transferrin in dialysed plasma from drunken drivers. Alcohol Alcohol 1987;22:271–6. [2] Iffland R, Grassnack F. Epidemiologic study of CDT (carbohydrate-deficient transferrin) and other indicators of alcohol problems in high blood alcohol German automobile drivers. Blutalkohol 1995;32:26–41 (in German). [3] Bortolotti F, Trettene M, Gottardo R, Bernini M, Ricossa MC, Tagliaro F. Carbohydratedeficient transferrin (CDT): a reliable indicator of the risk of driving under the influence of alcohol when determined by capillary electrophoresis. Forensic Sci Int 2007;170:175–8. [4] Morgan MY, Major MG. The use of serum carbohydrate-deficient transferrin in the assessment of “high risk offenders” in Great Britain. 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