Carbohydrate-deficient Transferrin Test Research Articles

A complement C4-derived glycopeptide is a biomarker for PMM2-CDG.

BACKGROUNDDiagnosis of PMM2-CDG, the most common congenital disorder of glycosylation (CDG), relies on measuring carbohydrate-deficient transferrin (CDT) and genetic testing. CDT tests have false negatives and may normalize with age. Site-specific changes in protein N-glycosylation have not been reported in sera in PMM2-CDG.METHODSUsing multistep mass spectrometry-based N-glycoproteomics, we analyzed sera from 72 individuals to discover and validate glycopeptide alterations. We performed comprehensive tandem mass tag-based discovery experiments in well-characterized patients and controls. Next, we developed a method for rapid profiling of additional samples. Finally, targeted mass spectrometry was used for validation in an independent set of samples in a blinded fashion.RESULTSOf the 3,342 N-glycopeptides identified, patients exhibited decrease in complex-type N-glycans and increase in truncated, mannose-rich, and hybrid species. We identified a glycopeptide from complement C4 carrying the glycan Man5GlcNAc2, which was not detected in controls, in 5 patients with normal CDT results, including 1 after liver transplant and 2 with a known genetic variant associated with mild disease, indicating greater sensitivity than CDT. It was detected by targeted analysis in 2 individuals with variants of uncertain significance in PMM2.CONCLUSIONComplement C4-derived Man5GlcNAc2 glycopeptide could be a biomarker for accurate diagnosis and therapeutic monitoring of patients with PMM2-CDG and other CDGs.FUNDINGU54NS115198 (Frontiers in Congenital Disorders of Glycosylation: NINDS; NCATS; Eunice Kennedy Shriver NICHD; Rare Disorders Consortium Disease Network); K08NS118119 (NINDS); Minnesota Partnership for Biotechnology and Medical Genomics; Rocket Fund; R01DK099551 (NIDDK); Mayo Clinic DERIVE Office; Mayo Clinic Center for Biomedical Discovery; IA/CRC/20/1/600002 (Center for Rare Disease Diagnosis, Research and Training; DBT/Wellcome Trust India Alliance).

An Unexpected Diagnosis of MAGT1 Deficiency in a Patient with CVID-like Features, Molluscum Contagiosum and Atopy

Case DescriptionA 6-year-old boy presented to the immunology clinic with recurrent infections. He started to have recurrent otitis media at the age of 18 months. He also had multiple sinus infections and 3–4 episodes of pneumonia. Immunologic evaluation revealed an IgG of 498 (ref 608–1229 mg/dL), an IgA of 20.8 (ref 33–200 mg/dL) and an impaired response to the Streptococcus pneumoniae vaccine. T and B lymphocyte counts were normal. He was started on subcutaneous immunoglobulin replacement therapy after which his IgG level normalized and his frequency of sinopulmonary infections improved. He also has a history of eczema, chronic cough and environmental allergies for which he receives allergen-specific subcutaneous immunotherapy in addition to treatment with an inhaled corticosteroid and omalizumab. The patient also had persistent skin warts on his hands and extensive molluscum contagiosum lesions. He underwent targeted genetic testing for a primary immunodeficiency which revealed a hemizygous pathogenic variant in the magnesium transporter 1 (MAGT1) gene (c.580dup; p.Ser194Phefs*3). This variant is a duplication at nucleotide position 580 that creates a premature stop codon 3 amino acids downstream from this location. The patient subsequently underwent a carbohydrate deficient transferrin test (CDT) for congenital disorders of glycosylation (CDG) that revealed moderately elevated transferrin mono-oligosaccharide to di-oligosaccharide and Apo CIII-1/Apo CIII-2 ratios, consistent with the diagnosis of MAGT1-CDG (XMEN; X-linked immunodeficiency with magnesium defect, EBV infection, and neoplasia). Additionally, NKG2D surface expression was reduced by ~90% on the CD8T cells, and by ~85% on the NK cells in the patient sample compared to the control sample assessed in parallel. The patient continues to test negative for EBV infection by PCR. ConclusionXMEN disease (OMIM number 300853) is a rare, combined immunodeficiency resulting from a mutation in the magnesium transporter 1 gene (MAGT1) located on the X chromosome. Only 36 patients have been reported to have this disease thus far. While the clinical phenotype is variable, the disease is often associated with EBV infection. This case is unique in that the patient was diagnosed due to a history of severe warts, atopy, and hypogammaglobulinemia in the absence of EBV infection.

Testing venous carbohydrate-deficient transferrin or capillary phosphatidylethanol with concurrent ethyl glucuronide and ethyl palmitate hair tests to assess historical and recent alcohol use.

Hair biomarkers, ethyl glucuronide (EtG) and ethyl palmitate (EtPa), together with blood biomarker tests, carbohydrate-deficient transferrin (CDT) or phosphatidylethanol (PEth), are commonly used in identifying patterns of alcohol consumption as they possess different windows of detection. The detection of EtG in hair samples is mainly used in combination with EtPa when hair cosmetic treatments such as hair colouring and bleaching affect EtG levels. The main purpose of our study was to investigate the differences in frequency distribution of positive CDT and PEth results indicating alcohol had been used, when EtG and EtPa were not detected, where evidence of abstinence is paramount. Of the total 602 cases, for 179 (29.7%), neither EtG nor EtPa markers were detected. Of these, 0.5% of the cases produced positive CDT. However, 18.6% produced positive PEth, a significantly higher proportion. A similar pattern emerges when results are evaluated according to whether hair had been either cosmetically treated or untreated. When hair was untreated, one case produced positive CDT, and 19.3% were positive for PEth (median of 51 ng/ml). No cases of positive CDT results, but 20.8% of PEth were positive (median of 106.5 ng/ml) when hair samples had been cosmetically treated. Whether EtG or EtPa markers were detected or not, significantly higher proportions of PEth than CDT were seen. The results of this study substantiate the case for using hair EtG in conjunction with a PEth test, rather than CDT test, for efficient monitoring of recent and historical alcohol consumption.

The Effect of Prolonged Storage Time on the Stability of Fatty Acid Ethyl Esters in Hair Samples.

The advantages of analysis of drugs in hair samples are recognized for the long window of detection, alongside easy sampling and long stability after sample collection. Alcohol markers, ethyl glucuronide (EtG) and total fatty acid ethyl esters (FAEEs) in hair, are widely used for monitoring alcohol consumption for clinical and forensic purposes. Although stability of drugs and EtG in hair samples is documented to a certain extent, stability of FAEEs in hair samples after collection has not been reported. This study covered hair samples that had been tested for FAEEs on the day of arrival at the laboratory and retested between 4 and 80months later. The statistical analysis of the data set reveals significant lower FAEEs levels including ethyl palmitate (EtPa) ester levels when samples were retested for the second time after 6days of storage under ideal conditions. Specifically, the results suggest that when measuring total FAEEs or solely EtPa in hair samples, the elapsed time between sample collection and analysis of the sample needs to be considered when interpreting the results. The recommendation is that whenever hair samples need to be tested for total FAEEs or EtPa, the analytical procedure needs to be performed within 1week after collection in order to obtain meaningful results. The study results substantiate the case for the use of hair samples solely for the analysis of EtG, in conjunction with other measurements such as full blood count, carbohydrate-deficient transferrin test, liver function test or phosphatidylethanol alongside clinical assessment for a more effective evaluation of alcohol consumption.

The characteristics of transferrin variants by carbohydrate-deficient transferrin tests using capillary zone electrophoresis.

Transferrin is the major plasma transport protein for iron. We aimed to investigate the characteristics of transferrin variant by carbohydrate-deficient transferrin (CDT) test using capillary zone electrophoresis. We retrospectively analyzed the CDT tests of 2449 patients from March 2009 to May 2017 at a tertiary hospital in Korea. CDT was quantified using a Capillarys 2 system (Sebia, Lisses, France) by capillary zone electrophoresis. The characteristics of variant transferrin patterns using electropherogram of CDT tests were analyzed. Seventy-seven (3.1%) patients were classified as variant transferrin. Mean age of these patients was 51.8years, and the male-to-female ratio was 3.5:1. The most common variants were the BC variants (n=37), followed by the CD variants (n=27), unclear patterns (n=7), BD variants (n=3), CC variants (n=2), misclassification (n=1). In the variant Tf group, the most common disease was alcoholic liver cirrhosis (n=22, 28.6%), followed by the toxic effects of substances (n=17, 22.1%), and mental and behavioral disorders attributable to alcohol (n=11, 14.3%). Nonvariant group showed a predominance of the toxic substance effects (n=880, 37.1%), a personal history of suicide attempts (n=634, 26.7%), and mental and behavioral disorders due to alcohol (n=336, 14.2%). We analyzed the basic characteristics of variant transferrin by CDT tests using capillary zone electrophoresis. The prevalence of variant transferrin was 3.1% of the study subjects. Male patients, alcohol abusers, and liver cirrhosis patients predominated in the variant transferrin population. Further prospective studies are warranted to elucidate variant transferrin in clinical practice.

First objective association between elevated carbohydrate-deficient transferrin concentrations and alcohol-related traffic accidents.

Carbohydrate-deficient transferrin (CDT) is a well-recognized highly specific marker of chronic alcohol abuse. The association of CDT with alcohol-related traffic accidents was evaluated to objectively validate the use of this marker for certifying the physical fitness for driving license regranting after its confiscation for drunk driving. The study was carried out on 468 injured drivers (InjDr), who underwent mandatory blood alcohol concentration (BAC) and drug analysis in biological fluids. The InjDr group was divided into 2 subgroups on the basis of BAC legal limit adopted in Italy (BAC ≤ 0.5 g/l: InjDr1 ; BAC >0.5 g/l: InjDr2 ). The control group (CntDr) included 236 subjects holding safety-sensitive job positions and undergoing mandatory toxicological analyses. The determination of BAC in blood and CDT in serum were performed using validated analytical methods based on head-space gas chromatography and high-performance liquid chromatography, respectively. The evaluation of CDT distribution in the 3 groups (CntDr, InjDr1 , InjDr2 ) showed that CDT distribution in the InjDr1 group was similar to that observed in the CntDr group (p = 0.159) and different from that observed in the InjDr2 group (p < 0.001). Partitioning the CDT data of each group into "CDT positives" and "CDT negatives" on the basis of the cut off (1.90%), it was possible to calculate the odds of the 3 groups and then the odds ratios. The odds ratio of InjDr1 versus CntDr was 4.56 (p = 0.158), whereas the odds ratio of InjDr2 versus CntDr was 132 (p < 0.001). Furthermore, a dose-response effect was found only when comparing InjDr2 with CntDr. The data of the present study strongly support the use of the CDT test to evaluate the risk of a subject to be involved in a road accident while driving under the influence of alcohol.

Carbohydrate-Deficient Transferrin Determination in a Clinical Setting: Consistency Between Capillary Electrophoresis Assays and Utility of HPLC as a Confirmatory Test.

Carbohydrate-deficient transferrin (CDT) is used to assess chronic alcohol consumption in administrative and forensic context. The aim of the present study was the optimization of the diagnostic strategy for CDT determination in a clinical laboratory setting. Two capillary zone electrophoresis (CZE) assays, the CEofix CDT (Analis, Suarlée, Belgium) run on single capillary MDQ instrument and the muticapillary (Sebia, Lisses, France), were compared as screening methods and a commercial high-performance liquid chromatography (HPLC) assay (Recipe, Munich, Germany) was used for confirmation. In total, 367 serum samples were analyzed by both CZE assays with concordant classification in 92% of cases. All discordant samples were classified as negative by HPLC, as did 2/3 of those that could not be classified by either CZE assay. Classification of samples with CDT values close to cut-off by CZE was confirmed by HPLC in 95-100% of negative samples but only in 28.6-33.3% of positive samples. Both CZE assays proved suitable for CDT screening. HPLC was useful for discriminating CDT value in most of samples that could not be interpreted by CZE due to analytical interferences. Considering the implication of CDT testing, HPLC assay may also be helpful for the confirmation of positive results close to the cut-off value of CZE assays.

Reflex Testing for Carbohydrate-Deficient Transferrin (CDT) in Insurance Applicants with Elevated High Density Lipoprotein Cholesterol (HDL)

Objectives .- Ascertain the utility of testing carbohydrate deficient transferrin (CDT) levels in insurance applicants with elevated high density lipoprotein cholesterol (HDL) levels. Background .- Chronic alcoholism is not uncommon and is a risk factor for health and longevity and thus of interest to providers of insurance. A number of tests serve as markers of alcohol use, eg, blood alcohol level, elevated liver enzymes, ethyl glucuronide in urine, whole blood associated aldehyde (WBAA), macrocytosis, elevated HDL, elevated CDT and others. WBAA and CDT are usually only done, if some other screening test suggests alcohol use. HDL testing is routinely done for assessing cardiac risk, however, chronic alcohol intake tends to raise HDL and some insurance providers reflex to CDT testing when HDL is elevated. Methods .- A number of the clients of Heritage Labs Inc. have rules in place to test for CDT levels in specimens showing elevated HDL levels. The commonest HDL level that serves as the trigger for reflex testing for CDT is 80mg/dL. The results of this practice were analyzed to assess the utility of reflex testing for CDT to identify chronic alcohol abusers among the applicants. Results .- In examining the results of CDT levels done as a reflex test due to elevated HDL levels, about 2% of the applicants, 0.7% of women and 3% of men, tested positive for elevated CDT levels. Conclusions .- The incidence of elevated CDT levels is high enough to warrant routinely testing for this analyte in applicants, especially men, with high HDL levels.

Determination of carbohydrate-deficient transferrin in a Han Chinese population

BackgroundCarbohydrate-deficient transferrin (CDT) is a widely used alcohol biomarker. Because of the high prevalence of chronic alcohol abuse in many countries, CDT plays an important role in the areas of traffic, clinical, and forensic medicine. However, CDT levels have not been determined in the Han Chinese population. Therefore, we investigated the frequency of genetic transferrin variants and the relationship between CDT levels and alcohol consumption in this population. From this data, we established a CDT cut-off for Han Chinese and evaluated the analytical performance of the CDT capillary zone electrophoresis system.ResultsThe prevalence of transferrin variants was 4.14%. The mean CDT level of the reference group was 0.73%. We recommended CDT level >1.5% as cut off standard of alcohol intake to ensuring the specificity was best. The CDT test total precision for 0.5%, 0.7%, and 1.55% was 14.4%, 11.5%, and 7.2%, respectively. The data showed good linearity in the studied range of 0.6% to 8.2%.ConclusionsThese results demonstrate that CDT is a useful marker to detect heavy daily alcohol consumption. We proposed and evaluated the first CDT cut-off for the Han Chinese population, and we showed that the CDT capillary zone electrophoresis system is a reliable analytic method.

Effect of Transferrin Glycation on the Use of Carbohydrate-Deficient Transferrin as an Alcohol Biomarker

Measurement of an alcohol-induced shift in the serum transferrin glycosylation pattern, termed carbohydrate-deficient transferrin (CDT), is used as a biomarker for sustained high alcohol consumption. The present work examined whether non-enzymatic reaction of transferrin with glucose (glycation) might interfere with the use of CDT as an alcohol biomarker. The blood specimens were leftover volumes from the routine sample pool. Plasma and serum were collected among samples submitted for hemoglobin A1c (HbA1c) and CDT testing. Quantification of individual transferrin glycoforms in percentage of total transferrin was performed by an HPLC candidate CDT reference method. Incubating serum spiked with 20 or 200 mmol/l glucose caused time- and dose-dependent changes in the chromatographic profile of transferrin glycoforms, resulting in gradually wider peaks and reduced relative amounts of disialo- and trisialotransferrin. No similar chromatographic effects were seen in samples collected from diabetic patients with elevated HbA1c (>68 mmol/mol) values. These samples instead showed slightly higher mean %disialotransferrin levels (1.21%) compared with low HbA1c (<44 mmol/mol) samples (mean 1.06%; P = 0.023), pointing at a higher alcohol consumption level in the former group. Altogether ∼5% of the CDT values exceeded the cutoff. There was no significant difference in phosphatidylethanol (PEth) levels between the high and low HbA1c samples, but several (∼14%) showed elevated PEth concentrations. Glycation of serum transferrin in vivo was indicated to differ from that in vitro, and suggested not to interfere with %CDT testing by the HPLC method. The results indicated that CDT and PEth are useful as objective, complementary alcohol biomarkers to identify risky drinking also in diabetic subjects.

N-Latex CDT Results in Liver Diseases

The aim of this study was to test whether liver diseases of alcoholic and non-alcoholic origin cause false-positive carbohydrate-deficient transferrin (CDT) results when the particle-enhanced immunonephelometry for CDT assays is used and to assess the effect of liver disease severity on N-Latex CDT results. Blood was sampled from 245 newly admitted patients suffering from liver diseases: alcoholic and non-alcoholic cirrhosis (AC), chronic viral (B and C) and non-viral hepatitis, toxic and autoimmune hepatitis (AIH), hepatocellular carcinoma and primary biliary cirrhosis (PBC). CDT was determined by particle-enhanced imunononephelometry using the N-Latex CDT test. There were significant differences in %CDT levels between liver diseases of various etiologies. The %CDT level in AC was higher than that in chronic hepatitis (non-viral and viral C). In turn, the %CDT level in chronic hepatitis C was lower than that in toxic hepatitis. The frequency of false-positive %CDT results in liver diseases of non-alcoholic origin was 13/146, and was highest in AIH (4/14). There were no CDT-positive results in PBC and chronic hepatitis B. The frequency of CDT-positive results in alcoholic liver diseases was 24/59 in cirrhosis and 10/34 in hepatitis. Serum levels of %CDT in cirrhotic patients are correlated with the severity of the disease assessed by the Child-Pugh score. We concluded that the liver diseases affect the relative but not absolute values of CDT when using the assay with the monoclonal antibodies directed against CDT. The CDT results from N-Latex CDT test reflect the severity of liver dysfunction.

Relationship between CDT and disease activity in rheumatoid arthritis

The aim of this study was to estimate the serum concentration of carbohydrate-deficient transferrin (CDT) in patients with rheumatoid arthritis (RA) and the relationship between the CDT level and disease activity in RA patients. Studies were carried out in 47female patients with RA and 32healthy women. Disease activity of RA was evaluated using the 28-joint count Disease Activity Score (DAS28). Serum CDT was determined by particle-enhanced immunononephelometry using the N Latex CDT test. Patients with RA had significantly lower serum concentrations of CDT compared with controls. The correlation study showed the significant negative relationship between CDT and DAS28 (r = - 0.483, p = 0.011). There were no correlations between serum CDT level and patient's age, disease duration, number of tender and swollen joints, and degree of disability evaluated by the Health Assessment Questionnaire. The level of CDT in patients with RA was significantly decreased and confirms the changes in transferrin glycosylation which are dependent on the disease activity. Therefore, measurement of CDT in the sera of patients with RA can be useful for the evaluation of disease activity in these patients.

The diagnostic power of direct carbohydrate-deficient transferrin immunoassay in alcoholics. Absolute or relative values?

The objective of this study was to compare the diagnostic power of direct carbohydrate-deficient transferrin (CDT) immunoassay in alcohol abuse expressed in relative units with the diagnostic power of the results expressed in absolute units. Serum CDT was determined in 127 alcoholics using N Latex CDT direct immunonephelometric assay (Siemens Healthcare Diagnostics, Marburg, Germany). The diagnostic sensitivity, specificity, negative and positive predictive value, and also the positive and negative likelihood ratios do not differ between results expressed in relative or absolute units independently of cutoff chosen. Finally, the area under the receiver operating characteristic (ROC) curves for N Latex CDT test expressed in absolute units does not differ from the area for results expressed in relative units. We conclude that the diagnostic usefulness of N Latex immunonephelometric assay using the relative or absolute values is the same.

Operating characteristics of carbohydrate-deficient transferrin (CDT) for identifying unhealthy alcohol use in adults with HIV infection

Unhealthy alcohol use (the spectrum of risky use through dependence) is common in HIV-infected persons, yet it can interfere with HIV medication adherence, may lower CD4 cell count, and can cause hepatic injury. Carbohydrate-deficient transferrin (CDT), often measured as %CDT, can detect heavy drinking but whether it does in people with HIV is not well established. We evaluated the operating characteristics of %CDT in HIV-infected adults using cross-sectional data from 300 HIV-infected adults with current or past alcohol problems. Past 30-day alcohol consumption was determined using the Timeline Followback (TLFB), a validated structured recall questionnaire, as the reference standard. Sensitivity and specificity of %CDT (at manufacturer's cut-off point of 2.6%) for detecting both “at-risk” (≥4 drinks in a day or >7 drinks per week for women, ≥5 drinks in a day or >14 per week for men) and “heavy” drinking (≥4 drinks in a day for women, ≥5 drinks in a day for men on at least seven days) were calculated. Receiver operating characteristic (ROC) curves were estimated to summarize the diagnostic ability of %CDT for distinguishing “at risk” and “heavy” levels of drinking. Exploratory analyses that stratified by gender and viral hepatitis infection were performed. Of 300 subjects, 103 reported current consumption at “at-risk” amounts, and 47 reported “heavy” amounts. For “at-risk” drinking, sensitivity of %CDT was 28% (95% confidence interval (CI) 19%, 37%), specificity 90% (95% CI 86%, 94%); area under the ROC curve (AUC) was 0.59. For “heavy” drinking, sensitivity was 36% (95% CI 22%, 50%), specificity 88% (95% CI 84%, 92%); AUC was 0.60. Sensitivity appeared lower among women and those with viral hepatitis; specificity was similar across subgroups. Among HIV-infected adults, %CDT testing yielded good specificity, but poor sensitivity for detecting “at-risk” and “heavy” alcohol consumption, limiting its clinical utility for detecting unhealthy alcohol use in this population.

Liver Disease and HPLC Quantification of Disialotransferrin for Heavy Alcohol Use: A Case Series

It had previously been suggested that individuals with cirrhosis may have a pattern of transferrin glycosylation that interferes with the interpretation of carbohydrate-deficient transferrin (CDT) testing for heavy alcohol use. The goal of this case series was to evaluate the prevalence of liver disease among individuals with poor resolution of transferrin glycoforms by high performance liquid chromatography. We reviewed the electronic medical records of 35 consecutive patients with poor chromatographic resolution of disialotransferrin from trisialotransferrin and recorded information on diagnosed liver disease, liver function testing, and other factors. Thirty of the 35 subjects with poor chromatographic resolution of the transferrin glycoforms had sufficient data in the medical record for some estimation of liver function. Of these 30 subjects, 25 had previously diagnosed liver pathology. Of the remaining 5 subjects, 2 had liver imaging results suggestive of benign tumor; the remaining 3 had mildly elevated bilirubin and aminotransferase activity, and low albumin. Liver abnormalities, but not necessarily cirrhosis, are common in individuals with poor chromatographic separation of transferrin glycoforms, which might lead to false-positive results on CDT testing. However, the chromatographic-based assay can detect this issue, minimizing the reporting of false positives, but not necessarily assisting in valid detection of heavy drinking.

Determination of Carbohydrate-deficient Transferrin Levels by Using Capillary Electrophoresis in a Korean Population

Carbohydrate-deficient transferrin (CDT) levels have rarely been determined in an Asian population. We evaluated the analytical performance of a test for measuring CDT levels by using capillary electrophoresis (EP). We determined the precision of CDT measurement by using capillary EP and nephelometry and compared the CDT values obtained using both the methods. We included healthy control subjects, abstinent patients with liver disease, and individuals consuming varying amounts of alcohol. The CDT measurement by using capillary EP were correlated well with those CDT measurement by using nephelometry, N Latex CDT assay, Y=0.5706X+1.581, R=0.930. The results obtained from both methods showed good qualitative agreement with each other (κ coefficient=0.61). Genetic variants of transferrin isoforms were detected in 4.1% of the tested population. Both the CDT and γ-glutamyl transpeptidase (GGT) levels in the abstinent patients with liver disease were significantly higher than those in healthy abstinent individuals (0.9% vs. 0.5%, 109.5 mg/dL vs. 28.5 mg/dL, respectively), but the difference in CDT values in the 2 groups was less pronounced for the CDT values. Individuals who had a mean daily alcohol intake of more than 60 g/day showed significantly higher CDT levels than those who had a mean daily alcohol intake of less than 60 g/day (1.9% vs. 0.7%, P=0.03). The CDT test using capillary EP showed good performance, and this method has several advantages such as automation and detection of variant forms. Thus, CDT can be a more useful marker than GGT for monitoring alcohol abstinence, especially in patients with liver disease.

Changes in transferrin glycosylation during pregnancy may lead to false-positive carbohydrate-deficient transferrin (CDT) results in testing for riskful alcohol consumption

Background An alcohol-induced change in serum transferrin glycosylation, termed carbohydrate-deficient transferrin (CDT), is widely used as a biomarker of heavy long-term drinking. This study examined the transferrin glycosylation profile and the risk for false-positive CDT results during pregnancy. Methods Serum samples were collected from 24 healthy pregnant women starting in gestation week 9–21, throughout pregnancy, and 8 or more weeks after delivery. Altogether 171 sera (5–9 samples/person) were analysed. Transferrin glycoforms were quantified as a percentage of total transferrin, using an HPLC candidate reference method for CDT. Results During pregnancy, the relative disialo-, pentasialo- and hexasialotransferrin levels increased gradually, whereas trisialo- and tetrasialotransferrin were reduced. This effect was most pronounced in the third trimester. For disialotransferrin, the main target in CDT testing, initial values of 1.07 ± 0.17% (mean ± SD) increased to 1.61 ± 0.23% before delivery (~ 50% increase). Nine (38%) pregnant women reached %disialotransferrin values ≥ 1.7% (97.5th percentile for controls) but all results were < 2.0%. In the postpartum samples, all glycoform levels had returned towards the starting values. Conclusions These results suggest that the cutoff for %disialotransferrin and %CDT employed to indicate heavy long-term drinking need to be raised slightly in pregnant women, to minimize the risk for false-positive results on CDT testing.

Gene expression profiling in blood: new diagnostics in alcoholism and addiction?

The successful treatment of most diseases relies heavily upon early detection. Biomarkers with diagnostic and prognostic value are critical to the addiction field. Most individuals with alcohol or drug dependence or use problems evade detection until severe medical, legal, or social consequences arise. The short half-life of alcohol in the blood after cessation of drinking eliminates the feasibility for using blood alcohol as a biomarker. Carbohydrate-deficient transferrin (CDT) is currently the most specific serum marker of chronic, heavy alcohol use (Reynaud et al, 2000), but the low sensitivity of the CDT test in the general population makes it an unreliable candidate for predicting either heavy alcohol use or for diagnosing alcohol abuse and/or dependence (Alte et al, 2004). Except for the drugs and their metabolites, there are not biomarkers for addiction. Advances in the field of genomics offer new diagnostic and screening potential for complex genetic diseases like addiction. The ability to simultaneously measure the level of all possible transcripts (mRNAs) provides an unbiased view of potential biomarkers. The importance of understanding gene expression changes in alcohol and drug dependence can be appreciated by the impact of expression profiling in other diseases, most notably cancer, where studies have led to improved pharmacotherapies and to a molecular classification of disease. Gene expression profiling is only beginning to be applied to psychiatric illnesses and may also provide an accurate means to diagnose these conditions. Human brain gene expression studies of alcoholics and cocaine abusers suggest that specific patterns of gene expression may underlie addiction-related phenotypes and provides evidence that a molecular classification of alcoholism may be feasible in the future (Liu et al, 2006; Mash et al, 2007). However, in order for expression profiling to be useful in the clinical screening of dependence and consumption, the tissues or cells under investigation need to be readily accessible. A wide variety of screening tests are available that relies upon peripheral blood samples for assaying biological markers. For example, blood tests allow early detection, and in some cases prevention, of conditions such as prostate cancer, diabetes, thyroid dysfunction, and heart disease. Blood samples offer advantages over procedures such as tissue biopsy as they are fast, noninvasive, and can be repeated many times on the same individual. Blood biomarkers also offer the potential to predict disease before any detrimental symptoms are manifested. Genomic profiling of peripheral blood samples could be of great value in identifying biomarkers for complex diseases including addiction. This idea is supported by studies utilizing white blood cells to identify discrete patterns of expression associated with modeled complex disease states in animals (Tang et al, 2001). In addition, patterns of gene expression have been identified from blood samples obtained from a large number of healthy individuals, revealing surprising consistency in expression of genes associated with age, gender, and blood composition (Tang et al, 2001). Thus, it’s feasible that nucleated blood cells of alcohol-dependent individuals could show changes in gene expression that will provide a ‘signature’ of the disease. Discovery of reliable blood-based molecular markers of alcohol dependence and use would mark a milestone for addiction research and offer a great benefit for predicting the disease even without knowing the role of the markers in the disease process. Once biomarkers are discovered, the opportunity for early detection and intervention as well as personalized therapeutics should lead to new treatments for the disease.

Insufficient Standardization of a Direct Carbohydrate-Deficient Transferrin Immunoassay

Measurement of carbohydrate-deficient transferrin (CDT) can reveal alcohol-related changes in the serum transferrin glycosylation pattern. CDT is a more alcohol-specific indicator than liver-function tests and is used for identification and follow-up of chronic high alcohol consumption(1). Therefore stable calibration of the assays is very important. Comparison of CDT results between methods has often been hindered by method-dependent discrepancies in the definition of the measurand (the transferrin glycoforms covered) and the way results are expressed. With some methods there has been an increased risk for false-positive results(2). The lack of CDT standardization prompted initiation of a working group under the International Federation of Clinical Chemistry and Laboratory Medicine, whose aim was to define the measurand, select and validate a reference method, and work out procedures for the production of reference materials. The first recommendation was that the fraction of disialotransferrin to total transferrin (%disialotransferrin) should be the primary target for CDT testing, with HPLC as the candidate reference method(3). The performance of individual laboratories and agreement of different methods can be determined through external quality assessment (EQA). A Swedish EQA scheme for CDT has been run by EQUALIS (External …

Determination of carbohydrate-deficient transferrin (CDT) in Italy

Carbohydrate-deficient transferrin (CDT) in serum is a biomarker of heavy alcohol consumption. In Italy, CDT testing is primarily used for matters of road safety by the commissions that reissue drivers' licenses after alcohol-related offences. The purpose of this study was to examine how CDT determinations are carried out by Italian laboratories. Public (hospital and university) laboratories, the companies producing CDT assays, and the organizers of two external quality assurance (EQA) programs were approached and telephone interviews were conducted. The study was carried out between October, 2006 and January, 2007, and considered the situation as of 31 December, 2006. In 2006, 142 Italian hospital and university laboratories performed CDT measurements and there were 67 license commissions using different protocols for the evaluation of alcohol abuse. Compared with 2005, the number of laboratories that assayed CDT had doubled in 2006. Several different CDT methods were in routine use and there were large differences in the ways results were expressed and in the cut-off limits applied, even for identical methods and instrumentations. Only approximately one-third of the laboratories participated in an EQA program for CDT. Despite that CDT testing is used almost exclusively for medico-legal purposes in Italy, many different methods, ways of expressing test results, and cut-off limits were routinely applied. This observation points at the urgent need for standardization of CDT measurement.