Reprint of Standardisation and use of the alcohol biomarker carbohydrate-deficient transferrin (CDT).

Carbohydrate-deficient transferrin (CDT) is a glycoform profile of serum transferrin that increases in response to sustained high alcohol intake and over the last decades has become an important alcohol biomarker with clinical and forensic applications. However, the wide range of CDT measurement procedures has resulted in lack of uniform results and reference limits, and hampered comparison of results. In 2005, the IFCC therefore founded a special working group (WG) aiming for standardisation of CDT measurement. This review summarises the history of CDT and the actions taken by the WG-CDT. Initial steps included the definition of the measurand (serum disialotransferrin to total transferrin fraction expressed in %), and the determination of a well-defined anion-exchange HPLC procedure as the candidate reference measurement procedure (cRMP). Subsequent achievements were the establishment of a network of reference laboratories to perform the cRMP, setting a reference interval, and development of a reference material based on human serum for which the laboratory network assign values. Using a set of reference materials for calibration allowed for achieving equivalence of results of all present CDT measurement procedures. The final steps of the WG-CDT have been a full validation of the cRMP to make it an IFCC approved RMP, and providing guidance for international standardisation of all CDT measurement procedures.

S100B maternal blood levels are gestational age- and gender-dependent in healthy pregnancies.

BACKGROUND: S100B is a well-established biomarker of central nervous system (CNS) development and damage in the perinatal period. Because the fetal CNS induces an overproduction of S100B measurable in the maternal bloodstream we evaluated S100B protein in healthy pregnancies in order to provide a reference curve of the protein in the second and third trimesters and to provide information on CNS development when standard monitoring procedures could be silent or unavailable. METHODS: Between July 2012 and December 2014 we conducted a prospective study in 1213 healthy pregnancies delivering healthy newborns. Maternal blood samples were collected for standard monitoring procedures and S100B assessment. S100B correlations with selected outcomes (gestational age at sampling, gender of fetus, gestational age and weight at birth, delivery mode) were calculated using multiple forward stepwise regression analysis. RESULTS: S100B concentrations in the second and third trimesters of pregnancy were found to be gestational age-, gender- and delivery mode-dependent (p<0.05, for all). Multiple forward stepwise regression analysis with S100B as the dependent variable and gestational age at sampling, gender, delivery mode, gestational age and weight at birth as independent variables, showed a significant correlation between S100B and gestational age at sampling (R=0.13; p<0.001). CONCLUSIONS: The present findings offering a S100B protein reference curve in maternal blood suggest that non-invasive fetal CNS monitoring is becoming feasible and open the way to further research in neuro-biomarker assessment in the maternal bloodstream.

IFCC approved HPLC reference measurement procedure for the alcohol consumption biomarker carbohydrate-deficient transferrin (CDT): Its validation and use.

Carbohydrate-deficient transferrin (CDT) is used as a biomarker of sustained high alcohol consumption. The currently available measurement procedures for CDT are based on various analytical techniques (HPLC, capillary electrophoresis, nephelometry), some differing in the definition of the analyte and using different reference intervals and cut-off values. The Working Group on Standardization of CDT (WG-CDT), initiated by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), has validated an HPLC candidate reference measurement procedure (cRMP) for CDT (% disialotransferrin to total transferrin based on peak areas), demonstrating that it is suitable as a reference measurement procedure (RMP) for CDT. Presented is a detailed description of the cRMP and its calibration. Practical aspects on how to treat genetic variant and so-called di-tri bridge samples are described. Results of method performance characteristics, as demanded by ISO 15189 and ISO 15193, are given, as well as the reference interval and measurement uncertainty and how to deal with that in routine use. The correlation of the cRMP with commercial CDT procedures and the performance of the cRMP in a network of laboratories are also presented. The performance of the CDT cRMP in combination with previously developed commutable calibrators allows for standardization of the currently available commercial measurement procedures for CDT. The cRMP has recently been approved by the IFCC and will be from now on be known as the IFCC-RMP for CDT, while CDT results standardized according to this RMP should be indicated as CDTIFCC.

Standardisation and use of the alcohol biomarker carbohydrate-deficient transferrin (CDT).

Carbohydrate-deficient transferrin (CDT) is a glycoform profile of serum transferrin that increases in response to sustained high alcohol intake and over the last decades has become an important alcohol biomarker with clinical and forensic applications. However, the wide range of CDT measurement procedures has resulted in lack of uniform results and reference limits, and hampered comparison of results. In 2005, the IFCC therefore founded a special working group (WG) aiming for standardisation of CDT measurement. This review summarises the history of CDT and the actions taken by the WG-CDT. Initial steps included the definition of the measurand (serum disialotransferrin to total transferrin fraction expressed in %), and the determination of a well-defined anion-exchange HPLC procedure as the candidate reference measurement procedure (cRMP). Subsequent achievements were the establishment of a network of reference laboratories to perform the cRMP, setting a reference interval, and development of a reference material based on human serum for which the laboratory network assign values. Using a set of reference materials for calibration allowed for achieving equivalence of results of all present CDT measurement procedures. The final steps of the WG-CDT have been a full validation of the cRMP to make it an IFCC approved RMP, and providing guidance for international standardisation of all CDT measurement procedures.

A comparison between serum carbohydrate-deficient transferrin and hair ethyl glucuronide in detecting chronic alcohol consumption in routine.

AIMS: In heavy alcohol consumption laboratory tests represent an objective evidence. In this study we compared older and newer biomarkers in blood and in hair. METHODS: Carbohydrate-deficient transferrin (CDT), ethyl glucuronide (EtG), AST, ALT, GGT, MCV were measured in a large sample (n = 562). All people declared no alcohol consumption within the last 3 months. Serum CDT was measured by the candidate HPLC reference method and expressed as relative amount of disialotransferrin (%DST: cutoff 1.7%). EtG was measured in hair by a validated in-house method by LC-MS/MS (cutoff 30 pg/mg). RESULTS: Respectively, 42 (7.5%) and 76 (13.5%) subjects were positive to CDT and EtG. In particular, 30 (5.3%) subjects were positive to both tests, 12 (2.1%) only to CDT, while 46 (8.2%) only to EtG. The agreement (positive and negative pairs) between CDT and EtG was 89.7%. Interestingly, 6 out of 12 (50%) CDT-positive subjects had EtG < 15 pg/mg, whereas 27 out of 46 (59%) EtG-positive subjects had CDT < 1.1%. Forty-one out of 76 (54%) EtG-positive subjects display EtG values within 30-50 pg/mg. CONCLUSION: Large variability exists between CDT and EtG in detecting chronic alcohol consumption. We suggest to use CDT, or a combination of different biomarkers, to identify alcohol abuse in a forensic context. EtG results close to the cutoff (30-50 pg/mg) should be cautiously considered before any sanction is assigned.

Multicenter comparison of automated procalcitonin immunoassays.

OBJECTIVES: A multicenter study to compare results of BRAHMS Kryptor PCT with those obtained using four BRAHMS-partnered procalcitonin (PCT) automated immunoassays (DiaSorin Liaison, BioMérieux Vidas, Roche Cobas E601 and Siemens Advia Centaur) and the Diazyme immunotubidimetric assay implemented on four clinical chemistry platforms (Abbott Architect c16000, Siemens Advia 2400, Roche Cobas C501 and Beckman Coulter AU5800). DESIGN AND METHODS: One hundred serum samples from in-patients with PCT values between 0.10 and 58.7 ng/mL were divided into aliquots and tested with the nine different reagents and analyzers. BRAHMS PCT Kryptor results were used as reference. RESULTS: Compared to BRAHMS PCT Kryptor, significant differences in results were observed on Vidas, Advia Centaur, Architect, Cobas C501 and AU5800. However, the correlation coeffiecients (r) with BRAHMS PCT Kryptor were between 0.899 and 0.988. The mean bias was less than ±1.02 ng/mL, except for Vidas (2.70 ng/mL). The agreement at three clinically relevant cut-offs was optimal: between 83-98% at 0.50 ng/mL, 90-97% at 2.0 ng/mL, and 98% at 10 ng/mL. The comparison of Diazyme PCT across the four clinical chemistry analyzers yielded high correlation coefficients (r between 0.952 and 0.976), a mean bias less than ±0.9 ng/mL, acceptable agreement at 0.5 ng/mL (>82%), and high concordance at the 2.0 ng/mL (>97%) and 10 ng/mL (>98%) cut-offs. CONCLUSIONS: The methods and applications evaluated in this multicenter study are aligned with BRAHMS PCT Kryptor and can be used for predicting the risk of progression to systemic inflammation in patients with bacterial infections using the conventional PCT diagnostic thresholds.

Harmonization of measurement results of the alcohol biomarker carbohydrate-deficient transferrin by use of the toolbox of technical procedures of the International Consortium for Harmonization of Clinical Laboratory Results.

BACKGROUND: The need for equivalent results of routine measurement procedures for the alcohol biomarker carbohydrate-deficient transferrin (CDT) has been recognized by the IFCC. This article describes a project to harmonize CDT as conducted by an IFCC working group initiated for this purpose. METHODS: We used procedures for achieving harmonization as developed by the Consortium for Harmonization of Clinical Laboratory Results to assess the suitability of a candidate reference measurement procedure (cRMP), candidate reference materials (cRMs), and the success of efforts to achieve harmonization. RESULTS: CDT measurement procedures in routine use showed good reproducibility (CV 1.1%-2.8%) and linearity (r > 0.990) with variable slopes (0.766-1.065) and intercepts (-0.34 to 0.92) compared to the cRMP. Heterogeneity after simulated harmonization was 4.7%. cRMs of frozen human native sera demonstrated commutability and 3-year stability for routine measurement procedures. The cRMP provided reproducible value assignment to cRMs with an expanded uncertainty (k = 2) of 0.03% at the 1.2% CDT level and 0.06% at the 4.4% CDT level. Harmonization efforts reduced the intermeasurement CV from 8.8% to 3.4%, allowed 99% recovery of the values assigned with the cRMP, and demonstrated 99% of results within the desirable allowable total error. Harmonization was less successful in samples with low CDT and high trisialotransferrin concentrations. CONCLUSIONS: Harmonization of CDT is possible with frozen human native sera as cRMs with values assigned by use of the cRMP. We propose the cRMP as a candidate international conventional reference measurement procedure and cRMs as candidate international calibrators.

Therapeutic drug monitoring of levetiracetam: comparison of a novel immunoassay with an HPLC method.

BACKGROUND: Therapeutic drug monitoring of the anticonvulsant levetiracetam may be indicated in patients with conditions that may alter pharmacokinetic characteristics, for tailoring individual dosage regimens or to investigate patient compliance. In this study, the Bio-Rad high-performance liquid chromatography (HPLC) method (in-use method) and the ARK immunoassay method (new method) for levetiracetam monitoring in serum were compared. METHODS: Levetiracetam concentrations were determined in 63 samples using: (1) "Levetiracetam by HPLC" kit by Bio-Rad (Hercules, CA) on the Agilent 1100 HPLC system, and (2) "ARK Levetiracetam" immunoassay by ARK Diagnostics Inc (Fremont, CA) on the CDx90 platform by ThermoFisher Scientific Inc. RESULTS: Within-laboratory imprecision and bias of the new method evaluated over a 20-day period were 7.4% and 0.5% at 7.5 mcg/mL, 4.5% and 1.9% at 30 mcg/mL, and 3.1% and 2.0% at 75 mcg/mL. Passing-Bablok regression analysis (X:Bio-Rad; Y:Ark) showed a nonsignificant intercept of 0.16 [95% confidence interval (CI), -0.55 to 0.72] and a slope marginally significantly different from unity of 0.95 (95% CI, 0.90-0.99), which suggested minimum proportional systematic error. In agreement, Bland-Altman analysis showed minimum systematic bias of 1.0 mcg/mL (95% CI, 0.32-1.69) with 95% of the HPLC-Ark differences ranging from -4.3 (95% CI, -5.52 to -3.16) to 6.3 (95% CI, 5.16-7.52). Our data showed that the 2 methods were identical both within inherent imprecision and analytical quality specifications (maximum allowable error 15%). CONCLUSIONS: The new Ark method on the CDx platform is acceptable and may be used to measure serum levetiracetam concentrations routinely.

False negativity to carbohydrate-deficient transferrin and drugs: a clinical case.

INTRODUCTION: In this work we report on the possible effect of the medical therapy on CDT concentration in a chronic alcohol abuser, with known medical history (July 2007 - April 2012) and alcohol abuse confirmed by relatives. CASE HISTORY: At the end of 2007, patient displayed the following laboratory results: AST 137 U/L, ALT 120 U/L, GGT 434 U/L, MCV 101 fL and CDT 3.3%. On December 2007, after double coronary artery bypass surgery, he began a pharmacological treatment with amlodipine, perindopril, atorvastatin, isosorbide mononitrate, carvedilol, ticlopidine and pantoprazole. In the next months, until may 2011, the patient resumed alcohol abuse, as confirmed by relatives; however, CDT values were repeatedly found negative (0.8% and 1.1%) despite elevated transaminases and GGT, concurrent elevated ethyl glucuronide concentration (> 50 mg/L) and blood alcohol concentration (> 1 g/L). Alcohol consumption still continued despite increasing disulfiram doses ordered by an Alcohol Rehab Center. On May 2011, the patient was transferred to a private medical center where he currently lives. CONCLUSIONS: This study suggests the possibility that a medical therapy including different drugs may hamper the identification of chronic alcohol abusers by CDT.

Toward standardization of carbohydrate-deficient transferrin (CDT) measurements: III. Performance of native serum and serum spiked with disialotransferrin proves that harmonization of CDT assays is possible.

Carbohydrate-deficient transferrin (CDT) is a generic term that refers to the transferrin glycoforms whose concentration in blood is temporarily increased by sustained alcohol consumption. Due to high clinical specificity, CDT was proposed as a biomarker of heavy alcohol use and has been available for about 20 years. A number of methods have been developed for CDT measurement based on different analytical techniques and principles and without any harmonization or calibration to a reference method. As a consequence, neither the reference limits nor the cut-off values have been similar across assays, hampering understanding of the diagnostic value of CDT and its routine use. This prompted the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) to initiate a Working Group on Standardization of CDT (WG-CDT). This third publication of the WG-CDT is devoted to testing the commutability of native and disialotransferrin-spiked serum panels as candidate secondary reference materials, in order to prove the harmonization potential of commercial CDT methods. The results showed that assay harmonization reduced the inter-laboratory imprecision in a network of reference laboratories running the HPLC candidate reference method. In the seven commercial methods evaluated in this study, the use of multi-level secondary calibrators of human serum origin significantly reduced the between-method imprecision. Thus, harmonization of CDT measurements by different methods can be achieved using this calibration system, opening the way for a full standardization of commercial methods against a reference method by use of certified reference materials.

High performance liquid chromatography evaluation of serum carbohydrate-deficient transferrin and more sialylated transferrin glycoforms in children.

OBJECTIVES: The evaluation of the age-specific distribution of transferrin glycoforms in paediatric patients may help in defining reference intervals which are critical for an improved and earlier diagnosis. DESIGN AND METHODS: Serum samples from 224 children (age: 2 months-14 years) were analyzed by HPLC (Bio-Rad CDT/HPLC kit) and glycoforms expressed as percentage of the total area of transferrin (Tf). RESULTS: Asialo- and Monosialo-Tf were not detectable in any patient. Medians (IQR) were respectively 0.92% (0.80-1.04%) for Disialo-Tf; 3.47% (2.69-4.18%) for Trisialo-Tf; 82.54% (81.32-83.53%) for Tetrasialo-Tf; 12.73% (11.91-14.09%) for Pentasialo-Tf. Statistically significant differences in Trisialo-Tf (p < 0.0005), Tetrasialo-Tf (p = 0.001), Pentasialo-Tf (p < 0.0005), but not in Disialo-Tf, were observed between the age groups. CONCLUSIONS: Age-specific Disialo-Tf cut-offs are not necessary. In children 1.3% and 6.4% may be suggested as upper limits of normal range to detect increases of Disialo- and Trisialo-Tf. The presence of Asialo- and Monosialo-Tf should be considered an abnormal finding and prompt further investigations.

Pregnancy and variations of carbohydrate-deficient transferrin levels measured by the candidate reference HPLC method.

AIMS: Contrasting data are available on the diagnostic accuracy of carbohydrate-deficient transferrin (CDT) during pregnancy. These differences may depend in part on how CDT was evaluated and expressed. Here, we report on variations of CDT levels in pregnant women using the high performance liquid chromatography (HPLC) candidate reference method. METHODS: Alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, mean corpuscular volume, serum transferrin, urine and serum ethyl glucuronide and CDT were measured in 64 women, self-reporting as non-alcohol abusers (age: median 34, IQR: 28-38), at different stages of normal pregnancy (gestational weeks: median 28, IQR: 8-33). CDT was expressed as percentage of disialotransferrin to total transferrin (%CDT). RESULTS: Transferrin was associated with both %CDT (r = 0.66; P < 0.001) and gestational week (r = 0.68; P < 0.001). Interestingly, %CDT was highly correlated with gestational week (r = 0.77; P < 0.001), even after controlling for the effect of transferrin. Moreover, statistically significant differences in %CDT were also evident between women grouped for pregnancy trimester (first trimester: mean 1.01% (SD 0.19); second trimester: 1.30% (SD 0.14); third trimester: 1.53% (SD 0.22); ANOVA P < 0.001). Trend analysis confirmed a proportional increase of %CDT along with pregnancy trimesters (P < 0.001). CONCLUSIONS: %CDT, measured with the HPLC candidate reference method, is independently associated with gestational week. Differently from what has been previously reported or expected, the relationship between pregnancy and CDT could be more complex. The diagnostic accuracy of CDT for detecting alcohol abuse in a legal context may be limited in pregnant women and the effect of gestational age should be considered.

Use of carbohydrate-deficient transferrin (CDT) and a combination of GGT and CDT (GGT-CDT) to assess heavy alcohol consumption in traffic medicine.

AIMS: Carbohydrate-deficient transferrin (CDT) has become widely used in traffic medicine to detect chronic alcohol abuse among subjects applying for driving-license renewal or regranting. By defining cut-off values in a large population of abstainers and moderate drinkers, we report on CDT, GGT-CDT (a combination of gamma-glutamylaminotransferase (GGT) and CDT) and the association between blood alcohol concentration (BAC) and CDT among Italian drivers. METHODS: CDT was evaluated by a high performance liquid chromatography (HPLC)-based commercial kit in 652 abstainers or moderate drinkers, 603 drivers applying for driving-license regranting after a rehabilitation programme and 105 drivers involved in car accidents with blood alcohol concentration higher than the legal limit used in Italy (BAC >0.5g/l). GGT-CDT was calculated according to Sillanaukee and Olsson and Niemelä. BAC has been assessed by gas chromatography-mass spectrometry. RESULTS: A common CDT cut-off (1.8%) and gender-specific GGT-CDT cut-off (4.15% for males, 3.56% for females) were calculated as 99.9th percentiles of the control population. Also, 3% and 27% of subjects were classified as CDT positive respectively among drivers applying for license regranting and drivers involved in car accidents. A significant association between BAC and both CDT values and CDT positivity was found, with a frequency up to 49% of CDT samples, suggesting chronic alcohol abuse, among drivers with BAC >2.5g/l. Concordance between CDT and GGT-CDT was only moderate (kappa = 0.44), with CDT performing better than GGT-CDT. CONCLUSIONS: A relevant proportion of drivers with high BAC are chronic abusers. GGT-CDT, previously validated with CDT immunoassays, should not be applied to traffic medicine in its current form and its performances re-evaluated with CDT measured by HPLC.

Determination of carbohydrate-deficient transferrin (CDT) in Italy.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.