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.

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.

Usefulness of suPAR in the risk stratification of patients with sepsis admitted to the emergency department.

To investigate the role of suPAR in patients with sepsis admitted to the Emergency Department (ED). We performed multicentre prospective trial including patients admitted to the ED of three different Italian hospitals. Patients were studied upon admission on day 1, 2, 4 and 7. They were subdivided into two groups: sepsis (group 1) and severe sepsis or septic shock (group 2). The two groups were comparable for age, gender and CRP level on day 1. Patients with severe sepsis or septic shock displayed significantly higher baseline levels of suPAR, PCT and lactate. In both groups, suPAR decreased across the time (p < 0.0005). Group 1 was not different from group 2 (p = 0.545) in mortality at 7 days, while group 2 had higher mortality at 30 days than group 1 (p = 0.022). At the multivariate analysis, lactate1 (p = 0.012) and age (p = 0.019) were independent predictors of mortality at 7 days, whereas suPAR1 (p = 0.023) and age (p = 0.032) were independent predictors of mortality at 30 days. Lactate and suPAR resulted the most predictive biomarkers in the risk stratification of patients with suspected infection initially admitted to the ED, according to their role in predicting 7- and 30-day mortality, respectively.

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.