Survey of total error of precipitation and homogeneous HDL-cholesterol methods and simultaneous evaluation of lyophilized saccharose-containing candidate reference materials for HDL-cholesterol

BACKGROUND: Standardization of HDL-cholesterol is needed for risk assessment. We assessed for the first time the accuracy of HDL-cholesterol testing in The Netherlands and evaluated 11 candidate reference materials (CRMs). METHODS: The total error (TE) of HDL-cholesterol measurements was assessed in native human sera by 25 Dutch clinical chemistry laboratories. Concomitantly, the suitability of lyophilized, saccharose-containing CRMs (n = 11) for HDL-cholesterol was evaluated. RESULTS: In the precipitation method group, which included 25 laboratories and four methods, the mean (minimum-maximum) TE was 11.5% (2.7-25.2%), signifying that 18 of 25 laboratories satisfied the TE goal of </=13% issued by the National Cholesterol Education Program (NCEP). In the homogeneous HDL-cholesterol method group, which included five laboratories, each performing two different methods, the mean (minimum-maximum) TE was 9.5% (6.0-17.3%) for the Boehringer assay and 15.7% (3.3-30.7%) for the Genzyme assay. For the Boehringer homogeneous assay, one of five laboratories did not meet the TE criterion, whereas for the Genzyme homogeneous assay, three of five laboratories exceeded the 13% criterion. The biases on the HDL-cholesterol values found by various precipitation methods were highly variable in all CRMs, irrespective of the quality, whereas the biases found by the homogeneous method from Boehringer were far less than +/-5% for the highest-quality CRMs (CRMs 4-6). CONCLUSIONS: The NCEP goal was met by 24 of 35 laboratories assessed by use of native human sera. Selectively pooled, lyophilized CRMs that are cryoprotected with 200 g/L saccharose have ample potential for use in the standardization of homogeneous HDL-cholesterol methods

Optimization of apolipoprotein(a) genotyping with pulsed field gel electrophoresis

BACKGROUND: Increased lipoprotein(a) is a risk factor for atherosclerosis, and its concentration in serum is inversely correlated with the size of the apoliprotein(a) [apo(a)] component. The size of the apo(a) gene is determined mainly by the Kringle IV size polymorphism. We have optimized and characterized pulsed field gel electrophoresis (PFGE) for apo(a) genotyping. METHODS: Established PFGE protocols were adjusted. The changes included the following: (a) increased DNA yields by the use of all leukocytes for isolation from either 3 mL of fresh EDTA whole blood or 250 microL of frozen buffy coats; (b) increased efficiency of Kpn1 digestion by the inclusion of a digestion buffer wash; (c) reduction of assay time by the use of capillary blotting; (d) increased sensitivity by the use of four digoxigenin-labeled apo(a) probes; and (e) identification using a single film by the inclusion of a digoxigenin-labeled lambda marker probe in addition to apo(a) probes in the hybridization mix. RESULTS: In older Caucasians, 93% (buffy coats, n=468) were heterozygous for apo(a) gene size. An inverse correlation between serum lipoprotein(a) and the sum of Kringle IV alleles was found (y = -23x + 1553; r = -0.442; n = 468). Gel-to-gel variation was minimal (3%). Imprecision (SD) was one Kringle IV repeat (control sample containing eight fragments of 72-233 kb; n=34 electrophoretic runs). CONCLUSIONS: The practicality and sensitivity of the apo(a) genotyping technique by PFGE were improved, and accuracy and reproducibility were preserved. The optimized procedure is promising for apo(a) genotyping on frozen buffy coats from large epidemiological studies

Reference standardization and triglyceride interference of a new homogeneous HDL-cholesterol assay compared with a former chemical precipitation assay

A homogeneous HDL-c assay (HDL-H), which uses polyethylene glycol-modified enzymes and sulfated alpha-cyclodextrin, was assessed for precision, accuracy, and cholesterol and triglyceride interference. In addition, its analytical performance was compared with that of a phosphotungstic acid (PTA)/MgCl2 precipitation method (HDL-P). Within-run CVs were < or = 1.87%; total CVs were < or = 3.08%. Accuracy was evaluated in fresh normotriglyceridemic sera using the Designated Comparison Method (HDL-H = 1.037 Designated Comparison Method + 4 mg/L; n = 63) and in moderately hypertriglyceridemic sera by using the Reference Method (HDL-H = 1.068 Reference Method - 17 mg/L; n = 41). Mean biases were 4.5% and 2.2%, respectively. In hypertriglyceridemic sera (n = 85), HDL-H concentrations were increasingly positively biased with increasing triglyceride concentrations. The method comparison between HDL-H and HDL-P yielded the following equation: HDL-H = 1.037 HDL-P + 15 mg/L; n = 478. We conclude that HDL-H amply meets the 1998 NCEP recommendations for total error; its precision is superior compared with that of HDL-P, and its average bias remains below +/-5% as long as triglyceride concentrations are < or = 10 g/L and in case of moderate hypercholesterolemia

Close monitoring of hepatitis B surface antigen levels helps classify flares during peginterferon therapy and predicts treatment response

Background. Alanine aminotransferase (ALT) flares occur frequently during peginterferon (PEG-IFN) therapy. We related occurrence of flares to presence of precore (PC) and/or basal core promoter (BCP) mutants and studied kinetics of hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) levels during flares.Methods. Fifty of 214 (23%) patients treated with PEG-IFN ± lamivudine for 52 weeks experienced flares. Flares were host-induced (ALT elevation followed by HBV DNA decline, n = 19), virus-induced (HBV DNA increase with subsequent ALT elevation, n = 17) or indeterminate (n = 14

Hepatitis B e antigen levels and response to peginterferon: Influence of precore and basal core promoter mutants

WOS: 000317709400013PubMed ID: 23274785Hepatitis B e antigen (HBeAg) levels may predict response to peginterferon (PEG-IFN) but are also influenced by presence of precore (PC) and core promoter (BCP) mutants. HBeAg was measured in 214 patients treated with PEG-IFN +/- lamivudine for 52 weeks. Patients were classified at baseline as wildtype (WT) or non-WT (detectable PC/BCP mutants). Combined response (HBeAg loss with HBV DNA 2000 IU/mL) or non-response was assessed at week 78. Mean baseline HBeAg levels were 2.65 logIU/mL in combined responders, 2.48 in non-responders and 2.24 in HBeAg responders (p F 0.034). Baseline HBeAg levels were not associated with combined response after stratification by WT/non-WT. Within the PEG-IFN monotherapy group (n = 104), patients with HBeAg 1 logIU/mL, p < 0.001), whereas no difference in response rates was observed in non-WT patients (p = 0.848). The relationship between HBeAg levels and response to PEG-IFN depends upon the presence of PC/BCP mutants. HBeAg levels should therefore not be routinely used to select patients for PEG-IFN, nor for monitoring of therapy. (C) 2013 Elsevier B.V. All rights reserved.RocheRoche Holding; Gilead SciencesGilead Sciences; Bristol Myers SquibbBristol-Myers Squibb; NovartisNovartis; MerckMerck & Company; Innogenetics; Foundation for Liver Research (SLO), Rotterdam, the NetherlandsNetherlands GovernmentMilan J. Sonneveld has received speaker's fee from Roche. Vincent Rijckborst is a consultant for Roche. Roeland Zoutendijk has received speaker's honoraria from Bristol Myers Squibb. Krzysztof Simon, Ulus S. Akarca, Louwerens Zwang, Suzan Pas and Bettina E. Hansen have nothing to disclose. Stefan Zeuzem is a consultant for: BMS, Biolex, HGS, Merck/Schering-Plough, Novartis and Roche. E. Jenny Heathcote received funds for research and fees for consulting from Roche and Gilead Sciences. Prof. Dr. H.L.A. Janssen received grants from and is a consultant for: Bristol Myers Squibb, Gilead Sciences, Novartis, Roche, Merck, Innogenetics.; Study initiator and sponsor: Foundation for Liver Research (SLO), Rotterdam, the Netherlands. Kits for HBeAg quantification were kindly provided by Roche Diagnostics, the Netherlands

The Association of the Dopamine Transporter Gene and the Dopamine Receptor 2 Gene With Delirium, a Meta-Analysis

Delirium is the most common neuropsychiatric syndrome in elderly ill patients. Previously, associations between delirium and the dopamine transporter gene (solute carrier family 6, member 3 (SLC6A3)) and dopamine receptor 2 gene (DRD2) were found. The aim of this study was to validate whether markers of the SLC6A3 and DRD2 genes are were associated with delirium in independent populations. Six European populations collected DNA of older delirious patients. Associations were determined per population and results were combined in a meta-analysis. In total 820 medical inpatients, 185 cardiac surgery patients, 134 non-cardiac surgery patients and 502 population-based elderly subjects were included. Mean age was 82 years (SD 7.5 years), 598 (36%) were male, 665 (41%) had preexisting cognitive impairment, and 558 (34%) experienced delirium. The SLC6A3 rs393795 homozygous AA genotype was more frequent in patients without delirium in all populations. The meta-analysis showed an Odds Ratio (OR) for delirium of 0.4 (95% confidence interval (C.I.) 0.2-0.6, P= 0.0003) for subjects with AA genotype compared to the AG and GG genotypes. SLC6A3 marker rs1042098 showed no association with delirium. In meta-analysis the DRD2 rs6276 homozygous GG genotype showed an OR of 0.8 for delirium (95% C.I. 0.6-1.1, P=0.24). When subjects were stratified for cognitive status the rs6276 GG genotype showed ORs of 0.6 (95% C.I. 0.4-1.0, P=0.06) and 0.8 (95% C.I. 0.5-1.5, P=0.51) for delirium in patients with and without cognitive impairment, respectively. In independent cohorts, a variation in the SLC6A3 gene and possibly the DRD2 gene were found to protect for delirium. (C) 2009 Wiley-Liss, Inc