Substantial variation in the hepatitis B surface antigen (HBsAg) in hepatitis B virus (HBV)-positive patients from South Africa: Reliable detection of HBV by the Elecsys HBsAg II assay.

BACKGROUND: It is essential that hepatitis B surface antigen (HBsAg) diagnostic assays reliably detect genetic diversity in the major hydrophilic region (MHR) of HBsAg to avoid false-negative results. Mutations in this domain display marked ethno-geographic variation and may lead to failure to diagnose hepatitis B virus (HBV) infection. OBJECTIVES: Evaluate diagnostic performance of the Elecsys® HBsAg II Qualitative assay in a cohort of South African HBV-positive blood donors. STUDY DESIGN: A total of 179 South African HBsAg- and HBV DNA > 100 IU/mL-positive blood donor samples were included. Samples were sequenced for genetic variation in HBsAg MHR using next-generation ultra-deep sequencing. HBsAg seropositivity was determined using the Roche Elecsys HBsAg II Qualitative assay. Mutation rates were compared between the first (amino acids 124-137) and second (amino acids 139-147) loops of the immunodominant MHR 'a' determinant region. Frequency of occult HBV infection-associated Y100C mutations was also determined. RESULTS: We observed a total of 279 MHR mutations (117 variants) in 102 (57%) samples, of which 91 were located in the 'a' determinant region. The major vaccine-induced escape mutation G145R was observed in two samples. All occult HBV infection-associated Y100C and common diagnostic and vaccine-escape-associated P120T, G145R, K122R, M133L, M133T, Q129H, G130N, and T126S mutations were reliably detected by the assay, which consistently detected the presence of HBsAg in all 179 samples including samples with 11 novel mutations. CONCLUSIONS: Despite substantial variation in HBsAg MHR, the Elecsys HBsAg II Qualitative assay robustly detects HBV infection in this South African cohort.

Frequency of hepatitis B surface antigen variants (HBsAg) in hepatitis B virus genotype B and C infected East- and Southeast Asian patients: Detection by the Elecsys® HBsAg II assay.

BACKGROUND: To avoid false negative results, hepatitis B surface antigen (HBsAg) assays need to detect samples with mutations in the immunodominant 'a' determinant region, which vary by ethnographic region. OBJECTIVE: We evaluated the prevalence and type of HBsAg mutations in a hepatitis B virus (HBV)-infected East- and Southeast Asian population, and the diagnostic performance of the Elecsys® HBsAg II Qualitative assay. STUDY DESIGN: We analyzed 898 samples from patients with HBV infection from four sites (China [Beijing and Guangzhou], Korea and Vietnam). HBsAg mutations were detected and sequenced using highly sensitive ultra-deep sequencing and compared between the first (amino acids 124-137) and second (amino acids 139-147) loops of the 'a' determinant region using the Elecsys® HBsAg II Qualitative assay. RESULTS: Overall, 237 distinct amino acid mutations in the major hydrophilic region were identified; mutations were present in 660 of 898 HBV-infected patient samples (73.5%). Within the pool of 237 distinct mutations, the majority of the amino acid mutations were found in HBV genotype C (64.8%). We identified 25 previously unknown distinct mutations, mostly prevalent in genotype C-infected Korean patients (n = 18) followed by Chinese (n = 12) patients. All 898 samples were correctly identified by the Elecsys® HBsAg II Qualitative assay. CONCLUSIONS: We observed 237 distinct (including 25 novel) mutations, demonstrating the complexity of HBsAg variants in HBV-infected East- and Southeast Asian patients. The Elecsys® HBsAg II Qualitative assay can reliably detect HBV-positive samples and is suitable for routine diagnostic use in East and Southeast Asia.

Thyroid hormone replacement for central hypothyroidism: a randomized controlled trial comparing two doses of thyroxine (T4) with a combination of T4 and triiodothyronine.

BACKGROUND: Dosage of T(4) in central hypothyroidism is primarily guided by the free serum T(4) level (fT4). However, the optimum fT4 range is ill defined, and subtle hypothyroidism might be missed using this approach. OBJECTIVES: Our aim was to investigate the effects of a body weight (bw)-adapted T(4) treatment, alone or in combination with T(3), on metabolism, well-being, and cognitive function in comparison to a regimen leading to normal fT4. DESIGN: This was a placebo-controlled trial (double-blind, crossover). PATIENTS: A total of 29 patients (age 52 +/- 2 yr; females/males, 8/21) with hypopituitarism, including TSH deficiency, participated in the study. INTERVENTIONS: Three regimens were compared (5 wk each): "EMPIRICAL-T4," empirical T(4) dosage (1 +/- 0.05 microg/kg bw) leading to normal fT4; BW-ADAPTED-T4 (1.6 microg/kg bw T(4)); and "BW-ADAPTED-T3T4," bw-adapted combination of T(3) and T(4) (ratio of 1:10). RESULTS: BW-ADAPTED-T4 administration increased mean fT4 concentrations to the upper limit of the normal range (peak levels). Compared with EMPIRICAL-T4, BW-ADAPTED-T4 treatment resulted in a lower body mass index (BMI) (29.0 +/- 0.7 vs. 29.5 +/- 0.7 kg/m(2); P < 0.03), lower total cholesterol (198 +/- 9 vs. 226 +/- 7 mg/dl; P < 0.01), and lower low-density lipoprotein (LDL) cholesterol (116 +/- 5 vs. 135 +/- 7 mg/dl; P < 0.01). BW-ADAPTED-T3T4 treatment was associated with additional beneficial effects on ankle reflex time and working memory but resulted in supraphysiological free serum T(3) (fT(3)) levels. LIMITATIONS: Long-term side effects may have been missed. CONCLUSIONS: Using a dose of 1.6 microg/kg bw improved markers commonly associated with central hypothyroidism. This suggests that T(4) dosage based on bw and aiming at fT4 in the upper reference range is superior to titration of T(4) aiming at middle normal fT4 concentrations in those patients.

Adhesion molecules in pediatric intensive care patients with organ dysfunction syndrome.

OBJECTIVE: To determine serum concentrations of the soluble forms of vascular cell adhesion molecule 1 (VCAM-1), intracellular adhesion molecule 1 (ICAM-1), and E-selectin in ventilated neonatal and pediatric intensive care patients with varying severity of multiorgan dysfunction syndrome (MODS) with or without infection-triggered organ failure. DESIGN AND SETTING: Prospective pilot study, a level III neonatal and pediatric intensive care unit at a University children's Hospital. PATIENTS: We studied 22 ventilated pediatric (n=15) and neonatal (n=7) intensive care patients (aged 3 days-16 years). Inclusion criteria were mechanical ventilation and signs of at least one additional organ dysfunction (cardiovascular, respiratory, neurological, hematological, or renal). MEASUREMENTS AND RESULTS: Serum concentrations of the adhesion molecules were analyzed on the day of maximum organ dysfunction score and were quantitated by a sandwich ELISA technique. The overall mortality rate was 36% (8/22). Dysfunction of three or more organ systems was defined as MODS and was associated with a significant increase in VCAM-1 serum levels relative to dysfunction of three or fewer organ systems [median 1239 ng/ml (IQR 928-1615) vs. 766 ng/ml (644-915)]. A significant difference in E-selectin serum levels was found between organ failure of infectious (median 131 ng/ml, IQR 112-146) and noninfectious origin (68 ng/ml 49-105). CONCLUSIONS: Determination of adhesion molecules in pediatric intensive care patients raises the possibility of more specific pathophysiological understanding. E-selectin showed significantly different serum levels between infectious and noninfectious causes of organ failure.

Ultra-deep sequencing reveals high prevalence and broad structural diversity of hepatitis B surface antigen mutations in a global population.

The diversity of the hepatitis B surface antigen (HBsAg) has a significant impact on the performance of diagnostic screening tests and the clinical outcome of hepatitis B infection. Neutralizing or diagnostic antibodies against the HBsAg are directed towards its highly conserved major hydrophilic region (MHR), in particular towards its "a" determinant subdomain. Here, we explored, on a global scale, the genetic diversity of the HBsAg MHR in a large, multi-ethnic cohort of randomly selected subjects with HBV infection from four continents. A total of 1553 HBsAg positive blood samples of subjects originating from 20 different countries across Africa, America, Asia and central Europe were characterized for amino acid variation in the MHR. Using highly sensitive ultra-deep sequencing, we found 72.8% of the successfully sequenced subjects (n = 1391) demonstrated amino acid sequence variation in the HBsAg MHR. This indicates that the global variation frequency in the HBsAg MHR is threefold higher than previously reported. The majority of the amino acid mutations were found in the HBV genotypes B (28.9%) and C (25.4%). Collectively, we identified 345 distinct amino acid mutations in the MHR. Among these, we report 62 previously unknown mutations, which extends the worldwide pool of currently known HBsAg MHR mutations by 22%. Importantly, topological analysis identified the "a" determinant upstream flanking region as the structurally most diverse subdomain of the HBsAg MHR. The highest prevalence of "a" determinant region mutations was observed in subjects from Asia, followed by the African, American and European cohorts, respectively. Finally, we found that more than half (59.3%) of all HBV subjects investigated carried multiple MHR mutations. Together, this worldwide ultra-deep sequencing based genotyping study reveals that the global prevalence and structural complexity of variation in the hepatitis B surface antigen have, to date, been significantly underappreciated.

Platelet-activating factor acetylhydrolase activity indicates angiographic coronary artery disease independently of systemic inflammation and other risk factors: the Ludwigshafen Risk and Cardiovascular Health Study.

BACKGROUND: Platelet-activating factor acetylhydrolase (PAF-AH), also denoted as lipoprotein-associated phospholipase A2, is a lipoprotein-bound enzyme that is possibly involved in inflammation and atherosclerosis. This study investigates the relationship of PAF-AH activity to angiographic coronary artery disease (CAD), the use of cardiovascular drugs, and other established risk factors. METHODS AND RESULTS: PAF-AH activity, lipoproteins, sensitive C-reactive protein (sCRP), fibrinogen, serum amyloid A, and white blood cell count were determined in 2454 subjects with angiographically confirmed CAD and in 694 control subjects. PAF-AH activity was highly correlated with LDL cholesterol (r=0.517), apolipoprotein B (r=0.644), and non-HDL cholesterol (r=0.648) but not with sCRP or fibrinogen. PAF-AH activity was lower in women than in men and was affected by the intake of lipid-lowering drugs (-12%; P<0.001), aspirin (-6%; P<0.001), beta-blockers (-6%; P<0.001), and digitalis (+7%; P<0.001). Unlike sCRP, fibrinogen, and serum amyloid A, PAF-AH activity was not elevated in unstable angina, non-ST-elevation myocardial infarction, or ST-elevation myocardial infarction. When nonusers of lipid-lowering drugs were examined, PAF-AH activity was associated with the severity of CAD and the number of coronary vessels with significant stenoses. In individuals not taking lipid-lowering drugs and after adjustment for use of aspirin, beta-blocker, and digitalis, the odds ratio for CAD associated with increasing PAF-AH activity was 1.39 (95% CI 1.26 to 1.54, P<0.001), a finding that was robust against further adjustments. CONCLUSIONS: PAF-AH activity is not an indicator of the systemic inflammation that accompanies acute coronary syndromes. PAF-AH activity is affected by a number of cardiovascular drugs; however, after such medication use was accounted for, PAF-AH activity was associated with angiographic CAD, complementary to sCRP and independently of established risk factors such as LDL cholesterol.

Primary biliary cirrhosis is not a clinical condition for increased carbohydrate-deficient transferrin: experience with four independent CDT analysis methods.

BACKGROUND: Primary biliary cirrhosis (PBC) is considered as an important cause for increased carbohydrate-deficient transferrin (CDT). The underlying pathomechanism is difficult to explain by the pathogenesis and/or consequences of PBC. We tested whether PBC causes increased CDT results with current CDT analysis methods and, if so, whether this depends on the CDT analysis principle. METHODS: 48 serum samples from PBC patients were analyzed by HPLC, microcolumn CDT and non-CDT fractionation followed by a turbidimetric immunoassay, particle-enhanced immunonephelometry with monoclonal CDT antibodies, and capillary electrophoresis. The test-specific decision limits were used for categorization of the CDT analysis results into normal and increased values. RESULTS: HPLC: 47 normal/1 increased, microcolumn+TIA: 46 normal/2 increased, particle-enhanced immunonephelometry: 41 normal/7 increased, capillary electrophoresis: 48 normal CDT results. After combining an immunological CDT test (microcolumn+TIA or particle-enhanced immunonephelometry) as the screening method with a physico-chemical CDT test (HPLC or electrophoresis) as the confirmatory method, 1 case remained with increased CDT values by the screening (value 2.6%, cut-off 2.5%, particle-enhanced immunonephelometry) and confirmatory (value 1.8%, cut-off 1.75%, HPLC) analysis. CONCLUSIONS: PBC should no longer be overstressed as an important cause for false-positive CDT results regarding chronic alcohol abuse. In the presence of odd CDT results, PBC should be considered in the anamnestic exploration. However, PBC is not by itself a cause for increased CDT values.

Adiponectin serum concentrations in men with coronary artery disease: the LUdwigshafen RIsk and Cardiovascular Health (LURIC) study.

BACKGROUND: Adiponectin, the most abundant adipocytokine of adipose tissue cells, has recently been found to be decreased in coronary artery disease (CAD). Data concerning adiponectin in different stages of CAD are rare, and it was not investigated if adiponectin levels are influenced by the severity of angina pectoris. METHODS: Thus, we measured adiponectin serum levels by means of ELISA in 1626 male probands, including 273 control subjects, 367 subjects with silent CAD, 608 patients with stable, and 378 patients with unstable angina. RESULTS: As compared to controls (8.56; 5.85 to 12.85 microg/ml) and subjects with silent CAD (8.60; 5.99 to 12.64 microg/ml), adiponectin was significantly decreased in patients with stable (7.22; 5.06 to 10.41 microg/ml; p < 0.001 for both) and unstable angina (6.72; 4.08 to 10.08 microg/ml; p < 0.001 for both). By a logistic regression analysis, low adiponectin levels were identified as a significant independent predictor for stable and unstable angina (p < 0.001 for both). No significant differences of adiponectin were observed, neither between the stable and unstable angina group, nor between any classes of angina according to the Canadian Cardiovascular Society (CCS) Angina Score for stable angina. CONCLUSIONS: These results suggest, that decreased adiponectin levels are indicative for symptomatic CAD, but are not further influenced by the progression of this disease.

Association of angiotensinogen haplotypes with angiotensinogen levels but not with blood pressure or coronary artery disease: the Ludwigshafen Risk and Cardiovascular Health Study.

Angiotensinogen and its cleaved forms angiotensin I and angiotensin II are important regulators of blood pressure. The gene for angiotensinogen (AGT) carries two common polymorphisms, T207M and M268T (previously described as T174M and M235T). To investigate the role of haplotypes formed by these polymorphisms for angiotensinogen levels we examined blood pressure, coronary artery disease (CAD), myocardial infarction (MI), and AGT genotypes and haplotypes in 2,575 patients with angiographically documented CAD and 731 individuals in whom CAD had been ruled out by angiography. Three haplotypes, designated as Hap1 (T207, M268), Hap2 (T207, T268) and Hap3 (M207, T268), accounted for more than 99% of alleles. The AGT Hap2 haplotype was significantly associated with angiotensinogen levels; one additional Hap2 allele accounted for an approx. 8% increase in angiotensinogen. This association was stronger than that of either single polymorphism. AGT genotypes or haplotypes were not related to hypertension, CAD or MI. We conclude that a common haplotype of the angiotensinogen gene is linked to angiotensinogen levels but has no major impact on blood pressure, hypertension, or cardiovascular risk.

Low-density lipoprotein triglycerides associated with low-grade systemic inflammation, adhesion molecules, and angiographic coronary artery disease: the Ludwigshafen Risk and Cardiovascular Health study.

BACKGROUND: Markers of systemic inflammation and LDL cholesterol (LDL-C) have been considered independent risk factors of coronary artery disease (CAD). We examined whether alterations of LDL metabolism not reflected by LDL-C were associated with low-grade inflammation, vascular injury, and CAD. METHODS AND RESULTS: We studied 739 subjects with stable angiographic CAD and 570 matched control subjects in which CAD had been ruled out by angiography. The association of LDL triglycerides (LDL-TGs) (odds ratio [OR], 1.30; 95% CI, 1.19 to 1.43; P<0.001) with CAD was stronger than that of LDL-C (OR, 1.10; 95% CI, 1.00 to 1.21; P=0.047). The predictive value of LDL-TG for CAD was independent of LDL-C. "Sensitive" C-reactive protein (CRP), serum amyloid A, fibrinogen, interleukin 6, intercellular adhesion molecule-1 (ICAM-1), and vascular adhesion molecule-1 (VCAM-1) increased in parallel to LDL-TG. CRP, ICAM-1, and VCAM-1 were inversely related to LDL-C. To examine whether LDL-TGs were associated with the distribution of LDL subfractions, we studied 114 individuals with impaired fasting glucose, impaired glucose tolerance, or type 2 diabetes mellitus. In subjects with high LDL-TG, LDLs were depleted of cholesteryl esters (CEs), and VLDLs, IDLs, and dense LDLs were significantly elevated. CONCLUSIONS: Alterations of LDL metabolism characterized by high LDL-TG are related to CAD, systemic low-grade inflammation, and vascular damage. High LDL-TGs are indicative of CE-depleted LDL, elevated IDL, and dense LDL. LDL-TG may better reflect the atherogenic potential of LDL than LDL-C.

G(-30)A polymorphism in the pancreatic promoter of the glucokinase gene associated with angiographic coronary artery disease and type 2 diabetes mellitus.

BACKGROUND: Type 2 diabetes mellitus (T2DM) increases the risk of coronary artery disease (CAD). A G(-30)A polymorphism in the beta-cell-specific promoter of glucokinase (GK-30PM) has been implicated in reduced pancreatic beta-cell function. Its impact on CAD has not been examined. METHODS AND RESULTS: The glucokinase G(-30)A variant was determined in 2567 patients with angiographic CAD and in 731 individuals in whom CAD had been ruled out by angiography. In carriers of the A allele, the adjusted OR of CAD was 1.39 (95% CI, 1.15 to 1.70). Corresponding ORs were 1.27 (95% CI, 1.02 to 1.59) and 1.92 (95% CI, 1.26 to 2.93) in individuals without and with T2DM, respectively. The prevalence of the A allele increased in parallel with the Friesinger coronary score. Patients with T2DM were more frequent among carriers of > or =1 A allele (OR, 1.17; 95% CI, 1.00 to 1.28). This association was stronger if CAD patients only were considered. The A allele was associated with higher glucose (fasting, P=0.002; 2 hours after oral glucose, P=0.017) and glycohemoglobin (HbA1c; P=0.002). Furthermore, presence of 1 A allele was negatively related to beta-cell function, estimated by beta percent (P=0.012) and by the ratios of proinsulin to insulin (P=0.025) and proinsulin to C peptide (P=0.019). CONCLUSIONS: The A allele of the pancreatic promoter of glucokinase increases the risk of CAD in individuals with and without T2DM. Furthermore, at least in CAD, it is associated with an augmented prevalence of T2DM.

The interleukin-6 G(-174)C promoter polymorphism in the LURIC cohort: no association with plasma interleukin-6, coronary artery disease, and myocardial infarction.

IL-6 plasma levels are predictive of major cardiovascular events. Recently a G/C polymorphism at position -174 in the promoter of the IL-6 gene has been associated with differences in both the IL-6 transcription rate in vitro and IL-6 levels in vivo. We examined the association of this polymorphism with coronary artery disease (CAD) and previous myocardial infarction (MI) in 2559 patients with angiographically documented CAD with ( n=1365) and without ( n=1194) MI and in a control group of 729 individuals in whom CAD had been ruled out angiographically. Assuming dominant or recessive modes of inheritance, carriers of the G allele had odds ratios of 0.98 (95% CI 0.79 - 1.20) and 0.96 (95% CI 0.80 - 1.14), respectively, for CAD, and almost identical ones for previous MI. In subgroups stratified for low cardiovascular risk, the IL-6 promoter polymorphism was also not related to the risk of CAD or MI. In addition, the plasma concentration of IL-6 did not differ between groups with different IL-6 genotypes in 942 randomly selected individuals. We conclude that the IL-6 G(-174)C polymorphism is not associated with the risk of CAD or MI and does not contribute to cardiovascular risk stratification.

Effects of glucagon-like peptide 1 on counterregulatory hormone responses, cognitive functions, and insulin secretion during hyperinsulinemic, stepped hypoglycemic clamp experiments in healthy volunteers.

Glucagon-like peptide 1 (GLP-1) and analogues are being evaluated as a new therapeutic principle for the treatment of type 2 diabetes. GLP-1 suppresses glucagon secretion, which could lead to disturbances of hypoglycemia counterregulation. This has, however, not been tested. Nine healthy volunteers with normal oral glucose tolerance received infusions of regular insulin (1 mU x kg(-1) x min(-1)) over 360 min on two occasions in the fasting state. Capillary glucose concentrations were clamped at plateaus of 4.3, 3.7, 3.0, and 2.3 mmol/liter for 90 min each (stepwise hypoglycemic clamp); on one occasion, GLP-1 (1.2 pmol x kg(-1) x min(-1)) was administered i.v. (steady-state concentration, approximately 125 pmol/liter); on the other occasion, NaCl was administered as placebo. Glucagon, cortisol, GH (immunoassays), and catecholamines (radioenzymatic assay) were determined, autonomous and neuroglucopenic symptoms were assessed, and cognitive function was tested at each plateau. Insulin secretion rates were estimated by deconvolution (two-compartment model of C-peptide kinetics). At insulin concentrations of approximately 45 mU/liter, glucose infusion rates were similar with and without GLP-1 (P = 0.26). Only during the euglycemic plateau (4.3 mmol/liter), GLP-1 suppressed glucagon concentrations (4.1 +/- 0.4 vs. 6.5 +/- 0.7 pmol/liter; P = 0.012); at all hypoglycemic plateaus, glucagon increased similarly with GLP-1 or placebo, to maximum values greater than 20 pmol/liter (P = 0.97). The other counterregulatory hormones and autonomic or neuroglucopenic symptom scores increased, and cognitive functions decreased with decreasing glucose concentrations, but there were no significant differences comparing experiments with GLP-1 or placebo, except for a significant reduction of GH responses during hypoglycemia with GLP-1 (P = 0.04). GLP-1 stimulated insulin secretion only at plasma glucose concentrations of at least 4.3 mmol/liter. In conclusion, the suppression of glucagon by GLP-1 does occur at euglycemia, but not at hypoglycemic plasma glucose concentrations (< or = 3.7 mmol/liter). GLP-1 does not impair overall hypoglycemia counterregulation except for a reduction in GH responses, which is in line with other findings demonstrating pituitary actions of GLP-1. Below plasma glucose concentrations of 4.3 mmol/liter, the insulinotropic action of GLP-1 is negligible.

Effects of statins on C-reactive protein and interleukin-6 (the Ludwigshafen Risk and Cardiovascular Health study).

This report provides preliminary evidence that statins may lower C-reactive protein levels by interfering with the generation and/or release of C-reactive protein in the liver rather than by modulating inflammatory processes in the vessel wall.

Targeted next-generation sequencing identifies a homozygous nonsense mutation in ABHD12, the gene underlying PHARC, in a family clinically diagnosed with Usher syndrome type 3.

BACKGROUND: Usher syndrome (USH) is an autosomal recessive genetically heterogeneous disorder with congenital sensorineural hearing impairment and retinitis pigmentosa (RP). We have identified a consanguineous Lebanese family with two affected members displaying progressive hearing loss, RP and cataracts, therefore clinically diagnosed as USH type 3 (USH3). Our study was aimed at the identification of the causative mutation in this USH3-like family. METHODS: Candidate loci were identified using genomewide SNP-array-based homozygosity mapping followed by targeted enrichment and next-generation sequencing. RESULTS: Using a capture array targeting the three identified homozygosity-by-descent regions on chromosomes 1q43-q44, 20p13-p12.2 and 20p11.23-q12, we identified a homozygous nonsense mutation, p.Arg65X, in ABHD12 segregating with the phenotype. CONCLUSION: Mutations of ABHD12, an enzyme hydrolyzing an endocannabinoid lipid transmitter, cause PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and early-onset cataract). After the identification of the ABHD12 mutation in this family, one patient underwent neurological examination which revealed ataxia, but no polyneuropathy. ABHD12 is not known to be related to the USH protein interactome. The phenotype of our patient represents a variant of PHARC, an entity that should be taken into account as differential diagnosis for USH3. Our study demonstrates the potential of comprehensive genetic analysis for improving the clinical diagnosis.

Detection and quantitation of HBV DNA in miniaturized samples: multi centre study to evaluate the performance of the COBAS ® AmpliPrep/COBAS ® TaqMan ® hepatitis B virus (HBV) test v2.0 by the use of plasma or serum specimens.

Laboratory analysis of blood specimens is an increasingly important tool for rapid diagnosis and control of therapy. So, miniaturization of test systems is needed, but reduced specimens might impair test quality. For rapid detection and quantitation of HBV DNA, the COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HBV test has proved a robust instrument in routine diagnostic services. The test system has been modified recently for application of reduced samples of blood plasma and for blood serum, too. The performance of this modified COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HBV v2.0 (HBV v2.0 (this test is currently not available in the USA)) test was evaluated by comparison with the former COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HBV v1.0 (HBV v1.0) test. In this study a platform correlation of both assay versions was done including 275 HBV DNA positive EDTA plasma samples. Comparable results were obtained (R(2)=0.97, mean difference -0.03 log(10)IU/ml). The verification of equivalency of the sample matrix (plasma vs. serum samples tested in HBV v2.0 in the same run) showed comparable results for all 278 samples with a R(2)=0.99 and a mean difference of 0.06 log(10)IU/ml. In conclusion, the new test version HBV v2.0 is highly specific and reproducible and quantifies accurately HBV DNA in EDTA plasma and serum samples from patients with chronic HBV infection.

A recapping system for automatic, semiautomatic, and manual use.

CONTEXT: Closed sample tubes are needed for stored as well as in-process samples because evaporation significantly changes analyte concentrations. In modern laboratories, it is essential that the procedures for ordering tests on stored serum samples be simple and time-efficient. Therefore, recapping and reopening of tubes should be readily possible in laboratories to save resources and time. OBJECTIVE: To evaluate the recapping system MagCap, in comparison to established snap caps and unsealed tubes. DESIGN: Specific balls, with an iron core, fit tightly by their specific weight into openings of primary and secondary tubes as well as microtiter plates. Recapping of the tubes can be performed individually by a single dispenser or rackwise by a multidispenser. Balls can be removed individually by a pencillike magnet or rackwise. Balls can be reused after cleaning in a glassware washer or used as disposables. RESULTS: Balls seal the sample tubes as effectively as snap caps and decrease evaporation significantly in comparison to unsealed tubes. Evaporation effects of up to 13% were detected in unsealed tubes (0.5 mL) within 24 hours, whereas resealing of the tubes diminished the concentration effect to less than 1%. Open samples (0.5 mL) showed a concentration effect in the refrigerator within 7 days of up to 25%. The evaporation effect of the sealed tubes was less than 3%. Multiple reopenening and recapping procedures are easily possible using magnetic forces. CONCLUSIONS: The MagCap recapping system seals primary and secondary containers securely and cost-effectively with the ball cap and thus facilitates a necessary advance in sample quality.

Forensic analysis of carbohydrate-deficient transferrin (CDT) by HPLC--statistics and extreme CDT values.

BACKGROUND: Carbohydrate-deficient transferrin (CDT) is the most specific serum marker of chronic alcohol abuse so far. There is little knowledge about extreme CDT values of >20% and the more >30% CDT. METHODS: Serum CDT/transferrin ratios from 19,236 serum samples sent to our laboratory for routine CDT analysis were determined by HPLC. About 75% of these serum samples were from traffic or employment medicine investigations. A CDT value frequency histogram was computed and extreme CDT values were clinically validated. RESULTS: Fourteen thousand four hundred and sixty-one CDT results were normal (< or =1.7%) and 4775 increased (1.8-36.9% CDT). Most frequent normal and increased results were 0.9% CDT (n=1964) and 1.8% CDT (n=356). CA. 70% of the pathological results were between 1.8% and 5.0% CDT, ca. 88% between 1.8% and 10.0% CDT, and 98% between 1.8% and 20.0%. CDT values >20.0% appeared in 79 cases and results >30.0% in two cases (33.8% and 36.9%). In each case of CDT values >20%, chronic alcohol abuse was the underlying cause as confirmed by anamnestic exploration. CONCLUSIONS: CDT/transferrin ratios are usually <20%. Higher values can appear in rare cases. CDT results >30% can be due to alcohol abuse but should be considered as remarkable single observations. Visualization of the transferrin isoform patterns by HPLC allows the detection of pathological transferrin isoform patterns and of genetic transferrin variants. This is essential for a reliable interpretation of (extreme) CDT values. CDT analysis by immunoassays without physico-chemical confirmatory analysis is no longer acceptable.