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Biomedical subjects

N Blanckaert

Publications and source records attributed to N Blanckaert.

At least 19 recordsLinked to original sources
PubMed2004

Detection of single nucleotide polymorphisms in the mannose-binding lectin gene using minor groove binder-DNA probes.

Structural point mutations in exon 1 at codons 52, 54 and 57 and a promotor polymorphism at -221 bp of the mannose-binding lectin (MBL) gene are associated with increased susceptibility to various infectious diseases. We developed a genotyping method based on the 5' nuclease (TaqMan) assay in combination with the use of minor-groove-binder (MGB) probes in screening for these mutations/polymorphisms. In contrast to conventional probes, MGB probes have a short length and can be used for detection of mutations that are in close proximity to each other, as is the case for the structural mutations in exon 1 of the MBL gene. Results obtained with the 5' nuclease assay using MGB probes were identical with results obtained with classical techniques such as restriction fragment length polymorphism, allele-specific PCR, and sequencing. In conclusion, the 5' nuclease assay using MGB probes is useful for large-scale screening of point mutations/polymorphisms, even when they are in close proximity.

DNA Primers↗
PubMed2001

Differential regulation of UDP-GlcUA transport in endoplasmic reticulum and in Golgi membranes.

BACKGROUND: In the endoplasmic reticulum (ER), the stimulation of UDP-glucuronosyltransferase (UGT) by UDP-GlcNAc is based on the interaction of transport across the ER membrane of UDP-GlcUA with UDP-GlcNAc. Intramicrosomal UDP-GlcNAc stimulates influx of UDP-GlcUA and thereby enhances delivery of UDP-GlcUA to the catalytic center of UGT in the ER lumen. AIM: The aim of this study is to investigate whether the interactions between nucleotide sugars for transport across the ER membrane also occur in the Golgi apparatus, and thereby affect UGT activity in Golgi membranes. RESULTS: We found that Golgi membrane preparations display UGT activity which, unlike in ER membranes, is not stimulated by UDP-GlcNAc. Efflux of intravesicular UDP-GlcNAc and UDP-Xyl marginally enhanced uptake of UDP-GlcUA in Golgi vesicles; such trans-stimulation was much more pronounced in the ER. Efflux of intravesicular UDP-GlcNAc was strongly trans-stimulated by cytosolic UDP-GlcUA in ER-derived vesicles but less so in Golgi-derived vesicles. CONCLUSION: The interaction between transport of UDP-GlcUA and transport of UDP-GlcNAc or UDP-Xyl is different in Golgi vesicles compared with ER vesicles. This finding is consistent with the different effects of UDP-GlcNAc on glucuronidation in Golgi and ER.

Animals↗
PubMed2001

Inter-laboratory evaluation of the COBAS INTEGRA 400 analytical system.

COBAS INTEGRA 400 is a random-access analytical system consolidating assays for clinical chemistry analytes, electrolytes, serum proteins, drugs of abuse and therapeutic drugs. Analytical performance and practicability of the instrument were evaluated in seven laboratories over a 2-year period in parallel with system development. Good within-run and total imprecision for all assays was observed with a few exceptions for specimen pools with low concentration or activity. The coefficients of variation for total imprecision were well below 3.0% for clinical chemistry analytes and electrolytes, and below 5.0% for serum proteins and therapeutic drugs. Method comparisons demonstrated a good agreement with the various systems used for comparison, with slopes varying typically from 0.94 to 1.05, and Spearman correlation coefficient generally > 0.975. Accuracy was verified by recovery of controls and certified reference materials within 90 to 110% of target values. Assay ranges were linear within +/- 5%. No carry-over on reagent or sample pipetting systems was observed. Manufacturer-specified interference limits and onboard stabilities of reagents were confirmed. A time study for calculating direct personnel times and total processing time was carried out in three laboratories under different conditions including consolidated, STAT and dedicated use. On a scenario-independent basis, the total working time was shorter on the COBAS INTEGRA 400 than on routine systems in all three laboratories. Personnel time, in particular, was significantly reduced when compared to routine instruments. In general, system practicability was judged very positively in all laboratories. Owing to its versatility, the instrument is best placed as a consolidated workstation in small- to medium-sized laboratories or as an instrument for special determinations such as serum proteins, drugs, urinalysis or emergency analyses in large laboratories.

Blood Proteins↗
PubMed2000

Screening for autoantibodies to SS-A/RO by indirect immunofluorescence using HEp-2000 cells.

We evaluated indirect immunofluorescence (IF) using HEp-2000 slides, which are transfected with SS-A cDNA, for screening for anti-SS-A antibodies, by comparing it with counterimmunoelectrophoresis (CIE). A total of 2427 specimens were screened for IF reactivity and for SS-A precipitins, of which 1033 (43%) were negative on both IF and CIE. There were 1271 SS-A precipitin-negative specimens (52%) which were IF-positive but lacked the distinctive SS-A staining pattern. One precipitin-negative serum was IF-positive with the distinctive SS-A pattern in the HEp-2000 system. One hundred and twenty-two specimens (5%) were positive for anti-SS-A precipitins on CIE, 107 showed the distinctive SS-A fluorescence staining pattern, whereas 15 of these precipitin-positive samples (12%) were IF-positive but did not display the distinctive SS-A pattern on the transfected cells. Fourteen of the 15 samples in which the distinctive SS-A pattern was not observed displayed other significant antinuclear antibody (titre equal or >1:320) patterns. In conclusion, the presence of the typical 'distinctive' SS-A pattern on IF using the HEp-2000 slides is highly specific for the presence of autoantibodies to SS-A and has a sensitivity of 88% for detecting these antibodies.

Antibodies, Antinuclear↗
PubMed1999

Analytical and clinical performance of two cardiac troponin I immunoassays.

Cardiac troponin I assays for Axsym (Abbott Diagnostics, Abbott Park, IL, USA) and Immuno 1 (Bayer Corporation, Tarrytown, NY, USA) analysers were evaluated. Heparin plasma or serum could be used for both assays. Samples were stable for 24 h at ambient temperature, 3 days at 4-8 degrees C and 3 months at -20 degrees C. After 10 months' storage at -80 degrees C, the recoveries were well above 100% by both assays. Total coefficients of variation for Axsym assay were 9.0%, 5.8% and 5.3% at concentrations of 2.6 microg/l, 9.83 microg/l and 34.3 microg/l respectively; for Immuno 1 these were 4.4 %, 1.6% and 1.8% at 2.3 microg/l, 6.27 microg/l and 44.35 microg/l respectively. It was > or =20% at concentration of < or =0.5 microg/l for Axysm assay and < or =0.15 microg/l for Immuno 1 assay. Recoveries were < or =90% at < or =0.22 microg/l on Axsym and at < or =1.47 microg/l on Immuno 1. Neither method showed significant interference with haemoglobin, bilirubin, triglycerides or rheumatoid factor. Correlation between the two methods was excellent (r = 0.997, Y (Axsym) = 4.2X (Immuno 1) +3.2). The highest concentrations detected in 50 healthy subjects were 0.3 microg/l and 0.1 microg/l by Axsym and Immuno 1 methods, respectively. Twelve out of 43 renal failure patients had troponin I 0.13-0.9 microg/l using Axsym method and 4 had levels of 0.07-0.13 microg/l using Immuno 1. In muscle trauma patients, troponin I was undetectable.

Adult↗
PubMed1999

Evaluation of interferences in rate and fixed-time nephelometric assays of specific serum proteins.

We performed interference studies for IgG, IgA, IgM, haptoglobin, and alpha1-antitrypsin assayed in serum, using either fixed-time nephelometry on the BN 100 from Behring or rate nephelometry on two analyzers from Beckman Instruments. For clear serum samples, results for IgG, IgA, IgM, and haptoglobin obtained with the three nephelometers showed good agreement. Values for alpha1-antitrypsin in clear sera were lower with the BN 100 than with the Array 360 or Immage. In lipemic samples, the BN 100 gave higher values than the Array 360 or Immage for all analytes except IgG. Addition of Intralipid to serum produced atypical reactions with the BN 100 (fixed-time nephelometry) but not with the Array 360 or Immage (rate nephelometry). The interference of lipemia on the BN 100 was also seen when the Beckman antibody was used, indicating that the effect was reagent-independent. For hemolyzed samples, the BN 100 gave higher values than the Array 360 or Immage for haptoglobin but not for the other analytes. Addition of increasing amounts of a hemolysate to serum revealed a negative interference in all assay systems. This effect was more pronounced with the Beckman reagent than with the Behring reagent in all three nephelometers and was independent of the type of instrument (fixed-time vs rate nephelometry).

Blood Proteins↗
PubMed1998

Disappointing dipstick screening for urinary tract infection in hospital inpatients.

AIM: To compare the performance of leucocyte esterase and nitrite dipstick tests with microscopic examination and culture of first morning urines (n = 420) of hospital inpatients. RESULTS: The sensitivity, specificity, and negative predictive value of the leucocyte esterase test for the cutoff of > 10 WBC/microliter were 57%, 94%, and 68%, respectively. For > 5 WBC per high power field (HPF) these variables were 84%, 90%, and 93%. For > 10(5) colony counts/ml, the sensitivity of the nitrite test was 27%, specificity 94%, and negative predictive value 87%. When either leucocyte esterase or nitrite positivity was accepted as a marker of urinary tract infection, the sensitivity was 78%, specificity 75%, and negative predictive value 94%, and there were 22% false negative results. Semiquantitative microscopic estimation of bacteria per HPF yielded 40% false positives. CONCLUSIONS: Leucocyte esterase and nitrite dipstick tests are not suitable for screening for urinary tract infections.

Adolescent↗
PubMed1998

Serum protein electrophoresis by CZE 2000 clinical capillary electrophoresis system.

We compared the automated Paragon 2000 clinical capillary zone electrophoresis (CZE) system with two manual methods, agarose electrophoresis (AGE) and cellulose acetate electrophoresis (CAE). Reference intervals in healthy adults were determined for each method. When compared with AGE and CAE, CZE gave substantially higher reference values for the alpha1-globulin fraction. With CZE, within-run precision for fraction quantitation was between 0.5% (albumin) and 4.1% (alpha1-globulin). Total precision was between 0.8% (albumin) and 5.3% (beta-globulin). Data obtained from CZE showed poor linear correlation with results obtained by AGE but good linear correlation with data from CAE. Analysis of serum from patients with inter alia inflammation, nephrotic syndrome, or polyclonal gammopathy showed that clinical information obtained by CZE is comparable with information obtained by AGE and CAE. We conclude that CZE offers a clinically reliable alternative to AGE and CAE and has the advantages of automation, higher precision, and faster turnaround time.

Acute-Phase Reaction↗
PubMed1998

Detection and classification of paraproteins by capillary immunofixation/subtraction.

A selection of 58 specimens with a monoclonal component identified by immunoelectrophoresis and/or immunofixation was analyzed with the immunosubtraction procedure on the Paragon 2000 capillary electrophoresis system. The capillary system detected 93% of the paraproteins and, using immunosubtraction, correctly identified 91% of the paraproteins. Paraproteins that were detected by immunofixation and/or immunoelectrophoresis but not by capillary electrophoresis were also missed by agarose electrophoresis and cellulose acetate electrophoresis. Cellulose acetate electrophoresis was the least sensitive method for detection of paraproteins. Only 74% of the monoclonal components were detected by this technique, whereas 86% were revealed by agarose electrophoresis. In addition to monoclonal paraproteins, we also studied biclonal paraproteins and oligoclonal banding. Capillary electrophoresis and immunosubtraction correctly detected and identified three specimens containing biclonal paraproteins. In one specimen, capillary zone electrophoresis detected only one band, whereas agarose gel electrophoresis detected two bands. The sensitivity for detection and identification of oligoclonal banding by capillary electrophoresis was inferior to immunofixation.

Electrophoresis, Agar Gel↗
PubMed1998

Serum protein electrophoresis and immunofixation by a semiautomated electrophoresis system.

Semiautomated agarose electrophoresis and immunofixation performed with Hydrasys-Hyrys (Sebia) were compared with conventional, manually performed methods, including cellulose acetate electrophoresis, immunoelectrophoresis, and immunofixation. Reference intervals for agarose electrophoresis with Hydrasys-Hyrys were determined. Within-run imprecision (CV) for fraction quantitation with the semiautomated system was between 1% (albumin) and 4.5% (beta-globulin). Total imprecision (CV) was between 2.7% (albumin) and 7.3% (beta-globulin). Semiautomated agarose electrophoresis showed linear correlation with cellulose acetate electrophoresis. Thirty-four specimens with monoclonal components were analyzed by manual immunoelectrophoresis and immunofixation and by Hydrasys. In one case, a light-chain disease was missed with Hydrasys when the sample was diluted 1:3 (the routine dilution) but not when the sample was assayed undiluted. In another case, the Hydrasys system revealed a small IgGA monoclonal component in addition to the IgA monoclonal component detected by the manual methods. In the other cases, no differences between the manual methods and the semiautomated method were seen with respect to paraprotein identification.

Bence Jones Protein↗
PubMed1997

Carrier-mediated transport of uridine diphosphoglucuronic acid across the endoplasmic reticulum membrane is a prerequisite for UDP-glucuronosyltransferase activity in rat liver.

UDP-glucuronosyltransferases (EC 2.4.1.17) is an isoenzyme family located primarily in the hepatic endoplasmic reticulum (ER) that displays latency of activity both in vitro and in vivo, as assessed respectively in microsomes and in isolated liver. The postulated luminal location of the active site of UDP-glucuronosyltransferases (UGTs) creates a permeability barrier to aglycone and UDP-GlcA access to the enzyme and implies a requirement for the transport of substrates across the ER membrane. The present study shows that the recently demonstrated carrier-mediated transport of UDP-GlcA across the ER membrane is required and rate-limiting for glucuronidation in sealed microsomal vesicles as well as in the intact ER of permeabilized hepatocytes. We found that in both microsomes and permeabilized hepatocytes a gradual inhibition by N-ethylmaleimide (NEM) of UDP-GlcA transport into the ER produced a correspondingly increasing inhibition of 4-methylumbelliferone glucuronidation. That NEM selectively inhibited the UDP-GlcA transporter, without affecting intrinsic UGT activity, was demonstrated by showing that NEM had no effect on glucuronidation in microsomes or hepatocytes with permeabilized ER membrane. Additional evidence that UDP-GlcA transport is rate-limiting for glucuronidation in sealed microsomal vesicles as well as in the intact ER of permeabilized hepatocytes was obtained by showing that gradual selective trans-stimulation of UDP-GlcA transport by UDP-GlcNAc, UDP-Xyl or UDP-Glc in each case produced correspondingly enhanced glucuronidation. Such stimulation of transport and glucuronidation was inhibited completely by NEM, which selectively inhibited UDP-GlcA transport.

Animals↗
PubMed1997

Time course of serum lipids and apolipoproteins after acute myocardial infarction: modification by pravastatin.

Pravastatin was administered to patients suffering an acute myocardial infarction starting with a dose of 10 mg/day at day 3 and continuing with a dose of 20 mg/day up to a period of 3 months. The study was performed on the basis of a randomized placebo-controlled double-blind trial. At the dosage used pravastatin significantly lowered the total cholesterol and LDL-cholesterol levels and increased the HDL-cholesterol levels compared to placebo. A prospective clinical trial in order to examine the possible clinical relevance of these findings is recommended.

Anticholesteremic Agents↗