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PubMed2004

HRCT diagnosis of diffuse parenchymal lung disease: inter-observer variation.

BACKGROUND: This study was designed to measure inter-observer variation between thoracic radiologists in the diagnosis of diffuse parenchymal lung disease (DPLD) using high resolution computed tomography (HRCT) and to identify areas of difficulty where expertise, in the form of national panels, would be of particular value. METHODS: HRCT images of 131 patients with DPLD (from a tertiary referral hospital (n = 66) and regional teaching centres (n = 65)) were reviewed by 11 thoracic radiologists. Inter-observer variation for the first choice diagnosis was quantified using the unadjusted kappa coefficient of agreement. Observers stated differential diagnoses and assigned a percentage likelihood to each. A weighted kappa was calculated for the likelihood of each of the six most frequently diagnosed disease entities. RESULTS: Observer agreement on the first choice diagnosis was moderate for the entire cohort (kappa = 0.48) and was higher for cases from regional centres (kappa = 0.60) than for cases from the tertiary referral centre (kappa = 0.34). 62% of cases from regional teaching centres were diagnosed with high confidence and good observer agreement (kappa = 0.77). Non-specific interstitial pneumonia (NSIP) was in the differential diagnosis in most disagreements (55%). Weighted kappa values quantifying the likelihood of specific diseases were moderate to good (mean 0.57, range 0.49-0.70). CONCLUSION: There is good agreement between thoracic radiologists for the HRCT diagnosis of DPLD encountered in regional teaching centres. However, cases diagnosed with low confidence, particularly where NSIP is considered as a differential diagnosis, may benefit from the expertise of a reference panel.

Adult↗

PubMed1982

New radioimmunoadsorbent method for detection of hepatitis B surface antigen.

A radioimmunoadsorbent assay for the detection of hepatitis B surface antigen (HBsAG) is described. The method uses small DEAE-cellulose columns to adsorb HBsAg from serum or plasma samples, and it uses 125 I-labeled antibody to HBsAg to detect the adsorbed antigen. A single 2-h incubation at 45 degrees C is used in the procedure. The sensitivity of the method was determined with the Bureau of Biologics HBsAg reference panels and was shown to be equivalent to other third-generation test methods. The specificity of the method was evaluated by testing specimens from blood donors, dialysis patients, and hospital staff in parallel with commercial radioimmunoassay kits for HBsAg. In the tests performed on 2,868 blood donor specimens, 6 positive specimens were identified by both the radioimmunoadsorbent method and the commercial radioimmunoassay kits. These positive specimens were confirmed by a counterimmunoelectrophoresis procedure. One nonconfirmable, repeatably positive specimen was observed with the radioimmunoadsorbent method. In testing 1,250 specimens from dialysis patients and hospital staff, 120 confirmable positive specimens were identified by both the radioimmunoadsorbent method and a commercial test. Overall, the false positive rate for the radioimmunoadsorbent method was found to be less than 1%.

Antigen-Antibody Complex↗

PubMed2001

Validation and standardisation of nucleic acid amplification technology (NAT) assays for the detection of viral contamination of blood and blood products.

Standardisation of NAT assays is necessary before the introduction of such assays for routine screening of blood and blood products for viral contaminants such as HBV, HCV, and HIV-1. Standardisation can be achieved by the use of well-characterised reference materials (working reagents) to validate each assay run. Working reagents for HCV, HIV-1, HBV, HAV, and human parvovirus B19 have been established by the NIBSC. Such reagents and reference panels are also available from other official medicinal control laboratories and commercial organisations. However, the nucleic acid content of these reagents are expressed in many different units, e. g. genome equivalents/ml, copies/ml, PCR detectable units/ml, making comparisons of results from laboratories using different reagents difficult. The establishment of internationally accepted standards against which all working reagents could be calibrated, using a common standard unit of measurement, IU, would overcome this major problem. The first International Standard for HCV RNA assays was established in 1997. This reagent, 96/790, is a lyophilised preparation of a genotype 1 isolate and the concentration of the standard is 10(5) IU/ml. Two further International Standards have since been established; for HIV-1 and HBV, containing 10(5) IU/ml and 10(6) IU/ml respectively. The establishment of the HCV International Standard has been critical in the introduction of mandatory testing. Since 1st July 1999, all batches of blood products marketed in Europe have to be prepared from plasma pools tested and found non-reactive for HCV RNA using a validated assay which can detect a sample containing 100 IU/ml of HCV RNA. In Germany, screening of blood donations for HCV RNA by NAT has been mandatory since 1st April 1999. The minimum sensitivity of assays should be 5000 IU/ml for a single donation.

DNA, Viral↗

PubMed1994

A small test of a sequence-based typing method: definition of the B*1520 allele.

Santamaria et al. (Human Immunology 1993 37: 39-50) describe a method of sequence-based typing (SBT) for HLA-A, B and C alleles said to give "unambiguous typing of any sample, heterozygous or homozygous, without requiring additional typing information". From SBT analysis, which involves determination of partial sequences of mixed alleles, these investigators reported that cell lines KT17 (HLA-B35,62) and OLGA (HLA-B62) from the reference panel of the 10th International Histocompatibility Workshop express novel variants of HLA-B15 (B1501-MN6) and HLA-B35 (B3501-MN7) respectively. To study further the novel alleles, we cloned and sequenced full-length HLA-B cDNA clones isolated from the KT17 and OLGA cell lines. We find that KT17 expresses B*3501, as assigned by SBT, and B*1501, the common allele encoding the B62 antigen. We were unable to confirm that KT17 expresses the novel B1501-MN6 variant identified by SBT. For OLGA our analysis confirms the partial sequences obtained by SBT. Thus OLGA expresses B*1501 and a novel HLA-B allele. The complete sequence of the latter shows it is a hybrid having exons 1 and 2 in common with B*1501 and other B15 subtypes and exons 3-7 in common with B*3501 and related molecules including B*5301 and B*5801. The novel allele has been designated B*1520 because of its sequence similarity with the B15 group; furthermore, serological analysis shows that the B*1520 product does not express epitopes in common with either B35, B53 or B58. The B*1520 heavy chain has a similar isoelectric point to A*3101; B*1520 was undetected by previous applications of isoelectric focusing because B*1520 and A31 are both expressed by OLGA. In conclusion, HLA-B typing of two cell lines by cDNA cloning and sequencing gives concordant results with SBT for three of the four alleles. The cause of the discrepancy for the fourth allele is unknown, however, this finding indicates that the novel HLA-A, B and C sequences emerging from SBT studies need independent verification.

Alleles↗

PubMed2025

Genetic Architecture of Idiopathic Inflammatory Myopathies From Meta-Analyses.

OBJECTIVE: Idiopathic inflammatory myopathies (IIMs, myositis) are rare systemic autoimmune disorders that lead to muscle inflammation, weakness, and extramuscular manifestations, with a strong genetic component influencing disease development and progression. Previous genome-wide association studies identified loci associated with IIMs. In this study, we imputed data from two prior genome-wide myositis studies and analyzed the largest myositis data set to date to identify novel risk loci and susceptibility genes associated with IIMs and its clinical subtypes. METHODS: We performed association analyses on 14,903 individuals (3,206 patients and 11,697 controls) with genotypes and imputed data from the Trans-Omics for Precision Medicine reference panel. Fine-mapping and expression quantitative trait locus colocalization analyses in myositis-relevant tissues indicated potential causal variants. Functional annotation and network analyses using the random walk with restart (RWR) algorithm explored underlying genetic networks and drug repurposing opportunities. RESULTS: Our analyses identified novel risk loci and susceptibility genes, such as FCRLA, NFKB1, IRF4, DCAKD, and ATXN2 in overall IIMs; NEMP2 in polymyositis; ACBC11 in dermatomyositis; and PSD3 in myositis with anti-histidyl-transfer RNA synthetase autoantibodies (anti-Jo-1). We also characterized effects of HLA region variants and the role of C4. Colocalization analyses suggested putative causal variants in DCAKD in skin and muscle, HCP5 in lung, and IRF4 in Epstein-Barr virus (EBV)-transformed lymphocytes, lung, and whole blood. RWR further prioritized additional candidate genes, including APP, CD74, CIITA, NR1H4, and TXNIP, for future investigation. CONCLUSION: Our study uncovers novel genetic regions contributing to IIMs, advancing our understanding of myositis pathogenesis and offering new insights for future research.

Humans↗

PubMed2003

Cold provocation testing and hand-arm vibration syndrome--an audit of the results of the Department of Trade and Industry scheme for the evaluation of miners.

BACKGROUND: Hand-arm vibration syndrome (HAVS) is a major industrial disease that causes considerable morbidity among workers exposed to vibration. Compensation is paid to those affected in civil claims against employers and (in the UK) in claims made under Social Security legislation rules for Prescribed Disease A11. Diagnostic tests have been proposed but most are not objective. The cold provocation test (CPT), which is objective, is often included in the evaluation of HAVS. METHODS: A continuous audit was made of the findings recorded at the 18 HAVS test centres in the UK established to evaluate miners. The audit, and its outcome, were monitored by the Medical Reference Panel who advise the Department of Trade and Industry. This audit report constitutes the second analysis of the findings relating to the CPT. RESULTS: The CPT, with measurement of digital rewarming times, is of no value in assessing vibration-induced damage to the hands. CONCLUSION: In its present format, the CPT should not be used for evaluating the vascular component of HAVS.

Adult↗

PubMed2006

Comparison of three commonly used PCR-based techniques to analyze MSI status in sporadic colorectal cancer.

Several retrospective studies have shown that a high level of microsatellite instability (MSI-H) is an important prognostic factor of a more favorable outcome in stage II and III colorectal cancer (CRC) patients. In this study, three commonly used polymerase chain reaction (PCR)-based MSI analysis techniques were compared (polyacrylamide gel electrophoresis followed by silver-staining [SSPAGE], fluorescence capillary electrophoresis [FCE], and denaturing high-performance liquid chromatography [DHPLC]) on a limited group of CRC patients, to identify the most optimal detection technique. Pathology blocks of 26 CRC patients were subjected to microdissection and the Bethesda reference panel was used for MSI analysis. Considering the samples analyzed by both SSPAGE and FCE, 8.7% were MSI-H, 8.7% were MSI-L, and 82.6% were MSS using SSPAGE. FCE resulted in 16% MSI-H, 4% MSI-L, and 80% MSS. Due to difficulties in analyzing the dinucleotide markers on DHPLC, we only analyzed the mononucleotide markers with this technique. The results were 100% concordant to those obtained by FCE. SSPAGE is time consuming, subjective, and less user-friendly and interpretable. DHPLC was not feasible due to interpretation difficulties for the dinucleotide markers. We recommend the use of FCE to analyze MSI status. This technique is sensitive, reproducible, user-friendly and leads to easy interpretation and high-throughput.

Chromatography, High Pressure Liquid↗

PubMed1993

Quantitative assessment of hepatitis B virus DNA in chronic hepatitis B: comparison of two solution hybridization assays.

We compared two solution hybridization assays, the AffiProbe assay (Orion Corporation) and the Abbott HBV-DNA assay (Abbott), for quantitative measurement of hepatitis B virus (HBV) DNA in serum samples obtained from chronic hepatitis B surface antigen (HBsAg) carriers. Forty transversally collected (group 1) and 83 serially collected (group 2) serum samples from chronic hepatitis B patients were tested with both assays. The serial serum samples were obtained from 6 patients who underwent alpha-interferon therapy with different outcomes (nonresponse, hepatitis B e antigen [HBeAg] seroconversion, HBeAg and HBsAg seroconversion). In group 1 a good correlation (r = 0.91; P < 0.001) was found between the HBV-DNA results of the two assays. Two samples (5%) were HBV-DNA positive according to the Abbott but negative according to the AffiProbe assay; for all other samples the HBV-DNA status corresponded. In group 2 the assays gave colinear HBV-DNA results during follow-up of 5 of the 6 patients (correlation for the total group: r = 0.90; P < 0.001). Nevertheless, in both groups the AffiProbe assay yielded about 5-10 times higher HBV-DNA levels than the Abbott HBV-DNA assay (P < 0.001). These discordant results were most probably due to standardization differences of the positive control samples of the two test systems. This observation underlines the need for international standardization of HBV-DNA and uniform reference panels.

Chronic Disease↗

PubMed2005

Use of mononucleotide repeat markers for detection of microsatellite instability in mouse tumors.

Tumors lacking DNA mismatch repair activity (MMR) from patients with Hereditary Nonpolyposis Colorectal Cancer (HNPCC) or those with sporadic colorectal cancer can be identified by the presence of high levels of instability in repetitive sequences known as microsatellites (MSI). The assessment of MSI phenotype in human tumors helps to establish a clinical diagnosis and is accomplished with a reference panel of five mononucleotide repeats. By contrast, detection of MSI in mouse tumors has proven to be problematic and lack of a uniform set of markers for classification of MSI has impeded comparison of results between studies. We tested for MSI in intestinal tumors from MMR-deficient mice with four mononucleotide repeats with polyA(24-37) tracts and three new markers with extended polyA(59-67) tracts. All seven markers were sensitive to MSI in MMR-deficient tumors, but those with extended mononucleotide tracts displayed larger deletions, which were easily distinguishable from the germline alleles. With a panel of the five most sensitive and specific mononucleotide repeats, a high level of MSI was detected in 100% of MMR-deficient tumors, but not in tumors with MMR activity. This novel panel is an improvement over existing combinations of mono- and dinucleotide repeat markers and should facilitate MSI screening and standardize results from different studies.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Quantitative proteome analysis of breast cancer cell lines using 18O-labeling and an accurate mass and time tag strategy.

Proteome comparison of cell lines derived from cancer and normal breast epithelium provide opportunities to identify differentially expressed proteins and pathways associated with specific phenotypes. We employed 16O/18O peptide labeling, FT-ICR MS, and an accurate mass and time (AMT) tag strategy to simultaneously compare the relative abundance of hundreds of proteins in non-cancer and cancer cell lines derived from breast tissue. A cell line reference panel allowed relative protein abundance comparisons among multiple cell lines and across multiple experiments. A peptide database generated from multidimensional LC separations and MS/MS analysis was used for subsequent AMT tag-based peptide identifications. This peptide database represented a total of 2299 proteins, including 514 that were quantified in five cell lines using the AMT tag and 16O/18O strategies. Eighty-six proteins showed at least a threefold protein abundance change between cancer and non-cancer cell lines. Hierarchical clustering of protein abundance ratios revealed that several groups of proteins were differentially expressed between the cancer cell lines.

Breast Neoplasms↗

PubMed1993

A microsatellite-based multipoint index map of human chromosome 22.

Utilizing the CEPH (Centre d'Etude du Polymorphism Humain) reference panel and genotyping data for 24 simple tandem repeat polymorphism (STRP) markers, we have constructed a 15-locus multipoint genetic framework map of human chromosome 22. The markers form a continuous linkage group of 51 cM in males and 81 cM in females. Likely genetic locations are provided for 9 additional STRP sequences. The map was constructed employing the CRIMAP computational methodology to build the multipoint map via a stepwise algorithm. The quality of the framework map was evaluated using a battery of statistical diagnostics that suggest a typing error frequency of 0.1% for markers within the map.

Base Sequence↗

PubMed1997

Genetic polymorphism and recombination in the subtelomeric region of chromosome 14q.

Subtelomeric regions of human chromosomes are the sites of increased meiotic recombination and have a male-to-female recombination ratio that is higher than elsewhere in the genome. We isolated two novel, polymorphic CA repeat markers from the distal part of the immunoglobulin heavy chain gene cluster, approximately 90 and 200 kb from the telomere of chromosome 14q. The 14q telomere was unambiguously located by physical mapping of telomeric YACs and Bal31 exonuclease digestion of genomic DNA. We then constructed haplotypes using genotype data from these markers and data from sCAW1 (D14S826) for use as a highly polymorphic genetic marker. Linkage analysis using the 40 pedigree CEPH reference panel and genotype data from these and other loci physically mapped to the terminal 1.5 Mb of chromosome 14q revealed an apparent increase in meiotic recombination within this region, relative to the average rate for the genome. Further, we found that recombination was higher in females than in males, indicating that the subtelomeric region of 14q differs from other human subtelomeric regions.

Chromosomes, Artificial, Yeast↗

PubMed1999

Molecular screening of potential HNPCC patients using a multiplex microsatellite PCR system.

Microsatellite instability (MSI) is a molecular hallmark of the H ereditary N on- P olyposis C olorectal C ancer (HNPCC) syndrome occurring in about 80-90% of the tumours and also in sporadic tumours of different organs, albeit at lower frequency. Highly unstable colorectal tumours (MSI-H) have different histopathological features and tend to have a better prognosis compared to neoplasms without (MSS) or with low levels of microsatellite instability (MSI-L). Since MSI classification allows the identification of potential HNPCC patients and might represent a valuable diagnostic parameter an increasing demand for high-throughput microsatellite analysis will arise. Therefore, we have adapted five diagnostic microsatellites, m(odified) ACTC, mBAT26, mD5S107, mD5S406 and mD13S153, to allow coamplification. Using this multiplex polymerase chain reaction (PCR) system 29 colorectal tumour tissues with known MSI status could be unambiguously identified as MSI-H (13 cases) or MSI-L/MSS (16 cases). Highly unstable colorectal tumour detection frequency of individual markers reached 77% (mD5S406), 85% (mACTC), 85% (mD5S107), 92% (D13S153) and 100% (mBAT26) showing similar sensitivity but improved specificity as compared with a microsatellite reference panel. In a prospective analysis of 31 colorectal tumours, the multiplex PCR system identified five MSI-H cases. Multiplex MSI PCR is a time saving and cost-effective method not restricted to specific technical equipment and applicable to a variety of microsatellite-based genotyping approaches.

Colorectal Neoplasms↗

PubMed2000

Identification of Staphylococcus aureus enterotoxins A and B genes by PCR-ELISA.

We developed PCR-enzyme linked immunosorbent (ELISA) assays to detect Staphylococcus aureus enterotoxins A and B genes. The assays use internal biotin-labelled oligonucleotides as capture probes for immobilizing and subsequently detecting target sequences on microtiter plates. The detection limits of the PCR-ELISAs were approximately 250 gene copies, versus 2500 gene copies by agarose gel analysis. The sensitivity of the assays, as determined from a reference panel of 46 coded samples that included DNA purified from 31 different bacterial species and strains, SEA and SEB plasmid controls, and no-template controls was 100%. No cross-reactivity was observed with DNA from non-staphylococcal species. Using 27 clinical isolates of S. aureus, the SEA PCR-ELISA identified the enterotoxin A (sea) gene in 26 samples, and the SEB PCR-ELISA identified the enterotoxin B (seb) gene in all 27 samples. Compared with conventional antigen capture ELISAs for SEA and SEB toxins, the PCR-ELISAs showed overall superior detection limits. The sensitivity and specificity levels of the SEA PCR-ELISA and the SEA toxin ELISA were comparable within their respective detection thresholds, but the sensitivity and specificity of the SEB PCR-ELISA was much greater than that of SEB toxin ELISA.

Bacteria↗

PubMed1993

DNA fingerprinting of trait-selected mouse lines and linkage analysis in reference families.

The first aim of the study was the molecular genetic characterization of long-term trait-selected (growth, adaptability and fertility) lines of mice using multilocus DNA fingerprinting with the simple tandem repetitive oligonucleotide probes (GAA)6 and (GACA)4. Secondly polymorphic markers were screened for association with growth performance based on DNA fingerprint analysis in reference families. Pooled DNA samples of ten unrelated mice of trait-selected mouse lines (over 40 generations) and the unselected control lines were analyzed. Resulting differences in band patterns were reanalyzed comparing individual fingerprints of the animals included in the pooled DNA samples of the different lines. Between the mouse lines about 30% of analyzed fingerprint bands were polymorphic. Individuals of long-term selected mouse lines show only a few individual-specific bands. Most polymorphic bands observed in DNA mixes appear in all animals included in the DNA mix of the corresponding line. Line-specific DNA fingerprint bands were analyzed for their inheritance and linkage with growth performance in reference families using animals with extreme growth performance of the first backcross after crossing of growth-selected with unselected mice. Scanning the distribution of line-specific bands in the reference panel few bands were identified which are associated with growth performance. They appear as useful markers for growth selection. Nevertheless most line-specific bands result from genetic drift rather than from selection.

Adaptation, Physiological↗

PubMed1993

Alleles and haplotypes of the MHC-encoded ABC transporters TAP1 and TAP2.

TAP1 and TAP2 are two major histocompatibility complex (MHC) genes, located between HLA-DP and -DQ, whose products form a transporter molecule involved in endogenous antigen processing. Polymorphic residues have been described in both genes and, in this study, we have identified another polymorphic site within the adenosine triphosphate (ATP)-binding domain of TAP2. We have used the amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) to characterize TAP1 and TAP2 alleles and haplotypes in a reference panel of 115 homozygous typing cell lines. Of four possible TAP1 alleles, we observed three, and of eight possible TAP2 alleles, we observed five. Among 88 (homozygous typing cells) (HTCs) homozygous at HLA-DR, -DQ and -DP, 80 were also homozygous at TAP1 and TAP2. Of 27 HTCs homozygous at HLA-DR and -DQ, but heterozygous at -DP, 14 were homozygous at TAP1 or TAP2 and 13 heterozygous, consistent with recombination taking place either side of the TAP loci. Of the fifteen possible combinations of TAP1 and TAP2 alleles, we observed eleven, each at a frequency similar to that predicted by individual allele frequencies. In this ethnically heterogenous panel there is no indication that particular combinations of TAP1 and TAP2 have been maintained together.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1980

HLA-A, B, C, DR alleles in congenital adrenal hyperplasia.

HLA markers (A, B, C, DR loci) were determined for the members of 52 unrelated families with at least one child suffering from congenital adrenal hyperplasia due to 21 hydroxylase deficiency, permitting genotyping. The gene frequencies of the 52 index cases were compared with those obtained from the patients' normal haplotypes and with those of a control reference panel. No significant differences were observed, except a clear decrease in the frequency of HLA-B8 among the haplotypes that carry the gene for congenital adrenal hyperplasia.

Adrenal Hyperplasia, Congenital↗

PubMed1994

A genetic and physical map of bovine chromosome 11.

A genetic map of bovine Chromosome (Chr) 11 (BTA11, synteny group U16) has been constructed from 330 animals belonging to 21 families, which constitute the international bovine reference panel (IBRP). This map is based on 13 polymorphic microsatellite markers, two of which were chosen in previously published maps. Three markers have been isolated from cosmids. Two of the three cosmids have been physically localized by fluorescence in situ hybridization (FISH), to anchor the genetic map on the chromosome. In addition, a biallelic polymorphism in the beta-lactoglobulin gene (LGB) has been genetically positioned relative to the microsatellite markers. The most probable order of the markers is: cen-INRA044-BM716-INRA177-(TGLA327, INRA198, INRA131)-INRA111-INRABERN169-(INRA115, INRA032)-INRA108-INRABERN162-INRA195-LGB. The total linkage group spans 126 cM, which probably corresponds to most of the chromosome length. The average intermarker distance is about 10.5 cM, allowing the potential detection of a genetic linkage with any Economic Trait Loci (ETL) of this chromosome. Seven of these markers have been previously published by Vaiman and coworkers (1994), two will be published as part of a set of markers (Eggen et al. in preparation), two are described in this paper, and two (BM716, TGLA327) were chosen from the published maps of BTA11 in order to integrate our data with existing maps. All these markers were assigned to synteny group U16 by use of a previously characterized panel of hamster/bovine somatic hybrid cell lines (Guérin et al. 1994).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗