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PubMed2002

Validation of the NucliSens Extractor combined with the AmpliScreen HIV version 1.5 and HCV version 2.0 test for application in NAT minipool screening.

BACKGROUND: Routine HCV NAT minipool screening (48 donations) of all blood donations was implemented in July 1999 and was combined with HIV NAT in November 2000. This report describes the validation of the NAT methods and the results of quality control testing. STUDY DESIGN AND METHODS: Nucleic acid was extracted from 2-mL plasma samples by using an automated silica-based extraction method (NucliSens Extractor, Organon Teknika). Eluates were tested with RT-PCR (AmpliScreen HIV-1 version 1.5 and AmpliScreen HCV version 2.0 test, Roche Diagnostic Systems). HIV-1 and HCV RNA reference panels and run controls (PeliCheck and PeliSpy, respectively, Sanquin-CLB) and human plasma minipools were used for NAT validation. RESULTS: The 95-percent detection limit (and 95% CI) for HIV-1 RNA genotype B, HIV-1 RNA genotype E, and HCV RNA genotype 1 was 32 (19-76), 30 (17-72), and 21 (13-44) genome equivalents (geq) per mL, respectively. During initial validation, 2332 samples for HIV-1 RNA and 2644 samples for HCV RNA were analyzed, with 13 (0.56%) and 12 (0.45%) invalid test results, respectively. Thereafter, over 19,600 samples (minipools and run controls) were analyzed during the first 11 months of routine screening. Invalid test results for HIV-1 RNA and HCV RNA were found in 1.1 and 1.07 percent of the samples tested, respectively. HIV-1 RNA minipool testing resulted in 27 (0.16%) initial false-positive results and 3 (0.02%) confirmed positive results. HCV RNA minipool testing resulted in four (0.02%) initial false-positive results and five (0.02%) confirmed positive results. CONCLUSION: Routine HIV and HCV NAT minipool screening using the NucliSens Extractor, AmpliScreen HIV-1 version 1.5, and AmpliScreen HCV version 2.0 meets the sensitivity criteria set by the regulatory bodies and provides sufficient specificity and robustness for timely release of blood donations.

Adsorption↗

PubMed2002

Evaluation of tumor microsatellite instability using five quasimonomorphic mononucleotide repeats and pentaplex PCR.

BACKGROUND & AIMS: The microsatellite instability (MSI) phenotype is a characteristic of the hereditary nonpolyposis colorectal cancer syndrome as well as approximately 15% of sporadic colon and gastric tumors. It is a valuable diagnostic marker for the identification of hereditary nonpolyposis colorectal cancer cases and may be a molecular predictive marker for the identification of colon cancer patients who benefit from chemotherapy. To evaluate MSI, a reference panel was proposed at an international consensus meeting, comprised of 2 mononucleotide (BAT-25, BAT-26) and 3 dinucleotide repeats. Analysis of BAT-26 is sufficient for detecting the MSI phenotype in most, but not all, cases. Additional results with dinucleotide markers can sometimes lead to incorrect classification of MSI tumors. METHODS: We describe here a single fluorescent multiplex system comprising 5 quasimonomorphic mononucleotide repeats for the detection of MSI tumors. RESULTS: None of 184 germline DNA samples, including 56 from African subjects, was found to contain allelic size variations in more than 2 of these markers. In contrast, all MSI tumors showed allelic size variations in 3 or more of the microsatellites. Using this assay, we confirmed (or reclassified in 6 cases) the MSI status of 124 colon and 50 gastric primary tumors and 16 colon cell lines. CONCLUSIONS: We propose that using a pentaplex polymerase chain reaction system allows accurate evaluation of tumor MSI status of DNA with 100% sensitivity and specificity without the need to match normal DNA. This assay is simpler to use than those involving dinucleotides and is more specific than using BAT-26 alone.

Africa↗

PubMed1995

Optically-navigable operating microscope for image-guided surgery.

In computer-assisted surgery (CAS), optical digitizing is state-of-the-art. Unfortunately standard navigation instruments, e.g. small, LED-equipped pointers are frequently shadowed by the operating microscope when the camera-array is ceiling- or rack-mounted. Thus, for microsurgery, a navigation module consisting of a reference panel mounted onto the housing of the microscope and an object distance measuring unit attached in front of the objective lens was developed. By means of this navigation module the microscope can be located in space with an accuracy of +/- 1-2 mm. Focusing errors due to the high depth of view of modern microscopes and individual refraction anomalies are eliminated by laser distance measurement. This device has been clinically tested since September 1994 and a typical case is reported.

Humans↗

PubMed2002

Characterization of the antigenic, immunogenic, and pathogenic variation of infectious bursal disease virus due to propagation in different host systems (bursa, embryo, and cell culture). II. Antigenicity at the epitope level.

Antigenic variation of infectious bursal disease virus (IBDV) due to propagation in different host systems (bursa of Fabricius, embryos, or cell cultures) was determined by enzyme-linked immunosorbent assay (indirect and antigen capture) and western blot analysis. To conduct this study, we used 27 non-neutralizing anti-VP(2) monoclonal antibodies, a reference panel of nine neutralizing monoclonal antibodies, and 13 neutralizing anti-IBDV chicken polyclonal antibodies. Changes occurred in neutralizing, cross-reactive, conformation-dependent epitopes on the VP(2) protein of IBDV. Interestingly, non-neutralizing, cross-reactive, conformation-dependent and confirmation-independent epitopes also changed on VP(2). These epitope changes were directly associated with the method used to propagate IBDV. These results demonstrate that different host systems may play an important role in the antigenicity of IBDV.

Animals↗

PubMed1998

Comparative and library epidemiological typing systems: outbreak investigations versus surveillance systems.

A number of high-resolution molecular typing systems have been developed in recent years. Their availability raises the new issues of selecting the method (s) best suited for a particular purpose and interpreting and communicating typing results. Most of the currently available methods are comparative only: they allow testing of a sample of isolates for delineation of those closely related from those markedly different in genomic backgrounds. This approach is adequate for outbreak investigation, allowing determination of clonal spread in a microenvironment and identification of the source of infection. Comparative methods with sufficient resolution for most pathogens include restriction fragment-length polymorphism (RFLP), pulsed-field gel electrophoresis (PFGE), and arbitrarily primed and randomly amplified polymorphic DNA-polymerase chain reaction (PCR) analysis. For surveillance systems, monitoring clonal spread and prevalence in populations over extended periods of time requires library typing systems. These must be standardized, must have a high throughput, and must use a uniform nomenclature. Promising or validated methods include serotyping, insertion sequence fingerprinting, ribotyping, PFGE, amplified fragment-length polymorphism (AFLP), infrequent-restriction-site amplification PCR, interrepetitive element PCR typing (rep-PCR) and PCR-RFLP of polymorphic loci. PCR methods generating arrays of size-specific amplicons (AFLP, rep-PCR) can be more reproducibly analyzed by using denaturing polyacrylamide gel or capillary electrophoresis with automated laser detection. Binary probe typing systems appear optimal and should be enhanced further through use of DNA chip technology. In these systems, amplification of polymorphic regions is followed by solid-phase hybridization with a reference panel of sequence-variant specific probes. The resulting binary type results allow determination of reproducible, numeric profiles. However, interpretation and nomenclature of typing results for large-scale surveillance purposes still require a better understanding of population structure and microevolution of most microbial pathogens.

Bacterial Infections↗

PubMed2002

Analysis of a conformational B cell epitope of human thyroid peroxidase: identification of a tyrosine residue at a strategic location for immunodominance.

Thyroid peroxidase (TPO) is involved in autoimmune thyroid diseases and high titers of TPO autoantibodies directed to various conformational B cell epitopes are frequently present in patients' sera. Deciphering these epitopes is a difficult task, but can give insight into the structural basis of autoimmune recognition. TPO is a membrane-bound enzyme with the extracellular part organized in three protein domains, but of unknown three-dimensional structure. We previously localized a TPO B cell epitope within amino acid residues 742-848, a region encompassing the two C-terminal, extracellular domains of the protein. We found that at least one of the three tyrosine residues of the peptide 742-848 might be involved in autoantibody binding. In this study, we show by site-directed mutagenesis that the autoepitope contains tyrosine 772 located near the hinge area between the two protein domains, suggesting they are both involved in the epitope structure. The B cell epitopes of TPO are clustered in two overlapping immunodominant regions. To map the newly localized epitope with respect of these regions, competition experiments were performed using a reference panel of TPO mAb and a further mAb previously found to be specific for the TPO peptide 742-848 at variance with all the other ones. Here, we show that the tyrosine 772-bearing epitope in the peptide 742-848 maps in a region that partly overlaps the reported two immunodominant regions. These results are suggestive of a complex TPO folding that involves all the three TPO protein domains to form a highly conformational immunodominant region.

Animals↗

PubMedDate not supplied

Microsatellite marker development and analysis in the eastern oyster (Crassostrea virginica): confirmation of null alleles and non-Mendelian segregation ratios.

Eighteen microsatellite markers were developed for the Crassostrea virginica nuclear genome, including di-, tri-, and tetranucleotide microsatellite repeat regions that included perfect, imperfect, and compound repeat sequences. A reference panel with DNA from the parents and four progeny of 10 full-sib families was used for a preliminary confirmation of polymorphism at these loci and indications of null alleles. Null alleles were discovered at three loci; in two instances, primer redesign enabled their amplification. Two to five representative alleles from each locus were sequenced to ensure that the targeted loci were amplifying. The sequence analysis revealed not only variation in the number of simple sequence repeat units, but also polymorphisms in the microsatellite flanking regions. A total of 3626 bp of combined microsatellite flanking region from the 18 loci was examined, revealing indels as well as nucleotide site substitutions. Overall, 16 indels and 146 substitutions were found with an average of 4.5% polymorphism across all loci. Eight markers were tested on the parents and 39-61 progeny from each of four families for examination of allelic inheritance patterns and genotypic ratios. Twenty-six tests of segregation ratios revealed eight significant departures from expected Mendelian ratios, three of which remained significant after correction for multiple tests. Deviations were observed in both the directions of heterozygote excess and deficiency.

Alleles↗

PubMed2026

Local ancestry inference identifies robust evidence of selection in Neolithic Europe.

During the European Neolithic, migrating Anatolian farmers admixed with local hunter-gatherers, coinciding with major shifts in diet, environment, and lifestyle that imposed strong selective pressures. Local ancestry inference is widely used to detect selection following admixture, but most methods were developed and validated on present-day populations. Their performance in ancient DNA - where reference panels are smaller, data are sparser, and admixture is more ancient - remains unresolved. We benchmark eight local ancestry inference methods on 176 imputed Neolithic genomes. While individual-level ancestry estimates are highly correlated across methods, inferred tract lengths and admixture time estimates vary by an order of magnitude. Overall, we recommend Gnomix or RFMix for general use. We also investigated our ability to detect natural selection using LAI. Integrating results across methods and replicating across methods and in two independent datasets (n=378 and 1,121) we identify a robust ancestry deviation at FADS1/2, consistent with adaptation on metabolism. We also identify IRAK4 (innate immunity) as a candidate locus, but with less consistent signal across methods. Finally, we replicate previous reports of excess hunter-gatherer ancestry at the HLA, but these results are inconsistent across methods and suggest that they may be affected by bias in local ancestry inference. Our findings demonstrate that while local ancestry inference recovers biologically meaningful signals in ancient genomes, results can be sensitive to the methods used for inference, particularly in complex regions like the HLA. Method choice critically influences inferred ancestry patterns and selection signals, underscoring the importance of multi-method validation.

Journal Article↗

PubMedDate not supplied

Genetic analysis of Père David's x red deer interspecies hybrids.

Interspecies hybrids provide unique opportunities for fundamental genetic analyses and for genetic improvement of farmed deer. We have bred F1 hybrids by artificial insemination of red deer hinds (Cervus elaphus) with semen from Père David's deer (Elaphurus davidianus). The male and female F1 Père David deer x red deer hybrids were fertile and in matings with red deer have produced over 300 viable backcross hybrids. DNA was collected from the backcross progeny as an international reference panel for gene linkage mapping and to investigate associations between segregating species-specific genetic markers and phenotypic traits. We have measured a range of phenotypic traits in the backcross hybrids and red deer. Several traits appear suitable for genetic analysis using mapped genetic markers, including gestation length, growth rate, live weight, head morphometrics, and tail length. Typically these traits show a large difference between Père David's deer and red deer and a high variance in the backcross so that many individuals have a phenotype outside the range observed in red deer.

Animals↗

PubMed2002

Development of the Knee Standardized Clinical Interview: a research tool for studying the primary care clinical epidemiology of knee problems in older adults.

OBJECTIVE: To develop a standardized clinical interview, incorporating the perspectives of general practitioners (GPs) and rheumatologists, for primary care clinical epidemiological studies of adults aged 50 yr and over with knee problems. METHODS: Two parallel, consensus development studies using a modified nominal group technique involving GPs (n=5) and consultant rheumatologists (n=4) from North Staffordshire with reference panels of GPs (n=28) and rheumatologists (n=11) from selected centres in Great Britain, respectively. A single standardized clinical interview was formed using the clinical history questions identified in the consensus development studies and its feasibility was tested in a small sample of patients. RESULTS: In the GP consensus development study, 115 clinical history questions were identified, of which 71 were of agreed importance following postal rating, face-to-face discussion and re-rating. In the rheumatologist study, 158 questions were identified, of which 47 were of agreed importance. There was considerable overlap in the clinical history questions independently developed by the two studies. A single standardized clinical interview containing 74 questions was formed. It contained questions on the history, onset and recent course of the complaint; nature, location and severity of current knee symptoms; impact of knee problem; past history of knee problems; family history; comorbidity; previous/current investigations and treatment; ideas, concerns and expectations. In preliminary testing it took 20-45 min to conduct and was comprehensible to patients. CONCLUSIONS: A research tool-the Knee Standardized Clinical Interview (KNE-SCI)-has been formed from consensus development studies involving GPs and rheumatologists. In preliminary testing, it is comprehensible to patients, and forms a coherent clinical interview for research data collection. However, further evaluation is required to determine its accuracy and reliability and its usefulness for clinical epidemiological research.

Aged↗

PubMed1982

Phase II evaluation of dibromodulcitol, ICRF-159, and maytansine for sarcomas.

Patients with objectively measurable soft tissue sarcomas, osteosarcomas, chondrosarcomas, and mesotheliomas were treated with dibromodulcitol (DBD) (180 mg/m2 p.o. days 1-10 q4 wks.). ICRF-159 (300 mg/m2 p.o. tid days 1-3 q4 wks), or maytansine (MAYT) (1.5 mg/m2 I.V. q3 wks.). Forty-five evaluable patients received DBD, 47 MAYT, and 37 ICRF-159. Only patients who had had their histopathologic diagnoses confirmed by a pathology reference panel were included in the final analysis. Two patients had objective partial responses: a patient with osteosarcoma who responded to DBD and a patient with fibrosarcoma who had a partial response of brief duration to ICRF-159. Approximately 70% of the patients treated with each drug were of ECOG performance status 0 or 1, and over half had moderate or worse toxicity. It seems unlikely that these drugs have significant therapeutic activity for common mesenchymal malignancies.

Adolescent↗

PubMed2004

Guidelines for preclerkship bioterrorism curricula.

PURPOSE: To develop medical school curriculum guidelines related to bioterrorism to ensure that future medical graduates are armed with the critical knowledge, skills, and attitudes to face this emerging threat. METHOD: An Internet-based Delphi survey was performed in 2002 under the auspices of the Association of Medical School Microbiology and Immunology Chairs involving 64 medical educators in microbiology, immunology, and infectious diseases representing 54 U.S. medical schools. A 12-member bioterrorism expert reference panel participated in the final phase of the survey. RESULTS: Study participants identified the top educational objectives for the following bioterrorism-related curriculum categories: general issues, biodefense, public health, infection control, infectious diseases, and toxins. CONCLUSION: The study focused on preparedness training through the development of curricular guidelines in predominantly preclerkship medical education by identifying basic science and clinical laboratory aspects of putative biologic weapons organisms and toxins, clinical manifestations of bioterrorist attacks, treatment strategies, epidemiology, and prophylaxis.

Allergy and Immunology↗

PubMed1982

Allospecific monoclonal antibodies recognizing rat class I and class II histocompatibility antigens.

Rat-mouse hybridomas have been developed producing monoclonal antibodies which recognize rat major histocompatibility complex specificities. Splenic lymphocytes from a LEW (RT1l) rat alloimmunized against ACI (Rt1a) splenocytes were used for hybridoma production. The six clones presented demonstrate two patterns of alloreactivity against ACI tissues: clone 36.3 produces an antibody which agglutinates ACI RBCs and binds to ACI splenic lymphocytes and RBCs. Five clones, 65.6, 79.7,1, 79.7.5, 155.3.1, and 189.3.5, from four independent wells produce monoclonal antibodies which bind to ACI splenic lymphocytes but not RBCs. Screening of these six monoclonal antibodies against a reference panel of RBCs and lymphocytes from 15 standard, congenic, and recombinant rat strains indicated that the monoclonal antibody 36.3 detects a RTI.Aa product ( a rat major histocompatibility complex (MHC) Class I antigen) and that 65.6, 79.7.1, 79.7.5, 155.3.1, and 189.3.5 are monoclonal antibodies which recognize a RT1.Ba private specificity (Class II or Ia antigens). These reagents are unique as they are rat monoclonal alloantibodies recognizing private Ia specificities of the rat MHC.

Animals↗

PubMed1996

Immunologic monitoring in lung allograft recipients.

To identify patients with increased risk of chronic lung allograft rejection, we assessed the utility of an in vitro biopsy-derived lymphocyte growth assay and serum anti-HLA antibody screening as a complement to currently available methods of monitoring lung allograft recipients. Lymphocyte growth assay was performed on bronchoscopic fragments of tissue cultured in medium with rIL-2. Seventy-nine biopsies from 31 lung transplant recipients were tested by lymphocyte growth assay, and results were correlated with histopathology findings. Positive lymphocyte growth was found in 12/26 (46%) episodes of acute rejection, 5/44 biopsies without rejection (11%), and 0/9 episodes of bronchitis. Positive lymphocyte growth was seen in 7/16 (44%) grade A1 rejections and in 5/10 (50%) grade A2 rejections, as opposed to only 5/44 (11%) grade A0 (no rejection) biopsies (P < 0.01 for both A1 and A2 with respect to A0). Actuarial probability of remaining free from obliterative bronchiolitis (OB)* tended to be higher in patients who did not exhibit lymphocyte growth in biopsies. Sequential samples of sera obtained at the time of the biopsy were screened for lymphocytotoxic anti-HLA antibodies. Twenty-two of 44 recipients (50%) developed anti-HLA antibodies during the first postoperative year, exhibiting greater than 10% reactivity to an HLA reference panel of lymphocytes in four or more consecutive serum samples. Actuarial survival of lung allograft recipients with anti-HLA antibodies (n = 22) was lower than in those without anti-HLA antibodies (n = 22; P = 0.03). Of the 22 antibody producers, 7/12 died as a consequence of OB. Of the 22 non-antibody-producers, 1/2 deaths occurred as a consequence of OB. Anti-HLA antibodies were present in 9/11 instances of OB (82% sensitivity) and in 13/33 patients without OB (61% specificity; P = 0.03). These data indicate that lung transplant recipients with positive lymphocyte growth and anti-HLA antibodies are at an increased risk of chronic allograft rejection.

Antibodies↗

PubMed1983

Bactericidal activity of serum from cystic fibrosis patients for Pseudomonas aeruginosa.

The bactericidal action of serum from 61 adult patients with cystic fibrosis (CF) against autologous and heterologous strains of Pseudomonas aeruginosa has been studied. CF serum had a similar bactericidal action to normal human serum (NHS) against a reference panel of strains. Six CF sera had a selective inability to kill autologous strains of pseudomonas, which were sensitive to NHS and to sera from other CF patients. The six sera had normal levels of complement and immunoglobulin and were bactericidal to other strains. A titratable blocking factor was present in these sera and it interfered with the bactericidal action of NHS on the appropriate strain. This factor was present in the IgG-containing fractions of serum obtained by ion-exchange chromatography, but was not removed from the serum by absorption with the pseudomonas strain. Some CF sera may fail to kill sensitive strains of pseudomonas because of the development of a blocking IgG antibody against naturally occurring bactericidal IgM antibody.

Adolescent↗

PubMed2025

Tractor Workflow Pipeline: A Scalable Nextflow Framework for Local Ancestry-Aware Genome-Wide Association Studies.

The routine exclusion of admixed individuals from traditional Genome-Wide Association Studies (GWAS) due to concerns about spurious associations has hindered genetic analyses involving multiple ancestries. Tractor GWAS addresses this issue by incorporating local ancestry into its analysis, empowering identification of ancestry-enriched hits and generating ancestry-specific summary statistics. However, Tractor requires accurate genomic phasing and local ancestry inference as prerequisite steps, which requires additional bioinformatics expertise and decision points regarding reference panel setup. To streamline, harmonize, and automate this process, we present a scalable Nextflow workflow that integrates all necessary steps, minimizing the need for manual intervention while remaining modular and customizable. The workflow supports multiple commonly used tools and offers flexibility in how Tractor is implemented. To demonstrate its utility, we applied this pipeline to analyze 32 blood biomarkers in 6,245 two-way AFR-EUR admixed individuals from the UK Biobank. This pipeline ran efficiently at scale, replicated known associations, and identified novel ancestry-specific loci. These novel associations were largely driven by variants present on African ancestral tracts but absent from European tracts, underscoring the value of local ancestry-aware methods in uncovering previously missed genetic signals. By enabling the efficient analysis of admixed individuals, our workflow facilitates Tractor use, paving the way for more broader genetic discovery.

Journal Article↗

PubMed1987

Determination of the HLA-DR profile of an HLA class II negative carcinoma cell line by restriction fragment length polymorphism (RFLP) analysis.

The human colonic adenocarcinoma cell line HT-29 does not normally express HLA class II molecules. By restriction fragment length polymorphism (RFLP) analysis of DNA with a DR beta-probe, we analysed the genomic DR beta polymorphism of this cell-line, and compared it with the RFLP patterns seen in DNA from a reference panel of different DR homozygous and heterozygous cells. The HT-29 cell-line expressed DR beta fragments similar to the sum of the fragments expressed by DR4 and DR7 homozygous cells. The DR4 and DR7 haplotypes of HT-29 was further confirmed by comparing the RFLP patterns of four DR4/7 heterozygotes with that of HT-29. Furthermore, the HT-29 cell line expressed a Hind III 9.3 kb fragment previously found to be strongly associated to DRw53. Following treatment with gamma-interferon, the HT-29 cells could be induced to express class II molecules. Serological typing revealed the presence of the DR4, DR7 and DRw53 antigenic determinants.

Adenocarcinoma↗

PubMed1995

Low resolution DNA typing of the HLA-B5 cross-reactive group by nested PCR-SSP.

We have established a DNA typing system for the HLA-B5 serologically cross-reactive group (CREG) by means of a two-step PCR amplification with nested sequence-specific primers (nPCR-SSP). The present study provides a low resolution definition of the HLA-B5 CREG, i.e. identifying polymorphism equivalent to serology. Two different primer combinations allow group-specific amplification of all HLA-B5 CREG alleles and other related HLA class I alleles from genomic DNA. The amplified DNA is subjected to a second amplification step using eleven nested primer pairs. This assay permits the detection of the HLA-B5 CREG specificities B35, B51, B52, B53, and B7801 in all homozygous and heterozygous combinations. Sensitivity and specificity as judged by a blind quality control study investigating a reference panel (n = 50) is 100%. Extension of this approach should allow rapid DNA typing of all serologically defined HLA-B specificities by nPCR-SSP.

Alleles↗