Publications (75) View all
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Article: Validation of a Solid-Phase Electrochemical Array for Genotyping Hepatitis C Virus.
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ABSTRACT: Hepatitis C viral infection is a major cause of progressive liver disease. HCV genotype is one of the most significant baseline predictor of response to HCV antiviral therapy. The objective was to evaluate an HCV genotyping method that targets the 5'-untranslated region (UTR) to detect genotypes/subtypes using the GenMark eSensor® XT-8 system. The HCV amplicon of major genotypes/subtypes from the Roche TaqMan® HCV assay served as a template for the nested PCR followed by a direct analysis on the XT-8 detection system. The assay was validated for limit of detection (LOD), specificity, accuracy and precision. The LOD determined was below 175IU/ml for all the subtypes except 6ab. The genotypes detected using this assay were in concordance with the LiPA assay. The high performance characteristics (LOD, specificity, intra- and inter-assay precision, and accuracy), make this assay particularly well suited for clinical HCV genotyping in order to guide antiviral therapy.Experimental and Molecular Pathology 04/2013; · 2.42 Impact Factor -
Article: A Clinical PCR Fragment Analysis Assay for TA Repeat Sizing in the UGT1A1 Promoter Region.
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ABSTRACT: BACKGROUND: A genetic TA repeat length polymorphism in the UGT1A1 promoter has been shown to affect UDP-glucuronosyltyransferase (UGT1A1) expression levels with significant clinical implications. The presence of 7 TA repeats has been associated with lowered UGT1A1 expression and the mild hyperbilinrubinemia manifested in Gilbert's syndrome. Furthermore, cancer patients carrying this variant exhibit irinotecan-related toxicity and require lower doses of this chemotherapeutic agent compared to patients carrying the 6 TA repeat allele. This polymorphism is very common and, therefore, necessitates the development of reliable means of detecting it in the clinical laboratory to deliver better personalized therapy regimens. METHODS: We used 45 whole blood samples from patients previously tested with the FDA-approved Invader UGT1A1 assay (Hologic, Madison, WI) to assess extraction method, analytical sensitivity, accuracy and precision of this assay. In addition, cell line controls were used to test for the common and rare alleles of this polymorphism. The assay was based on PCR amplification and capillary electrophoresis for accurate sizing of the TA repeat copy number. RESULTS: All samples tested and controls gave the expected results. CONCLUSIONS: We have developed and validated a simple and sensitive PCR fragment analysis assay for accurately determining TA repeat length in the UGT1A1 promoter. At our medical center, this testing is used primarily for guiding irinotecan dosing decisions for our cancer patients.Clinica chimica acta; international journal of clinical chemistry 03/2013; · 2.54 Impact Factor -
Article: Recent Advances in MicroRNA-Mediated Gene Regulation in Chronic Lymphocytic Leukemia.
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ABSTRACT: Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world and is a very clinically heterogeneous disease for which better prognostic biomarkers are needed. Current prognostic markers exhibit both biological and technical limitations. MicroRNAs (miRNAs) are small endogenous, non-coding 22-nucleotide regulatory RNAs that have been shown to modulate hematopoietic lineage differentiation and play important gene-regulatory roles in disease processes. In this manuscript, we review miRNA biology and the association of specific miRNAs with CLL.Clinical biochemistry 03/2013; · 2.02 Impact Factor -
Article: Establishment of a CYP2C19 Genotyping Assay for Clinical Use.
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ABSTRACT: Conversion of clopidogrel (Plavix) to its active metabolite is catalyzed largely by the P450 enzyme 2C19 (CYP2C19). Numerous allelic variants of CYP2C19 exist. The *1 allele is considered wild type, whereas the *2 and *3 alleles have no in vivo enzymatic activity. Conversely, the *17 allele has increased expression, resulting in increased clopidogrel activation. Poor metabolizers (*2/*2 and *2/*3 genotypes) experience higher rates of therapeutic failure. For this reason, we have validated a CYP2C19 genotyping assay for the *1, *2, *3, and *17 alleles. Genomic DNA extracted from 30 deidentified EDTA whole-blood samples from patients was analyzed at 2 independent facilities using specific TaqMan realtime polymerase chain reaction primers and probes. Concordant genotypes were generated on all samples tested. Of the 30 samples, 15 were CYP2C19*1/*1, 8 were CYP2C19*1/*17, 5 were CYP2C19*1/*2, and 2 were CYP2C19*2/*17. There were no CYP2C19*3 alleles or *2/*2 homozygous genotypes detected. This CYP2C19 genotyping assay is appropriate for clinical testing, demonstrating excellent interlaboratory concordance, enabling the selection of the most effective clopidogrel treatment regimen for patients undergoing percutaneous coronary intervention.American Journal of Clinical Pathology 02/2013; 139(2):202-7. · 2.60 Impact Factor -
Article: Automation of genomic DNA isolation from formalin-fixed, paraffin-embedded tissues.
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ABSTRACT: Isolation of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue remains a laborious task for clinical laboratories and researchers who need to screen several samples for genetic variants. The objective of this study was to evaluate DNA isolation methods from FFPE tissues and to choose an efficient method with less hands-on time to obtain DNA of optimum concentration and purity for use in routine molecular diagnostic assays. Three methods were compared in this study: Gentra Puregene Tissue Kit, EZ1 DNA Tissue Kit and QIAamp FFPE Tissue Kit. Samples consisted of FFPE tissues of head/neck and lung tumor resections. Quality control for the extraction process end product included determination of the concentration and purity of isolated DNA and the ability to amplify a housekeeping gene, GAPDH, using real-time PCR assay. The hands-on-time required was less for the EZ1 protocol compared to the other methods. The average DNA concentration obtained was 112, 61 and 40ng/μl, respectively, for the Gentra Puregene Tissue Kit, Qiagen EZ1 DNA Tissue Kit and QIAamp FFPE Tissue Kit. The purity and quality of samples obtained using the different DNA isolation methods were comparable. Comparative evaluation of three DNA isolation methods indicated that the Qiagen EZ1 method surpassed the other methods with reduced hands-on-time to produce optimum concentration of quality DNA for use in routine molecular analyses.Pathology - Research and Practice 10/2012; · 1.21 Impact Factor
