title: Diagnostic DHPLC Quality Assurance (DDQA): a collaborative approach to the generation of validated and standardized methods for DHPLC-based mutation screening in clinical genetics laboratories.
abstract: Genetic testing in a clinical diagnostic environment must be subject to rigorous quality control procedures, in order to ensure consistency and accuracy of results. Denaturing high performance liquid chromatography (DHPLC) has become a standard prescreening tool for mutation detection, offering very high efficiency and sensitivity of detection. Despite the relatively simple software-assisted assay setup, DHPLC is a complex assay, and quality control is reliant on ensuring optimal instrument performance, excellent assay design and validation, and sufficient user training and proficiency to interpret results. We describe here a unique collaborative effort by a group of diagnostic clinical genetics laboratories with DHPLC expertise who, together with the manufacturer of one of the most widely used DHPLC platforms, have generated standard operating procedures (SOPs) for instrument operation and maintenance, and for mutation detection by DHPLC. We also describe the validation of a disease-specific SOP for DHPLC assisted mutation screening of the MECP2 gene associated with Rett syndrome. The proposed SOP was validated, and used independently in two laboratories to introduce MECP2 testing. In addition, we provide empirically derived normal ranges for the WAVE System Mutation Standards, which are essential for optimal instrument performance. This effort was initiated to try to standardize DHPLC-based mutation screening procedures across laboratories, and so increase the overall quality of this testing method. This endeavor will thus save each laboratory from having to generate SOPs on their own, which is a lengthy and laborious task. In this respect, we define "generic" SOPs as procedures that are easily adaptable to the individual laboratories' quality systems.
authors.affiliation: Center for Human Genetics, University of Leuven, Leuven, Belgium.
authors.forename: Els
authors.initials: E
authors.lastname: Schollen
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Dequeker
authors.forename: Shirley
authors.initials: S
authors.lastname: McQuaid
authors.forename: Bruno
authors.initials: B
authors.lastname: Vankeirsbilck
authors.forename: Geneviève
authors.initials: G
authors.lastname: Michils
authors.forename: John
authors.initials: J
authors.lastname: Harvey
authors.forename: Eric
authors.initials: E
authors.lastname: van den Akker
authors.forename: Ron
authors.initials: R
authors.lastname: van Schooten
authors.forename: Zandra
authors.initials: Z
authors.lastname: Clark
authors.forename: Stephan
authors.initials: S
authors.lastname: Schrooten
authors.forename: Gert
authors.initials: G
authors.lastname: Matthijs
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003299:Cooperative Behavior; D004252:DNA Mutational Analysis; D005820:Genetic Testing; D005826:Genetics, Medical; D006801:Humans; D007753:Laboratories; D051783:Methyl-CpG-Binding Protein 2 / Q000235:genetics; D025202:Molecular Diagnostic Techniques / Q000592:standards*; D009154:Mutation / Q000235:genetics*; D009691:Nucleic Acid Denaturation / Q000235:genetics*; D011786:Quality Control; D015203:Reproducibility of Results; D012107:Research Design
chemical_list: D051783:Methyl-CpG-Binding Protein 2