Author:

Keywords:

Science & Technology, Life Sciences & Biomedicine, Pathology, 1108 Medical Microbiology, 3202 Clinical sciences, 3211 Oncology and carcinogenesis

Abstract:

Introduction: With the arrival of increasingly complex molecular tests we are forced to identify and create new ways to monitor and troubleshoot the daily performance of these multiplex assays. We have designed and validated a specific synthetic reference sample to augment a typical standard genomic DNA control set. This control was included as an evaluation sample in the European cystic fibrosis (CF) network External Quality Assessment (EQA) scheme in 2007 to assess its utility and performance in a large set of laboratories throughout the world. Materials and Methods: We designed, tested, and validated a collection of synthetic DNA molecules that may be used as a control for the simultaneous detection of 32 mutations associated with CF. Using the synthetic control in addition to a genomic mutant, a genomic wild-type, and a no template sample we have created a comprehensive quality control system for cystic fibrosis testing. This control, while designed specifically for the OLA platform, has application on other assays. To assess the utility of the control we submitted samples to the CF EQA / PT program of EuroGentest, which is a network of excellence for evaluation. Results: There were 105 laboratories in 19 countries that registered to participate in the evaluation of the synthetic control sample. From the initial 105 laboratories, 90 of them reported results identifying the mutations that they found. 68 laboratories reported the full complement of 32 mutations. Other reports consisted of mutation counts ranging from 31 mutants to just 5. The majority of testing methods consisted of OLA or reverse dot blot (Innogenetics), with considerable variability between laboratory procedures, equipment, reagents, and analysis methods based on the raw data that was submitted. In two specific cases the synthetic control identified procedural methodologies that block or hide the signal for the V520F mutation. Conclusions: Based on the reported results and raw data received from the EQA scheme, synthetic controls are a useful tool for monitoring quality control and troubleshooting underperforming assays. The results of this study illustrate the need for standardization, comprehensive validation, and greater collaboration between testing laboratories. While there is no single control that is ideal to answer every question, the thoughtful application of multiple tools and controls will allow for comprehensive and efficient quality control of complex genetic tests.