Abstract
We designed a laboratory developed test (LDT) by using an open platform for mutation/polymorphism detection. Using a 108-member (mutation plus variant) cystic fibrosis carrier screening panel as a model, we completed the last phase of LDT validation by using matrix-assisted laser desorption/ionization time of flight mass spectrometry. Panel customization was accomplished via specific amplification primer and extension probe design. Amplified genomic DNA was subjected to allele specific, single base extension endpoint analysis by mass spectrometry for inspection of the cystic fibrosis transmembrane regulator gene (NM_000492.3). The panel of mutations and variants was tested against 386 blinded samples supplied by "authority" laboratories highly experienced in cystic fibrosis transmembrane regulator genotyping; >98% concordance was observed. All discrepant and discordant results were resolved satisfactorily. Taken together, these results describe the concluding portion of the LDT validation process and the use of mass spectrometry to detect a large number of complex reactions within a single run as well as its suitability as a platform appropriate for interrogation of scores to hundreds of targets.