UCL  IRIS
Institutional Research Information Service
UCL Logo
Please report any queries concerning the funding data grouped in the sections named "Externally Awarded" or "Internally Disbursed" (shown on the profile page) to your Research Finance Administrator. Your can find your Research Finance Administrator at https://www.ucl.ac.uk/finance/research/rs-contacts.php by entering your department
Please report any queries concerning the student data shown on the profile page to:

Email: portico-services@ucl.ac.uk

Help Desk: http://www.ucl.ac.uk/ras/portico/helpdesk
Publication Detail
Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial.
  • Publication Type:
    Journal article
  • Publication Sub Type:
    Comparative Study
  • Authors:
    Kapp JR, Diss T, Spicer J, Gandy M, Schrijver I, Jennings LJ, Li MM, Tsongalis GJ, de Castro DG, Bridge JA, Wallace A, Deignan JL, Hing S, Butler R, Verghese E, Latham GJ, Hamoudi RA
  • Publication date:
    02/2015
  • Pagination:
    111, 118
  • Journal:
    J Clin Pathol
  • Volume:
    68
  • Issue:
    2
  • Status:
    Published
  • Country:
    England
  • PII:
    jclinpath-2014-202644
  • Language:
    eng
  • Keywords:
    LUNG CANCER, MELANOMA, MOLECULAR PATHOLOGY, PCR, diagnostic screening, Cell Line, Tumor, DNA Mutational Analysis, DNA, Neoplasm, Diagnostic Errors, ErbB Receptors, Fixatives, Fluorometry, Formaldehyde, Humans, Laboratory Proficiency Testing, Mutation, Observer Variation, Paraffin Embedding, Polymerase Chain Reaction, Predictive Value of Tests, Proto-Oncogene Proteins B-raf, Reproducibility of Results, Spectrophotometry, Tissue Fixation, Transfection, United Kingdom, United States, Workflow
Abstract
AIMS: Mutation detection accuracy has been described extensively; however, it is surprising that pre-PCR processing of formalin-fixed paraffin-embedded (FFPE) samples has not been systematically assessed in clinical context. We designed a RING trial to (i) investigate pre-PCR variability, (ii) correlate pre-PCR variation with EGFR/BRAF mutation testing accuracy and (iii) investigate causes for observed variation. METHODS: 13 molecular pathology laboratories were recruited. 104 blinded FFPE curls including engineered FFPE curls, cell-negative FFPE curls and control FFPE tissue samples were distributed to participants for pre-PCR processing and mutation detection. Follow-up analysis was performed to assess sample purity, DNA integrity and DNA quantitation. RESULTS: Rate of mutation detection failure was 11.9%. Of these failures, 80% were attributed to pre-PCR error. Significant differences in DNA yields across all samples were seen using analysis of variance (p<0.0001), and yield variation from engineered samples was not significant (p=0.3782). Two laboratories failed DNA extraction from samples that may be attributed to operator error. DNA extraction protocols themselves were not found to contribute significant variation. 10/13 labs reported yields averaging 235.8 ng (95% CI 90.7 to 380.9) from cell-negative samples, which was attributed to issues with spectrophotometry. DNA measurements using Qubit Fluorometry demonstrated a median fivefold overestimation of DNA quantity by Nanodrop Spectrophotometry. DNA integrity and PCR inhibition were factors not found to contribute significant variation. CONCLUSIONS: In this study, we provide evidence demonstrating that variation in pre-PCR steps is prevalent and may detrimentally affect the patient's ability to receive critical therapy. We provide recommendations for preanalytical workflow optimisation that may reduce errors in down-stream sequencing and for next-generation sequencing library generation.
Publication data is maintained in RPS. Visit https://rps.ucl.ac.uk
 More search options
UCL Researchers
Author
Div of Surgery & Interventional Sci
University College London - Gower Street - London - WC1E 6BT Tel:+44 (0)20 7679 2000

© UCL 1999–2011

Search by