Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial.
Author(s): Kapp JR, Diss T, Spicer J, Gandy M, Schrijver I, Jennings LJ, Li MM, Tsongalis GJ, Gonzalez de Castro D, Bridge JA, Wallace A, Deignan JL, Hing S, Butler R, Verghese E, Latham GJ, Hamoudi RA
Publication: J Clin Pathol, 2015, Vol. 68(2), Page 111-8
PubMed ID: 25430497 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of DNA extraction and quantification methods on PCR success and mutation detection using engineered formalin-fixed paraffin-embedded (FFPE) specimens and a single FFPE tonsil specimen.
Conclusion of Paper
Mutation analysis failed to identify introduced mutations for 11.9% of the engineered specimens. 3 of the 4 failures were due to insufficient DNA quantity with the fourth failure due to Sanger sequencing error. While the variability in DNA yield depended on the method of measurement, amplifiable DNA was obtained by all methods, and PCR inhibition was minimal. However, the measurement of DNA yield was 5.1 fold higher by Nanodrop than Qubit, and the use of Nanodrop resulted in quantification of DNA from negative controls containing no cells.
Studies
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Study Purpose
The purpose of this study was to determine the effects of DNA extraction and quantification methods on PCR success and mutation detection using engineered FFPE specimens and a single FFPE tonsil specimen. For consistency, specimens were comprised of MCF10A and SW48 cells which had known mutations introduced into BRAF and EGFR. Blocks were designed to have 160,000 cells/section. No specifics of the FFPE tonsil specimen(s) were provided. DNA extractions were done at 13 different laboratories blinded to specimen contents using one of 5 methods for extraction and one of two or both methods for quantification.
Summary of Findings:
The variation in DNA yield between laboratories for the engineered specimens was not significant, but quantified yields were 1.3 fold higher than expected based on the number of cells. Importantly, laboratories reported DNA yields averaging 235.8 ng from negative control specimens lacking cells, but this was only true when Nanodrop was used for quantification and not Qubit. Further there was an 11.9% failure rate for mutation detection which was attributed to the failure to obtain sufficient DNA from 2 of 2 and 1 of 4 specimens at two laboratories and a single missed call by Sanger sequencing at another laboratory. DNA extraction methods differed between the laboratories and resulted in variances in DNA yield (52-82%), but amplifiable DNA was obtained by all methods, and there was no evidence of substantial PCR inhibition. Importantly, while amplifiable DNA of 100-400 bp was obtained from engineered specimens, the tonsil specimen yielded DNA that was highly degraded and only allowed for reliable amplification of the 100 bp product, although weak amplification of other bands was possible in specimens from some laboratories. The measurement of DNA yield as assessed by Nanodrop (spectrophotometry) was only modestly correlated with that by Qubit (fluoremetry) (R=0.48), with Nanodrop reading averaging 5.1 fold higher than Qubit readings, and this difference was accentuated with the non-engineered specimen which had degraded DNA.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform DNA DNA sequencing DNA Spectrophotometry DNA Fluorometry DNA PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) PCR Specific Length of gene fragment 100 bp
200 bp
300 bp
400 bp
Analyte Extraction and Purification Analyte isolation method COBAS
Dneasy
RecoverAll
QIAamp
EZ1
Spectrophotometry Specific Technology platform Qubit