Validation of a standardized extraction method for formalin-fixed paraffin-embedded tissue samples.
Author(s): Lagheden C, Eklund C, Kleppe SN, Unger ER, Dillner J, Sundström K
Publication: J Clin Virol, 2016, Vol. 80, Page 36-9
PubMed ID: 27148635 PubMed Review Paper? No
Purpose of Paper
This paper compared human papillomavirus (HPV) detection in DNA extracted from formalin-fixed paraffin-embedded (FFPE) specimens by two different protocols.
Conclusion of Paper
Specimens that were deparaffinized using heat, digested at 65˚C for 16 h, and from which DNA was purified using DNeasy Mini Spin columns (heat/DNeasy method) were less likely to require dilution, had 2-fold higher copy numbers of β-globin and HPV, and a higher HPV detection rate and a lower HPV false negative rate than specimens that were only deparaffinized in xylene and digested at 37˚C for 24 h (xylene method); however, when HPV was detected in both specimens, there was 100% concordance in genotype. Specimens from large tumors required dilution more often than those from smaller tumors and a trend toward HPV negativity in large tumors was observed.
Studies
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Study Purpose
This study compared the effects of two different DNA extraction protocols on human papillomavirus (HPV) detection in formalin-fixed paraffin-embedded (FFPE) specimens from 50 patients with invasive cervical cancer or unspecified uterine cancer stored 5-14 years. For each specimen, two 5µm sections were placed in a tube and xylene deparaffinized, proteinase K digested at 37˚C for 24 h, and proteinase K inactivated by boiling for 10 min (Xylene method). Another two sections of each specimen were heated to 120°C in lysis buffer and proteinase K digested at 65˚C for 16 h before purification with the DNeasy Mini Spin columns (heat/DNeasy method). HPV detection and genotyping were done using real-time PCR with fluorescently-labeled probes.
Summary of Findings:
Copy numbers for β-globin and HPV were more than 2-fold higher when DNA was extracted using the method that combined heat deparaffinization and DNeasy purification (heat/DNeasy method) than when specimens were only xylene deparaffinized and digested (xylene method, P<0.0001 and P=0.036, respectively). Ten of fifty (20%) cases were found to be HPV positive when extraction was by the heat/DNeasy method but negative when extracted using the xylene method and 2 of 50 (4%) were found to be positive when extraction was with the xylene method but not with the heat/DNeasy method. Consequently, the percentage of HPV positive specimens was higher when specimens were extracted using the heat/DNeasy method than the xylene method (86% versus 70%, P=0.039); however, when HPV was detected in both specimens, there was 100% concordance in genotype. Specimens extracted using the heat/DNeasy method were less likely to require specimen dilution than those extracted with xylene for detection of β-globin by real-time PCR (1 of 50 versus 9 of 50 specimens, P=0.008), HPV by real-time PCR (1 of 50 versus 4 of 50 specimens) and HPV in the real-time PCR genotyping assay (0 versus 5 of the specimens, P=0.06). Specimens from large tumors required dilution more often than those from small tumors for detection of β-globin and HPV (P=0.015, both) and a trend toward HPV negativity in large tumors was observed but size cut-offs were unspecified.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method DNeasy spin column
None
Analyte Extraction and Purification Deparaffinization Xylene
120°C in lysis buffer
Real-time qPCR Specific Template/input amount Undiluted DNA
DNA diluted 1:10
Real-time qPCR Specific Targeted nucleic acid β-globin
HPV
HPV genotyping assay
Analyte Extraction and Purification Protein digestion 37°C for 24 h
65°C for 16 h