Details:

Alternative Title:
J Clin Virol
Publisher's site:
http://dx.doi.org/10.1016/j.jcv.2016.04.016
Personal Author:
Lagheden, Camilla ; Eklund, Carina ; Kleppe, Sara Nordqvist ; Unger, Elizabeth R. ; Dillner, Joakim ; ... More +
Lagheden, Camilla ; Eklund, Carina ; Kleppe, Sara Nordqvist ; Unger, Elizabeth R. ; Dillner, Joakim ; Sundström, Karin Less -
Description:
Background

Formalin-fixed paraffin-embedded (FFPE) samples can be DNA-extracted and used for human papillomavirus (HPV) genotyping. The xylene-based gold standard for extracting FFPE samples is laborious, suboptimal and involves health hazards for the personnel involved.

Objectives

To compare extraction with the standard xylene method to a xylene-free method used in an HPV LabNet Global Reference Laboratory at the Centers for Disease Control (CDC); based on a commercial method with an extra heating step.

Study design

Fifty FFPE samples were randomly selected from a national audit of all cervical cancer cases diagnosed in Sweden during 10 years. For each case-block, a blank-block was sectioned, as a control for contamination. For xylene extraction, the standard WHO Laboratory Manual protocol was used. For the CDC method, the manufacturers’ protocol was followed except for an extra heating step, 120°C for 20 minutes. Samples were extracted and tested in parallel with β-globin real-time PCR, HPV16 real-time PCR and HPV typing using modified general primers (MGP)-PCR and Luminex assays.

Results

For a valid result the blank-block had to be betaglobin-negative in all tests and the case-block positive for beta-globin. Overall, detection was improved with the heating method and the amount of HPV-positive samples increased from 70% to 86% (p=0.039). For all samples where HPV type concordance could be evaluated, there was 100% type concordance.

Conclusions

A xylene-free and robust extraction method for HPV-DNA typing in FFPE material is currently in great demand. Our proposed standardized protocol appears to be generally useful.


Subject:
[+]
DNA, Viral
Female
Formaldehyde
Genotype
Human Papillomavirus 16
Humans
Molecular Typing
Papillomavirus Infections
Paraffin Embedding
Sensitivity And Specificity
Tissue Fixation
Uterine Cervical Neoplasms
Xylenes

Source:
J Clin Virol. 80:36-39.
Pubmed ID:
27148635
Pubmed Central ID:
PMC5075962
Document Type:
Journal Article
Funding:
CC999999/Intramural CDC HHS/United States
Collection(s):
CDC Public Access
Main Document Checksum:
[+]
urn:sha256:4e5d976e9b654bf92ca54675c045f9b8a049483cc109e0949fbaf68038e88200
File Type:

Supporting Files

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