Validation of a standardized extraction method for formalin-fixed paraffin-embedded tissue samples☆
Section snippets
Background
Human Papillomavirus (HPV) infections are known to be a major cause of cervical cancer [1]. More than 200 types of HPV have been fully characterized [2]. HPV types are divided in high-risk and low-risk viruses, where the high-risk types, notably HPV16 and HPV18, predominate in HPV-related cancers [1]. In light of current developments in HPV-based screening and HPV vaccination, it is important to develop a robust method for HPV genotyping and detection in cervical tissue, to be able to analyze
Objectives
The aim of this study was to evaluate a xylene-free method for the extraction of HPV-DNA in FFPE samples. If found robust, this method could become a new standard method for FFPE extraction before HPV genotyping. We thus compared the xylene-based gold standard method to a commercial column-based extraction with an extra heating step [5].
FFPE samples
Fifty FFPE-samples from patients with cervical cancer were randomly selected from a national case-control audit encompassing all invasive cervical and unspecified uterine cancers diagnosed from 2002 until 2011 in Sweden, the Advancing Cervical Cancer Eradication Strategies, ACCES, study. The diagnostic slides and the formalin-fixed paraffin-embedded (FFPE) tissue blocks were collected from different pathology biobanks throughout Sweden. Here, we selected fifty samples from one biobank,
Results
One case-block out of 50 extracted with the Qiagen/heating was negative for undiluted beta-globin compared to 9/50 case-blocks extracted with the xylene method (p = 0.008). All the 50 case-blocks were valid with both extraction methods after 1/10 dilution. Requirement for dilution did correlate with size of tumor, with positivity only after dilution being found more often in large tissue specimens (p = 0.015).
In the HPV16 real-time PCR, one Qiagen/heating-extracted case-block became positive after
Discussion
In a systematic comparison of two extraction protocols designed for FFPE tissue; standard xylene-based and a method using Qiagen columns augmented with a heat treatment, we find that the Qiagen/heating extraction method was superior in all comparisons made, along with the added benefit of a less labor-intensive method and fewer health hazards due to the fact that no use of xylene is required. The Qiagen kit is faster to work with and requires less handling after extraction, making it more time
Funding
This study was supported by the Swedish foundation for strategic research.
Competing interest
None.
Ethical approval
Ethical approval for this study was granted by the Regional ethical review board of Stockholm, Sweden, Dnr: 2011/1026-31/4.
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Cited by (26)
Human papillomavirus negative high grade cervical lesions and cancers: Suggested guidance for HPV testing quality assurance
2024, Journal of Clinical VirologyEvaluation of the Novaplex II HPV28 Detection Assay for HPV Typing in Formalin-Fixed, Paraffin-Embedded Tissues
2023, Journal of Molecular DiagnosticsOptimization of RNA extraction protocol for long-term archived formalin-fixed paraffin-embedded tissues of horses
2019, Experimental and Molecular PathologyCitation Excerpt :Furthermore, xylol was kept as melting reagent for all methods tested, to maintain the similarities with in-house extraction methods. On the other hand, tests with other melting methods like by hot mineral oil and water are promising alternatives for an adequate xylol-free extraction, denotating lower toxic laboratory waste and improving laboratory staff health (Kalantari et al., 2016; Lagheden et al., 2016). Therefore, combination of the present protocol with these adaptations should be tested in further studies.
Sensitivity of tumor surface brushings to detect human papilloma virus DNA in head and neck cancer
2017, Oral OncologyCitation Excerpt :In these tumors, episomal, transcriptionally inactive HPV DNA may have been detected by PCR, but had not resulted in p16 overexpression [31]. In the 6 and 5 samples, which were only HPV positive in FFPE specimens or in the brush test, a contamination or erroneous amplification cannot be excluded [32],[33]. We assume that some of the 6 FFPE samples were p16 IHC negative due to episomal HPV localization and the brush test was negative, because the number of viral copies obtained by brushing was too low [32],[34].
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The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.