Practical tips to using formalin-fixed paraffin-embedded tissue archives for molecular diagnostics in a South African setting

Abstract

Background: Formalin-fixed paraffin-embedded (FFPE) tissue archives in hospitals, biobanks, and others offer a vast collection of extensive, readily available specimens for molecular testing. Unfortunately, the use of tissue samples for molecular diagnostic applications is challenging; thus, the forensic pathology FFPE tissue archives in Africa have been a largely unexploited genetic resource, with the usability of DNA obtainable from these samples being unknown.

Intervention: The study, conducted from January 2015 to August 2016, determined the usefulness of FFPE tissue as a reliable source of genetic material for successful post-mortem molecular applications and diagnostics. Formalin-fixed paraffin-embedded tissue samples were collected and archived from autopsies conducted over 13 years in the forensic medicine department of the University of Pretoria (Pretoria, South Africa). Deoxyribonucleic acid from FFPE tissue samples and control blood samples was amplified by high-resolution melt real-time polymerase chain reaction before sequencing. The procurement parameters and fixation times were compared with the quantity and quality of the extracted DNA and the efficiency of its subsequent molecular applications.

Lessons learnt: This study has shown that FFPE samples are still usable in molecular forensics, despite inadequate sample preparation, and offer immense value to forensic molecular diagnostics.

Recommendations: FFPE samples fixed in formalin for more than 24 h should still be used in molecular diagnostics or research, as long as the primer design targets amplicons not exceeding 300 base pairs.

Keywords: Autopsy; deoxyribonucleic acid; formalin-fixed paraffin-embedded tissue; formalin-fixed paraffin-embedded tissue archive; high-resolution melt analysis; molecular diagnostics; polymerase chain reaction; post-mortem genetic testing; sequencing.

FIGURE 1

Effect of amplicon size on PCR amplification success rate of DNA extracted from FFPE tissue samples, Pretoria, South Africa, June 2015 – May 2016 (online Supplementary Document Table 3).

FIGURE 2

Laboratory results showing the association between a prolonged formalin fixation period and a decline in PCR amplification success rate, Pretoria, South Africa, March 2015 – July 2016 (online Supplementary Document Table 2).