Toby Ward, PhD
Field Applications Scientist

The importance of proper formalin fixation of FFPE specimens

Tumor biopsies are often formalin-fixed and paraffin-embedded (FFPE) for histological staining and archival purposes. Formalin treatment preserves tissue, but also damages nucleic acids and poses a challenge to identifying true variants in tumor samples using next-generation sequencing (NGS).

Although FFPE treated samples have been notoriously challenging for NGS studies in the past (Hedegaard et al. 2014), more recent reports within the field suggest it may be possible to isolate high quality nucleic acids and to profile small variants from properly prepared FFPE samples. These latest studies have used a standard pathology protocol by fixing tissue with 10% neutral buffered formalin for 24 hours. However, many clinical research studies may be dependent upon archival tissue that has been fixed using variable methods and inconsistent procedures.

Based on results from our initial handling of FFPE samples, the scientists at Personalis hypothesized that varied sequencing results may be due to deviations from the standard fixation protocol. These could include inaccurate logging of the fixation protocol, variation in the fixation time, fixation temperature, and/or varied storage conditions of the samples.

To understand the role these parameters may have upon the sequencing quality of variants from formalin fixed tissue, we performed NGS DNA sequencing on fresh frozen (FF) and formalin-fixed samples using a standard reference cell line NA12878. We assessed raw DNA quality, library quality, sequencing QC metrics, and variant concordance profiles between FF and formalin treated samples.

We found that the quality of DNA isolated from formalin treated NA12878 cells varied considerably between different protocols. The traditional formalin fixation protocol of one day in 100% phosphate buffered saline (PBS)-buffered formalin at room temperature yielded DNA with high molecular weight and low fragmentation. The library constructed from this preparation was also of good quality. On the other hand, fixation protocols using unbuffered formalin at higher temperatures, and for prolonged periods of time resulted in highly fragmented, low molecular weight DNA. Input of this poor quality DNA also resulted in poor library efficiency. Based on these results, we found that fixation using buffered formalin at the proper temperature is crucial to preserve high-quality DNA for downstream NGS protocols.

Protocols employing long fixation times (>1 day) at high temperatures in unbuffered formalin adversely affect tissue DNA and can result in highly fragmented DNA and lower library quality. This, in turn, results in impaired alignment metrics and reduced sensitivity and specificity in somatic variant calling.

Given this massive variation in quality, it’s critical to communicate upfront with pathology labs which fix human tissue for histology and storage should that they must adhere to proper fixation protocols, so that tissues may be successfully used in both clinical cancer research and diagnostic NGS assays.

View our full results in Dr. Ward’s upcoming white paper, “Formalin Fixation Effects on Next-Generation Sequencing.”


Hedegaard J, Thorsen K, Lund MK, Hein A-MK, Hamilton-Dutoit SJ, Vang S, et al. (2014) Next-Generation Sequencing of RNA and DNA Isolated from Paired Fresh-Frozen and Formalin-Fixed Paraffin-Embedded Samples of Human Cancer and Normal Tissue. PLoS ONE 9(5): e98187.