FFPE is great for histology and terrible for molecular testing

Formalin-fixed, paraffin-embedded (FFPE) tissues in oncology diagnostics: is there a better way?

FFPE is great for histology and terrible for molecular testing

But, before we get to that, what is FFPE?

Formalin-fixation was discovered as a cellular preservation technique in the late 19th century and quickly became the most common embalming method throughout the 20th century.

While useful in preserving dead people and animals, it's also quite good at preserving tissues for scientific analysis because it chemically 'cross-links' proteins together making them much harder to degrade.

However, to prevent tissues from shriveling up and losing their morphology, the preservation technique requires a physical stabilizer.

Enter paraffin wax.

After formalin-fixation completes (~24hr), the tissue is embedded in a wax block.

This is followed by 'sectioning' with a microtome (cuts razor thin slices), and finally mounting the tissue sections on a microscope slide for viewing.

This process is great for understanding cellular pathology, but terrible for molecular analysis because getting all of the good bits out of an FFPE sample is very challenging.


Preservation of cellular morphology and proteins for imaging studies


Everything else

Formalin damages nucleic acids: the fixation process causes DNA and RNA to fragment and this degradation can continue even during cold storage. This can result in a significant reduction in signal, which is important in samples with low tumor fractions.

Fixation can introduce base modifications: deamination of cytosine to uracil and transitions (A>G,T>C) are most common. Base modifications are never ideal because they can result in the reporting of false positive results.

Loss of long range information: because formalin fixation breaks nucleic acid, long range sequence context is lost. Meaning, it's harder to detect gene fusions or chromosomal anomalies using traditional sequencing methods.

Sequence chimeras are prevalent: the damage caused during fixation creates sequence overhangs or single stranded DNA that ends up recombining to create new 'chimeric' sequence that didn't exist in the original sample.

De-paraffinizing a sample is not fun: this involves xylene or intense sample sonication with specialized equipment. One method is unpleasant, the other is expensive.

Treatment with enzymes during processing can reverse some of the damage, but they don't work miracles.

Fortunately, there are alternatives to FFPE and because we know formalin damages nucleic acid, we've been able to develop new methods that are more gentle on the molecular components we're most interested in testing.

These include:

*Processing fresh tissue
*Flash freezing in liquid nitrogen
*Preservation in a tissue stabilizer solution like AllProtect or RNAlater

However, the biggest hurdle is updating the clinical workflow to add the collection of a molecular friendly specimen (in addition to the ones that end up in the jars of formalin)!