FFPE or Formalin-Fixed Paraffin-Embedded is a method of preservation of cell tissues used extensively in profiling gene expression. FFPE Samples can be stored at room temperature and therefore avoid the complexities and risks of freezing.
Pre-analytical processing typically involves multiple steps, including sample fixation, DNA isolation and quantitation. At each of these stages, even minor differences in e.g. operator to operator sample handling, instrumentation and methodology can introduce significant variation, calling into question the quality of DNA and the reliability of downstream assay results.
Comparison of DNA Extraction Methods from FFPE Samples
Laboratories often assume that a failed assay is as a result of a failed analytical step, however root cause analysis often shows that problems with DNA extraction or issues with quantification are really at fault (e.g. over-estimating sample DNA concentration leading to the assay being under loaded). Analyzing the cause of a failed assay is a particular challenge for laboratories not used to handling FFPE tissues, or for laboratories using quantification methodologies that tend to overestimate the amount of DNA in a sample when measuring concentrations below 20 ng per microliter (e.g. NanoDrop).
|Figure 1. Using FFPE in the workflow to control variability|
In response to this need, Horizon Diagnostics has developed a range of HDx™ FFPE Reference Standards to enable effective end-to-end process validation.
Through custom Reference Standards we can offer the option of customizing reference material uniquely into multiple different analytes and formats.
The reference standards are highly consistent and reliable, yielding near identical quantities of DNA, enabling users to compare extraction kits and control for batch to batch variation, as seen in the figure below.
|Figure 2. The consistency between FFPE sections|
Applying Reference Standards in the workflow:
Reference Standards are prepared using highly homogeneous cell densities that yield a consistent and reproducible quantity of DNA. Laboratories are able to compare and establish the efficacy of DNA extraction through we benchmarked against the total theoretical yield of our reference material. The comparison of the different methods within the laboratory as can be seen above is an example of the variability.
Data from Horizon (below) shows that when DNA is extracted from these standards using five different commercially available protocols, significant variability in yield is observed.
|Figure 3. DNA recovery from total theoretical yield from reference standards.|
Following extraction, accurate FFPE DNA yield must be determined in order to input the appropriate amount of material for downstream sequencing library preparation, as well as to understand theoretical allele frequency thresholds (see below). While spectroscopic methods like UV/VIS provide adequate estimation at concentrations higher than 10 ng/µL, fluorometric quantification will always be more accurate and specific (measuring DNA only). Importantly, the method of quantification will greatly affect your results.
|Figure 4. across measurements collected by multiple laboratories, there is notable variability between DNA quantified spectrophotometrically by Nanodrop vs. fluorometrically by Qubit.|
FFPE products for: