FFPE

What is FFPE?

Definition: 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.

  • The use of formalin fixative stems from its ability to cross-link proteins, however it has also been reported to induce methylol modifications and acid-mediated hydrolysis which can confound genotyping analysis
  • Eextraction is usually performed using commercially available kits. Variation between manufacturers and even lot to lot from the same manufacturer is common
  • Quantitation is not performed by all laboratories but can be useful to ensure that DNA isolates meet minimum concentration for use in an assay. Measurement systems can be confounded by low sample yield and sample fragmentation.

Comparison of DNA Extraction Methods from FFPE Samples

Comparison of DNA extraction methods from FFPE samples

Isolation of genomic DNA from formalin fixed paraffin embedded (FFPE) tissues is a critical step for molecular diagnostic (MDx) assays. It is essential that the maximum amount of DNA is recovered from the FFPE tissues and its quality is sufficient to perform the necessary MDx assays (e.g. QPCR, Sanger Sequencing, Pyrosequencing™).

A custom FFPE cell line block was generated to investigate the most appropriate DNA extraction kit for recovering the optimum DNA yield and concentration from low cell numbers. DNA extraction was performed using five comparable methods; Promega Maxwell® 16 FFPE Tissue LEV DNA Purification Kit; Promega MagneSil® Genomic, Fixed Tissue System Kit; Promega ReliaPrep™ FFPE gDNA Miniprep System; Qiagen DNeasy Blood & Tissue Kit; Roche Cobas® Sample Preparation Kit.

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Ready-made & Custom Reference Material

Using FFPE in the workflow to control variability

Figure 1 Using FFPE in the workflow to control variability

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).

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.

Custom Reference Standards

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.

Making FFPE Reference Material

Development of Quantitative B-Raf, EGFR, K-Ras and PI3Kα FFPE molecular reference standards

Development of Quantitative B-Raf, EGFR, K-Ras and PI3Kα FFPE Molecular Reference Standards

Isolation of genomic DNA from formalin fixed paraffin embedded (FFPE) tissues is a critical step in many molecular diagnostic assays. Horizon Diagnostics has generated FFPE reference standards containing defined DNA quantities and mutant allelic frequencies, enabling the quality control of both DNA extraction and assay sensitivity.

How we manufacture the FFPE Reference Standards

 

FFPE Sections Graphs

Figure 2 The consistency between FFPE sections

 

Applying Reference Standards in the workflow

FFPE DNA Extraction

FFPE DNA Extraction Graph

Figure 3. DNA recovery from total theoretical yield from reference standards

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.

DNA Quantification

DNA Quantification Graph

Figure 4 across measurements collected by multiple laboratories, there is notable variability between DNA quantified spectrophotometrically by Nanodrop vs. fluorometrically by Qubit

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.

FFPE products

Sanger and qPCR Sequencing   Next Generation Sequencing

Also available as Genomic DNA.

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