Critical factors for successful molecular analysis of FFPE samples

Critical factors for successful molecular analysis of FFPE samples

Fixation of tissues involves placing specimens in a formalin solution which can vary in composition (a typical 10% formalin solution contains 3.7% formaldehyde and 1–1.5% methanol). For optimal results, use neutral-buffered formalin solution instead of unbuffered or acidic formalin solutions.
Also, pay equal attention to other factors that influence the extent of tissue fixation such as the thickness of the tissue specimen, the volume of formalin solution and the duration of fixation.
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Did you know? Formalin starts penetrating tissues at a rate of about 1 mm per hour. However, the rate decreases after the first millimeter.

Figure 2. Avoide the consequences of overfixation or underfixation.
  • Document all steps from sample collection to isolation of the analyte
  • Use standard-buffered formalin solution as fixative – check pH and concentration before use
  • Use thin tissue pieces, preferably ≤ 5 mm
  • Ensure at least 10:1 (V/V) formalin to tissue ratio for optimal fixation
  • Reduce time to fixation to the minimum
  • Keep fixation duration between 12 and 24 hours for 5 mm tissue pieces
  • Try not to exceed a temperature of 60°C during embedding
  • Use low-melting-temperature paraffin
  • Avoid sample staining – if absolutely required, avoid procedures that involve high pH or heavy metal ions, instead, use a nucleic acid-compatible stain such as cresyl violet
  • Store your FFPE samples at 4°C or below, if possible

Document the following:

  • ID of the specimen donor/patient, relevant health status, medical treatment, appropriate consent
  • Information about the specimen:
    • Start of ischemia within the body
    • Time, date and method of removal from the body
    • Description of tissue type, tissue conditions and organ of origin
  • ID of the person responsible for specimen collection
  • Time (cold ischemia) and storage conditions until fixation
  • Any additions or modifications to the specimen after removal from the body (e.g., further grossing, ink-marking, stitches, incisions)
  • Formalin used for fixation, ratio of formalin to tissue, temperature and duration
  • Dehydration and paraffin infiltration steps (temperature, duration, vacuum), quality and concentration of alcohol and paraffin
  • Storage of FFPE samples (time, temperature, humidity)
  • Sectioning for DNA/RNA extraction:
    • Number of sections and their thickness
    • Information about grossing into the block before removal of sections for DNA/RNA extraction
    • Extraction of DNA/RNA directly from sections of FFPE or from areas excised from sections mounted on slides

For standardization of the pre-examination process refer to:

ISO 20166-1 Molecular in vitro diagnostic examinations – Specifications for pre-examination processes for formalin-fixed and paraffin-embedded tissue – Part 1: Isolated RNA

ISO 20166-3 Molecular in vitro diagnostic examinations – Specifications for pre-examination processes for formalin-fixed and paraffin-embedded tissue – Part 3: Isolated DN

In FFPE samples, deamination, which occurs randomly during formalin fixation and with aging, turns cytosine into a uracil base. Deaminated cytosine pairs with adenine as uracil. In DNA sequencing reactions, this will manifest as a C>T|G>A transition.
Figure 3. Potential for false positives. Cytosine deatamination can lead to false positives during DNA sequencing. 
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Did you know? To improve the reliability of low-frequency variant detection by whole genome or targeted DNA sequencing, uracil- N-glycosylase (UNG) treatment is recommended during DNA sample preparation. UNG removes uracil to prevent mismatch amplification.
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