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BRAF+ melanoma
What makes it different, makes it vulnerable

Importance of the pre-analytical phase steps

The pre-analytical steps take place in the institution where the sample is taken and the closest histopathology laboratory, often in the same institution where the sample is acquired. Handling and pre-treatment of the tissue during transport to the pathology laboratory (see Step 2), as well as within the laboratory, are critical stages that can affect the quality of the tissue, proteins and nucleic acids available for further examination and testing.1

In the time between administration of the anaesthetic to the patient and ligation of the vessels during tumour excision, (in addition to sample handling and transport until fixation), there are changes that occur in gene expression, with degradation of biomolecules (autolysis), due to ischemia of the tissue/cells.2

  • Warm ischemia – during the surgical procedure
  • Cold ischemia – during the sample handling and transport

The tissue preservation procedures, including fixation and paraffin embedding have a significant impact on the quality of the tissue, thereby affecting testing, and must be performed based on accepted standards and recommendations.1–4

Pre-analytical phase of the diagnostic procedure

  • STEP 1
    Sample acquisition
  • STEP 2
    Transporting to pathology laboratory
  • STEP 3
    Gross examination
  • STEP 4
    Formalin fixation
  • STEP 5
    Paraffin embedding
  • STEP 6
    Slides preparation

Step 1: Sample acquisition

Surgery/biopsy procedure times

It is important that the surgery/biopsy procedure takes the minimum possible time after administration of the anaesthetic and ligation of the vessels. During excision of the tissue, changes in gene expression along with degradation of biomolecules (autolysis) occurs, this is due to ischemia of the tissue referred to as ‘warm ischemia’.1

Next article in this section >

For this slide:

  1. Stanta G (Ed). Guidelines for Molecular Analysis in Archive Tissues. Edition XVI, Springer-Verlag Berlin, Heidelberg 2011.


For this page:

  1. Groenen P, et al. Preparing pathology for personalized medicine: possibilities for improvement of the pre-analytical phase. Histopathology. 2011;59(1):1–7.
  2. Stanta G (Ed). Guidelines for Molecular Analysis in Archive Tissues. Edition XVI, Springer-Verlag Berlin, Heidelberg 2011.
  3. Cree IA, et al; European Society of Pathology Task Force on Quality Assurance in Molecular Pathology; Royal College of Pathologists. Guidance for laboratories performing molecular pathology for cancer patients. J Clin Pathol. 2014;67(11):923–31.
  4. Bussolati G, et al. Formalin Fixation at Low Temperature Better Preserves Nucleic Acid Integrity. PLoS ONE. 2011;6(6):e21043.

Step 2: Transporting to pathology laboratory

There are a number of ways in which tissue samples may be transported:

  • Fresh tissue samples: In special containers, small specimens are immersed in saline and kept at room temperature1
  • Tissue samples immersed in formalin: The toxicity of formalin means that operating theatres are not suitable for this process, (there is movement towards environmentally-friendly and ‘formalin-free’ hospitals).2 However, formalin fixation is considered a straightforward procedure and very common for biopsies, such as melanoma samples1
  • Tissue samples in specific cell culture media (mild isotonic [RPMI], or solutions with specific salts [Hanks]): Very good for preserving morphology and biomolecules, but it has not yet been fully validated or standardised2
  • Tissue samples in vacuum conditions and stored at 4⁰C up to 48 hours: Good for tissue preservation and especially useful for large tissue samples. Very environmentally-friendly and also allows for examination and testing on fresh tissue1

Next article in this section >

For this slide:

  1. Stanta G (Ed). Guidelines for Molecular Analysis in Archive Tissues. Edition XVI, Springer-Verlag Berlin, Heidelberg 2011.
  2. Groenen P, et al. Preparing pathology for personalized medicine: possibilities for improvement of the pre-analytical phase. Histopathology. 2011;59(1):1–7.


For this page:

  1. Groenen P, et al. Preparing pathology for personalized medicine: possibilities for improvement of the pre-analytical phase. Histopathology. 2011;59(1):1–7.
  2. Stanta G (Ed). Guidelines for Molecular Analysis in Archive Tissues. Edition XVI, Springer-Verlag Berlin, Heidelberg 2011.
  3. Cree IA, et al; European Society of Pathology Task Force on Quality Assurance in Molecular Pathology; Royal College of Pathologists. Guidance for laboratories performing molecular pathology for cancer patients. J Clin Pathol. 2014;67(11):923–31.
  4. Bussolati G, et al. Formalin Fixation at Low Temperature Better Preserves Nucleic Acid Integrity. PLoS ONE. 2011;6(6):e21043.

Step 3: Gross examination

What happens at the histopathology laboratory

Once the surgical specimen arrives in the histopathology laboratory, it is taken to a ‘grossing station’, where it is thoroughly inspected for disease, measured and otherwise described.1,2

The portion suspected of being diseased is dissected into small pieces and placed into plastic tissue cassettes ready for processing. These are then placed into formalin, and later processed in a tissue processor (for paraffin embedding).1,3

Next article in this section >

For this slide:

  1. Groenen P, et al. Preparing pathology for personalized medicine: possibilities for improvement of the pre-analytical phase. Histopathology. 2011;59(1):1–7.
  2. Stanta G (Ed). Guidelines for Molecular Analysis in Archive Tissues. Edition XVI, Springer-Verlag Berlin, Heidelberg 2011.
  3. Cree IA, et al; European Society of Pathology Task Force on Quality Assurance in Molecular Pathology; Royal College of Pathologists. Guidance for laboratories performing molecular pathology for cancer patients. J Clin Pathol. 2014;67(11):923–31.


For this page:

  1. Groenen P, et al. Preparing pathology for personalized medicine: possibilities for improvement of the pre-analytical phase. Histopathology. 2011;59(1):1–7.
  2. Stanta G (Ed). Guidelines for Molecular Analysis in Archive Tissues. Edition XVI, Springer-Verlag Berlin, Heidelberg 2011.
  3. Cree IA, et al; European Society of Pathology Task Force on Quality Assurance in Molecular Pathology; Royal College of Pathologists. Guidance for laboratories performing molecular pathology for cancer patients. J Clin Pathol. 2014;67(11):923–31.
  4. Bussolati G, et al. Formalin Fixation at Low Temperature Better Preserves Nucleic Acid Integrity. PLoS ONE. 2011;6(6):e21043.

Step 4: Formalin fixation

Overview of tissue fixation using formalin

This is a critical step in tissue pre-treatment for preserving adequate tissue quality for further analysis.1 Formalin is a formaldehyde and methanol solution; only neutral-buffered, 10% solutions should be used (not un-buffered or acidic). When the tissue is immersed in formalin, crosslinks between biomolecules (e.g. nucleic acids, proteins) are created, which preserves them and prevents degradation.

Most of the molecular pathology tests are validated with formalin-fixed, paraffin-embedded (FFPE) tissue. FFPE is considered the process of choice to preserve, transport and archive the tissue for future molecular pathology testing.2

Important factors that influence the fixation process:1,3,4

  • The thickness of the sample (ideally ≤5 mm, as formalin penetrates the tissue at a rate of about 1mm per hour and the speed decreases with the thickness of the tissue). For large surgical samples, to ensure the sample is evenly fixed throughout, injection of the solution directly into the tissue is performed to achieve faster infiltration of the central part
  • The volume of the formalin solution compared to tissue (ideally, should be a ratio of 10:1.)
  • The duration of fixation (ideally <24 hours). If the tissue is ‘over fixed’, the proportion of irreversible crosslinks is high, which may lead to problems with amplification and also reduce recovery of proteins from FFPE samples.

Next article in this section >

For this slide:

  1. Groenen P, et al. Preparing pathology for personalized medicine: possibilities for improvement of the pre-analytical phase. Histopathology. 2011;59(1):1–7.
  2. Bussolati G, et al. Formalin Fixation at Low Temperature Better Preserves Nucleic Acid Integrity. PLoS ONE. 2011;6(6):e21043.
  3. Stanta G (Ed). Guidelines for Molecular Analysis in Archive Tissues. Edition XVI, Springer-Verlag Berlin, Heidelberg 2011.
  4. Cree IA, et al; European Society of Pathology Task Force on Quality Assurance in Molecular Pathology; Royal College of Pathologists. Guidance for laboratories performing molecular pathology for cancer patients. J Clin Pathol. 2014;67(11):923–31.


For this page:

  1. Groenen P, et al. Preparing pathology for personalized medicine: possibilities for improvement of the pre-analytical phase. Histopathology. 2011;59(1):1–7.
  2. Stanta G (Ed). Guidelines for Molecular Analysis in Archive Tissues. Edition XVI, Springer-Verlag Berlin, Heidelberg 2011.
  3. Cree IA, et al; European Society of Pathology Task Force on Quality Assurance in Molecular Pathology; Royal College of Pathologists. Guidance for laboratories performing molecular pathology for cancer patients. J Clin Pathol. 2014;67(11):923–31.
  4. Bussolati G, et al. Formalin Fixation at Low Temperature Better Preserves Nucleic Acid Integrity. PLoS ONE. 2011;6(6):e21043.

Step 5: Paraffin embedding

After fixation

The fixed tissue is dehydrated and ‘cleared’, which is a process of immersing it in different percentage solutions of alcohol and xylene to prepare the sample for embedding in paraffin.1,2 This process is performed in high-throughput automatic tissue processors.

The next step is embedding in paraffin: the pieces of tissue in the cassettes are infiltrated and entirely surrounded by paraffin, creating a ‘block’ that can be thinly sliced ready for application to microscope slides and further testing.1,3

Next article in this section >

For this slide:

  1. Groenen P, et al. Preparing pathology for personalized medicine: possibilities for improvement of the pre-analytical phase. Histopathology. 2011;59(1):1–7.
  2. Stanta G (Ed). Guidelines for Molecular Analysis in Archive Tissues. Edition XVI, Springer-Verlag Berlin, Heidelberg 2011.
  3. Cree IA, et al; European Society of Pathology Task Force on Quality Assurance in Molecular Pathology; Royal College of Pathologists. Guidance for laboratories performing molecular pathology for cancer patients. J Clin Pathol. 2014;67(11):923–31.


For this page:

  1. Groenen P, et al. Preparing pathology for personalized medicine: possibilities for improvement of the pre-analytical phase. Histopathology. 2011;59(1):1–7.
  2. Stanta G (Ed). Guidelines for Molecular Analysis in Archive Tissues. Edition XVI, Springer-Verlag Berlin, Heidelberg 2011.
  3. Cree IA, et al; European Society of Pathology Task Force on Quality Assurance in Molecular Pathology; Royal College of Pathologists. Guidance for laboratories performing molecular pathology for cancer patients. J Clin Pathol. 2014;67(11):923–31.
  4. Bussolati G, et al. Formalin Fixation at Low Temperature Better Preserves Nucleic Acid Integrity. PLoS ONE. 2011;6(6):e21043.

Step 6: Slides preparation

Tissue sample slides

These are prepared during the sectioning process. Very thin sections (2–10 micrometres) are sliced from the tissue block using a microtome.1,2 These slices are laid on a glass slide and can be stained for further analysis and microscopic examination.

Next article in this section >

For this slide:

  1. Cree IA, et al; European Society of Pathology Task Force on Quality Assurance in Molecular Pathology; Royal College of Pathologists. Guidance for laboratories performing molecular pathology for cancer patients. J Clin Pathol. 2014;67(11):923–31.
  2. Groenen P, et al. Preparing pathology for personalized medicine: possibilities for improvement of the pre-analytical phase. Histopathology. 2011;59(1):1–7.


For this page:

  1. Groenen P, et al. Preparing pathology for personalized medicine: possibilities for improvement of the pre-analytical phase. Histopathology. 2011;59(1):1–7.
  2. Stanta G (Ed). Guidelines for Molecular Analysis in Archive Tissues. Edition XVI, Springer-Verlag Berlin, Heidelberg 2011.
  3. Cree IA, et al; European Society of Pathology Task Force on Quality Assurance in Molecular Pathology; Royal College of Pathologists. Guidance for laboratories performing molecular pathology for cancer patients. J Clin Pathol. 2014;67(11):923–31.
  4. Bussolati G, et al. Formalin Fixation at Low Temperature Better Preserves Nucleic Acid Integrity. PLoS ONE. 2011;6(6):e21043.
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