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Ensuring sample quality for BRAF testing
Ensuring sample quality, integrity and quantity
BRAF mutation testing is usually carried out on formalin-fixed paraffin-embedded (FFPE) melanoma tissue, but can also be performed on fresh or fresh frozen tissue.
Depending on the analytical method chosen, there are specific requirements for the quality, integrity and quantity of the tissue to be used, which are specified in the validated protocol for each test.1
Inadequate quality or quantity of the tissue sample can cause failure of the test, leading to an invalid result, with the need to test again on a different sample and therefore can critically delay therapy initiation.
General requirements:1–3
- The sample should not include necrotic tissue, fatty tissue, haemorrhagic tissue or non-tumour melanin-rich areas.
- It is also important that the sample has sufficient tumour cell content (neoplastic cellularity).
This is the percentage content of the true tumour, which includes neoplastic cells in the tumour area, (excluding the normal tissue cells and tumour microenvironment [intratumoural blood vessels, tumour-infiltrating immune cells and tumour stroma]). This is calculated by dividing the number (or surface) of neoplastic cells in the tumour area by the total number (or surface) of cells (neoplastic cells + normal cells + tumour microenvironment) in the tumour area.
The histopathologist is responsible for estimating the neoplastic cell content and applying appropriate techniques, such as manual micro/macro dissection, to ensure that a sufficient amount of neoplastic cells are scribed into the tube from which the nucleic acid will be isolated. Another reason for possible failure of the test due to quality of the tissue is inappropriate sample handling, mainly fixation, of the sample during the pre-analytical phase.
