BRAF testing workflow

Step 2 : Sample acquisition and initial preparation

Tissue from melanoma patients is acquired by surgical removal of the initial tumour, metastasis or biopsy.¹

What happens to the tissue samples when they reach the pathology laboratory?

When the tumour samples arrive in the pathology laboratory they are examined and then cut in to small pieces that are fixed, embedded in paraffin and prepared as tissue blocks ready to be sectioned and placed onto microscope slides for further analysis.^1,2 All these steps are part of the pre-analytical phase and are important in order to preserve good quality tissue samples and biomolecules for analysis.

Step 3: Sample transport

Where BRAF testing is done

The initial sample preparation is done in the closest pathology laboratory, often in the same institution where the sample is acquired. The BRAF mutation test is a specialised and technically demanding procedure and cannot be performed in every pathology laboratory; therefore, in some cases, the sample (in the form of a tissue block or microscope slide) is sent to a specialised testing centre.¹

How samples should be sent to BRAF testing centres

Formalin-fixed, paraffin-embedded (FFPE) samples, as tissue blocks or slides, can be sent at room temperature to the specialised testing centre.

What can affect the time taken for samples to reach testing centres?

Transportation of samples is a critical step that can cause delays in the overall diagnostic process, as depending on the particular case and workflow setting, it may take some time for the sample to reach the testing laboratory.

For instance, significant additional workload, cost and potential time delays can occur if a test is requested for a patient with metastatic melanoma, and the original sample from the time of diagnosis has to be retrieved and shipped from the archives of the institution where the original sample was taken.¹ If the local pathology or molecular biology laboratory where the test is requested has the original sample, then this step involves only retrieval of the sample from the archive. Alternatively, if the test is performed at the initial diagnosis of melanoma, on the primary tumour sample, delays due to sample retrieval can be completely eliminated.¹

Step 4: Sample preparation

How tissue samples are prepared for BRAF testing¹

Using microscopy, the pathologist identifies the tumour area on the slide by assessing the cellularity (percentage of neoplastic cells within the sample, which also consists of tumour stroma and other cell types), and eliminating the necrotic, fatty, and haemorrhagic tissue. An appropriate amount of the tumour tissue is taken from the slide and placed into a tube ready for nucleic acid extraction for the BRAF mutation test. Guidance on the protocol for this step is usually part of the instruction for use of the BRAF mutation test and/or validated by the testing laboratory.

Step 5: Target isolation

How DNA is analysed¹

The actual target for mutation analysis is DNA from melanoma cells. During this step, DNA is isolated from the tissue which was taken from the slide. The paraffin in which the sample is embedded is removed, and the sample is lysed and then heated to reverse formalin crosslinking.

The DNA is membrane-bound so washing is required, after which concentrated DNA is eluted from the membrane.

Step 6: Mutation detection¹

How BRAF gene mutations are detected

During this step the DNA sample is analysed using whatever reagents and instrumentation is required for the testing method that has been selected. The presence of targeted BRAF gene mutations is detected, and a report is generated.

The available technologies are discussed in BRAF mutation testing methods.

Step 7: Reporting

How to complete the molecular pathology report

The pathology report is the final stage of the diagnostic process, and it is important to accurately convey the information the clinician needs to treat the patient.¹ Sufficient information is needed to allow for correct interpretation of the result.¹ Ensuring that the report is in an appropriate format that is easy to interpret, and reaches the clinicians in a timely manner is critical to patient care.¹