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How Q-PCR testing works in practice
Q-PCR testing extracts the available mRNA in a blood or marrow sample. A test result is expressed as a percentage showing the ratio between of mRNA from normal control gene transcripts, for example ABL1 or BCR, compared to mRNA from the abnormal BCR-ABL1 gene transcripts present in a sample.
To perform the test, samples of blood or bone marrow are sent to the molecular pathology laboratory where mRNA is extracted from the white cells. There are a variety of ways of doing this and methods vary to a greater or lesser degree between laboratories.
To ensure results that more accurately reflect the number of Ph positive cells present in an individual, samples taken from patients must contain adequate numbers of copies of a control (normal) gene.
At least 10,000 copies of a control gene, such as ABL1 or BCR, are needed in every sample sent for testing if the percentage ratio of BCR- ABL1 is to be correctly assessed.
The control genes most commonly used are ABL1, BCR or GUSB. There is no consensus as to which of these normal genes is the best control to use. The choice lies entirely with the laboratory performing the test.
Common factors affecting the suitability of a sample sent for q-PCR testing
- If a blood sample has been in transit for several days, or has been stored for too long after collection, the cells it contains will already be in the process of dying and the mRNA will have started to degrade. This means there is a greater chance of an inaccurate result and many labs will not report results from such samples if the control gene is at too low a level.
- The lowest acceptable level for a control gene in any one sample is 10,000 copies (transcripts).
- Test results will show a relative proportion, (expressed as a percentage) of how many BCR-ABL1 transcripts are present over the total number of cells analysed in the blood sample.
- A sample taken from a patient who is responding well to therapy is more likely to contain a good amount of normal ABL1 gene transcripts and a much lower amount of abnormal BCR-ABL1 transcripts. This is because cells containing the abnormal fusion gene will have been killed during therapy and would be very few in number.