Samples may be tested at sites remote from where patients are seen, and the results generated by different laboratories may vary for a number of reasons, including:
- method of isolating white blood cells (e.g. red blood cell lysis, density gradient centrifugation)
- method of extracting RNA (e.g. phenol–chloroform, column purification)
- choice of reverse transcriptase and DNA polymerase enzymes
- choice of primers
- source of fluorescence (Sybr Green, Taqman® probes)
- RT-qPCR machine used
- quantification method (e.g. standard curve, delta-delta cycle threshold).
Standardisation is achieved by normalising results to the International Scale (IS), which is based on a set of standards from patients in the IRIS trial of imatinib in CML. Laboratories either calibrate their equipment based on RNA standards from an IS laboratory or use a conversion factor. In the UK, the National External Quality Assessment Services is responsible for standardisation between laboratories, and results are normally calibrated to the IS Laboratory at Sheffield Teaching Hospitals NHS Foundation Trust.
