25. Importance of internal controls to monitor performance of sensitive qPCR JAK2 V617F test kits

      The JAK2 V617F mutation is commonly detected in myeloproliferative neoplasms (MPN) accounting for >95% of cases of polycythemia vera and approximately 50% of cases of both essential thrombocythemia and primary myelofibrosis. JAK2 V617F mutation screening is routinely done to support a diagnosis of MPN and rule out other entities in the differential diagnosis. Sensitive, allele-specific quantitative PCR kits are available that can detect the V617F allele at <1% allele frequency. Kit calibrators and kit controls are provided to ensure highly sensitive and reliable results. Testing at UCLA showed the importance of having an internal control to help understand and monitor the performance of these kits. Through the use of an internal 5% V617F control, a period of time was identified where the measurements of the 5% control were outside of a monitored control range. Raw copy numbers calculated without the use of calibrators were stable for the 5% control and showed an overall normalization shift for the kit calibrators. A new process was developed where copy numbers are calculated independent of kit calibrators and a set of 1.3%, 5%, and 50% controls are used to monitor stability and reliability. This process has led to significant noise reduction and more accurate delineation between negative and positive results at the limit of detection. This strategy supports the use of extra internal controls and can be applied to other sensitive qPCR test kits such as BCR-ABL1 quantitation.
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