Quality Control in qPCR

Mario Cunha, Luis Martins, Carmo Ornelas
Clinical Pathology, Lab. Virology, Portuguese Institute of Oncology, Lisbon, Portugal


With the introduction of Real Time PCR (qPCR) in most clinical laboratories, we have been able to combine the kinetics of PCR with fluorescent probes, in order to monitor the PCR product in Real Time. Besides the excellent sensibility and specificity, qPCR is easy to execute and has a low risk of contamination. With all these capabilities, it has become an excellent alternative to traditional methods like cell culture and immunoassays. When a new assay is implemented in a clinical laboratory, precise and exact results are an absolute requirement, two of the main characteristics of Quality Control. Since qPCR has been introduced very recently in the clinical laboratory, we must normalize the steps we have to take regarding its validation and implementation in routine. The Accreditation procedures (ISO 15189) demand validation of every assay implemented. Despite the emission, by Portuguese Institute of Accreditation (IPAC), of some guidelines, these lack some of the important steps to validation. However, there are scientific articles and international guides that describe the necessary steps to take, in order to implement qPCR assays in the clinical laboratory. Once validated and implemented, the qPCR assays must be monitored routinely in order to check for shifts/deviations from the parameters set initially. The current methodology is the Internal Quality Control (IQA) and External Quality Control (EQA). In the IQA procedure, we must introduce a Non Template Control (NTC) in every run, with or without sample replicas, using one or several Positive Controls, expressed in Cq, Copies/ml or IU/ml. The values can be monitored in Quality Control Charts (MultiQC). We can also monitor the regression parameters, establishing limits for the Slope, Y Intercept, Detection/Quantification Limit and Efficiency. Concerning EQA, the aim of this methodology is to evaluate the Laboratory’s capacity to produce good quality results. For every qPCR assay that its implemented, we must participate in an EQA program. In Virology there are several programs available, namely QCMD, NEQAS, and Instand. Both results, from IQA and EQA, are used to measure the performance of the laboratory through the determination of Total Error and Sigma. For the Total Error, we apply the following formula: TE=|Bias|+ZxCV% The Bias can be obtained when we compare our EQA results with those obtained by other laboratories; CV% can be measured by our Positive Controls, in Quality Control Charts; and Z is constant: 1,65 for CI of 95%. If we have established an Allowed Total Error (TEa), we can determine the Sigma of our qPCR assay: Sigma = (TEa – |Bias|)/CV%. The Sigma has a scale of evaluation: 0 is bad, 6 or above is perfect.

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