Standardization and Quality Control
Although companies compete on achieving the highest reproducibility on their qPCR instruments, the qPCR measurement for molecular diagnostics is hardly ever the most confounding step of the testing protocol, provided an adequately performing instrument is used.
Collection, transport, and storage of samples, nucleic acid extraction, and reverse transcription (in case of RNA) are steps that contribute much more confounding variation than qPCR. For example, blood collected in unstabilized matrices, such as EDTA tubes, shows seriously perturbed expression profiles due to the cells’ response to the foreign environment in the test tube. Serious preanalytical problems are also experienced in preserved tissue samples, where common fixation reagents such as formalin, selected primarily to preserve sample morphology, cause serious damage to the RNA.
In Europe a major effort has been undertaken by the SPIDIA consortium to tackle the standardization and improvement of pre-analytical procedures for in vitro diagnostics. The program covers steps from the development of evidence-based guidelines to the identification of quality biomarkers and tools for the pre-analytical phase.
Recently, SPIDIA presented an improved procedure for tissue preservation that maintains RNA at high quality, while preserving morphology. SPIDIA also coordinates proficiency ring trials in Europe helping routine laboratories evaluating their performance in the handling of and analyzing biological specimens with qPCR.
The European Committee for Standardization (CEN) is a SPIDIA member and the consortium interacts closely with the Office of Biorepositories and Biospecimen Research (OBBR) of the National Cancer Institute and the Clinical Laboratory Standards Institute (CLSI).
CLSI has released several guidelines relevant for molecular testing, many of which has been adapted by the FDA. These initiatives are most important to establish qPCR as a robust and reliable platform for routine diagnostics.
For research applications the “MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments” have been developed to encourage better experimental practice and allow for more reliable and unequivocal interpretation of qPCR results.
Several leading journals enforce MIQE and many more endorse them. Guidelines are good but not sufficient; we also need standards. Recently, the U.S. National Institute of Standards and Technology released the first viral DNA Standard Reference Material—for cytomegalovirus (CMV). The CMV was quantified as genome copies/volume using digital PCR (dPCR).
In dPCR a sample is aliquoted into a large number of reaction chambers such that only some contain targeted DNA and give rise to product upon PCR amplification. Counting the number of positive reactions essentially corresponds to counting single DNA molecules, resulting in absolute quantification and traceability to the SI unit, the mole.
qPCR technology is developing rapidly, although most current efforts are on upstream (pre-analytic) and downstream (data mining) processes, rather than on the qPCR technique itself which, I believe, has almost reached perfection.
A lot of work remains to be done until the entire workflow is standardized. Even more challenging will be to develop standards for various important DNA and RNA targets. The guidelines under development now are an important step in the right direction.