Fundamentals of Quality Assessment of Molecular Amplification Methods in Clinical Diagnostics (Special Report‪)‬

The following recommendations on quality assessment of molecular biology methods in clinical diagnostics refer to the preanalytical and analytical steps, particularly those of amplification techniques. In particular, the polymerase chain reaction (PCR) and methods based thereon are those primarily used for developing laboratory tests that have potential for future routine applications. We have not attempted to deal with individual applications, which are at present in constant change and evolution and for many of which the clinical significance has yet to be established. Because of the special requirements associated with amplification techniques, the aspects of good laboratory practice dealt with in depth are those regarding preanalytical and analytical aspects of nucleic acids amplifications. Many of the recommendations are aimed at avoiding contaminations and facilitating timely recognition of contaminants, should they occur. Although every amplification assay is prone to contamination, the technical effort associated with different types of nucleic analyses varies widely. For example, genotyping patients usually does not require optimization of the amplification conditions for improved detection limits. On the other hand, for detection of minimal residual disease or for virus detection, a very low detection limit is prerequisite. Accordingly, contamination risk may be not a major problem in one application but obviously can be critical in another. Also, if different enzymes have to be used in subsequent steps of a given test, as in reverse transcriptase (RT) PCR, the handling of the additionally required material must be considered a potential contamination hazard.