Quality Control And Quality Assurance Of The Testing Process

Every molecular diagnostics laboratory should develop a comprehensive written quality assurance program. The objective of the quality assurance program is to objectively and systematically monitor and evaluate the quality and appropriateness of the test results. The quality assurance program should address every aspect of the testing process: preanalytical, analytical, and post-analytical processes. The program must include written policies and documentation for the education and training of personnel, continuing medical education, proficiency testing, internal and external inspections, including documentation of corrective actions for deficiencies cited, quality control programs for the clinical testing, equipment performance, and safety. Establishment of molecular diagnostic tests, particularly amplification assays, requires many considerations at every stage, including reagent preparation, specimen collection, specimen aliquoting, and performance of the actual assay. There are a number of considerations that apply to all molecular assays and that important to guarantee reliable results. Quality assurance programs must be developed to address the testing process during all phases of the testing process: preanalytical, analytical, and postanalytical. The amount of quality assurance and quality control performed for any molecular assay will depend on the type of test employed, FDA cleared, "for research use only," off-label, or ASR. The preanalytic and postanalytic phases for FDA-cleared and research use only tests are very similar. As part of the preanalytical phase, special attention should be given to the specimen collection, transport, and storage. This is critical because in many situations, the results reported could influence treatment decisions for a particular patient; this decision should be reflective of the patient status and not of specimen handling or mishandling. Quality control of reagents is another critical aspect that needs to be considered for all laboratory programs. Laboratories should determine critical reagents for the overall testing procedure and these reagents should be tested for composition, concentration, purity, and functionality. All new reagents should be tested in parallel using new reagents and should be compared to those generated using current reagents in crossover studies and proven to be comparable before the new reagents are used in assays with clinical samples.

As with any laboratory tests, a well-thought-out and well-written laboratory procedure is a key factor for the reproducibility of the assay. It is one of the most important aids during hands-on training of new personnel. The procedure protocol should be written according specific guidelines set up by the National Committee for Clinical Laboratory Standards. Performance of nucleic-acid based methods usually requires specific setup and/or workflow.

A careful selection of controls is vital for the interpretation of results. Several types of control are used through out the execution of the assays in order to assure the appropriate performance of a specific assay. Negative and positive controls are required by CLIA'88 regulations and must be processed in every clinical test. Failure to obtain the correct result for any of the controls invalidates the entire test and requires retesting of all samples. Whenever possible, positive and negative controls should resemble a patient's specimen as much as possible. Furthermore, a positive control should represent a clinically relevant range of the nucleic acid target sequence in a background of a negative nucleic acid target sequence. The negative control should represent a negative nucleic acid target. In addition to the negative and positive controls, a blank control should be included with every assay, containing all of the components of the reaction mixture but nucleic acid. In addition to these controls, in some instances it is imperative to add an internal positive control. There are many circumstances in which the presence or absence of an amplicon makes the diagnosis. In these circumstances, when a negative result is obtained, it is not clear if the absence of such amplicon is the result of the absence of the target sequence in the patient specimen or, for example, the result of the presence of inhibitors. In order to avoid this situation, it is recommended to perform the amplification of an internal positive control. The addition of sensitivity controls in which the last control is not detected is very valuable for monitoring not only the performance of the assay over time but also for monitoring the presence of amplicon contamination.

It is extremely important for LDTs to implement a quality control program for validating the robustness, purity, and performance of every critical reagent of the testing process. Every critical reagent should be tested before being approved for clinical testing. Tolerance limits for every critical reagent should be established. Whenever possible, tolerance limits should be established using a quantitative measurement in order to avoid subjective evaluation of the quality of the critical reagent.

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