scope:

Multiplex assays generate multiple, simultaneous results on a single sample, such that two or more mutations are simultaneously examined in either a single gene or many genes; examples include cystic fibrosis transmembrane conductance regulator (CFTR) mutation analysis, a panel of several loci with common mutations in the Ashkenazi Jewish population, cytochrome P-450 genotyping, deep vein thrombosis panels, and hepatitis C virus (HCV) genotyping. Multiplex testing provides significant challenges to the laboratory with regards to appropriate verification and validation. A variety of technologies and instrument platforms are available for performing multiplex assays. It is important to note that acquisition of appropriate biological control materials can be extremely difficult or impossible. Moreover, the complexity of data analysis and reporting of results is increased relative to uniplex assays.

This guideline provides recommendations for analytic verification and validation of qualitative multiplex assays, as well as a review of different types of biologic and synthetic reference materials (RM). Multiplex assays are defined as those in which two or more targets are simultaneously detected through a common process of sample preparation, target or signal amplification, allele discrimination, and collective interpretation. An overview of currently available technologies, as well as recommendations for evaluating new ones, is provided. An extensive review of the design, acquisition, and appropriate use of different types of control materials, including blood samples, residual patient samples, products of whole genome amplification, synthetic oligonucleotide uniplex and multiplex controls, and plasmids, is provided. Current assay formats are used to illustrate proper verification and validation protocols, and appropriate data analysis and result reporting for multiplex assays are described.

This guideline does not address assays that measure individual targets that are then evaluated simultaneously, and is limited to a discussion of analytic validation and verification of qualitative multiplex assays for genotyping and pathogen detection/identification. Extensive discussion of validation and verification of gene expression assays, which may be a subject of a separate guideline, is excluded; however, many aspects of this guideline may also apply to expression assays, such as specimen handling, sample preparation, nucleic acid isolation, amplification and detection technologies, and instrument platforms. This guideline focuses on analytical performance and only includes a general discussion on clinical performance. A general overview of multiplex technologies is provided to the extent of discussing unique aspects and special considerations for multiplex assays, but does not discuss in detail the basic technologies, which are covered in other Molecular Methods guidelines (ie, this guideline does not specifically address many microarray-based detection platforms, which are the subject of a separate CLSI document MM12).¹ See CLSI documents MM1, MM3, and MM12.^1-3