scope:

This guideline provides recommendations for qualitative multiplex nucleic acid assay validation and verification. This guideline focuses primarily on analytical validation, analytical verification, and QC and briefly discusses establishing clinical validation and clinical verification for these assays. The intended audience includes laboratory directors, medical microbiologists, laboratory technologists, QA personnel, and assay manufacturers. This guideline is not intended to be regulatory guidance but to provide current best practice recommendations. Additional regulatory and/or accreditation requirements may apply.

The design, acquisition, and appropriate use of different control materials are extensively reviewed. Current assay formats are used to illustrate proper validation and verification protocols, and appropriate data analysis and results reporting for multiplex assays are described. Because traditional single-measurand protocols are difficult or impossible to perform with multiplex assays, an error-based approach to validation and verification is presented. This error-based approach may be applicable to multiplex assays performed with a single test method, for which the performance characteristics for different measurands are expected to be similar.

This guideline describes general considerations and recommendations for multiplex testing platforms but does not discuss some basic technologies covered in detail in other CLSI molecular methods guidelines (eg, this guideline does not specifically discuss many microarray-based detection platforms or nextgeneration sequencing). Appendix A provides an overview of some currently available multiplex testing technologies. For additional information, see CLSI documents MM01,¹ MM03,² MM06,³ MM09,⁴ MM21,⁵ MM22,⁶ and MM23.⁷

This guideline discusses multiplex assays for genotyping and pathogen detection and excludes gene expression assays. This guideline also does not cover assays measuring individual targets that are then evaluated together to yield a composite score or classifier as a result.