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Review
. 2025 May;108(3):252-266.
doi: 10.1002/cyto.b.22202. Epub 2024 Aug 21.

Flow cytometry assay modifications: Recommendations for method validation based on CLSI H62 guidelines

Sara A Monaghan  1 , Steven Eck  2 , Silvia Bunting  3 , Xiangyang X Dong  4 , Robert J Durso  5 , Christele Gonneau  6 , Amanda Hays  7 , Andrea Illingworth  8 , Stacy C League  9 , Eleni Linskens  10 , Megan McCausland  11 , Thomas W McCloskey  12 , Nina Rolf  13 , Min Shi  14 , Paul K Wallace  15 , Virginia Litwin  16 , Wolfgang Kern  17 , George Deeb  18 , Veronica Nash  19 , Horatiu Olteanu  14
Affiliations
Review

Flow cytometry assay modifications: Recommendations for method validation based on CLSI H62 guidelines

Sara A Monaghan et al. Cytometry B Clin Cytom. 2025 May.

Abstract

The Clinical and Laboratory Standards Institute (CLSI) H62-Validation of Assays Performed by Flow Cytometry guideline, released in 2021, provides recommendations for platform workflow and quality system essentials, instrument setup and standardization, assay development and optimization and fit-for-purpose analytical method validation. In addition, CLSI H62 includes some recommendations for the validation strategies after a validated flow cytometric method has been modified. This manuscript builds on those recommendations and discusses the impact of different types of assay modifications on assay performance. Recommendations regarding which validation parameters to evaluate depending on the type of modification are provided. The impact of assay modification on the assay's intended use is discussed. When recommending minor deviations from the CLSI H62 process for a laboratory-initiated assay revision (e.g., specimen numbers for sensitivity, specificity, or precision studies), a rationale based on expert opinion is provided with the understanding that not every laboratory, assay type, and circumstance can be comprehensively addressed in this paper. These recommendations are meant as a practical recommendation and are not intended to be restrictive, prescriptive, or understood as necessarily sufficient to meet every specific requirement from regulatory bodies (e.g., FDA or New York State Department of Health).

Keywords: CLSI; H62; assay modification; flow cytometry; validation.

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References

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