Validation of cell‐based fluorescence assays: Practice guidelines from the ICSH and ICCS – part V – assay performance criteria

Abstract

(Table 1). Definitive quantitative assays include calibrators fit to a regression model to calculate absolute values and reference standards that are well characterized and fully representative of the endogenous measurand. Definitive quantitative assays can be both accurate and precise. Relative quantitative assays utilize responseconcentration calibration, however in this scenario the reference standards are not fully characterized or truly representative of the endogenous measurand. As such, imprecision can be demonstrated for a relative quantitative method, but accuracy can only be estimated. With quasi-quantitative assays there is a relationship between the response and the measurand but calibration standards are not used. Thus, quasi-quantitative methods can be validated for imprecision, but not accuracy. Qualitative methods generate categorical data. Flow cytometric methods largely fall in the two latter categories and are essentially therefore quasi-quantitative or qualitative.Multi-color flow cytometry is a unique technology, which enables the analysis of heterogeneous cellular systems and provides multiparametric information at a cellby-cell level. The strength of flow cytometry lies not only in the ability to simultaneously measure multiple parameters, but also in the flexibility to report them in different ways. The appropriate data output depends on the biology of the system being investigated, the analytical or scientific question being asked, and the intended use of the results. A wide variety of data outputs can be reported usually expressed in terms of several characteristics of cells, or cell subsets, in the sample tested for example, percentage of positive events, absolute counts, median fluorescence intensity, quantitative antigen expression levels, ratiometric indices, markers coexpression, or relative nucleic acid content.

Consensus guidelines for myeloma minimal residual disease sample staining and data acquisition

“…Specific antibodies must be used in fully validated combinations since the information they provide when measured simultaneously in such combinations is clearly superior than when they are used in other less informative antibody combinations, even when only the fluorochrome positions are modified (1,2). Panel design is a key but difficult process and results of a given antibody-fluorochrome reagent combination must be objectively validated in sequential and numerous rounds of testing using approaches such as those previously described (1,(3)(4)(5)(6). Also, distinct sample preparation protocols may impact differently on the performance of combinations of different antibodies and fluorochrome conjugates (4,7,8).…”

“…Panel design is a key but difficult process and results of a given antibody-fluorochrome reagent combination must be objectively validated in sequential and numerous rounds of testing using approaches such as those previously described (1,(3)(4)(5)(6). Also, distinct sample preparation protocols may impact differently on the performance of combinations of different antibodies and fluorochrome conjugates (4,7,8). Consequently, the panels should be evaluated with the predicated sample preparation technique they will be used with.…”

Validation of a flow cytometry‐based assay to assess C5aR receptor occupancy on neutrophils and monocytes for use in drug development

“…Formal well-defined guidance for the validation of flow cytometric assays is not currently available; however, various advisory documents can serve as practical guidelines (16)(17)(18)22).…”

“…Validation parameters were evaluated using fit-forpurpose acceptance criteria (15)(16)(17)(18). Twenty-four reportable results were evaluated in total and are depicted in Table 2; frequency (%), MFI, and MESF values were obtained for CD451CD161 neutrophils and CD451 CD141 monocytes.…”

Recommendations for the development and validation of flow cytometry‐based receptor occupancy assays

“…Similar to all methods utilized in drug development, validation plans for RO must consider the intended use of the data and follow a fit-for-purpose approach specific to flow cytometric methods (37)(38)(39). By utilizing an iterative approach, the validation activities can build upon one another in order to provide comprehensive evidence of the integrity of the data as the assay follows the compound throughout the drug development lifecycle.…”