Despite the growing use of flow cytometry, there are currently no official regulatory guidance documents governing its validation. Having recognized the gap, stakeholders from the pharmaceutical industry and clinical testing laboratories have proactively published recommendations.
Scientists helping scientists with guidelines
In 2005, biomarker research was gaining momentum but the lack of clear validation guidelines made biomarker data difficult to interpret, hampering its usage. Existing validation paradigms applied only to PK data. Scientists from the American Association of Pharmaceutical Scientists (AAPS) realized that one set of rules could not fit all and that new standards were needed. They issued Fit-for-Purpose papers, addressing the need for accuracy, compliance and fitness for intended use and introducing the concept of iterative method validation to track biomarker development phases.
In 2011, members of the AAPS Action Program Committee (APC) published recommendation papers on instrument and method validation, specifically for flow cytometry in drug development. These highlighted the challenges unique to flow cytometers. The same year, experts from two separate organizations joined forces and in 2013 published draft guidelines in the Journal of Clinical Cytometry for validating flow cytometry assays in clinical labs. These were submitted to the FDA for consideration as official guidance. The FDA subsequently expressed interest in LDTs and organized a workshop, covering standards for CLL and flow cytometry standardization and focusing on instruments and validation among other topics.
In 2014, the International Society for Advancement of Cytometry (ISAC) also held a workshop on understanding analytical method validation as applied to cell cytometry. The FDA later distributed Flow Cytometry Devices Draft Guidance but subsequently withdrew it.
However, those earlier recommendations provide a robust starting point for any lab intent on standardizing policies and performing productive validation.
Rigorous flow instrument validation delivers reliable results
As assay validation should be conducted only on a validated instrument. Not only does it ensure the reliability and precision of all data generated throughout the life cycle of the flow assays, it is also mandatory for regulated labs and strongly recommended for non-regulated labs.
After determining the instrument's intended use and compliance requirements to define the validation scope, it is recommended that you assemble a cross-functional team comprised of a system owner, quality assurance and IT personnel. Their role is to develop and execute an instrument validation plan outlining deliverables and acceptance criteria.
Installation Qualification (IQ) and Operational Qualification (OQ) are designed to demonstrate that the instrument and associated software are installed and functioning per manufacturer's specifications and user's requirements. Vendors frequently provide such packages and can perform the testing to save qualified specialists' time. IQ testing might include environment and utilities. OQ would cover software functionality, optical precision or sample acquisition.
Performance Qualification (PQ) takes up the lion's share of validation and is usually executed by internal qualified staff. Again, many vendors provide integrated packages but expert users determine optimum settings and track instrument performance. Reports typically cover optical alignment, sensitivity, and linearity, for generating quantitative data.
Extended performance qualification should also be carried out during inter-instrument, inter-lab and longitudinal studies, to minimize variability and demonstrate comparability.
Robust method validation maximizes cutting edge flow potential
Using flow cytometry results for diagnostic or treatment purposes in regulated labs is different from using custom, cutting edge flow for more exploratory endpoints and parmacodynamic information in non-regulated labs. However, regardless of usage, designing and developing robust and specific assays remains paramount. Planning is critical and many parameters govern method validation as applied to flow cytometry.
Method development revolves around selecting reagents, the key determinants of assay specificity. You can buy commercial antibodies but, if you resort to custom reagents, you should ensure adequate quality control. Do not attempt to save money on reagents. It would simply compromise specificity and ultimately jeopardize the quality of your assay data.
Having selected reagents, how do you prove assay specificity? A number of options exist, including in vitro drug treatment experiments; using blocking reagents; isolating subpopulations of interest to verify markers, and using disease state samples.
Stability and Logistics
Early on, consider sample stability and logistics issues: where is the sample collected? How much can be collected? Will it need to be split for several assays? Will the assays be performed on-site, or in a central lab, the recommended option for greater consistency? In the latter case, logistics come into play for preserving the integrity of your sample. Following these expert validation recommendations can help harness the power of cutting edge flow and deliver precise, compliant results.
Conducting such a thorough validation of flow cytometry assays might appear time-consuming and requiring highly technical expertise. However, given the potential rewards flowing from delivering such rich data toward the development of novel therapies for patients in need, it is worth the investment. Official guidance may remain ambiguous but it is evolving. By pressing for greater clarity and offering expert recommendations, scientists can maximize cutting edge flow cytometry for the benefit of all users.
Linsen Du, PhD. Dr. Du received her PhD in Biological Sciences from National University of Singapore and specialized in using flow cytometry technology for supporting biomedical research and clinical trials. She has extensive experience in cytometry platform management and assay development. She works with pharmaceutical companies to design and implement custom flow assays in drug development.