Going With the Flow (Cytometry): New Guidance From Experts

This first edition document addresses everything from instrumentation to data reporting

Last year, the Clinical and Laboratory Standards Institute (CLSI) published a new guidance document dedicated to the validation of flow cytometric methods (CLSI H62 - Validation of Assays Performed by Flow Cytometry). This document brings us closer than ever to official guidance for flow cytometry. The target audience for CLSI H62 is very broad and includes laboratories conducting academic research, clinical laboratories, the biopharmaceutical sector, instrument and reagent manufacturers, and regulatory agencies. The driving force behind creating CLSI H62 was flow cytometrists from the American Association of Pharmaceutical Scientists (AAPS) and the International Clinical Cytometry Society (ICCS) who recognized both the need for guidance on the subject and the requirement that such guidance should be written by experts in the field. The 37-member document development committee (DDC) included at least two representatives from each of three constituencies (professional, industry and government), as required by CLSI. The diverse committee included many members of the drug development industry, adding their perspective on this crucial process.

CLSI H62 is a comprehensive document addressing all aspects of conducting flow cytometry, including: instrument characterization, standardization, and monitoring; assay development and validation; and data review and reporting.

The document includes detailed discussions addressing the unique validation requirements for flow cytometric methods and how specific validation parameters can be addressed in flow cytometry. It presents a variety of validation strategies based on the context-of-use. Given that every validation study is unique, these suggested minimum validation plans will not cover every possible application, but they represent a reasonable starting place for designing and conducting an analytical method validation and will be of value to the flow cytometry community. In addition, the document addresses the specific aspects of quality management systems, quality assurance, and quality control processes unique to flow cytometry.

Why does flow cytometry require dedicated validation guidelines?

There are many reasons why the validation guidelines specific to other technologies are not fully applicable to flow cytometry. These include the complexity of live cellular measurands, the differences that can exist between the cellular populations in healthy compared with disease-state samples, and the lack of true reference materials. The fact that the data, although quantitative, are not derived from a calibration curve and the fact that the instruments themselves are highly configurable contribute to the unique challenges associated with the method validation and quality management systems for flow cytometric methods. An additional contributing factor is that several software packages are usually used in generating the data for each assay.

What is the relevance of CLSI H62 to Drug Discovery and Development?

The role of flow cytometry in drug discovery and development has been steadily increasing for many years. This trajectory began in the early 2000’s when the industry shifted to a translational science approach by incorporating biomarkers into the drug development process, and accelerated dramatically with the advent of immunotherapies. The success of cell therapies then resulted in a further increase in not only the application of flow cytometry but also the importance of the technology as flow cytometry was also being used even in the manufacturing stage. Most recently, flow cytometry has become an essential tool in evaluating new therapies and vaccines for SARS-CoV2.

Along with the expanded application of flow cytometry and increased importance of the data comes the need to generate robust results. It is widely recognized that the standardization of laboratory practices, robust assay development, and stringent performance assessments are critical elements in generating the type of high-quality, combinable data required in drug development.

Are CLSI documents relevant to drug discovery and development?

Yes! CLSI documents are applicable to a wide variety of settings outside of traditional clinical testing laboratories. In addition, CLSI is accredited by the American National Standards Institute (ANSI) and aligned with the International Organization for Standardization (ISO). The preparation of CLSI consensus documents follows an ANSI compliant review process. Moreover, the FDA often recognizes CLSI guidelines and participates in their development.

Virginia Litwin, Ph.D. is a Research Scientist in Immunology at Charles River Laboratories. Virginia was also the chair of the Document Development Committee for the new CLSI Guideline, H62- Validation of Assays Performed by Flow Cytometry. She was a councilor for both the International Society for the Advancement of Cytometry (ISAC) and the International Clinical Cytometry Society (ICCS). In addition, she serves on the ICCS Advocacy Committee whose mission is to interface with regulatory agencies.