Flow cytometry is an elegant and powerful tool that has been critical to understanding the immune system and advancing the development of immune-based therapies. Critical to many studies, and essential for FDA filings, is the development and documentation of a validated assay. While most flow cytometric assays fall into the “quasi-quantitative” category according to FDA guidelines, there are some assays that can be quantitative and even qualitative. It is critical to know what category your assay falls into in order to determine what is necessary to develop a validated test. While appropriate validation is essential for those collecting data for FDA approval or for clinical use, it is also important at the early research stages. In order to reach the goal of a validated flow cytometry assay, there are several steps you must take:

Step 1: What is Your Intended Use?

The first step in determining a validation plan is to clearly define the intended use of the assay.  By doing this, you will determine what type of data you will collect and how it will be used and that will guide you to the selection of the appropriate category: quasi-quantitative, quantitative, qualitative.

Step 2: Design a Fit-For-Purpose Validation Plan

Once you have defined your intended use and classification of your assay, you then design a Fit-For-Purpose assay validation plan to assure that the assay is specific, accurate, precise, sensitive, and stable. This encompasses all aspects from pre-analytical to instrument standardization to data analytic methods.  Key determinations as to whether you must use fresh cells, or will frozen work; are the antibodies stable independently or in a cocktail, are certain clones more reliable, does your instrument perform the same each day, what is your lower limit of quantitation and lower limit of detection, each criteria will be determined by your Fit-for-Purpose Plan. Flow cytometry contract research organizations are very familiar with these requirements and are leaders in the field of standardization. They are ideal partners for the development of a regulatory-compliant flow cytometry assay.

Step 3: Document, Document, Document!!

The number one thing to remember is that documentation is critical! There is no step or no protocol change that is so small that it should not be documented. When you go the final steps for validation whether it is for the FDA or general clinical use or even publication of a research project, your goal is to provide documentation that you have followed your validation plan and that your assay does indeed do what it was intended to do.

Assay validation can take time and may lead to changes in your protocol. Nonetheless, assay validation helps you work out these speed bumps, and working alongside experts in validation and regulation assure that you check all the right boxes.