Flow Cytometric Analyses of Lymphocyte Markers in Immune Oncology: A Comprehensive Guidance for Validation Practice According to Laws and Standards

Abstract

Many anticancer therapies such as antibody-based therapies, cellular therapeutics (e.g., genetically modified cells, regulators of cytokine signaling, and signal transduction), and other biologically tailored interventions strongly influence the immune system and require tools for research, diagnosis, and monitoring. In flow cytometry, in vitro diagnostic (IVD) test kits that have been compiled and validated by the manufacturer are not available for all requirements. Laboratories are therefore usually dependent on modifying commercially available assays or, most often, developing them to meet clinical needs. However, both variants must then undergo full validation to fulfill the IVD regulatory requirements. Flow cytometric immunophenotyping is a multiparametric analysis of parameters, some of which have to be repeatedly adjusted; that must be considered when developing specific antibody panels. Careful adjustments of general rules are required to meet legal and regulatory requirements in the analysis of these assays. Here, we describe the relevant regulatory framework for flow cytometry-based assays and describe methods for the introduction of new antibody combinations into routine work including development of performance specifications, validation, and statistical methodology for design and analysis of the experiments. The aim is to increase reliability, efficiency, and auditability after the introduction of in-house-developed flow cytometry assays.

Comparison of Laboratory Methods for the Clinical Follow Up of Checkpoint Blockade Therapies in Leukemia: Current Status and Challenges Ahead

“…Suggested parameters for this approach is presented in Table 3 . Yet, due to the complex nature of flow cytometric analyses, standardized procedures are required for reproducibility ( 61 ).…”

Making the most of high‐dimensional cytometry data

“…Under these conditions, Poisson statistics describe the relationship between the frequency of a population of interest and the number of cells required for a given level of precision of a measurement of that cell population. In turn, this dictates the number of total events from a sample that need to be acquired with larger cell numbers in a population leading to greater ability to determine differences between experimental groups 13–15 . Key parameters influencing power that are under control of the investigator are group sample size and the number of events collected per sample (an important practical note here is to incorporate cell losses during processing, acquisition and preliminary gating).…”

“…In turn, this dictates the number of total events from a sample that need to be acquired with larger cell numbers in a population leading to greater ability to determine differences between experimental groups. 13 , 14 , 15 Key parameters influencing power that are under control of the investigator are group sample size and the number of events collected per sample (an important practical note here is to incorporate cell losses during processing, acquisition and preliminary gating). By contrast, parameters that are outside the control of the investigator, but which should be incorporated when estimating power, include the relative abundance of the rarest population of interest, between‐subject variability of populations (either abundance or staining intensity) and intrapopulation marker staining variability (if testing for effects on marker expression).…”

Detection of Immune Checkpoint Receptors – A Current Challenge in Clinical Flow Cytometry

“…This is a challenge in clinical practice (2). For validation, recent publications give support (3). Reference values are often not known and must be established in-house (4).…”