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.

Keywords: accreditation; flow cytometry; laboratory diagnostics; procedures; quality control; validation.

Figure 1

Demonstration of a statistically proof using confidence intervals (A). When this problem is formulated as a statistical test, it refers to the two 1-sided test approach (TOST) (B).

Figure 2

Result of 1,000 simulation of results of repeatability experiment when 3, 5, 10, 20, and 50 replicates are used, with mean=10 and standard deviation =2, shown as dot-plots with overlying Box-whisker plots.

Figure 3

Presentation of the structure proposed for the accreditation documents. A generic form is to record and report all common information (including environment, material, management, manpower) and method characteristics that cannot be tested for each panel. Then specific forms should be written individually per panel (several parameters, several assays). Technical details (antibodies, clones, conjugates, gating strategy, risks of error, and guidelines for interpretation) should be presented in an easy-to-update SOP. Results with technical and reference information should be managed by the laboratory informatics system to be published for correct interpretation. Any redundancy should be avoided for safety and management reasons.