Clinical and Laboratory Standards Institute document I/LA26-A2—Performance of Single Cell Immune Response Assays; Approved Guideline—Second Edition describes assays that measure antigen-specific cellular immune responses in the context of clinical trials and in the management of subjects with immune-mediated diseases. Immune therapeutic approaches are being applied in various fields of medicine, including infectious diseases, transplantation, autoimmune disease, cancer, and allergies. Assays are required to measure the cellular effects of such therapeutic approaches. This guideline focuses on the methods of intracellular cytokine evaluation, major histocompatibility complex multimer quantitation, enzyme-linked immunospot technology, and carboxyfluorescein succinimidyl ester tracking dye staining. The document covers basic aspects of specimen collection, transport, and preparation, in addition to QA and method validation approaches. Data acquisition, data analysis, and reporting aspects for these assays are also summarized.

The second edition of I/LA26 includes the following changes that have been made since the first approved edition: 

• The references were revised to include recent experience with each of the assays in terms of their new applications, inclusions in clinical trials, and assay optimizations. 

– For the flow cytometry–based assays, new and more complex gating algorithms (eg, doublet removal, inclusion, exclusion gating) are described, which serve to increase the signal-to-noise ratio and improve the sensitivity and precision of detecting rare events. New figures illustrating the improved gating algorithms are included. 

– Information related to newly available methods of assessing proficiency is included. 

– The features and impact of improved and harmonized ELISPOT assays are reflected, along with more detailed information regarding troubleshooting, and figures illustrating common problems. 

• The specimen handling guidelines were revised according to Centers for Disease Control and Prevention recommendations. 

• An entirely new section for assessment of antigen-specific proliferation using the tracking dye CFSE was added. 

• Additional information was added on pentamers and Dextramers® (or the equivalent), in addition to new information on multimer products in general. 

• Information was added on new cell preparation tubes, which contain a premade gel barrier for density gradient separation for the isolation of peripheral blood mononuclear cells with a single centrifugation step. 

• Modifications were made to the intracellular cytokine staining section (formerly cytokine flow cytometry), which include polychromatic flow and more versatility in the preparation and storage of samples that reflects the experience with the assays since the last version of this document. These changes are accompanied by new figures. 

• Information was added on new fixable stable viability marker dyes compatible with intracellular staining protocols. 

• A new appendix (see Appendix B) was added that describes the statistics of rare-event analysis. 

• The terminology was revised to reflect current practice.

This document provides guidance for the performance of single cell immune response assays within the clinical context of infectious diseases (especially HIV), cancer, transplantation, autoimmune disease, and allergies. This guideline focuses on antigen-specific functional assays within CD4 and CD8 T-cell subsets in response to the recognition that markers of immune competency are increasingly required in clinical trials and for the approval of new immune-based therapies by regulatory agencies. The assays in this document include antigen-stimulated intracellular cytokine production measured by flow cytometry, the quantification of antigen-specific CD4 and CD8 T-cells using major histocompatibility complex (MHC) multimers and flow cytometry, antigen-specific cell quantification using the enzyme-linked immunospot (ELISPOT) assay, and lastly the flow cytometric assessment of changes in the level of fluorescence of carboxyfluorescein succinimidyl ester (CFSE)–stained cells as a measure of antigen-induced lymphocyte proliferation. The document covers details of the procedure and data interpretation as well as issues such as specimen collection, transport, sample preparation, QC, test validation, and troubleshooting. 

The guideline provides laboratorians with methods for clinical research application in the growing field of immune-based therapy, as well as guidance to pharmaceutical manufacturers in the laboratory evaluation of new products before submission to regulatory agencies. It is also a valuable resource for academic investigators developing these assays for the evaluation of antigen-specific responses in their own research and for coordinating the improved implementation and assessment of these assays within and between laboratories participating in multicenter/multinational clinical trials. Overall, this guideline establishes consensus methods for a rapidly evolving field of single cell immune functional assays. 

Clinical applications of single cell response assays have not been approved by the US Food and Drug Administration (FDA) to date. 

This guideline: 

• Is not intended to be used “as is” for clinical use by diagnostic laboratories; nor is it intended to be a clinical diagnostic procedure manual. It is not intended to be formatted according to CLSI document QMS021 for writing clinical laboratory procedures for adoption by diagnostic laboratories. 

• Is designed to address the general procedures and those particular components involved in each of the four procedures that have been observed to be important in their successful application and interpretation, and is not intended to provide detailed step-by-step instructions for any specific stimuli or for specific lymphocyte subsets. However, these limitations do not preclude its use as a guide in the development of future clinical laboratory procedures. 

• Does not address any specific application within any specific patient population.