scope:

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.

¹ CLSI. Quality Management System: Development and Management of Laboratory Documents; Approved Guideline-Sixth Edition. CLSI document QMS02-A6. Wayne, PA: Clinical and Laboratory Standards Institute; 2013.