Performance Verification and Establishment of Reference Intervals for Dendritic Cells, γδT Cells, and Follicular Helper T Cells Using Flow Cytometry.

Abstract

Context.—: Dendritic cells (DCs), gamma-delta T (γδT) cells, and T follicular helper (Tfh) cells are key players in immune responses and clinical surveillance. While flow cytometry (FCM) provides precise quantification of these subsets with their subpopulations, its clinical application remains limited, partly due to lack of standardized performance verification and reference intervals.

Objective.—: To facilitate the standardization and clinical application of peripheral DCs, γδT cells, and Tfh cells with their subpopulations, based on FCM.

Design.—: Peripheral whole blood from healthy individuals was collected to measure the frequency of DCs, γδT cells, and Tfh cells with their subpopulations via FCM. Sample stability, reportable range, intra-assay/interassay precision, and linearity were evaluated from best practice documents, followed by establishing reference intervals.

Results.—: For sample stability, samples were stable up to 24 hours post collection at 4°C for DCs with their subpopulations; samples were stable up to 96 hours at both 4°C and room temperature for γδT cells with their subpopulations; samples showed instability after 24 hours at both 4°C and room temperature for Tfh cells with their subpopulations. For the reportable range, the minimum cell numbers of upper gate for target cells that could meet the detection requirements of DCs, γδT cells, and Tfh cells with their subpopulations were 5000, 250, and 1000. Both intra-assay/interassay precision and linearity fulfilled the acceptance criteria. Furthermore, we established the reference intervals for DCs, γδT cells, and Tfh cells with their subpopulations.

Conclusions.—: We conducted systematic performance verification and established reference intervals for DCs, γδT cells, and Tfh cells with their subpopulations via FCM, promoting the standardization and clinical application of these assays.