Approaches for Performance Verification Toward Standardization of Peripheral Blood Regulatory T-Cell Detection by Flow Cytometry

A wide range of immunologic methods is available for monitoring the immune system to aid in the diagnosis and clinical management of immune-mediated diseases, such as autoimmune diseases, infectious diseases, [...]
* Context.--Regulatory T-cell (Treg) detection in peripheral blood, based on flow cytometry, is invaluable for diagnosis and treatment of immune-mediated diseases. However, there is a lack of reliable methods to verify the performance, which is pivotal toward standardization of the Tregs assay. Objective.--To conduct standardization studies and verify the performance of 3 commercially available reagent sets for the Tregs assay based on flow cytometry and agreement analysis for Treg detection across the different reagent sets. Design.--The analytical performance of Tregs assay using reagent sets supplied by 3 manufacturers was evaluated after establishing the gating strategy and determining the optimal antibody concentration. Postcollection sample stability was evaluated, as well as the repeatability, reproducibility, reportable range, linearity, and assay carryover. Agreement between the different assays was assessed via Bland-Altman plots and linear regression analysis. The relationship between the frequency of [CD4.sup.+][CD25.sup.+][CD127.sup.low/-] Tregs and [CD4.sup.+][CD25.sup.+][Foxp3.sup.+] Tregs was evaluated. Results.--The postcollection sample stability was set at 72 hours after collection at room temperature. The accuracy, repeatability, reproducibility, and accuracy all met the requirements for clinical analysis. Excellent linearity, with [R.sup.2] [greater than or equal to] 0.9 and no assay carryover, was observed. For reportable range, a minimum of 1000 events in the [CD3.sup.+][CD4.sup.+] gate was required for Tregs assay. Moreover, the results for Tregs labeled by antibodies from the 3 manufacturers were in good agreement. The percentage of [CD4.sup.+][CD25.sup.+][CD127.sup.low/-] Tregs was closely correlated with [CD4.sup.+][CD25.sup.+][Foxp3.sup.+] Tregs. Conclusions.--This is the first study to evaluate systematically the measurement performance of Tregs in peripheral blood by flow cytometry, which provides a practical solution to verifying the performance of flow cytometry-based immune monitoring projects in clinical practice. (Arch Pathol Lab Med. 2024;148:1234-1243; doi: 10.5858/arpa.2023-0284-OA)