Peripheral Blood Lymphocyte-Gated Flow Cytometry Parameters and 24-Month Mortality in COPD: An Exploratory Cohort...

Background: Chronic obstructive pulmonary disease (COPD) is associated with substantial long-term morbidity and mortality. Peripheral blood flow cytometry may provide exploratory information regarding immune-cell distributions and activation-related markers. However, careful interpretation is required when flow cytometry outputs are derived from lymphocyte-gated percentages rather than marker-specific mean fluorescence intensity or sequential lineage-confirmed gating. We investigated whether specific lymphocyte-gated flow cytometry parameters are associated with mortality during follow-up in COPD patients. Methods: In this single-center observational cohort study, 51 consecutive clinically stable outpatients with COPD were enrolled in November 2023 and followed for 24 months. Baseline peripheral blood flow cytometry results were verified against archived original instrument reports. The principal exploratory flow cytometry-derived variables were CD45/SSC-defined lymphocyte-gate percentage and lymphocyte-gated CD138+ events; HLA-DR positivity was evaluated as a secondary exploratory variable. Group comparisons and descriptive receiver operating characteristic (ROC) analyses were performed. Multiplicity was assessed using a hierarchical Benjamini-Hochberg false discovery rate (FDR) framework that separated the two biologically prioritized principal variables from the remaining exploratory screening variables. For transparency, a more conservative pooled FDR correction across all ten flow cytometry-derived variables was also reported. A two-variable analysis was performed only as exploratory signal aggregation, with descriptive internal assessment using leave-one-out cross-validation (LOO-CV) and bootstrap optimism correction. Results: During the 24-month follow-up, 13 of 51 patients died (25.5%). In unadjusted analyses, non-survivors had lower arterial oxygen tension and nominally lower CD45/SSC-defined lymphocyte-gate percentages (median 13.08% vs. 22.63%, p = 0.008) and lymphocyte-gated CD138+ event percentages (median 0.07% vs. 0.39%, p = 0.026) than survivors. Within the hierarchical analytical-family framework, both CD45/SSC-defined lymphocyte-gate percentage and lymphocyte-gated CD138+ events retained significance in the principal-variable family (within-family q = 0.016 and 0.026), whereas no secondary-family parameter, including HLA-DR (within-family q = 0.50), did; significance was not retained under a single correction across all ten parameters (CD45 q = 0.081; CD138 q = 0.129). Descriptive AUCs were 0.749 for CD45/SSC-defined lymphocyte-gate percentage and 0.710 for CD138+ events. The two-variable signal-aggregation analysis yielded an apparent AUC of 0.858, an LOO-CV AUC of 0.796, and a bootstrap optimism-corrected AUC of 0.832. NLR was available for all 51 patients; NLR-adjusted analyses did not establish clinical incremental utility. Conclusions: Lower CD45/SSC-defined lymphocyte-gate percentage and lower lymphocyte-gated CD138+ event percentage showed within-cohort associations with 24-month mortality in this small COPD cohort. These observations should be regarded solely as hypothesis-generating signals. Neither principal finding was retained after pooled correction across all ten flow cytometry-derived parameters, and no incremental prognostic value beyond routine inflammatory indices or established clinical predictors was demonstrated. External validation was absent; prospective replication in larger, appropriately adjusted cohorts is required.