Recovery efficiency and functional characterization of T cells and NK cells from leukocyte filters.

Objective: This study aimed to investigate the recovery efficiency and functional characteristics of lymphocytes isolated from leukocyte filters, compared with those isolated directly from whole blood sample, and to assess their potential as an alternative immune cell source for therapeutic applications. Methods: From December 2023 to 2024, leukocyte filters and 5 mL whole blood samples were collected from healthy donors. Cell recovery was optimized by testing different flushing methods (forward/reverse), solutions (1 × PBS/NS), and volumes (20 - 80 mL). Peripheral blood mononuclear cells (PBMCs) were isolated via density gradient centrifugation, and lymphocyte subsets (CD3+CD56+ NK, CD3+CD4+ T, CD3+CD8+ T cells) were analyzed by flow cytometry. Transcriptome sequencing was conducted to evaluate functional differences. Results: Reverse flushing with 40 mL normal saline (NS) achieved cost-effective lymphocyte recovery, with cell counts correlating with flushing method and volume. The proportion of CD3+CD4+ T cells in 40 mL NS was higher than that in whole blood samples (P < 0.05), while CD3+CD8+ T cells was lower (P < 0.01). CD3+CD4+ T cells exhibited the highest recovery efficiency, and CD3+CD8+ T cells the lowest. Transcriptome analysis identified 634 differentially expressed genes, however, GO/KEGG enrichment analysis revealed no significant pathways related to NK or T cell functions (P-adjust > 0.05). Conclusion: Reverse flushing of leukocyte filters with 40 mL NS enables cost-effective recovery of lymphocytes, particularly CD3+CD4+ T cells. The recovered NK and T cells exhibit functional equivalence to those from those from whole blood samples, offering a low-cost and sustainable cell source for allogeneic cell therapy.