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Background Clinical trials rely on high-dimensional immune profiling to evaluate treatment responses. However, inconsistencies in blood collection, shipping, and processing lead to a loss of roughly 10-15% of samples. An estimated 80% of samples are processed live, but blood degradation during transit under variable conditions leads to a 49% reduction in cell count, with uneven loss across major immune populations illustrating the need for reliable preservation methods. Teiko’s current two-step preservation kit (TokuKit) has demonstrated strong performance in clinical workflows with R > 0.97 when compared to live sample processing. Here, we aimed to compare one-step stabilization methods against TokuKit to explore opportunities for streamlining immune monitoring logistics while maintaining sample quality.Methods Whole blood was collected from three healthy donors in sodium heparin vacutainers and split across several preservation conditions: live processing, TokuKit fixation, and stabilization in CytoChex® or Transfix® tubes. Samples were subjected to variable lysis methods, temperature conditions (4°C, RT, 37°C), and storage durations (Days 1, 3, and 7). All samples were stained with a 41-marker in-house mass cytometry panel and acquired on a Helios® mass cytometer. Live samples were RBC-lysed using ACK buffer prior to staining. Fixed samples were cryopreserved and processed post-thaw following standard workflows.Results Transfix demonstrated ~8% better cell preservation than CytoChex, but performed 15% worse than TokuKit. Moreover preservation with CytoChex significantly affected B cell and granulocyte preservation with ~70% and ~48% reductions compared to TokuKit, respectively. Freeze/thaw recovery was highest in TokuKit samples, followed by Transfix and Cytochex. Antibody signal correlation to live-processed controls was highest with R > 0.93 in Transfix samples stored at 4°C and lysed with ACK on Days 1–3. Several markers, including Ki-67, T-bet, CD16, and CCR7, showed variable staining under Transfix and Cytochex fixed conditions.Conclusions Transfix enables short-term stabilization (≤3 days at 4°C) but is not optimal for storage longer than 3 days. CytoChex underperforms in preserving key immune cell populations. TokuKit remains the best option, preserving surface, intracellular, granulocyte, and B cell markers across long-term storage conditions. If storage beyond three days is required, TokuKit is currently the most reliable solution. This study highlights the need for further optimization of one-step preservation strategies. Until then, TokuKit offers the strongest performance for immune monitoring in multicenter clinical trials requiring delayed processing or centralized analysis.