Lehle Sarah, Völkl Simon, Seitz Katharina, Goossens Chloë, Emons Julius, Ruebner Matthias, Uhrig Sabrina, Ziegler Philipp, Theuser Anna-Katharin, Beckmann Matthias W, Fasching Peter A, Huebner Hanna
Department of Gynecology and Obstetrics, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany.
Comprehensive Cancer Center Erlangen-EMN (CCC ER-EMN), Erlangen, Germany.
BMC Immunol. 2025 Mar 15;26(1):21. doi: 10.1186/s12865-025-00701-y.
Peripheral blood mononuclear cells (PBMCs) are valuable biomarkers, providing crucial insights into the patients' immune system. Reliable biobanking of PBMCs is essential to minimize heterogeneity. In multicenter trials, blood sample transportation to central laboratories can increase the time between blood collection and PBMC isolation. This study evaluated the effect of prolonged blood hold time on PBMC viability and cytotoxicity.
From July 2021 to May 2023, 104 patients with early HER2-positive breast cancer were enrolled in the NeoOn trial, of whom 49 patients were included in this subproject. PBMCs were isolated ≤ 6 hours (h) or ≥ 20 h after blood collection. PBMC yield and viability were determined using the LUNA-II Automated Cell Counter. Flow cytometry was used to quantify in vitro cytotoxicity, the percentage of natural killer (NK) and T cells, as well as apoptotic and necrotic cells.
Isolating PBMCs ≥ 20 h resulted in a higher cell yield, but lower NK cell viability compared to PBMCs ≤ 6 h. PBMCs ≥ 20 h were less robust to thawing and showed higher loss during recovery. Compared to PMBCS ≤ 6 h, PBMCs ≥ 20 h exhibited lower antibody-mediated cytotoxicity (p ≤ 0.0001) and antibody-dependent phagocytosis (p < 0.0051). While the percentage of T and NK cells and the T cell viability remained unaffected by hold time, the percentage of apoptotic NK cells was higher for PBMCs ≥ 20 h (41.0 ± 12.9% vs. 23.8 ± 13.4%; p = 0.0364).
Extended blood storage time caused increased apoptosis and necrosis of NK cells, adversely affecting PBMC quality and reducing NK cell related functionality. Hence, blood hold time should be minimized to maintain PBMC integrity and NK cell functionality for in vitro biomarker assays.
Trial registration number: EudraCT 2020-001943-21. Date of registration: December 29th 2020.
外周血单个核细胞（PBMCs）是有价值的生物标志物，能为了解患者免疫系统提供关键见解。可靠地保存PBMCs对于减少异质性至关重要。在多中心试验中，将血样运输到中心实验室会增加采血与PBMC分离之间的时间。本研究评估了延长血液保存时间对PBMC活力和细胞毒性的影响。
2021年7月至2023年5月，104例早期HER2阳性乳腺癌患者参加了NeoOn试验，其中49例患者被纳入该子项目。在采血后≤6小时（h）或≥20小时分离PBMCs。使用LUNA-II自动细胞计数器测定PBMC产量和活力。采用流式细胞术定量体外细胞毒性、自然杀伤（NK）细胞和T细胞的百分比以及凋亡和坏死细胞。
与≤6小时的PBMCs相比，≥20小时分离PBMCs可获得更高的细胞产量，但NK细胞活力较低。≥20小时的PBMCs对解冻的耐受性较差，复苏过程中的损失更高。与≤6小时的PBMCs相比，≥20小时的PBMCs表现出较低的抗体介导的细胞毒性（p≤0.0001）和抗体依赖性吞噬作用（p<0.0051）。虽然T细胞和NK细胞的百分比以及T细胞活力不受保存时间的影响，但≥20小时的PBMCs中凋亡NK细胞的百分比更高（41.0±12.9%对23.8±13.4%；p=0.0364）。
延长血液储存时间会导致NK细胞凋亡和坏死增加，对PBMC质量产生不利影响，并降低NK细胞相关功能。因此，应尽量缩短血液保存时间，以维持PBMC完整性和NK细胞功能，用于体外生物标志物检测。
试验注册号：EudraCT 2020-001943-21。注册日期：2020年12月29日。
