Division of Pediatric Cardiology, University of Alberta, Edmonton, Alberta, Canada; Canadian Donation and Transplant Research Program, Edmonton, Alberta, Canada.
Canadian Donation and Transplant Research Program, Edmonton, Alberta, Canada; Division of Pediatric Nephrology, University of British Columbia, Vancouver, Canada.
Hum Immunol. 2024 Sep;85(5):110837. doi: 10.1016/j.humimm.2024.110837. Epub 2024 Jul 15.
Flow-cytometric immune phenotyping is influenced by cryopreservation and inter-laboratory variability limiting comparability in multicenter studies. We assessed a system of optimized, pre-mixed dry-antibody panel tubes requiring small amounts of whole blood for validity, reliability and challenges in a Canadian multicenter study (POSITIVE) with long-distance sample shipping, using standardized protocols. Thirty-seven children awaiting solid-organ transplant were enrolled for parallel immune-phenotyping with both validated, optimized in-house panels and the dry-antibody system. Samples were collected before, 3 and 12 months post-transplant. Quality-assurance measures and congruence of phenotypes were compared using Bland-Altman comparisons, linear regression and group comparisons. Samples showed excellent lymphocyte viability (mean 94.8 %) and recovery when processed within 30 h. Comparing staining methods, significant correlations (Spearman correlation coefficient >0.6, p < 0.05), mean difference <5 % and variation 2SD <25 % were found for natural-killer, T and B cells, including many immunologically important cell subsets (CD8+, naïve, memory CD4+ T; switched-memory, transitional B). Some subgroups (plasmablasts, CD1d+CD5hi B cells) showed weak correlations, limiting interpretation reliability. The dry-antibody system provides a reliable method for standardized analysis of many immune phenotypes after long-distance shipping when processed within 30 h, rendering the system attractive for pediatric studies due to small blood amounts required and highly standardized processing and analysis.
流式细胞免疫表型分析受到冷冻保存和实验室间变异性的影响，限制了多中心研究的可比性。我们评估了一种优化的、预混合的干抗体面板管系统，该系统需要少量全血，在一项具有长途样本运输的加拿大多中心研究（POSITIVE）中具有有效性、可靠性和挑战，使用标准化方案。37 名等待实体器官移植的儿童同时进行了经过验证的优化内部面板和干抗体系统的并行免疫表型分析。在移植前、移植后 3 个月和 12 个月采集样本。使用 Bland-Altman 比较、线性回归和组间比较比较了质量保证措施和表型的一致性。在 30 小时内处理时，样本显示出优异的淋巴细胞活力（平均 94.8%）和恢复。比较染色方法时，对于自然杀伤细胞、T 细胞和 B 细胞，发现了具有显著相关性（Spearman 相关系数>0.6，p<0.05）、平均差异<5%和变异 2SD<25%的方法，包括许多免疫上重要的细胞亚群（CD8+、幼稚、记忆 CD4+T；记忆 B 细胞、过渡 B 细胞）。一些亚组（浆母细胞、CD1d+CD5hi B 细胞）显示出较弱的相关性，限制了解释的可靠性。当在 30 小时内处理时，干抗体系统为经过长途运输后的许多免疫表型提供了一种可靠的标准化分析方法，由于所需的血量少且高度标准化的处理和分析，该系统对儿科研究具有吸引力。
