Division of Infectious Diseases, Department of Medicine Solna, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden; CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.
Division of Immunology and Allergy, Department of Medicine Solna, Center for Molecular Medicine, Karolinska Institutet, 171 64 Stockholm, Sweden.
J Immunol Methods. 2020 Jun-Jul;481-482:112792. doi: 10.1016/j.jim.2020.112792. Epub 2020 May 6.
Whole-blood fixation provides a rapid and simplified method for cell preservation compared to isolation of peripheral blood mononuclear cells (PBMCs). This can be especially important for sample acquisition and storage in resource-limited settings. However, some caveats have been reported, such as reduced cell marker recognition. Here, we evaluated the whole-blood proteomic stabilizer PROT1 and compared recognition of 53 common cell markers in fixed buffy coats and cryopreserved PBMCs isolated from the same donor. Several antibodies completely lost their binding to the cells, while others presented with partial loss of marker recognition or no effect at all. Based on the screened antibodies, we designed two antibody panels allowing phenotyping of B cells, monocytes, and dendritic cells and also T cells and NK cells in both fixed and non-fixed material. Taken together, our observations suggest that antibodies intended to be used with fixed blood first need to be evaluated for marker recognition and staining intensity, in comparison with fresh samples or cryopreserved PBMCs.
与分离外周血单核细胞（PBMCs）相比，全血固定提供了一种快速简化的细胞保存方法。在资源有限的环境中，这对于样本采集和存储尤为重要。然而，据报道，全血固定存在一些注意事项，例如减少细胞标志物识别。在这里，我们评估了全血蛋白稳定剂 PROT1，并比较了从同一供体分离的固定血涂片和冷冻保存的 PBMCs 中 53 种常见细胞标志物的识别情况。一些抗体完全失去了与细胞的结合能力，而其他抗体则表现出部分标志物识别丧失或完全没有影响。基于筛选出的抗体，我们设计了两个抗体面板，允许对固定和非固定材料中的 B 细胞、单核细胞和树突状细胞以及 T 细胞和 NK 细胞进行表型分析。综上所述，我们的观察结果表明，与新鲜样本或冷冻保存的 PBMCs 相比，用于固定血液的抗体首先需要评估其标志物识别和染色强度。
