Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA.
Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA.
Sci Rep. 2022 Mar 7;12(1):3667. doi: 10.1038/s41598-022-07455-2.
Neutrophils are the most abundant circulating leukocyte population with critical roles in immune defense, regulation of innate and adaptive immune systems, and disease pathogenesis. Our progress in understanding precise mechanisms of neutrophil activation, recruitment, and function has been hampered by the lack of optimized and standardized methods for the characterization and phenotyping of this readily activated population. By comparing eight methods of neutrophil characterization, we demonstrate that the level of neutrophil activation and degranulation is associated with specific experimental conditions and the number and type of manipulation steps employed. Staining whole blood at 4 °C and removal of remaining unbound antibodies prior to one-step fixation and red blood cell lysis minimizes neutrophil activation, decreases phenotypic alterations during processing, and prevents nonspecific antibody binding. The effects of anticoagulants used for collection, processing delays, and time and temperature during sample analysis on neutrophil phenotype are addressed. The presented data provide a foundation for higher quality standards of neutrophil characterization improving consistency and reproducibility among studies.
中性粒细胞是循环白细胞中最丰富的群体，在免疫防御、固有和适应性免疫系统的调节以及疾病发病机制中具有关键作用。我们在理解中性粒细胞激活、募集和功能的精确机制方面取得的进展受到缺乏优化和标准化方法的阻碍，这些方法用于中性粒细胞的特征描述和表型分析。通过比较八种中性粒细胞特征描述方法，我们证明中性粒细胞的激活和脱颗粒水平与特定的实验条件以及所采用的操作步骤的数量和类型有关。在 4°C 下对全血进行染色，并在一步固定和红细胞裂解之前去除剩余的未结合抗体，可最大程度减少中性粒细胞的激活，减少处理过程中的表型改变，并防止非特异性抗体结合。还讨论了用于收集的抗凝剂、处理延迟以及样品分析过程中的时间和温度对中性粒细胞表型的影响。所提供的数据为中性粒细胞特征描述提供了更高的质量标准基础，从而提高了研究之间的一致性和可重复性。
