This is especially problematic for neutrophils since they cannot be cryopreserved for delayed analysis and must be processed fresh. In clinical research, blood collection often occurs at locations distant from the site of analysis, making delays in sample processing unavoidable. This presents a significant obstacle for accurate characterization of neutrophil phenotype and complicates comparison of results among studies, impeding progress in the field. Individual studies utilize specific preparation methods, type of anticoagulant for blood collection, and timeframes for sample processing resulting in reports with varying levels of activation and artifactual changes in phenotype. Currently used methods for neutrophil characterization often rely on combinations of density gradient centrifugation and RBC removal by sedimentation and/or lysis 15, 16, 17, 18, 19, 20, 21. Despite the discrepancies between studies, prior publications have not systematically compared characterization methods to determine approaches minimizing neutrophil activation. Low-frequency subpopulations are typically identified based on the levels of specific surface proteins that may be altered by activation during preparation, making accurate characterization of neutrophil subsets challenging. ![]() Several neutrophil subpopulations have recently been described in homeostatic and pathological conditions based on phenotypic, transcriptional, and functional properties 2, 4, 5, 9, 10, 11, 12, 13, 14. Neutrophils are known to become rapidly activated by many common methods of preparation including red blood cell (RBC) sedimentation 6, RBC lysis 7, and density gradient centrifugation 8. Comprehensive characterization of neutrophils and neutrophil subsets is hampered by their inherent ex vivo instability, their tendency to form multiplets with other cell types, and a high level of nonspecific background staining likely due to the exposure of cationic proteins during activation. Neutrophils constitute 50–70% of circulating leukocytes and are increasingly recognized as a heterogeneous population with critical roles in immune regulation 1, 2, 3 and disease pathogenesis 4, 5. The presented data provide a foundation for higher quality standards of neutrophil characterization improving consistency and reproducibility among studies. The effects of anticoagulants used for collection, processing delays, and time and temperature during sample analysis on neutrophil phenotype are addressed. Staining whole blood at 4 ☌ 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. ![]() 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. 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. Neutrophils are the most abundant circulating leukocyte population with critical roles in immune defense, regulation of innate and adaptive immune systems, and disease pathogenesis.
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