Optimization of methods for the accurate characterization of whole blood neutrophils

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.