Graduate Student Literature Review: Standardized approaches for neutrophil functional assessment in dairy cattle—A practical guide

Polymorphonuclear leukocytes (neutrophils; PMN) serve as critical indicators of innate immune function in dairy cattle. Accurate assessment of their functional capacities is essential for understanding the status of the innate immune system in health and disease; or following immunomodulatory treatment. However, the lack of standardized protocols for evaluating PMN functional capacity in dairy cattle has led to inconsistencies in research methodologies and hindered cross-laboratory comparisons. To address this issue, we propose systematic, evidence-based methodologies for assessing PMN function in dairy cattle. It begins with establishing standardized protocols for blood sample collection, processing, storage, PMN isolation and culture: protocols specifically designed to ensure PMN viability and generate reproducible outcomes across different laboratory settings. Building upon this foundation, we describe existing laboratory methodologies for assessing the 4 cardinal PMN functions: chemotaxis, phagocytosis, oxidative burst, and extracellular trap formation. We then elaborate on the advantages, limitations, and practical considerations of each approach by comparing natural PMN behavior in vivo with the corresponding in vitro assay. By establishing standardized methodological frameworks, this review aims to facilitate meaningful cross-laboratory comparisons and ensure that PMN functional measurements are accurate and reproducible, thereby allowing them to properly reflect the innate immune status across diverse experimental and laboratory settings.