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Updated: Aug 9, 2026
A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release
Published on: November 21, 2016
Evaluation in vitro of canine neutrophil function
S Comazzi1, S Paltrinieri, V Spagnolo
1Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Sezione di Patologia Generale Veterinaria e Parassitologia, via Celoria 10, 20133 Milano, Italy. stex@mailserver.unimi.it
Abstract:
Polymorphonuclear granulocyte (PMN) phagocytosis may be affected by many pathological changes. A panel of tests requiring relatively small volumes of blood was applied to 16 healthy dogs in order to obtain normal values and to standardize techniques. PMNs were isolated by discontinuous Percoll gradients; chemotaxis was tested in a modified Boyden chamber using the leading front method; fluorescinated yeast uptake was evaluated on a slide and superoxide (SO) production and adherence was carried out on a microtitre plate. The different aspects of phagocytosis showed no correlation with one another. Better results were obtained using a 60 min incubation period using interleukin-8 (25 ng/mL) as an activator for chemotaxis, and incubating plates for 30 min with phorbol myristate acetate (10(-6)mol/L) to assess SO production.
Insights
This study established normal values for canine polymorphonuclear granulocyte (PMN) phagocytosis tests. Standardized methods were developed for assessing PMN function in dogs, crucial for diagnosing various diseases.
Area of Science:
- Veterinary Immunology
- Canine Hematology
- Cellular Immunology
Background:
- Polymorphonuclear granulocyte (PMN) phagocytosis is vital for immune response.
- Pathological changes can significantly impact PMN function.
- Establishing normal canine PMN phagocytosis values is essential for disease diagnosis.
Purpose of the Study:
- To obtain normal values for canine PMN phagocytosis assays.
- To standardize techniques for evaluating PMN function in dogs.
- To assess various aspects of phagocytosis, including chemotaxis, yeast uptake, superoxide production, and adherence.
Main Methods:
- Isolation of PMNs using discontinuous Percoll gradients.
- Chemotaxis assessed via modified Boyden chamber (leading front method).
- Fluorescinated yeast uptake evaluated on a slide.
- Superoxide (SO) production and adherence measured using microtitre plates.
- Optimized incubation periods and activators (interleukin-8 for chemotaxis, phorbol myristate acetate for SO production) were determined.
Main Results:
- Normal values for canine PMN phagocytosis assays were established.
- Standardized protocols for PMN isolation and functional testing were developed.
- No significant correlation was found between different phagocytic functions tested.
- Optimized incubation times (60 min for chemotaxis, 30 min for SO production) yielded better results.
Conclusions:
- The study successfully standardized and obtained normal values for key canine PMN phagocytosis assays.
- These standardized methods and values are crucial for accurate interpretation of PMN function in clinical veterinary diagnostics.
- The lack of correlation between different phagocytic aspects suggests they measure distinct functional capabilities.
