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A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

Iowa State University

Low albumin to globulin ratio has been found previously to have a high positive predictive value for feline infectious peritonitis (FIP) in cats with clinical signs highly suggestive of the disease. However, FIP can have a more vague clinical presentation. This retrospective study found that the positive predictive value of an albumin:globulin (A:G) ratio of <0.8 and <0.6 was only 12.5% and 25%, respectively, in a group of 100 cats with one or more clinical signs consistent with FIP. The negative predictive value was 100% and 99% for an A:G ratio of <0.8 and A:G<0.6%, respectively. Therefore, when the prevalence of FIP is low, the A:G ratio is useful to rule out FIP but is not helpful in making a positive diagnosis of FIP.

Positive predictive value of albumin: globulin ratio for feline infectious peritonitis in a mid-western referral hospital population.

Science and practice.

Neutrophil extracellular traps (NETs) are webs of DNA and protein with both anti-microbial and pro-thrombotic properties which have not been previously reported in dogs. To confirm dog neutrophils can form NETs, neutrophils were isolated from healthy dogs, and stimulated in vitro with 2μM, 8μM, 31μM, and 125μM platelet activating factor (PAF) or 0.03μM, 0.1μM, 0.4μM, 1.6μM and 6.4μM phorbol-12-myristate-13-acetate (PMA). Extracellular DNA was measured using the cell impermeable dye Sytox Green every hour for 4h. At 4h, extracellular DNA was significantly greater than non-stimulated cells at concentrations ≥31μM and ≥0.1μM for PAF and PMA, respectively. Cells stimulated with 31.25μM PAF reached maximal fluorescence by 1h, whereas maximal fluorescence was not achieved until 2h for cells stimulated with 0.1μM PMA. Immunofluorescent imaging using DAPI and anti-elastase antibody confirmed that extracellular DNA is released as NETs. As NETs have been implicated in thrombosis, nucleosomes, a marker correlated with NET formation, were measured in the serum of dogs with the thrombotic disorder primary immune-mediated hemolytic anemia (IMHA) (n=7) and healthy controls (n=20) using a commercially available ELISA. NETs were significantly higher in IMHA cases than controls (median 0.12 and 0.90, respectively, p=0.01), but there were large positive interferences associated with hemolysis and icterus. In summary, the study is the first to describe NET generation by canine neutrophils and provides preliminary evidence that a marker associated with NETs is elevated in IMHA. However, this apparent elevation must be interpreted with caution due to the effect of interference, emphasizing the need for a more specific and robust assay for NETs in clinical samples.

Dogs cast NETs too: Canine neutrophil extracellular traps in health and immune-mediated hemolytic anemia.

Thrombosis is a major cause of mortality and morbidity in humans and dogs; however, anti-thrombotic drugs carry a risk of bleeding and increase the cost of patient care. The ability to identify individuals at high risk of thrombosis would allow targeting of anti-coagulant therapy at those most likely to derive a net benefit. Significant advances have been made towards predicting thrombotic risk in humans using laboratory tests individually and as part of risk prediction models. Assays that have shown potential in humans include D-dimers, activated partial thromboplastin time and viscoelastic testing, all of which are available to veterinarians. This review discusses the extent to which these assays are likely to predict thrombosis in dogs, and introduces new research techniques which may have future clinical value.

Using the laboratory to predict thrombosis in dogs: An achievable goal?

OBJECTIVE To evaluate accuracy and reliability of 3 novel goniometers for measurement of canine stifle joint angles and compare the results with those obtained with a universal goniometer (UG). SAMPLE 8 pelvic limbs from 4 canine cadavers. PROCEDURES Each limb was secured to a wooden platform at 3 arbitrarily selected fixed stifle joint angles. Goniometry was performed with 2 smartphone-based applications (novel goniometers A and B), a digital goniometer (novel goniometer C), and a UG; 3 evaluators performed measurements in triplicate for each angle with each device. Results were compared with stifle joint angle measurements on radiographs (used as a gold standard). Accuracy was determined by calculation of bias and total error, coefficients of variation were calculated to estimate reliability, and strength of linear association between radiographic and goniometer measurements was assessed by calculation of correlation coefficients. RESULTS Mean coefficient of variation was lowest for the UG (4.88%), followed by novel goniometers B (7.37%), A (7.57%), and C (12.71%). Correlation with radiographic measurements was highest for the UG (r = 0.97), followed by novel goniometers B (0.93), A (0.90), and C (0.78). Constant bias was present for all devices except novel goniometer B. The UG and novel goniometer A had positive constant bias; novel goniometer C had negative constant bias. Total error at 50° and 100° angles was > 5% for all devices. CONCLUSIONS AND CLINICAL RELEVANCE None of the devices accurately represented radiographically measured stifle joint angles. Additional veterinary studies are indicated prior to the use of novel goniometers in dogs.

Unity Jeffery

Department of Veterinary Microbiology and Preventative Medicine,
College of Veterinary Medicine,
Department of Veterinary Microbiology and Preventative Medicine, College of Veterinary Medicine

Iowa State University

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

Unity Jeffery

Department of Veterinary Microbiology and Preventative Medicine,College of Veterinary Medicine,Department of Veterinary Microbiology and Preventative Medicine, College of Veterinary Medicine

Iowa State University

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

Department of Veterinary Microbiology and Preventative Medicine,
College of Veterinary Medicine,
Department of Veterinary Microbiology and Preventative Medicine, College of Veterinary Medicine