Diagnostic Accuracy of a Direct Panfungal Polymerase Chain Reaction Assay Performed on Stained Cytology Slides.

Molecular techniques are increasingly being applied to stained cytology slides for the diagnosis of neoplastic and infectious diseases. Such techniques for the identification of fungi from stained cytology slides have not yet been evaluated. This study aimed to assess the diagnostic accuracy of direct (without nucleic acid isolation) panfungal polymerase chain reaction (PCR) followed by sequencing for identification of fungi and oomycetes on stained cytology slides from dogs, cats, horses, and other species. Thirty-six cases were identified with cytologically identifiable fungi/oomycetes and concurrent identification via fungal culture or immunoassay. Twenty-nine controls were identified with no cytologically or histologically visible organisms and a concurrent negative fungal culture. Direct PCR targeting the internal transcribed spacer region followed by sequencing was performed on one cytology slide from each case and control, and the sensitivity and specificity of the assay were calculated. The sensitivity of the panfungal PCR assay performed on stained cytology slides was 67% overall, 73% excluding cases with oomycetes, and 86% when considering only slides with abundant fungi. The specificity was 62%, which was attributed to amplification of fungal DNA from control slides with no visible fungus and negative culture results. Direct panfungal PCR is capable of providing genus- or species-level identification of fungi from stained cytology slides. Given the potential of panfungal PCR to amplify contaminant fungal DNA, this assay should be performed on slides with visible fungi and interpreted in conjunction with morphologic assessment by a clinical pathologist.

Neutrophil-Extracellular Traps, Cell-Free DNA, and Immunothrombosis in Companion Animals: A Review.

Immunothrombosis is a potentially beneficial physiological process that aids innate immunity and host defense against pathogen invasion. However, this process can also be damaging when it occurs to excess or in critical blood vessels. Formation of extracellular traps by leukocytes, particularly neutrophils, is central to our understanding of immunothrombosis. In addition to degranulation and phagocytosis, extracellular traps are the third mechanism by which neutrophils combat potential pathogens. These traps consist of extracellular DNA decorated with bactericidal cellular proteins, including elastase, myeloperoxidase, and cathepsins. Neutrophils can release these structures as part of a controlled cell-death process or via a process termed vital NETosis that enables the cells to extrude DNA but remain viable. There is accumulating evidence that NETosis occurs in companion animals, including dogs, horses, and cats, and that it actively contributes to pathogenesis. Numerous studies have been published detailing various methods for identification and quantification of extracellular trap formation, including cell-free DNA, measurements of histones and proteins such as high-mobility group box-1, and techniques involving microscopy and flow cytometry. Here, we outline the present understanding of these phenomena and the mechanisms of extracellular trap formation. We critically review the data regarding measurement of NETosis in companion animals, summarize the existing literature on NETosis in veterinary species, and speculate on what therapeutic options these insights might present to clinicians in the future.

SYSTEMATIC EVALUATION OF 106 LABORATORY REFERENCE DATA ARTICLES FROM NONDOMESTIC SPECIES PUBLISHED FROM 2014 TO 2016: ASSESSING COMPLIANCE WITH REFERENCE INTERVAL GUIDELINES.

Population-based reference intervals (RIs) are vital tools used to characterize health and disease based on laboratory values. The science and statistical basis for RI generation have evolved over the past 50 yr. Current veterinary-specific guidelines by the American Society of Veterinary Clinical Pathology exist for establishing RIs from nondomestic and wild animals. A list of 35 items that should be included during generation and publication of reference data was distilled from the currently available RI guidelines. The archives of five peer-reviewed journals were searched and 106 articles presenting laboratory reference data from nondomestic or wildlife species were identified and each reviewed by two authors to determine compliance with the list of 35 items. A compliance score was calculated as the number of articles that fulfilled the item out of the number where it would have been appropriate to fulfill the item. Most articles reported the number of reference individuals (compliance score 0.98), their partitioning demographics (compliance score 0.95), and sample collection and handling practices (compliance scores 0.97 and 0.96, respectively). Common deficiencies included omitting discussion of the validation status of the analytical methods for the species being evaluated (compliance score 0.12), documentation of use of exclusion criteria (compliance score 0.51), outlier detection (compliance score 0.43), appropriate statistical methods for the reference population (compliance score 0.34), and calculation and presentation of confidence intervals around the reference limits (compliance score 0.35). Compliance scores were not statistically different when stratified on the number of individuals in the largest and smallest evaluated group or the format of the article (full vs short format). Articles that cited RI generation guidelines fulfilled more of the required steps and provided a more complete description of their data (compliance score 0.74) than those that did not cite guidelines (compliance score 0.58). Additional attention to the science of and recommendations for RI generation is recommended to strengthen the utility of published data.

When neuroscience met clinical pathology: partitioning experimental variation to aid data interpretation in neuroscience.

In animal experiments, neuroscientists typically assess the effectiveness of interventions by comparing the average response of groups of treated and untreated animals. While providing useful insights, focusing only on group effects risks overemphasis of small, statistically significant but physiologically unimportant, differences. Such differences can be created by analytical variability or physiological within-individual variation, especially if the number of animals in each group is small enough that one or two outlier values can have considerable impact on the summary measures for the group. Physicians face a similar dilemma when comparing two results from the same patient. To determine whether the change between two values reflects disease progression or known analytical and physiological variation, the magnitude of the difference between two results is compared to the reference change value. These values are generated by quantifying analytical and within-individual variation, and differences between two results from the same patient are considered clinically meaningful only if they exceed the combined effect of these two sources of 'noise'. In this article, we describe how the reference change interval can be applied within neuroscience. This form of analysis provides a measure of outcome at an individual level that complements traditional group-level comparisons, and therefore, introduction of this technique into neuroscience can enrich interpretation of experimental data. It can also safeguard against some of the possible misinterpretations that may occur during analysis of the small experimental groups that are common in neuroscience and, by illuminating analytical error, may aid in design of more efficient experimental methods.

Canine Neutrophil Extracellular Traps Enhance Clot Formation and Delay Lysis.

Autoimmune diseases increase the risk of thrombosis. Neutrophil extracellular traps (NETs) are webs of DNA and protein that may mediate thrombosis in autoimmune diseases. Human and murine studies show NET-releasing neutrophils within a thrombus promote its growth, but it is unclear to what extent NET fragments released into circulation during inflammation are prothrombotic. This study hypothesized that canine NETs promote clot formation and impair lysis even in the absence of neutrophils. NETs were prepared from PMA-stimulated neutrophils and added to fibrinogen and thrombin or to recalcified pooled canine platelet-poor plasma, tissue factor, and tissue plasminogen activator. Clot formation and lysis were measured spectrophotometrically. NETs did not alter fibrin clot formation, but NETs increased maximum clot formation velocity ( P = .001) and delayed lysis ( P = .009) of plasma clots compared with supernatants from nonstimulated neutrophils. DNase digestion of NETs reduced their effect on clot lysis but not maximum clot formation velocity. This suggested impaired lysis was principally mediated by DNA within NETs but that NET proteins were principally responsible for increased speed of clot formation. Previous reports suggested elastase or histones might be responsible for the effect of NETs on clot formation. Elastase activity was greatly reduced by plasma, and addition of histones to plasma did not increase formation velocity, suggesting these proteins were not responsible for increasing maximum formation velocity. This study showed that NETs enhanced clot formation and impaired clot lysis in canine platelet-poor plasma. These in vitro findings suggest both NET proteins and DNA may contribute to thrombosis in inflammatory disease.

DNA methylation and chromatin accessibility predict age in the domestic dog.

Across mammals, the epigenome is highly predictive of chronological age. These "epigenetic clocks," most of which have been built using DNA methylation (DNAm) profiles, have gained traction as biomarkers of aging and organismal health. While the ability of DNAm to predict chronological age has been repeatedly demonstrated, the ability of other epigenetic features to predict age remains unclear. Here, we use two types of epigenetic information-DNAm, and chromatin accessibility as measured by ATAC-seq-to develop age predictors in peripheral blood mononuclear cells sampled from a population of domesticated dogs. We measured DNAm and ATAC-seq profiles for 71 dogs, building separate predictive clocks from each, as well as the combined dataset. We also use fluorescence-assisted cell sorting to quantify major lymphoid populations for each sample. We found that chromatin accessibility can accurately predict chronological age (R2 ATAC = 26%), though less accurately than the DNAm clock (R2 DNAm = 33%), and the clock built from the combined datasets was comparable to both (R2 combined = 29%). We also observed various populations of CD62L+ T cells significantly correlated with dog age. Finally, we found that all three clocks selected features that were in or near at least two protein-coding genes: BAIAP2 and SCARF2, both previously implicated in processes related to cognitive or neurological impairment. Taken together, these results highlight the potential of chromatin accessibility as a complementary epigenetic resource for modeling and investigating biologic age.

Response of the VCMVet viscoelastic coagulation monitor to veterinary environmental simulation challenges.

BACKGROUND: Traditional viscoelastic clotting tests are significantly impacted by the operator and environmental variation. The VCMVet coagulation monitor could provide a more user-friendly alternative for veterinary practices. OBJECTIVES: We aimed to determine if environmental vibration commonly encountered in veterinary practice alters the results of a point-of-care viscoelastic device, the VCMVet. METHODS: Nine fresh whole blood samples from healthy dogs were evaluated simultaneously using VCMVet instruments under four environmental conditions: (normal) alone and undisturbed on a raised tabletop, (centrifuge) on a countertop 6 inches from a centrifuge that operated at 12 000 rpm for 10 minutes every 20 minutes, (workspace) on a tabletop workspace in proximity to two heavy-use computers, and (gurney) on a rolling gurney at a walking pace for 10 minutes every 20 minutes. Results were compared between conditions using a Friedman test, and if this was significant (P-value < .05), it was followed by a Wilcoxon test for paired samples. RESULTS: Analysis of samples on a rolling gurney created obvious movement artifacts, and this condition was excluded from statistical analysis. The centrifuge condition resulted in a significantly higher alpha angle (median 49 degrees, interquartile range 4) than the normal condition (median 46 degrees, interquartile range 5, P = .0078). Other viscoelastic parameters were not significantly different between the normal, centrifuge, and workspace conditions. CONCLUSIONS: The VCMVet is suitable for use in a busy veterinary environment but should be protected from vibration. The instrument does not produce reliable results when operated on a moving gurney, and it should be stationary during sample analysis.

Decreased antithrombin activity and inflammation in cats.

OBJECTIVES: The objective of this study was to determine if inflammatory markers are associated with antithrombin activity in cats. METHODS: For a retrospective population of 231 cats admitted to a referral hospital, antithrombin activity was classified as decreased (n = 77), intermediate (n = 97) or in the upper quartile (n = 57). Odds ratios (ORs) were calculated for an association between decreased or upper quartile activity and hypoalbuminemia, hyperfibrinogenemia, band neutrophilia and toxic change. Multiple logistic regression was performed to determine if an association between band neutrophilia and decreased antithrombin activity was independent of decreased hepatic synthesis, consumptive coagulopathy or protein loss. RESULTS: Cats with decreased antithrombin activity were more likely than cats with intermediate-to-upper quartile activity to have band neutrophilia (OR 2.85, P = 0.0050), hypoalbuminemia (OR 12.1, P <0.0001) or toxic neutrophils (OR 4.47, P <0.0001). Cats with antithrombin activity in the upper quartile were less likely than those with intermediate-to-low activity to have hypoalbuminemia (OR 0.31, P = 0.0023) or toxic neutrophils (OR 0.44, P = 0.033). In a regression model that included other mechanisms for decreased antithrombin, band neutrophilia remained associated with decreased antithrombin activity (adjusted OR 2.62, P = 0.013). CONCLUSIONS AND RELEVANCE: Contrary to previous studies suggesting antithrombin is a feline positive acute phase protein, this study demonstrates an association between decreased antithrombin activity and inflammation. Further studies are needed to determine the mechanistic basis of this association.

Evaluation of two benchtop blood gas analyzers for measurement of electrolyte concentrations in venous blood samples from dogs.

OBJECTIVE: To assess agreement between 2 benchtop blood gas analyzers developed by 1 manufacturer (BGA 1 and BGA 2 [a newer model with reduced maintenance requirements]) and a reference chemistry analyzer for measurement of electrolyte (sodium, chloride, and potassium) in blood samples from dogs. ANIMALS: 17 healthy staff- and student-owned dogs and 23 client-owned dogs admitted to an emergency and intensive care service. PROCEDURES: Blood collected by venipuncture was placed in lithium heparin-containing tubes. Aliquots were analyzed immediately with each BGA. Samples were centrifuged, and plasma was analyzed with the reference analyzer. Results for each BGA were compared with results for the reference analyzer by Passing-Bablok regression analysis. Percentage differences between BGA and reference analyzer results were compared with published guidelines for total allowable error. RESULTS: Proportional bias was detected for measurement of chloride concentration (slope, 0.7; 95% CI, 0.7 to 0.8), and constant positive bias was detected for measurement of chloride (y-intercept, 34, mmol/L; 95% CI, 16.9 to 38 mmol/L) and potassium (y-intercept, 0.1 mmol/L; 95% CI, 0.1 to 0.2 mmol/L) concentrations with BGA 1. There was no significant bias for measurement of potassium or chloride concentration with BGA 2 or sodium concentration with either BGA. Differences from the reference analyzer result exceeded total allowable error guidelines for ≥ 1 sample/analyte/BGA, but median observed measurement differences between each BGA and the reference analyzer did not. CONCLUSIONS AND CLINICAL RELEVANCE: Good agreement with reference analyzer results was found for measurement of the selected electrolyte concentrations in canine blood samples with each BGA.

Blood loss and coagulation profile in pregnant and non-pregnant queens undergoing elective ovariohysterectomy.

OBJECTIVES: The aims of this study were to determine if there is increased risk of intraoperative bleeding in pregnant cats undergoing elective ovariohysterectomy (OHE), and to compare coagulation in queens in various stages of estrus and pregnancy subjected to elective OHE using a whole-blood viscoelastic assay. METHODS: Intraoperative blood loss was compared between non-pregnant and pregnant cats undergoing elective OHE. Viscoelastic evaluations of whole blood drawn pre- and postoperatively were performed using a point-of-care device measuring clot time (CT), clot formation time (CFT), alpha angle, maximum clot formation (MCF), amplitude at 10 and 20 mins (A10 and A20, respectively), and lysis index at 30 and 45 mins after MCF (LI30 and LI45, respectively). RESULTS: One hundred and ninety-three cats underwent OHE by a ventral midline approach. Median blood loss was greater for pregnant cats (2.0 ml, range <0.5-13 ml) than non-pregnant cats (<0.5 ml, range <0.5-15 ml; P <0.0001). Preoperatively, pregnant cats had a shorter median CFT (165 s vs 190.5 s), increased median A10 (31 from 25.5 VCM units) and A20 (38 from 35 VCM units), and a lower median LI45 (99% from 100%) than non-pregnant cats. Postoperatively, A10 and A20 increased, and LI30 and LI45 decreased in both non-pregnant and pregnant queens. In pregnant queens, mean CT also increased postoperatively. CONCLUSIONS AND RELEVANCE: Pregnant cats were relatively hypercoagulable and had an increased rate of clot lysis than non-pregnant cats. Intraoperative blood loss was increased in pregnant vs non-pregnant cats, but no clinically relevant bleeding conditions occurred.

Systemic mastocytosis with subcutaneous hemorrhage and edema in a Greyhound dog: case report and review of diagnostic criteria.

Systemic mastocytosis, characterized by infiltration of multiple organs by neoplastic mast cells, is a well-described entity in human medicine with specific criteria for diagnosis, but is ill defined in veterinary literature. Hemostatic disorders are reported in humans affected by systemic mastocytosis but have not been well described in veterinary literature. A 5-y-old, spayed female Greyhound dog had a 1-mo history of progressive ventral cutaneous edema, hemorrhage, and pain. Cytology of an antemortem aspirate from the subcutis of the ventral abdomen was suggestive of mast cell neoplasia, but no discrete mass was present. The dog was euthanized and submitted for autopsy; marked subcutaneous edema and hemorrhage were confirmed. The ventral abdominal panniculus and dermis superficial to the panniculus carnosus were infiltrated by a dense sheet of neoplastic mast cells. The neoplastic cells contained toluidine blue-positive granules and formed aggregates within the bone marrow and several visceral organs, including the liver, spleen, heart, and kidney. Diffuse edema and hemorrhage is an unusual presentation of mast cell tumors in dogs. Antemortem tests, including complete blood count, coagulation profile, and viscoelastic coagulation testing, were suggestive of a primary hemostatic defect. We discuss here the diagnostic criteria used in humans, how these can be applied to veterinary patients, and the limitations of the current diagnostic framework.

Comparison of viscoelastic test results from blood collected near simultaneously from the jugular and saphenous veins in cats.

OBJECTIVES: The aim of this study was to compare viscoelastic test results from samples collected from a jugular vein using a 20 G needle and a medial saphenous vein using a 22 G needle in cats presenting for elective ovariohysterectomy (OHE) or castration. METHODS: Forty apparently healthy cats (20 males and 20 females) presenting for elective OHE or castration were included in a prospective study observing viscoelastic test results from central and peripherally collected whole blood. Cats were anesthetized during blood collection with a standardized protocol including buprenorphine, ketamine, dexmedetomidine and isoflurane. Blood samples from jugular and saphenous veins were collected near simultaneously. Viscoelastic evaluations of whole blood were performed using a point-of-care device measuring clot time (CT), clot formation time (CFT), alpha angle (α), maximum clot formation (MCF), and amplitude at 10 and 20 mins (A10 and A20, respectively). Viscoelastometry continued post-clot time to determine a lysis index at 30 and 45 mins (LI30 and LI45, respectively) to assess fibrinolysis. RESULTS: Studied cats had a median age of 18 months (range 5 months to 5 years) and a median weight of 3.6 kg (range 2.7-5.9 kg). A total of 80 samples were available for analysis. While lysis indices were not different, viscoelastic measures of coagulation differed between sampling sites (CT, P <0.005; CFT, P = 0.01; α, P <0.05; MCF, P <0.0005; A10, P <0.0005; A20, P <0.0005). CONCLUSIONS AND RELEVANCE: Viscoelastic results from jugular venous blood samples appear to be more hypercoagulable than those collected from the medial saphenous vein, suggesting that the same site should be used consistently for serial monitoring or for collecting study data.

Variation in biochemistry test results between annual wellness visits in apparently healthy Golden Retrievers.

BACKGROUND: Annual wellness testing is widely recommended for apparently healthy dogs, but there is little data to assist with distinguishing normal variation from clinically important changes. OBJECTIVES: To define variability in biochemistry analytes between annual wellness tests in healthy Golden Retrievers. ANIMALS: Four hundred thirty-four Golden Retrievers undergoing annual health assessments by their primary care veterinarians as part of a prospective cohort study. METHODS: Changes in 23 biochemistry analytes were calculated between year 1 and year 2 health checks for 196 dogs classified as healthy for ≥3 consecutive years. Using a direct nonparametric method, annual change intervals were constructed to define normal variability. A validation cohort of 238 dogs without a diagnosis of systemic disease for ≥3 consecutive years were compared with the reference and annual change intervals, and the proportions of dogs outside annual change intervals and a population-based reference interval were compared by using a McNemar test. RESULTS: Annual change intervals were calculated based on 190 dogs after outlier removal. For all 23 analytes, >90% of dogs in the validation cohort were within the annual change interval. There were no significant differences in the classification by reference versus annual change intervals. CONCLUSIONS AND CLINICAL IMPORTANCE: The annual change intervals met performance requirements for classification of dogs that did not develop systemic disease in the year following wellness testing as normal.

Effect of dilution of canine blood samples on the specificity of saline agglutination tests for immune-mediated hemolysis.

BACKGROUND: Saline agglutination tests (SATs) are widely recommended for diagnosis of immune-mediated hemolytic anemia in dogs, but there are frequent false-positive results. OBJECTIVES: Specificity of SATs will improve at higher saline-to-blood ratios. ANIMALS: One hundred fifty dogs treated at a veterinary referral hospital with hematocrits ≤30%. METHODS: Prospective diagnostic accuracy study. Immune-mediated hemolysis (IMH) was considered present if a gel direct antiglobulin test (DAT) was positive and there was clinical evidence of hemolysis (n = 9), absent if another mechanism for anemia was identified and the DAT was negative or there was no hemolysis (n = 138), and if IMH status was unclear, dogs were excluded (n = 3). Saline agglutination tests were prepared at 1 : 1, 4 : 1, 9 : 1, and 49 : 1 saline-to-blood ratios, and microscopic agglutination was considered a positive result. RESULTS: Specificity for IMH increased from 29% (95% confidence interval 20-38) at a 1 : 1 dilution to 97% (93-99) at a 49 : 1 dilution. Sensitivity was 88% (47-100) at 1 : 1 and 4 : 1 dilutions and 67% (30-93%) at 9 : 1 and 49 : 1 dilutions. Diagnostic accuracy increased from 33% (24-42) at 1 : 1 dilution to 95% (90-98) at 49 : 1 dilution. CONCLUSIONS AND CLINICAL IMPORTANCE: If performed using a 49 : 1 saline-to-blood ratio, SATs achieve high specificity for IMH. Based on a gold standard of positive DAT and evidence of hemolysis, lower saline-to-blood ratio results should not be used because false-positive results are common.

Comparison of the prothrombin time-derived and Clauss assays for the measurement of plasma fibrinogen in hospitalized dogs.

BACKGROUND: Accurate measurement of fibrinogen is necessary for detecting bleeding tendencies and inflammation. The Clauss assay determines fibrinogen concentration from its inverse relationship with thrombin-induced clot times. PT-derived assays determine fibrinogen concentrations from changes in the optical density during a routine prothrombin assay and allow determination of fibrinogen without additional reagents. This method has not been assessed in clinically ill dogs. OBJECTIVES: We aimed to determine the agreement between the Clauss and PT-derived fibrinogen assays and compare the ability of the assays to predict surgery-associated transfusions and discriminate between dogs with and without bleeding. METHODS: Retrospective medical record review identified 200 dogs with a variety of underlying diseases with results from both assays. The two assays were compared using Passing-Bablok regression, and the ability of the assays to identify bleeding and predict the need for transfusions was assessed with receiver operating characteristic (ROC) curve analyses. RESULTS: The PT-derived assay displayed constant (y-intercept, 32 mg/dL; 95% CI 18-41) and proportional (slope, 0.79; 95% CI 0.75-0.82) bias compared with the Clauss assay. The Clauss assay reported lower values than the PT-derived assay at lower fibrinogen concentrations and higher values at higher concentrations. Comparing the area under the ROC curve did not detect significant differences in the ability of the two assays to discriminate between dogs with and without bleeding or predict the need for surgery-associated transfusions. CONCLUSIONS: The PT-derived and Clauss assays are not interchangeable, and the Clauss assay could be more sensitive to hypofibrinogenemia in dogs.

Variability in Open-Field Locomotor Scoring Following Force-Defined Spinal Cord Injury in Rats: Quantification and Implications.

Spinal cord injury research in experimental animals aims to define mechanisms of tissue damage and identify interventions that can be translated into effective clinical therapies. Highly reliable models of injury and outcome measurement are essential to achieve these aims and avoid problems with reproducibility. Functional scoring is a critical component of outcome assessment and is currently commonly focused on open field locomotion (the "BBB score"). Here we analyze variability of observed locomotor outcome after a highly regulated spinal cord contusion in a large group of rats that had not received any therapeutic intervention. Our data indicate that, despite tight regulation of the injury severity, there is considerable variability in open-field score of individual rats at 21 days after injury, when the group as a whole reaches a functional plateau. The bootstrapped reference interval (that defines boundaries that contain 95% scores in the population without regard for data distributional character) for the score at 21 days was calculated to range from 2.3 to 15.9 on the 22-point scale. Further analysis indicated that the mean day 21 score of random groups of 10 individuals drawn by bootstrap sampling from the whole study population varies between 9.5 and 13.5. Wide variability between individuals implies that detection of small magnitude group-level treatment effects will likely be unreliable, especially if using small experimental group sizes. To minimize this problem in intervention studies, consideration should be given to assessing treatment effects by comparing proportions of animals in comparator groups that attain pre-specified criterion scores.

An Analysis of Variability in "CatWalk" Locomotor Measurements to Aid Experimental Design and Interpretation.

Preclinical studies in models of neurologic injury and disease rely on behavioral outcomes to measure intervention efficacy. For spinal cord injury, the CatWalk system provides unbiased quantitative assessment of subtle aspects of locomotor function in rodents and so can powerfully detect significant differences between experimental and control groups. Although clearly of key importance, summary group-level data can obscure the variability within and between individual subjects and therefore make it difficult to understand the magnitude of effect in individual animals and the proportion of a group that may show benefit. Here, we calculate reference change intervals (RCIs) that define boundaries of normal variability for measures of rat locomotion on the CatWalk. Our results indicate that many commonly-used outcome measures are highly variable, such that differences of up to 70% from baseline value must be considered normal variation. Many CatWalk outcome variables are also highly correlated and dependent on run speed. Application of calculated RCIs to open access data (https://scicrunch.org/odc-sci) on hindlimb stride length in spinal cord-injured rats illustrates the complementarity between group-level (16 mm change; p = 0.0009) and individual-level (5/32 animals show change outside RCI boundaries) analysis between week 3 and week 6 after injury. We also conclude that interdependence among CatWalk variables implies that test "batteries" require careful composition to ensure that different aspects of defective gait are analyzed. Calculation of RCIs aids in experimental design by quantifying variability and enriches overall data analysis by providing details of change at an individual level that complement group-level analysis.

Inaccurate point-of-care blood glucose measurement in a dog with secondary erythrocytosis.

BACKGROUND: Point-of-care (POC) portable blood glucose meters (PBGMs) are convenient and inexpensive tools for assessing patient blood glucose concentrations. They are often used to quickly diagnose hypoglycemia or collect serial glucose readings in diabetic patients. However, POC meters have been previously identified in human and veterinary literature to be inaccurate when utilized in patients with abnormal HCT. This problem may not be reflected in manufacturer guidelines referenced by practitioners in the POC setting. KEY FINDINGS: A 1.5-year-old dog, previously diagnosed with multiple congenital cardiac malformations, right-to-left cardiac shunting and secondary erythrocytosis, presented to a veterinary emergency center minimally responsive and without detectable pulses. PBGM measurement identified hypoglycemia. Following stabilization of the dog, serial glucose assessments showed discordant results between PBGMs and the reference laboratory biochemistry analyzer. A pathological cause for hypoglycemia was not identified and PBGM readings were determined to be erroneously low due to the dog's abnormally high HCT. SIGNIFICANCE: This case demonstrates the limitations of using PBGMs to assess blood glucose in a dog with secondary erythrocytosis. The report emphasizes the need for judicious use of PBGMs in critically ill patients and that these glucometers may not be reliable in patients with abnormal HCT values.

Effects of marked hypertriglyceridemia and lipid clearance techniques on canine biochemistry testing.

Triglyceride concentrations in dogs with hyperlipidemic disorders can exceed concentrations used by assay manufacturers for interference testing. High-speed centrifugation or the polar solvent LipoClear reduce triglyceride concentrations, but efficacy requires evaluation in veterinary species. We determined the effect of marked hypertriglyceridemia on canine biochemistry testing; assessed the ability of high-speed centrifugation or LipoClear to correct lipemic interferences; and determined if LipoClear introduces inaccuracy into biochemistry assays. Fifteen pooled canine serum samples were aliquoted and spiked with equal volumes of water or Intralipid [triglyceride concentration 33.9 mmol/L (3,000 mg/dL)]. Intralipid aliquots underwent lipid removal by high-speed centrifugation or LipoClear treatment, and a water-spiked aliquot underwent LipoClear treatment. Biochemistry panels were performed using a Vitros 4600 chemistry analyzer. Results were compared by paired t-test or Wilcoxon test. Total observed errors were considered clinically acceptable if below veterinary allowable total error (TEa) guidelines. Statistically significant (p ≤ 0.05) interferences were introduced by Intralipid for 15 of 15 analytes. Median observed error exceeded TEa for potassium and enzymatic carbon dioxide, neither of which were identified by the manufacturer as susceptible to lipemic interference. After centrifugation, median observed error exceeded TEa for potassium and chloride. LipoClear treatment resulted in median errors that exceeded TEa for total protein, chloride, and phosphorus. Given that severe lipemia can occur in dogs with primary or secondary hyperlipidemia, veterinary laboratories should perform their own interference testing at triglyceride concentrations relevant to their patient population and provide this information to clinicians to ensure optimal case management.

ACVIM consensus statement on the diagnosis of immune-mediated hemolytic anemia in dogs and cats.

Immune-mediated hemolytic anemia (IMHA) is an important cause of morbidity and mortality in dogs. IMHA also occurs in cats, although less commonly. IMHA is considered secondary when it can be attributed to an underlying disease, and as primary (idiopathic) if no cause is found. Eliminating diseases that cause IMHA may attenuate or stop immune-mediated erythrocyte destruction, and adverse consequences of long-term immunosuppressive treatment can be avoided. Infections, cancer, drugs, vaccines, and inflammatory processes may be underlying causes of IMHA. Evidence for these comorbidities has not been systematically evaluated, rendering evidence-based decisions difficult. We identified and extracted data from studies published in the veterinary literature and developed a novel tool for evaluation of evidence quality, using it to assess study design, diagnostic criteria for IMHA, comorbidities, and causality. Succinct evidence summary statements were written, along with screening recommendations. Statements were refined by conducting 3 iterations of Delphi review with panel and task force members. Commentary was solicited from several professional bodies to maximize clinical applicability before the recommendations were submitted. The resulting document is intended to provide clinical guidelines for diagnosis of, and underlying disease screening for, IMHA in dogs and cats. These should be implemented with consideration of animal, owner, and geographical factors.