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.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Because the SAT is intended as a patient‐side screening test, we opted to use a simple protocol which we believe, based on text book descriptions and anecdotal experience, is consistent with that ...used in many clinical pathology laboratories and by veterinarians in primary care and emergency practice. ...we would advocate for the use of DAT as a confirmatory test in most cases of suspected immune‐mediated hemolytic anemia (IMHA), but we recognize many veterinarians cannot obtain a laboratory DAT result at initial presentation. ...we would emphasize that while the 49 : 1 dilution achieved high specificity for IMHA, occasional (4/138) false positives did occur. ...as for any diagnostic test for IMHA, SAT results should be evaluated as part of the full clinical picture, with a particular emphasis on establishing if there is evidence of hemolysis.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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.
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.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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.
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NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
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.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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.
Outcome measurements in neuroscience vary because of intra‐ and interanimal variability and experimenter error. Sometimes, when analysed at a group level, these sources of variation combine to suggest misleading conclusions regarding the impact of an intervention. Derivation of individual‐level outcomes by partitioning and measuring sources of variability avoids this pitfall and enriches experimental interpretation.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
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.
17 healthy staff- and student-owned dogs and 23 client-owned dogs admitted to an emergency and intensive care service.
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.
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.
Good agreement with reference analyzer results was found for measurement of the selected electrolyte concentrations in canine blood samples with each BGA.
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.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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