Study on biological variation of haemostatic parameters in clinically healthy dogs

Thromboelastography (TEG) may be a valuable supplement to the coagulation assays activated partial thromboplastin time (aPTT), prothrombin time (PT), thrombin time (TT), fibrinogen, antithrombin (AT) and D-Dimer currently used in most clinical pathology laboratories. Allowable imprecision and bias reference limits for analytical tests can be calculated based on measurements of biological variation. No studies to date have examined the effect of biological variation on these haemostasis parameters in the same group of dogs. Plasma samples were collected after a set protocol once weekly for five consecutive weeks from eight healthy dogs (four males and four females) and stored at -80 degrees C until analysis. Randomized duplicate coagulation tests and TEG analyses were performed on all plasma samples within one run. The data were analyzed for outliers and subsequently subjected to nested analysis of variance to obtain the coefficient of analytical, intra-individual and inter-individual variation. From these objective analytical performance standards for imprecision, critical difference, total error and the index of individuality were calculated to assess the utility of conventional population-based reference ranges. All the clotting times (aPTT, PT and TT), fibrinogen, AT and D-Dimer showed a degree of individuality, which may make the use of population-based reference ranges alone an insensitive interpretation criterion, whereas a population-based reference interval seems to be sensitive for interpreting all TEG parameters. Analytical performance standards for imprecision were only met for one of the coagulation assays, whereas all TEG parameters except the alpha angle, alpha achieved this analytical goal.     

Current and emerging concepts in biological and analytical variation applied in clinical practice

A single laboratory result actually represents a range of possible values, and a given laboratory result is impacted not just by the presence or absence of disease, but also by biological variation of the measurand in question and analytical variation of the equipment used to make the measurement. Biological variation refers to variability in measurand concentration or activity around a homeostatic set point. Knowledge of biological and analytical variation can be used to facilitate interpretation of patient clinicopathologic data and is particularly useful for interpreting serial patient data and data at or near reference limits or clinical decision thresholds. Understanding how biological and analytical variation impact laboratory results is of increasing importance, because veterinarians evaluate serial data from individual patients, interpret data from multiple testing sites, and use expert consensus guidelines that include decision thresholds for clinicopathologic data interpretation. The purpose of our report is to review current and emerging concepts in biological and analytical variation and discuss how biological and analytical variation data can be used to facilitate clinicopathologic data interpretation. Inclusion of veterinary clinical pathologists having expertise in laboratory quality management and biological variation on research teams and veterinary practice guideline development teams is recommended, to ensure that various considerations for clinicopathologic data interpretation are addressed.     

Breed‐specific variation of hematologic and biochemical analytes in healthy adult Bernese Mountain dogs

Background: Hematology and serum biochemistry reference intervals in dogs may be affected by internal factors, such as breed and age, and external factors, such as the environment, diet, and lifestyle. In humans, it is well established that geographic origin and age may have an impact on reference intervals and, therefore, more specific reference intervals are sought for subpopulations. Objective: The objective of this study was to validate and transfer standard laboratory reference intervals for healthy Bernese Mountain dogs and to create new intervals for analytes where the established laboratory reference intervals were rejected. Methods: The procedure was performed using the human Clinical and Laboratory Standards Institute‐approved model modified for veterinary use. Thirty‐two dogs were included in the study using a direct a priori method, as recommended. Results: While 23 of the standard laboratory reference intervals were readily validated, 7 of the analytes (eosinophils, MCHC, alkaline phosphatase [ALP], γ‐glutamyltransferase, total bilirubin, amylase, and cholesterol) required new reference intervals according to the standard. These were calculated using the robust method. In particular, the new reference range for ALP was wide compared with the established laboratory reference interval. No clinical causes were found for differences in the results of these analytes. Conclusion: We found significant differences in 7 hematologic and serum biochemical analytes for which a breed‐specific variation appears to be the most plausible explanation. Breed‐specific reference intervals for Bernese Mountain dogs will help avoid misinterpretation of laboratory results in the diagnostic process.     

Evaluation of biological variance of cystatin C in comparison with other endogenous markers of glomerular filtration rate in healthy dogs

BACKGROUND: The aim of the study was to investigate and compare components of variance of endogenous markers of glomerular filtration rate (GFR) in healthy dogs and impact on the interpretation of results. HYPOTHESIS: Cystatin C (cysC) in the dog is superior to creatinine (crea) and urea in detecting decreased renal function because of a high index of individuality (IoI). ANIMALS AND METHOD: Variance components of cysC, crea (2 methods: creaE, creaJ), urea, and inorganic phosphate (Pi) in plasma were determined in a longitudinal study over 6 months in 24 healthy dogs (6 German Shorthair Pointers, 18 Beagles). IoI and critical differences (CD) were calculated, as well as the numbers of measurements necessary to determine the individual's homeostatic set point. Results: Mean concentrations of cysC, creaJ, creaE, urea, and Pi (mean +/- SD) were 0.93 +/- 0.19 mg/L, 0.94 +/- 0.17 mg/dL, 0.76 +/- 0.18 mg/dL, 35.34 +/- 9.08 mg/dL, and 3.74 +/- 0.68 mg/dL, respectively. The IoI for cysC, creaJ, creaE, urea, and Pi were 0.96, 0.89, 0.80, 0.90, and 1.16, respectively. The C(D) for cysC, creaJ, creaE, urea, and Pi were 0.37 mg/L, 0.26 mg/dL, 0.27 mg/dL, 16.94 mg/dL, and 1.45 mg/dL, respectively. CONCLUSION: In dogs, components of biological variance of cysC and crea are in the same range. Analytical precision requirements were fulfilled by crea(both), urea, and Pi. All parameters had an intermediate IoI, which allowed the application of population-based reference limits. The application of the CD for crea or cysC might be useful in detecting a decrease of GFR, when sequential measurements in an individual reveal an increase exceeding the CD but not the upper reference limit.     

The immunophenotype of peripheral blood lymphocytes in clinically healthy dogs and dogs with lymphoma in remission

Lymphoma is a common cancer of dogs that frequently is treated with chemotherapy or radiation therapy. Response to therapy is variable and currently available diagnostic tests do not reliably predict response to therapy. Treatment for lymphoma often results in lymphopenia, but it is unknown whether the changes in circulating lymphocytes result from generalized or specific reduction of lymphocytes. In this study, blood lymphocytes from 12 clinically healthy dogs, 10 dogs in remission because of treatment for B-cell lymphoma, and 8 dogs in remission from T-cell lymphoma were analyzed by flow cytometry by using a panel of 20 antibodies reactive with canine leukocyte antigens. Results identified similar lymphocyte parameters in treated dogs regardless of the type of lymphoma. Treated dogs had >50% reduction in blood lymphocyte concentration, and an absolute decrease in most subsets of lymphocytes. Both groups of treated dogs had relative increases in the proportion of CD3+, T-cell receptor (TCR)αβ+, and CD90+ lymphocytes, and a decreased proportion of CD45RA+ cells. In addition, dogs with T-cell lymphoma in remission had a significant increase in the proportion of CD49d+ lymphocytes. These findings were interpreted as representing likely suppression of lymphocyte regeneration by chemotherapy, with a relative increase in the proportion of memory over naïve lymphocytes. Lack of correlation with the T- or B-cell origin of the initial lymphoma suggested that, by using flow cytometric methods, residual circulating neoplastic cells could not be detected. However, the changes in the lymphocyte profile of dogs treated with chemotherapy may have relevance to their immunocompetence.     

Interbreed variation in serum serotonin (5-hydroxytryptamine) concentration in healthy dogs

Introduction

Serotonin (5-hydroxytryptamine [5-HT]) has several biological functions. In different species, excessive 5-HT has been linked to valvular lesions, similar to those seen in dogs with myxomatous mitral valve disease. Previous studies suggest higher 5-HT in healthy Cavalier King Charles Spaniels (CKCSs), a breed highly affected by myxomatous mitral valve disease, compared to other breeds.

Validation of heart rate spot-check protocol to measure circadian variation and heart rate in healthy dogs and dogs with atrial fibrillation

IntroductionAlternatives for out-of-clinic heart rate (HR) measurement are required to optimise the management of atrial fibrillation (AF) in dogs. Additionally, the presence of circadian variation (CV) in HR in pet dogs remains unknown.We aimed to identify the number and duration of spot-checks required for an accurate estimation of 24-hour HR in canine AF. Circadian variation in HR was examined in healthy dogs and dogs with AF, and spot-check-derived HR was compared with a CV-derived gold standard.Materials and methodsAmbulatory electrocardiogram data from healthy dogs and dogs with AF were retrospectively analysed. Heart rate was calculated from the entire recording and pre-defined periods (spot-checks) of one hour to 30 and 60 s in duration. Circadian variation in HR was determined by cosinor analysis. Bias and limits of agreement of means and median HR with mesor HR were determined by correlation and Bland–Altman analysis.ResultsCircadian variation in HR was identified not only in 18/22 healthy dogs and 14/21 AF dogs but only on ambulatory electrocardiogram recordings.Four-hourly spot-checks provided the most accurate estimate of mesor HR in healthy dogs (bias of the median over 30 s 7.70, limits of agreement 7.48), whereas, in dogs with AF, four, six and eight-hourly spot-checks provided reliable estimates of mesor HR (bias within −1.29 and −29.5).ConclusionsFour, six and eight-hourly HR spot-checks can estimate 24-hourly HR in dogs with AF. There was CV in HR in most healthy pet dogs and dogs with AF. Spot-check protocols cannot identify CV in HR.     

Correlates of objectively measured physical activity in dogs

To increase physical activity (PA) levels in dogs and to better evaluate their energy requirements, there is a need to understand which factors or correlates are associated with PA and/or sedentary behaviour. Improving our understanding of these correlates also has implications for prescribed energy requirements in dogs. PA was measured using accelerometry in 62 dogs from two common breeds (Labrador retrievers and Cocker spaniels). Five potential correlates (age, sex, breed, neuter status, body condition score) were tested for associations with total volume of PA, light-moderate intensity PA, vigorous intensity PA and sedentary behaviour. Age and breed were associated with total volume of PA, light-moderate intensity PA and sedentary behaviour in the final models. Age was associated with vigorous intensity PA. The final models explained 60%, 40%, 63% and 44% of variance in total volume of PA, light-moderate intensity PA, vigorous intensity PA and sedentary behaviour, respectively. These results should improve understanding of the variation in energy requirements of dogs, as well as the development of age and breed-specific diets and the prevention and treatment of canine obesity.     

Two-dimensional echocardiographic estimates of left atrial function in healthy dogs and dogs with myxomatous mitral valve disease

Objectives

To provide reference intervals for 2-dimensional linear and area-based estimates of left atrial (LA) function in healthy dogs and to evaluate the ability of estimates of LA function to differentiate dogs with subclinical myxomatous mitral valve disease (MMVD) and similarly affected dogs with congestive heart failure (CHF).

Evaluation of tricuspid annular plane systolic excursion measured by two-dimensional echocardiography in healthy dogs: repeatability,reference intervals,and comparison with M-mode assessment

Introduction

We sought to determine the feasibility, measurement variability, and within-day repeatability of tricuspid annular plane systolic excursion (TAPSE) measured by two-dimensional echocardiography (2D TAPSE), generate reference intervals for 2D TAPSE, assess agreement and correlation between 2D TAPSE and the conventional TAPSE measured by M-mode echocardiography (MM TAPSE), and to assess the ability of 2D TAPSE to track a drug-induced decrease in right ventricular (RV) function compared with MM TAPSE.

Biologic variability of N-terminal pro-brain natriuretic peptide in healthy dogs and dogs with myxomatous mitral valve disease

Introduction

To determine the biologic variability of N-terminal pro-brain natriuretic peptide (NTproBNP) in healthy dogs and dogs with various stages of myxomatous mitral valve disease (MMVD).

Effect of spironolactone on diuresis and urine sodium and potassium excretion in healthy dogs

ObjectivesTo document the diuretic effect of different oral doses of spironolactone (SP) in healthy dogs.BackgroundSP is currently mentioned as a diuretic agent in the dog. However, the recommended doses were empirically defined and their corresponding diuretic effect has never been documented in dogs.Animals, materials and methodsEight adult Beagle dogs were used for two separate 2 * 2 cross-over designs. In the first cross-over, 4 dogs received SP orally for 8 days at 1 and 2 mg/kg per day. In the second cross-over the 4 other dogs received SP similarly, but at 4 and 8 mg/kg per day. Dogs were weighed on the first and last day of each period. Plasma SP and canrenone (the main active metabolite of SP) were assayed by high performance liquid chromatography (HPLC). Daily water consumption, urine weight, urine specific gravity, and urine excretion of sodium and potassium were measured during the SP treatment.ResultsTwo hours after SP administration, SP was metabolized into canrenone. A significant 14 and 22% decrease in urine potassium excretion was observed at 1 and 2 mg/kg, respectively, but not at the two other dose levels. Daily water consumption, urine weight, urine specific gravity, and urine excretion of sodium were not significantly altered by the SP treatment regardless of dose.ConclusionsRepeated oral administration of SP at 1, 2, 4 or 8 mg/kg for 8 days had no effect on water and sodium diuresis in healthy dogs.     

Genotypic and phenotypic characterization of Clostridium perfringens and Clostridium difficile in diarrheic and healthy dogs

The objectives of this study were to examine the potential roles of Clostridium difficile and enterotoxigenic Clostridium perfringens in diarrhea in dogs by comparison of isolation, determination of toxin status via enzyme-linked immunosorbent assay (ELISA), and application of multiplex polymerase chain reaction (PCR). These techniques were used to evaluate fecal specimens in 132 healthy and diarrheic dogs. These dogs were prospectively evaluated by grouping them into the following 3 categories: hospitalized dogs with diarrhea (n = 32), hospitalized dogs without diarrhea (n = 42), and apparently healthy outpatient dogs without diarrhea (n = 58). All fecal specimens were cultured using selective media for C difficile, Salmonella spp., and Campylobacter spp. and selective media after heat shock for C perfringens. No significant difference was found in the isolation of C perfringens or C difficile among the 3 groups. A significant association was found between the presence of diarrhea and detection of C perfringens enterotoxin (CPE) or toxin A via ELISA for both C perfringens and C difficile, respectively. PCR performed on C difficile isolates for toxin A and toxin B genes revealed no significant differences among the 3 groups, but diarrheic dogs were significantly more likely to be positive for the enterotoxin gene of C perfringens. Based on the results of this study, the use of ELISA for detection of CPE in feces combined with the detection of enterotoxigenic fecal isolates obtained via heat shock provides the strongest evidence for the presence of C perfringens-associated diarrhea.     

Biological variability of C-reactive protein and specific canine pancreatic lipase immunoreactivity in apparently healthy dogs

Background: C‐reactive protein (CRP) and specific canine pancreatic lipase immunoreactivity (Spec cPL) are biomarkers of generalized or nonspecific inflammation and pancreatic inflammation in dogs, respectively. The extent of inter‐ and intraindividual variation over time of these analytes is not well defined in dogs. The minimal critical difference for sequential determinations of these markers (ie, the smallest change necessary to represent physiological change rather than biological variation), has not been defined. Objectives: To determine the inter‐ and intraindividual variability (CV_G and CV_I) and minimal critical difference for sequential determinations of serum CRP and Spec cPL concentrations in apparently healthy dogs. Animals: Eleven apparently healthy dogs owned by staff or students at a veterinary teaching hospital. Methods: Blood was collected repeatedly at varying intervals over 12 weeks. CRP and Spec cPL concentrations were determined with commercially available assays. Indices of inter‐, intraindividual, and assay variability and 1‐sided minimal critical differences for sequential concentrations were calculated. Results: For CRP, CV_G was 90.8%, CV_I was 115.5%, and the analytical variability (CV_A) was 6.3%; the index of individuality was 0.74, and 1‐sided critical difference was 269.9%. For Spec cPL, CV_G= 49.48%, CV_I= 193.8%, CV_A= 8.4%, index of individuality = 0.24, and 1‐sided critical difference was 452.6%. Conclusions and Clinical Importance: A population‐based reference range is appropriate for Spec cPL, but questionable for CRP in dogs. Large changes in serial measurements of Spec cPL are necessary to infer clinical importance, more modest changes in CRP are likely to be meaningful.