Stomoxys calcitrans, mechanical vector of virulent Besnoitia besnoiti from chronically infected cattle to susceptible rabbit.

Cattle besnoitiosis caused by Besnoitia besnoiti (Eucoccidiorida: Sarcocystidae) is a re-emerging disease in Europe. Its mechanical transmission by biting flies has not been investigated since the 1960s. The aim of this study was to re-examine the ability of Stomoxys calcitrans (Diptera: Muscidae) to transmit virulent B. besnoiti bradyzoites from chronically infected cows to susceptible rabbits. Three batches of 300 stable flies were allowed to take an interrupted bloodmeal on chronically infected cows, followed by an immediate bloodmeal on three rabbits (Group B). A control group of rabbits and a group exposed to the bites of non-infected S. calcitrans were included in the study. Blood quantitative polymerase chain reaction (qPCR) analyses, and clinical, serological and haematological surveys were performed in the three groups over 152 days until the rabbits were killed. Quantitative PCR analyses and histological examinations were performed in 24 tissue samples per rabbit. Only one rabbit in Group B exhibited clinical signs of the acute phase of besnoitiosis (hyperthermia, weight loss, regenerative anaemia and transient positive qPCR in blood) and was seroconverted. Parasite DNA was detected in four tissue samples from this rabbit, but no cysts were observed on histological examination. These findings indicate that S. calcitrans may act as a mechanical vector of B. besnoiti more efficiently than was previously considered.

Hematologic reference intervals in Cynomolgus (Macaca fascicularis) monkeys.

BACKGROUND: Reference intervals are important aids for interpreting clinical pathology laboratory data especially in Cynomolgus monkey (Macaca fascicularis), the non-human primate species most widely used in biomedical research. The purpose of this study was to establish hematologic reference intervals for Cynomolgus according to the International Federation of Clinical Chemistry and Clinical and Laboratory Standards Institute guidelines using the databank at a primatology center. METHODS: Blood specimens from 272 healthy Cynomolgus imported from Mauritius, the Philippines and Vietnam, were analyzed. Reference intervals were established by nonparametric method. Effects of sex, age, body weight, and breeding origin were investigated. RESULTS: Hemoglobin, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration decreased slightly and mean corpuscular volume increased slightly with age. Lower red blood cell concentration, hemoglobin, and hematocrit were observed in monkeys from the Philippines. CONCLUSIONS: These hematology reference intervals, established according to international recommendations, can be used in settings using similar animals and analyzers.

Phosphocalcic metabolism and its potential association with biomarkers of kidney disease in dogs with spontaneous hyperadrenocorticism.

The pathogenesis of increased serum phosphate concentration and proteinuria in dogs with spontaneous hyperadrenocorticism (HAC) is unclear. A potential link between proteinuria and calcium/phosphate metabolism has never been studied in dogs with HAC. The aims of the study were: (1) To evaluate calcium/phosphate metabolism in dogs with spontaneous HAC and compare to healthy dogs as well as to dogs with non-HAC illness; (2) to look for associations between markers of calcium/phosphate metabolism and biomarkers of kidney disease in dogs with HAC. Fifty-four dogs were included in the study, classified as HAC (n=27), non-HAC disease (n=17), and healthy (n=10). Serum calcium, phosphate, 25(OH)Vitamin D, 1,25(OH)2Vitamin D, plasma intact parathyroid hormone concentration (iPTH), FGF23, and urinary fractional excretion of calcium and phosphate were evaluated in all dogs at diagnosis and compared between each group. The correlation between these variables and urine protein-to-creatinine ratio (UPC) and urinary N-acetylglucosaminidase-to-creatinine ratio (uNAG/C) was evaluated in the HAC group. Medians [range] of serum phosphate concentration, urinary fractional excretion of calcium (FE(Ca)), and iPTH were significantly higher in dogs with HAC than in dogs with non-HAC illness (P<0.01) and healthy dogs (P<0.01). Increased 1,25(OH)2Vitamin D/25(OH)Vitamin D was also observed (P<0.001). In HAC group, UPC was significantly negatively correlated with 25(OH)Vitamin D (r(s): -0.54; P<0.01). Urinary NAG/C was significantly positively correlated with serum phosphate (r(s): 0.46; P=0.019). Increased serum phosphate, urinary excretion of calcium, and hyperparathyroidism were observed in dogs with HAC. Vitamin D metabolism may be shifted towards increased 1-alpha hydroxylation.

Description of serum symmetric dimethylarginine concentration and of urinary SDS-AGE pattern in dogs with ACTH dependent hyperadrenocorticism.

Serum symmetric dimethylarginine (SDMA) and patterns of urinary protein separated by sodium dodecyl sulfate agarose gel electrophoresis (SDS-AGE) have not been investigated as biomarkers in dogs with ACTH-dependent hyperadrenocorticism (ADHAC). This exploratory prospective study aimed to evaluate SDMA, serum creatinine (sCR), and SDS-AGE in dogs with ADHAC with and without proteinuria (ADHAC-P and ADHAC-nP, respectively). Thirty-five pet dogs classified as ADHAC-P (n=16), ADHAC-nP (n=6) and healthy (n=13) were included. Renal biomarkers were evaluated in all dogs at diagnosis. Baseline concentration of SDMA was not significantly different between the three groups (P = 0.15) whereas sCr was significantly lower in dogs in ADHAC dogs compared to healthy dogs (88.0 µmol/L [70.4-132.6; 79.2-114.4]) whether they had proteinuria or not (P = 0.014 and 0.002, respectively). However, baseline concentrations of sCr and SDMA were not significantly different between dogs with ADHAC-P dogs (SDMA, 8 µg/dL [5-12; 7-9]; sCr, 57.2 µmol/L [35.2-212.2; 52.8-92.4]) and ADHAC-nP dogs (SDMA, 8.5 µg/dL [7-13; 8-10]; sCr, 70.4 µmol/L [61.6-79.2; 61.6-70.4]) (P = 0.35 and P = 0.41, respectively). Proteinuria in dogs with ADHAC-P was mainly of glomerular origin (SDS-AGE pattern: glomerular in 10/16 dogs; mixed glomerular/tubular in four dogs). In our study, SDMA was neither significantly different in dogs with ADHAC whether they were proteinuric or not, nor between ADHAC and healthy dogs. Urinary electrophoresis provides additional information to the UPC and further investigations are needed to determine whether it may help identify dogs with ADHAC-P requiring specific antiproteinuric treatment.

Short course of immune-suppressive doses of prednisolone, evaluated through a prospective double-masked placebo-controlled clinical trial in healthy Beagles, is associated with sustained modifications in renal, hydration, and electrolytic status.

OBJECTIVE: To investigate the effects and duration of orally administered prednisolone on renal function evaluated by glomerular filtration rate (GFR) determination and creatinine (Cr) and symmetric dimethylarginine (SDMA) concentrations as well as on urinalysis, electrolytes, and hydric status in healthy dogs. ANIMALS: 14 healthy Beagles. PROCEDURES: In this prospective double-masked placebo-controlled study, dogs were randomized after baseline evaluation to receive a 7-day course of either prednisolone (1.5 to 2.0 mg/kg, PO, q 12 h) or a placebo. A repeated-measure design was performed, each dog participating in 4 successive sampling sessions. Clinical data, systolic blood pressure, CBC, and biochemical analyses including serum SDMA concentration, GFR determination, urine output quantification, and complete urinalysis were performed for all dogs the day before (D0) and at the end of steroid administration (D7) as well as 2 weeks (D21) and 4 weeks (D35) after the end of treatment. RESULTS: At D7, when compared with baseline, GFR increased significantly in treated dogs, whereas creatinine and SDMA concentrations decreased significantly. GFR and Cr but not SDMA modifications persisted significantly at D21. None of the variables differed significantly from baseline at D35. The OR of presenting an albumin band on urine electrophoresis was 2.4 times as high in treated versus control dogs (OR, 36; 95% CI, 1.8 to 719.4; P = 0.02). CLINICAL RELEVANCE: A short-term course of immune-suppressive prednisolone treatment in healthy dogs leads to a sustained but reversible renal hyperfiltration state. Modification in electrolytic variables can affect the clinical interpretation of blood work in such patients.

Comparison of measurements of canine plasma glucose, creatinine, urea, total proteins, alanine aminotransferase, and alkaline phosphatase obtained with the APOLOWAKO and Vitros 250 analyzers.

The APOLOWAKO is an entirely automatic benchtop biochemistry analyzer that uses stabilized liquid reagents. It was tested for canine blood and plasma glucose, creatinine, urea, total proteins, alanine aminotransferase, and alkaline phosphatase. The APOLOWAKO gave very similar results for whole blood and the corresponding plasma (n=32). Within-laboratory imprecision was below 2.2% and 5.8% for substrates and enzymes, respectively. Comparison of results with whole blood by APOLOWAKO and with the corresponding plasma by Vitros 250 (n=139) showed very good correlations. Passing-Bablok's regression slopes ranged from 0.83 to 1.12 and intercepts were close to zero, except for ALP where the results obtained by APOLOWAKO were approximately 1.5 times higher than by Vitros. The APOLOWAKO system can be a reliable instrument in veterinary practices where larger systems are not available but it should be further validated and reference intervals should be determined.

Treatment of 11 dogs with meningoencephalomyelitis of unknown origin with a combination of prednisolone and cytosine arabinoside.

The records of 11 dogs with evidence of meningoencephalomyelitis of unknown origin were reviewed. Two of them had had a focal form of the disease and the other nine a disseminated form. The forebrain was involved in five of the nine dogs with disseminated disease, the brainstem in all nine and the cerebellum in one. They had been treated with courses of cytosine arabinoside every three weeks and immunosuppressive doses of prednisolone. Their response to the treatment, in terms of quality of life, was judged by their owners and referring veterinarians to have been excellent in five, good in five and poor in one; their survival times ranged from 78 days to more than 603 days. The cumulative probability of survival at two years was 58.4 per cent. No signs of myelosuppression or other side effects associated with cytosine arabinoside were observed.

A multicentric retrospective study of serum/plasma urea and creatinine concentrations in dogs using univariate and multivariate decision rules to evaluate diagnostic efficiency.

BACKGROUND: Urea and creatinine are the most frequently used indirect markers in plasma and serum of glomerular filtration rate in dogs. Both have been shown to lack sensitivity but their diagnostic efficiency for the diagnosis of kidney disease has been minimally investigated. OBJECTIVE: The purpose of this retrospective study was to investigate the influence of possible factors of variation on both analytes and to determine whether specific decision rules should be drawn up for subpopulations of dogs. METHODS: The results of urea and creatinine measurements, breed, sex, age, and health status (healthy, renal disease, or nonrenal disease) of 3822 dogs were collected from the archives of 5 veterinary clinics. Data were analyzed with univariate and multivariate decision rules with and without adjustment. RESULTS: There were significant effects and interactions of almost all of the sources of variation. Slight improvements in diagnostic efficiency were obtained by adjusting the decision rules to these sources of variations. Univariate decision rules gave approximately the same diagnostic efficiency for urea and creatinine concentrations, with sensitivity and specificity in the range of 70% and 90%, respectively, using the upper limit of the reference interval as the threshold value. Multivariate decision rules provided only minor improvements in diagnostic efficiency. CONCLUSION: Simultaneous measurement of both urea and creatinine is of limited diagnostic value over the analysis of a single variable. Creatinine is the preferred analyte as it is affected by fewer extrarenal factors of variation.

Comparison of measurements of canine plasma creatinine, glucose, proteins, urea, alanine aminotransferase, and alkaline phosphatase obtained with Spotchem SP 4430 and Vitros 250 analyzers.

The suitability of the Spotchem 4430 benchtop biochemistry analyzer for canine blood samples was tested for creatinine, glucose, proteins, urea, alkaline phosphatases and alanine aminotransferase. Results obtained from whole blood and corresponding heparin plasma were identical except for proteins which were higher in plasma (n=10). Between series imprecision (n=10) was <5% for substrates and <10% for enzymes. Comparison of results from 100 Li-heparin samples with those measured with a Vitros 250 analyzer showed good correlation (r>0.93). The slopes of the Passing-Bablock's regression ranged from 0.90 to 1.20 and intercepts were low. The mean biases were low, except for creatinine for which the results obtained by Spotchem (Jaffe reaction) were about 20 micromol/L higher than with the Vitros (enzymatic reaction). The results of this study show that the Spotchem analyzer is suitable for use in canine whole blood or plasma when small numbers of tests are to be performed and large analyzers are not available.

Pharmacokinetics and pharmacodynamics of a therapeutic dose of unfractionated heparin (200 U/kg) administered subcutaneously or intravenously to healthy dogs.

BACKGROUND: Heparin treatment has been recommended for dogs in hypercoagulable states such as disseminated intravascular coagulation, however, potential benefits have to be balanced against the bleeding risk if overdosage occurs. A better understanding of the pharmacology of heparin and tests to monitor heparin therapy in dogs may help prevent therapeutic hazards. OBJECTIVES: The purpose of this study was to evaluate the effects of 200 U/kg of sodium unfractionated heparin (UFH) on coagulation times in dogs after intravenous (IV) and subcutaneous (SC) administration and to compare these effects with plasma heparin concentrations assessed by its antifactor Xa (aXa) activity. METHODS: 200 U/kg of UFH were administered IV and SC to 5 healthy adult Beagle dogs with a washout period of at least 3 days. Activated partial thromboplastin time (APTT), prothrombin time (PT), and plasma aXa activity were determined in serial blood samples. RESULTS: After IV injection, PT remained unchanged except for a slight increase in 1 dog; APTT was not measurable (>60 seconds) for 45-90 minutes, and then decreased gradually to baseline values between 150 and 240 minutes. High plasma heparin concentrations were observed (maximal concentration = 4.64 +/-1.4 aXa U/mL) and decreased according to a slightly concave-convex pattern on a semilogarithmic curve, but returned to baseline slightly more slowly (t240-t300 minutes) than did APTT. After SC administration, APTT was moderately prolonged (by a ratio of 1.55 +/-0.28 APTT t0, range 1.35-2.01) between 1 and 4 hours after administration. Plasma aXa activity reached a maximum of 0.56 +/-0.20 aXa U/mL (range 0.42-0.9 U/mL) after 132 +/-26.8 minutes; this lasted for 102 +/-26.8 minutes. Prolongation of APTTs of 120-160% corresponded to plasma heparin concentrations of 0.3-0.7 aXa U/mL. CONCLUSIONS: As in humans, the pharmacokinetics of UFH in dogs was nonlinear. Administration of 200 U/kg of UFH SC in healthy dogs resulted in sustained plasma heparin concentrations in accordance with human recommendations for thrombosis treatment or prevention, without excessively increased bleeding risks. In these conditions, APTT can be used as a surrogate to assess plasma heparin concentrations. These findings need to be confirmed in diseased animals.

Phosphoinositide 3-kinase inhibition reverses platelet aggregation triggered by the combination of the neutrophil proteinases elastase and cathepsin G without impairing alpha(IIb)beta(3) integrin activation.

Neutrophil elastase (NE) upregulates the fibrinogen binding activity of the platelet integrin alpha(IIb)beta(3) through proteolysis of the alpha(IIb) subunit. This cleavage allows a strong potentiation of platelet aggregation induced by low concentrations of cathepsin G (CG), another neutrophil serine proteinase. During this activation process, we observed a strong fibrinogen binding and aggregation-dependent phosphatidylinositol 3,4-bis-phosphate (PtdIns(3,4)P(2)) accumulation. PtdIns(3,4)P(2) has been suggested to play a role in the stabilization of platelet aggregation, possibly through the control of a maintained alpha(IIb)beta(3) integrin activation. Here we show that inhibition of phosphoinositide 3-kinase (PI 3-K) by very low concentrations of wortmannin or LY294002 transformed the irreversible platelet aggregation induced by a combination of NE and low concentrations of CG into a reversible aggregation. However, although inhibition of PI 3-K was very efficient in inducing platelet disaggregation, it did not modify the level of alpha(IIb)beta(3) activation as assessed by binding of an activation-dependent antibody. These results indicate that PI 3-K activity can control the irreversibility of platelet aggregation even under conditions where alpha(IIb)beta(3) integrin remains activated.

Platelet aggregation induced by the C-terminal peptide of thrombospondin-1 requires the docking protein LAT but is largely independent of alphaIIb/beta3.

Thrombospondin-1 (TSP1) is abundantly secreted during platelet activation and plays a role in irreversible platelet aggregation. A peptide derived from the C-terminal domain of TSP1, RFYVVMWK (RFY) can activate human platelets at least in part via its binding to integrin-associated protein. Although integrin-associated protein is known to physically interact with alphaIIb/beta3, we found that this major platelet integrin had only a partial implication in RFY-mediated platelet aggregation. Accordingly, RFY induced a significant Glanzmann type I thrombasthenic platelet aggregation. The alphaIIb/beta3-dependent part of platelet aggregation induced by RFY was mainly due to secreted ADP and thromboxane A2. In the absence of alphaIIb/beta3 and fibrinogen, RFY stimulated a rapid tyrosine phosphorylation of a set of proteins, including Syk, linker for activation of T cells (LAT) and phospholipase Cgamma2. This signaling pathway was critical for RFY-mediated platelet activation as revealed by the use of pharmacological inhibitors as well as LAT-deficient mouse platelets. Phosphoinositide 3-kinase activation was also required for RFY-mediated platelet aggregation. Our results unravel a new alphaIIb/beta3 and fibrinogen-independent mechanism for platelet aggregation in response to the active peptide from the C-terminal domain of TSP1.

The integrin alpha IIb/beta 3 in human platelet signal transduction.

Platelets are critical for the maintenance of the integrity of the vascular system and are the first line of defence against haemorrhage. When they encounter a subendothelial matrix exposed by injury to a vessel, platelets adhere, are activated, and become adhesive for other platelets so that they aggregate. alpha IIb/beta 3, a platelet-specific integrin, is largely prominent amongst the adhesion receptors and is essential for platelet aggregation. The ligands for alpha IIb/beta 3 are the multivalent adhesive proteins fibrinogen and von Willebrand factor. In resting platelets, alpha IIb/beta 3 is normally in a low activation state, unable to interact with soluble fibrinogen. Stimulation of platelets with various agonists will induce a conformational change in alpha IIb/beta 3 (inside-out signalling), which is then able to bind soluble fibrinogen resulting in the onset of platelet aggregation. However, fibrinogen binding to its membrane receptor is not simply a passive event allowing the formation of intercellular bridges between platelets. Indeed, a complex signalling pathway triggered by integrin ligation and clustering (outside-in signalling) will regulate the extent of irreversible platelet aggregation and clot retraction. Amongst the signalling enzymes activated downstream of alpha IIb/beta 3 engagement, phosphoinositide 3-kinase plays an important role in the control of the irreversible phase of aggregation.

Comparison of whole blood and plasma potassium concentrations in dogs using the Reflovet system.

BACKGROUND: The Reflovet system is designed for chemical analysis of whole blood. However, plasma or serum is recommended for potassium analysis because of possible interference from RBC potassium. Because RBC potassium concentration is low in most canine erythrocytes, however, there should be little or no interference. OBJECTIVE: The objective of this study was to compare potassium results obtained in whole blood and in plasma from dogs using the Reflovet system. METHODS: Blood samples were collected from 104 dogs into lithium-heparin tubes. The potassium concentration was measured in whole blood, and subsequently the PCV was measured. Samples were centrifuged and the potassium concentration was measured in plasma. Comparisons were made using Deming's regression and Bland-Altman difference plots. RESULTS: There was very good correlation between results of potassium measurements in whole blood and plasma (r = 0.93). Potassium values were moderately lower in whole blood: Potassium(blood) = 0.912 x Potassium(plasma)+ 0.119. Hemolysis had a negligible effect on the results, but the difference increased with the PCV value. In more than 90% of samples, the difference between the 2 measurements was

Assessment of a pyrogallol red technique for total protein measurement in the cerebrospinal fluid of dogs.

The measurement of protein concentration in the cerebrospinal fluid is a basic analytical method in neurology. In this study, a pyrogallol red technique using a human albumin calibrator previously validated in human medicine was tested for canine samples, and the results were compared with those obtained using urine test strips. Pyrogallol red significantly (P<0.05) but moderately underestimated purified dog albumin and globulins. The imprecision of the technique was low: intra- and between-series coefficients of variation were 1.6 and 4.3 per cent at protein concentrations of about 0.3 g/litre. Over 49 samples, there was good agreement between the pyrogallol red and test strip results (r=0.63), especially for low and high protein concentrations, but misclassifications were observed with '+' test strip readings.

Breed-specific hematologic reference intervals in healthy adult Dogues de Bordeaux.

BACKGROUND: There is an increasing interest for breed-specific reference intervals in veterinary medicine. In a previous study, breed-specific biochemical reference intervals (RIs) have been established for Dogues de Bordeaux (DDBs). This breed is predisposed to familial juvenile glomerulonephropathy and hypothyroidism, and would benefit from hematologic RI. OBJECTIVE: The purpose was de novo establishment of breed-specific hematologic RIs for the DDB in accordance with the International Federation of Clinical Chemistry and Clinical and Laboratory Standards Institute guidelines. METHODS: One hundred and twenty DDBs from France and Belgium were recruited. CBCs were determined with the Sysmex XT-2000iV analyzer within 12 hours of blood collection. RIs were determined using the nonparametric method. Effects of sex, age, and face mask color were studied. RESULTS: RIs were determined in 58 healthy dogs. DDBs had higher RIs for HGB, HCT, MCV, MCHC, and mean platelet volume, and lower RIs for reticulocytes counts, platelets by impedance (PLT-I) and optical count (PLT-O), and plateletcrit when compared with generic canine RIs. Age significantly affected RIs for HGB, HCT, MCHC, WBC, neutrophil, lymphocyte, and monocyte counts. CONCLUSION: The generic canine RIs established in the same laboratory with analogous preanalytical and analytical variations did not differ significantly from breed-specific RIs, and thus have no significant impact on clinical decision making; however, breed-specific RIs are advised for some RBC and all platelet-related variables to avoid erroneous suspicion of polycythemia and thrombocytopenia when using general canine RIs for evaluation of DDB.

Breed-specific biochemical reference intervals for the adult Dogue de Bordeaux.

BACKGROUND: Breed-specific reference intervals are of increasing interest in veterinary medicine. The health monitoring of the Dogue de Bordeaux, a breed predisposed to familial juvenile glomerulonephropathy and hypothyroidism, would benefit from specific reference intervals. OBJECTIVE: The purpose of this study was to establish breed-specific biochemical reference intervals for the Dogue de Bordeaux in accordance with the International Federation of Clinical Chemistry and Clinical and Laboratory Standards Institute guidelines. METHODS: One hundred and twenty Dogues de Bordeaux from France and Belgium were recruited. Complete urinalysis and chemistry panels, venous blood gas variables, total thyroxin and thyroid stimulating hormone, and fibrinogen and antithrombin were measured for each dog. Reference intervals were determined using the non-parametric method. Confounding variables such as sex, age and color of facial mask were analyzed. RESULTS: Due to pre-defined criteria for exclusion, 62 healthy dogs were finally selected for the reference intervals determination. Using the instrument manufacturer's generic canine RI for most analytes did not have a significant impact on potential clinical decisions, except for total proteins, ALT, AST, total cholesterol, lipase and total thyroxin, for which possible clinically relevant differences were noted. CONCLUSION: Specific reference intervals for biochemical analytes in the Dogue de Bordeaux were determined under controlled pre-analytical and analytical conditions, and according to international recommendations. The use of these breed-specific reference intervals is recommended when using the specified analytic instruments, especially for the 6 analytes for which the reference intervals differed considerably from those provided by manufacturers.

Confidence intervals of reference limits in small reference sample groups.

BACKGROUND: Reference intervals are the most common tool used to interpret results of laboratory tests. However, in veterinary clinical pathology, the number of available reference individuals is often small. OBJECTIVES: The purpose of this study was to investigate the effects of small reference sample groups on the imprecision of the reference limits. METHODS: Gaussian and log-Gaussian distributions of 10 ≤ n ≤ 750 values were analyzed. Reference limits and 90% confidence interval of limits (90% CI) were calculated. Imprecision of limits was estimated by the ratio of the width of the 90% CI: width of the reference interval (WCI/WRI). RESULTS: For Gaussian distributions, the WCI/WRI ratio cannot be expected to be lower than 0.2 when n < 55. In log-Gaussian distributions, the ratio greatly increases for the upper limit with skewness toward high values, whereas it moderately decreases for the lower limit. CONCLUSION: Independent of the size of the reference sample group, it is very important to report the CIs of the reference limits, which can be very large for small reference sample groups. When the sample size is very small (n < 20), calculations maybe misleading and it is better to instead report all values.

Estimation of reference intervals from small samples: an example using canine plasma creatinine.

BACKGROUND: According to international recommendations, reference intervals should be determined from at least 120 reference individuals, which often are impossible to achieve in veterinary clinical pathology, especially for wild animals. When only a small number of reference subjects is available, the possible bias cannot be known and the normality of the distribution cannot be evaluated. A comparison of reference intervals estimated by different methods could be helpful. OBJECTIVE: The purpose of this study was to compare reference limits determined from a large set of canine plasma creatinine reference values, and large subsets of this data, with estimates obtained from small samples selected randomly. METHODS: Twenty sets each of 120 and 27 samples were randomly selected from a set of 1439 plasma creatinine results obtained from healthy dogs in another study. Reference intervals for the whole sample and for the large samples were determined by a nonparametric method. The estimated reference limits for the small samples were minimum and maximum, mean +/- 2 SD of native and Box-Cox-transformed values, 2.5th and 97.5th percentiles by a robust method on native and Box-Cox-transformed values, and estimates from diagrams of cumulative distribution functions. RESULTS: The whole sample had a heavily skewed distribution, which approached Gaussian after Box-Cox transformation. The reference limits estimated from small samples were highly variable. The closest estimates to the 1439-result reference interval for 27-result subsamples were obtained by both parametric and robust methods after Box-Cox transformation but were grossly erroneous in some cases. CONCLUSION: For small samples, it is recommended that all values be reported graphically in a dot plot or histogram and that estimates of the reference limits be compared using different methods.