Effect of storage conditions on prothrombin time, activated partial thromboplastin time and fibrinogen concentration on canine plasma samples

The present study was to assess the effect of storage conditions on prothrombin time (PT), activated partial thromboplastin time (aPTT) and fibrinogen concentration in blood samples of healthy dogs. Thirty-five dogs of various breeds were included in the study. Citrated blood samples were obtained and plasma was divided into four aliquots to assess selected clotting parameters by means of a coagulometer. The first aliquot was analysed within 1 h after collection, while the remaining 3 were stored at 8℃ for 4, 8 and 24 h, respectively. One-way repeated measures analysis of variance documented a significant decreasing effect on PT at 24 h compared to 8 h and on fibrinogen concentration after 8 and 24 h compared to sampling time and at 4 and 24 h compared to 8 h post sampling. In conclusion, the results of this study indicate that only fibrinogen appears prone to significant decrease. In fact, aPTT is not substantially affected by refrigeration for at least 24 h post sampling and PT showed a statistical difference that does not necessary indicate biological significance as the results obtained were within reference intervals for the dog.     

Determination of plasma albumin concentration in healthy and diseased turtles: a comparison of protein electrophoresis and the bromcresol green dye‐binding method

Background: In reptile medicine, plasma chemistry analysis is widely used for the evaluation of an individual's health status. The standard method for the determination of plasma albumin concentration is protein electrophoresis combined with the determination of total protein concentration, but the bromcresol green (BCG) dye‐binding method is also used. The reliability of the BCG method for the measurement of albumin concentration in reptiles is unknown. Objective: The aim of this study was to compare the plasma albumin values of turtles obtained by protein electrophoresis and the BCG method. Methods: Between March 2008 and September 2008, heparinized plasma samples from 16 clinically healthy and 10 diseased turtles of different species were collected. Plasma albumin concentrations were measured by protein electrophoresis and by the BCG method. The results of the 2 methods were compared using Passing–Bablok regression and Bland–Altman plots. Results: Albumin concentration measured by BCG was weakly correlated with the corresponding protein electrophoretic values in all turtles (r_s=.610, P<.001) and in healthy turtles evaluated separately (r_s=.700, P=.003), whereas in diseased turtles no such correlation was found (r_s=.374, P=.287). The albumin concentration measured with the 2 different methods differed significantly in all turtles (P=.009; Wilcoxon's test) and in healthy turtles (P=.005) but not in diseased animals (P=.241). In the Bland–Altman plot a systematic error was found between the 2 methods in diseased turtles. Conclusion: Measurement of albumin by the BCG dye‐binding method may lead to inaccurate results for plasma albumin concentration, especially in ill turtles. Therefore, for health assessment in turtles, albumin should be measured by protein electrophoresis.     

Measurement of ionized calcium in canine blood samples collected in prefilled and self-filled heparinized syringes using the i-STAT point-of-care analyzer

BACKGROUND: Heparinized syringes are commonly used with point-of-care analyzers (eg, i-STAT) to measure ionized calcium (iCa(2+)); however there is little information about the validity of their use in canine patients. OBJECTIVE: To examine the suitability of prefilled (40 IU heparin/mL) and self-filled (150 IU heparin/mL) heparinized syringes for iCa(2+) measurements using the i-STAT analyzer. METHODS: Forty-seven blood samples were collected from 41 canine patients. Two milliliters of blood were collected into a 2-mL nonanticoagulated (NA) syringe, a commercially available preheparinized (PH) syringe (dry calcium-balanced lithium heparin, 40 IU/mL), and a 2-mL self-filled heparinized (SH) syringe (liquid sodium heparin, 150 IU/mL). iCa(2+) was measured in the sample using the i-STAT analyzer and a wet-reagent analyzer (KoneLab 30i) used as the reference instrument. Data were analyzed using paired t-tests, Pearson correlation coefficient, and Bland-Altman difference plots. RESULTS: There was no significant difference between the results obtained from NA and PH syringes using the i-STAT analyzer, and the correlation was excellent (r =.97). The i-STAT values from the SH syringes (mean, 1.07 mmol/L) were significantly lower (P<.001) than those from the NA syringes (mean, 1.38 mmol/L). iCa(2+) was significantly higher with the i-STAT analyzer than with the KoneLab analyzer for both the PH (mean i-STAT, 1.38 mmol/L vs mean KoneLab, 1.30 mmol/L) and SH (mean i-STAT, 1.07 mmol/L vs mean KoneLab, 1.03 mmol/L) samples. CONCLUSIONS: Blood samples collected in the PH syringes used in this study can be used interchangeably with those collected in NA syringes for measuring iCa(2+) using the i-STAT analyzer. SH syringes with high-concentration heparin products (5000 IU/mL) are unsuitable for measuring iCa(2+) because they cause clinically significant underestimations. Although there was good correlation between the i-STAT and KoneLab analyzers, the results should be interpreted using analyzer-specific reference intervals.     

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.     

Effect of repeated freeze-thaw cycles on routine plasma biochemical constituents in canine plasma

BACKGROUND: The effect of repeated freeze-thaw cycles on plasma constituents has not been assessed in dogs, although such a procedure is not uncommon to use in routine laboratory practice. OBJECTIVE: To assess the effect of freeze-thaw cycles on routine plasma constituents in healthy dogs. METHODS: Six healthy adult dogs were used. Blood was sampled and placed in heparinized tubes. After centrifugation, plasma was separated into 5 aliquots. One aliquot was considered as the reference aliquot and used immediately for the assay of all of the biochemical constituents. All of the other aliquots were stored at 20 degrees C. Three aliquots underwent 1, 2, or 3 freeze-thaw cycles during a 1- to 3-day period. The last aliquot remained at 20 degrees C throughout the study and was thawed on the third day. The following biochemical constituents were assayed: glucose, urea, creatinine, total proteins, sodium, potassium, chloride, calcium, phosphates, aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), and alkaline phosphatase (ALP). RESULTS: No clinically relevant change was observed between the different aliquots for all of the constituents. CONCLUSION: Repeated freeze-thaw cycles do not cause changes in the biochemical constituents studied in canine plasma.     

Inaccuracy of routine creatinine measurement in canine urine

BACKGROUND: Urine creatinine concentration often is used in ratios such as urine protein:creatinine to compensate for dilution or concentration of spot urine samples. OBJECTIVE: The purpose of this study was to compare the accuracy of different techniques of urine creatinine measurement currently available for veterinary practitioners. METHODS: In 104 samples of canine urine diluted 1:20 with distilled water, creatinine concentration was measured using a kinetic Jaffé reaction assay, and an enzymatic technique on an automatic analyzer (Elimat) and 3 benchtop analyzers (Reflovet, Scil; Vitros DT2, Ortho-Clinical Diagnostics; Vettest 8008, IDEXX) used in veterinary practice. RESULTS: The Jaffé and enzymatic techniques on the Elimat were not significantly different, and their inaccuracy tested with human control urines was <5%. The benchtop analyzers underestimated creatinine concentration, especially at concentrations >2000 mg/L. Inaccuracy was higher with multilayer slide technology systems (Vitros and Vettest) than with the Reflovet system. Results were approximately 25% and 2% lower, respectively, than with the Elimat at urine creatinine concentrations about 2000 mg/L. CONCLUSION: Inaccuracy in urine creatinine measurements using benchtop analyzers should be taken into account when defining decision thresholds, which should be corrected according to the method used to avoid misinterpretations.     

Evaluation of a chromogenic assay to measure the factor Xa inhibitory activity of unfractionated heparin in canine plasma

BACKGROUND: Unfractionated heparin (UFH) has a complex pharmacologic profile that necessitates patient monitoring to prevent inadequate anticoagulation or overdosage and hemorrhage. Factor Xa inhibitory assays (to measure anti-Xa activity) are used to adjust UFH dosage and define safe and effective regimens for specific thrombotic disorders in humans. OBJECTIVE: In this study, the accuracy, linearity, and clinical utility of a chromogenic assay were assessed for monitoring UFH anti-Xa activity in canine plasma samples. METHODS: A commercial assay (Rotachrom Heparin, Diagnostica Stago, Parsippany, NJ, USA) was used to measure anti-Xa activity in canine plasma samples spiked with different concentrations of UFH. Background absorbance and assay linearity were compared for canine and human plasmas. Percentage recovery of UFH anti-Xa activity and intra- and interassay imprecisions were investigated by multiple measurements of canine plasma to which known amounts of UFH were added. The spiked plasma samples also were used to determine the heparin sensitivity of an activated partial thromboplastin time (aPTT) test. RESULTS: Canine plasma samples were assayed at a higher dilution than were human plasma samples (3:8 versus 4:8) to eliminate higher background anti-Xa activity in canine plasma. Using this modification, the recovery of anti-Xa activity in canine plasma was linear (R2 > .9) at concentrations of 0 - 0.75 U/mL UFH. Intra- and interassay imprecisions for plasma samples containing 0.5 U/mL UFH were <10%, whereas samples containing 0.25 U/mL UFH had imprecisions of 13% and 24%, respectively. The anti-Xa activity range of 0.5 - 0.75 U/mL caused prolongation of aPTTs to 1.5 - 2.5 times the assay mean. CONCLUSION: Plasma anti-Xa activity of dogs treated with UFH can be accurately monitored using this commercially available chromogenic assay.     

Various protein and albumin corrections of the serum fructosamine concentration in the diagnosis of canine diabetes mellitus

Fructosamines are formed when glucose reacts non-enzymatically with amino groups on proteins, and previous studies have indicated that the serum fructosamine concentration could be of importance in the diagnosis of canine diabetes mellitus. Owing to the connection between the protein/albumin concentration and serum fructosamine concentration, it has been suggested that the serum fructosamine concentration should be corrected for the protein/albumin concentration. Thus, the purpose of the present study was to evaluate the uncorrected serum fructosamine concentration and various protein and albumin corrections of the serum fructosamine concentration in the separation of dogs with diabetes mellitus from dogs with other diseases that presented with clinical signs suggestive of diabetes mellitus. The evaluation was assisted by relative operating characteristic curves (ROC curves), which may be used to compare various diagnostic tests under equivalent conditions (equal true positive ratios or false positive ratios) and over the entire range of cutoff values. A total of 58 dogs (15 dogs with diabetes mellitus and 43 dogs with other diseases) were included in the study. Serum fructosamine concentration, serum total protein concentration and serum albumin concentration were measured in each dog, and various corrections of the serum fructosamine concentration for protein or albumin concentration were made. Comparing the ROC curves of the uncorrected and each corrected serum fructosamine concentration indicated that there was no decisive difference between the uncorrected and the corrected serum fructosamine concentrations in discriminating between dogs with and without diabetes mellitus. Hence, correcting the serum fructosamine concentration as a routine procedure cannot be advocated from the results of the study. Moreover, the sensitivity and specificity of the uncorrected serum fructosamine concentration were very high, 0.93 and 0.95, respectively, further evidence of the value of the uncorrected serum fructosamine concentration in the diagnosis of canine diabetes mellitus.Abbreviations SFC serum fructosamine concentration - SFC-P serum fructosamine concentration corrected for the actual serum total protein concentration - SFC-A serum fructosamine concentration corrected for the actual serum albumin concentration - SFC-Po serum fructosamine concentration corrected for the actual serum total protein concentration, only when the serum total protein concentration is outside the reference interval - SFC-Ao serum fructosamine concentration corrected for the actual serum albumin concentration, only when the serum albumin concentration is outside the reference interval - SFC-K serum fructosamine concentration corrected according to Kawamotoet al. (1992)     

Validation of a commercially available enzyme-linked immunosorbent assay (ELISA) for the determination of cortisol in canine plasma samples

Samples were obtained from clinically normal dogs before and after ACTH stimulation and dexamethasone suppression tests. The test kit Enzymun-Test (Boehringer Mannheim) for determining cortisol concentrations in human plasma was used in connection with the analyser system Enzymun-Test (Boehringer Mannheim) System ES300 following the manufacturer's instructions. The intra-assay and inter-assay coefficients of variation were 1.28% and 5.64%, respectively. The mean recovery when assaying samples with a cortisol content of more than 100 nmol/L was 95.41%, but this percentage decreased in samples with lower cortisol levels. The sensitivity of the assay was 2.76 nmol/L. The results of the ACTH stimulation and dexamethasone suppression tests were similar to those published previously. The ELISA method evaluated allows a precise and sensitive determination of cortisol concentrations in canine plasma samples. The major drawback observed was the loss of accuracy at low cortisol concentrations. Since the assay tends then to report lower cortisol concentrations, the generally accepted concentration of 40 nmol/L may not be suitable as the cutoff value in dexamethasone suppression tests.     

Cytologic interpretation of canine cerebrospinal fluid samples with low total nucleated cell concentration,with and without blood contamination

Background: Cerebrospinal fluid (CSF) is potentially altered by iatrogenic blood contamination at the time of sampling due to the addition of blood‐associated leukocytes and protein. Objectives: The objective of this study was to assess whether protein concentration, neutrophil percentage, and the presence of activated macrophages, reactive lymphocytes, or eosinophils in CSF samples with low total nucleated cell concentration (TNCC) are affected by blood contamination or associated with central nervous system (CNS) disease. Methods: Case records from the Royal Veterinary College Diagnostic Laboratory were searched retrospectively for dogs with CSF having ≤5 TNCC/μL. TNCC, RBC, and protein concentrations; neutrophil percentage; and the presence of activated macrophages, reactive lymphocytes, and eosinophils were recorded. Results of magnetic resonance imaging (MRI) also were recorded as a marker of CNS disease. Results: Of 906 cases evaluated, 106 (12%) had blood contamination (>500 RBCs/μL) in CSF. Protein concentration and neutrophil percentage were significantly higher and the presence of eosinophils was more likely in blood contaminated vs noncontaminated samples. Non‐blood‐contaminated samples with activated macrophages or reactive lymphocytes had higher protein concentrations and neutrophil percentages, and those with activated macrophages were more likely to have a positive finding on MRI. Conclusions: Protein concentration, neutrophil percentage, and the presence of eosinophils are significantly affected by blood contamination in canine CSF having low TNCC. Activated macrophages and reactive lymphocytes are not affected by blood contamination, however, and may be useful in identifying dogs with CNS abnormalities.     

Comparison of quantitative immunoturbidimetric and semiquantitative latex‐agglutination assays for D‐dimer measurement in canine plasma

Background: D‐dimer measurement in dogs is considered the most reliable test for detecting disseminated intravascular coagulation or thromboembolism. Objectives: The purposes of this study were to compare 2 D‐dimer assays, a quantitative immunoturbidimetric and a semiquantitative latex agglutination assay, and to assess the effect of hemolysis and storage conditions on D‐dimer concentration using the quantitative assay. Methods: The immunoturbidimetric assay was validated using canine citrated plasma samples containing different concentrations of D‐dimer. The effect of storage at various temperatures and times was assessed. Hemolysis was produced by adding lysed RBCs to the samples for a final hemoglobin concentration of 0.35 g/dL. Results: For clinically relevant values (>250 μg/L), intra‐assay and interassay coefficients of variation were 6.8% and 7.2%. The assay was linear (r²=1.00), and the tests had good agreement (κ=0.685, P<.001). Storage at 4 °C and ?20 °C and hemolysis had no significant effect on D‐dimer concentrations. In hemolyzed samples stored at room temperature for ≥48 hours, fine clots were noted and often resulted in falsely increased D‐dimer concentrations. Conclusions: Our findings suggest that the immunoturbidimetric assay validated in this study is reliable and accurate for the measurement of D‐dimer in canine plasma. Samples can be stored for up to 1 month at ?20 °C and moderate hemolysis does not significantly affect the D‐dimer concentration in frozen or refrigerated samples.     

Fecal alpha1-proteinase inhibitor concentration in dogs with chronic gastrointestinal disease

Background: Fecal α₁‐proteinase inhibitor (α₁‐PI) clearance is a reliable, noninvasive marker for protein‐losing enteropathy in human beings. An assay for use in dogs has been developed and validated. Objective: The aim of this study was to evaluate fecal α₁‐PI concentration in dogs with chronic gastrointestinal disease, compared with healthy dogs, and to assess its correlation with serum albumin concentration. Methods: Fecal samples were collected from 2 groups of dogs. Group 1 consisted of 21 clinically healthy client‐owned dogs without signs of gastrointestinal disease. Group 2 consisted of 16 dogs referred for investigation of suspected gastrointestinal disease. On the basis of gastric and duodenal biopsies, group 2 was further subdivided into dogs with normal histology (n = 9) and those with histologic abnormalities (n = 7: inflammatory bowel disease, n = 3; lymphangiectasia, n = 4). An ELISA was used to measure α₁‐PI concentrations in fecal extracts. Results: Fecal α₁‐PI concentrations, expressed as μg/g of feces, were not significantly different between groups 1 and 2 as a whole. However, fecal α₁‐PI concentrations (median, minimum‐maximum) were significantly higher in dogs with gastrointestinal diseases associated with histologic abnormalities (60.6 μg/g, 7.4–201.7 μg/g) compared with dogs with normal histology (3.8 μg/g, 0.7–74.0 μg/g) and control dogs (9.9 μg/g, 0.0–32.1 μg/g). There was no significant correlation between fecal α₁‐PI and serum albumin concentrations in dogs with gastrointestinal disease. Conclusions: Increased fecal α₁‐PI concentration may signal the need to obtain gastrointestinal biopsies for a final diagnosis. Fecal α₁‐PI concentration may be a useful test for early detection of protein‐losing enteropathy before decreases in serum albumin concentration can be detected.     

Creatinine concentration in plasma from dog, rat, and mouse: a comparison of 3 different methods

BACKGROUND: There are numerous methods for analyzing creatinine concentration in plasma, including the Jaffé alkaline picrate method in various modifications, enzymatic tests, and chromatographic methods. OBJECTIVE: The purpose of this study was to evaluate whether an enzymatic method could replace a Jaffé method for routine creatinine measurements in plasma from dogs, rats, and mice. The enzymatic method and a compensated Jaffé method were tested against a high-pressure liquid chromatography (HPLC) method, regarded as the gold standard for creatinine measurement. METHODS: Heparinized plasma samples were obtained from 20 beagle dogs, 20 Wistar rats, and 20 CD1-strain mice. The 2 test kits (Roche Diagnostics), Creatinine Jaffé Compensated and the enzymatic Creatinine Plus Version 2 reagent, were used on a Cobas Integra 400. The Jaffé compensated method used a calibration adjustment of 18 micromol/L to correct for the protein matrix in serum and plasma. The HPLC method was an isocratic method using a weak cation-exchange column following protein precipitation. RESULTS: Creatinine concentrations obtained using the enzymatic and the Jaffé methods differed significantly from the results obtained by the HPLC method. For dog plasma, mean values of 61.2, 61.8, and 67.8 micromol/L were obtained by the compensated Jaffé, enzymatic and HPLC methods, respectively. In the rat, respective mean values were 26.7, 21.9, and 23.0 micromol/L, and in the mouse, respective mean values were 14.2, 5.4, and 9.2 micromol/L. CONCLUSION: The enzymatic method can replace the Jaffé method for plasma creatinine determination in dogs, rats, and mice because results from the enzymatic method were closer to HPLC values than were those of the Jaffé method.     

Outbreak of ocular disease associated with naturally-acquired canine herpesvirus-1 infection in a closed domestic dog colony

Objective  To describe clinical and virological findings of an outbreak of ocular disease attributed to naturally-acquired primary canine herpesvirus-1 (CHV-1) infection in a closed domestic dog colony.
Animals studied  Twenty-seven 10- to 16-week-old laboratory Beagles.
Procedure  Complete ophthalmic examinations were performed and ocular samples collected for CHV-1 polymerase chain reaction and virus isolation.
Results  The prevalence of ocular morbidity was 100% in examined dogs. Lesions were restricted to the ocular surface and included bilateral conjunctivitis (100% of dogs); punctate, dendritic, or geographic ulcerative keratitis (26% of dogs); and non-ulcerative keratitis (19% of dogs). Conjunctival petechiae were detected in 22% of dogs. Punctate and dendritic corneal ulcers were frequently organized into discrete groups or linear arrangements. Non-ulcerative keratitis appeared clinically as a perilimbal ring of superficial corneal vascularization and leukocyte infiltration. CHV-1 was detected in ocular samples by polymerase chain reaction or virus isolation in all dogs sampled.
Conclusions  In susceptible populations of domestic dogs, CHV-1 may be associated with outbreaks of highly contagious ocular infection in the absence of concurrent overt systemic disease. This naturally-acquired outbreak of CHV-1 infection provides an opportunity to report the spectrum and prevalence of ocular lesions associated with primary ocular CHV-1 infection in dogs. Conjunctivitis was the most frequent ocular lesion detected. Ulcerative and non-ulcerative keratitis were less prevalent and of variable clinical appearance. Dendritic ulcerative keratitis, a classic and relatively specific ocular lesion associated with alphaherpesvirus infection, was detected in < 20% of dogs.