Both rabbits (Oryctolagus cuniculus) and ferrets (Mustela putorius furo) are susceptible to several diseases that result in azotemia, and BUN concentration is frequently measured to screen for, diagnose, or monitor these diseases.1 It would be helpful, therefore, for veterinarians who provide medical care for rabbits and ferrets to have a fast, accurate method of measuring BUN concentration.
Point-of-care testing is common in human medicine and becoming increasingly so in veterinary medicine.2,3 Because of the small sample volume needed, use of reagent test strips for point-of-care testing can be particularly advantageous for small animals such as rabbits and ferrets and for animals requiring repeated measurements (eg, to monitor trends). However, the clinical importance of a point-of-care test result in a particular species is unknown until the test has been determined to produce valid results in that species. Commercially available reagent test strips have been validated for estimation of BUN concentration in dogs and cats,4–6 wild fruit bats (Rousettus aegyptiacus),7 and captive black-tailed prairie dogs (Cynomys ludovicianus).8 To our knowledge, they have not been validated for use in rabbits or ferrets.
The American Society for Veterinary Clinical Pathology has published guidelines for validation of point-of-care tests for use in a new species. These guidelines include, among other steps, documentation of analytic performance of the test in the new species and evaluation of test performance with respect to an existing reference (gold-standard) method.9 The purpose of the study reported here was to evaluate the utility of commercially available reagent test strips for estimation of BUN concentration and detection of azotemia in pet rabbits and ferrets. We hypothesized that the test strips would accurately estimate BUN concentration and detect azotemia when compared with results obtained by use of a standard clinical laboratory biochemical analyzer.
Materials and Methods
All procedures related to the use of animals in the study conformed to guidelines approved by the Kansas State University College of Veterinary Medicine's Institutional Animal Care and Use Committee (protocol No. 3510.1). Blood samples obtained from pet rabbits and ferrets by the Exotic and Zoo Animal Medicine Service, Kansas State University Veterinary Health Center, as part of a routine wellness examination or because of clinical disease were considered for inclusion in the study. Rabbit and ferret blood samples were excluded if they were of insufficient volume or if sample processing was inconsistent with the study protocol. Because blood samples were obtained as part of routine care for the animal, no client consent was required.
For blood sample collection from ferrets, anesthesia was first induced with 5% isoflurane in oxygen (5.0 L/min delivered via a chamber) and maintained with 2% isoflurane in oxygen (2.0 L/min delivered via a small face mask and nonrebreathing circuit). A 1-mL heparinized syringe (flushed with heparin sodium [1,000 U/mL]) attached to a 25-gauge, 0.625-inch needle was then used to obtain a blood sample from the cranial vena cava. For blood sample collection from rabbits, rabbits were manually restrained, and a blood sample was obtained from a lateral saphenous vein with a heparinized syringe and needle of the same sizes as for ferrets.
Within 5 minutes after each blood sample was collected, BUN concentration was estimated with a commercially available reagent test stripa in accordance with the manufacturer's guidelines.10 Briefly, a large drop of blood was placed over the reagent pad of the test strip, then rinsed off with tap water after exactly 60 seconds. Results were read immediately by comparing the color of the reagent pad with the manufacturer-provided color chart. All results were read under white fluorescent light by a single individual (DE). Test strips were stored in their original containers under conditions consistent with the manufacturer's guidelines (ie, in a dark cabinet drawer, away from light and humidity, and in a temperature-controlled room [22°C to 24°C {72°F to 75°F}]). Test strips in all open containers were used prior to their expiration date, and containers were replaced every other month.
Test strips provided a semiquantitative measurement of BUN concentration, with the degree of color change in the reagent pad (from yellow to green) indicative of BUN concentration in the sample. Results were assigned to categories ranging from 1 to 4. According to the manufacturer, category 1 corresponded to a BUN concentration of 5 to 15 mg/dL, category 2 corresponded to a BUN concentration of 15 to 26 mg/dL, category 3 corresponded to a BUN concentration of 30 to 40 mg/dL, and category 4 corresponded to a BUN concentration of 50 to 80 mg/dL.
The remainder of each blood sample was placed in a blood-collection tube containing lithium heparinb and immediately submitted to the Kansas State Veterinary Diagnostic Laboratory for analysis with an automated clinical laboratory biochemical analyzer.c All samples were analyzed within 2 hours after collection. For determination of BUN concentration, samples were centrifuged for 10 minutes at 3,000 × g, and the plasma was transferred to a microcentrifuge tube.
The biochemical analyzer was maintained and calibrated according to the manufacturer's instructions and standard operating protocols for the laboratory. Instrument performance was monitored daily by use of commercial quality control serad with 12s or 13s rules, depending on the analyte measured. External quality control for the biochemical analyzer was performed quarterly through the Veterinary Laboratory Association Quality Assurance Program. All analyses were performed by trained laboratory medical technologists.
For comparison with test strip performance, BUN concentrations measured with the biochemical analyzer were assigned to categories ranging from 1 to 4 that generally corresponded to the manufacturer's assigned test strip categories. Samples with a BUN concentration of 5 to 14 mg/dL were assigned to category 1, samples with a BUN concentration of 15 to 26 mg/dL were assigned to category 2, samples with a BUN concentration of 27 to 40 mg/dL were assigned to category 3, and samples with a BUN concentration ≥ 41 mg/dL were assigned to category 4.
To evaluate precision of the test strips, BUN concentration was estimated 3 times each (with 3 test strips from the same package) for blood samples from a convenience sample (samples of sufficient volume collected during the last month of the study period) consisting of 20 samples from 19 rabbits and 18 samples from 15 ferrets.
Statistical analysis
Descriptive statistics (ie, median and interquartile [25th to 75th percentile] range) were calculated with standard software.e The Shapiro-Wilk expanded test was used to determine whether interval data for the test strip BUN category (ie, categories 1 to 4) and biochemical analyzer BUN category (ie, categories 1 to 4) and continuous data for BUN concentration measured with the biochemical analyzer were normally distributed. Because these data were not normally distributed, the Wilcoxon signed rank test was used to compare the distribution of the test strip BUN categories with that of the biochemical analyzer BUN categories. In addition, agreement between test strip and biochemical analyzer BUN categories was assessed by calculating the weighted Cohen κ coefficient; agreement was interpreted as poor (κ < 0.00), slight (κ = 0.00 to 0.20), fair (κ = 0.21 to 0.40), moderate (κ = 0.41 to 0.60), substantial (κ = 0.61 to 0.80), or almost perfect (κ = 0.81 to 0.99).11
To determine the utility of the test strips for detecting azotemia, animals were classified as having or not having azotemia on the basis of BUN concentration measured with the biochemical analyzer. Rabbits with a BUN concentration ≥ 27 mg/dL12 and ferrets with a concentration ≥ 41 mg/dL13 were considered to have azotemia. Sensitivity, specificity, and accuracy of the test strips for detection of azotemia were then calculated by use of standard equations.14 For rabbit samples, a test strip BUN category of 3 or 4 was considered positive for azotemia; for ferret samples, a test strip BUN category of 4 was considered positive for azotemia.
To evaluate precision of the test strips, the precision for each sample was calculated as the number of concordant test strip BUN category results divided by 3. Overall precision was calculated as the mean of the individual sample precision values.
Results
During the study period, 194 rabbits and 90 ferrets were evaluated for a routine wellness examination or because of clinical disease and had a blood sample collected; samples from 141 rabbits and 40 ferrets were excluded because they were of insufficient volume or sample processing was not consistent with the study protocol. Included blood samples were obtained from 53 rabbits (16 castrated males, 14 spayed females, 12 sexually intact females, and 11 sexually intact males) and 50 ferrets (28 spayed females, 21 castrated males, and 1 sexually intact male). Ten rabbits and 13 ferrets had blood samples collected during > 1 visit (maximum, 3 samples/rabbit and 4 samples/ferret), so that a total of 65 rabbit blood samples and 71 ferret blood samples were included in the study. Each sample was treated as independent.
For the 65 rabbit blood samples, BUN concentrations measured with the biochemical analyzer ranged from 9 to 269 mg/dL (median, 19 mg/dL; interquartile range, 16 to 24 mg/dL). Test strip and biochemical analyzer BUN categories were concordant (ie, assigned to the same category) for 46 of the 65 (71%) rabbit blood samples (Figure 1). For 13 of the 65 (20%) samples, the test strip BUN category was 1 category higher than the biochemical analyzer BUN category, and for 6 (9%) samples, the test strip BUN category was 1 category lower than the biochemical analyzer BUN category. Distributions for the test strip and biochemical analyzer BUN categories did not differ significantly (P = 0.16) from each other, and there was substantial agreement between categories assigned with the 2 methods (κ = 0.65).
Overall, 13 of the 65 (20%) rabbit blood samples were classified as azotemic (ie, biochemical analyzer BUN concentration ≥ 27 mg/dL). Test strips yielded false-positive results for azotemia for 11 of 23 (48%) rabbit blood samples, with biochemical analyzer BUN concentration for these 11 samples ranging from 18 to 24 mg/dL. Test strips yielded a false-negative result for azotemia for 1 of 42 (2%) rabbit blood samples; biochemical analyzer BUN concentration for this sample was 30 mg/dL. For detection of azotemia in rabbits, sensitivity of the test strips was 92% (12/13; 95% confidence interval, 64% to 100%), specificity was 79% (41/52; 95% confidence interval, 65% to 89%), and accuracy was 82% (53/65; 95% confidence interval, 70% to 90%).
For the 20 rabbit blood samples tested 3 times each with test strips, 18 samples had concordant results for all 3 tests (ie, sample precision of 100%) and 2 samples had concordant results for 2 of 3 tests (ie, sample precision of 67%). The overall precision (ie, mean of sample precision values) was 97%.
For the 71 ferret blood samples, BUN concentration determined with the biochemical analyzer ranged from 8 to 267 mg/dL (median, 26 mg/dL; interquartile range, 20 to 39 mg/dL). Test strip and biochemical analyzer BUN categories were concordant (ie, assigned to the same category) for 58 of the 71 (82%) ferret blood samples (Figure 2). For 6 of the 71 (8%) ferret blood samples, the test strip BUN category was 1 category higher than the biochemical analyzer BUN category; for another 6 (8%) samples, the test strip BUN category was 1 category lower than the biochemical analyzer BUN category. For the remaining sample with discordant results, the test strip BUN category was 2 categories lower than the biochemical analyzer BUN category. Distributions for the test strip and biochemical analyzer BUN categories did not differ significantly (P = 0.65) from each other, and there was substantial agreement between categories assigned with the 2 methods (κ = 0.80).
Overall, 15 of the 71 (21%) ferret blood samples were classified as azotemic (ie, biochemical analyzer BUN concentration ≥ 41 mg/dL). Test strips did not yield any false-positive results for azotemia; however, they yielded false-negative results for azotemia for 3 of 59 (5%) ferret blood samples; biochemical analyzer BUN concentrations for these 3 samples were 54, 55, and 59 mg/dL. For detection of azotemia in ferrets, sensitivity of the test strips was 80% (12/15; 95% confidence interval, 52% to 96%), specificity was 100% (56/56; 95% confidence interval, 94% to 100%), and accuracy was 96% (68/71; 95% confidence interval, 88% to 99%).
For the 18 ferret blood samples tested 3 times each with test strips, all samples had concordant results for all 3 tests. The overall precision was 100%.
Discussion
Results of the present study indicated that, for rabbit and ferret blood samples, the commercially available reagent test strips yielded BUN category results that were in substantial agreement with BUN categories assigned on the basis of concentrations determined with a biochemical analyzer. Accuracy of the test strips for detection of azotemia was good for both rabbit and ferret samples, as was precision of the test strip results.
Of the 42 rabbit blood samples with a negative test strip result for azotemia (ie, a test strip BUN category of 1 or 2), 41 (98%; 95% confidence interval, 86% to 100%) were truly not azotemic (ie, BUN concentration determined with the biochemical analyzer was < 27 mg/dL), suggesting that the test strip results were generally a reliable indicator that azotemia was not present. However, only 12 of the 23 (52%; 95% confidence interval, 39% to 65%) samples with a positive test strip result for azotemia (ie, a test strip BUN category of 3 or 4) were truly azotemic, with the remaining positive test strip results for azotemia classified as false-positive results. Together, these findings suggested that test strips should be used primarily as a screening test to rule out azotemia in rabbits and that blood samples with a positive test strip result for azotemia should be further tested with a biochemical analyzer to confirm whether azotemia is present.
Fifty-six of the 59 (95%; 95% confidence interval, 87% to 98%) ferret blood samples with a negative test strip result for azotemia (ie, a test strip BUN category of 1, 2, or 3) were truly not azotemic (ie, BUN concentration determined with the biochemical analyzer was < 41 mg/dL), and all 12 (100%) samples with a positive test strip result for azotemia (ie, a test strip BUN category of 4) were truly positive for azotemia. Thus, although a positive test strip result for ferret blood samples was generally a reliable indicator that azotemia was present, 3 of the 15 (20%) ferret samples that were truly positive for azotemia (as determined with the biochemical analyzer) had negative test strip results for azotemia. Accordingly, there was a risk of not detecting azotemia in ferret samples if test strips had been used as the sole indicator of azotemia. As with rabbit blood samples, testing with a biochemical analyzer remains the gold standard for measurement of BUN concentration and detection of azotemia in ferret samples.
For 13 of the 65 (20%) rabbit blood samples in the present study, the test strip BUN category was higher than the biochemical analyzer BUN category. In contrast, test strips underestimate BUN concentrations in human, dog, and cat samples.4,15 The test strip sensitivity (92%) for detection of azotemia in rabbit blood samples of the present study was similar to values previously reported for dog (95%) and cat (87%) samples4 but was lower than that previously reported for prairie dog samples (100%).8 The test strip specificity (79%) for detection of azotemia in rabbit blood samples of the present study was lower than values previously reported for dog, cat, and prairie dog samples (99%, 100%, and 95%, respectively).4,8
In contrast, the test strips were equally likely to over- or underestimate BUN concentration in the ferret blood samples of the present study, similar to reported findings for black-tailed prairie dog samples.8 There were more false-negative test strip results (3/59) than false-positive results (0/12) for azotemia in the ferret blood samples of the present study, resulting in lower sensitivity (80%) than reported for dog, cat, and prairie dog samples (95%, 87%, and 100%, respectively).4,8 The test strip specificity (100%) for detection of azotemia in ferret samples of the present study was equivalent to that previously reported for cat samples (100%) and higher than values reported for dog (99%) and prairie dog (95%) samples.4,8
Only a limited number of studies4,5 have been reported in which the use of commercially available reagent test strips was evaluated for detection of azotemia in companion animals, and care must be taken when comparing the results of the present study with results of those previous studies because of differences in methods used to store, handle, and process blood samples. For the present study, blood samples were collected with a heparinized syringe, applied to test strips, then placed in blood-collection tubes containing lithium heparin prior to testing with the biochemical analyzer. Conversely, in previous studies,4,5 blood without additives or blood anticoagulated with EDTA was applied to test strips, and serum samples were tested with the biochemical analyzer. Additionally, different biochemical analyzers were used in previous studies,4,16 which is an important consideration given that biochemical analyzer–specific reference intervals for BUN concentration were not available to classify samples as azotemic. Another consideration is the potential for subjectivity among individuals reading and interpreting the color change on the test strips.16 In the present study, a single individual read all strip test results under white fluorescent light to control variability. For future studies, the use of an automated test strip reader should be considered to further increase reproducibility of results.17
A potential limitation of the present study was the slight difference in BUN concentrations between the test strip and biochemical analyzer BUN categories. This was required to ensure that all BUN concentrations obtained with the biochemical analyzer could be assigned to a single category. Biochemical analyzer BUN concentrations for 10 of 65 rabbit samples and 11 of 71 ferret samples could otherwise not have been assigned to a single category because of overlap between the manufacturer-provided test strip categories or the gap in BUN concentrations between manufacturer-provided test strip categories 3 and 4. This adjustment effectively resulted in assignment of biochemical analyzer BUN concentrations that could have been assigned to 2 categories to the higher category, as in previous studies.4,8 However, it is unclear how this adjustment may have affected results of the present study.
Sample sizes in the present study (53 rabbits and 50 ferrets) met the minimum sample size recommended previously18 for method comparison and were similar to those used in previous studies of dogs (n = 50),6 cats (58),4 Egyptian fruit bats (41),7 and black-tailed prairie dogs (47).8 However, larger sample sizes are more likely to be representative of the target population and would be of benefit in future studies, especially when evaluating the performance of test strips in a subset of animals with confirmed azotemia. In the present study, azotemia in rabbits and ferrets was defined on the basis of a single reference interval for each species.12,13 Although we considered these previously described12,13 reference intervals to be the most appropriate given the study design and study population characteristics of those previous studies, it is important to note that healthy animals were used to establish these reference intervals. Therefore, BUN concentrations used to define azotemia in the present study were not necessarily associated with clinical abnormalities or disease. Furthermore, blood samples included in the present study were from rabbits and ferrets with clinical disease as well as those undergoing routine wellness examinations (ie, apparently healthy animals). Validation studies in separate populations of healthy animals and animals with confirmed disease are warranted.
Additional methods for evaluating point-of-care tests include Bland-Altman plots and regression analyses to identify systematic and proportional biases2 and analysis of allowable total error.19 However, these assessments rely on continuous data sets, and the categorical nature of test strip results in the present study precluded the application of these other methods of evaluation.
In conclusion, commercially available reagent test strips could potentially be useful as a point-of-care method for detecting azotemia in rabbits and ferrets. For the rabbit blood samples of the present study, test strips provided reasonable estimates of BUN concentration but were more appropriate for ruling out than for ruling in azotemia. For the ferret blood samples of the present study, test strips also provided reasonable estimates of BUN concentration, but false-negative results were a concern. Biochemical analyzers should always be used to confirm a positive test strip result for azotemia in rabbit samples and should still be considered the most reliable method for measurement of BUN concentration and detection of azotemia in rabbits and ferrets.
Acknowledgments
No external funding was used in this study. The authors declare that there were no conflicts of interest.
Footnotes
Azostix, Siemens Healthcare Diagnostics, Tarrytown, NY.
Lithium heparin BD microtainer tube, Becton, Dickinson and Company, Franklin Lakes, NJ.
Cobas c 501, Roche Diagnostics, Indianapolis, Ind.
PreciNorm and Precipath control sera, Roche Diagnostics, Indianapolis, Ind.
Microsoft Excel 2013, Microsoft Corp, Redmond, Wash.
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