Measurement of concentrations of cTnI, a myofibril protein that regulates contraction of myocardium, is considered the gold standard diagnostic test for detection of cardiac injury in humans.1,2 There is > 96% homology between cTnI of humans and that of cattle3; therefore, antibodies against human cTnI in commercially available assays are expected to cross-react with bovine cTnI. Cardiac troponin has been used as a biomarker of cardiac injury attributable to various diseases in dogs, cats, and horses.4–11 Also, cTnI concentration increases during various diseases of cows, including idiopathic pericarditis,12 traumatic reticulopericarditis,13,14 foot and mouth disease,15 monensin toxicosis,16 and experimentally induced endotoxemia.17 Results of another study18 indicate that cattle with various cardiac diseases or noncardiac intrathoracic diseases have high serum cTnI concentrations, compared with concentrations for healthy control animals.
Recently, a benchtop immunoassay has been validated for measurement of bovine cTnI concentrations.19 Although that assay is not portable, it could be used to evaluate the precision of a point-of-care diagnostic assay for measurement of bovine cTnI concentrations in field settings. A commercially available point-of-care device and disposable cartridge have been used to measure plasma cTnI concentrations for animals including dogs,6 alpacas,20 and horses.21 The objective of the study reported here was to determine the repeatability of results of a point-of-care diagnostic test for determination of cTnI concentrations in plasma samples obtained from calves and to compare results with those determined by use of a previously validated diagnostic test.19 We hypothesized that the point-of-care diagnostic test would detect bovine cTnI and the test would be precise for measurement of cTnI concentrations.
Materials and Methods
Samples—For determination of point-of-care cTnI assay precision, standards with various concentrations of cTnI were prepared with bovine plasma obtained from a university-owned, healthy mature Holstein steer. In addition, blood and plasma samples were obtained from 28 healthy Holstein calves (approx age, 2.5 months) for cTnI testing; whole blood sample cTnI concentrations determined by use of a point-of-care assay were compared with plasma cTnI concentrations determined by use of a validated immunoassay. Calves were purchased from a commercial calf-rearing facility located in Iowa. Calves were housed together in a pen (25 × 25 m) outdoors during the study. The study protocol was approved by the Kansas State University Animal Care and Use Committee.
Point-of-care cTnI assay precision—The precision of a commercially available point-of-care devicea and disposable cartridgeb was determined via measurement of various cTnI concentrations in standards prepared with bovine plasma containing lithium heparin and purified bovine cTnI.c The assay was repeated 3 times for standards without cTnI (cTnI concentration, 0 ng/mL [negative control sample]) and 5 times for standards with various concentrations of cTnI (1, 0.5, 0.25, 0.1, 0.05, 0.025, and .01 ng/mL). Precision of the assay for measurement of cTnI concentrations in bovine plasma samples was determined via calculation of CVs.
Comparison of cTnI point-of-care assay results with immunoassay results—Results obtained with a point-of-care assaya and disposable cartridgeb were compared with those obtained by use of a validated19 immunoassayd for measurement of cTnI in whole blood and plasma samples obtained from calves. A venous blood sample (5 mL) was collected from a jugular vein of each calf into a tube containing lithium heparin by use of a 1.5-inch × 20-gauge needle. Whole blood samples were analyzed for determination of whole blood cTnI concentrations once with the point-of-care assaya and disposable cartridgeb on the same day of sample collection.a Then, each sample was centrifuged (2,350 × g) for 10 minutes at 22°C, plasma was harvested, and plasma samples were stored at −80°C for up to 14 days until determination of cTnI concentrations by use of a validated19 immunoassayd; results of another study19 indicate cTnI concentrations do not decrease in bovine plasma samples stored at −80°C for 14 days.
Data and statistical analysis—Precision of the point-of-care assay for determination of cTnI concentrations in prepared bovine plasma standards was determined via calculation of CVs. A 1-sample t test was used to identify differences between known and measured concentration means. Outlier values were defined as values > the 99th-percentile value for each known cTnI concentration22; such data (1 value each for 0.01 and 0.05 ng/mL cTnI concentration standards) were attributed to faulty cartridges and were not included in the statistical analysis. The whole blood cTnI concentration values determined by use of the point-of-care assay for calves were compared with plasma cTnI concentrations determined by use of the validated immunoassay via a Student t test for paired data. For statistical analysis, plasma cTnI concentration values determined by use of the immunoassay that were < 0.01 ng/mL were considered to be 0 ng/mL. Values of P < 0.05 were considered significant. All analyses were performed with commercially available computer software.e
Results
Point-of-care cTnI assay precision—The cTnI concentrations determined by use of the point-of-care assay were typically higher than the known concentrations of cTnI in prepared bovine plasma standards (Table 1); values were significantly different for all cTnI concentration standards except the 0.01 ng/mL standard. However, the CV for the point-of-care assay was < 20% for all prepared bovine plasma cTnI standards except for the lowest cTnI concentration (0.01 ng/mL; CV, 38%). Concentrations of cTnI in negative control samples determined by use of the point-of-care assay were 0 ng/mL for all 3 repeated tests; therefore, use of the bovine plasma as a diluent for the cTnI concentration standards was considered appropriate.
Concentrations of cTnI In prepared bovine plasma standard samples determined by use of a point-of-care assay.
| Known cTnI concentration in standard (ng/mL) | Mean ± SD measured concentration (ng/mL) | P value* | CV (%) |
|---|---|---|---|
| 0 | 0 ± 0 | ND | ND |
| 0.01 | 0.015 ± 0.006 | 0.136 | 38 |
| 0.025 | 0.068 ± 0.010 | 0.001 | 19 |
| 0.05 | 0.078 ± 0.010 | 0.003 | 12 |
| 0.1 | 0.120 ± 0.020 | 0.03 | 16 |
| 0.25 | 0.296 ± 0.030 | 0.03 | 11 |
| 0.5 | 0.702 ± 0.050 | 0.001 | 7 |
| 1 | 1.502 ± 0.050 | 0.009 | 10 |
Determined by use of a 1-sample t test.
ND = Not determined because all results were 0 ng/mL.
Comparison of cTnI point-of-care assay results with immunoassay results—The cTnI concentrations in whole blood samples determined by use of the point-of-care assay (mean ± SD, 0.01 ± 0.01 ng/mL; median, 0 ng/mL; range, 0 to 0.04 ng/mL) were similar to plasma concentrations determined by use of the validated immunoassay (mean ± SD, 0.006 ± 0.006 ng/mL; median, 0.01 ng/mL; range, 0 to 0.02 ng/mL). Results of statistical analysis performed with a Student t test for paired data indicated concentrations determined via the 2 assays were not significantly different. Of the 28 whole blood and harvested plasma samples, 12 (43%) had identical cTnI concentration test results, 13 (46%) had a concentration difference of 0.01 ng/mL between the 2 tests, 2 (7%) had a concentration difference of 0.02 ng/mL between the 2 tests, and 1 (4%) had a concentration difference of 0.03 ng/mL between the 2 tests.
Discussion
The results of the present study suggested that the point-of-care assay may be appropriate for determination of concentrations of cTnI in blood samples obtained from cattle. This point-of-care assay has been used to measure circulating cTnI concentrations for healthy alpacas20; however, investigators of that study could not validate the test for that species because purified alpaca cTnI was not available. The point-of-care assay was validated for measurement of bovine cTnI in the present study via testing of standards prepared with bovine plasma and purified bovine cTnI. To obtain accurate results with this assay, whole blood samples must be collected into tubes containing lithium heparin. Initially, we attempted to prepare cTnI standards with bovine plasma containing acid-citrate-dextrose solution, but results had high variability, and cTnI concentrations were markedly lower than the known concentrations in standard samples. Conclusions of this study may have been stronger if high cTnI concentrations had been detected in blood and plasma samples; this would have necessitated obtaining blood samples from cattle with myocardial disease. Cattle with myocardial disease were not available for use in this study, and induction of cardiac injury in calves was not feasible. Future research is warranted to determine the clinical usefulness of the point-of-care assay for measurement of high concentrations of cTnI in blood samples obtained from cattle. On the basis of results of this study, we concluded that clinicians would be unlikely to misdiagnose myocardial injury for calves with low cTnI concentrations. Further research is warranted to determine the likelihood that cattle with myocardial injury would have clinically normal cTnI concentrations as determined by use of the point-of-care assay.
Results of other studies3,19 indicate cTnI assays have minimal cross-reactivity with proteins in noncardiac bovine muscle homogenates; those results suggest that assays for measurement of bovine cTnI are likely specific for myocardial proteins. In the present study, we used an immunoassay and a point-of-care analyzer that were designed for detection of cTnI in blood and plasma samples of humans. These assays were expected to be useful for measurement of cTnI in bovine blood and plasma samples because bovine and human cTnI are > 96% homologous.3 This assumption was supported by results of another study19 in which known concentrations of bovine cTnI were analyzed by use of an assay designed for measurement of human cTnI.
Results of the present study indicated that the precision of the point-of-care assay for measurement of cTnI concentrations > 0.01 ng/mL was good because the CV of data for such concentrations was < 20%. The CV was high (38%) for measurement of a low cTnI concentration (0.01 ng/mL) in prepared plasma standards; this finding suggested that results for low cTnI concentrations were not precise. Results of other studies16,17,19 indicate the reference range for cTnI concentrations in blood samples of cattle is < 0.05 to < 0.08 ng/mL. Therefore, the point-of-care analyzer evaluated in the present study may be useful for identification of cattle with myocardial disease, although further research is warranted.
The point-of-care assay evaluated in the present study overestimates cTnI concentrations.22 This suggests that calves having myocardial injury are unlikely to be classified as clinically normal on the basis of results of this assay. Results of the present study suggested the analytic sensitivity of the point-of-care device for measurement of cTnI concentrations in bovine blood samples was 0.02 ng/mL (the lowest value that could be distinguished from a concentration of 0 ng/mL); therefore, a cTnI concentration of 0.01 ng/mL could not be reliably differentiated from a concentration of 0 ng/mL. The estimated 20% and 10% functional sensitivities of the point-of-care assay determined via calculation of CV percentages for cTnI concentrations are 0.07 and 0.10 ng/mL, respectively.22 This information may help explain the finding of a high CV for measurement of cTnI at a concentration of 0.01 ng/mL and the substantially lower CVs for concentrations > 0.10 ng/mL. The point-of-care diagnostic test has higher accuracy for measurement of abnormal concentrations of cTnI, which are considered to be > 0.05 to > 0.08 ng/mL on the basis of previous reports.16,17,19 Low cTnI concentration results may not be precise but would rarely cause a misclassification of a concentration associated with disease.
The concentrations of cTnI determined with the point-of-care assay for blood samples obtained from calves in the present study were similar to those determined via a different method for cattle in another study.19 This assay may be useful for point-of-care determination of cTnI concentrations for cattle. Further research may be warranted to determine the usefulness of this assay for identification of cattle with cardiopulmonary disease. Use of the point-of-care assay evaluated in the present study may allow rapid determination of cTnI concentrations and aid management of cattle with cardiopulmonary disease.
ABBREVIATIONS
| cTnI | Cardiac troponin I |
| CV | Coefficient of variation |
i-STAT 1 portable clinical analyzer, Abaxis North America, Union City, Calif.
i-STAT cTnI cartridge, Abaxis North America, Union City, Calif.
Purified bovine cTnI, Life Diagnostics, West Chester, Pa.
ADVIA Centaur TnI-Ultra, Siemens Medical Solutions Diagnostics, New York, NY.
Excel for Mac 2008, Microsoft Corp, Redmond, Wash.
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