Blood samples for biochemical analysis may be collected from animals at remote sites, and delays may occur from collection to analysis.1 The duration of storage and storage conditions for the blood samples during this period may affect their quality and results of analyses, which therefore might also influence clinical decisions. The preanalytic phase of clinical pathology (steps from specimen collection to analysis) is the phase at which most laboratory variability occurs for human clinical pathology, and this probably is also true for veterinary clinical pathology.2 Biological and physiologic factors (eg, feeding, stress, and biological and endocrine patterns) may affect the validity of results but may not be controllable. Technical factors (eg, the anticoagulant, venipuncture site, duration of storage, and conditions for specimen handling) can be better controlled.2
Compared with the amount of literature for human clinical pathology, there is a paucity of veterinary reports regarding the stability of blood biochemical analyses following refrigeration of samples and the effects of freeze-thaw cycles on sample results, with only a few reports3–7 on the effects of storage on samples obtained from reptilian species. Stability during storage can differ among veterinary species.8 Therefore, the purpose of the study reported here was to examine the stability of selected plasma biochemical analytes in samples obtained from red-eared sliders (Trachemys scripta elegans) and stored for various amounts of time at various storage temperatures in an attempt to simulate short-term delayed-testing scenarios. Red-eared sliders are native to North America, are a prolific invading species in multiple geographic locations on other continents, and are commonly kept as privately owned pets; thus, plasma biochemical analyses are often indicated for use in health evaluations. To the authors' knowledge, no studies have been conducted to evaluate the stability of blood samples over time and at various storage temperatures for this species. The study hypothesis was that both time and storage temperature would not significantly affect results of biochemical analyses.
Materials and Methods
Animals
Eight healthy adult female red-eared sliders (median body weight, 1,042 g; range, 551 to 1,636 g) were included in the study. Turtles were caught in the wild and were group housed at an outdoor pond at a zoological garden.a All turtles had been at the zoo and monitored for up to a year before the beginning of the study. They were fed a mixture of freshly cut fish daily (0.5% of total body weight). The study was approved by the ethics committee of the participating zoological garden and the Institutional Animal Care and Use Committee of Kansas State University (No. 3611.1).
Blood sample collection
The red-eared sliders were brought to the zoo's veterinary clinic for the purpose of collection of a blood sample; they were immediately returned to their original holding area once the blood collection procedure was completed. Each turtle was manually restrained, and a physical examination was performed. A venous blood sample (3.0 mL) was aseptically collected from the subcarapacial venous plexus of each turtle by use of a 23-gauge, 1.5-inch needleb attached to a 6.0-mL syringec; samples were placed into heparin-coated blood collection tubes.d Lithium heparin was used because it is the anticoagulant recommended for use with chelonian blood.4 The PCV was determined routinely by use of the microhematocrit method. Heparinized blood samples in capillary tubes were centrifuged at 12,000 × g for 5 minutes. The value for total solids was obtained with a handheld refractometer. Lymph hemodilution, hemolysis, and lipemia were not apparent in any of the samples.
Plasma sample analysis
The remainder of each blood sample was centrifuged (3,000 × g for 10 minutes). Plasma was harvested, and aliquots (0.15 mL) were placed into individual 0.5-mL collection tubes.e Samples were stored as designated (room temperature [approx 23°C], 4°C, and −20°C) and tested at scheduled time points. The original sample was at room temperature and analyzed (time = 1 hour); an aliquot stored at room temperature was also analyzed at 4 hours. Samples refrigerated at 4°C were analyzed at 8, 24, and 48 hours, whereas samples frozen at −20°C were analyzed at 48 and 72 hours and 7 days. All analyses were performed by use of a veterinary bench-top analyzerf with a predetermined reptile biochemical panel.g Analytes evaluated were AST, bile acids, CK, uric acid, glucose, total protein, albumin, calcium, phosphorus, potassium, and sodium. Globulin concentration was reported as a calculated analyte.
We chose to use this analyzer in the study because it was commonly used for samples obtained from reptile species and could provide test results immediately (approx 15 minutes).5,9–11 For the biochemical analysis, a 100-μL aliquot of plasma was removed from each 0.5-mL collection tube by use of an analyzer pipetteh and placed into a plastic biochemical rotorg as recommended by the manufacturer. Each plastic biochemical rotor was used within 15 minutes after it was removed from a refrigerator and immediately after its protective pouch was opened. Samples were analyzed immediately after the plastic biochemical rotor was filled; analysis was performed in accordance with the manufacturer's directions. The biochemical analyzer was used routinely and functioned appropriately before the study, and analyzer software was updated regularly as provided by the manufacturer. All samples were analyzed by the same investigator (DE).
Statistical analysis
For each time point within each storage temperature, bias from results for the original sample was calculated by subtracting those values from the value obtained for the room temperature sample at 1 hour. Bias (instead of the actual values) was used to assess agreement over time, and that technique was deemed more meaningful than mean changes. Bias was modeled by use of a linear mixed model, with time, storage temperature, and the time-by-storage temperature interaction as fixed effects and turtle as a random effect. When variances were unequal among storage temperatures, a heteroscedastic model was fitted. Assumptions of the model (lack of outliers, linearity, normality of residuals, and homoscedasticity of residuals) and goodness of fit were assessed on residual plots and quantile plots. Globulin concentration was not evaluated because it was a calculated analyte. A type III ANOVA was then performed on the fixed effects. When a significant (P < 0.05) effect was detected, post hoc analyses were performed with a Tukey adjustment. A statistical programi and nonlinear mixed effects modelj were used for statistical analysis.
Goals for the allowable total error indicated in the ASVCP guidelines12 were used to determine accuracy (or bias) of the results for each time point within a storage temperature, compared with the value at 1 hour. Observed total observed error was calculated by obtaining the mean of the ratio for the bias to the reference value (ie, value at 1 hour). In accordance with ASVCP quality-assurance guidelines,13 acceptable clinical agreement was defined as observed total error minus the allowable total error.
Results
Blood samples were obtained from all turtles; no obvious adverse effects were evident. Mean ± SD PCV of the blood samples was 24 ± 3.6%, which was consistent with results for non-lymph-diluted samples from red-eared sliders (PCV > 20%).14 There were no technical issues with use of the analyzer. Values for the analytes were determined (Figure 1; Table 1); results for all turtles and analytes were within the predetermined cutoff values for this analyzer, except for concentrations of bile acids (< 35 mg/dL; n = 8) and calcium (> 20 mg/dL; 2). Because the concentration of bile acids was consistently 0 mg/dL for all time points and storage temperatures, no statistical analysis could be conducted. Statistical analysis of globulin concentration was not performed because it was a calculated value.
Box-and-whisker plots of the bias for AST activity (A), potassium concentration (B), and uric acid concentration (C) in plasma of 8 red-eared sliders (Trachemys scripta elegans) after storage at various temperatures. Room temperature was approximately 23°C. Each box represents the interquartile range, the horizontal line in each box is the median, whiskers are the minimum and maximum values, and circles are outliers.
Citation: American Journal of Veterinary Research 79, 8; 10.2460/ajvr.79.8.852
Box-and-whisker plots of the bias for AST activity (A), potassium concentration (B), and uric acid concentration (C) in plasma of 8 red-eared sliders (Trachemys scripta elegans) after storage at various temperatures. Room temperature was approximately 23°C. Each box represents the interquartile range, the horizontal line in each box is the median, whiskers are the minimum and maximum values, and circles are outliers.
Citation: American Journal of Veterinary Research 79, 8; 10.2460/ajvr.79.8.852
Box-and-whisker plots of the bias for AST activity (A), potassium concentration (B), and uric acid concentration (C) in plasma of 8 red-eared sliders (Trachemys scripta elegans) after storage at various temperatures. Room temperature was approximately 23°C. Each box represents the interquartile range, the horizontal line in each box is the median, whiskers are the minimum and maximum values, and circles are outliers.
Citation: American Journal of Veterinary Research 79, 8; 10.2460/ajvr.79.8.852
Reliability statistics for the effects of storage conditions on biochemical analytes in plasma of 8 red-eared sliders (Trachemys scripta elegans).
| Analyte | Storage temperature (°C) | Mean bias | 95% limits of agreement | TEa (%) | TEobs (%) | TEobs within clinical allowable error limits |
|---|---|---|---|---|---|---|
| AST (U/L) | 4 −20 | −10.0* −9.5* | −25.0 to 5.0 −23.4 to 4.3 | 30 30 | 6 5 | Yes Yes |
| CK (U/L) | 4 −20 | −8.7 −64.8 | −69.4 to 52.0 −256.1 to 126.5 | 30 30 | 3 27 | Yes Yes |
| Uric acid (mg/dL) | 4 −20 | −0.3* −0.3* | −1.1 to 0.5 −0.6 to 0.0 | 10 10 | ND ND | Yes ND |
| Glucose (mg/dL) | 4 −20 | 0.7 0.6 | −2.9 to 4.4 −1.2 to 2.4 | 20 20 | 2 2 | Yes Yes |
| Calcium (mg/dL) | 4 −20 | 0.1 −2.7 | −1.5 to 1.7 −19.7 to 14.4 | 10 10 | 2 12 | Yes No |
| Phosphorus (mg/dL) | 4 −20 | −0.2 0.4 | −0.7 to 0.1 −0.7 to 1.5 | 15 15 | 4 7 | Yes Yes |
| Total protein (g/dL) | 4 −20 | −0.2*† −0.1 | −0.4 to 0.1 −0.3 to 0.2 | 10 10 | 4 1 | Yes Yes |
| Albumin (g/dL) | 4 −20 | −0.1 0 | −0.2 to 0.1 −0.2 to 0.2 | 15 15 | 4 1 | Yes Yes |
| Potassium (mmol/L) | 4 −20 | −0.2* −0.3* | −0.4 to 0.1 −0.6 to 0.0 | 5 5 | 4 8 | Yes No |
| Sodium (mmol/L) | 4 −20 | −0.5 −1.2 | −4.2 to 3.2 −7.0 to 4.6 | 5 5 | 1 1 | Yes Yes |
Values represent comparison with results for the initial sample at room temperature (approx 23°C) analyzed approximately 1 hour after the initial sample was obtained.
Mean bias differs significantly (P < 0.05) from results at 1 hour.
Difference was significant only for samples stored at 4°C for > 24 hours.
ND = Not determined; observed total error (TEobs) was not calculated because most of the results for uric acid at 1 hour were 0 mg/dL. TEa = Allowable total error.
Storage temperature had a significant effect on AST bias. Samples stored at 4° and −20°C had AST activity that was significantly (P < 0.001) higher (by approx 11 U/L), compared with the value for the initial sample at 1 hour. Amount of time in storage did not affect bias. Therefore, storage temperature, rather than the amount of time in storage, was responsible for increases in AST bias. Variability in bias was similar between storage at 4° and −20°C, which was higher than variability for storage at room temperature for 1 hour.
For CK, neither storage temperature nor time in storage affected the mean bias. However, variability of the bias increased dramatically with storage at 4° and −20°C (approx 3 and 10 times as much variability, respectively, as the value for storage at room temperature for 1 hour). Also, bias was more unpredictable for samples stored at 4° and −20°C.
For uric acid, storage temperature had a significant (P < 0.001) effect on bias, which increased approximately 0.2 and 0.3 mg/dL for samples stored at 4° and −20°C, respectively, compared with results for storage at room temperature for 1 hour. Amount of time in storage for each temperature did not have further effects on bias. Therefore, storage temperature, rather the amount of time in storage, was responsible for increases in bias. The variability in bias was similar between storage at 4° and −20°C, which was higher than variability for storage at room temperature for 1 hour.
For glucose, neither storage temperature nor time in storage affected mean bias. However, variability of the bias increased dramatically for samples stored at 4° and −20°C, for which there was approximately 5 and 2 times as much variability, respectively, as the value for storage at room temperature for 1 hour. Also, bias was more unpredictable for samples stored at 4° and −20°C.
For phosphorus, neither storage temperature nor time in storage affected mean bias. However, variability of the bias increased dramatically for samples stored at 4° and −20°C, for which there was approximately 3 and 7 times as much variability, respectively, as the value for storage at room temperature for 1 hour. Also, bias was more unpredictable for samples stored at 4° and −20°C.
For total protein, the time-by-storage temperature interaction significantly (P = 0.015) affected bias, with samples stored at 4°C for > 24 hours increasing by approximately 0.1 to 0.3 g/dL, compared with the value for storage at room temperature for 1 hour. No difference from the value for storage at room temperature for 1 hour was observed for samples stored at 4° or −20°C for < 8 hours. The variability in bias was similar among the 3 storage temperatures.
For potassium, storage temperature significantly (P = 0.02) affected bias, with samples stored at 4° and −20°C increasing by approximately 0.1 to 0.2 and 0.2 to 0.3 mg/dL, respectively, compared with values for storage at room temperature for 1 hour. Amount of time in storage did not affect the bias. Therefore, storage temperature, rather than the amount of time in storage, was responsible for the increases in bias. The variability in bias was similar between storage at 4° and −20°C, but values for both were higher (approx 4 to 5 times as high) than the value for storage at room temperature for 1 hour.
For calcium, albumin, and sodium, neither storage temperature nor time in storage significantly affected the mean bias.
Results did not differ significantly between plasma samples stored at room temperature for 1 or 4 hours. There were significant differences in results attributable to storage temperature but not to amount of time in storage for plasma samples stored at 4° and −20°C for up to 48 hours, compared with results for storage at room temperature for 1 hour.
Differences in measurements obtained after storage were mostly within acceptable limits for allowable total error, except for calcium and potassium concentrations in plasma stored at −20°C (Table 1).
Discussion
For the study reported here, storage temperature but not the amount of time in storage had effects (mainly an increase in and greater variability of measured concentrations) on several plasma biochemical analytes of red-eared sliders, which suggested that these effects should be taken into consideration. Analyses of calcium, albumin, and sodium concentrations revealed no major changes, compared with concentrations for the room temperature sample at 1 hour.
In general, AST activity and uric acid, potassium, and total protein concentrations were higher after storage at 4° and −20°C in the present study. Aspartate aminotransferase is an intracellular enzyme, and AST activity could have increased as a result of cell destruction.15 Uric acid concentrations increase in human plasma after storage at −4°C for 48 hours14 and on the basis of the amount of storage time for whole blood of humans15 and loggerhead sea turtles (Caretta caretta).4 Plasma potassium concentrations can increase as a result of cellular destruction but are also dependent on temperature because they are more stable at 25°C.15 At lower temperatures, activity of the cellular Na-K-ATPase pump is decreased and leads to an increased efflux of potassium from cells.16 Total protein analyses revealed an increase in the concentration for turtles in the present study; however, total protein concentrations are stable in plasma of horned vipers (Vipera ammodytes ammodytes)3 and loggerhead sea turtles6 after storage at −20°C.
In the present study, there was great variability in CK activity after storage at 4° and −20°C, and it is possible that CK was damaged as a result of storage because the enzyme is found in mitochondrial membranes. Phosphorus (a major intracellular anion in mammals) in this study also had great variability (more for plasma stored at −20°C than for plasma stored at 4°C) because phosphatases and changes in cell membrane integrity can cause increased efflux of phosphate from cells.16
The measured increase in the plasma AST activity and potassium concentration as well as the great variability for CK activity and phosphorus concentration in the present study can be attributed to intra-sample hemolysis as a result of storage.4,17,18 Although the plasma was immediately separated and none of the samples had evidence of apparent hemolytic discoloration, it is possible that there was some degree of hemolysis that could have affected the samples.17,18 Fast freezing and slow thawing can cause severe damage to proteins and other cellular components, which thus artifactually increases their measured concentrations.14,17 In the study reported here, the small-volume sample (0.15 mL) stored at −20°C underwent a fast freeze and fast thaw, which potentially could have led to the observed results. Sample dehydration during freezing19 or thawing can also cause an increase in measured concentrations or greater variability in results because each plasma sample (and perhaps also individual analytes) are affected in a different manner. For example, glucose is required for cellular metabolism, and the rate at which glucose is depleted is dependent on temperature and time as well as the cell count.4,20 Sample dehydration can perhaps explain the great variability in the measured plasma concentrations of glucose in the present study despite the separation of plasma from the cellular component of the blood sample. Future investigations can be conducted to evaluate the potential effect of the volume of stored samples on results of biochemical analyses.
Clinicians and researchers are interested in the accuracy and reliability of biochemical analyses; thus, it is imperative to have knowledge about the imprecision of a testing method.21 In the study reported here, the stability (and bias) of each analyte after storage was assessed by making comparisons with published values for allowable total error,12 and most of the analytes were within an acceptable range, except for potassium and calcium stored at −20°C. However, the present study involved only female turtles, and it is possible that some were in the reproductive period given the initial high plasma concentrations of calcium.22 Because the allowable total error suggested in the ASVCP guidelines12 is general, it is possible that it may not reflect an acceptable biological range suitable for calcium concentration in turtles in the reproductive phase, as has also been suggested for humans.21,23 Determination of seasonal and sex-based allowable total error for reptilian species is needed.
In the present study, we attempted to simulate various realistic immediate and short-term delayed testing time points and storage conditions. However, this precluded direct matching of time points for the various storage conditions, except for the 48-hour time point. Only a small number of turtles were used in the study, and this could have affected the results. However, similar numbers of subjects have been used in comparable studies of humans24 and other veterinary species.1,4,7,25–30 Also, it is possible that the results were accurate for this testing method. Further studies on samples from this species should include use of other testing methods.
The stability of blood samples differs among species, and information on the manner in which storage conditions affect plasma of reptiles is limited. Analysis of data from the study reported here indicated significant changes in some biochemical analytes that were associated with storage temperature but not with the amount of time in storage. Results for plasma samples kept at room temperature for 4 hours did not differ significantly from results for plasma analyzed at 1 hour. Results for plasma samples stored at 4° and −20°C for up to 48 hours indicated significant differences, which were attributed to storage temperature. With the knowledge that storage conditions can substantially affect the plasma of red-eared sliders, appropriate handling and storage guidelines for plasma samples obtained from this species can be developed. This will allow for better sample quality and a more accurate evaluation of analytes used in health evaluations for this species. Further studies are necessary to assess alterations in results of biochemical analyses for plasma samples stored for > 7 days at various storage temperatures.
Acknowledgments
Supported by Abaxis Veterinary Diagnostics.
The authors thank Dr. Avital Paz for technical assistance.
ABREVIATIONS
| AST | Aspartate aminotransferase |
| ASVCP | American Society for Veterinary Clinical Pathology |
| CK | Creatine kinase |
Footnotes
The Tisch Family Zoological Gardens in Jerusalem, Derech Aharon Shulov 1, Jerusalem, Israel.
Exelint International Co, Los Angeles, Calif.
Kendall Monoject, Tyco Healthcare Group, Mansfield, Mass.
Becton Dickinson Co, Franklin Lakes, NJ.
Eppendorf, Brinkmann Instruments Inc, Westbury, NY.
Vetscan VS2, provided by Abaxis Inc, Union City, Calif.
Avian/reptilian Profile Plus, provided by Abaxis Veterinary Diagnostics, Union City, Calif.
Minipipette, provided by Abaxis Inc, Union City, Calif.
R development core team, R Foundation for Statistical Computing, Vienna, Austria.
Pinheiro J, Bates D, DebRoy S, et al. nlme: linear and nonlinear mixed effects models. R package, version 3.1–103 for mixed modeling, R-Core. Available at CRAN.R-project.org/package=nlme. Accessed MONTH, DAY 2018.
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