• View in gallery

    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.

  • 1. Hulme-Moir KL, Clark P, Spencer PB. Effects of temperature and duration of sample storage on the haematological characteristics of western grey kangaroos (Macropus fuliginosus). Aust Vet J 2006;84:143147.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2. Braun JP, Bourgès-Abella N, Geffré A, et al. The preanalytic phase in veterinary clinical pathology. Vet Clin Pathol 2015;44:825.

  • 3. Proverbio D, Giorgi GB, Pepa AD, et al. Preliminary evaluation of total protein concentration and electrophoretic protein fractions in fresh and frozen serum from wild horned vipers (Vipera ammodytes ammodytes). Vet Clin Pathol 2012;41:582586.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Eisenhawer E, Courtney CH, Raskin RE, et al. Relationship between separation time of plasma from heparinized whole blood on plasma biochemical analytes of loggerhead sea turtles (Caretta caretta). J Zoo Wildl Med 2008;39:208215.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Atkins A, Jacobson E, Hernandez J, et al. Use of a portable point-of-care (Vetscan VS2) biochemical analyzer for measuring plasma biochemical levels in free-living loggerhead sea turtles (Caretta caretta). J Zoo Wildl Med 2010;41:585593.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6. Alberghina D, Marafioti S, Spadola F, et al. Influence of short-term storage conditions on the stability of total protein concentrations and electrophoretic fractions in plasma samples from loggerhead sea turtles. Caretta caretta. Comp Clin Pathol 2015;24:10911095.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Hamilton MT, Finger JW, Winzeler ME, et al. Evaluating the effect of sample type on American alligator (Alligator mississippiensis) analyte values in a point-of-care blood analyser. Conserv Physiol 2016;4:17.

    • Search Google Scholar
    • Export Citation
  • 8. Cray C, Rodriguez M, Zaias J, et al. Effects of storage temperature and time on clinical biochemical parameters from rat serum. J Am Assoc Lab Anim Sci 2009;48:202204.

    • Search Google Scholar
    • Export Citation
  • 9. Eshar D, Gancz AY, Avni-Magen N, et al. Hematologic, plasma biochemistry, and acid-base analysis of adult Negev Desert tortoises (Testudo werneri) in Israel. J Zoo Wildl Med 2014;45:979983.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Eshar D, Gancz AY, Avni-Magen N, et al. Selected plasma biochemistry analytes of healthy captive sulcata (African spurred) tortoises (Centrochelys Sulcata). J Zoo Wildl Med 2016;47:993999.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11. Stoot LJ, Cairns NA, Cull F, et al. Use of portable blood physiology point-of-care devices for basic and applied research on vertebrates: a review. Conserv Physiol 2014;2:121.

    • Search Google Scholar
    • Export Citation
  • 12. Harr KE, Flatland B, Nabity M, et al. ASVCP guidelines: allowable total error guidelines for biochemistry. Vet Clin Pathol 2013;42:424436.

  • 13. Flatland B, Freeman KP, Friedrichs KR, et al. ASVCP quality assurance guidelines: control of general analytical factors in veterinary laboratories. Vet Clin Pathol 2010;39:264277.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Crawshaw GJ, Holz P. Comparison of plasma biochemical values in blood and blood-lymph mixtures from red-eared sliders. Trachemys scripta elegans. Bull Assoc Reptil Amphib Vet 1996;6:79.

    • Search Google Scholar
    • Export Citation
  • 15. Cuhadar S, Koseoglu M, Atay A, et al. The effect of storage time and freeze-thaw cycles on the stability of serum samples. Biochem Med (Zagreb) 2013;23:7077.

    • Search Google Scholar
    • Export Citation
  • 16. Tanner M, Kent N, Smith B, et al. Stability of common biochemical analytes in serum gel tubes subjected to various storage temperatures and times pre-centrifugation. Ann Clin Biochem 2008;45:375379.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17. Thomas L. Haemolysis as influence and interference factor. Biochim Clin 2002;26:9598.

  • 18. Sonntag O. Haemolysis as an interference factor in clinical chemistry. J Clin Chem Clin Biochem 1986;24:127139.

  • 19. Braceland M, Houston K, Ashby A, et al. Technical pre-analytical effects on the clinical biochemistry of Atlantic salmon (Salmo salar L.). J Fish Dis 2017;40:2940.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20. Oddoze C, Lombard E, Portugal H. Stability study of 81 analytes in human whole blood, in serum and in plasma. Clin Biochem 2012;45:464469.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21. Oosterhuis WP. Gross overestimation of total allowable error based on biological variation. Clin Chem 2011;57:13341336.

  • 22. Andreani G, Carpenè E, Cannavacciuolo A, et al. Reference values for hematology and plasma biochemistry variables, and protein electrophoresis of healthy Hermann's tortoises (Testudo hermanni ssp.). Vet Clin Pathol 2014;43:573583.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23. Oosterhuis WP, Theodorsson E. Total error vs. measurement uncertainty: revolution or evolution? Clin Chem Lab Med 2016;54:235239.

  • 24. Zander J, Bruegel M, Kleinhempel A, et al. Effect of biobanking conditions on short-term stability of biomarkers in human serum and plasma. Clin Chem Lab Med 2014;52:629639.

    • Search Google Scholar
    • Export Citation
  • 25. Divya PD, Jayavardhanan KK. Effect of temperature and storage time on hepatobiliary enzyme activities in goat serum. Vet World 2010;3:277279.

    • Search Google Scholar
    • Export Citation
  • 26. Peng TC, Hsu BG, Yang FL, et al. Stability of blood biochemistry levels in animal model research: effects of storage condition and time. Biol Res Nurs 2010;11:395400.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27. Thoresen SI, Havre GN, Morberg H, et al. Effects of storage time on chemistry results from canine whole blood, heparinized whole blood, serum and heparinized plasma. Vet Clin Pathol 1992;21:8894.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28. Hawkins MG, Kass PH, Zinkl JG, et al. Comparison of biochemical values in serum and plasma, fresh and frozen plasma, and hemolyzed samples from orange-winged Amazon parrots (Amazona amazonica). Vet Clin Pathol 2006;35:219225.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29. Singh M, Pandya R, Chandra S, et al. Stability of clinical chemistry and hematological analytes in preserved plasma and blood obtained from Wistar rats. Scand J Lab Anim Sci 2015;41:16.

    • Search Google Scholar
    • Export Citation
  • 30. Thoresen SI, Tverdal A, Havre G, et al. Effects of storage time and freezing temperature on clinical chemical parameters from canine serum and heparinized plasma. Vet Clin Pathol 1995;24:129133.

    • Crossref
    • Search Google Scholar
    • Export Citation

Advertisement

Effects of time and storage temperature on selected biochemical analytes in plasma of red-eared sliders (Trachemys scripta elegans)

View More View Less
  • 1 Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506.
  • | 2 Tisch Family Zoological Gardens in Jerusalem, Derech Aharon Shulov 1, Jerusalem 91008, Israel.
  • | 3 Tisch Family Zoological Gardens in Jerusalem, Derech Aharon Shulov 1, Jerusalem 91008, Israel.
  • | 4 Department of Clinical Studies, Ontario Veterinary College, University of Guelph, ON N1G2W1, Canada.

Abstract

OBJECTIVE To investigate effects of storage duration and temperature on biochemical analytes in plasma from red-eared sliders (Trachemys scripta elegans).

ANIMALS 8 red-eared sliders.

PROCEDURES Blood samples were collected. Plasma was harvested and analyzed at room temperature (approx 23°C; time = 1 hour) and then fractioned into 0.1-mL aliquots that were stored at room temperature or were refrigerated (4°C) or frozen (−20°C). Biochemical analysis of stored samples was performed at 4 (room temperature), 8 (4°C), 24 (4°C), 48 (4° and −20°C), and 72 (−20°C) hours and at 7 days (−20°C). For each time point for each storage temperature, bias was calculated by subtracting values from the value obtained at 1 hour. Bias was modeled by use of a linear mixed model.

RESULTS Storage temperature had a significant effect on several plasma biochemical analytes. In general, aspartate aminotransferase activity and uric acid, total protein, and potassium concentrations increased after storage at 4° and −20°C. Differences in values after storage were mostly within the acceptable range for allowable total error, except for calcium and potassium concentrations for samples stored at −20°C. Both storage temperatures increased variability of measurement results. Results for samples stored at room temperature for 4 hours did not differ significantly from values at 1 hour. Results differed significantly between refrigerated and frozen samples stored for 48 hours.

CONCLUSIONS AND CLINICAL RELEVANCE Short-term storage conditions influenced results for some biochemical analytes. These effects should be considered when performing biochemical analyses of plasma samples obtained from red-eared sliders.

Abstract

OBJECTIVE To investigate effects of storage duration and temperature on biochemical analytes in plasma from red-eared sliders (Trachemys scripta elegans).

ANIMALS 8 red-eared sliders.

PROCEDURES Blood samples were collected. Plasma was harvested and analyzed at room temperature (approx 23°C; time = 1 hour) and then fractioned into 0.1-mL aliquots that were stored at room temperature or were refrigerated (4°C) or frozen (−20°C). Biochemical analysis of stored samples was performed at 4 (room temperature), 8 (4°C), 24 (4°C), 48 (4° and −20°C), and 72 (−20°C) hours and at 7 days (−20°C). For each time point for each storage temperature, bias was calculated by subtracting values from the value obtained at 1 hour. Bias was modeled by use of a linear mixed model.

RESULTS Storage temperature had a significant effect on several plasma biochemical analytes. In general, aspartate aminotransferase activity and uric acid, total protein, and potassium concentrations increased after storage at 4° and −20°C. Differences in values after storage were mostly within the acceptable range for allowable total error, except for calcium and potassium concentrations for samples stored at −20°C. Both storage temperatures increased variability of measurement results. Results for samples stored at room temperature for 4 hours did not differ significantly from values at 1 hour. Results differed significantly between refrigerated and frozen samples stored for 48 hours.

CONCLUSIONS AND CLINICAL RELEVANCE Short-term storage conditions influenced results for some biochemical analytes. These effects should be considered when performing biochemical analyses of plasma samples obtained from red-eared sliders.

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.

Figure 1—
Figure 1—

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

Table 1—

Reliability statistics for the effects of storage conditions on biochemical analytes in plasma of 8 red-eared sliders (Trachemys scripta elegans).

AnalyteStorage temperature (°C)Mean bias95% limits of agreementTEa (%)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

a.

The Tisch Family Zoological Gardens in Jerusalem, Derech Aharon Shulov 1, Jerusalem, Israel.

b.

Exelint International Co, Los Angeles, Calif.

c.

Kendall Monoject, Tyco Healthcare Group, Mansfield, Mass.

d.

Becton Dickinson Co, Franklin Lakes, NJ.

e.

Eppendorf, Brinkmann Instruments Inc, Westbury, NY.

f.

Vetscan VS2, provided by Abaxis Inc, Union City, Calif.

g.

Avian/reptilian Profile Plus, provided by Abaxis Veterinary Diagnostics, Union City, Calif.

h.

Minipipette, provided by Abaxis Inc, Union City, Calif.

i.

R development core team, R Foundation for Statistical Computing, Vienna, Austria.

j.

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.

References

  • 1. Hulme-Moir KL, Clark P, Spencer PB. Effects of temperature and duration of sample storage on the haematological characteristics of western grey kangaroos (Macropus fuliginosus). Aust Vet J 2006;84:143147.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2. Braun JP, Bourgès-Abella N, Geffré A, et al. The preanalytic phase in veterinary clinical pathology. Vet Clin Pathol 2015;44:825.

  • 3. Proverbio D, Giorgi GB, Pepa AD, et al. Preliminary evaluation of total protein concentration and electrophoretic protein fractions in fresh and frozen serum from wild horned vipers (Vipera ammodytes ammodytes). Vet Clin Pathol 2012;41:582586.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Eisenhawer E, Courtney CH, Raskin RE, et al. Relationship between separation time of plasma from heparinized whole blood on plasma biochemical analytes of loggerhead sea turtles (Caretta caretta). J Zoo Wildl Med 2008;39:208215.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Atkins A, Jacobson E, Hernandez J, et al. Use of a portable point-of-care (Vetscan VS2) biochemical analyzer for measuring plasma biochemical levels in free-living loggerhead sea turtles (Caretta caretta). J Zoo Wildl Med 2010;41:585593.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6. Alberghina D, Marafioti S, Spadola F, et al. Influence of short-term storage conditions on the stability of total protein concentrations and electrophoretic fractions in plasma samples from loggerhead sea turtles. Caretta caretta. Comp Clin Pathol 2015;24:10911095.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Hamilton MT, Finger JW, Winzeler ME, et al. Evaluating the effect of sample type on American alligator (Alligator mississippiensis) analyte values in a point-of-care blood analyser. Conserv Physiol 2016;4:17.

    • Search Google Scholar
    • Export Citation
  • 8. Cray C, Rodriguez M, Zaias J, et al. Effects of storage temperature and time on clinical biochemical parameters from rat serum. J Am Assoc Lab Anim Sci 2009;48:202204.

    • Search Google Scholar
    • Export Citation
  • 9. Eshar D, Gancz AY, Avni-Magen N, et al. Hematologic, plasma biochemistry, and acid-base analysis of adult Negev Desert tortoises (Testudo werneri) in Israel. J Zoo Wildl Med 2014;45:979983.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Eshar D, Gancz AY, Avni-Magen N, et al. Selected plasma biochemistry analytes of healthy captive sulcata (African spurred) tortoises (Centrochelys Sulcata). J Zoo Wildl Med 2016;47:993999.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11. Stoot LJ, Cairns NA, Cull F, et al. Use of portable blood physiology point-of-care devices for basic and applied research on vertebrates: a review. Conserv Physiol 2014;2:121.

    • Search Google Scholar
    • Export Citation
  • 12. Harr KE, Flatland B, Nabity M, et al. ASVCP guidelines: allowable total error guidelines for biochemistry. Vet Clin Pathol 2013;42:424436.

  • 13. Flatland B, Freeman KP, Friedrichs KR, et al. ASVCP quality assurance guidelines: control of general analytical factors in veterinary laboratories. Vet Clin Pathol 2010;39:264277.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Crawshaw GJ, Holz P. Comparison of plasma biochemical values in blood and blood-lymph mixtures from red-eared sliders. Trachemys scripta elegans. Bull Assoc Reptil Amphib Vet 1996;6:79.

    • Search Google Scholar
    • Export Citation
  • 15. Cuhadar S, Koseoglu M, Atay A, et al. The effect of storage time and freeze-thaw cycles on the stability of serum samples. Biochem Med (Zagreb) 2013;23:7077.

    • Search Google Scholar
    • Export Citation
  • 16. Tanner M, Kent N, Smith B, et al. Stability of common biochemical analytes in serum gel tubes subjected to various storage temperatures and times pre-centrifugation. Ann Clin Biochem 2008;45:375379.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17. Thomas L. Haemolysis as influence and interference factor. Biochim Clin 2002;26:9598.

  • 18. Sonntag O. Haemolysis as an interference factor in clinical chemistry. J Clin Chem Clin Biochem 1986;24:127139.

  • 19. Braceland M, Houston K, Ashby A, et al. Technical pre-analytical effects on the clinical biochemistry of Atlantic salmon (Salmo salar L.). J Fish Dis 2017;40:2940.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20. Oddoze C, Lombard E, Portugal H. Stability study of 81 analytes in human whole blood, in serum and in plasma. Clin Biochem 2012;45:464469.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21. Oosterhuis WP. Gross overestimation of total allowable error based on biological variation. Clin Chem 2011;57:13341336.

  • 22. Andreani G, Carpenè E, Cannavacciuolo A, et al. Reference values for hematology and plasma biochemistry variables, and protein electrophoresis of healthy Hermann's tortoises (Testudo hermanni ssp.). Vet Clin Pathol 2014;43:573583.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23. Oosterhuis WP, Theodorsson E. Total error vs. measurement uncertainty: revolution or evolution? Clin Chem Lab Med 2016;54:235239.

  • 24. Zander J, Bruegel M, Kleinhempel A, et al. Effect of biobanking conditions on short-term stability of biomarkers in human serum and plasma. Clin Chem Lab Med 2014;52:629639.

    • Search Google Scholar
    • Export Citation
  • 25. Divya PD, Jayavardhanan KK. Effect of temperature and storage time on hepatobiliary enzyme activities in goat serum. Vet World 2010;3:277279.

    • Search Google Scholar
    • Export Citation
  • 26. Peng TC, Hsu BG, Yang FL, et al. Stability of blood biochemistry levels in animal model research: effects of storage condition and time. Biol Res Nurs 2010;11:395400.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27. Thoresen SI, Havre GN, Morberg H, et al. Effects of storage time on chemistry results from canine whole blood, heparinized whole blood, serum and heparinized plasma. Vet Clin Pathol 1992;21:8894.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28. Hawkins MG, Kass PH, Zinkl JG, et al. Comparison of biochemical values in serum and plasma, fresh and frozen plasma, and hemolyzed samples from orange-winged Amazon parrots (Amazona amazonica). Vet Clin Pathol 2006;35:219225.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29. Singh M, Pandya R, Chandra S, et al. Stability of clinical chemistry and hematological analytes in preserved plasma and blood obtained from Wistar rats. Scand J Lab Anim Sci 2015;41:16.

    • Search Google Scholar
    • Export Citation
  • 30. Thoresen SI, Tverdal A, Havre G, et al. Effects of storage time and freezing temperature on clinical chemical parameters from canine serum and heparinized plasma. Vet Clin Pathol 1995;24:129133.

    • Crossref
    • Search Google Scholar
    • Export Citation

Contributor Notes

Address correspondence to Dr. Eshar (deshar@vet.k-state.edu).