Knowledge of factors influencing blood sample integrity is important to interpret results correctly. Effects of storage duration at different temperatures on porcine uncentrifuged blood samples are poorly described.The objective of this study were to elucidate effects of storage time and temperature on different porcine biochemical analytes. Multiple clotted, uncentrifuged blood samples from ten gilts were collected and samples were centrifuged 2h after sampling in the laboratory. Remaining samples were stored chilled (at 4±2°C, CT) or at room temperature (20 ± 2°C, RT). Samples were centrifuged 6, 12, 24, 48 or 72hours after arriving at the laboratory. Storage characteristics and stability of albumin (Alb), calcium (Ca), creatinine (Creat), iron (Fe), gamma-glutamyltransferase (GGT), glutamate dehydrogenase (GLDH), glucose (Gluc), magnesium (Mg), inorganic phosphate (P), total protein (TP) and urea were studied. Test repeatability was within acceptable diagnostic limits. A significant time effect was found for Alb (P<0.001), Ca (P<0.0001), Creat (P<0.0001), GLDH (P<0.0001), Gluc (P<0.0001), Mg (P<0.0001), P (P<0.0001) and TP (P<0.0001). Over time, Alb, GLDH, Mg, P and TP increase while Ca, Creat and Gluc decrease. A temperature effect was identified for Ca (P<0.05), GGT (P<0.05), GLDH(P<0.0001), Gluc (P<0.0001), Mg (P<0.0001) and P (P<0.0002). All values, except Gluc, were higher when stored at RT. Gluc values were lower when stored at RT. This study highlights the importance of standardized storage routines of porcine clotted blood samples to ensure the validity of biochemical analyses.

Keywords: pig, biochemistry, storage, clotted, uncentrifuged, time, temperature

Correct interpretation of biochemical analytes is crucial for precise diagnosis and treatment of different diseases in production animals including pigs. Given the considerable distances between some farms and clinical laboratories in some countries like e.g. Norway, blood samples are sometimes delayed during shipment. If sample shipment should include a delay over weekend, transport duration may exceed 72hours. During the summertime, temperatures in Norway can easily reach 25°C. On the other hand, winter temperatures in certain districts can occasionally drop below -30°C.¹ Previous studies confirm that storage conditions and transport duration of blood samples have a significant influence on biochemical values. Several studies have focused on storage duration and environmental effects on samples of human origin.^2‒7 Both normal serum activity and isoenzyme patterns differ between species and therefore it is not advisable to uncritically draw parallels between species.⁸ A few studies have looked at environmental and storage duration consequences on biochemical analytes in animal samples like sheep,^8,9 cattle,^10,11 horses¹² and dogs.^13,14 Some studies have described effects on porcine blood fractions,^8,15‒17 a couple of these focused on the serum fraction.^8,15 To the authors’ knowledge, only one study has been conducted on uncentrifuged clotted blood from pigs¹⁵ and this investigation looked at samples stored at room temperature. The aim of this study was to determine the analyte stability of porcine clotted, uncentrifuged blood samples stored at chilled temperature (CT) or at room temperature (RT) prior to centrifugation 2hours to 72hours post sampling. The wider objective was to be able to recommend standardized routines for sample storage and processing of blood samples from pigs.

Animals, sample collection and processing

Ten clinically healthy, five months old Norsv in Topigs gilts from a commercial sow pool center were used for this study. The gilts’ body condition score (BCS) was evaluated and all gilts showed BCS between 3 and 3.5 (1 to 5 grading system). For blood sampling, the gilts were fixed with a rope around the upper jaw and blood samples were collected from the V. jugularis externa. Twelve tubes of 9ml with micronized silica particles to activate efficient clotting (Vacuette® 9 ml, Med-Kjemi AS, Asker, Norway) were obtained from each animal. Tubes with separator gels were not used since gels can contain trace metals.¹⁸ The blood samples were transported in a closed, dark container to the laboratory, and arrival at the laboratory was within twohours after sampling. The samples from each animal were divided in two and stored either in a dark environment either at 4 or 20°C. The first set of samples were centrifuged (3500xg for 15minutes (Megafuge 1.0 R, Heraeus SEPATECH, USA)) directly after arriving at the laboratory (2h, RT). The remaining samples were centrifuge dafter storage durations of 6h, 12h, 24h, 48h and 72h, at CT or RT. To prevent thawing, freezing and thawing of the same sample, four serum aliquots of 2mL from each tube were frozen at -20°C in micro tubes (VWR, Hanover, Germany) until analyzed. In order to avoid concentration gradients during freezing and thawing,^19,20 samples were mixed before analysis. The biochemical analyses were performed within two months after freezing.

Measurements of biochemical analytes

The following analytes were measured by applying the ABX Pentra 400 Analyzer (Horiba) for colorimetric analysis: albumin (Alb), creatinine (Creat), iron (Fe), glutamate dehydrogenase (GLDH), glucose (Gluc), gamma-glutamyl transferase (GGT), inorganic phosphate (P), total protein (TP), and urea. Calcium (Ca) and Magnesium (Mg) were measured by Atomic absorption (Analyst 300 Perkin Elmer). An overview of analytes, article numbers, methods and manufacturers is listed in Table 1.

Data analyses were performed using the commercially available statistical software Jmp (Jmp® Pro version 12.1.0, Cary, NC, USA). Normality and homogeneity of variance assumptions were graphically assessed by evaluating histograms and scatterplots, skewness and kurtosis, and by the Shapiro-Wilks test. Biochemical analytes in a reference sample were measured repeatedly (n=18) and the coefficient of variation (CV) was calculated to assess test repeatability. All results were within specified limits of acceptance. The sample stability under different storage conditions for parametric data was evaluated by applying analysis of repeated measures (MANOVA, multivariate analysis of variance). The sample stability under different storage conditions for non-parametric data was determined using Kruskal-Wallis. Post-hoc testing by Tukey’s HSD test was used for multiple comparisons of the variables where the MANOVA was significant(P<0.05).Values of the biochemical constituents centrifuged twohours after sampling at RT were set to 100%. Values measured at 6h, 12h, 24h, 48h and 72h, at CT or RT after centrifugation were calculated as a percentage of the values achieved on samples 2h after centrifugation (Table 2).

The measured biochemical constituents after 6h, 12h, 24h, 48h and 72h, at CT and RT were evaluated relative to values at 2h (RT). Values at 2h (RT) were set to 100%. The numbers are discussed below and shown in Table 2. Repeated measurement analysis showed an overall significant time and temperature effect (P<0.0001). The time and temperature effects for each parameter are listed in Table 3.

Total protein (TP) and albumin (Alb)

A significant time effect was found for TP (P<0.0001) and Alb (P<0.001). TP serum mean concentrations in samples stored at CT or RT ranged from 89% to 110% of the mean concentration measured 2h post sampling while Alb mean concentrations ranged from 97% to 106%.

Calcium (Ca), iron (Fe), magnesium (Mg) and inorganic phosphate (P)

Significant time and temperature effects were found for Ca (P<0.0001). The values remained more stable when stored at RT compared to CT, ranging from 90% to 100% of the mean value measured at 2h post sampling. Significant time and temperature effects were found for Mg (P<0.0001). Mg concentrations changed significantly with increasing storage duration at CT and RT. However, less Mg changes were registered when samples were stored at CT. Storage of both 48 and 72 hat CT gave Mg concentrations of 113%. When stored at RT, a rise to 175% and 213% were found after 48h and 72h, respectively. Significant time and temperature effects were found for P (P<0.0001). Serum samples stored at both CT and RT, showed a significant increase in P concentrations when stored over time. Again, less P concentration changes were registered when samples were stored at CT. No significant changes in Fe values were found.

Creatinine (Creat), glucose (Gluc), and urea

A significant time effect was found for Creat (P<0.0001). Samples stored at CT and RT for 72h showed Creat mean levels at 83% and 88% of the values measured2 h post sampling, respectively. Significant time and temperature effects were found for porcine Gluc concentrations (P<0.0001). A storage period of 6h at CT led to mean Gluc concentrations that were 95% of the values measured at 2h. When stored at RT, the mean values were down to 86%. At CT and storage duration of 12h, the mean value was 88%. The corresponding mean value at RT was 67%. Finally, after 72h, Gluc concentrations of 71% and 12%were found at CT and RT, respectively. No significant changes in urea mean concentrations were found.

Gamma-glutamyl transferase (GGT) and glutamate dehydrogenase (GLDH)

A significant time and temperature effect was found for GLDH (P<0.0001). A rise in enzyme levels was found during the experimental period, both at CT and RT. However, enzyme levels reached higher levels when stored at RT. A temperature effect was found for GGT (P<0.05), and greater alterations in enzyme levels were found when samples were stored at RT. A total of 10 gilts were included in this study to provide robust scientific data. This number is not unlike that used by other researchers in both human and animal blood sample stability studies.^{11,14,15,21‒24} The study clearly shows that the stability of different relevant porcine blood analytes depend on both temperature levels and storage durations. Clotted blood samples were centrifuged2 h after sampling or left at 4°C or 20°C for time intervals of 6h, 12h, 24h, 48h and 72h to simulate potential storage conditions and durations of porcine blood samples before reaching the laboratory. In general, few reports describe effects of temperature and storage duration on biochemical analytes in clotted blood of farm animal species,^{11,15‒17,25} a couple of these include pigs.^15‒17 Dissimilarities in e.g. Gluc uptake and metabolism by erythrocytes exist across different species,^{11,12,26‒30} demonstrating the importance of drawing up species-specific recommendations. Based on previous reports, clinically acceptable limits (CAL) in changes by storage for the analytes Mg, Creat, urea, Gluc, Fe, P and GGT can be set to 10%. With regards to Ca, Alb and TP, a CAL of 5% can be defined.^3,15 To the authors’ knowledge, only one published study describe the effects of storage duration and the anticoagulant additive heparine on analytes in uncentrifuged porcine blood samples, but this work was only done at room temperature (RT).¹⁵ The general trends in several biochemical analytes analyzed in the study of Framstad et al.¹⁵ and this study are similar. After a period of 48 h storage without cooling, GGT values obtained in this study or by Framstad et al.¹⁵ exceeded the defined CAL of 10%.

The increase of the mainly membrane bound GGT in serum samples stored at room temperature is due to oxidative damage of RBC during storage, both liberating cytoplasmic GGT and membrane bound GGT. Lower storage temperature will delay metabolic processes,³¹ slowing down cell membrane damaging processes. Storage at CT for 48h in this study led to a value decrease of 3%, within the defined CAL set for GGT. GLDH values were influenced both by time and temperature conditions. Even though GLDH values did rise with more than 300% when stored at RT for 72h, the absolute GLDH levels were within the reference range.³² A strong Gluc decrease was found in samples stored non-cooled both in this study and by Framstad et al.¹⁵ This study gave a mean value of 46% after 24h storage at RT while their study gave a value of 67%, both far beyond the predetermined CAL of 10%. Although the mature erythrocyte of the pig has been observed to possess the slowest metabolic rate of any mammalian cell type, pig erythrocytes metabolize low but appreciable amounts of glucose to meet energy requirements,^33,34 which explains why uncentrifuged samples will drop rapidly in Gluc concentrations post sampling. Again, as mentioned above, lower storage temperature will slow down metabolic processes. Gluc concentrations in samples stored at CT dropped slower than samples stored at RT. This study also shows that P and Mg increase more rapidly when stored at RT. This is in accordance with previous reports, highlighting the importance of slowing down metabolic processes by cooling down uncentrifuged, clotted blood samples from pigs prior to processing at the laboratory¹⁵ since erythrocytes will leak P into the serum post sampling.¹⁸ Differences in the degree of analyte increase or decrease between the study of Framstad et al.¹⁵ and this study may be due to the fact that temperature conditions may have been somewhat different and that the samples in this study were frozen before analysed. The authors acknowledge that freezing the samples prior to the analyses deviates from the routine practice of many laboratories. Additionally, tubes, laboratory equipment and reagents used for analyses have changed. Despite some differences in the degree of analyte increase or decrease, the general trends are very similar.

The results from this study show a significant time and temperature influence on analytical results in uncentrifuged, clotted blood samples from pigs. This study gives helpful guidelines for Veterinary practitioners and researchers in handling clotted blood samples from pigs. Laboratories can also benefit from the results, although samples were frozen after centrifugation. Serum tubes with separator gels can prevent metabolism effects by forming a barrier between the cellular and the serum fraction in the tube, which can be of advantage if the serum cannot be separated prior to shipment of the samples.

If no serum tubes with separator gels are available and the RT storage time before analysis is anticipated to exceed 24hours, the samples should indeed be centrifuged before shipment. If no centrifuge is available, the addition of cooling elements can slow down alterations. Some parameters like Gluc, Mg, P crossed the predetermined CAL already after 6h, 12h and 24h at RT, respectively, and such alterations may lead to misinterpretations of results. Finally, analytical results from uncentrifuged porcine blood samples should always be interpreted with caution according to the shipment duration.

No financial interests or conflict of interests exist.

1. Statistics Norway Luftens temperatur, Celsiusgrader (SÅ 24). 2016.
2. Taylor EC, Sethi B. Stability of 27 biochemistry analytes in storage at a range of temperatures after centrifugation. Br J Biomed Sci. 2011;68:147‒157.
3. 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(4):375‒379.
4. Cuhadar S, Atay A, Koseoglu M, et al. Stability studies of common biochemical analytes in serum separator tubes with or without gel barrier subjected to various storage conditions. Bio chem Medica. 2012;22:202‒214.
5. Henriksen LO, Faber NR, Moller MF, et al. Stability of 35 biochemical and immunological routine tests after 10 hours storage and transport of human whole blood at 21°C. Scand J Clin Lab Invest. 2014;74:603‒610.
6. Heins M, Heil W, Withold W. Storage of serum or whole blood samples? Effects of time and temperature on 22 serum analytes. Eur J Clin Chem Clin Biochem 1995;33:231‒238.
7. Donnelly JG, Soldin SJ, Nealon DA, et al. Stability of twenty-five analytes in human serum at 22 degrees C, 4 degrees C, and -20 degrees C. Pediatr Pathol Lab Med. 1995;15:869‒874.
8. Tollersrud S. Stability of some serum enzymes in sheep, cattle, and swine during storage at different temperatures. Acta Vet Scand. 1969;10:359‒371.
9. Jones DG. Stability and storage characteristics of enzymes in sheep blood. Res Vet Sci. 1985;38:307‒311.
10. Jones DG. Stability and storage characteristics of enzymes in cattle blood. Res Vet Sci. 1985;38:301‒306.
11. Ehsani A, Afshari A, Bahadori H, et al. Serum constituents analyses in dairy cows: effects of duration and temperature of the storage of clotted blood. Res Vet Sci. 2008;85:473‒475.
12. Rendle DI, Heller J, Hughes KJ, et al. Stability of common biochemistry analytes in equine blood stored at room temperature. Equine Vet J. 2009;41(5):428‒432.
13. 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 ClinPathol Am Soc. 1992;21(3):88‒94.
14. 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 ClinPathol Am Soc. 1995;24(4):129‒133.
15. Framstad T, Havre GN, Morberg H. Effekt av lagring, separering og heparin på klinisk-kjemiske analyseresultater av griseblod. Nor Veterinærtidsskrift. 1989;101:237‒244.
16. Ihedioha JI, Onwubuche RC. Artifactual changes in PCV, hemoglobin concentration, and cell counts in bovine, caprine, and porcine blood stored at room and refrigerator temperatures. Vet ClinPathol Am Soc. 2007;36(1):60‒63.
17. Serp B Die Verwendung von Schweineblut in der Blutspurenmusteranalyse. Hämatologische und hämorheologische Eigenschaften von alterndem Schweineblut. Bachelor degree. Fachhochschule FH Campus Wien; 2015.
18. Humann Ziehank E, Ganter M. Pre-analytical factors affecting the results of laboratory blood analyses in farm animal veterinary diagnostics. Animal. 2012;6(7):1115‒1123.
19. Livesey JH, Ellis MJ, Evans MJ. Pre-Analytical Requirements. ClinBiochem Rev. 2008;1:11‒15.
20. Omang SH, Vellar OD. Concentration gradients in biological samples during storage, freezing and thawingKonzentrationsgradienten in biologischenProbenwährend der Lagerung, demEinfrieren und Auftauen. Fresenius Z Für Anal Chem. 1974;269:177‒181.
21. Thiers RE, Wu GT, Reed AH, et al. sSample stability: a suggested definition and method of determination. Clin Chem. 1976;22(2):176‒183.      
22. Clark S, Youngman LD, Palmer A, et al. Stability of plasma analytes after delayed separation of whole blood: implications for epidemiological studies. Int J Epidemiol. 2003;32(1):125‒130.
23. Ono T, Kitaguchi K, Takehara M, et al. Serum-constituents analyses: effect of duration and temperature of storage of clotted blood. Clin Chem. 1981;27(1):35‒88.
24. Cramb R, French J, Mackness M, et al. Lipid external quality assessment: commutability between external quality assessment and clinical specimens. Ann ClinBiochem. 2008;45(3):260‒265.
25. Fazio F, Giangrosso G, Marafioti S, et al. Blood haemogram in Ovisaries and Capra hyrcus: effect of storage time. Can J Anim Sci. 2016;96(1):32‒36.
26. Willard MD, Tvedten H. Introduction to Serum Chemistries: artifacts in Biochemical Determinations. In: Small Animal Clinical Diagnosis by Laboratory Methods. St. Louis, USA: Saunders Elsevier; 2004:113‒116.
27. Young D, Bermes E, Haverstick D. Separation and storage of specimens. In: Tietz Fundamentals of Clinical Chemistry. 6th ed. St. Louis, USA: Saunders Elsevier; 2008.
28. Kaneko JJ. Carbohydrate metabolism and its diseases. In: Clinical Biochemistry of Domestic Animals. 6th ed. Burlington: Academic Press; 2008:45‒80 p.
29. Stockham SL, Scott MA. Glucose, Ketoamines, and Related Regulatory Hormones. In: Fundamentals of Veterinary Clinical Pathology. Ames, Iowa: Blackwell Publishing; 2008:707‒737 p.
30. Weiser G. Sample collection, Processing, and Analysis of Laboratory Service Options. In: Veterinary Hematology and Clinical Chemistry. Ames, Iowa: Blackwell Publishing; 2006:39‒44.
31. Stoll C, Wolkers WF. Membrane Stability during Biopreservation of Blood Cells. Transfus Med Hemotherapy. 2011;38(2):89‒97.
32. Klem TB, Bleken E, Morberg H, et al. Hematologic and biochemical reference intervals for Norwegian crossbreed grower pigs. Vet ClinPathol. 2010;39(2):221‒226.
33. Dixon E, Wilson BA. Erythrocyte metabolism: kinetic and electrophoretic analyses of pig red cell hexokinase. J Exp Zool. 1981;215(1):63‒76.
34. Magnani M, Stocchi V, Serafini N, et al. Pig red blood cell hexokinase: regulatory characteristics and possible physiological role. Arch Biochem Biophys. 1983;226(1):377‒387.
35. Horiba Medical. Documentation data base Albumin CP.
36. Horiba Medical. Documentation data base Creatinine CP. A93A01230AEN.
37. Horiba Medical. Documentation data base Iron CP. A11A01637. 2007.
38. Horiba Medical. Documentation data base, ABX pentraggt cp. a93a00072hen. 201AD.
39. Horiba Medical. Documentation data base, ABX Glucose HK CP. A11A01667. 2006.
40. VWR. Magnesium ≥99.5%, ribbon TECHNICAL. Magnesium ≥99.5%, ribbon TECHNICAL.
41. Horiba Medical. Documentation data base Phosphorus CP. A11A01665. 2007.
42. Horiba Medical. Documentation data base total protein CP. A11A01669. 2005.
43. Horiba Medical. Documentation data base Urea CP. A11A01641. 2006.