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Acta Vet Scand
Acta Vet Scand
Acta Veterinaria Scandinavica
0044-605X
1751-0147
BioMed Central London

764
10.1186/s13028-024-00764-8
Brief Communication
A preliminary study on effects of storage time and repeated freezing-thawing on the stability of avian serum amyloid A
Rhim Haerin 1
Kwag Chaeyoung 1
http://orcid.org/0000-0001-8471-4761
Han Jae-Ik jihan@jbnu.ac.kr

12
1 https://ror.org/05q92br09 grid.411545.0 0000 0004 0470 4320 Laboratory of Wildlife Medicine, College of Veterinary Medicine, Jeonbuk National University, Iksan, 54596 Republic of Korea
2 https://ror.org/05q92br09 grid.411545.0 0000 0004 0470 4320 Jeonbuk Wildlife Center, Jeonbuk National University, Iksan, 54596 Republic of Korea
2 9 2024
2 9 2024
2024
66 4212 2 2024
16 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Within the field of clinical research, reports on the stability of avian serum amyloid A (SAA) under varying storage conditions are currently scarce. In this study, avian plasma samples were evaluated for SAA, a major acute-phase protein in birds, to assess how varying storage periods and repeated freeze-thaw cycles impact the stability of SAA in the frozen samples. Seven plasma samples from two species and six plasma samples from three species stored at ‒20 °C were used to evaluate the time and temperature effects accordingly. A chicken-specific SAA ELISA kit was used for the measurements. Statistical analysis was performed using SPSS, and the Kruskal-Wallis test and Spearman’s correlation coefficient were applied, with statistical significance set at P < 0.05. The SAA concentrations measured daily for 30 days showed no statistically significant differences over time. Freezing-thawing was repeated five times, and a significant negative relationship was confirmed over the cycles (r=‒0.8857, P < 0.05). Although no significance was observed between a decreased concentration and the number of cycles, a decrease in the concentration of > 10% was observed after the fourth cycle in four out of six samples. However, one to three freeze-thaw cycles did not result in a significant decline. Taken together, the results indicate that a negative correlation existed between the mean concentration and multiple freeze-thaw cycles, indicating that these should be avoided where possible.

Keywords

Acute phase protein
Bird
ELISA
Long-term
SAA
Wildlife
National Institute of Wildlife Diseases Control and Prevention2021 Han Jae-Ik issue-copyright-statement© The Danish Veterinary Association 2024
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pmcFindings

The acute phase reaction (APR) serves as a non-specific defensive response to a range of internal or external stimuli, playing a crucial role in maintaining physical homeostasis [1]. In response to adverse immunological events, the expression of acute phase proteins (APP) is significantly elevated through the action of cytokines [2]. While these proteins typically exist in the blood at minimal levels, their expression undergoes substantial changes during the APR, leading to their widespread use in medical and veterinary screenings [3]. Although APP are not in themselves indicative of a specific disease, they are employed, alongside other diagnostic tests, to narrow down potential causes.

Major APP have been identified in both humans and various animals, and their measurement assay is a routine procedure in a clinical setting for humans and animals alike. This testing is extending its application beyond companion animals and livestock to various wild animals [4–8]. In the avian context, proteins like serum amyloid A (SAA), alpha-1-acid glycoprotein (AGP), haptoglobin, and transferrin have been mainly described in chickens, falcons, and geese [9–13]. Among these, SAA stands out as a hydrophobic apolipoprotein of high-density lipoprotein with several isoforms [14, 15]. It exhibits high sensitivity, increasing rapidly during APR, up to 100–1000 times in both humans and animals [16–19]. While the precise maintenance period varies among animals, peaks tend to occur within 24–72 h, followed by a rapid decrease [20, 21]. SAA plays a crucial role in inflammation regulation, although it was initially identified as a fibrillogenic precursor of Amyloid A protein [3, 22].

Despite these insights, reports on the use of SAA as a clinical indicator in wild birds remains lacking. To date, there are still no commercially available reagents or testing kits that can be utilized in various birds, and the only ELISA kit available is for chickens. While testing fresh samples immediately is ideal, real-time testing with a single sample is limited due to the technique’s nature, necessitating storage. Also, stored samples can be tested in various cases, including validation of a method and tracking changes by comparing past samples with the present [23, 24]. This study aimed to fill this gap by evaluating the stability of SAA under varying storage conditions before its widespread application in wild birds. Our hypotheses were: (1) SAA concentrations would not change significantly during 30 days at ‒20 °C; (2) SAA concentration would decrease with repeated freeze-thaw cycles.

Several studies have been based on reports that APP, including human SAA, remain stable when stored at ‒20 °C [25, 26]. However, freezing can damage proteins due to surface denaturation and increase turbidity after thawing which may interfere with analysis [27]. In another study, AGP was found to be not affected by freezing [28]. Similarly, serum amyloid P, known for its role in human amyloidosis, showed no significant change when stored at 4 ℃ or ‒30 ℃ for four weeks, even after three freeze-thaw cycles [29]. In horses, one study found no significant change in SAA values of samples stored in a refrigerator for two months [30]. Another study reported consistent concentrations of equine SAA in both room-temperature and refrigerated samples for up to 17 days after collection [31]. On the contrary, frozen bovine serum samples exhibited a significant decrease in SAA levels starting from the second day of storage, suggesting refrigeration over freezing [32].

Plasma samples were collected from rescued wild birds (n = 13) at the Jeonbuk Wildlife Center during intake examinations. A manual complete blood count was performed immediately, followed by biochemical tests after plasma centrifugation. To avoid any changes being masked by a low baseline value, birds with inflammation confirmed by blood test results, including elevated white blood cell count and toxic changes predicting high SAA, were selected. The SAA was measured using an anti-chicken SAA ELISA kit (Eagle Biosciences, Amherst, NH, USA). All samples were diluted 50:1 prior to measurement, according to the manufacturer’s instructions. This kit was validated in our previous study and was used after preliminary tests were performed to evaluate whether the same sample in the three species yielded a consistent measurement across serial dilution ratios [23, 33]. All measurements were performed in duplicate and blinded to sample information. The intra- and inter-assay variations were 3.35% (range 0.22–16.23%) and 8.68% (range 8.3–9.06%), respectively. The lowest detection limit (mean + 2*SD) was 0.072 ng/mL.

To evaluate the effect of storage time on SAA, seven samples from feral pigeons (Columba livia) and Eurasian eagle-owls (Bubo bubo) were examined. Plasma was divided into microtubes and stored at ‒20 °C, with the first test conducted before freezing. Measurements were performed at the same time daily for 30 days at room temperature. For the effect of freeze-thaw cycles, six samples from common kestrels (Falco tinnunculus) and feral pigeons were examined. Plasma was divided into microtubes in aliquots and stored at ‒20 °C, and the first test was performed before freezing. The freeze–thaw cycle was repeated five times. The samples were frozen for at least two days and kept at room temperature for 30 min to be completely thawed before measurement.

Storage stability was evaluated using Spearman’s correlation coefficient and the Kruskal-Wallis test. Post-hoc tests were performed using the Mann-Whitney U test with Bonferroni correction. The Mann-Whitney U test was also applied separately to compare the days. Statistical significance was set at P < 0.05, using SPSS V27 (IBM SPSS, Armonk, NY, USA) and GraphPad Prism V9 (GraphPad Software, San Diego, CA, USA).

No statistically significant change was observed in the SAA concentrations of the seven plasma samples during the 30-day storage period (Fig. 1). Their variance was within the CV range of 10%. A significant negative relationship was observed between the mean SAA concentration in response to freezing and thawing, according to Spearman’s correlation coefficient (r=‒0.8857, P < 0.05) (Fig. 2). The same result was confirmed in four out of six samples when analyzed individually (‒0.935 < r<‒0.651, P < 0.05). A tendency to decrease with repetitions was found in the freeze-thaw processes from the third to the fifth cycle. The value decreased by over 10% from the second cycle in one sample to the third cycle in one sample and to the fourth cycle in three samples, which was out of the acceptable range for the method used. However, the analysis of which cycle showed a significant decline was not statistically effective for all samples.

Fig. 1 Plots of SAA concentrations for 30 days storage at ‒20 ℃. The graph (a) shows each value in the seven samples over time. No significant change according to the days was observed. (b) Plot of the median SAA concentration of all seven samples. The median (dots) and interquartile range (bars) are shown. No significant change between the days was observed. EO, Eurasian eagle owl; FP, feral pigeon. SAA: Serum amyloid A

Fig. 2 Scatter diagram and linear regression of SAA concentrations following repeated freezing and thawing cycles. (a) Two samples with high values; (b) four samples with moderate values. (c) Plot of the median SAA concentrations of all six samples over cycles. The median (dots), interquartile range (bars), and simple linear regression (dotted line) are shown (r = ‒0.8857, P < 0.05). Asterisks (*) denote samples showing a significant negative correlation with mean concentration after repeated freeze-thaw cycles. CK: common kestrel; FP: feral pigeon; SAA: serum amyloid A

Plasma or serum samples are often stored before testing due to limited in-clinic equipment, inability to run a test immediately, or having only one sample. However, the choice of storage temperature and period has been reported to affect the samples and their subsequent analysis results [34]. Changes in many serum biochemical parameters in response to storage conditions have been studied in both humans and animals [35–39]. In general, serum is stored frozen because the levels of many analytes can be significantly altered when refrigerated for an extended period. Frozen samples stored at ‒20 ℃ did not show any significant alterations in SAA values during the 30-day study, unlike one study in bovine SAA [32]. This might be due to the different effects on protein structure depending on the speed of the freezing and thawing process [40]. Whether this is due to differences in animals, requires further investigation as the sample numbers in both studies were small.

A previous study reported marked fractional changes in plasma protein electrophoresis from the second day of refrigeration of psittacine samples, reflecting a much weaker stability than freezing [41]. However, it was also reported that refrigerated samples were also stable for several days, suggesting that a comparative study at different storage temperatures is warranted to determine whether SAA is affected by refrigeration or freezing [30, 31]. Additionally, since samples older than several months or years might be used in practical situations, samples stored for a longer period still need to be warranted.

On the contrary, we could confirm the negative relationship between the freezing-thawing cycle and avian SAA. Although not all samples showed a statistically significant negative correlation in individual analyses, a decrease of > 10% from the initial value was observed in all of them after the fourth cycle. Nevertheless, when the number of cycles was less than four, the values were within the acceptable variation (< 10%). Similarly, no significant change was observed in the fraction percentages of psittacine plasma and canine C-reactive protein stored at − 20 °C according to repeated freezing and thawing up to three and four times, respectively [24, 41]. The fact that decreases after the fourth cycle were observed in five out of six samples were indicative that repeated freeze-thaw should be avoided as far as possible. However, freeze-thaw from one to three cycles did not appear to have a significant effect on the concentration of avian SAA.

As a limitation of our study, we used birds with confirmed inflammatory responses in this study. Even though there was an inflammatory reaction confirmed by blood smears, it was unknown how much the SAA level would have risen. However, given that the CV of ELISA methods is within 10%, low SAA levels could mask detectable changes, even if the daily variation was not predictable. It was confirmed in a later study that SAA values used in this study increased from mild to severe [33], but due to sampling bias, a broader survey including healthy birds should be addressed in the future.

In conclusion, SAA was found to be stable for one month and 1–3 times of freeze-thaw cycles in plasma frozen at ‒20 °C. However, a negative correlation was observed in samples over repeated freeze-thaw cycles. These findings contribute to the understanding of SAA’s reliability in avian health assessments and emphasize appropriate sample handling for accurate results.

Abbreviations

AA Amyloid A

AGP Alpha-1-acid glycoprotein

APP Acute phase protein

APR Acute phase reaction

SAA Serum amyloid A

Acknowledgements

We would like to express our deepest gratitude to the staff and students who helped with patient care and sample collection at Jeonbuk Wildlife Center.

Authors’ contributions

RH, KC, and HJI conceived and designed the study; RH and KC conducted experiments; RH analysed the data and wrote the original draft. HJI supervised and received funding; RH, KC, and HJI reviewed and revised the manuscript. All authors have read and approved the final version of the manuscript.

Funding

This work was supported by the National Institute of Wildlife Diseases Control and Prevention as a “Specialized Graduate School Support Project for Wildlife Disease Specialists.”

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval

Because this study used archived samples, institutional ethical approval was not required. These samples were collected for basic health screening of all animals at the wildlife center. Animals were treated in accordance with high ethical standards and national legislation.

Consent for publication

Not applicable.

Prior publication

Data have not been published previously.

Competing interests

The authors declare that they have no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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References

1. Petersen HH Nielsen JP Heegaard PMH Application of acute phase protein measurements in veterinary clinical chemistry Vet Res 2004 35 163 87 10.1051/vetres:2004002 15099494
Petersen HH, Nielsen JP, Heegaard PMH. Application of acute phase protein measurements in veterinary clinical chemistry. Vet Res. 2004;35:163–87. 10.1051/vetres:2004002.15099494 10.1051/vetres:2004002
2. Eckersall PD The time is right for acute phase protein assays Vet J 2004 168 3 5 10.1016/j.tvjl.2003.09.003 15158201
Eckersall PD. The time is right for acute phase protein assays. Vet J. 2004;168:3–5. 10.1016/j.tvjl.2003.09.003.15158201 10.1016/j.tvjl.2003.09.003
3. Cray C Zaias J Altman NH Acute phase response in animals: a review Comp Med 2009 59 517 26 20034426
Cray C, Zaias J, Altman NH. Acute phase response in animals: a review. Comp Med. 2009;59:517–26.20034426
4. Bertelsen MF Kjelgaard-Hansen M Grøndahl C Heegaard PM Jacobsen S Identification of acute phase proteins and assays applicable in nondomesticated mammals J Zoo Wildl Med 2009 40 199 203 10.1638/2007-0125.1 19368263
Bertelsen MF, Kjelgaard-Hansen M, Grøndahl C, Heegaard PM, Jacobsen S. Identification of acute phase proteins and assays applicable in nondomesticated mammals. J Zoo Wildl Med. 2009;40:199–203. 10.1638/2007-0125.1.19368263 10.1638/2007-0125.1
5. Depauw S Delanghe J Whitehouse-Tedd K Kjelgaard-Hansen M Christensen M Hesta M Serum protein capillary electrophoresis and measurement of acute phase proteins in a captive cheetah (Acinonyx jubatus) population J Zoo Wildl Med 2014 45 497 506 10.1638/2013-0111R1.1 25314816
Depauw S, Delanghe J, Whitehouse-Tedd K, Kjelgaard-Hansen M, Christensen M, Hesta M, et al. Serum protein capillary electrophoresis and measurement of acute phase proteins in a captive cheetah (Acinonyx jubatus) population. J Zoo Wildl Med. 2014;45:497–506. 10.1638/2013-0111R1.1.25314816 10.1638/2013-0111R1.1
6. Harr KE Rember R Ginn PE Lightsey J Keller M Reid J Serum amyloid A (SAA) as a biomarker of chronic infection due to boat strike trauma in a free-ranging Florida manatee (Trichechus manatus latirostris) with incidental polycystic kidneys J Wildl Dis 2011 47 1026 31 10.7589/0090-3558-47.4.1026 22102678
Harr KE, Rember R, Ginn PE, Lightsey J, Keller M, Reid J, et al. Serum amyloid A (SAA) as a biomarker of chronic infection due to boat strike trauma in a free-ranging Florida manatee (Trichechus manatus latirostris) with incidental polycystic kidneys. J Wildl Dis. 2011;47:1026–31. 10.7589/0090-3558-47.4.1026.22102678 10.7589/0090-3558-47.4.1026
7. Stanton JJ Cray C Rodriguez M Arheart KL Ling PD Herron A Acute phase protein expression during elephant endotheliotropic herpesvirus-1 viremia in Asian elephants (Elephas maximus) J Zoo Wildl Med 2013 44 605 12 10.1638/2012-0174R1.1 24063088
Stanton JJ, Cray C, Rodriguez M, Arheart KL, Ling PD, Herron A. Acute phase protein expression during elephant endotheliotropic herpesvirus-1 viremia in Asian elephants (Elephas maximus). J Zoo Wildl Med. 2013;44:605–12. 10.1638/2012-0174R1.1.24063088 10.1638/2012-0174R1.1
8. Krogh AKH Lundsgaard JFH Bakker J Langermans JA Verreck FA Kjelgaard-Hansen M Acute-phase responses in healthy and diseased rhesus macaques (Macaca mulatta) J Zoo Wildl Med 2014 45 306 14 10.1638/2013-0153R.1 25000691
Krogh AKH, Lundsgaard JFH, Bakker J, Langermans JA, Verreck FA, Kjelgaard-Hansen M, et al. Acute-phase responses in healthy and diseased rhesus macaques (Macaca mulatta). J Zoo Wildl Med. 2014;45:306–14. 10.1638/2013-0153R.1.25000691 10.1638/2013-0153R.1
9. Kovács BM Toussaint MJ Gruys E Fábián IB Szilágyi L Janan J Evaluation of goose serum amyloid a acute phase response by enzyme-linked immunosorbent assay Acta Vet Hung 2007 55 349 57 10.1556/AVet.55.2007.3.9 17867462
Kovács BM, Toussaint MJ, Gruys E, Fábián IB, Szilágyi L, Janan J, et al. Evaluation of goose serum amyloid a acute phase response by enzyme-linked immunosorbent assay. Acta Vet Hung. 2007;55:349–57. 10.1556/AVet.55.2007.3.9.17867462 10.1556/AVet.55.2007.3.9
10. Caliendo V McKinney P Bailey T Kinne J Wernery U Serum amyloid A as an indicator of health status in falcons J Avian Med Surg 2013 27 83 9 10.1647/2011-026 23971216
Caliendo V, McKinney P, Bailey T, Kinne J, Wernery U. Serum amyloid A as an indicator of health status in falcons. J Avian Med Surg. 2013;27:83–9. 10.1647/2011-026.23971216 10.1647/2011-026
11. Fischer D Van Waeyenberghe L Cray C Gross M Usleber E Pasmans F Comparison of diagnostic tools for the detection of aspergillosis in blood samples of experimentally infected falcons Avian Dis 2014 58 587 98 10.1637/10831-032714-Reg 25619004
Fischer D, Van Waeyenberghe L, Cray C, Gross M, Usleber E, Pasmans F, et al. Comparison of diagnostic tools for the detection of aspergillosis in blood samples of experimentally infected falcons. Avian Dis. 2014;58:587–98. 10.1637/10831-032714-Reg.25619004 10.1637/10831-032714-Reg
12. Chamanza R Toussaint MJM Van Ederen AM van Veen L Hulskamp-Koch C Fabri TH Serum amyloid A and transferrin in chicken. A preliminary investigation of using acute-phase variables to assess diseases in chickens Vet Q 1999 21 158 62 10.1080/01652176.1999.9695012 10568007
Chamanza R, Toussaint MJM, Van Ederen AM, van Veen L, Hulskamp-Koch C, Fabri TH. Serum amyloid A and transferrin in chicken. A preliminary investigation of using acute-phase variables to assess diseases in chickens. Vet Q. 1999;21:158–62. 10.1080/01652176.1999.9695012.10568007 10.1080/01652176.1999.9695012
13. Chamanza R van Veen L Tivapasi MT Toussaint MJM Acute phase proteins in the domestic fowl Worlds Poult Sci J 1999 55 61 71 10.1079/WPS19990005
Chamanza R, van Veen L, Tivapasi MT, Toussaint MJM. Acute phase proteins in the domestic fowl. Worlds Poult Sci J. 1999;55:61–71. 10.1079/WPS19990005.10.1079/WPS19990005
14. Jensen LE Whitehead AS Regulation of serum amyloid A protein expression during the acute-phase response Biochem J 1998 334 489 503 10.1042/bj3340489 9729453
Jensen LE, Whitehead AS. Regulation of serum amyloid A protein expression during the acute-phase response. Biochem J. 1998;334:489–503. 10.1042/bj3340489.9729453 10.1042/bj3340489
15. Landman WJM Gruys E Gielkens ALJ Avian amyloidosis Avian Pathol 1998 27 437 49 10.1080/03079459808419367 18484028
Landman WJM, Gruys E, Gielkens ALJ. Avian amyloidosis. Avian Pathol. 1998;27:437–49. 10.1080/03079459808419367.18484028 10.1080/03079459808419367
16. Vandenplas ML Moore JN Barton MH Roussel AJ Cohen ND Concentrations of serum amyloid A and lipopolysaccharide-binding protein in horses with colic Am J Vet Res 2005 66 1509 16 10.2460/ajvr.2005.66.1509 16261823
Vandenplas ML, Moore JN, Barton MH, Roussel AJ, Cohen ND. Concentrations of serum amyloid A and lipopolysaccharide-binding protein in horses with colic. Am J Vet Res. 2005;66:1509–16. 10.2460/ajvr.2005.66.1509.16261823 10.2460/ajvr.2005.66.1509
17. Kushner I The phenomenon of the acute phase response Ann N Y Acad Sci 1982 389 39 48 10.1111/j.1749-6632.1982.tb22124.x 7046585
Kushner I. The phenomenon of the acute phase response. Ann N Y Acad Sci. 1982;389:39–48. 10.1111/j.1749-6632.1982.tb22124.x.7046585 10.1111/j.1749-6632.1982.tb22124.x
18. Christensen M Jacobsen S Ichiyanagi T Kjelgaard-Hansen M Evaluation of an automated assay based on monoclonal anti-human serum amyloid A (SAA) antibodies for measurement of canine, feline, and equine SAA Vet J 2012 194 332 7 10.1016/j.tvjl.2012.05.007 22704135
Christensen M, Jacobsen S, Ichiyanagi T, Kjelgaard-Hansen M. Evaluation of an automated assay based on monoclonal anti-human serum amyloid A (SAA) antibodies for measurement of canine, feline, and equine SAA. Vet J. 2012;194:332–7. 10.1016/j.tvjl.2012.05.007.22704135 10.1016/j.tvjl.2012.05.007
19. Uhlar CM Whitehead AS Serum amyloid A, the major vertebrate acute-phase reactant Eur J Biochem 1999 265 501 23 10.1046/j.1432-1327.1999.00657.x 10504381
Uhlar CM, Whitehead AS. Serum amyloid A, the major vertebrate acute-phase reactant. Eur J Biochem. 1999;265:501–23. 10.1046/j.1432-1327.1999.00657.x.10504381 10.1046/j.1432-1327.1999.00657.x
20. Tape C Kisilevsky R Apolipoprotein A-I and apolipoprotein SAA half-lives during acute inflammation and amyloidogenesis Biochim Biophys Acta 1990 1043 295 300 10.1016/0005-2760(90)90030-2 2108727
Tape C, Kisilevsky R. Apolipoprotein A-I and apolipoprotein SAA half-lives during acute inflammation and amyloidogenesis. Biochim Biophys Acta. 1990;1043:295–300. 10.1016/0005-2760(90)90030-2.2108727 10.1016/0005-2760(90)90030-2
21. Yamada T Serum amyloid A (SAA): a concise review of biology, assay methods and clinical usefulness Clin Chem Lab Med 1999 37 381 8 10.1515/CCLM.1999.063 10369107
Yamada T. Serum amyloid A (SAA): a concise review of biology, assay methods and clinical usefulness. Clin Chem Lab Med. 1999;37:381–8. 10.1515/CCLM.1999.063.10369107 10.1515/CCLM.1999.063
22. Levin M Pras M Franklin EC Immunologic studies of the major nonimmunoglobulin protein of amyloid: I. Identification and partial characterization of a related serum component J Exp Med 1973 138 373 80 10.1084/jem.138.2.373 4198200
Levin M, Pras M, Franklin EC. Immunologic studies of the major nonimmunoglobulin protein of amyloid: I. Identification and partial characterization of a related serum component. J Exp Med. 1973;138:373–80. 10.1084/jem.138.2.373.4198200 10.1084/jem.138.2.373
23. Kjelgaard-Hansen M Jacobsen S Assay validation and diagnostic applications of major acute-phase protein testing in companion animals Clin Lab Med 2011 31 51 70 10.1016/j.cll.2010.10.002 21295722
Kjelgaard-Hansen M, Jacobsen S. Assay validation and diagnostic applications of major acute-phase protein testing in companion animals. Clin Lab Med. 2011;31:51–70. 10.1016/j.cll.2010.10.002.21295722 10.1016/j.cll.2010.10.002
24. Hillström A Hagman R Tvedten H Kjelgaard-Hansen M Validation of a commercially available automated canine-specific immunoturbidimetric method for measuring canine C-reactive protein Vet Clin Pathol 2014 43 235 43 10.1111/vcp.12150 24798319
Hillström A, Hagman R, Tvedten H, Kjelgaard-Hansen M. Validation of a commercially available automated canine-specific immunoturbidimetric method for measuring canine C-reactive protein. Vet Clin Pathol. 2014;43:235–43. 10.1111/vcp.12150.24798319 10.1111/vcp.12150
25. McDonald TL Weber A Smith JW A monoclonal antibody sandwich immunoassay for serum amyloid A (SAA) protein J Immunol Methods 1991 144 149 55 10.1016/0022-1759(91)90081-p26 1720442
McDonald TL, Weber A, Smith JW. A monoclonal antibody sandwich immunoassay for serum amyloid A (SAA) protein. J Immunol Methods. 1991;144:149–55. 10.1016/0022-1759(91)90081-p26.1720442 10.1016/0022-1759(91)90081-p26
26. Cerón JJ Eckersall PD Martýnez-Subiela S Acute phase proteins in dogs and cats: current knowledge and future perspectives Vet Clin Pathol 2005 34 85 99 10.1111/j.1939-165x.2005.tb00019.x 15902658
Cerón JJ, Eckersall PD, Martýnez-Subiela S. Acute phase proteins in dogs and cats: current knowledge and future perspectives. Vet Clin Pathol. 2005;34:85–99. 10.1111/j.1939-165x.2005.tb00019.x.15902658 10.1111/j.1939-165x.2005.tb00019.x
27. Gislefoss RE Lauritzen M Langseth H Mørkrid L Effect of multiple freeze-thaw cycles on selected biochemical serum components Clin Chem Lab Med 2017 55 967 73 10.1515/cclm-2016-0892 27987362
Gislefoss RE, Lauritzen M, Langseth H, Mørkrid L. Effect of multiple freeze-thaw cycles on selected biochemical serum components. Clin Chem Lab Med. 2017;55:967–73. 10.1515/cclm-2016-0892.27987362 10.1515/cclm-2016-0892
28. Ganz PA Shell WE Tökés ZA Evaluation of a radioimmunoassay for α1-acid glycoprotein to monitor therapy of cancer patients J Natl Cancer Inst 1983 71 25 30 6575206
Ganz PA, Shell WE, Tökés ZA. Evaluation of a radioimmunoassay for α1-acid glycoprotein to monitor therapy of cancer patients. J Natl Cancer Inst. 1983;71:25–30.6575206
29. Millar DJ Hutchinson WL Pepys MB Immunoradiometric assay for human serum amyloid P component J Immunol Methods 2011 371 18 24 10.1016/j.jim.2011.06.010 21708157
Millar DJ, Hutchinson WL, Pepys MB. Immunoradiometric assay for human serum amyloid P component. J Immunol Methods. 2011;371:18–24. 10.1016/j.jim.2011.06.010.21708157 10.1016/j.jim.2011.06.010
30. Pepys MB Baltz ML Tennent GA Kent J Ousey J Rossdale PD Serum amyloid A protein (SAA) in horses: objective measurement of the acute phase response Equine Vet J 1989 21 106 9 10.1111/j.2042-3306.1989.tb02108.x 2539996
Pepys MB, Baltz ML, Tennent GA, Kent J, Ousey J, Rossdale PD. Serum amyloid A protein (SAA) in horses: objective measurement of the acute phase response. Equine Vet J. 1989;21:106–9. 10.1111/j.2042-3306.1989.tb02108.x.2539996 10.1111/j.2042-3306.1989.tb02108.x
31. Hillström A Tvedten H Lilliehöök I Evaluation of an in-clinic serum amyloid A (SAA) assay and assessment of the effects of storage on SAA samples Acta Vet Scand 2010 52 8 10.1186/1751-0147-52-8 20122257
Hillström A, Tvedten H, Lilliehöök I. Evaluation of an in-clinic serum amyloid A (SAA) assay and assessment of the effects of storage on SAA samples. Acta Vet Scand. 2010;52:8. 10.1186/1751-0147-52-8.20122257 10.1186/1751-0147-52-8
32. Tóthová C, Nagy O, Seidel H, Kováč G. The effect of storage temperature and time on the concentrations of bovine serum amyloid A and its mammary associated isoform. Vet Med Int. 2012;861458. 10.1155/2012/861458.
33. Rhim H Kim M Gim S Han JI Diagnostic value of serum amyloid A in differentiating the inflammatory disorders in wild birds Front Vet Sci 2024 11 1284113 10.3389/fvets.2024.1284113 38379926
Rhim H, Kim M, Gim S, Han JI. Diagnostic value of serum amyloid A in differentiating the inflammatory disorders in wild birds. Front Vet Sci. 2024;11:1284113. 10.3389/fvets.2024.1284113.38379926 10.3389/fvets.2024.1284113
34. Boyanton BL Jr Blick KE Stability studies of twenty-four analytes in human plasma and serum Clin Chem 2002 48 2242 7 10.1093/clinchem/48.12.2242 12446483
Boyanton BL Jr, Blick KE. Stability studies of twenty-four analytes in human plasma and serum. Clin Chem. 2002;48:2242–7. 10.1093/clinchem/48.12.2242.12446483 10.1093/clinchem/48.12.2242
35. Cray C Rodriguez M Zaias J Altman NH Effects of storage temperature and time on clinical biochemical parameters from rat serum J Am Assoc Lab Anim Sci 2009 48 202 4 19383219
Cray C, Rodriguez M, Zaias J, Altman NH. Effects of storage temperature and time on clinical biochemical parameters from rat serum. J Am Assoc Lab Anim Sci. 2009;48:202–4.19383219
36. Reynolds B Taillade B Médaille C Palenché F Trumel C Lefebvre HP Effect of repeated freeze-thaw cycles on routine plasma biochemical constituents in canine plasma Vet Clin Pathol 2006 35 339 40 10.1111/j.1939-165x.2006.tb00144.x 16967422
Reynolds B, Taillade B, Médaille C, Palenché F, Trumel C, Lefebvre HP. Effect of repeated freeze-thaw cycles on routine plasma biochemical constituents in canine plasma. Vet Clin Pathol. 2006;35:339–40. 10.1111/j.1939-165x.2006.tb00144.x.16967422 10.1111/j.1939-165x.2006.tb00144.x
37. Thoresen SI Tverdal A Havre G Morberg H Effects of storage time and freezing temperature on clinical chemical parameters from canine serum and heparinized plasma Vet Clin Pathol 1995 24 129 33 10.1111/j.1939-165x.1995.tb00954.x 12664427
Thoresen SI, Tverdal A, Havre G, Morberg H. Effects of storage time and freezing temperature on clinical chemical parameters from canine serum and heparinized plasma. Vet Clin Pathol. 1995;24:129–33. 10.1111/j.1939-165x.1995.tb00954.x.12664427 10.1111/j.1939-165x.1995.tb00954.x
38. Hawkins MG Kass PH Zinkl JG Tell LA 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 35 219 25 10.1111/j.1939-165x.2006.tb00118.x 16783717
Hawkins MG, Kass PH, Zinkl JG, Tell LA. 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;35:219–25. 10.1111/j.1939-165x.2006.tb00118.x.16783717 10.1111/j.1939-165x.2006.tb00118.x
39. Jakubowski J Aebischer V Luetzelschwab J Vogel B Donatsch P Cordier A Stability of clinical chemistry parameter values in minipig serum under different storage conditions Scand J Lab Anim Sci 1998 25 197 204
Jakubowski J, Aebischer V, Luetzelschwab J, Vogel B, Donatsch P, Cordier A. Stability of clinical chemistry parameter values in minipig serum under different storage conditions. Scand J Lab Anim Sci. 1998;25:197–204.
40. Cao E Chen Y Cui Z Foster PR Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions Biotechnol Bioeng 2003 82 684 90 10.1002/bit.10612 12673768
Cao E, Chen Y, Cui Z, Foster PR. Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions. Biotechnol Bioeng. 2003;82:684–90. 10.1002/bit.10612.12673768 10.1002/bit.10612
41. Cray C Rodriguez M Zaias J Protein electrophoresis of psittacine plasma Vet Clin Pathol 2007 36 64 72 10.1111/j.2042-3306.1989.tb02108.x 17311197
Cray C, Rodriguez M, Zaias J. Protein electrophoresis of psittacine plasma. Vet Clin Pathol. 2007;36:64–72. 10.1111/j.2042-3306.1989.tb02108.x.17311197 10.1111/j.2042-3306.1989.tb02108.x
