One of the key advantages for using VMRD’s SAA test for equine and feline is the accuracy and consistency provided by the calibration system.
SAA exists in varying configurations and changes structure frequently, including binding with cholesterol in the bloodstream, making quantification a challenge. Different assays have different components that affect recognition of SAA, therefore it is not unexpected that absolute numbers may vary between assays.1 This is very similar to how reference ranges for biochemistry values often vary between labs and different machines when running standard bloodwork. Even if specific values may vary, the overall trend observed should be the same with SAA increasing after an acute systemic inflammatory insult (such as infection) and decreasing as the issue resolves.
There is no existing test that can provide exact quantitation of SAA, therefore it is necessary to understand the reference standard and its importance. During development of VMRD SAA, we chose to calibrate against the Eiken LZ-SAA assay as it has historically been considered the “gold standard” 2 used in most veterinary publications3-31, and specifically validated for equine32 and feline33 samples. As intended, the equine VMRD SAA assay correlates very well with Eiken LZ-SAA, demonstrated in the independent validation study performed by Dr. Nicola Pusterla at UC Davis.34
At the time of this article, Eiken stopped producing LZ-SAA and switched to a different assay for the veterinary market (VET-SAA) that could also be used for canine samples. This naturally led to a change in the assay used by university labs such as Cornell and the University of Miami to Eiken VET-SAA. In our experience, and that of others, VET-SAA results are often half the values seen with LZ-SAA. This may be related to the fact that at least 3 isoforms of SAA exist in horses3, and our understanding is that VET-SAA only recognizes 1 of these (SAA-1). Since use of the LZ-SAA assay has been much more extensively documented than VET-SAA, and the observed results between the two have been so different, VMRD elected to continue calibrating to LZ-SAA so that we could maintain the wide range of results historically found in horses. This enables more effective monitoring of trends as a broader range allows a greater change in SAA value associated with changes in clinical condition.
To calibrate VMRD SAA, we use clinical samples from real animals as standards, with the same samples tested in each lot to ensure consistency. The SAA values of these samples were established by thorough testing with Eiken LZ-SAA, and they were then stored at -80°C in single-use aliquots for long-term use. During manufacturing, these standards are used as reference points to ensure that each lot/batch of tests is performing as expected and generating consistent results from one lot to the next.
When each lot of tests is completed, we use the results from the calibration samples to create a standard curve, which plots the known SAA concentration for each sample against the raw data obtained when the reader reads the intensity of the red lines on the test cartridge. This lot-specific and test-specific standard curve is programmed onto the calibration card, which communicates the information to the VMRD reader via RFID technology when the card is attached to the reader.
When a patient sample is run, the red lines on the test develop according to SAA concentration. The reader measures the intensity of the 3 lines (2 test lines and 1 control) and uses the standard curve from the calibration card to translate those raw numbers into an SAA value. In this manner, the reader is able to adjust interpretation of the test cartridge based on specifications for that individual lot and test type. All diagnostic assays have some degree of unavoidable variability between lots that occurs during manufacturing. However, this process of lot-specific calibration using a standard curve mitigates any existing variability to provide consistency and reliability over time and between readers.
1 Jacobsen S, Andersen PH. The acute phase protein serum amyloid A (SAA) as a marker of inflammation in horses. Equine Vet Educ 2007;19(1):38-46.
2 Canisso IF, Loux SC, and Lima FS. Biomarkers for placental disease in mares. Theriogenology 2020;150:302-307.
3 Jacobsen S, Niewold TA, Halling-Thomsen M, et al. Serum amyloid A isoforms in serum and synovial fluid in horses with lipopolysaccharide-induced arthritis. Vet Immunol Immunopathol 2006;110(3-4):325-330.
4 Belgrave RL, Dickey MM, Arheart KL. Assessment of serum amyloid A testing of horses and its clinical application in a specialized equine practice. J Am Vet Med Assoc 2013;243(1):113-119.
5 Borba LDA, Nogueira CEW, Bruhm FRP, et al. Peripheral blood markers of sepsis in foals born from mares with experimentally induced ascending placentitis. Vet Rec 2020;187(1):29-37.
6 Christoffersen M, Baagoe CD, Jacobsen S, et al. Evaluation of the systemic acute phase response and endometrial gene expression of serum amyloid A and pro- and anti-inflammatory cytokines in mares with experimentally induced endometritis. Vet Immunol Immunopathol 2010;138:95-105.
7 Canisso IF, Vall BA, Cray C, et al. Use of a qualitative hose-side test to measure serum amyloid A in mares with experimentally induced ascending placentitis. J Equine Vet Sci 2015;35:54-59.
8 Daniel AJ, Leise BS, Burgess BA, et al. Concentrations of serum amyloid A and plasma fibrinogen in horses undergoing emergency abdominal surgery. J Vet Emerg Crit Care 2016;26(3):344-351.
9 Duran MC, Dumrath CAC, Bartmann CP, et al. Serum amyloid A (SAA) concentration after vaccination in horses and mules. J Equine Vet Sci 2020;92:103165.
10 Escribano D, Bustillo AO, Marin LP, et al. Analytical validation of two point-of-care assays for serum amyloid A measurements in cats. Animals 2021;11:2518.
11 Falomo ME, Del Re C, Rossi M, et al. Relationship between postpartum uterine involution and biomarkers of inflammation and oxidative stress in clinically healthy mares (Equus caballus). Heliyon 2020;6:e03691.
12 Gluck K, Mohrs S, Hazuchova K, et al. Impact of radioiodine treatment on acute phase proteins in hyperthyroid cats. J Feline Med Surg 2022;24(4):359-365.
13 Haltmayer E, Schendenwein I, Licka TF. Course of serum amyloid A (SAA) plasma concentrations in horses undergoing surgery for injuries penetrating synovial structures, an observational clinical study. BMC Vet Res 2017;13:137-147.
14 Hooijberg EH, van der Hoven R, Tichy A, et al. Diagnostic and predictive capability of routine laboratory tests for the diagnosis and staging of equine inflammatory disease. J Vet Intern Med 2014;38:1587-1593.
15 Howard J and Graubner C. Comparison of paired serum and lithium heparin plasma samples for the measurement of serum amyloid A in horses using an automated turbidometric immunoassay. Vet J 2014;199:457-460.
16 Lilliehook I, Brojer J, Nostell K, et al. Hematologic, Prostaglandin F2α-metabolite, serum amyloid A, and serum iron changes in horses with experimentally induced endotoxemia. Vet Clin Pathol 2020;49:319-325.
17 Pihl TH, Andersen PH, Kjelgaard-Hansen M, et al. Serum amyloid A and haptoglobin concentrations in serum and peritoneal fluid of healthy horses and horses with acute abdominal pain. Vet Clin Pathol 2013;42(2):177-183.
18 Pihl TH, Scheepers E, Sanz M, et al. Influence of disease process and duration on acute phase proteins in serum and peritoneal fluid of horses with colic. J Vet Intern Med 2015;29:651-658.
19 Pihl TH, Scheepers E, Sanz M, et al. Acute-phase proteins as diagnostic markers in horses with colic. J Vet Emerg Crit Care 2016;26(5):664-674.
20 Robinson CS, Singer ER, Piviani M, et al. Are serum amyloid A or D-lactate useful to diagnose synovial contamination or sepsis in horses? Vet Rec 2017;181(16):425-429.
21 Rossi H, Raekallio M, Maatta M, et al. Effects of general anesthesia in dorsal recumbency with and without vatinoxan on bronchoalveolar lavage cytology of healthy horses. Vet J 2019;251:105352.
22 Rossi G, Meazzi S, Giordano A, et al. Serum paraoxonase 1 activity in cats: analytical validation, reference intervals, and correlation with serum amyloid A and alpha-1-acid glycoprotein. J Vet Diagn Invest 2020;32(6):844-855.
23 Sinovich M, Villarino NF, Singer E, et al. Can blood serum amyloid A concentrations in horses differentiate synovial sepsis from extrasynovial inflammation and determine response to treatment? Vet Rec 2020; 187(6):235-240.
24 Vandenplas ML, Moore JN, Barton MH, et al. Concentrations of serum amyloid A and lipopolysaccharide binding protein in horses with colic. Am J Vet Res 2005;66(9):1509-1516.
25 Vilhena H, Figueiredo M, Ceron, JJ, et al. Acute phase proteins and antioxidant responses in queens with pyometra. Theriogenology 2018;115:30-37.
26 Vilhena H, Tvarijonaviciute A, Ceron JJ, et al. Acute phase responses in cats naturally infected by hemotropic mycoplasmas. Comp Immunol Microbiol Infect Dis 2018;56:1-5.
27 Vilhena H, Tvarijonaviciute A, Ceron JJ, et al. Acute phase proteins and biomarkers of oxidative status in feline spontaneous malignant mammary tumors. Vet Comp Oncol 2019;17(3):394-406.
28 Westerman TL, Tornquist SJ, Foster CM, et al. Evaluation of serum amyloid A and haptoglobin concentrations as prognostic indicators for horses with inflammatory disease examined at a tertiary care hospital. Am J Vet Res 2015;76(10):882-888.
29 Westerman TL, Foster CM, Tornquist SJ, et al. Evaluation of serum amyloid A and haptoglobin concentrations as prognostic indicators for horses with colic. J Am Vet Med Assoc 2016;248(8):935-940.
30 Tamamoto T, Ohno K, Ohmi A, et al. Time-course monitoring of serum amyloid A in a cat with pancreatitis. Vet Clin Pathol 2009;38(1):83-86.
31 Tamamoto T, Ohno K, Takahashi M, et al. Serum amyloid A as a prognostic marker in cats with various diseases. J Vet Diagn Invest 2013;25(3):428-432.
32 Jacobsen S, Kjelgaard-Hansen M, Petersen HH, et al. Evaluation of a commercially available human serum amyloid A (SAA) turbidometric immunoassay for determination of equine SAA concentrations. Vet J 2006;172(2):315-319.
33 Tamamoto T, Ohno K, Ohmi A, et al. Verification of measurement of the feline serum amyloid A (SAA) concentration by human SAA turbidimetric immunoassay and its clinical application. J Vet Med Sci 2008;70(11):1247-1252.
34 Karam B, Hines S, Skipper L, et al. Whole-blood validation of a new point-of-care equine serum amyloid A assay. J Equine Vet Sci 2020;94:103222.