Serum Amyloid A
Serum amyloid A (SAA) is an acute phase protein and α-globulin that is produced in the liver in response to inflammatory cytokines. It is considered a major acute phase protein in domestic species, except for the pig, in that low values are present in normal animals with marked increases (100-1000 fold) occurring within 24-48 hours with acute inflammation. Concentrations also rapidly decrease after resolution of inflammation, making SAA measurement a useful tool for monitoring the course of inflammation in an individual animal.
Serum amyloid A is produced in the liver and is highly conserved across species. It functions as an inflammatory and immunomodulatory protein, inducing inflammatory cytokine secretion, chemotaxis of neutrophils and mast cells and modulates immune responses, through the inflammasome. SAA mediates these effects by binding to several receptors on cells, including Toll like receptors (2 and 4), CD36 (a scavenger receptor) and an ADP receptor (P2X7). It is also involved in lipid metabolism and transport and in humans, circulates bound to high-density lipoproteins. Although the liver is the main site of synthesis, extrahepatic production of SAA does occur (e.g. in lungs, mammary gland, uterus, gastrointestinal system – based on mRNA expression in tissues; Berg et al 2011). Hepatic synthesis occurs in response to inflammatory cytokines (such as interleukins-1 and 6) and is considered part of the innate immune response. In animals, testing for SAA is predominantly done as a sensitive marker of inflammation.
Serum amyloid A is measured at Cornell University using an automated latex bead-based immunoturbidometric assay (SAA-VET, Eiken Chemical Co.). We previously used the LZ-SAA method, which utilized different antibodies than the current SAA-VET method. Previous studies have shown that LZ-SAA assay detects SAA in horses (Jacobsen et al 2006), wildlife (musk ox, impala, Asian elephants; Bertelsen et al 2009) and primates (chimpanzee and macaques; Bertelsen et al 2009, Krogh et al 2014). The newer VET-SAA method may not cross-react with the same species. An enzyme-linked immunosorbent assay is also available and detects SAA in dogs, horses and cattle, however this assay is less useful in a diagnostic laboratory setting and is prone to more imprecision than the automated immunoturbidometric assay. The SAA-VET assay detects SAA in horses (Jacobson et al 2019) and dogs (internal studies at Cornell University), and shows a much broader working range versus the LZ-SAA. The two methods provide comparable results up to about 4,000 mg/L and concentrations as high as 9,000 mg/L can be obtained with the VET-SAA (Jacobson et al 2019). There is also a point-of-care assay for SAA, but results do not always correlate with the immunoturbidometric assay, particularly at higher SAA concentrations, and there is batch-to-batch variation (Schwartz et al 2018).
- SAA-VET method: The reagent contains latex beads with bound rat anti-human SAA antibodies. The beads bind SAA in the patient’s sample, forming a precipitate, which alters the turbidity of the sample. The change in turbidity is measured spectrophotemetrically, after calibration with a standard with known SAA concentrations (in this case, a human standard). The lower limit of detection is 1, with a limit of quantification of 10 ug/mL with the Cobas 501 analyzer (Behling-Kelly et al 2022). The upper limit of detection is 2500 ug/mL (mg/L). A similar lower limit of detection and quantification was seen with a different analyzer (Waugh et al 2022).
Units of measurement
SAA concentration is measured in ug/mL (conventional units) and mg/L (SI units). The conversion equation is shown below:
ug/mL x 1 = mg/L
Serum, heparin plasma, body cavity fluids (synovial, peritoneal)
A study showed no significant difference in SAA concentrations in equine heparinized plasma and serum (Howard and Graubner 2014).
Previous studies have shown that equine SAA is more stable at room temperature than refrigerated. A small study at Cornell University confirmed this finding with the LZ-SAA method. Two equine samples with high values of 481 and 177 ug/ml showed a 2-3% decrease in concentration at 4ºC with storage for 24 hours versus a 3-5% decrease at room temperature. With storage for 72 hours, the drop at 4ºC was greater than at room temperature (7% versus 3-6% at 48 hours and 8-9% versus 2-4% at 72 hours). Very high values (>60 ug/dL) were still abnormal after 10 days of storage at either temperature (Hillstrom et al 2010). SAA concentrations are stable frozen (-20ºC), thus it is recommended that for storage >24 hours, the samples should be frozen and shipped ensuring that they remain frozen. If freezing is not an option, then the samples should be maintained at room temperature. This also suggests that mildly increased concentrations may drop to within reference intervals (<20 ug/mL in horses) with storage. Stability is unknown in other species.
Canine SAA concentrations measured with SAA-VET are stable for 6 months frozen at -20C and for 5 days refrigerated (Behling-Kelly et al 2022).
- Lipemia, hemolysis, and icterus: Unknown effect. Per manufacturer, lipemia and hemolysis should not interfere.
- Drugs: Corticosteroids may increase SAA concentrations in humans but not dogs (Martinez-Subiela et al 2004).
In horses, a reported reference interval for VET-SAA is 0.5-24 ug/mL or mg/L (Jacobsen et al 2019), but more than 90% of samples are below 5 ug/mL. The lower limit of detection of the VET-SAA is 1.1 ug/mL with the ADVIA 1800 analyzer (this may differ between analyzers). Low concentrations are not clinically relevant. In one study of 22 clinically healthy dogs, VET-SAA results were all below the lower limit of detection (0 ug/mL) (Behling-Kelly et al 2022). VET-SAA concentrations in 54 clinically healthy cats ranged from 0-5.4 ug/mL (Waugh et al 2022).
Increased SAA concentration
- Physiologic: Higher concentrations of SAA (using the ELISA assay) may be seen in newborn foals. Values are highest just after birth and then decline by 7 days of age (but still may be higher than reference intervals, even if clinically healthy) (Paltrinieri et al 2008). Similar results were seen in one study of newborn lambs, with highest SAA concentrations seen at 1-3 days of birth (Dinler et al 2020), which may be secondary to absorption of colostral endotoxins of pro-inflammatory mediators.
- Pathophysiologic: As indicated above, measurement of SAA is usually used to detect subclinical inflammation and the resolution thereof.
- Systemic inflammation: High SAA concentrations are seen in horses, dogs (Christensen et al 2014, Behling-Kelly et al 2022), cats (Waugh et al 2022) and other species with induced or spontaneously occurring inflammation and concentrations decline with resolution. SAA is not specific for the cause of inflammation, with increases being seen in dogs with inflammation due to septic and non-septic causes (Behling-Kelly et al 2022). After intravenous infusion of 500 ng/kg LPS, SAA concentrations increased after 6 hours and were still increased (>3000 mg/L) at 72 hours. The increases in SAA lagged behind decreased serum iron concentrations, which occurred within 6 hours of infusion (Lillehöök et al 2020).
- Localized inflammation: High SAA concentrations have been detected in synovial fluid of horses with various inflammatory joint and tendon conditions, including bacterial infection (Jacobsen et al 2006). High SAA concentrations are not, however, specific for bacterial infection. High concentrations of SAA were also seen in the peritoneal fluid and serum of horses with colic (Pihl et al 2013).
- Amyloidosis: Persistently high SAA concentrations can lead to the syndrome of systemic amyloidosis.