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.

Physiology

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.

Methodology

Serum amyloid A is measured at Cornell University using an automated latex bead-based immunoturbidometric assay (LZ-SAA, Eiken Chemical Co.). Previous studies have shown that this 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). 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.

Reaction type

End-point

Procedure

  • LZ-SAA method: The reagent contains latex beads with bound polyclonal rabbit and murine monoclonal 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 and upper limits of detection are 5 and 2,500 ug/mL, respectively.

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

Sample considerations

Sample type

Serum, heparin plasma, body cavity fluids (synovial, peritoneal)

Anticoagulant

A study showed no significant difference in SAA concentrations in equine heparinized plasma and serum (Howard and Graubner 2014).

Stability

Previous studies have shown that equine SAA is more stable at room temperature than refrigerated. A small study at Cornell University confirmed this finding. 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.

Interferences

  • 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).

Test interpretation

In horses, a reported reference interval for SAA is 0.5-20 ug/mL (Jacobsen et al 2006). Since the reference interval spans the lower limit of detection (<5 ug/mL), low concentrations are not relevant.

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).
  • 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) and other species with induced or spontaneously occurring inflammation and concentrations decline with resolution. SAA is not specific for the cause of inflammation.
    • 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.

 

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