B-130 Quantification of the Sensitive Inflammatory Marker Serum Amyloid A (SAA) by Electrochemiluminescence Immunoassay

Abstract Background Serum Amyloid A (SAA) is an acute phase response (APR) protein. As a marker of both acute and chronic inflammation. SAA has been used to investigate common systemic autoimmune diseases and infections like SARS-CoV-2 as well as less common inflammatory conditions like familial Mediterranean fever (FMF) and Behcet’s disease. Similar to but more sensitive than C-reactive protein (CRP), SAA levels rise quickly more than 100-fold during episodes of inflammation and then fall as inflammation resolves. SAA is also the soluble precursor of Amyloid A which can accumulate in tissues such as heart, kidney, liver, spleen, and nervous system as amyloidosis. Amyloidosis affects FMF and Behcet’s patients who have not only episodic inflammation but also prolonged subclinical inflammation. Importantly, monitoring SAA levels allows for the detection of subclinical inflammation, and better control of subclinical inflammation holds the promise of long-term benefits in growth, intellectual function, cardiovascular health and reduced amyloidosis with reduced end-stage kidney disease. Here, we developed an electrochemiluminescence immunoassay (ECLIA) to quantify SAA with robust analytical performance for such diverse clinical applications as evaluation and monitoring of patients with chronic inflammatory diseases. Methods Detection and quantification of SAA was accomplished with a lab developed test (LDT) two-step ECLIA. The analytical performance of the assay was validated for the parameters of accuracy, precision, lower limit of quantitation (LLOQ), upper limit of quantitation (ULOQ), and dilutional linearity. We established a reference interval cut point using serum samples from >120 individuals with no history of inflammatory disease and no recent evidence of inflammation, infection, or injury. Calculation of reference interval cut point was determined using a transformed parametric test. Results The analytical measurement range (AMR) of the assay was determined to be from 0.4 µg/mL to 625 µg/mL. Samples diluted linearly >1,000-fold (recovery within 80 – 120%) allowing for a maximum reportable value of 625,000 µg/mL and a lower limit of quantitation (LLOQ) of 0.4 µg/mL. Within the AMR, standard bias was = ±8.5% and standard imprecision was = 9.1% CV. Sample imprecision was = 10.9% CV. The reference interval cut point was determined to be < 5 µg/mL (95% CI, 3.78 – 5.51) with a confidence interval of 0.21. Of the 120 samples screened, < 15% fell below the assay LLOQ and < 1.7% were above the assigned reference interval cut point. Conclusion SAA is a highly sensitive marker of both acute and chronic inflammation. Levels of SAA in serum have clinical utility in identifying inflammation, especially subclinical inflammation, in FMF and other diseases that lack other more specific biomarkers. We report our validation of this highly sensitive LDT electrochemiluminescence assay to quantify SAA with the necessary analytic range and precision to allow proactive management of diverse inflammatory conditions aimed toward amelioration of their long-term sequelae.