Laboratory testing for anti-synthetase antibodies: a novel algorithm to improve diagnostic accuracy

Anti-synthetase syndrome (ASyS) is an autoimmune disorder that presents specific autoantibodies against aminoacyl-tRNA synthetases (ARS). Although numerous commercial assays are available to detect anti-ARS, their accuracy is highly variable. The aim of the present study is to evaluate the assays most commonly used in Spanish clinical laboratories.

We evaluated 99 serum samples from five Spanish laboratories. All samples tested positive for anti-ARS by line blot (LB) and were examined by indirect immunofluorescence (IIF) on HEp-2 cells. The samples were subsequently evaluated at a single central laboratory by dot blot (DB) and RNA immunoprecipitation (RNA-IP). If DB or RNA-IP confirmed the original finding, the test result was labeled as confirmed, and thus, a true positive.

Of the 99 samples (2 double-positive excluded), 26 (27%) were confirmed. The confirmation rate for 1+ and 2+ LB intensity was only 5%. If the LB cut-off is raised to 3+ and the IIF pattern is considered, the confirmed rate reached 100%. RNA-IP was the most sensitive confirmatory technique for anti-PL-12, anti-EJ, and anti-OJ, whereas DB was more sensitive for the detection of anti-Jo-1 and anti-PL-7.

Commercial LB methods demonstrate significant variability, as evidenced by the high unconfirmed rate (73%) in this study. We found that specificity can be significantly increased by using a LB signal intensity cut-off of 3+ together with the presence of a diagnosis-compatible IIF pattern. Based on these findings, we have developed a diagnostic algorithm to improve the reliability of anti-ARS detection in routine laboratory practice.