When IIF and the third technique were positive and consistent with the immunodot, the result was considered as confirmed. confirmed results and nonconfirmed EUROLINE immunodots. Results PNS+2 blot was positive in 128/1,658 (7.7%) sera and confirmed in 47/128 (36.7%). IFNB1 EUROLINE was positive in 186/3,626 (5.1%) and confirmed in 56/186 (30.1%). Confirmation was highly variable among the antibodies tested, from 7.2% (PNS+2 blot) and 5.8% (EUROLINE) for anti-Yo to 88.2% (PNS+2 blot) and 65.0% (EUROLINE) for anti-Hu. None of the 27 weak positive sera by EUROLINE was confirmed. Band Efonidipine hydrochloride monoethanolate intensity in confirmed cases was variable among the antibodies from strong positive for all anti-Yo (n = 3) and anti-Hu (n = 11) to positive (n = 19) or strong positive (n = 9) for anti-SOX1. Among patients with a nonconfirmed EUROLINE result and available clinical information, all had an alternative diagnosis, and only 6.7% had cancer. Conclusions Immunodots may be useful for PNS screening, but a threshold should be established for each antibody, and clinical information and confirmation by other techniques are essential. Classification of evidence The study provides Class IV evidence that immunodot assays for onconeural antibodies accurately Efonidipine hydrochloride monoethanolate identify patients with paraneoplastic neurologic syndromes. Paraneoplastic neurologic syndromes (PNSs) are rare but now well-characterized immune-mediated neurologic diseases triggered by cancer and diagnosed by the presence of circulating autoantibodies.1 Among them, autoantibodies directed against intracellular neural antigens (also known as onconeural antibodies) are strongly associated with the presence of an underlying cancer, and its detection is a cornerstone of PNS diagnosis. Indirect immunofluorescence (IIF) Efonidipine hydrochloride monoethanolate on rat brain slices is the preferred screening test for identification of onconeural antibodies, but the result should be confirmed by a second technique, either Western blot or for some cases such as anti-delta/notch-like epidermal growth factorCrelated receptor (anti-Tr/DNER) by cell-based assays (CBAs).2,3 These techniques have been developed mainly in research laboratories and are not available for routine analysis. However, 2 commercial immunodot assays are currently marketed: PNS+2 blot (Ravo Diagnostika, Freiburg, Germany) and EUROLINE PNS 12 Ag (Euroimmun, Lbeck, Germany). These immunodot assays present the advantage to be easily and quickly performed as they are fully automated; they also screen several antibodies at the same time. However, very little is known about the reliability of these immunodot assays, as only a few published studies have analyzed the sensitivity for the detection of anti-CV2/CRMP5 (collapsin response-mediator protein-5) antibodies,4 and the sensitivity and specificity for anti-Ma2 antibodies,5 and anti-SOX1 antibodies.6 In our laboratory, we use commercial immunodot assays as the first step of biological PNS diagnosis for all onconeural antibodies. Herein, we studied the diagnostic yield of 2 commercial immunodots by investigating the proportion of positive results confirmed by alternative techniques, taking also into account the clinical information when it was available. Methods This study is a single-center retrospective analysis of samples (sera) from patients with suspicion of PNS that were analyzed at the French Reference Center on Paraneoplastic Neurological Syndromes (Lyon, France). First, sera were screened by commercial immunodot assays, using PNS+2 blot (Ravo Diagnostika), from January 2016 to May 2017, and EUROLINE PNS 12 Ag (Euroimmun), from July 2017 to November 2018. Only the sera that were found positive by the immunodot assay for at least one of the onconeural antibodies were further analyzed by 2 in-house techniques: IIF followed by a technique using recombinant protein, either a Western blot for anti-CV2/CRMP5 and anti-amphiphysin antibodies or a CBA for the other antibodies. When a positive immunodot result was also found positive using the 2 2 different confirmatory techniques (IIF and Western blot/CBA), the case was considered as confirmed. When both IIF and the third Efonidipine hydrochloride monoethanolate technique were negative, the immunodot result was considered as nonconfirmed. All confirmed cases were included in the database of the French Reference Center, along with clinical information. For the current study, we also collected clinical data (including clinical phenotype, cancer association, and final diagnosis) for patients whose serum was tested using the EUROLINE PNS 12 Ag (Euroimmun) but were nonconfirmed; these data were not available for patients whose serum was tested using the PNS+2 blot (Ravo Diagnostika). When a tumor was detected, it was.