Diagnostic yield of commercial immunodots to diagnose paraneoplastic neurologic syndromes

Abstract

Objective: To investigate the diagnostic yield of commercial immunodots to detect onconeural antibodies associated with paraneoplastic neurologic syndromes (PNSs), we analyzed the proportion of confirmed positive results using alternative techniques.

Methods: Sera (n = 5,300) of patients with suspected PNS were tested by PNS+2 blot (Ravo Diagnostika; January 2016-May 2017) or EUROLINE PNS 12 Ag (Euroimmun; July 2017-November 2018). Positive samples were further explored by in-house indirect immunofluorescence and a third in-house technique (Western blot or cell-based assay) using recombinant protein. Those found negative by these 2 techniques were considered as nonconfirmed. We analyzed the relationship between band intensity and final confirmation. Clinical data were collected for all 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%). 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 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 identify patients with paraneoplastic neurologic syndromes.

Figure 1. Alternative techniques used for confirmation of commercial immunodots

EUROLINE PNS 12 Ag (Euroimmun) positive for anti-Ma2 (A), and anti-Yo (C), not confirmed by indirect immunofluorescence (IIF) on the rat cerebellum (left), and cell-based assay (CBA, right). A confirmed case for anti-Ma2 (B) and anti-Yo (D) is also provided for comparison. Immunodot band and scan value on the top of each case. For IIF, nuclei are stained with DAPI and anti-human IgG in green. For CBA, nuclei are also stained with DAPI, the protein of interest (Ma2 or Yo) in green as transfection control (GFP), and human IgG in red; a merged image is shown at the end of each row. GFP = green fluorescent protein.

Figure 2. Diagnostic algorithm followed in our laboratory to confirm positive immunodots

All positive sera by immunodots (except those positive for anti-glutamic acid decarboxylase [GAD65], anti-recoverin, and anti-titin, which were excluded from the current study) were analyzed using indirect immunofluorescence (IIF) and a technique using recombinant protein, either a Western blot for anti-amphiphysin and anti-CV2/CRMP5 (collapsin response-mediator protein-5) antibodies or a cell-based assay for the other antibodies. The results from the 2 confirmatory tests were always concordant. When IIF and the third technique were positive and consistent with the immunodot, the result was considered as confirmed. When IIF and the third technique were both negative, the immunodot result was considered as nonconfirmed.