Milano Chiara, Businaro Pietro, Papi Claudia, Arlettaz Lionel, Marmolejo Laura, Naranjo Laura, Gastaldi Matteo, Iorio Raffaele, Saiz Albert, Planagumà Jesús, Aguilar Esther, Pizzanelli Chiara, Martinez-Hernandez Eugenia, Armangué Thaís, Guasp Mar, Ruiz-García Raquel, Sabater Lidia, Dalmau Josep O, Graus Francesc, Spatola Marianna
Neuroimmunology Program, Fundació de Recerca Clínic Barcelona-Institut d'Investigacions Biomèdiques August Pi i Sunyer (FRCB-IDIBAPS), University of Barcelona, Spain.
Caixa Research Institute (CRI), Barcelona, Spain.
Neurol Neuroimmunol Neuroinflamm. 2025 Jul;12(4):e200410. doi: 10.1212/NXI.0000000000200410. Epub 2025 May 20.
Current strategies to detect autoantibodies against intracellular neural antigens (IC-Abs) include tissue-based assays (TBAs) alongside line blots or cell-based assays (CBAs). Many clinical laboratories use commercially available TBAs as a screening test, but their diagnostic yield has not been assessed. We determined the performance of 2 commercial TBAs in detecting IC-Abs.
We analyzed samples from 100 patients with autoimmune or paraneoplastic neurologic syndromes harboring IC-Abs (confirmed by in-house TBAs and line blots or CBAs) and 50 negative controls. IC-Abs samples included serum (10 each for Hu, Yo, Ri, SOX1, CV2, Ma2, Tr, amphiphysin, and GAD65 antibodies) or CSF (10 with GFAP antibodies) samples. Two commercial indirect immunofluorescence (IIF) TBAs (INOVA and EUROIMMUN) were assessed by 2 experienced investigators and 3 less experienced raters, all blinded to antibody status. Discordant results were re-evaluated through interrater discussion and assessed using Cohen's kappa.
The 2 experienced raters showed substantial agreement (85% for INOVA, 83% for EUROIMMUN) on negative/positive results, which increased to >95% after interrater discussion (Cohen's kappa 0.95 and 0.93, respectively). With IIF-INOVA, they correctly identified 118 of 150 samples (79%) and misclassified 28 of 150 (19%, 2 false positives and 26 false negatives) while results remained discordant in the remaining 4 of 150 samples (2%). With IIF-EUROIMMUN, they correctly identified 105 of 150 samples (70%) and misclassified 40 of 150 (27%, 6 false positives, 34 false negatives), with discordance in 5 of 150 samples (3%). Overall, the sensitivity was 73% for IIF-INOVA and 66% for IIF-EUROIMMUN. The specificity was 96% for IIF-INOVA and 88% for IIF-EUROIMMUN. Both TBAs showed low sensitivity in detecting CV2, SOX1, and amphiphysin antibodies while Ma2 antibodies were missed mainly by IIF-EUROIMMUN and Hu/Ri antibodies by IIF-INOVA. Antibody-specific immunostaining patterns were correctly identified in 62 of 100 positive samples with IIF-INOVA and 55 of 100 with IIF-EUROIMMUN ( = 0.34). Less experienced raters showed higher rates of false-positive results (up to 22% with IIF-EUROIMMUN).
The performance of commercial IIF-TBAs for IC-Abs detection is suboptimal, exhibiting high false-negative rates of 25%-35%. Therefore, commercial TBAs should not be used alone for IC-Abs screening, but alongside more sensitive techniques, such as line blots. Discordant results between 2 techniques should prompt retesting in reference centers with in-house TBAs, particularly when the suspicion of an autoimmune or paraneoplastic syndrome is high.
目前检测针对细胞内神经抗原的自身抗体（IC-Abs）的策略包括基于组织的检测方法（TBAs）以及线性印迹法或基于细胞的检测方法（CBAs）。许多临床实验室使用市售的TBAs作为筛查试验，但尚未评估其诊断率。我们测定了两种市售TBAs检测IC-Abs的性能。
我们分析了100例患有IC-Abs的自身免疫性或副肿瘤性神经综合征患者（经内部TBAs及线性印迹法或CBAs确诊）的样本以及50例阴性对照样本。IC-Abs样本包括血清（分别含10份针对Hu、Yo、Ri、SOX1、CV2、Ma2、Tr、 amphiphysin和GAD65抗体的样本）或脑脊液（含10份针对GFAP抗体的样本）。两名经验丰富的研究人员和三名经验较少的评分者对两种市售间接免疫荧光（IIF）TBAs（INOVA和EUROIMMUN）进行评估，他们均不知晓抗体状态。通过评分者间讨论对不一致的结果进行重新评估，并使用Cohen's kappa进行评估。
两名经验丰富的评分者在阴性/阳性结果上显示出高度一致性（INOVA为85%，EUROIMMUN为83%），经评分者间讨论后一致性提高至>95%（Cohen's kappa分别为0.95和0.93）。使用IIF-INOVA时，他们正确识别了150份样本中的118份（79%），将150份中的28份误分类（19%，2例假阳性和26例假阴性），而在其余150份样本中的4份（2%）结果仍不一致。使用IIF-EUROIMMUN时，他们正确识别了150份样本中的105份（70%），将150份中的40份误分类（27%，6例假阳性，34例假阴性），在150份样本中的5份（3%）结果不一致。总体而言，IIF-INOVA的敏感性为73%，IIF-EUROIMMUN的敏感性为66%。IIF-INOVA的特异性为96%，IIF-EUROIMMUN的特异性为88%。两种TBAs在检测CV2、SOX1和amphiphysin抗体时均显示出低敏感性，而Ma2抗体主要被IIF-EUROIMMUN漏检，Hu/Ri抗体主要被IIF-INOVA漏检。在100份阳性样本中，IIF-INOVA正确识别了6份的抗体特异性免疫染色模式，IIF-EUROIMMUN正确识别了55份（κ = 0.34）。经验较少的评分者显示出较高的假阳性率（IIF-EUROIMMUN高达22%）。
市售IIF-TBAs检测IC-Abs的性能欠佳，假阴性率高达25%-35%。因此，市售TBAs不应单独用于IC-Abs筛查，而应与更敏感的技术（如线性印迹法）一起使用。两种技术之间的不一致结果应促使在参考中心使用内部TBAs进行重新检测，特别是在高度怀疑自身免疫性或副肿瘤性综合征时。
