Comparison of commercial and in-house tissue-based and cell-based assays for the detection of autoantibodies targeting neuronal surface proteins: a prospective cohort study

Abstract

Introduction: The detection of antibodies targeting neuronal antigens is a keystone for the diagnosis of autoimmune encephalitis (AE) and paraneoplastic neurological syndromes (PNS). This study aimed to compare the performance of a commercial tissue-based immunofluorescence assay (cIFA) to that of an inhouse IFA (hIFA) for the screening of autoantibodies targeting neuronal surface proteins in the cerebrospinal fluid (CSF) and to compare the performance of commercial cell-based assays (cCBA) to that of in-house CBA (hCBA) in serum samples.

Methods: Between March and June 2021, 2135 CSF samples and 524 serum samples from 2283 patients referred to the French Reference Center on PNS and AE were prospectively included. CSF samples were all tested using 3 different assays: cIFA, hIFA, and cCBA. Serum samples were all tested using at least 1 cCBA and 1 hCBA for the detection of the following autoantibodies: CASPR2, GABABR, and LGI1.

Results & discussion: Among the 2135 CSF tested, 93 (4.4%) were positive using both cIFA and hIFA, 1 (0.05%) was positive using only cIFA, and 6 (0.3%) were positive using only hIFA. Among the double-positive samples, 37 (39.8%) were positive using cCBA for the following autoantibodies: anti-NMDAR (n=16), -LGI1 (n=8), -CASPR2 (n=7), -GABABR (n=5), and -DPPX (n=1) autoantibodies. The remaining 56 (60.2%) double-positive samples were negative using cCBA and additional tests were performed to identify the autoantibodies according to the pattern observed on the IFA. The only sample positive using cIFA but negative using hIFA was positive for anti-LGI1 autoantibodies using cCBA. Among the 6 samples negative using cIFA but positive using hIFA, only one sample was positive with cCBA for anti-NMDAR autoantibodies. These data indicate that, in CSF, cIFA and hIFA performed similarly for the detection of autoantibodies targeting neuronal surface proteins.Regarding serum samples, cCBA and hCBA were both positive in 3 patients for CASPR2, 4 patients for LGI1, and 1 patient for GABABR. A positive cCBA and negative hCBA was observed in 2 patients for LGI1 and 4 patients for GABABR. A lack of specificity of GABABR cCBA is suspected as CSF explorations were negative in 3 of these patients and none presented clinical features highly suggestive of AE.

Keywords: autoantibodies; autoimmune encephalitis; cell-based assay; diagnostic test; immunofluorescence assays; paraneoplastic neurological syndromes; tissue-based assay.

Figure 1

Flow diagram of autoantibody detection in cerebrospinal fluid (CSF) samples of patients with a suspicion of autoimmune encephalitis (AE) or paraneoplastic neurological syndrome (PNS). Add, additional tests; AGO2, argonaute protein 2; AK5, adenylate kinase 5; CASPR2, contactin-associated protein-like 2; cCBA, commercial cell-based assay; cIFA, commercial tissue-based immunofluorescence assay; DNER, Tr/delta/notchlike epidermal growth factor-related receptor; DPPX, dipeptidyl-peptidase 6; GABABR, gamma-aminobutyric B receptor; GAD, glutamic acid decarboxylase; GFAP, glial fibrillary acidic protein; hIFA, in-house tissue-based immunofluorescence assay; IGLON5, Ig-like domain-containing protein 5; LGI1, leucine-rich glioma inactivated protein 1; NMDAR, N-methyl-D-aspartate receptor; SOX1, Sry-like high-mobility group box 1.

Figure 2

Typical pattern on cIFA of anti-NMDAR, -CASPR2, -LGI1, -GABABR aAbs and a negative control. For anti-NMDAR aAbs, in the hippocampus, the molecular layer of the dentate gyrus is stained following a gradient, with a more intense fluorescence near the dentate granule cell layer; in cerebellum, the granular layer is stained. For anti-CASPR2 aAbs, in the hippocampus, the molecular layer of the dentate gyrus and the dentate hilus are stained homogenously; in the cerebellum, the granular and molecular layers are stained with the same intensity. For anti-LGI1 aAbs, in the hippocampus, the molecular layer of the dentate gyrus and the dentate hilus are stained with a decrease in signal in the inner layer of the molecular layer; in the cerebellum, the molecular layer shows an intense staining while the granular layer staining is mild. For anti-GABABR aAbs, in the hippocampus, the molecular layer of the dentate gyrus and the dentate hilus are stained homogenously; in the cerebellum, the molecular layer shows an intense staining while the granular layer staining is mild. CASPR2, contactin-associated protein-like 2; GABABR, gamma-aminobutyric B receptor; LGI1, leucine-rich glioma inactivated protein 1; NMDAR, N-methyl-D-aspartate receptor.

Figure 3

Typical pattern on cIFA of anti-GFAP, -GAD, -IGLON5, -Hu, and -Yo aAbs. For anti-GFAP aAbs, in the hippocampus, there is a filamentous staining associated with an astrocyte staining in the dentate hilus; in the cerebellum, the radial glia of Bergmann in the molecular layer and astrocytes in the granular layer are stained. For anti-GAD aAbs, in the hippocampus, there is a strong staining of all the layers due to the cytoplasmic expression of GAD; in the cerebellum, the molecular layer shows a granular staining with dots while the granular layer and Purkinje cells show an intense cytoplasmic staining. For anti-IGLON5 aAbs, in the hippocampus, the molecular layer of the dentate gyrus and the dentate hilus are stained homogenously; in the cerebellum, the granular and molecular layers are stained with the same intensity. For anti-Hu aAbs, in the hippocampus, the nuclei of the dentate granule cells are stained while the cytoplasm and the nuclei of cells in the stratum pyramidale are stained; in the cerebellum, the nuclei of cells in all layers are stained as well as Purkinje cell cytoplasms. For anti-Yo aAbs, in the hippocampus, the cytoplasms of some cells in the hilus are stained; in the cerebellum, Purkinje cell cytoplasms are stained. GAD, glutamic acid decarboxylase; GFAP, glial fibrillary acidic protein; IGLON5, Ig-like domain-containing protein 5.

Figure 4

Flow diagram of autoantibody detection in serum samples of patients with a suspicion of autoimmune encephalitis (AE) or paraneoplastic neurological syndrome (PNS). CASPR2, contactin-associated protein-like 2; cCBA, commercial cell-based assay; GABABR, gamma-aminobutyric B receptor; hCBA, in-house cell-based assay; LGI1, leucine-rich glioma inactivated protein 1.