Neuronal surface antigen-specific immunostaining pattern on a rat brain immunohistochemistry in autoimmune encephalitis

Abstract

A variety of neuronal surface (NS) antibodies (NS-Ab) have been identified in autoimmune encephalitis (AE). Tissue-based assay (TBA) using a rodent brain immunohistochemistry (IHC) is used to screen NS-Ab, while cell-based assay (CBA) to determine NS antigens. Commercial rat brain IHC is currently available but its clinical relevance remains unclear. Immunostaining patterns of NS antigens have not been extensively studied yet. To address these issues, we assessed a predictive value of "neuropil pattern" and "GFAP pattern" on commercial IHC in 261 patients, and characterized an immunostaining pattern of 7 NS antigens (NMDAR, LGI1, GABAaR, GABAbR, AMPAR, Caspr2, GluK2). Sensitivity and specificity of "neuropil pattern" for predicting NS-Ab were 66.0% (95% CI 55.7-75.3), and 98.2% (95% CI 94.8-99.6), respectively. False-positive rate was 1.8% (3/164) while false-negative rate was 34.0% (33/97). In all 3 false-positive patients, neuropil-like staining was attributed to high titers of GAD65-Ab. In 33 false-negative patients, NMDAR was most frequently identified (n=18 [54.5%], 16/18 [88.9%] had low titers [< 1:32]), followed by GABAaR (n=5). Of 261 patients, 25 (9.6%) had either GFAP (n=21) or GFAP-mimicking pattern (n=4). GFAP-Ab were identified in 21 of 31 patients examined with CBA (20 with GFAP pattern, 1 with GFAP-mimicking pattern). Immunostaining pattern of each NS antigen was as follows: 1) NMDAR revealed homogenous reactivity in the dentate gyrus molecular layer (DG-ML) with less intense dot-like reactivity in the cerebellar granular layer (CB-GL); 2) both GABAaR and GluK2 revealed intense dot-like reactivity in the CB-GL, but GABAaR revealed homogenous reactivity in the DG-ML while GluK2 revealed intense reactivity along the inner layer of the DG-ML; and 3) LGI1, Caspr2, GABAbR, and AMPAR revealed intense reactivity in the cerebellar ML (CB-ML) but LGI1 revealed intense reactivity along the middle layer of the DG-ML. Whereas, Caspr2, GABAbR, and AMPAR revealed similar reactivity in the DG-ML but some difference in other regions. TBA is useful not only for screening NS- or GFAP-Ab but also for estimating NS antigens; however, negative results should be interpreted cautiously because "neuropil pattern" may be missed on commercial IHC when antibody titers are low. Antigen-specific immunoreactivity is a useful biomarker of AE.

Keywords: autoantibodies; autoimmune encephalitis; cell-based assay; immunohistochemistry; neuronal surface antigens; tissue-based assay.

Figure 1

This figure shows a diagram of the study conducted including Part I and Part II. In Part I, a predictive value of a “neuropil pattern” suggesting NS-Ab and a pattern suggesting GFAP-Ab were mainly investigated, whereas in Part II, an immunostaining pattern of the pre-selected 7 NS antigens are evaluated based on the subjects listed here. The number of patients is shown in parentheses. AE, autoimmune encephalitis; Ab, antibodies; GFAP, glial fibrillary acidic protein; IHC, immunohistochemistry; NS, neuronal surface.

Figure 2

This figure shows the results of immunostaining pattern using in-house IHC (A–C) and commercial kit, which consists of 4 biochips per field containing the NMDAR-transfected cells (D), control-transfected cells (E), cerebellum (F), and hippocampus (G). Note intense reactivity with NS antigen in the DG-ML (A, B, G), less intense dot-like reactivity in the CB-GL but no apparent reactivity on the CB-ML (C, F). This staining pattern is consistent with NMDAR. (D) shows intense staining on NMDAR-transfected cells but not on control-transfected cells (E), confirming the diagnosis of anti-NMDAR encephalitis. Antibody assay was performed using CSF (diluted 1:2) obtained from a patient with anti-NMDAR encephalitis (antibody titer 1:2048), with in-house IHC adapted to NS antigens (A–C), commercial fixed CBA (D, E), and commercial IHC (F, G). See Text. CBA, cell-based assay; CB-GL, cerebellar granular layer; CB-ML, cerebellar molecular layer; DGC, dentate granule cells; DG-ML, dentate gyrus molecular layer; IHC, immunohistochemistry; NMDAR, NMDA receptor; NS, neuronal surface; SLM, stratum lacunosum moleculare; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; WM, white matter.

Figure 3

(A) does not show apparent “neuropil pattern” with negative control, but (B) reveals some reactivity mimicking “neuropil pattern” in the middle and outer layers of the DG-ML. Reactivity in the surroundings of the DGC, Purkinje cells, and CB-GL are consistent with a pattern of GAD65 reactivity. (C, D) did not reveal apparent “neuropil pattern” on commercial IHC but visually recognizable reactivity on in-house IHC. (E, F) reveal a cactus thorn-like or filamentous staining consistent with a pattern of GFAP reactivity. Note that GFAP pattern is more clearly shown on commercial IHC than on in-house IHC (E). (F) shows intense reactivity with GFAP and MOG but not apparent “neuropil pattern”. CB-GL, cerebellar granular layer; CB-ML, cerebellar molecular layer; DGC, dentate granule cells; DG-ML, dentate gyrus molecular layer; WM, white matter.

Figure 4

TBA reveals an immunostaining pattern highly characteristic of the individual NS antigen. (See Text). These preselected 7 NS antigens reveals intense reactivity with NS antigen “neuropil pattern”, among NS antigens, two reveal antigen-specific laminar reactivity, along the inner layer in GluK2 (D, arrows) and the middle layer in LGI1 (E, arrows). The first 3 NS antigens (NMDAR, GABAaR, GluK2) have dot-like reactivity on the CB-GL (B–D), while the other 4 (LGI1, Caspr2, GABAbR, AMPAR) have homogenous reactivity on the CB-ML with some different immunoreactivity on the hilus, CB-GL, or Purkinje cells (see Text). CB-GL, cerebellar granular layer; CB-ML, cerebellar molecular layer; DGC, dentate granule cells; DG-ML, dentate gyrus molecular layer; ML, molecular layer; WM, white matter.

Figure 5

A series of illustrations are created as simple as possible to make it easy to follow individual staining pattern at the level of hippocampus and cerebellum. See Text and Figure 4 . CB-GL, cerebellar granular layer; CB-ML, cerebellar molecular layer; DGC, dentate granule cells; DG-ML, dentate gyrus molecular layer; PC, Purkinje cells; SLM, stratum lacunosum moleculare; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; WM, white matter.