The last two decades of research have led to remarkable advances in the clinical and electrophysiological categorization of acute and chronic immune-mediated neuropathies (IN) that was paralleled with the identification of multiple novel autoantibodies (autoAb) and their corresponding targets. Chronic inflammatory demyelinating polyneuropathy (CIDP) is a rare autoimmune disorder of the peripheral nervous system and can clinically distinguished between typical CIDP and atypical variants, such as distal acquired-demyelinating polyneuropathy (DADS), multifocal-acquired demyelinating sensory and motor polyneuropathy (MADSAM) and sensory-CIDP , . Its heterogeneous clinical manifestation suggests different autoimmune targets. Previously, we and others detected autoreactive T-cell responses against myelin antigenic epitopes , . Further, autoAbs against the paranodal protein neurofascin NF155 have been identified in patients suffering from combined central and peripheral demyelination  and in a subset of CIDP patients with distinct clinical features . In addition, autoAbs against the nodal neurofascin NF186 have been found in CIDP .
In contrast to acute IN, the value of anti-ganglioside autoantibody (aGAAb) testing has been established for only a minority of chronic IN yet. This holds particularly true for autoAbs against GM1 (aGM1) in multifocal motor neuropathy (MMN) and to the myelin-associated glycoprotein (aMAG) in DADS , . In addition, increased titers of anti-sulfatide (aSF) IgM are found in patients with neuropathy where they are often associated with a concomitant reactivity to MAG. A selective reactivity to sulfatide (SF), however, was identified only in 1% of CIDP patients  so that a diagnostic value is still in doubt. In a retrospective analysis of altogether 172 patients, aGM1 IgM was found in 46% of patients with MMN but in only 3% of CIDP patients .
Thus, in most IN, the diagnostic value of aGAAb is still elusive. Thin-layer chromatography is supposed to be the gold standard assay technique for aGAAb analysis, although it is not applicable for routine use. Enzyme immunoassays with highly purified gangliosides are not cost-effective. Dot or line immunoassays (LIA) may be a good alternative for the multiparametric determination of aGAAbs, provided that an optimal autoantigenic epitope-preserving binding of gangliosides on membranes can be achieved , . We developed a novel semiquantitative evaluation method for aGAAb detection by LIA and tested its performance for the analysis of GAAb in patients with chronic IN.
Materials and methods
Standard protocol approvals, registrations and patient consents
The study was approved by the Ethical Committee of Charité – Universitätsmedizin Berlin (approval number EA1/025/11), and clinical investigations were conducted according to the Declaration of Helsinki. All patients were recruited in the outpatient clinic of Charité Department of Neurology. All patients gave their written informed consent to the study. HC were recruited by Medipan Company.
For our study, 61 patients with IN including typical CIDP (n=27), DADS (n=12), MADSAM (n=6), sensory-CIDP (n=10), focal-CIDP (n=1) and five MMNs were recruited. Patients included in this study did not show any sign of clinical infection. Diagnoses were made according to criteria of European Federation of Neurological Societies/ Peripheral Nerve Society (EFNS/PNS) . Clinical conditions of patients were assessed by Medical Research Council (MRC)  and inflammatory neuropathy cause and treatment (INCAT) disability score . For classification, the clinical disease activity status was used. Accordingly, unstable active and improving status was summarized as unstable stage, whereas stable active and remission statuses were summarized as stable stage . Symptoms stated in case history were used for analyses of clinical features. Positive treatment response was defined as an improvement of two or more points on the MRC sum score in two different muscle groups, or an improvement of one point or more on the INCAT score, or an improvement of the walking distance of more than 50% compared to baseline results , . Forty patients with other neuromuscular disorders (OND) comprising 15 non-immune polyneuropathies, and 25 myasthenia gravis as well as 29 patients with relapsing-remitting multiple sclerosis (MS) according to revised McDonald criteria  and 54 healthy controls (HC) were included as controls. In patients treated with intravenous immunoglobulins (IVIg), blood was obtained on the first day of IVIg therapy before starting the infusion. Standardized patient sample collection (in BD vacutainer SST II Advance tubes) and processing (10 min centrifugation, 2000g, 20°C, storage of sera at −80°C until shipment) as well as documentation of preanalytical conditions were supported and monitored via NeuroHub biomarker management platform and LabVantage 7.0 software at the NeuroCure Clinical Research Center.
Statistical analyses were performed using Prism 6 software (GraphPad, La Jolla, CA, USA). For group differences with regard to sex, typical manifestation, diagnosis certainty, tremor, sensory ataxia, neuropathic pain, positive cerebrospinal fluid, secondary axonal degeneration and all electrophysiological parameters prior treatment Fisher’s exact test was used. For normally distributed age and INCAT score, unpaired t-test was used. Antibody reactivity was tested by the two-tailed Mann-Whitney and Kruskal-Wallis tests followed by Dunn’s multiple comparison tests in two and more groups, respectively. Level of significance was defined as p<0.05 for all comparative tests.
Determination of aGAAbs
A novel line immunoassay using 11 different gangliosides (GM1, GM2, GM3, GM4, GD1a, GD1b, GD2, GD3, GT1a, GT1b and GQ1b) and SF was used to analyze aGAAb profiles in sera of patients with clinically defined CIPD according to the EFNS/PNS criteria, patients with OND healthy blood donors according to the instructions of the manufacturer. Briefly, patients’ and control sera, stored at −30°C, were agitated gently after thawing in order to ensure homogeneity. Sera were diluted at 1:100 and incubated 2 h at 4°C to allow sufficient binding of autoAbs to the gangliosides immobilized on the solid phase (membrane). Unbound sample components were removed by the following wash step. The bound autoAbs reacted specifically with anti-IgG or anti-IgM conjugated to horseradish peroxidase during a second incubation step of 60 min at 4°C. Excessive conjugate was separated from the solid-phase immune complexes by an additional wash step. After substrate addition (tetramethyl benzidine) and incubation for 10 min at room temperature, stripes were dried for at least 30 min and read off by a scanner. Obtained images were assessed with the interpretation software DotBlot Analyzer using a grayscale calibration card for standardization. The grayscale calibration card was provided on the template of the kit. Values were read off as optical density (OD) units. A sample was considered to be positive in respect to one of the gangliosides or SF if the OD of the test line was higher than 20 OD units.
Sixty-one patients with IN, 40 patients with OND, 29 patients with MS and 54 patients HC were included into this study. There was no statistical difference between the cohorts with regard to age at the time of inclusion, sex or proportion of patients receiving immunosuppressive treatment apart from patients with MS (Table 1). The latter were younger (p<0.001), more often female (p=0.04) and were less frequently treated with immunotherapy (p=0.001) (Table 1).
Intra- and interassay variability of the line immunoassay (LIA)
Binding of aGAAbs was quantified for each target as OD values by the DotBlot Analyzer software using a grayscale calibration. The intra-assay (Figure 1A) and inter-assay (Figure 1B) coefficients of variation (CVs) for aGAAb testing by LIA were analyzed for three aGAAb-positive sera in accordance with the CLSI-protocol EP15-A2 (https://clsi.org/media/1431/ep15a3_sample.pdf). Thus, intra-assay CV was determined by eight measurements for each serum, whereas interassay CV was assessed by analyzing eight determinations for each serum on five different days. At the cutoff value of 20 OD, the intra-assay CV reached 18% (Figure 1A) and the interassay CV 32% (Figure 1B).
Elevated frequencies of aSF, aGM1 and aGD1b IgM in INs
In order to evaluate whether aGAAbs were more frequent in IN compared to the control groups of OND, MS and HC, we screened the sera for aGAAbs IgG as well as IgM against 11 gangliosides and SF. For IgG aGAAbs, there were low frequencies in OND and IN (Figure 2A) with a positive aGAAb against at least one IgG aGAAb in 2/61 (3.3%) of IN patients, in 0/40 (0%) of OND, in 0/29 (0%) of MS patients compared with 7/54 (12.9%, p=0.01) of HC. By contrast, aGAAb IgM was generally more frequent and diverse in IN compared to OND, MS and HC (Figure 2B). Positive IgM reactivity against at least one ganglioside/SF was found in 17/61 (27.9%) of IN patients compared to 2/40 (5.0%) of OND, 2/29 (6.9%) of MS and 4/54 (7.4%) of HC, which was statistically relevant (p=0.002, χ2-test). Analyzing IgM aGAAbs, there were statistically higher frequencies of aSF IgM in IN compared to OND (Figure 3A; p=0.08). Anti-GM1 IgM was elevated in IN compared to OND and HC (Figure 3B; p=0.009). aGD1b IgM was statistically elevated in IN compared to HC (Figure 3C; p=0.04). The prevalence of aGM1 IgM in CIDP was lower (12%) compared to MMN (60%, p=0.027, Fisher’s exact test) (Figure 4).
Correlation between aGAAbs and clinical characteristics
Patient’s characteristics and clinical parameters were stratified by positive reactivity against SF, GM1, GD1b as well as reactivity to at least two gangliosides/SF. Patients showing aSF, aGM1 and aGM2 IgM were younger compared to those of the autoAb-negative group (Table 2). Interestingly, patients demonstrating aSF IgM presented significantly more frequently a typical clinical presentation for CIDP and MMN according to established EFNS criteria , . Further, they revealed significantly more frequently conduction blocks (p=0.002; Table 2). For aGD1b IgM, all five positively tested patients fulfilled definite diagnostic criteria according to EFNS . Of them, four patients showed also aGM1 IgM (80%). The analysis of all patients with at least two aGAAbs also revealed an association with diagnosis certainty (p=0.002, Table 2). There was no difference between IVIG-treated patients and patients who did not receive IVIG treatment. Interestingly, we found no association with disease duration (time since diagnosis), disease activity (unstable disease) and disease severity as measured by INCAT and axonal degeneration nor distinct clinical features such as tremor, sensory ataxia and neuropathic pain (Table 2).
By using a novel multiplex LIA for the analysis of aGAAb, we found an elevated frequency of at least one aGAAb IgM in patients suffering from CIDP or MMN compared to controls including patients with OND and HC. Of all tested autoAbs, aSF, aGM1 as well as aGD1b IgM were most frequently detected in IN compared to the control groups.
Anti-SF autoAbs were associated with different subtypes of peripheral neuropathy most of them of axonal type. However, also a demyelinating type with a lower prevalence was identified . A meta-analysis of MS patients identified higher aSF titers in patients suffering from secondary axonal degeneration . In accordance, MS control patients tested in the present study did not show a significantly increased frequency of aSF IgM antibodies, and most of the patients remained below cut off levels.
The assessment of the assay variability based on OD values obtained in a standardized scanning procedure revealed reliable data supporting the usability of quantitative autoAb analysis by LIA as demonstrated for antiphospholipid antibody testing by LIA, too . The obtained interassay CV of 32% at the cutoff of 20 OD units appears satisfactory and confirms earlier data of antibody analysis for sera with higher OD units by LIA . Further, the hydrophobic LIA-solid phase has already proven its usefulness for the specific analysis of auto/Abs to amphiphatic molecules like lipopolysaccharides and glycolipids exhibiting similar physicochemical characteristics , , .
Interestingly, aSF-positive patients in our cohort were younger and showed typical manifestation of clinical symptoms compared to the negative tested group. However, we found no association with axonal degeneration as described , . We also did not find any association with hematological disease such as monoclonal gammopathy IgM nor combined with aMAG autoAbs as described recently , .
In contrast to the aforementioned studies, we included patients fulfilling diagnostic criteria of CIDP or MMN. The fact, that aSF-positive patients were younger and responded well to immunomodulatory treatment may support the autoimmune relevance of SF, which is predominantly expressed within the non-compact myelin . Beside aSF IgM, we detected higher levels (16%) of aGM1 IgM in CIDP and MMN patients compared to controls. This rate is higher than the one shown recently (7%) where IgM to glycolipid complexes containing GM1 and SF were the most frequently observed autoAbs of CIDP patients using combinatorial thin layer chromatography . Interestingly, those patients demonstrated more frequently motor disturbances than antibody-negative ones did . Accordingly, patients of our study demonstrated typical clinical manifestation with predominantly distal and motor disturbances more frequently in aSF and, by trend, aGM1-IgM-positive patients. Moreover, patients with positive aSF autoAbs showed a higher rate of conduction blocks in nerve conduction studies (Table 2). The affection of primarily motor functions may be explained by the experimental model that SF depletion leads to dramatically reduction of myelin proteins, especially in the paranodal region affecting neurofascin 155 . It is also in accordance with the fact that the GM1 epitope is highly expressed on the membranes of motor nerves and on the surface of Schwann cells. Additionally, binding of the antibodies to the axon at the nodes of Ranvier on to Schwann cells may cause complement activation and disruption of sodium channel clusters, resulting in conduction abnormalities , .
In the present study, aGD1b IgM was found in patients with the highest diagnostic certainty fulfilling EFNS criteria of definite CIDP. GD1b is a disialoganglioside with two sialic acids in its structure. Because they share a same terminal Gal-GalNAc residue, cross-reactivity is often seen between autoAbs to GM1, GD1b and GD1a , . Here, we also found cross-reactivity between GD1b and GM1 in 80% of all positive tested patients.
In contrast to acute immune neuropathies such as Guillain-Barré syndrome, here we found aGAAbs of IgM isotype. This might be due to the fact that these autoantibodies are secondary to a predominant T cell-mediated demyelination. Indeed, previous studies of our working group found evidence for a T-cell-driven pathomechanism in CIDP (Klehmet et al. 2015). In order to exclude an unspecific secondary effect, we recruited MS patients as a control for demyelinating disease. Using LIA as novel assay technique, we did not find increased frequencies of IgM nor IgG aGAAbs in MS compared to IN which was all the more remarkable because MS patients were younger and less frequently treated with immunotherapy.
Patients included in this study are part of a very well characterized cohort. We therefore correlated a range of clinical features with antibody specificity but were not able to characterize a distinct subtype as shown in recently published neurofascin NF155-specific CIDP subtype .
The early diagnosis of CIDP and treatment initiation is essential in order to prevent irreversible axonal damage and thus disability. However, the correct diagnosis of CIDP is still challenging due to its very heterogeneous manifestation. A diagnostic marker could help to support early diagnosis. In addition, biomarkers could help to predict treatment response and differentiate between clinical phenotypes. It would be therefore of high relevance to validate our findings in larger multicentric studies.
Advantages of our study are a well-defined patient cohort with high diagnostic certainty and age-matched controls, which is essential to assess IgM reactivity. However, our study has also certain limitations. Even though this study is comparatively large with a total number of 130 patients, it is still too small to firmly identify autoAb specificities in correlation with clinical features or specific CIDP subtypes. Another limitation may be the fact that the included patients were not treatment-naive and had rather long disease duration.
In conclusion, we provide evidence of a pathogenetic relevance of aSF, aGM1 and aGD1b IgM in CIDP and MMN. Patients with at least two aGAAbs showed a strong association with fulfilling diagnostic EFNS criteria. Anti-SF, aGM1 and aGM2 IgM were associated with younger age supporting the autoimmune relevance. In addition, aSF and aGM1 IgM were associated with predominant motor disturbances. Further multicentric studies are required to validate these promising biomarkers with reliable cutoffs as useful diagnostic tools for CIDP, its subtypes and for treatment guidance.
The authors thank Arun Prakash Singh (NeuroHub) and Salomé Kiessling for their support in sample management.
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About the article
Published Online: 2018-01-12
Published in Print: 2018-05-24
Author contributions: Statistical analysis conducted by Juliane Klehmet, MD, Charité – Universitätsmedizin Berlin, Germany and Dirk Roggenbuck, MD, Institute of Biotechnology, Senftenberg, Germany. All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: This work was supported by the German Research Foundation (DFG; Funder Id: 10.13039/501100001659; EXC 257 NeuroCure) and by IBB TransBonus project funding of the investment bank Berlin.
Employment or leadership: DR has a management role and is a shareholder of GA Generic Assays GmbH and Medipan GmbH. Both companies are diagnostic manufacturers. TB is employed at GA Generic Assays.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
Citation Information: Clinical Chemistry and Laboratory Medicine (CCLM), Volume 56, Issue 6, Pages 919–926, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/cclm-2017-0792.
©2018 Juliane Klehmet et al., published by De Gruyter, Berlin/Boston. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License. BY-NC-ND 4.0