A total of 245 patients with ALS in the Department of Neurology in Xiangya Hospital at Central South University (CSU) in China from April 2013 to April 2019 were enrolled in the study. All patients were diagnosed with definite, probable, or probable-laboratory-supported ALS by at least two neurologists according to the revised El Escorial criteria 2015 . Their demographic features and clinical data, including sex, age, site at onset, age at onset, disease duration, and the ALSFRS-R score, were collected. All data were collected when the blood tests were performed.
Two control groups were included in the study. A total of 82 sex- and age-matched healthy individuals without any neurological diseases were recruited from the Health Management Center in Xiangya Hospital at CSU as HCs. In addition, 65 sex- and age-matched patients with MSA and 60 matched patients with PD recruited from the same location as the ALS patients were enrolled as neurological disease controls. All patients with PD were diagnosed according to the Movement Disorder Society (MDS) clinical diagnostic criteria . All patients with MSA were diagnosed according to the current consensus criteria established by Gilman and colleagues .
Subjects with systemic inflammation, monoclonal gammopathy, non-malignant endocrine abnormalities, neoplastic disorders, auto-antibodies, and infection, or those who had a history of using anti-inflammatory drugs, acetylsalicylic acid, steroids, and statins within the 2 months before enrolment were excluded from this study. This study was approved by the Ethics Committee of Xiangya Hospital at CSU in China, and all subjects included provided written informed consent prior to participation.
Ten milliliters of peripheral venous blood samples were collected from all subjects in accordance with the study protocols. Then, each blood sample that was collected was sent to the Clinical Laboratory at Xiangya Hospital, and the levels of immunoglobulin and complement were measured.
All analyses were performed using SPSS 22.0 (SPSS, Chicago, IL, USA) and GraphPad Prism 5.03 (® GraphPad Software, Inc.). Differences with p<0.05 were considered statistically significant, and multiple comparisons were performed with Bonferroni correction. All continuous data, including those of age, disease duration, age at onset, the ALSFRS-R scores, and the levels of IgG, IgA, IgM, C3, and C4 are presented as means ± standard deviations. The Chi-square test was used to analyse the differences in the sex distributions among different groups. A one-way analysis of variance (ANOVA) was used to analyse the differences in age, disease duration, age at onset, the ALSFRS-R scores, and the levels of IgG, IgA, IgM, C3, and C4 among more than two groups; then, Dunn’s multiple comparison test was performed. The correlations between serum immune variables and ALS clinical characteristics, including age at onset, disease duration, and the ALSFRS-R score, were analysed using Spearman’s correlation analysis.
The methods of search strategy, inclusion and exclusion criteria, outcome measures, and statistical analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement . The Embase, Pubmed, and the Cochrane Library primary databases were searched using the following search terms: “amyotrophic lateral sclerosis” OR “ALS” OR “motor neuron disease” OR “MND” combined with the terms “immunoglobulin” OR “IgG” OR “IgM” OR “IgA” OR “complement” OR “C3” OR “C4”. The language of the articles was restricted to English, and all searches were performed prior to April 6, 2020.
All the articles were carefully read and evaluated. Studies were included if they met the following criteria: 1) observational studies (including case-control studies or cohorts) that evaluated the serum levels of immunoglobulin or complement between patients with ALS and subjects without neurological diseases; 2) the ALS diagnostic criteria were clearly stated; and 3) met at least six points of the Newcastle-Ottawa Scale criteria (NOS) , which was used to evaluate the methodological quality of case-control and cohort studies. Review articles, editorials, commentaries, case reports, animal experiments, hypothesis papers, letters that reported no new data, meta-analyses, and abstracts were excluded.
Two investigators independently selected studies and extracted data according to the inclusion criteria, and a third researcher was asked to resolve disputes. The following data were extracted: the first author, publication year, country, sample size, mean age of the study group, mean and standard derivations of the serum levels of immunoglobulin and complement, methods used to measure the levels of immunoglobulin or complement measurement.
In the meta-analysis, the primary summary measure was the standardized mean difference (SMD) in serum immunoglobulin and complement levels between patients with ALS and control subjects. The SMDs with 95% CIs were calculated using a random-effects model and presented in a forest plot for each immunity-related variable. The level of heterogeneity across studies was evaluated using the I2 statistic  and Galbraith graph, and sensitivity analysis was performed by study-by-study exclusion. Publication bias was assessed using funnel plots, as well as Egger’s and Begg’s tests. A two-tailed p value of less than 0.05 was considered statistically significant. All data analyses were performed using STATA (version 13).