B-094 Assessment of a Live Cell-based Flow Cytometry Assay for MuSK-IgG4 Testing in Myasthenia Gravis
Authors: Wu J , Nam Y , Gorsh A , Mills J R 
Published: October 2024

Abstract

Background
Myasthenia gravis (MG) is caused by disrupting the function of postsynaptic acetylcholine receptors (AChRs) at the neuromuscular junction. 85% of MG patients have autoantibodies targeting AChRs, while 6% of MG patients have autoantibodies targeting the muscle-specific receptor tyrosine kinase (MuSK). MuSK is located on the muscle membrane and is activated by the low-density lipoprotein receptor-related protein 4 (LRP4) upon the binding with ligand Agrin. Activated MuSK undergoes autophosphorylation and stimulates downstream signaling to cluster AChRs, which is important for the formation and maintenance of neuromuscular synapses. IgG4 antibodies that bind MuSK are thought to directly disrupt the ability of MuSK to cluster AChR. The gold standard methodology for MuSK antibody testing is radioimmunoprecipitation assay (RIA) using anti-human IgG secondary antibodies. Performing RIA methods in the clinical laboratory is a burden due to radioactive biohazard concerns and the analytical limitations of these assays. This study is to assess the analytical and clinical performance of a live cell-based flow cytometry assay as a potential replacement for the MuSK RIA.

Methods
HEK293T cells were transiently transfected with a vector co-expressing human MuSK protein and GFP. Patient sera were incubated with a mix of HEK293 cells (GFP-positive cells that express MuSK protein on the cell surface and GFP-negative cells that do no express MuSK) followed by incubation with anti-human IgG4 secondary antibody conjugated with alexa fluor 647 (AF647). An IgG binding index (IBI) was calculated for each sample as the ratio of the median AF647 fluorescence intensity (MFI) of the MuSK-expressing cells (GFP+) to the MFI of the non-MuSK expressing cells (GFP-). An arbitrary positive IBI cut-off of 2.0 was selected. A preliminary assessment of assay imprecision was performed for the flow cytometry assay, as well as a method comparison study to compare this assay to RIA utilizing a cohort of 130 (34 positive and 96 negative) residual samples previously tested by the gold-standard assay. 133 disease control samples were also tested to assess the clinical specificity of the flow cytometry-based assay.

Results
To assess the imprecision, eight samples with varying IBIs were tested with 5 replicates in each assay over five days. The coefficient of variation (CV) for intra-assay replicates ranged from 1% to 7% for all samples, while CV for inter-assay replicates ranged from 2% to 15%. Based on the 130 samples previously tested by RIA, the Positive Percentage Agreement (PPA) between these two assays was 76.5% and the negative Percentage Agreement (NPA) was 100%. Of the discordant samples (n=8), clinical information was available for only 1 sample (positive by RIA, negative by flow). This patient had a final non-MG diagnosis (ALS). All control samples were negative on the flow cytometry-based assay.

Conclusions
The live cell-based flow cytometry MuSK IgG4 assay demonstrated encouraging preliminary analytical performance that warrants further development and validation.