Effects of the Positive Threshold and Data Analysis on Human MOG Antibody Detection by Live Flow Cytometry

Abstract

Human autoantibodies targeting myelin oligodendrocyte glycoprotein (MOG Ab) have become a useful clinical biomarker for the diagnosis of a spectrum of inflammatory demyelinating disorders. Live cell-based assays that detect MOG Ab against conformational MOG are currently the gold standard. Flow cytometry, in which serum binding to MOG-expressing cells and control cells are quantitively evaluated, is a widely used observer-independent, precise, and reliable detection method. However, there is currently no consensus on data analysis; for example, seropositive thresholds have been reported using varying standard deviations above a control cohort. Herein, we used a large cohort of 482 sera including samples from patients with monophasic or relapsing demyelination phenotypes consistent with MOG antibody-associated demyelination and other neurological diseases, as well as healthy controls, and applied a series of published analyses involving a background subtraction (delta) or a division (ratio). Loss of seropositivity and reduced detection sensitivity were observed when MOG ratio analyses or when 10 standard deviation (SD) or an arbitrary number was used to establish the threshold. Background binding and MOG ratio value were negatively correlated, in which patients seronegative by MOG ratio had high non-specific binding, a characteristic of serum that must be acknowledged. Most MOG Ab serostatuses were similar across analyses when optimal thresholds obtained by ROC analyses were used, demonstrating the robust nature and high discriminatory power of flow cytometry cell-based assays. With increased demand to identify MOG Ab-positive patients, a consensus on analysis is vital to improve patient diagnosis and for cross-study comparisons to ultimately define MOG Ab-associated disorders.

Keywords: MOG antibody; antibody detection; demyelination; flow cytometry analysis; myelitis; optic neuritis (ON); patient diagnosis.

Figure 1

Assessment of patient MOG Ab serostatus by flow cytometry live cell-based assay. (1) MOG-expressing cells (MOG+) and empty vector or untransduced/untransfected control cells (MOG-) were gated. MOG- cells can be either seeded together with or separate from MOG+ cells. (2) The mean, median, or geometric mean fluorescence intensity of the MOG+ and MOG- cells can be determined. (3) MOG Ab binding to MOG is quantified by subtraction (ΔMOG) or division (MOG ratio) of MOG+ and MOG- cells. (4) The threshold of seropositivity can be determined by an arbitrary number or calculated at 3–10 standard deviations (SD) above a control cohort. A breakdown of the analyses is shown in Table 1. Recommended methods of analysis are indicated by green dots. Analyses that demonstrated reduced seropositive outcomes and detection sensitivity are indicated by a red dot.

Figure 2

High serum background binding reduced seropositivity detection in MOG ratio analysis. Patients negative (filled red) by mean (A,B), median (C), and geometric mean (D) MOG ratio analysis had high background binding. There was a negative correlation between background binding and mean or median MOG ratio values (P < 0.0001). (E) ΔMOG values of MOG Ab+ patients reported negative in MOG ratio analysis by 3 or 4 SD (red, Analysis 6a and 7a, respectively) or 10 SD (orange, Analysis 7b). Children (left) and adults (right) negative by MOG ratio analysis had a broad range of MOG Ab titers and fell within the range of ΔMOG values of patients who were positive by MOG ratio analysis. Dotted lines represent the ΔMOG positive threshold 3 SD above controls. Representative data from three experiments are shown. (F) Patients reported negative by MOG ratio median analysis (4 SD, Analysis 7a) clinically presented with MOG Ab-associated phenotypes. P, pediatric patients; A, adults; ADEM, acute disseminated encephalomyelitis; BON, bilateral optic neuritis; CIS, clinically isolated syndrome; LETM, longitudinally extensive transverse myelitis; ON mixed, combination of BON and UON; ON/TM, simultaneous ON and transverse myelitis; relapsing ADEM, multiphasic ADEM (41); TM, transverse myelitis; UON, unilateral optic neuritis.