The clinical need for clustered AChR cell-based assay testing of seronegative MG

Masi, Gianvito; Li, Yingkai; Karatz, Tabitha; Pham, Minh; Oxendine, Seneca R.; Nowak, Richard J.; Guptill, Jeffrey T.; O’Connor, Kevin

doi:10.1016/j.jneuroim.2022.577850

Cited by 11 publications

(12 citation statements)

References 18 publications

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“… 16 Alternatively, live CBAs can be assessed through flow cytometry, which could represent a technical improvement providing a quantification of Ab binding. 17 The FACS results shown here need to be confirmed in a larger cohort of patients and controls. Finally, it is relevant to point out that although Ab titers and their changes, as assessed by RIA, have been shown to correlate with the MG clinical course, 18 , 19 the possible application of CBA to clinical monitoring of patients with MG during their follow-up has yet to be demonstrated.…”

Section: Discussionmentioning

confidence: 75%

Comparison of Fixed and Live Cell-Based Assay for the Detection of AChR and MuSK Antibodies in Myasthenia Gravis

Spagni

Gastaldi

Businaro

et al. 2023

Neurol Neuroimmunol Neuroinflamm

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Background and ObjectivesLive cell-based assay (CBA) can detect acetylcholine receptors (AChRs) or muscle-specific tyrosine kinase (MuSK) antibodies (Abs) in a proportion of patients with radioimmunoassay (RIA)-double seronegative myasthenia gravis (dSN-MG). A commercial fixed CBA for AChR and MuSK Abs has recently become available; however, comparative studies on fixed and live CBAs are lacking. In this study, we compared the performance of fixed and live CBAs in patients with RIA-dSN MG and assessed their sensitivity in RIA-positive MG samples and their specificity.MethodsAChR and MuSK Abs were tested in 292 serum samples from 2 Italian MG referral centers by live and fixed CBAs: 192 from patients with MG and 100 from controls. All samples had been previously assessed by RIA: 66 were AChR positive, 40 MuSK positive, and 86 dSN. All controls were negative. Two independent raters assessed the CBA results. Fixed and live CBAs were compared with the McNemar test; interrater and interlaboratory agreement were assessed with Cohen's kappa or interclass correlation coefficient (ICC), as appropriate.ResultsIn 86 RIA-dSN samples, fixed CBA detected Abs in 10 cases (11.6%, 95% CI 5.7–20.3), whereas live CBA detected Abs in 16 (18.6%, 95% CI 11.0–28.5) (p= 0.0143). Of these sera, those positive by fixed CBA were also positive by live CBA. In addition, live CBA could detect MuSK Abs in 4 and AChR Abs in 2 samples that were negative by fixed CBA, providing an 8% (95% CI 2.9–16.6) further increase in the Ab detection rate. These results were confirmed by flow cytometry. In the RIA-positive cohort, the sensitivity for AChR Abs was 98.5% (95% CI 91.9%–99.9%) for fixed CBA and 100% (95% CI 94.6–100) for live CBA (p= 0.1573). For both assays, the sensitivity for MuSK Abs was 100% (95% CI 91.2–100), and the specificity was 100% (95% CI 96.4–100). Interrater agreement was almost perfect for live and fixed CBAs (Cohen's kappa 0.972 and 0.978, respectively), alike interlaboratory agreement. Interrater agreement for the CBA score ranged from good to excellent (ICC: 0.832–0.973).DiscussionFixed CBA represents a valuable alternative to RIA for AChR and MuSK Ab detection in patients with MG and could be considered as a first-step diagnostic test. Live CBA can be useful in the serologic evaluation of RIA- and fixed CBA-negative samples.

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Section: Discussionmentioning

confidence: 75%

Comparison of Fixed and Live Cell-Based Assay for the Detection of AChR and MuSK Antibodies in Myasthenia Gravis

Spagni

Gastaldi

Businaro

et al. 2023

Neurol Neuroimmunol Neuroinflamm

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“…For example, antibodies binding to acetylcholine receptor or muscle‐specific tyrosine kinase were found in ~80% and 1–10% of myasthenia gravis patients, respectively. The acetylcholine receptor is a membrane protein with multiple transmembrane domains and it is difficult to produce the recombinant acetylcholine receptor while persevering its native conformation [ 40 , 41 ], which limits the accuracy of the related assays [ 42 , 43 ]. Among the rest of the seronegative patients, autoantibodies to acetylcholine receptor were detected using a cell‐based assay requiring technical expertise and specialized equipment, which limits its general use [ 43 ] .…”

Section: Discussionmentioning

confidence: 99%

Optimization of peripheral blood volume for in silico reconstitution of the human B‐cell receptor repertoire

et al. 2022

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B cells recognize antigens via membrane‐expressed B‐cell receptors (BCR) and antibodies. Similar human BCR sequences are frequently found at a significantly higher frequency than that theoretically calculated. Patients infected with SARS‐CoV2 and HIV or with autoimmune diseases share very similar BCRs. Therefore, in silico reconstitution of BCR repertoires and identification of stereotypical BCR sequences related to human pathology have diagnostic potential. Furthermore, monitoring changes of clinically significant BCR sequences and isotype conversion has prognostic potential. For BCR repertoire analysis, peripheral blood (PB) is the most convenient source. However, the optimal human PB volume for in silico reconstitution of the BCR repertoire has not been studied in detail. Here, we sampled 5, 10, and 20 mL PB from the left arm and 40 mL PB from the right arm of two volunteers, reconstituted in silico PB BCR repertoires, and compared their composition. In both volunteers, PB sampling over 20 mL resulted in slight increases in functional unique sequences (FUSs) or almost no increase in repertoire diversity. All FUSs with a frequency above 0.08% or 0.03% in the 40 mL PB BCR repertoire were detected even in the 5 mL PB BCR repertoire from each volunteer. FUSs with a higher frequency were more likely to be found in BCR repertoires from reduced PB volume, and those coexisting in two repertoires showed a statistically significant correlation in frequency irrespective of sampled anatomical site. The correlation was more significant in higher‐frequency FUSs. These observations support the potential of BCR repertoire analysis for diagnosis.

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“…All samples were tested on live clustered AChR, MuSK, and low‐density lipoprotein receptor‐related protein 4 (LRP4) CBAs using flow cytometry, as previously described 9 . For the LRP4 CBA, human embryonic kidney (HEK) 293T cells were transfected with human LRP4 plasmid (a generous gift from Dr. Stephan Kröger) 10 .…”

Section: Methodsmentioning

confidence: 99%

Clinicoserological insights into patients with immune checkpoint inhibitor‐induced myasthenia gravis

Masi

Pham

Karatz

et al. 2023

Ann Clin Transl Neurol

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To compare the immunopathology of immune checkpoint inhibitor-induced myasthenia gravis (ICI-MG) and idiopathic MG, we profiled the respective AChR autoantibody pathogenic properties. Of three ICI-MG patients with AChR autoantibodies, only one showed complement activation and modulation/blocking potency, resembling idiopathic MG. In contrast, AChR autoantibody-mediated effector functions were not detected in the other two patients, questioning the role of their AChR autoantibodies as key mediators of pathology. The contrasting properties of AChR autoantibodies in these cases challenge the accuracy of serological testing in establishing definite ICI-MG diagnoses and underscore the importance of a thorough clinical assessment when evaluating ICI-related adverse events.

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The clinical need for clustered AChR cell-based assay testing of seronegative MG

Cited by 11 publications

References 18 publications

Comparison of Fixed and Live Cell-Based Assay for the Detection of AChR and MuSK Antibodies in Myasthenia Gravis

Comparison of Fixed and Live Cell-Based Assay for the Detection of AChR and MuSK Antibodies in Myasthenia Gravis

Optimization of peripheral blood volume for in silico reconstitution of the human B‐cell receptor repertoire

Clinicoserological insights into patients with immune checkpoint inhibitor‐induced myasthenia gravis

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