Detecting ectopic thymus in thymoma-associated myasthenia gravis through flow cytometry analysis of CD3mediumTCRvβmediumCD4+CD8+ T cells and its clinical significance.

Background: Traditional histological methods for identifying ectopic thymus (ET) have significant limitations including high risk of false negatives. This study aims to evaluate the effectiveness of flow cytometry in detecting ETs in patients undergoing total thymectomy.

Methods: We analyzed 864 samples from 103 patients using flow cytometry and hematoxylin and eosin (H&E) staining. ETs were identified by the presence of CD3^mediumTCRvβ^mediumCD4⁺CD8⁺ T cells in flow cytometry or Hassall's corpuscles in H&E staining.

Results: In the discovery set, flow cytometry detected ETs in 69.2% of samples, compared to 23.6% by histological methods. The validation set showed a higher incidence of ETs in myasthenia gravis (MG) patients than in non-MG patients (73.5% vs. 58.0%, P < 0.0001) and in those with thymic epithelial tumors versus normal thymus (68.1% vs. 58.1%, P = 0.0088). MG patients exhibited a higher prevalence of active ETs, characterized by a high proportion of CD4⁺CD8⁺ T cells, indicating robust thymopoiesis, compared to those without MG (P = 0.0001). Specific regions, such as the left cervical root, areas along the right and left phrenic nerves, and the left innominate vein, showed significantly higher activity (P < 0.05). Additionally, ETs were more frequently found in the cervical region than in the mediastinum (75.0% vs. 60.8%, P = 0.0012), and in patients aged 40 years or younger compared to those older than 40 years (73.0% vs. 60.6%, P = 0.0027).

Conclusions: Flow cytometry is a viable alternative for ET detection, providing a novel distribution map that enhances surgical decision-making in MG treatment.