Does Re-referencing Matter? Large Laplacian Filter Optimizes Single-Trial P300 BCI Performance

Electroencephalography (EEG) provides a non-invasive window into brain activity, enabling Brain-Computer Interfaces (BCIs) for communication and control. However, their performance is limited by signal fidelity issues, among which the choice of re-referencing strategy is a pervasive but often overlooked preprocessing bias. Addressing controversies about its necessity and optimal choice, we adopted a quantified approach to evaluate four strategies - no re-referencing, Common Average Reference (CAR), small Laplacian, and large Laplacian - using 62-channels EEG (31 subjects, 2,520 trials). To our knowledge, this is the first study systematically quantifying their impact on single-trial P300 classification accuracy. Our controlled pipeline isolated re-referencing effects for source-space reconstruction (eLORETA with Phase Lag Index) and anatomically constrained classification. The large Laplacian resolves distributed P3b networks while maintaining P3a specificity, achieving the best P300 peak classification accuracy (81.57% hybrid method; 75.97% majority regions of interest). Performance follows a consistent and statistically significant hierarchy: large Laplacian > CAR > no re-reference > small Laplacian, providing a foundation for unified methodological evaluation.