Mapping Anti-HLA Class I Cross-Reactivity for Transplantation Using Interpretable Embedding and Clustering of SAB MFI

Background: Mapping anti–HLA class I cross-reactivity from single-antigen bead (SAB) mean fluorescence intensity (MFI) data supports donor selection. However, interpretation is complicated by analytical choices and assay variability. Methods: A total of 4327 SAB assays were analyzed (antigen × test matrix) using an interpretable, distance-based workflow. Antigen profiles were z-scored across tests. Multidimensional scaling (MDS) was used for visualization and hierarchical clustering analysis (HCA) for grouping, and complemented these with a common PCA space for model selection (K-Means via Silhouette; Gaussian Mixture Models via BIC), agglomerative (Ward and average-link on 1–correlation), spectral clustering on correlation-derived affinities, and a graph approach (k-NN ∪ minimum-spanning-tree with modularity-based communities). Non-linear embeddings (t-SNE/UMAP) and density-based HDBSCAN were used only for visual analytics, not for primary inference. Results: The pipeline revealed coherent reactivity neighborhoods that partially overlapped known cross-reactive antigen groups (CREGs) and eplet-based expectations while also highlighting less-documented relationships. Robustness was confirmed through bootstrap resampling, graph modularity, and consensus clustering across methods. Conclusions: This unified, auditable workflow converts descriptive maps into method-robust summaries of antibody reactivity and cross-reactivity. While exploratory and performed on a single dataset without linked outcomes, the approach provides a reproducible structure for comparing cohorts and prioritizing hypotheses that could be prospectively validated for clinical decision support in transplantation.