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The number of clusters (k=5) was chosen objectively by maximizing a combined score of: Silhouette coefficient on a discovery split: measures how well-separated the clusters are internally Adjusted Rand Index (ARI) on a held-out validation split: measures whether an independent reclustering of new data produces the same structure This two-stage validation approach guards against over-fitting the cluster count to a single sample

Inter-Assay Precision (Precision between assays): Three samples of known concentration were tested in twenty separate assays to assess inter-assay precision