CAP validation set should reflect a range from no expression to high expression.

• A. Only high expression samples
• B. No expression samples only
• C. Range from no expression to high expression
• D. Inclusion of low-expression samples only

Answer: (C)

Including a spectrum from no expression to high expression is essential because a CAP validation set must test how the assay performs across the full range of staining that can occur in real samples. If you only include high-expression cases, you won’t see how the assay handles weak or borderline staining, nor how the scoring system interprets low signals or background. Conversely, using only negative samples wouldn’t reveal the assay’s sensitivity or its ability to distinguish true positives from non-specific staining. By spanning from no expression through low to high expression, the validation assesses the assay’s dynamic range, the robustness of positivity thresholds, and the consistency of interpretation across different staining intensities, providing a realistic and comprehensive evaluation.