In semi-quantitative IHC scoring (0-3+), which element calibrates the scoring system?

• A. Antibody affinity
• B. Reference controls
• C. Tissue morphology
• D. Staining duration

Answer: (B)

Calibrating a semi-quantitative IHC score relies on reference controls to establish a stable benchmark under the current assay conditions. These controls are slides with known levels of target expression that are run alongside samples in every batch. They define what 0, 1+, 2+, and 3+ look like given the specific reagents, fixation, antigen retrieval, instrument settings, and staining chemistry. By comparing sample staining to the reference, you maintain consistent interpretation across runs and guard against drift from reagent lots or protocol changes.

Antibody affinity affects how tightly the reagent binds its target, influencing signal strength but does not provide a standardized scale for interpretation. Tissue morphology can alter how staining appears (density of cells, background), but it doesn’t set the scoring thresholds themselves. Staining duration changes overall intensity but is a procedural parameter; calibration requires a fixed reference point to map intensity to a score reliably.