Controls, Validation, and Quality Assurance/Quality Control
Control types
The candidate must distinguish four controls. A positive tissue control is tissue known to express the antigen and confirms the whole reagent chain worked. A negative tissue control is tissue known not to express it and demonstrates specificity. A negative reagent control (buffer or irrelevant antibody replacing the primary) exposes nonspecific background and endogenous peroxidase or biotin effects. An on-slide control places control tissue on the same slide as the patient section, which best captures slide-level variables such as retrieval and drying.
Validation and verification
Laboratory-developed tests require documented initial validation, typically at least 20 positive and 20 negative cases per CAP expectations, plus comparison with expected results and correlation with morphology. Every new antibody clone, new lot number, new detection system, new retrieval protocol, or new platform requires verification before clinical use. Antibodies sold as IVD, analyte-specific reagents, or research-use-only carry different regulatory weight; RUO reagents cannot be reported without local validation.
Routine QC
Run controls with every staining batch. Log temperatures, reagent expiration, and lot numbers. Perform daily and monthly equipment checks, participate in proficiency testing, investigate failures with documented corrective action, and verify competency of technologists and interpreting pathologists.
Traps
A control that is overwhelmingly strongly positive can mask a loss of sensitivity - choose moderately expressing control tissue. If the positive control fails, the whole run is invalid regardless of patient staining. A negative reagent control is not the same as a negative tissue control, and questions frequently swap them.
Rule of thumb
Positive control negative: the run failed, do not report. Positive control positive but the patient block negative: look at the patient's preanalytics and biology, not at the reagents first.