QIHC key terms

The process of preserving tissue by cross-linking proteins, most often with 10% neutral buffered formalin, to maintain morphology and antigenicity before processing and sectioning.

The interval between tissue removal from the patient and the start of fixation, during which enzymatic degradation and antigen loss can occur if the specimen is not promptly handled.

Prolonged exposure to formalin that creates excessive protein cross-linking, masking epitopes and often causing weak or absent immunohistochemical staining that may require stronger antigen retrieval.

Insufficient formalin exposure that leaves tissue inadequately preserved, producing poor morphology, unreliable antigen localization, and inconsistent staining results across the section.

The concealment of antibody binding sites caused by formalin cross-linking and tissue processing, requiring antigen retrieval to restore antibody access.

The thickness at which paraffin blocks are cut, typically 4-5 micrometers for IHC; thicker sections can increase background and alter apparent staining intensity.

Treatment of bone or calcified tissue with acid or chelating agents to allow sectioning; strong acids can destroy antigens and cause false-negative IHC results.

Attachment of tissue sections to charged or coated glass slides so they resist detachment during heat retrieval and repeated washing steps.

Any pretreatment that unmasks epitopes hidden by fixation so antibodies can bind; includes heat-based and enzymatic methods and is a major determinant of staining success.

A pretreatment in which slides are heated in a specific buffer using a water bath, pressure cooker, or microwave to break cross-links and expose epitopes.

Antigen retrieval using proteases such as trypsin or proteinase K to cleave proteins and uncover epitopes; overdigestion damages morphology and can cause false-negative staining.

The acidity or alkalinity of the solution used during heat retrieval; different epitopes require different pH values, commonly citrate at pH 6 or Tris-EDTA at pH 9.

Systematic testing of retrieval method, buffer pH, temperature, and time to find conditions that give strong specific staining with minimal background for a given antibody.

A detection method using a polymer backbone carrying many enzyme molecules and secondary antibodies, giving strong signal without biotin and reducing endogenous biotin interference.

A detection method in which biotinylated secondary antibodies bind avidin-biotin-enzyme complexes, amplifying signal but potentially reacting with endogenous biotin in liver and kidney.

A pretreatment, usually with hydrogen peroxide, that inactivates native tissue peroxidase enzymes so they do not produce false-positive signal when a peroxidase-based detection system is used.

A colorless compound that becomes an insoluble colored deposit where the detection enzyme is located, allowing antibody binding to be seen under the microscope, for example DAB or fast red.

An amplification technique in which peroxidase converts tyramide-labeled molecules into reactive species that deposit near the target, greatly increasing signal for weakly expressed antigens.

Staining several antigens on one section using different chromogens or fluorophores, allowing spatial relationships between markers to be assessed in the same tissue.

An antibody produced by a single clone of cells that binds one specific epitope, giving consistent and reproducible staining with low lot-to-lot variability.

A mixture of antibodies from different B-cell clones that recognize multiple epitopes of an antigen, often giving stronger signal but greater background and variability.

The concentration at which a primary antibody is applied, determined by titration to achieve optimal specific staining without excessive background or nonspecific binding.

A pale dye, commonly hematoxylin, applied after chromogen development to reveal tissue morphology and help localize the specific staining signal.

A tissue known to contain the target antigen, processed alongside the patient slide to confirm that reagents and retrieval worked and expected staining was obtained.

A slide treated identically but with primary antibody omitted or replaced by nonimmune serum, used to detect nonspecific background or detection system problems.

An antibody of the same class and concentration as the primary but with irrelevant specificity, used to assess nonspecific binding of the antibody reagent itself.

Documented verification that a test performs as intended for its clinical use, including accuracy, precision, sensitivity, and appropriate control performance before patient reporting.

An external program in which a laboratory tests unknown samples and compares results with peer laboratories to demonstrate ongoing competency and detect systematic problems.

Routine monitoring of reagents, equipment, controls, and staining runs to ensure results stay within acceptable limits and to detect problems before they affect patient care.

The subcellular distribution of a positive stain, such as nuclear, cytoplasmic, or membranous, which is essential for correct interpretation of the marker.

A semiquantitative scoring method combining staining intensity with the percentage of positive cells to produce a value between 0 and 300.

A scoring system that adds a proportion score for positive cells to an intensity score, giving a total from 0 to 8, commonly used for hormone receptors.

Normal cells or tissue elements within the patient section that should stain and thus confirm that the assay worked even when the tumor is negative.

Unwanted color across the tissue caused by nonspecific antibody binding, inadequate blocking, or insufficient washing, which can obscure true signal.

Staining that is not due to the target antigen, arising from antibody cross-reactivity, endogenous enzymes, or biotin, and often distinguished by its diffuse or inappropriate distribution.

A pattern of uneven or strong staining at tissue edges, caused by drying, uneven fixation, or uneven reagent coverage, which can mimic true positivity.

Failure to obtain expected signal, often due to overfixation, inadequate retrieval, expired reagents, or protocol errors, requiring systematic troubleshooting.

A written, approved protocol describing each step of a laboratory procedure so that staff perform it consistently and the process can be audited.

Handling and exposure precautions for formaldehyde, a known carcinogen and irritant, including ventilation, personal protective equipment, and proper waste disposal.