QIHC flashcards

32 cards. Read the term and the definition here. The deck at the top of this page is optional.

Cold ischemia time

Interval from tissue devascularization to fixation. Prolonged cold ischemia degrades proteins and phosphoepitopes, causing weak or false-negative immunohistochemical staining.

10% neutral buffered formalin

Standard fixative that cross-links proteins. Most tissues require about 6–24 hours; underfixation causes central antigen loss and overfixation can mask epitopes.

Acid decalcification

Strong acids such as hydrochloric or nitric acid hydrolyze proteins and nucleic acids. For IHC, EDTA chelation at neutral pH better preserves antigens in bone sections.

IHC section thickness

Formalin-fixed paraffin sections are usually cut at 3–5 µm. Thicker sections increase background and nuclear overlap; thinner sections risk tissue loss during retrieval.

HIER buffer pH

Citrate pH 6.0 suits many cytoplasmic and membranous epitopes, while Tris-EDTA pH 9.0 often improves nuclear antigens. Each antibody requires validation of retrieval pH.

Pressure cooker retrieval

High-temperature steam heats slides uniformly. Time and temperature depend on the vessel and must be validated with positive tissue to avoid under- or over-retrieval.

Enzymatic antigen retrieval

Proteases such as trypsin, pepsin, or proteinase K digest proteins to expose epitopes. Overdigestion destroys antigen and increases background, so time and temperature require validation.

Retrieval cool-down

After heating, slides are cooled in retrieval buffer before staining. Gradual cooling reduces section detachment and helps produce uniform, reproducible staining.

No-retrieval antibody

Some antibody clones stain adequately without antigen retrieval. Applying unnecessary heat can reduce signal or increase background, so the validated protocol should be followed.

Direct detection

The primary antibody carries the label directly. It is simple and less prone to cross-reactivity but generally less sensitive than indirect or polymer methods.

Polymer detection

HRP- or AP-conjugated polymers bind secondary antibodies and amplify signal. They offer high sensitivity and avoid avidin-biotin background but require proper blocking.

Endogenous biotin

Liver, kidney, and some tumors contain biotin that binds avidin-biotin detection systems, causing false-positive staining. Biotin blocking or polymer detection prevents this interference.

DAB chromogen

Horseradish peroxidase substrate that forms a brown, alcohol-insoluble precipitate. It is suitable for permanent mounting and sequential multiplex immunohistochemistry.

AEC chromogen

Horseradish peroxidase substrate that forms a red precipitate soluble in alcohol and organic solvents. It requires aqueous mounting and cannot be dehydrated through alcohols.

Positive tissue control

Known antigen-expressing tissue processed like patient specimens. It confirms that antibody, retrieval, and detection worked and must be acceptable before patient slides are interpreted.

Negative reagent control

Same-species, same-isotype IgG at matching concentration replaces the primary antibody. It identifies nonspecific binding and detection-system background.

Lot-to-lot validation

New antibody or detection lots are compared with the current lot using positive and negative tissues. Acceptable concordance is documented before clinical use.

Control intensity grading

Controls are recorded as weak, moderate, or strong. A shift from the established baseline indicates reagent, retrieval, or detection drift requiring investigation.

Proficiency testing

External challenge samples assess laboratory performance. Failures require root-cause analysis, corrective action, and documentation under accreditation requirements.

H-score

H-score equals 3 times percent strong plus 2 times percent moderate plus 1 times percent weak staining, giving a range of 0–300.

Allred score

The Allred score adds a proportion score from 0–5 to an intensity score from 0–3, producing a total from 0–8; it is commonly used for ER and PR.

HER2 IHC scoring

HER2 IHC is scored 0 or 1+ negative, 2+ equivocal requiring ISH, and 3+ positive. Scoring depends on complete membranous staining intensity and percentage of tumor cells.

Ki-67 hotspot

Ki-67 is the percentage of tumor nuclei with nuclear staining in a defined hotspot or representative area. The antibody clone and scoring method should be specified.

PD-L1 CPS

Combined positive score equals stained tumor cells plus stained immune cells divided by viable tumor cells, multiplied by 100. Assay-specific rules define eligible cells.

Edge drying artifact

Evaporation at section edges during staining increases nonspecific background or causes false-negative areas. Adequate reagent volume and a humid chamber prevent it.

Underfixation artifact

Insufficient formalin fixation leaves central tissue unfixed, causing weak or absent staining in the center. Adequate fixation time before processing prevents this pattern.

Endogenous peroxidase

Red blood cells and neutrophils contain peroxidase that can produce DAB signal. Hydrogen peroxide blocking before primary antibody reduces this false-positive staining.

Nonspecific background

Insufficient washing, excessive antibody concentration, protein precipitation, or hydrophobic interactions cause background. Titrate antibody and increase wash stringency to reduce it.

Formalin safety

Formalin is a carcinogen and irritant. Use ventilation, gloves, and eye protection; manage spills with a hazardous spill kit and collect formalin waste separately.

DAB hazard

DAB is a suspected carcinogen. Avoid powdered stock, use ready-to-use reagent when possible, wear PPE, and collect contaminated waste as hazardous.

CAP/CLIA IHC requirements

Laboratories must maintain written SOPs, validated antibodies, documented quality control, proficiency testing, and corrective action for failed controls or proficiency samples.

Reagent lot tracking

Record lot numbers and expiration dates for antibodies, detection systems, and retrieval buffers. Linking lots to patient slides enables recall if a problem is found.