QIHC cheat sheet

Cold ischemia time from devascularization to fixation should be minimized; delay degrades proteins and phosphoepitopes and can produce weak or false-negative IHC.

10% neutral buffered formalin is standard; most tissues fix 6–24 h, with small biopsies shorter and fatty or fibrotic tissue longer; underfixation and overfixation both alter epitopes.

Strong acid decalcifiers such as hydrochloric or nitric acid hydrolyze antigens; EDTA at neutral pH is preferred when IHC is planned on bone or calcified tissue.

Cut IHC sections at 3–5 µm on positively charged slides; bake at about 60°C before deparaffinization to improve adhesion.

Avoid prolonged block storage, repeated freeze-thaw, and excessive drying; antigenicity declines with age and warm storage.

Necrotic, autolyzed, or cauterized areas can stain nonspecifically or lose antigen; select viable representative areas for interpretation.

Cytology specimens require validated fixation, such as alcohol or formalin, and cell-block processing; air-drying before fixation can distort antigens.

Record fixation type and duration; ischemic time and processing schedule are part of the preanalytic record used for troubleshooting.

HIER buffers: citrate pH 6.0 suits many cytoplasmic and membranous epitopes; Tris-EDTA pH 9.0 often improves nuclear antigens; pH and molarity must be verified.

Heat sources include pressure cooker, microwave, steamer, and water bath; each has a distinct time-temperature profile and must be validated per antibody.

Optimization varies retrieval pH, heating time, antibody dilution, and detection; change one variable at a time and compare with known positive tissue.

Enzymatic pretreatment with trypsin, pepsin, or protease is used for some epitopes and frozen sections; overdigestion destroys antigen and raises background.

Slides should not boil dry; maintain buffer level and even heating; dry sections show edge artifact and irreversible antigen loss.

Cool slides in retrieval buffer before staining; abrupt transfer can detach sections and uneven cooling can affect staining.

Some antibodies require no retrieval; unnecessary heat can reduce signal or increase background.

Verify retrieval buffer pH before each run; drifted pH changes staining intensity and must be corrected.

Direct detection labels the primary antibody; indirect detection uses a labeled secondary antibody and amplifies signal but adds cross-reactivity risk.

Polymer and compact polymer systems use HRP- or AP-conjugated backbones; they provide high sensitivity and low avidin-biotin background.

Avidin-biotin complex detection is sensitive but can bind endogenous biotin in liver, kidney, and some tumors; use biotin block or polymer detection.

HRP substrates: DAB gives a brown alcohol-insoluble precipitate; AEC gives a red alcohol-soluble precipitate requiring aqueous mounting.

AP substrates include Fast Red and BCIP/NBT; levamisole blocks endogenous alkaline phosphatase.

Endogenous peroxidase is blocked with hydrogen peroxide; endogenous phosphatase is blocked with levamisole or reduced substrate exposure.

Multiplex IHC uses sequential staining, antibody stripping or elution, or tyramide amplification; verify channel separation and cross-reactivity.

Species cross-reactivity and secondary antibody host must match; mouse-on-mouse blocking is needed when using mouse primary antibody on mouse tissue.

Every IHC run needs a positive tissue control with known antigen expression and a negative reagent control matched for species and IgG concentration.

Positive controls should be processed and fixed like patient tissue; control staining must be acceptable before patient results are reported.

New antibody, detection system, retrieval method, or lot requires validation against the current method with sensitivity and concordance data.

Daily QC includes control slides, reagent checks, temperature logs, and review of background and counterstain quality.

Control intensity should be graded weak, moderate, or strong; a shift from baseline signals reagent, retrieval, or detection drift.

External proficiency testing and periodic internal audits are required under CAP/CLIA; failures need root cause analysis and corrective action.

Document antibody clone, lot, dilution, retrieval, detection, and control results; records enable traceability and recall.

Store control blocks and slides under conditions that preserve antigen; avoid repeated sectioning of exhausted blocks.

Report localization as nuclear, cytoplasmic, membranous, or mixed; the expected compartment determines whether staining is true positivity.

H-score = 3×%strong + 2×%moderate + 1×%weak, range 0–300; report the formula and staining compartment.

Allred score combines proportion (0–5) and intensity (0–3), total 0–8, commonly used for ER and PR.

HER2 IHC: 0 and 1+ negative, 2+ equivocal requiring ISH, 3+ positive; assess complete membranous staining in viable tumor cells.

Ki-67 is reported as percentage of tumor nuclei stained in a defined hotspot or representative area; specify antibody and scoring method.

PD-L1 CPS = (stained tumor cells + stained immune cells) / viable tumor cells ×100; scoring rules are assay-specific.

Do not interpret crushed, necrotic, or edge-dried areas; compare with internal normal tissue when available.

Report clone, platform, retrieval, controls, and any limitations; for equivocal results state recommended follow-up.

Strong background: check endogenous peroxidase or biotin, nonspecific protein binding, antibody concentration, wash steps, and detection system.

Weak or absent staining: verify positive control, antigen preservation, retrieval pH and time, antibody titer and expiry, and detection reagent.

Edge artifact: drying, uneven fixation, thick sections, bubbles, or insufficient reagent volume; use a humid chamber and full reagent coverage.

Necrosis and crush artifact can bind antibodies nonspecifically; interpret only viable representative tissue.

Brown granular pigment such as hemosiderin, melanin, or lipofuscin can mimic DAB; use a negative control and alternate chromogen.

Frozen sections may need different fixation and retrieval than FFPE; do not transfer protocols without validation.

Troubleshoot by changing one variable at a time and comparing with known positive and negative tissue controls.

If controls fail, do not report patient results; repeat the run after correcting the identified cause.

Formalin is a carcinogen and irritant; use ventilation, gloves, eye protection, and hazardous spill procedures.

DAB is a suspected carcinogen; avoid powdered stock, use ready-to-use when possible, and segregate contaminated waste.

Xylene and alcohol wastes are hazardous; segregate halogenated, nonhalogenated, and aqueous waste per local rules.

CAP/CLIA require written SOPs, validated IHC methods, documented QC, proficiency testing, and corrective action.

Track reagent lot numbers, expiration dates, and storage temperatures; link lots to patient slides for recall.

Monitor refrigerator, freezer, and oven temperatures; document daily or continuously as required.

Treat all specimens as biohazardous; use universal precautions and disinfect work surfaces.

Retain validation, QC, maintenance, and training records for the period required by the accrediting agency.