12.2 Control Tissue Selection and Use

12.2 Control Tissue Selection and Use

Quick Answer: Controls tell you whether the stain on this slide, with these reagents, detected the antigen at the intensity the method claims. Place positive and negative tissue on the same glass as the patient when you can. For predictive markers, the positive control must include low expressors near the clinical cutoff—not only a blazing tonsil. Read internal tissue controls. Use leftover diagnostic tissue under laboratory ethics rules; do not empty a small biopsy.

This OpenExamPrep section is independent teaching on IHC control tissues covering published QIHC laboratory-operations topic areas. It is not an ASCP or CAP publication and does not claim Board or College approval.

What a control is for

A control is the experiment's known so you can interpret the unknown. If the positive control is blank, you cannot call the patient negative. If the negative control is dirty, you cannot call the patient positive without a hard look at nonspecific staining. If the only positive control is a 3+ blast of signal, you cannot know whether a weak-but-clinically-important patient stain would still have been detected.

Controls come in layers:

1. External positive tissue — known to contain the target, processed like clinical tissue when possible
2. External negative tissue — known not to contain the target, or to sit below the reporting threshold
3. Internal tissue control — residual normal structures in the patient section that should (or should not) label
4. Negative reagent control — same slide handling with primary antibody omitted or replaced by an irrelevant primary, used when the procedure requires it (especially on tissues prone to biotin, endogenous enzyme, or pigment artifact)

CAP 2024 Goldsmith published practice (Section 12.3) asks that validation sets include high and low expressors for positives when appropriate and that they span the expected clinical range for scored assays. Daily QC should not be philosophically weaker than that validation idea. A method that was challenged with weak positives on day one should not be babysat forever by a single 3+ cell line.

Match fixative and processing to clinical work when you can. An NBF FFPE control does not prove an alcohol-fixed cytology cell block received a working assay. A frozen control does not prove an FFPE run. Section 12.3 treats those mismatches as validation problems; this section treats them as daily control-selection problems.

On-slide versus batch controls

On-slide controls are preferred, especially for predictive markers, because the control tissue travels through the same dewax, retrieval, antibody, detection, and chromogen as the patient. A separate batch slide can look perfect while a single patient slide sat on a dry pad or missed a dispense. Batch controls still have a role on large nonpredictive runs when the SOP allows them, but they do not prove that every position on the instrument was wet.

Some laboratories place a small control core or a sliver of control tissue at the end of the patient section (an edge mount or "sandwich"). That is still on-slide control, provided the control actually saw the same droplets. A control stuck on with a different adhesive that lifts during retrieval is a false failed positive, not proof that the patient antibody vial is empty.

If the laboratory must use a batch control, the SOP should say so, and screening should still look for internal controls on each patient. A perfect batch slide plus a blank internal epithelium on every patient on positions 8–12 is an instrument-position story, not a run of keratin-negative carcinomas.

Positive controls must include low expressors for predictive work

For a binary nonpredictive marker (cytokeratin present or absent), a strongly positive appendix or skin may be an adequate daily positive if the SOP says so—though even then, a weak internal control in the patient (residual epithelium) is extra evidence.

For predictive scored assays (HER2, ER, PR, PD-L1, and similar), a strongly positive-only control is a poor-only control. Mechanism:

• Clinical cutoffs often live in the weak range (HER2 1+ versus 2+, a PD-L1 percentage near a drug-specific threshold, weak nuclear ER).
• An assay that has drifted—lower antibody activity, shorter retrieval, dying detection lot—may still paint a 3+ cell block a deep brown while losing the faint positives.
• Daily QC then "passes," and patients near the cutoff are under-called.

Build or buy multi-tissue controls that contain negative, low-positive, and high-positive cores or slices. Include the expression levels you actually report, not only the photograph you would put on a poster.

Why a strongly positive tonsil is a poor-only control for a faint cutoff

Tonsil is a superb multi-antigen block for many lineage markers: squamous epithelium, germinal centers, endothelium, and plasma cells live on one cheap, leftover-friendly piece of tissue. It is still a poor sole control for a faint clinical cutoff if the relevant cells in that tonsil are uniformly strong (or uniformly negative) for the scored antigen.

Worked example. PD-L1 on a tumor type is reported with a cutoff at a low percentage of weak membrane staining. The laboratory's only daily positive is tonsil, in which crypt epithelium and macrophages label strongly with that clone. For months the tonsil is an intense brown, so QC is checked off. A new detection lot quietly lowers sensitivity. Strong tonsil is still strong. Patient tumors with true 2–5% weak staining now look negative. The assay did not fail the tonsil; the tonsil never challenged the cutoff.

The same story applies to HER2 if the only control is a 3+ cell line, and to ER if the only control is a strongly positive tumor without a weak nuclear-positive challenge. A HER2 3+ breast tumor plus a HER2-negative tumor, without a 1+/2+ challenge, is the same mistake in two cores.

Fix: add characterized low-positive tumor, a scored cell-line set, or a multi-core TMA with 0, low, and high; read internal immune cells when the scoring system uses them; and treat a sudden loss of weak patient positives—while the 3+ control still shouts—as a method problem, not as a sudden epidemic of negative tumors.

Select controls for the decision you report, not for the prettiest photograph.

Negative controls

Negative tissue controls show that the assay does not stain what it should not stain. Choose tissue that is processed like clinical work and that is a realistic look-alike (normal liver for a marker that should be negative in hepatocytes; a known HER2-negative breast for HER2). A negative control that is a completely different specimen type is not the same test.

Negative reagent controls (omit primary, or use an irrelevant primary of the same isotype when the SOP says so) detect detection-system background, endogenous enzyme, and pigment that can be mistaken for chromogen. They do not prove the primary antibody is specific; they prove the downstream chemistry is clean. Use them when the SOP and the tissue (liver, kidney, heavily pigmented skin, biotin-rich kidney if an avidin-biotin method is still in play) require them.

Do not call a dirty omit-primary slide a "positive patient." Do not call a clean omit-primary slide proof that a poorly characterized primary is specific. Those are different questions.

Internal tissue controls

The patient slide often carries its own control structures. Read them before you believe a surprising negative or a surprising dirty positive.

Internal controls do not replace external controls. Heterogeneous tumors and small cores may lack the internal structure. Internal controls also cannot show you a low-expressor challenge if the residual normal is strong. Use both. If the internal control is present and appropriate and it is blank, you have a failed stain on that slide even if a batch control on another piece of glass looked fine.

Tissue microarrays

A tissue microarray (TMA) packs many cores—often 2 mm or smaller—into one block so one slide carries dozens of characterized tissues. TMAs are efficient for validation sets and for multi-tissue daily controls. A TMA mounted on the patient slide is still an on-slide control.

Limits to name on an exam stem:

• Sampling. A "negative" core may have missed the positive clone of a heterogeneous tumor.
• Edge and core dropout. Small cores lift during retrieval more than whole sections.
• Fixation history of donated cores may not match your clinical stream unless you built the TMA from your own processed leftovers.
• You still need low expressors, not 40 cores of 3+ tumor.

A TMA is a filing cabinet of tiny tissues. It is not automatically a better control than a well-chosen whole section that includes negative, weak, and strong areas.

Leftover-tissue ethics at a high level

Most IHC validation and control blocks come from residual human tissue originally removed for diagnosis. That leftover is not a free chemical.

• Do not exhaust a small biopsy or a block that may be needed for future predictive, molecular, or legal review. Control cores come from cases with adequate remaining tumor, under pathologist or medical-director rules.
• Laboratory quality activities (validation, controls, troubleshooting) are not the same as human-subjects research. Research on leftover tissue may need IRB review and consent under institutional policy. QA use still needs a written policy.
• Privacy: treat donor identity as protected health information. Extra-clinical sharing is de-identified or handled under the institution's rules.
• Respect the diagnosis. Do not harvest control tissue in a way that compromises margins or residual tumor measurement.
• Follow institutional and applicable U.S. rules. This guide does not invent a consent form and does not claim a single nationwide leftover-tissue statute for every hospital.

If a stem asks whether you may shave an entire 4 mm core biopsy into a TMA for extra HER2 controls, the answer is no: that block is the patient's archive. If a stem asks whether leftover tissue from a large resection, released by the pathologist after diagnostic needs are met, may become a multi-tissue control under laboratory policy, that is the usual ethical path—documented, not casual.

Choose controls that share processing with patients, sit on the same slide when possible, and challenge the faint end of the report you sign.