10.3 Preliminary Screening and Expected Patterns
Key Takeaways
- QIHC preliminary screening means the technologist verifies that controls worked, chromogen is in the expected compartment, on-slide internal controls are present when they should be, and predictive-marker slides are technically score-ready for the pathologist—not that the technologist issues the diagnosis.
- ASCO/CAP breast HER2 IHC (2018 criteria, affirmed 2023): 0 / 1+ / 2+ / 3+; 3+ is complete intense circumferential membrane staining in more than 10% of invasive tumor cells; 2+ (weak to moderate complete membrane in more than 10%) is equivocal and reflex ISH is required.
- ASCO/CAP 2020 ER/PR: nuclear staining; 1% or more tumor nuclei is positive; ER with 1–10% nuclei is reported as ER Low Positive; less than 1% is negative.
- PD-L1 TPS (tumor-cell membranes), CPS (tumor cells plus lymphocytes and macrophages, capped at 100), and IC (percent of tumor area occupied by PD-L1-positive immune cells) are different readouts; CAP 2024 Statement 6 requires each assay–scoring-system combination to be validated or verified as a pair (minimum 20 positive and 20 negative tissues).
- MMR proteins are nuclear; pair loss is typically MLH1 with PMS2 or MSH2 with MSH6, and loss counts only when internal-control nuclei remain positive.
10.3 Preliminary Screening and Expected Patterns
Quick Answer: Preliminary screening on the QIHC topic outline means you verify that the stain is technically interpretable: external and on-slide internal controls, expected compartment, adequate invasive tumor, and no fatal artifact. For HER2, ER, PR, PD-L1, and MMR, you present score-ready slides and know the published pattern rules so you can flag problems—not so you can issue the final result. HER2 3+ is complete intense membrane in more than 10%; 2+ goes to ISH. ER/PR are nuclear at ≥1% (ASCO/CAP 2020). PD-L1 TPS, CPS, and IC are different scores tied to clone and indication (CAP 2024 Statement 6). MMR pair loss is MLH1+PMS2 or MSH2+MSH6 with intact internal nuclei.
This OpenExamPrep section is independent teaching on preliminary screening and expected staining patterns covering published QIHC staining topics IV.B.2.a (patterns) and IV.F (preliminary screening). It is not an ASCP publication and does not claim Board, ASCO, or CAP approval. Predictive-marker numbers below are from published ASCO/CAP breast guidance and the 2024 CAP analytic-validation guideline; they are applications of pattern reading, not an invented sixth official domain such as “predictive biomarkers 15%.”
What “preliminary screening” means in the IHC laboratory
The pathologist diagnoses. The QIHC-level technologist screens the stained slide for technical truth before it is scored:
- The run worked. On-slide or batch external controls show the expected positive and negative elements for that antibody.
- The patient tissue can show antigen. If a relevant internal control is present (normal breast epithelium for ER, residual lymphocytes for CD45 or MMR, non-neoplastic nerve for S100), it stains in the correct compartment.
- Chromogen is in the biologically expected place. HER2 is membrane, not dirty cytoplasm. ER/PR/MMR are nuclear. PD-L1 tumor-cell signal for TPS is membrane. A beautiful wrong-compartment stain is not ready to score.
- The target population is on the slide. Invasive carcinoma for breast HER2/ER/PR—not only DCIS, not only cautery edge, not only 20 crushed cells.
- Artifact is not being offered as signal. Drying rings, folded tissue, pigment, edge burn, and carryover floaters were covered in morphology teaching; they still fail a predictive slide.
If any of those fail, the slide is not score-ready. Restain, recut, or document why interpretation is blocked. Do not “average in” a failed internal control and hand the pathologist a number.
| Check | Pass | Fail (do not treat as a negative tumor) |
|---|---|---|
| External positive control | Expected cells stain in the expected compartment | Blank or wrong-compartment control |
| External negative / omit-primary | No specific chromogen | Dirty background that matches the patient “signal” |
| On-slide internal control (when present) | Normal elements stain as they should | Internal epithelium or lymphocytes blank while tumor is called negative |
| Localization | Membrane vs nucleus vs cytoplasm matches the analyte | HER2 scored on cytoplasmic blush; ER scored on membrane |
| Adequacy | Enough well-fixed invasive tumor for that assay | Crush, empty block, only in-situ disease when invasive scoring is required |
On-slide internal control is the strongest proof that this piece of tissue still has detectable antigen after fixation and retrieval. An external control on a different slide proves the reagent dispenser worked that hour; it does not prove the patient core was not over-fixed, burnt, or decalcified into antigen loss. ASCO/CAP ER guidance specifically wants internal-control status mentioned when staining is 0–10%.
Preanalytic locks that still apply (ASCO/CAP breast biomarker handling, not ASCP outline text): cold ischemia preferably ≤1 hour; 10% NBF for 6–72 hours. A technically pretty HER2 slide on a specimen fixed 2 hours after a 6-hour cold delay is still a compromised predictive assay.
Breast HER2 IHC: 0 / 1+ / 2+ / 3+ (ASCO/CAP 2018, affirmed 2023)
Score invasive carcinoma. Use membrane pattern and extent. The 2023 focused update affirmed the 2018 IHC definitions; it did not invent a new 0–3+ scale. HER2-low is a reporting construct for IHC 1+ or IHC 2+ with ISH-negative—it does not rewrite the 0/1+/2+/3+ criteria.
| IHC score | Staining of invasive tumor cells | Category | Screening implication |
|---|---|---|---|
| 0 | No staining, or incomplete faint/barely perceptible membrane staining in ≤10% of cells | Negative (IHC 0) | Not the classic HER2-positive pathway |
| 1+ | Incomplete faint/barely perceptible membrane staining in >10% | Negative (IHC 1+) | Not reflex ISH for overexpression; may be reported in a HER2-low framework |
| 2+ | Weak to moderate complete membrane staining in >10% | Equivocal | Reflex ISH required |
| 3+ | Complete, intense, circumferential membrane staining in >10%, readily seen at low power in a cohesive invasive population | Positive | IHC-positive; ISH is not required to establish IHC 3+ positivity |
Unusual patterns (for example intense complete membrane in ≤10%, or intense but incomplete/basolateral staining in some subtypes) are treated as 2+ equivocal in published ASCO/CAP discussion, not as home-made 3+. Cytoplasmic blush that hides the membrane is not a 3+.
Technologist screening: Is the stain membranous? Is the invasive component mapped on the H&E? Did the on-slide 3+ and 0/1+ controls (or cell-line controls, if that is the validated method) behave? Then the pathologist assigns 0/1+/2+/3+. You do not upgrade 1+ to 3+ because the oncologist “needs it,” and you do not skip ISH on a 2+ because the membrane “looks almost strong.”
Breast ER and PR: nuclear, ≥1% (ASCO/CAP 2020)
Estrogen receptor (ER) and progesterone receptor (PR/PgR) are nuclear transcription factors. Membrane or cytoplasmic-only chromogen is not a positive receptor result.
ASCO/CAP 2020:
- Positive: 1% to 100% of tumor nuclei immunoreactive.
- ER Low Positive: 1% to 10% ER nuclear staining—still in the positive group for endocrine-therapy eligibility, but reported with the published limited-benefit comment. This Low Positive category is specified for ER, not as a separate official PR name.
- Negative: 0% or <1% nuclear staining.
- Report percentage and intensity; report control status, especially when the result is 0–10%.
- If tumor is negative or low and no internal epithelial control is present, the published comment notes that external controls worked and that another specimen with internal controls may be needed.
PR follows the same 1% nuclear positive / <1% negative idea and is used mainly as a prognostic adjunct in ER-positive disease. DCIS is tested for ER; PR on DCIS is optional in that guideline.
Screening: normal ducts and lobules (if present) should show nuclear ER. A tumor called “ER 0%” on a slide whose residual normal epithelium is also blank is not a negative result—it is an uninterpretable or failed patient stain until explained. Do not score DCIS as if it were the invasive cancer when invasive scoring was requested.
PD-L1: TPS versus CPS versus IC, and why clone plus scoring system are a pair
PD-L1 IHC is not one number. Three common readouts exist; they count different cells and use different denominators. Cutoffs are indication- and assay-specific—this section teaches the definitions, not a table of every drug cutoff.
Tumor Proportion Score (TPS): percentage of viable tumor cells with partial or complete membrane PD-L1 staining at any intensity, divided by all viable tumor cells, times 100. Immune-cell staining is excluded. Classic pairing example: 22C3 pharmDx with TPS in non-small cell lung carcinoma. Necrosis, pigment, and alveolar macrophages are not tumor-cell membrane.
Combined Positive Score (CPS): number of PD-L1-staining tumor cells + lymphocytes + macrophages, divided by total viable tumor cells, times 100. The calculated value may exceed 100; reported CPS is capped at 100. Same clone (for example 22C3) may be used with CPS in other tumor types. You cannot take a TPS percentage and relabel it CPS.
Immune Cell (IC) score: percentage of the tumor area occupied by PD-L1-positive tumor-infiltrating immune cells (any intensity). This is an area readout, not a tumor-cell percentage. A common pairing example is SP142 with IC in specified indications (historically including some triple-negative breast and urothelial algorithms). IC is not TPS with a different name.
CAP 2024 analytic validation, Statement 6 (strong recommendation): for laboratory-developed assays and for verification of FDA-approved/cleared predictive IHC assays that have distinct scoring schemes (examples given: HER2 and PD-L1), laboratories should separately validate or verify each assay–scoring-system combination with a minimum of 20 positive and 20 negative tissues, including challenges that match intended use. That is why clone + scoring system are a pair: 22C3-TPS is not automatically 22C3-CPS; SP142-IC is not SP263-TPS. Swapping a clone onto another drug’s cutoff without that paired validation is not a screening shortcut.
Minimum viable tumor cells (often 100 for 22C3 CPS/TPS protocols) and on-slide control cell lines are kit- and SOP-specific; follow the validated method. The technologist flags macrophage-only brown on a TPS assay, missing membrane on tumor cells, and control failure. The pathologist assigns the numeric score used for treatment.
MMR: nuclear pair loss (MLH1+PMS2 or MSH2+MSH6)
The four mismatch-repair proteins form heterodimers: MutLα = MLH1 + PMS2; MutSα = MSH2 + MSH6. PMS2 is unstable without MLH1; MSH6 is unstable without MSH2. That biochemistry is why pair loss is the screening pattern.
| IHC pattern in tumor nuclei (internal control nuclei retained) | Screening meaning |
|---|---|
| All four intact nuclear | MMR-proficient pattern (does not entirely exclude rare antigen-positive but nonfunctional protein) |
| MLH1 loss + PMS2 loss, MSH2/MSH6 intact | Classic pair loss; often sporadic MLH1 silencing in colorectal cancer, but the pathologist directs BRAF / methylation / germline work-up |
| MSH2 loss + MSH6 loss, MLH1/PMS2 intact | Classic pair loss pointing toward MSH2 (Lynch-type work-up is a pathologist/genetics step) |
| Isolated PMS2 or isolated MSH6 loss | Also abnormal; not a failed dimer partner by definition |
| Tumor and lymphocytes/stroma/crypts all negative for one antibody | Failed stain or wrong retrieval, not pair loss |
Loss means absent nuclear staining in the tumor with intact nuclear staining in internal controls (lymphocytes, stroma, non-neoplastic crypts). Cytoplasmic MMR chromogen without nuclei is not “retained.” Heterogeneous or clonal loss is a pathologist problem, not a technologist “average.”
Do not diagnose Lynch syndrome from the stainer. Do not call MLH1/PMS2 loss “the patient has Lynch” (many colorectal cases are sporadic promoter methylation). Do not treat intact IHC as a license to skip the pathologist’s integrated report.
Putting IV.F next to IV.B.2.a without inventing a domain
Staining-pattern knowledge (lineage markers in 10.1–10.2, epithelial markers in Chapter 9) and preliminary screening (this section) are how you know what the slide should look like before anyone assigns a clinical category. HER2 membrane scores, ER nuclear percentages, PD-L1 system-specific readouts, and MMR pair loss are expected-pattern applications. They are not an official ASCP content area titled predictive biomarkers at 15%.
Worked example
A breast core has HER2 IHC with weak-to-moderate complete membrane staining in about 40% of invasive cells: that is 2+ equivocal, and the SOP sends ISH, even if someone wishes it were 3+. Adjacent ER shows nuclear staining in 5% of invasive nuclei at weak intensity; residual normal lobules are nuclear ER-positive: that is ER Low Positive by ASCO/CAP 2020, not ER-negative, and not a 3+ HER2 equivalent. A lung PD-L1 22C3 slide requested for TPS shows strong macrophage cytoplasm and no tumor-cell membrane: macrophages do not enter TPS—do not convert the slide to an SP142 IC number. A colon tumor shows complete nuclear loss of MLH1 and PMS2 with MSH2/MSH6 retained in tumor, while lymphocytes next to the crypts are nuclear-positive for all four: that is MLH1/PMS2 pair loss with a valid internal control, ready for the pathologist—not a technologist Lynch diagnosis, and not a failed MLH1 if the lymphocytes stain.
If the same colon slide had blank MLH1 in tumor and in lymphocytes, you restain; you do not report pair loss.
The QIHC candidate’s product is a technically honest, pattern-correct slide. The final HER2 category, ER percentage used for therapy, PD-L1 cutoff for a named drug, and Lynch versus sporadic algorithm belong to the pathologist and the validated report.
A breast core shows weak-to-moderate complete membrane HER2 staining in more than 10% of invasive carcinoma cells. External and internal controls are appropriate. What is the ASCO/CAP screening-level category and next technical step?
Which statement matches published screening rules for ER/PR, PD-L1, or MMR without turning the technologist into the diagnosing pathologist?