Immunohistochemical staining

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Immunohistochemical staining, also immunostaining, is a powerful tool. It is abbreviated IHC.

CK20 staining of normal small intestinal mucosa.

Utility

Use of immunohistochemistry:[1]

  1. Unknown primary tumours.
  2. Poorly differentiated tumours.
  3. Prognostic markers, e.g. ERBB2 (HER2).
  4. Proving clonality - in the context of hematologic malignancies.
  5. Mutation specific antibodies, eg. IDH-1 R132H.

Method was introduced in 1941 by Coons.[2]

Theory

  • Antigen retrieval.
  • Antibody binds to the antigen.
  • Amplification - needed as the signal is usually too weak.

Signaling systems

  • Avidin-biotin complex.
    • Older.[3]
    • May suffer from endogenous avidin-biotin activity.[4]
      • Higher false positive rates than with polymer based methods.
  • Polymer based methods.
    • Newer.
    • Less prone to false positives.
      • Negative controls not needed or infrequently required.[5]

Signal assessment

  • Manual (Morphology) vs. automated (Speed) counting.
  • Choice of proper chromogen.
    • Dynamic range of DAB is 1-2logs vs. fluorescent probes 2-3logs.
    • Protein expression range can be up to 4 logs in gene amplification (information is missed in IHC).
    • FastRed stains are more suited in melanocytic tumors than DAB.
  • Use of established cutoffs for "intensity" and for "positive" staining.
  • Visual pattern recogniton vs. detecting spatial subtle changes.
  • Detecting differences at low intensity with human eye is less accurate.

Quality control

This is an evolving area in pathology that has been ignored for a surprisingly long time.

It is touched upon the in the quality article in the immunohistochemistry section.

There are at least 62 pre-analytical variables to be considered, that may affect staining results.[6]

Interpretation

To determine whether a stain is (1) done correctly, and (2) positive, one needs to know:

  1. What tissues it stains:
    • Tumour.
    • Normal tissue.
  2. How it stains the various tissues:
    • Patchy.
    • Diffuse.
  3. Where it stains the various tissues:
    • Nucleus.
    • Cytoplasm.
    • Membrane.
    • A combination of the above.

Generally, interpretations can be subjective, and this is especially true when the staining is weak and focal. In other words, "... your weak [positive] stain might be somebody else’s negative."[7]

The cynical might say it is an unwritten rule that: "... if the stain is weak and focal it can be anything you want to make it -- positive or negative -- so it fits perfectly with your diagnosis!"

In cases where the morphology is unclear, it is judicious to have two or more immunostains that support the diagnosis, and negative stains for important entities in the differential diagnosis.

Publications with contradicting results are not uncommon. Differences can arise from the fixation, processing protocol, antibody clone and interpretation.

According to Galloway, one third pathologists substantially overestimate the diagnostic significance of unexpected immunohistochemical staining results.[8]

General (malignant) differential diagnosis

 
 
 
 
 
 
 
 
 
 
Malignancy
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Epithelial
(Carcinoma)
 
Mesenchymal
(Sarcoma)
 
Germ cell
tumour
 
Neuroendocrine
carcinoma
 
Hematologic
 
Malignant
melanoma
  • Carcinoma.
  • Sarcoma.
    • Vimentin.
      • Many pathologists think this stain is totally useless.
  • Germ cell tumours.
  • Neuroendocrine carcinoma.
    • Chromogranin A.
    • Synaptophysin.
    • CD56.
    • CD57.[10]
  • Melanoma.
    • S-100, HMB-45, Melan A (MART-1).
      • Additional: melanoma cocktail (HMB-45, MART-1)[11], microphthalmia,[12] tyrosinase.[13]
  • Hematologic.
    • Lymphoma/leukemia.
      • CD45 (common leukocyte antigen).
      • CD30.
    • Plasma cell:
      • Kappa.
      • Lambda.
      • CD138.

Keratins

Mark epithelial cells. Are typically seen in carcinomas.

Organ specific

Thyroid and lung

Image: Adenocarcinoma with nuclear TTF-1 positivity (WC).

Breast markers

  • GCDFP-15 (AKA BRST-2) -- specific, but NOT sensitive.
  • ER (estrogen receptor) - in normal breast.
  • PR (progesterone receptor) - in normal breast.
  • HER2/neu - pathological, assoc. with worse prognosis.

Prostate gland

Colorectal carcinoma markers

  • CK20.
  • CDX2.
    • Uncommon in primary lung, breast, pancreas, kidney, gallbladder, liver, urinary bladder, thyroid gland.[20]
  • CEA.

Small bowel

Kidney

Renal cell carcinoma:

  • RCC, EMA, CD10.
  • CK7 -ve in clear cell RCC.
  • AMACR +ve in papillary RCC.
  • D2-40 +ve in ChRCC.

Xanthogranulomatous pyelonephritis:

  • CD68 (for macrophages).

Ovary

  • CA125, CK7+, CK20-.
  • WT1 -- 90% in serous +ve.

Serous markers

Liver

HCC vs. cholangiocarcinoma:

  • TTF-1: ~90-100% +ve (cytoplasmic) in HCC vs. ~10% in cholangiocarcinoma.[23]

Mesothelium

Panel:[24]

  • Mesothelial markers:
  • Carcinoma markers:

Note:

  • One should use two mesothelial markers and two carcinoma markers.[24]

Pancreas

Neuropathology

General:

  • S-100.

Glial:

  • GFAP.

Neuronal:

  • Synaptophysin.
  • Chromogranin.

Glial/Neuronal:

Specific entities:

Tumour (low-grade gliomas):

Miscellaneous

Macrophages

  • CD68.

Special:

Special, less common:

One organ versus another

Cervix versus uterus

  • Cervix (typically): CEA +ve,[33] p16 +ve.
    • ... and ER -ve, PR -ve, vimentin -ve.
  • Uterus (typically): vimentin +ve, ER +ve, PR +ve.[34]
    • ... and CEA -ve, p16 -ve.

Liver versus bile duct

Intrahepatic cholangiocarcinoma (ICC) vs. hepatocellular carcinoma (HCC):[35]

  • ICC: CK19 (92.5%), MUC-1 (73.8%) +ve.
  • HCC: HepPar-1 (85.6%), CD34 (87.8%) +ve.

Prostate versus bladder

Prostate adenocarcinoma vs. urothelial carcinoma:

  • Prostate adenocarcinoma: PSA +ve, PSAP +ve, AR +ve, CK7 -ve, CK20 -ve, GATA3 -ve.
  • Urothelial carcinoma: GATA3 +ve, CK7 +ve, CK20 +ve, PSA -ve, PSAP -ve, AR -ve.

Breast versus ovary

Breast carcinoma versus ovarian carcinoma:

Lymphomas

This is covered more extensively in the lymphoma article.

Lymphocytes

  • CD45 (AKA common leukocyte antigen).

B-cells

  • CD20.
  • CD19 (flow only).
  • PAX5.
  • CD79a.

T-cells

  • CD3 - general T-cell marker (marks both CD4 +ve and CD8 +ve cells).
  • CD4.
  • CD8.
  • CD7.
  • CD43.

Specific entities

Follicular lymphoma

  • CD10 +ve, BCL6 +ve.

CLL

Mantle cell lymphoma

Hodgkin's lymphoma

This is covered more extensively in the Hodgkin lymphoma article.

Classic types:

  • CD30 Reed-Sternberg cells (RSCs) +ve ~98%.[39]
  • CD15 Reed-Sternberg cells +ve ~80%, stains neutrophils.

Germ cell tumours

Seminoma

  • D2-40 +ve.[9]
  • OCT4 +ve.

Embryonal carcinoma

  • CD30 +ve - cytoplasm, cell membrane, Golgi.
    • Rarely positive in seminoma.
  • CK7 +ve.[40]
  • AE1/AE3 +ve.

Yolk sac tumour (endodermal sinus tumour)

  • AFP (alpha fetoprotein).

Choriocarcinoma

  • beta-hCG.

Bare bones mnemonic for GCTs

The germ cell tumour (GCT) IHC mnemonic ABCD:

  • AFP = yolk sac tumour.
  • Beta-hCG = choriocarcinoma.
  • CD30 = embryonal carcinoma.
  • D2-40 = seminoma.

Spindle cell lesions

Abbreviated spindle cell panel (memory device SCADS):

  • S100.
  • CD34.
  • AE1/AE3.
  • Desmin.
  • SMA.

A MFH panel:

Muscle markers

  • Desmin - all three types.
  • H-caldesmon - smooth muscle - most specific.
  • Smooth muscle actin (SMA) - smooth muscle.
  • MyoD1 - skeletal muscle.
  • Smooth muscle myosin (abbreviated SMMS).

Proliferation markers

  • MIB1 - an antibody against the protein Ki-67 (a protein expressed in proliferating cells).

Notes:

  • Ki-67 is found in lymphocytes - these are a positive control; lymphocytes should not be confused for (positive) tumour cells.[43]
  • MIB1 should not be confused with mindbomb homolog 1 (MIB-1), a gene that regulates apoptosis.[44]

Carcinomas

CK7 and CK20

CK7+ CK20-

Mnemonic: OBE + lung x2 + H&N x2

CK7- CK20+

CK7+ CK20+

Mnemonic: POOTE.

Note:

CK7- CK20-

Mnemonic: NASH TURP.

Vimentin and cytokeratin positivity

A few tumours are positive for both vimentin and cytokeratins.

Sarcomas and cytokeratins

Most sarcomas are cytokeratin negative.

Exceptions - classic:

Others:

See also

References

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