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.


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]


  • 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]


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

Germ cell
  • 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.


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


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).


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

Serous markers


HCC vs. cholangiocarcinoma:

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



  • Mesothelial markers:
  • Carcinoma markers:


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




  • S-100.


  • GFAP.


  • Synaptophysin.
  • Chromogranin.


Specific entities:

Tumour (low-grade gliomas):



  • CD68.


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:


This is covered more extensively in the lymphoma article.


  • CD45 (AKA common leukocyte antigen).


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


  • 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.


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


  • 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).


  • 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).


  • 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]


CK7 and CK20

CK7+ CK20-

Mnemonic: OBE + lung x2 + H&N x2

CK7- CK20+

CK7+ CK20+

Mnemonic: POOTE.


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:


See also


  1. Mitchell, Richard; Kumar, Vinay; Fausto, Nelson; Abbas, Abul K.; Aster, Jon (2011). Pocket Companion to Robbins & Cotran Pathologic Basis of Disease (8th ed.). Elsevier Saunders. pp. 175. ISBN 978-1416054542.
  2. Coons, AH. (Jun 1971). "The development of immunohistochemistry.". Ann N Y Acad Sci 177: 5-9. PMID 4400556.
  3. Vosse, BA.; Seelentag, W.; Bachmann, A.; Bosman, FT.; Yan, P. (Mar 2007). "Background staining of visualization systems in immunohistochemistry: comparison of the Avidin-Biotin Complex system and the EnVision+ system.". Appl Immunohistochem Mol Morphol 15 (1): 103-7. PMID 17536316.
  4. Vosse, BA.; Seelentag, W.; Bachmann, A.; Bosman, FT.; Yan, P. (Mar 2007). "Background staining of visualization systems in immunohistochemistry: comparison of the Avidin-Biotin Complex system and the EnVision+ system.". Appl Immunohistochem Mol Morphol 15 (1): 103-7. PMID 17536316.
  5. Torlakovic, EE.; Francis, G.; Garratt, J.; Gilks, B.; Hyjek, E.; Ibrahim, M.; Miller, R.; Nielsen, S. et al. (Apr 2014). "Standardization of negative controls in diagnostic immunohistochemistry: recommendations from the international ad hoc expert panel.". Appl Immunohistochem Mol Morphol 22 (4): 241-52. doi:10.1097/PAI.0000000000000069. PMID 24714041.
  6. Engel, KB.; Moore, HM. (May 2011). "Effects of preanalytical variables on the detection of proteins by immunohistochemistry in formalin-fixed, paraffin-embedded tissue.". Arch Pathol Lab Med 135 (5): 537-43. doi:10.1043/2010-0702-RAIR.1. PMID 21526952.
  7. URL: Accessed on: 1 September 2012.
  8. Galloway, M. (2011). "Base-rate error in the interpretation of immunohistochemistry.". Patholog Res Int 2011: 636495. doi:10.4061/2011/636495. PMID 21660231.
  9. 9.0 9.1 Iczkowski KA, Butler SL, Shanks JH, et al (February 2008). "Trials of new germ cell immunohistochemical stains in 93 extragonadal and metastatic germ cell tumors". Hum. Pathol. 39 (2): 275-81. doi:10.1016/j.humpath.2007.07.002. PMID 18045648.
  10. Kurokawa, M.; Nabeshima, K.; Akiyama, Y.; Maeda, S.; Nishida, T.; Nakayama, F.; Amano, M.; Ogata, K. et al. (May 2003). "CD56: a useful marker for diagnosing Merkel cell carcinoma.". J Dermatol Sci 31 (3): 219-24. PMID 12727026.
  11. Jani P, Chetty R, Ghazarian DM (April 2008). "An unusual composite pilomatrix carcinoma with intralesional melanocytes: differential diagnosis, immunohistochemical evaluation, and review of the literature". Am J Dermatopathol 30 (2): 174–7. doi:10.1097/DAD.0b013e318165b8fe. PMID 18360125.
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External links