Difference between revisions of "Immunohistochemical staining"

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Generally, interpretations are 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."<ref>URL: [http://bitesizebio.com/articles/immunohistochemistry-getting-the-stain-you-want/ http://bitesizebio.com/articles/immunohistochemistry-getting-the-stain-you-want/]. Accessed on: 1 September 2012.</ref>   
Generally, interpretations are 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."<ref>URL: [http://bitesizebio.com/articles/immunohistochemistry-getting-the-stain-you-want/ http://bitesizebio.com/articles/immunohistochemistry-getting-the-stain-you-want/]. Accessed on: 1 September 2012.</ref>   


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


==General (malignant) differential diagnosis==
==General (malignant) differential diagnosis==

Revision as of 03:07, 1 September 2012

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

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.

Theory

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

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.

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 tissued:
    • Nucleus.
    • Cytoplasm.
    • Membrane.
    • A combination of the above.

Generally, interpretations are 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."[2]

The cynical might say it is 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!"

General (malignant) differential diagnosis

 
 
 
 
 
 
 
 
 
 
Malignancy
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Epithelial
(Carcinoma)
 
Mesenchymal
(Sarcoma)
 
Germ cell
tumour
 
Neuroendocrine
carcinoma
 
Hematologic
 
Malignant
melanoma
  • Carcinoma.
    • AE1/AE3 - pankeratin.
    • Others: EMA, HMWK, LMWK.
  • Sarcoma.
    • Vimentin.
      • Many pathologists think this stain is totally useless.
  • Germ cell tumours.
    • PLAP (placental-like alkaline phosphatase) - not very sensitive.[3]
    • Others: D2-40, OCT 3/4.
  • Neuroendocrine carcinoma.
    • Chromogranin A.
    • Synaptophysin.
    • CD56.
    • CD57.[4]
  • Melanoma.
    • S-100, HMB-45, Melan A (MART-1).
      • Additional (UHN): melanoma cocktail (HMB-45, MART-1)[5], microphthalmia,[6] tyrosinase.[7]
  • Hematologic.
    • Lymphoma/leukemia.
      • CD45 (common leukocyte antigen).
      • CD30.
    • Plasma cell:
      • Kappa.
      • Lambda.
      • CD138.

Keratins

Classification:[8]

  • Low molecular weight keratins (LMWK): 7, 8/18, 19, 20.
  • High molecular weight keratins (HWMK): 4, 10, 13, 14, 17.

Uses:

Organ specific

Thyroid and lung

  • TTF-1 (thyroid transcription factor-1) -- +ve in thyroid gland malignancies.
    • Very good for breast vs. lung.[11]
      • Often negative in squamous cell carcinoma of the lung (as with CK7 & CK20), though HMWK is usually positive.
  • Thyroglobulin usu. +ve in the thyroid.[12]

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

  • PSA - prostatic-specific antigen.
  • PSAP - prostatic-specific acid phosphatase.
  • p63 - stains nuclei of basal cell in normal prostate.
  • 34betaE12 - stains basal cells in normal prostate.
  • AMACR (racemase, P504S[15]) - present in adenocarcinoma (NOT in normal prostate).
  • AR - usually present in prostate confined cancers.[16]
  • CAP cocktail - AKA CAP, AKA PIN-4, AKA PIN.

Colorectal carcinoma markers

  • CEA.

Small bowel

  • CDX2.
  • Villin.

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

  • WT-1, CA-125, D2-40.

Liver

HCC vs. cholangiocarcinoma:

  • TTF-1: ~90-100% +ve (cytoplasmic) in HCC vs. ~10% in cholangiocarcinoma.[19]

Mesothelium

Panel:[20]

  • Mesothelial markers:
    • Calretinin.
    • WT-1.
    • D2-40.
    • CK5/6.
  • Carcinoma markers:
    • CEA (monoclonal and polyclonal).
    • TTF-1.
    • Ber-EP4.
    • MOC-31.

Note:

  • One should use two mesothelial markers and two carcinoma markers.[20]

Pancreas

Neuropathology

General:

  • S-100.

Glial:

  • GFAP.

Neuronal:

  • Synaptophysin.
  • Chromogranin.

Specific entities:

Tumour (low-grade gliomas):

Miscellaneous

Macrophages

Main article: Histocytoses
  • CD68.

Special:

Special, less common:

One organ versus another

Cervix versus uterus

  • Cervix (typically): CEA +ve,[29] p16 +ve.
    • ... and ER -ve, PR -ve, vimentin -ve.
  • Uterus (typically): vimentin +ve, ER +ve, PR +ve.[30]
    • ... and CEA -ve, p16 -ve.

Liver versus bile duct

Intrahepatic cholangiocarcinoma (ICC) vs. hepatocellular carcinoma (HCC):[31]

  • 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 adenocarcioma: PSA +ve, PSAP +ve, AR +ve, CK7 -ve, CK20 -ve.
  • Urothelial carcinoma: CK7 +ve, CK20 +ve, PSA -ve, PSAP -ve, AR -ve.

Breast versus ovary

Breast carcinoma versus ovarian carcinoma:

  • Ovary: WT-1 +ve, PAX8 +ve.[32]
  • Breast: mammaglobin +ve,[33] BRST2 +ve.

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

  • CD5 +ve, CD23 +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%.[35]
  • CD15 Reed-Sternberg cells +ve ~80%, stains neutrophils.

Germ cell tumours

Main article: Germ cell tumours

Seminoma

  • D2-40 +ve.[3]
  • OCT 3/4 +ve.

Embryonal carcinoma

  • CD30 +ve - cytoplasm, cell membrane, Golgi.
    • Rarely positive in seminoma.
  • CK7 +ve.[36]
  • 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

Main article: 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).

Note: MIB1 should not be confused with mindbomb homolog 1 (MIB-1), a gene that regulates apoptosis.[39]

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 & cytokeratin

A few tumours are positive for both vimentin and cytokeratins.

Common tumours:

Common tumours that uncommonly have the pattern:

Rare tumours:

Sarcomas cytokeratins

Most sarcomas are cytokeratin negative.

Exceptions - classic:

Others:

References

  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. ↑ URL: http://bitesizebio.com/articles/immunohistochemistry-getting-the-stain-you-want/. Accessed on: 1 September 2012.
  3. ↑ 3.0 3.1 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.
  4. ↑ 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.
  5. ↑ 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.
  6. ↑ URL: http://www.ncbi.nlm.nih.gov/omim/156845. Accessed on: 18 August 2010.
  7. ↑ Roma, AA.; Magi-Galluzzi, C.; Zhou, M. (Jan 2007). "Differential expression of melanocytic markers in myoid, lipomatous, and vascular components of renal angiomyolipomas.". Arch Pathol Lab Med 131 (1): 122-5. doi:10.1043/1543-2165(2007)131[122:DEOMMI]2.0.CO;2. PMID 17227112.
  8. ↑ http://www.nordiqc.org/Epitopes/Cytokeratins/cytokeratins.htm
  9. ↑ Murata T, Nakashima Y, Takeuchi M, Sueishi K, Inomata H (September 1993). "The diagnostic use of low molecular weight keratin expression in sebaceous carcinoma". Pathol. Res. Pract. 189 (8): 888–93. PMID 7508102.
  10. ↑ URL: http://www.aruplab.com/guides/ug/tests/2003978.jsp. Accessed on: 17 March 2011.
  11. ↑ Jagirdar J (March 2008). "Application of immunohistochemistry to the diagnosis of primary and metastatic carcinoma to the lung". Arch. Pathol. Lab. Med. 132 (3): 384–96. PMID 18318581. http://journals.allenpress.com/jrnlserv/?request=get-abstract&issn=0003-9985&volume=132&page=384.
  12. ↑ Dralle, H.; Böcker, W. (1982). "[Thyroglobulin immunohistochemistry: new aspects of pathophysiology and differential diagnosis of benign and malignant goitre (author's transl)].". Langenbecks Arch Chir 356 (3): 205-12. PMID 7070163.
  13. ↑ de Micco, C.; Chapel, F.; Dor, AM.; Garcia, S.; Ruf, J.; Carayon, P.; Henry, JF.; Lebreuil, G. (Mar 1993). "Thyroglobulin in medullary thyroid carcinoma: immunohistochemical study with polyclonal and monoclonal antibodies.". Hum Pathol 24 (3): 256-62. PMID 8454270.
  14. ↑ Azumi, N.; Traweek, ST.; Battifora, H. (Aug 1991). "Prostatic acid phosphatase in carcinoid tumors. Immunohistochemical and immunoblot studies.". Am J Surg Pathol 15 (8): 785-90. PMID 1712549.
  15. ↑ http://www.antibodies-online.com/antibody/125649/P504S+alphaMethylacylCoA+Racemace+AMACR+Human/
  16. ↑ Fleischmann, A.; Rocha, C.; Schobinger, S.; Seiler, R.; Wiese, B.; Thalmann, GN. (Apr 2011). "Androgen receptors are differentially expressed in Gleason patterns of prostate cancer and down-regulated in matched lymph node metastases.". Prostate 71 (5): 453-60. doi:10.1002/pros.21259. PMID 20878946.
  17. ↑ Anatelli F, Chuang ST, Yang XJ, Wang HL. (2008). "Value of glypican 3 immunostaining in the diagnosis of hepatocellular carcinoma on needle biopsy". Am J Clin Pathol. 130 (2): 219-23?. doi:10.1309/WMB5PX57Y4P8QCTY. PMID 18628090.
  18. ↑ The diagnostic value of hepatocyte paraffin antibody 1 in differentiating hepatocellular neoplasms from nonhepatic tumors: a review. Lamps LW, Folpe AL. Adv Anat Pathol. 2003 Jan;10(1):39-43. Review. PMID 12502967.
  19. ↑ Lei JY, Bourne PA, diSant'Agnese PA, Huang J (April 2006). "Cytoplasmic staining of TTF-1 in the differential diagnosis of hepatocellular carcinoma vs cholangiocarcinoma and metastatic carcinoma of the liver". Am. J. Clin. Pathol. 125 (4): 519–25. doi:10.1309/59TN-EFAL-UL5W-J94M. PMID 16627262.
  20. ↑ 20.0 20.1 Marchevsky AM (March 2008). "Application of immunohistochemistry to the diagnosis of malignant mesothelioma". Arch. Pathol. Lab. Med. 132 (3): 397-401. PMID 18318582. http://journals.allenpress.com/jrnlserv/?request=get-abstract&issn=0003-9985&volume=132&page=397.
  21. ↑ Goldstein NS, Bassi D (May 2001). "Cytokeratins 7, 17, and 20 reactivity in pancreatic and ampulla of vater adenocarcinomas. Percentage of positivity and distribution is affected by the cut-point threshold". Am. J. Clin. Pathol. 115 (5): 695–702. doi:10.1309/1NCM-46QX-3B5T-7XHR. PMID 11345833.
  22. ↑ Geller SA, Dhall D, Alsabeh R (March 2008). "Application of immunohistochemistry to liver and gastrointestinal neoplasms: liver, stomach, colon, and pancreas". Arch. Pathol. Lab. Med. 132 (3): 490–9. PMID 18318589.
  23. ↑ Perry, Arie; Brat, Daniel J. (2010). Practical Surgical Neuropathology: A Diagnostic Approach: A Volume in the Pattern Recognition series (1st ed.). Churchill Livingstone. pp. 12. ISBN 978-0443069826.
  24. ↑ Pernick NL, DaSilva M, Gangi MD, Crissman J, Adsay V (November 1999). ""Histiocytic markers" in melanoma". Mod. Pathol. 12 (11): 1072–7. PMID 10574605.
  25. ↑ URL: http://www.ncbi.nlm.nih.gov/omim/605545. Accessed on: 3 February 2011.
  26. ↑ URL: http://www.abcam.com/Macrophage-antibody-MAC387-FITC-ab7429.html. Accessed on: 3 February 2011.
  27. ↑ URL: http://www.abcam.com/Macrophage-antibody-MAC387-ab49408.html. Accessed on: 3 February 2011.
  28. ↑ URL: http://www.ncbi.nlm.nih.gov/omim/604862. Accessed on: 2 February 2011.
  29. ↑ 29.0 29.1 Alkushi A, Irving J, Hsu F, et al. (March 2003). "Immunoprofile of cervical and endometrial adenocarcinomas using a tissue microarray". Virchows Arch. 442 (3): 271-7. doi:10.1007/s00428-002-0752-4. PMID 12647218.
  30. ↑ URL: http://www.nature.com/modpathol/journal/v19/n8/full/3800620a.html
  31. ↑ [Evaluation of immunohistochemical markers for differential diagnosis of hepatocellular carcinoma from intrahepatic cholangiocarcinoma] Dong H, Cong WL, Zhu ZZ, Wang B, Xian ZH, Yu H. Zhonghua Zhong Liu Za Zhi. 2008 Sep;30(9):702-5. Chinese. PMID 19173916.
  32. ↑ Nonaka, D.; Chiriboga, L.; Soslow, RA. (Oct 2008). "Expression of pax8 as a useful marker in distinguishing ovarian carcinomas from mammary carcinomas.". Am J Surg Pathol 32 (10): 1566-71. doi:10.1097/PAS.0b013e31816d71ad. PMID 18724243.
  33. ↑ Kanner, WA.; Galgano, MT.; Stoler, MH.; Mills, SE.; Atkins, KA. (Oct 2008). "Distinguishing breast carcinoma from Müllerian serous carcinoma with mammaglobin and mesothelin.". Int J Gynecol Pathol 27 (4): 491-5. doi:10.1097/PGP.0b013e31817d5340. PMID 18753974.
  34. ↑ URL: http://atlasgeneticsoncology.org/Genes/BCL1.html. Accessed on: 17 December 2010.
  35. ↑ Humphrey, Peter A; Dehner, Louis P; Pfeifer, John D (2008). The Washington Manual of Surgical Pathology (1st ed.). Lippincott Williams & Wilkins. pp. 567. ISBN 978-0781765275.
  36. ↑ Cheville JC, Rao S, Iczkowski KA, Lohse CM, Pankratz VS (April 2000). "Cytokeratin expression in seminoma of the human testis". Am. J. Clin. Pathol. 113 (4): 583–8. doi:10.1309/3QLC-5MF1-JYXU-A5XX. PMID 10761461.
  37. ↑ Tadrous, Paul.J. Diagnostic Criteria Handbook in Histopathology: A Surgical Pathology Vade Mecum (1st ed.). Wiley. pp. 107. ISBN 978-0470519035.
  38. ↑ URL: http://ajp.amjpathol.org/cgi/content/full/160/3/759. Accessed on: 3 August 2010.
  39. ↑ http://www.genenames.org/data/hgnc_data.php?hgnc_id=21086
  40. ↑ Tazi, EM.; Lalya, I.; Tazi, MF.; Ahellal, Y.; M'rabti, H.; Errihani, H. (2010). "Transitional cell carcinoma of the ovary: a rare case and review of literature.". World J Surg Oncol 8: 98. doi:10.1186/1477-7819-8-98. PMID 21073751.
  41. ↑ URL: http://cat.inist.fr/?aModele=afficheN&cpsidt=2504165. Accessed on: 26 April 2011.
  42. ↑ Llombart-Bosch A, Lopez-Guerrero JA, Peydro-Olaya A (2002). "Synovial sarcoma (SS): new perspectives supported by modern technology". Arkh. Patol. 64 (3): 39–47. PMID 15338724.
  43. ↑ Itakura E, Tamiya S, Morita K, et al. (September 2001). "Subcellular distribution of cytokeratin and vimentin in malignant rhabdoid tumor: three-dimensional imaging with confocal laser scanning microscopy and double immunofluorescence". Mod. Pathol. 14 (9): 854–61. doi:10.1038/modpathol.3880401. PMID 11557780. http://www.nature.com/modpathol/journal/v14/n9/full/3880401a.html.
  44. ↑ Miettinen M, Fanburg-Smith JC, Virolainen M, Shmookler BM, Fetsch JF (August 1999). "Epithelioid sarcoma: an immunohistochemical analysis of 112 classical and variant cases and a discussion of the differential diagnosis". Hum. Pathol. 30 (8): 934–42. PMID 10452506.

External links

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