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Immunohistochemical staining (view source)
Revision as of 11:49, 30 November 2020
, 11:49, 30 November 2020→Signal assessment: Update
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#Prognostic markers, e.g. ERBB2 (HER2). | #Prognostic markers, e.g. ERBB2 (HER2). | ||
#Proving clonality - in the context of hematologic malignancies. | #Proving clonality - in the context of hematologic malignancies. | ||
#Mutation specific antibodies, eg. [[IDH-1]] R132H. | |||
Method was introduced in 1941 by Coons.<ref>{{Cite journal | last1 = Coons | first1 = AH. | title = The development of immunohistochemistry. | journal = Ann N Y Acad Sci | volume = 177 | issue = | pages = 5-9 | month = Jun | year = 1971 | doi = | PMID = 4400556 }}</ref> | |||
==Theory== | ==Theory== | ||
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**Older.<ref>{{Cite journal | last1 = Vosse | first1 = BA. | last2 = Seelentag | first2 = W. | last3 = Bachmann | first3 = A. | last4 = Bosman | first4 = FT. | last5 = Yan | first5 = P. | title = Background staining of visualization systems in immunohistochemistry: comparison of the Avidin-Biotin Complex system and the EnVision+ system. | journal = Appl Immunohistochem Mol Morphol | volume = 15 | issue = 1 | pages = 103-7 | month = Mar | year = 2007 | doi = | PMID = 17536316 }}</ref> | **Older.<ref>{{Cite journal | last1 = Vosse | first1 = BA. | last2 = Seelentag | first2 = W. | last3 = Bachmann | first3 = A. | last4 = Bosman | first4 = FT. | last5 = Yan | first5 = P. | title = Background staining of visualization systems in immunohistochemistry: comparison of the Avidin-Biotin Complex system and the EnVision+ system. | journal = Appl Immunohistochem Mol Morphol | volume = 15 | issue = 1 | pages = 103-7 | month = Mar | year = 2007 | doi = | PMID = 17536316 }}</ref> | ||
**May suffer from endogenous avidin-biotin activity.<ref name=pmid17536316>{{Cite journal | last1 = Vosse | first1 = BA. | last2 = Seelentag | first2 = W. | last3 = Bachmann | first3 = A. | last4 = Bosman | first4 = FT. | last5 = Yan | first5 = P. | title = Background staining of visualization systems in immunohistochemistry: comparison of the Avidin-Biotin Complex system and the EnVision+ system. | journal = Appl Immunohistochem Mol Morphol | volume = 15 | issue = 1 | pages = 103-7 | month = Mar | year = 2007 | doi = | PMID = 17536316 }}</ref> | **May suffer from endogenous avidin-biotin activity.<ref name=pmid17536316>{{Cite journal | last1 = Vosse | first1 = BA. | last2 = Seelentag | first2 = W. | last3 = Bachmann | first3 = A. | last4 = Bosman | first4 = FT. | last5 = Yan | first5 = P. | title = Background staining of visualization systems in immunohistochemistry: comparison of the Avidin-Biotin Complex system and the EnVision+ system. | journal = Appl Immunohistochem Mol Morphol | volume = 15 | issue = 1 | pages = 103-7 | month = Mar | year = 2007 | doi = | PMID = 17536316 }}</ref> | ||
***Higher false positive rates than with polymer based methods. | |||
*Polymer based methods. | *Polymer based methods. | ||
**Newer. | **Newer. | ||
**Less prone to false positives. | |||
***Negative controls not needed or infrequently required.<ref name=pmid24714041>{{Cite journal | last1 = Torlakovic | first1 = EE. | last2 = Francis | first2 = G. | last3 = Garratt | first3 = J. | last4 = Gilks | first4 = B. | last5 = Hyjek | first5 = E. | last6 = Ibrahim | first6 = M. | last7 = Miller | first7 = R. | last8 = Nielsen | first8 = S. | last9 = Petcu | first9 = EB. | title = Standardization of negative controls in diagnostic immunohistochemistry: recommendations from the international ad hoc expert panel. | journal = Appl Immunohistochem Mol Morphol | volume = 22 | issue = 4 | pages = 241-52 | month = Apr | year = 2014 | doi = 10.1097/PAI.0000000000000069 | PMID = 24714041 }}</ref> | |||
===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== | ==Quality control== | ||
This is an evolving area in pathology that has been ignored for a surprisingly long time. | 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 ''[[Quality#Immunohistochemistry|immunohistochemistry]]'' section. | It is touched upon the in the ''[[quality]]'' article in the ''[[Quality#Immunohistochemistry|immunohistochemistry]]'' section. | ||
There are at least 62 pre-analytical variables to be considered, that may affect staining results.<ref>{{Cite journal | last1 = Engel | first1 = KB. | last2 = Moore | first2 = HM. | title = Effects of preanalytical variables on the detection of proteins by immunohistochemistry in formalin-fixed, paraffin-embedded tissue. | journal = Arch Pathol Lab Med | volume = 135 | issue = 5 | pages = 537-43 | month = May | year = 2011 | doi = 10.1043/2010-0702-RAIR.1 | PMID = 21526952 }}</ref> | |||
==Interpretation== | ==Interpretation== | ||
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#*A combination of the above. | #*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."<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 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."<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 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!" | 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. | 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.<ref name=pmid21660231>{{Cite journal | last1 = Galloway | first1 = M. | title = Base-rate error in the interpretation of immunohistochemistry. | journal = Patholog Res Int | volume = 2011 | issue = | pages = 636495 | month = | year = 2011 | doi = 10.4061/2011/636495 | PMID = 21660231 }}</ref> | |||
==General (malignant) differential diagnosis== | ==General (malignant) differential diagnosis== | ||
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*Carcinoma. | *Carcinoma. | ||
**AE1/AE3 - pankeratin. | **[[AE1/AE3]] - pankeratin. | ||
**Others: [[EMA]], HMWK, LMWK. | **Others: [[EMA]], HMWK, LMWK. | ||
*Sarcoma. | *Sarcoma. | ||
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==Keratins== | ==Keratins== | ||
{{Main|Keratins}} | |||
Mark epithelial cells. Are typically seen in [[carcinoma]]s. | |||
==Organ specific== | ==Organ specific== | ||
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===Breast markers=== | ===Breast markers=== | ||
*[[GCDFP-15]] ([[AKA]] BRST-2) -- specific, but NOT sensitive. | *[[GCDFP-15]] ([[AKA]] BRST-2) -- specific, but NOT sensitive. | ||
*ER (estrogen receptor) - in normal [[breast]]. | *[[Estrogen receptor|ER]] (estrogen receptor) - in normal [[breast]]. | ||
*PR (progesterone receptor) - in normal breast. | *PR (progesterone receptor) - in normal breast. | ||
*HER2/neu - pathological, assoc. with worse prognosis. | *HER2/neu - pathological, assoc. with worse prognosis. | ||
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===[[Small bowel]]=== | ===[[Small bowel]]=== | ||
*[[CDX2]]. | *[[CDX2]]. | ||
*Villin. | *[[Villin]]. | ||
===[[Kidney tumours|Kidney]]=== | ===[[Kidney tumours|Kidney]]=== | ||
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===[[Ovarian tumours|Ovary]]=== | ===[[Ovarian tumours|Ovary]]=== | ||
*CA125, CK7+, CK20-. | *[[CA125]], [[CK7]]+, CK20-. | ||
*WT1 -- 90% in serous +ve. | *WT1 -- 90% in serous +ve. | ||
====Serous markers==== | ====Serous markers==== | ||
*[[WT-1]], | *[[WT-1]], [[CA125]], D2-40. | ||
===[[Liver neoplasms|Liver]]=== | ===[[Liver neoplasms|Liver]]=== | ||
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*Glypican-3. | *Glypican-3. | ||
**[[Hepatocellular carcinoma]] (HCC) stains with glypican 3, while [[liver]] with dysplastic changes and/or [[cirrhosis|cirrhotic changes]] does not.<ref>{{cite journal | author=Anatelli F, Chuang ST, Yang XJ, Wang HL. |title=Value of glypican 3 immunostaining in the diagnosis of hepatocellular carcinoma on needle biopsy |journal=Am J Clin Pathol. |volume=130 |issue= 2 |pages= 219-23? |year= 2008 |pmid= 18628090 |doi= 10.1309/WMB5PX57Y4P8QCTY }}</ref> | **[[Hepatocellular carcinoma]] (HCC) stains with glypican 3, while [[liver]] with dysplastic changes and/or [[cirrhosis|cirrhotic changes]] does not.<ref>{{cite journal | author=Anatelli F, Chuang ST, Yang XJ, Wang HL. |title=Value of glypican 3 immunostaining in the diagnosis of hepatocellular carcinoma on needle biopsy |journal=Am J Clin Pathol. |volume=130 |issue= 2 |pages= 219-23? |year= 2008 |pmid= 18628090 |doi= 10.1309/WMB5PX57Y4P8QCTY }}</ref> | ||
*HepPar-1 (hepatocytes paraffin antibody 1) - labels hepatocellular mitochondria.<ref name=pmid12502967>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.</ref> | *[[HepPar-1]] (hepatocytes paraffin antibody 1) - labels hepatocellular mitochondria.<ref name=pmid12502967>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.</ref> | ||
[[HCC]] vs. [[cholangiocarcinoma]]: | [[HCC]] vs. [[cholangiocarcinoma]]: | ||
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**CEA (monoclonal and polyclonal). | **CEA (monoclonal and polyclonal). | ||
**[[TTF-1]]. | **[[TTF-1]]. | ||
**Ber-EP4. | **[[Ber-EP4]]. | ||
**MOC-31. | **MOC-31. | ||
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*S-100 - neural differentiation, melanoma. | *S-100 - neural differentiation, melanoma. | ||
*Desmin - smooth muscle. | *Desmin - smooth muscle. | ||
*MIB1 - proliferation marker (target is Ki-67 protein). | *[[MIB1]] - proliferation marker (target is [[Ki-67]] protein). | ||
*CD99 - blue small cell tumours, membranous staining [[EWS]]. | *CD99 - blue small cell tumours, membranous staining [[EWS]]. | ||
*BCL2 - [[synovial sarcoma]], [[small cell lymphomas]], spindle cell lipoma.<ref name=Ref_DCHH107>{{Ref DCHH|107}}</ref><ref>URL: [http://ajp.amjpathol.org/cgi/content/full/160/3/759 http://ajp.amjpathol.org/cgi/content/full/160/3/759]. Accessed on: 3 August 2010.</ref> | *BCL2 - [[synovial sarcoma]], [[small cell lymphomas]], spindle cell lipoma.<ref name=Ref_DCHH107>{{Ref DCHH|107}}</ref><ref>URL: [http://ajp.amjpathol.org/cgi/content/full/160/3/759 http://ajp.amjpathol.org/cgi/content/full/160/3/759]. Accessed on: 3 August 2010.</ref> | ||
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==Vimentin and cytokeratin positivity== | ==Vimentin and cytokeratin positivity== | ||
{{Main|Vimentin#Vimentin_and_cytokeratin_positivity}} | |||
A few tumours are positive for both vimentin and cytokeratins. | A few tumours are positive for both vimentin and cytokeratins. | ||
==Sarcomas and cytokeratins== | |||
{{Main|Keratins}} | |||
Most sarcomas are cytokeratin negative. | Most sarcomas are cytokeratin negative. | ||