Immunohistochemistry: Difference between revisions

(17 intermediate revisions by the same user not shown)

Line 1:

{{Top

<noinclude>{{Top

|author1=[[User:Mikael Häggström|Mikael Häggström]]

|author2=

}}

}}</noinclude>

When [[learning pathology]], the percentages by which immunohistochemistry results are positive or negative for various diseases are generally easily looked up when needed, so what a pathologist needs to learn is mainly '''how to select''' the optimal immunohistochemistry panels in the first place for various presentations where the diagnosis is unknown.

[[File:Main staining patterns on immunohistochemistry.jpg|thumb|270px|Main staining patterns.]]

[[File:Immunohistochemistry for p16 in uterine papillary serous adenocarcinoma showing both nuclear and cytoplasmic staining.jpg|thumb|160px|'''"Block" staining''': strong nuclear and cytoplasmic expression in a continuous segment of cells.<ref>Image by Mikael Häggström, MD. Reference for terminology: {{cite web|url=https://www.pathologyoutlines.com/topic/stainsp16.html|title=p16|website=Pathology Outlines|author=Anjelica Hodgson, M.D., Carlos Parra-Herran, M.D.}} Last staff update: 25 January 2024</ref>]]

When [[learning pathology]], the percentages by which immunohistochemistry (IHC) results are positive or negative for various diseases are generally easily looked up when needed, so what a pathologist needs to learn is mainly '''how to select''' the optimal immunohistochemistry panels in the first place for various presentations where the diagnosis is unknown.

==Immunohistochemistry ordering==

The main approaches to immunohistochemistry ordering are:

*Look for a specified '''panel''' for the presentation at hand. For example, for an undifferentiated tumor with no clear lineage differentiation, an initial panel of CK, S100, vimentin and LCA can be used.<ref name="pmid25427040">{{cite journal| author=Lin F, Liu H| title=Immunohistochemistry in undifferentiated neoplasm/tumor of uncertain origin. | journal=Arch Pathol Lab Med | year= 2014 | volume= 138 | issue= 12 | pages= 1583-610 | pmid=25427040 | doi=10.5858/arpa.2014-0061-RA | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=25427040 }} </ref>

*If there is '''insufficient tissue''' left for immunohistochemistry after standard (usually H&E) staining, you can potentially ask a histotechnologist to destain a glass slide and subsequently perform IHC on that slide. For small specimens, if you predict that there will be several stainsin the near future, order extra unstained slides upfront (since there is always a bit of a waste of tissue at the microtome if the paraffin block needs to be remounted and cut later).

*Look for a specified '''panel''' for the presentation at hand. For example, for an undifferentiated tumor with no clear lineage differentiation, an initial panel of cytokeratin (CK), S100, vimentin and LCA (CD45) can be used.<ref name="pmid25427040">{{cite journal| author=Lin F, Liu H| title=Immunohistochemistry in undifferentiated neoplasm/tumor of uncertain origin. | journal=Arch Pathol Lab Med | year= 2014 | volume= 138 | issue= 12 | pages= 1583-610 | pmid=25427040 | doi=10.5858/arpa.2014-0061-RA | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=25427040 }} </ref> {{further|Tumor evaluation}}<br>In cancer cases, also be familiar with local routines for reflex tests by various cancer types, which may include both immunohistochemistry and other molecular tests.

*Coming up with the most relevant '''differential diagnoses''' for the case at hand, and find the immunohistochemistry stains that best distinguish them. Immunohistochemistry profiles for diseases and conditions, as well as their main differential diagnoses, is generally found at '''[https://www.pathologyoutlines.com/ Pathology Outlines]''', or you can pay for a subscription to '''[https://www.immunoquery.com/ ImmunoQuery]'''{{ImmunoQuery COI declaration}}:

Line 92:

Line 95:

GRPR is the top result when specifically comparing lung versus anus squamous cell carcinoma, both at sensitivity settings 1 and 3, with no major difference between them, and thus anus origin is favored in case of either diffuse or focal cytoplasmic staining. DLK showed as N/A for anus SCC.

==~~Immunohistochemistry evaluation~~==

The main methods for ~~evaluating~~ immunohistochemistry results are:

===Chromogen color===

Request a red rather than brown chromogen in heavily pigmented lesions, such as in some [[melanoma]]s.

==Evaluation==

[[File:Positive and negative controls in immunohistochemistry.png|thumb|270px|Confirm that you see the expected results for positive and negative controls. For this purpose, "internal" means tissue from the target patient, and "external" means that the tissue is from another patient.<ref>Image by Mikael Häggström, MD. Reference: {{cite journal| author=Torlakovic EE, Francis G, Garratt J, Gilks B, Hyjek E, Ibrahim M | display-authors=etal| title=Standardization of negative controls in diagnostic immunohistochemistry: recommendations from the international ad hoc expert panel. | journal=Appl Immunohistochem Mol Morphol | year= 2014 | volume= 22 | issue= 4 | pages= 241-52 | pmid=24714041 | doi=10.1097/PAI.0000000000000069 | pmc=4206554 | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=24714041 }}</ref>]]

First be familiar with which cells on the slide are being evaluated, such as first looking at a slide with standard staining (usually H&E) to avoid counting background cells.

When the relevant cells take up stains, confirm whether their staining pattern actually counts as positive. For example, a cytoplasmic staining for p63 still counts as "negative (cytoplasmic)" when suspecting squamous, myoepithelial and prostatic basal cells (where only nuclear stain counts as positive). On the other hand, the same cytoplasmic staining for p63 counts as "positive (cytoplasmic)" when suspecting muscle differentiation.<ref name="pmid21623385">{{cite journal| author=Martin SE, Temm CJ, Goheen MP, Ulbright TM, Hattab EM| title=Cytoplasmic p63 immunohistochemistry is a useful marker for muscle differentiation: an immunohistochemical and immunoelectron microscopic study. | journal=Mod Pathol | year= 2011 | volume= 24 | issue= 10 | pages= 1320-6 | pmid=21623385 | doi=10.1038/modpathol.2011.89 | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=21623385 }} </ref>

==Interpretation==

The main methods for interpreting immunohistochemistry results are:

*Looking up each differential diagnosis at for example '''[https://www.pathologyoutlines.com/ Pathology Outlines]''' and comparing their expected staining to see which entity is most likely.

*Paying for a subscription to '''ImmunoQuery'''~~<ref group=notes name=ImmunoQueryCOI/>~~, where you can enter immunohistochemistry results and generate a list of most likely conditions with that profile.

*Paying for a subscription to '''ImmunoQuery''',{{ImmunoQuery COI declaration}} where you can enter immunohistochemistry results and generate a list of most likely conditions with that profile.

Preferably, immunohistochemistry results will be very specific or sensitive for a suspected condition, thereby confirming it if positive, or excluding it if negative, respectively. Even when that is not the case, immunohistochemistry can at least '''alter the likelihoods''' of different differential diagnoses. In practice, clinicians or pathologists do not state exact or even approximate numbers of likelihoods of differential diagnoses ({{further|Reporting}}<includeonly>see the [[Reporting]] chapter for phrasing uncertainty</includeonly>), since reality is too complex for that, but to demonstrate the general principle of how immunohistochemistry results can be calculated, the following formula can be used:

Line 113:

Line 126:

In the same way, the corresponding gross likelihood of pleomorphic rhabdomyosarcoma is calculated as:

*70% x 95% x (100% - 71%) = 19%

As a result in this case, immunohistochemistry resulted in pleomorphic rhabdomyosarcoma going from about twice as likely compared to pleomorphic liposarcoma to about 4 times as likely. Although results like these do not make major differences individually, detailed calculations of results from a large panel of immunohistochemistry stains can have a major impact when taken together, even if each stain has relatively low sensitivity and specificity.<ref group=~~notes~~>More detailed explanations about likelihood calculations on differential diagnoses in general can be read at:<br>-{{cite journal | last=Häggström | first=Mikael | title=An epidemiology-based and a likelihood ratio-based method of differential diagnosis | journal=WikiJournal of Medicine | publisher=Wikiversity Journal of Medicine | volume=1 | issue=1 | year=2014 | issn=2002-4436 | doi=10.15347/wjm/2014.002}}</ref>

As a result in this case, immunohistochemistry resulted in pleomorphic rhabdomyosarcoma going from about twice as likely compared to pleomorphic liposarcoma to about 4 times as likely. Although results like these do not make major differences individually, detailed calculations of results from a large panel of immunohistochemistry stains can have a major impact when taken together, even if each stain has relatively low sensitivity and specificity.<ref group=note>More detailed explanations about likelihood calculations on differential diagnoses in general can be read at:<br>-{{cite journal | last=Häggström | first=Mikael | title=An epidemiology-based and a likelihood ratio-based method of differential diagnosis | journal=WikiJournal of Medicine | publisher=Wikiversity Journal of Medicine | volume=1 | issue=1 | year=2014 | issn=2002-4436 | doi=10.15347/wjm/2014.002}}</ref>

</noinclude>

@@ Line 1: / Line 1: @@
-{{Top
+<noinclude>{{Top
 |author1=[[User:Mikael Häggström|Mikael Häggström]]
 |author2=
-}}
+}}</noinclude>
-When [[learning pathology]], the percentages by which immunohistochemistry results are positive or negative for various diseases are generally easily looked up when needed, so what a pathologist needs to learn is mainly '''how to select''' the optimal immunohistochemistry panels in the first place for various presentations where the diagnosis is unknown.
+[[File:Main staining patterns on immunohistochemistry.jpg|thumb|270px|Main staining patterns.]]
+[[File:Immunohistochemistry for p16 in uterine papillary serous adenocarcinoma showing both nuclear and cytoplasmic staining.jpg|thumb|160px|'''"Block" staining''': strong nuclear and cytoplasmic expression in a continuous segment of cells.<ref>Image by Mikael Häggström, MD. Reference for terminology: {{cite web|url=https://www.pathologyoutlines.com/topic/stainsp16.html|title=p16|website=Pathology Outlines|author=Anjelica Hodgson, M.D., Carlos Parra-Herran, M.D.}} Last staff update: 25 January 2024</ref>]]
+When [[learning pathology]], the percentages by which immunohistochemistry (IHC) results are positive or negative for various diseases are generally easily looked up when needed, so what a pathologist needs to learn is mainly '''how to select''' the optimal immunohistochemistry panels in the first place for various presentations where the diagnosis is unknown.
 ==Immunohistochemistry ordering==
 The main approaches to immunohistochemistry ordering are:
-*Look for a specified '''panel''' for the presentation at hand. For example, for an undifferentiated tumor with no clear lineage differentiation, an initial panel of CK, S100, vimentin and LCA can be used.<ref name="pmid25427040">{{cite journal| author=Lin F, Liu H| title=Immunohistochemistry in undifferentiated neoplasm/tumor of uncertain origin. | journal=Arch Pathol Lab Med | year= 2014 | volume= 138 | issue= 12 | pages= 1583-610 | pmid=25427040 | doi=10.5858/arpa.2014-0061-RA | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=25427040  }} </ref>
+*If there is '''insufficient tissue''' left for immunohistochemistry after standard (usually H&E) staining, you can potentially ask a histotechnologist to destain a glass slide and subsequently perform IHC on that slide. For small specimens, if you predict that there will be several stainsin the near future, order extra unstained slides upfront (since there is always a bit of a waste of tissue at the microtome if the paraffin block needs to be remounted and cut later).
+*Look for a specified '''panel''' for the presentation at hand. For example, for an undifferentiated tumor with no clear lineage differentiation, an initial panel of cytokeratin (CK), S100, vimentin and LCA (CD45) can be used.<ref name="pmid25427040">{{cite journal| author=Lin F, Liu H| title=Immunohistochemistry in undifferentiated neoplasm/tumor of uncertain origin. | journal=Arch Pathol Lab Med | year= 2014 | volume= 138 | issue= 12 | pages= 1583-610 | pmid=25427040 | doi=10.5858/arpa.2014-0061-RA | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=25427040  }} </ref> {{further|Tumor evaluation}}<br>In cancer cases, also be familiar with local routines for reflex tests by various cancer types, which may include both immunohistochemistry and other molecular tests.
 *Coming up with the most relevant '''differential diagnoses''' for the case at hand, and find the immunohistochemistry stains that best distinguish them. Immunohistochemistry profiles for diseases and conditions, as well as their main differential diagnoses, is generally found at '''[https://www.pathologyoutlines.com/ Pathology Outlines]''', or you can pay for a subscription to '''[https://www.immunoquery.com/ ImmunoQuery]'''{{ImmunoQuery COI declaration}}:
@@ Line 92: / Line 95: @@
 GRPR is the top result when specifically comparing lung versus anus squamous cell carcinoma, both at sensitivity settings 1 and 3, with no major difference between them, and thus anus origin is favored in case of either diffuse or focal cytoplasmic staining. DLK showed as N/A for anus SCC.
 {{Question-end}}
-==Immunohistochemistry evaluation==
-The main methods for evaluating immunohistochemistry results are:
+===Chromogen color===
+Request a red rather than brown chromogen in heavily pigmented lesions, such as in some [[melanoma]]s.
+==Evaluation==
+[[File:Positive and negative controls in immunohistochemistry.png|thumb|270px|Confirm that you see the expected results for positive and negative controls. For this purpose, "internal" means tissue from the target patient, and "external" means that the tissue is from another patient.<ref>Image by Mikael Häggström, MD. Reference: {{cite journal| author=Torlakovic EE, Francis G, Garratt J, Gilks B, Hyjek E, Ibrahim M | display-authors=etal| title=Standardization of negative controls in diagnostic immunohistochemistry: recommendations from the international ad hoc expert panel. | journal=Appl Immunohistochem Mol Morphol | year= 2014 | volume= 22 | issue= 4 | pages= 241-52 | pmid=24714041 | doi=10.1097/PAI.0000000000000069 | pmc=4206554 | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=24714041  }}</ref>]]
+First be familiar with which cells on the slide are being evaluated, such as first looking at a slide with standard staining (usually H&E) to avoid counting background cells.
+When the relevant cells take up stains, confirm whether their staining pattern actually counts as positive. For example, a cytoplasmic staining for p63 still counts as "negative (cytoplasmic)" when suspecting squamous, myoepithelial and prostatic basal cells (where only nuclear stain counts as positive). On the other hand, the same cytoplasmic staining for p63 counts as "positive (cytoplasmic)" when suspecting muscle differentiation.<ref name="pmid21623385">{{cite journal| author=Martin SE, Temm CJ, Goheen MP, Ulbright TM, Hattab EM| title=Cytoplasmic p63 immunohistochemistry is a useful marker for muscle differentiation: an immunohistochemical and immunoelectron microscopic study. | journal=Mod Pathol | year= 2011 | volume= 24 | issue= 10 | pages= 1320-6 | pmid=21623385 | doi=10.1038/modpathol.2011.89 | pmc= | url=https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=21623385  }} </ref>
+==Interpretation==
+The main methods for interpreting immunohistochemistry results are:
 *Looking up each differential diagnosis at for example '''[https://www.pathologyoutlines.com/ Pathology Outlines]''' and comparing their expected staining to see which entity is most likely.
-*Paying for a subscription to '''ImmunoQuery'''<ref group=notes name=ImmunoQueryCOI/>, where you can enter immunohistochemistry results and generate a list of most likely conditions with that profile.
+*Paying for a subscription to '''ImmunoQuery''',{{ImmunoQuery COI declaration}} where you can enter immunohistochemistry results and generate a list of most likely conditions with that profile.
 Preferably, immunohistochemistry results will be very specific or sensitive for a suspected condition, thereby confirming it if positive, or excluding it if negative, respectively. Even when that is not the case, immunohistochemistry can at least '''alter the likelihoods''' of different differential diagnoses. In practice, clinicians or pathologists do not state exact or even approximate numbers of likelihoods of differential diagnoses ({{further|Reporting}}<includeonly>see the [[Reporting]] chapter for phrasing uncertainty</includeonly>), since reality is too complex for that, but to demonstrate the general principle of how immunohistochemistry results can be calculated, the following formula can be used:
@@ Line 113: / Line 126: @@
 In the same way, the corresponding gross likelihood of pleomorphic rhabdomyosarcoma is calculated as:
 *70% x 95% x (100% - 71%) = 19%
-As a result in this case, immunohistochemistry resulted in pleomorphic rhabdomyosarcoma going from about twice as likely compared to pleomorphic liposarcoma to about 4 times as likely. Although results like these do not make major differences individually, detailed calculations of results from a large panel of immunohistochemistry stains can have a major impact when taken together, even if each stain has relatively low sensitivity and specificity.<ref group=notes>More detailed explanations about likelihood calculations on differential diagnoses in general can be read at:<br>-{{cite journal | last=Häggström | first=Mikael | title=An epidemiology-based and a likelihood ratio-based method of differential diagnosis | journal=WikiJournal of Medicine | publisher=Wikiversity Journal of Medicine | volume=1 | issue=1 | year=2014 | issn=2002-4436 | doi=10.15347/wjm/2014.002}}</ref>
+As a result in this case, immunohistochemistry resulted in pleomorphic rhabdomyosarcoma going from about twice as likely compared to pleomorphic liposarcoma to about 4 times as likely. Although results like these do not make major differences individually, detailed calculations of results from a large panel of immunohistochemistry stains can have a major impact when taken together, even if each stain has relatively low sensitivity and specificity.<ref group=note>More detailed explanations about likelihood calculations on differential diagnoses in general can be read at:<br>-{{cite journal | last=Häggström | first=Mikael | title=An epidemiology-based and a likelihood ratio-based method of differential diagnosis | journal=WikiJournal of Medicine | publisher=Wikiversity Journal of Medicine | volume=1 | issue=1 | year=2014 | issn=2002-4436 | doi=10.15347/wjm/2014.002}}</ref>
 <noinclude>
 {{General notes}}
 {{Bottom}}
 </noinclude>

Immunohistochemistry: Difference between revisions

Navigation menu

Search