Difference between revisions of "Cytogenetics"

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This article deals with '''cytogenetics'''.
This article deals with '''cytogenetics'''.  An introduction to molecular pathology is found in the ''[[molecular pathology]]'' article.


==General==
=General=
*Large changes (chromosomal).
*Detects "large" changes (in chromosomes).
**Maximum resolution 3-4 megabase pairs (3-4 million base pairs); may be less - dependent on band density.<ref>{{Ref WMSP|695}}</ref>
**Conventional karyotyping has a maximum resolution 3-4 megabase pairs (3-4 million base pairs); may be less - dependent on band density.<ref name=Ref_WMSP695>{{Ref WMSP|695}}</ref>
*Morphologic data.
*Cytogenetics is morphologic (data); thus, the interpretation has a greater degree of subjectivity vis-à-vis genetic sequencing.


==Techniques==
=Techniques (overview)=
*ISH = in situ hybridization.
*ISH = in situ hybridization.
**FISH = fluorescent in situ hybridization.  
**FISH = fluorescent in situ hybridization.  
Line 12: Line 12:
*Karyotyping.
*Karyotyping.


===Karyotyping===
==Karyotyping==
What?
What?
*Metaphase nuclei.
*Metaphase nuclei.
**The number of metapase nuclei (in samples) is enhanced by added a microtubule inhibitor (colcemid), that prevents progression to anaphase.
**Cells are bathed in a hypotonic solution to make 'em larger and spread out the nuclear material.


How?
How?
Line 23: Line 25:
***Several different techniques (stains) are used:<ref>URL: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC355922/ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC355922/]. Accessed on: 10 May 2011.</ref>
***Several different techniques (stains) are used:<ref>URL: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC355922/ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC355922/]. Accessed on: 10 May 2011.</ref>
****Examples: Q-banding (Q=Quinacrine), C-banding (C=constitutive heterchromatin), G-banding (G=Giemsa), R-banding (R=reverse).
****Examples: Q-banding (Q=Quinacrine), C-banding (C=constitutive heterchromatin), G-banding (G=Giemsa), R-banding (R=reverse).
*****Q-banding is the standard at UHN.


Analysis:
Analysis:
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***The detail of an ideogram is given in "band levels"; a 850 band ideogram has a higher resolution than a 400 band ideogram.  The typical range for band level is 300-850.  The band level, for a given specimen, is determined by an empirical standard and based on the number of bands an observer sees in a subset of the chromosomes.<ref name=pmid8291552>{{Cite journal  | last1 = Welborn | first1 = JL. | last2 = Welborn | first2 = R. | title = Banding resolution of human chromosomes: a method of accuracy and simplicity. | journal = Am J Med Genet | volume = 47 | issue = 8 | pages = 1180-3 | month = Dec | year = 1993 | doi = 10.1002/ajmg.1320470810 | PMID = 8291552 }}
***The detail of an ideogram is given in "band levels"; a 850 band ideogram has a higher resolution than a 400 band ideogram.  The typical range for band level is 300-850.  The band level, for a given specimen, is determined by an empirical standard and based on the number of bands an observer sees in a subset of the chromosomes.<ref name=pmid8291552>{{Cite journal  | last1 = Welborn | first1 = JL. | last2 = Welborn | first2 = R. | title = Banding resolution of human chromosomes: a method of accuracy and simplicity. | journal = Am J Med Genet | volume = 47 | issue = 8 | pages = 1180-3 | month = Dec | year = 1993 | doi = 10.1002/ajmg.1320470810 | PMID = 8291552 }}
</ref>
</ref>
 
*Typically done with karyotyping software (e.g. ''Ikaros'' from ''MetaSystems''<ref>URL: [http://www.metasystems-international.com/index.php?option=com_content&view=article&id=46:ikaros-general&catid=34:ikarosisis&Itemid=119 http://www.metasystems-international.com/index.php?option=com_content&view=article&id=46:ikaros-general&catid=34:ikarosisis&Itemid=119]. Accessed on: 12 May 2011.</ref>).
Image:
*[http://commons.wikimedia.org/wiki/File:NHGRI_human_male_karyotype.png Karyotype (WC)].


Notes:
Notes:
*Quinacrine dye - AT-rich regions brighter than GC-rich regions.
*Quinacrine dye - AT-rich regions brighter than GC-rich regions.


===ISH===
====Image====
*Usu. interphase nuclei.
<gallery>
Image:NHGRI_human_male_karyotype.png | Karyotype (WC)
</gallery>
===Virtual karyotyping===
*An evolving technique based on SNP microarrays<ref name=pmid20736741>{{Cite journal  | last1 = Alvarez | first1 = K. | last2 = Kash | first2 = SF. | last3 = Lyons-Weiler | first3 = MA. | last4 = Kim | first4 = HJ. | last5 = Peterson | first5 = LE. | last6 = Mathai | first6 = B. | last7 = Hagenkord | first7 = JM. | last8 = Monzon | first8 = FA. | title = Reproducibility and performance of virtual karyotyping with SNP microarrays for the detection of chromosomal imbalances in formalin-fixed paraffin-embedded tissues. | journal = Diagn Mol Pathol | volume = 19 | issue = 3 | pages = 127-34 | month = Sep | year = 2010 | doi = 10.1097/PDM.0b013e3181d527c5 | PMID = 20736741 }}
</ref> or comparative genomic hybridization (CGH).
 
==In situ hybridization==
*Typically abbreviated ''ISH''.
 
===Overview===
*ISH is a relatively high resolution cytogenetic technique (vis-à-vis karyotyping) used to identify specific DNA sequences and, with multiple probes, determine their relative location.
**Usually done on interphase nuclei.
**DNA probe size ~20-1000 base pairs.
 
Comes in different flavours:
*FISH = fluorescent in situ hybridization.
*SISH = silver in situ hybridization.<ref>URL: [http://www.immunoportal.com/modules.php?name=News&file=article&sid=186 http://www.immunoportal.com/modules.php?name=News&file=article&sid=186]. Accessed on: 2 May 2011.</ref>


May be prepared from:
May be prepared from:
Line 46: Line 62:
*Cultured cells.
*Cultured cells.


====Probe types====
===Probe types===
Types:
Types:
*Fusion (two colours).
*Fusion - usually two colours , e.g. [[translocations|IGH/BCL2 translocation]], BCR/ABL translocation.
*Breakapart (two colours).
**Two colour - two different probes with different colours.
*Deletion/duplication (e.g. trisomy).
**Two colour fusion with extra signal - similar two colour fusion.<ref name=pmid12764379>{{Cite journal  | last1 = Primo | first1 = D. | last2 = Tabernero | first2 = MD. | last3 = Rasillo | first3 = A. | last4 = Sayagués | first4 = JM. | last5 = Espinosa | first5 = AB. | last6 = Chillón | first6 = MC. | last7 = Garcia-Sanz | first7 = R. | last8 = Gutierrez | first8 = N. | last9 = Giralt | first9 = M. | title = Patterns of BCR/ABL gene rearrangements by interphase fluorescence in situ hybridization (FISH) in BCR/ABL+ leukemias: incidence and underlying genetic abnormalities. | journal = Leukemia | volume = 17 | issue = 6 | pages = 1124-9 | month = Jun | year = 2003 | doi = 10.1038/sj.leu.2402963 | PMID = 12764379 | url = http://www.nature.com/leu/journal/v17/n6/full/2402963a.html }}</ref>
**Three-colour † - two probes (e.g. red and blue) straddle the break point.<ref>{{Cite journal  | last1 = Sinclair | first1 = PB. | last2 = Green | first2 = AR. | last3 = Grace | first3 = C. | last4 = Nacheva | first4 = EP. | title = Improved sensitivity of BCR-ABL detection: a triple-probe three-color fluorescence in situ hybridization system. | journal = Blood | volume = 90 | issue = 4 | pages = 1395-402 | month = Aug | year = 1997 | doi =  | PMID = 9269756 | url= http://bloodjournal.hematologylibrary.org/content/90/4/1395.full.html }}</ref>
***One of the (true) translocations (e.g. presence of a Philadelphia chromosome) will have only two colours (red and green or yellow).
***The derivative chromosome (e.g. derivative 9 chromosome in Philadelphia chromosome cells), which may be lost, will have all three colours.
***A (non-pathologic) coincidental colocalization (or overlap) of probes will consist of all three colours.
*Break apart - two colour, e.g. [[translocations|c-MYC rearrangement]].
*Deletion/duplication (e.g. trisomy) - one colour.
 
Notes:
* † This could be considered a break apart ''and'' fusion probe; it has both elements.
 
====Image====
<gallery>
Image:Bcrablmet.jpg| Fusion signal suggesting a BCR-ABL translocation. (WC/Pmx)
</gallery>
 
===Tests===
====ERBB2====
*[[AKA]] HER2,<ref>{{OMIM|164870}}</ref> AKA HER2/neu.
*Important in [[invasive breast cancer]].


Image:
Protocol:
*[http://commons.wikimedia.org/wiki/File:Bcrablmet.jpg Bcr-Abl translocation (WC)].
*ISH label for HER2.
*ISH label for CEP17 (D17Z1 -- labels centromere of chromosome 17).<ref>URL: [http://www.medscape.com/viewarticle/546925_3 http://www.medscape.com/viewarticle/546925_3]. Accessed on: 10 May 2011.</ref>
*In 60 nuclei:
**Count number of HER2 signals.
**Count number of CEP17 signals.
*Calculate ratio HER2/D17Z1 signals:<ref>ASCO/CAP 2007 guidelines. URL: [http://www.cap.org/apps/cap.portal?_nfpb=true&cntvwrPtlt_actionOverride=%2Fportlets%2FcontentViewer%2Fshow&_windowLabel=cntvwrPtlt&cntvwrPtlt%7BactionForm.contentReference%7D=committees%2Fimmunohistochemistry%2Fher2_index.html http://www.cap.org/apps/cap.portal?_nfpb=true&cntvwrPtlt_actionOverride=%2Fportlets%2FcontentViewer%2Fshow&_windowLabel=cntvwrPtlt&cntvwrPtlt%7BactionForm.contentReference%7D=committees%2Fimmunohistochemistry%2Fher2_index.html]. Accessed on: 10 May 2011.</ref>
**>2.2 --> HER2 amplification.
**1.8-2.2 --> equivocal.


==See also==
=See also=
*[[Molecular pathology]].
*[[Molecular pathology]].
*[[Chromosomal translocations]].
*[[Chromosomal translocations]].
*[[Cytogenetic tests]].


==References==
=References=
{{Reflist|2}}
{{Reflist|2}}


==External links==
=External links=
*[http://homepage.mac.com/wildlifeweb/cyto/human/ Cytogenetics (homepage.mac.com)] - has ideograms (banding maps) of the human chromosomes.
*[http://homepage.mac.com/wildlifeweb/cyto/human/ Cytogenetics (homepage.mac.com)] - has ideograms (banding maps) of the human chromosomes.
*[http://ccr.coriell.org/sections/support/global/iscn_help.aspx?PgId=263 ISCN symbols and abbreviated terms (coriell.org)] - cytogenetics nomenclature.
*[http://www.sickkids.ca/paediatriclabmedicinems/test-catalogue/cytogenetics-listing.html Cytogenetics catalogue (sickkids.ca)].


[[Category:Molecular pathology]]
[[Category:Molecular pathology]]

Latest revision as of 12:51, 11 May 2016

This article deals with cytogenetics. An introduction to molecular pathology is found in the molecular pathology article.

General

  • Detects "large" changes (in chromosomes).
    • Conventional karyotyping has a maximum resolution 3-4 megabase pairs (3-4 million base pairs); may be less - dependent on band density.[1]
  • Cytogenetics is morphologic (data); thus, the interpretation has a greater degree of subjectivity vis-à-vis genetic sequencing.

Techniques (overview)

  • ISH = in situ hybridization.
    • FISH = fluorescent in situ hybridization.
    • SISH = silver in situ hybridization.[2]
  • Karyotyping.

Karyotyping

What?

  • Metaphase nuclei.
    • The number of metapase nuclei (in samples) is enhanced by added a microtubule inhibitor (colcemid), that prevents progression to anaphase.
    • Cells are bathed in a hypotonic solution to make 'em larger and spread out the nuclear material.

How?

  • Chromosomes are identified by:
    • Size (chromosome 1 = largest, chromosome 22 = smallest).
    • Position of centromere.
    • Banding pattern - using (special) stains:
      • Several different techniques (stains) are used:[3]
        • Examples: Q-banding (Q=Quinacrine), C-banding (C=constitutive heterchromatin), G-banding (G=Giemsa), R-banding (R=reverse).

Analysis:

  • Based on comparison to maps of the chromosomes (ideograms).
    • Detailed ideograms are in ISCN 2009.[4]
      • The detail of an ideogram is given in "band levels"; a 850 band ideogram has a higher resolution than a 400 band ideogram. The typical range for band level is 300-850. The band level, for a given specimen, is determined by an empirical standard and based on the number of bands an observer sees in a subset of the chromosomes.[5]
  • Typically done with karyotyping software (e.g. Ikaros from MetaSystems[6]).

Notes:

  • Quinacrine dye - AT-rich regions brighter than GC-rich regions.

Image

  • Karyotype (WC)

Virtual karyotyping

  • An evolving technique based on SNP microarrays[7] or comparative genomic hybridization (CGH).

In situ hybridization

  • Typically abbreviated ISH.

Overview

  • ISH is a relatively high resolution cytogenetic technique (vis-à-vis karyotyping) used to identify specific DNA sequences and, with multiple probes, determine their relative location.
    • Usually done on interphase nuclei.
    • DNA probe size ~20-1000 base pairs.

Comes in different flavours:

  • FISH = fluorescent in situ hybridization.
  • SISH = silver in situ hybridization.[8]

May be prepared from:

  • In situ paraffin sections (section 4 micrometers thick).
  • Isolated nuclei (section 10 micrometers thick).
  • Cytospin.
  • Cultured cells.

Probe types

Types:

  • Fusion - usually two colours , e.g. IGH/BCL2 translocation, BCR/ABL translocation.
    • Two colour - two different probes with different colours.
    • Two colour fusion with extra signal - similar two colour fusion.[9]
    • Three-colour † - two probes (e.g. red and blue) straddle the break point.[10]
      • One of the (true) translocations (e.g. presence of a Philadelphia chromosome) will have only two colours (red and green or yellow).
      • The derivative chromosome (e.g. derivative 9 chromosome in Philadelphia chromosome cells), which may be lost, will have all three colours.
      • A (non-pathologic) coincidental colocalization (or overlap) of probes will consist of all three colours.
  • Break apart - two colour, e.g. c-MYC rearrangement.
  • Deletion/duplication (e.g. trisomy) - one colour.

Notes:

  • † This could be considered a break apart and fusion probe; it has both elements.

Image

  • Fusion signal suggesting a BCR-ABL translocation. (WC/Pmx)

Tests

ERBB2

Protocol:

  • ISH label for HER2.
  • ISH label for CEP17 (D17Z1 -- labels centromere of chromosome 17).[12]
  • In 60 nuclei:
    • Count number of HER2 signals.
    • Count number of CEP17 signals.
  • Calculate ratio HER2/D17Z1 signals:[13]
    • >2.2 --> HER2 amplification.
    • 1.8-2.2 --> equivocal.

See also

References

  1. ↑ Humphrey, Peter A; Dehner, Louis P; Pfeifer, John D (2008). The Washington Manual of Surgical Pathology (1st ed.). Lippincott Williams & Wilkins. pp. 695. ISBN 978-0781765275.
  2. ↑ URL: http://www.immunoportal.com/modules.php?name=News&file=article&sid=186. Accessed on: 2 May 2011.
  3. ↑ URL: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC355922/. Accessed on: 10 May 2011.
  4. ↑ Shaffer, Lisa G.; Slovak, Marilyn L.; Campbell, Lynda J. (2009). ISCN 2009: an international system for human cytogenetic nomenclature (2009 ed.). S. Karger AG. ISBN 978-3805589857.
  5. ↑ Welborn, JL.; Welborn, R. (Dec 1993). "Banding resolution of human chromosomes: a method of accuracy and simplicity.". Am J Med Genet 47 (8): 1180-3. doi:10.1002/ajmg.1320470810. PMID 8291552.
  6. ↑ URL: http://www.metasystems-international.com/index.php?option=com_content&view=article&id=46:ikaros-general&catid=34:ikarosisis&Itemid=119. Accessed on: 12 May 2011.
  7. ↑ Alvarez, K.; Kash, SF.; Lyons-Weiler, MA.; Kim, HJ.; Peterson, LE.; Mathai, B.; Hagenkord, JM.; Monzon, FA. (Sep 2010). "Reproducibility and performance of virtual karyotyping with SNP microarrays for the detection of chromosomal imbalances in formalin-fixed paraffin-embedded tissues.". Diagn Mol Pathol 19 (3): 127-34. doi:10.1097/PDM.0b013e3181d527c5. PMID 20736741.
  8. ↑ URL: http://www.immunoportal.com/modules.php?name=News&file=article&sid=186. Accessed on: 2 May 2011.
  9. ↑ Primo, D.; Tabernero, MD.; Rasillo, A.; Sayagués, JM.; Espinosa, AB.; Chillón, MC.; Garcia-Sanz, R.; Gutierrez, N. et al. (Jun 2003). "Patterns of BCR/ABL gene rearrangements by interphase fluorescence in situ hybridization (FISH) in BCR/ABL+ leukemias: incidence and underlying genetic abnormalities.". Leukemia 17 (6): 1124-9. doi:10.1038/sj.leu.2402963. PMID 12764379. http://www.nature.com/leu/journal/v17/n6/full/2402963a.html.
  10. ↑ Sinclair, PB.; Green, AR.; Grace, C.; Nacheva, EP. (Aug 1997). "Improved sensitivity of BCR-ABL detection: a triple-probe three-color fluorescence in situ hybridization system.". Blood 90 (4): 1395-402. PMID 9269756. http://bloodjournal.hematologylibrary.org/content/90/4/1395.full.html.
  11. ↑ Online 'Mendelian Inheritance in Man' (OMIM) 164870
  12. ↑ URL: http://www.medscape.com/viewarticle/546925_3. Accessed on: 10 May 2011.
  13. ↑ ASCO/CAP 2007 guidelines. URL: http://www.cap.org/apps/cap.portal?_nfpb=true&cntvwrPtlt_actionOverride=%2Fportlets%2FcontentViewer%2Fshow&_windowLabel=cntvwrPtlt&cntvwrPtlt%7BactionForm.contentReference%7D=committees%2Fimmunohistochemistry%2Fher2_index.html. Accessed on: 10 May 2011.

External links

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