Cytogenetics

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This article deals with cytogenetics.

General

  • Large changes (chromosomal).
    • Maximum resolution 3-4 megabase pairs (3-4 million base pairs); may be less - dependent on band density.[1]
  • Morphologic data.

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).
          • Q-banding is the standard at UHN.

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]).

Image:

Notes:

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

Virtual karyotyping

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

In situ hybridization

  • Typically abbreviated ISH.

Overview

  • Usually done on interphase nuclei.

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 - two colour (two different probes with different colours).
  • Breakapart - two colour.
  • Deletion/duplication (e.g. trisomy) - one colour.

Image:

Tests

ERBB2

Protocol:

  • ISH label for HER2.
  • ISH label for CEP17 (D17Z1 -- labels centromere of chromosome 17).[10]
  • In 60 nuclei:
    • Count number of HER2 signals.
    • Count number of CEP17 signals.
  • Calculate ratio HER2/D17Z1 signals:[11]
    • >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. Online 'Mendelian Inheritance in Man' (OMIM) 164870
  10. URL: http://www.medscape.com/viewarticle/546925_3. Accessed on: 10 May 2011.
  11. 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