Cytogenetics

From Libre Pathology
(Redirected from SISH)
Jump to navigation Jump to search

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

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

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