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


  • 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.



  • 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.


  • 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).


  • 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]).


  • 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.


  • 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


  • 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.


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





  • 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


  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: Accessed on: 2 May 2011.
  3. URL: 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: 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: 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.
  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.
  11. Online 'Mendelian Inheritance in Man' (OMIM) 164870
  12. URL: Accessed on: 10 May 2011.
  13. ASCO/CAP 2007 guidelines. URL: Accessed on: 10 May 2011.

External links