Molecular Cytogenetics

Fluorescent in-situ hybridisation (FISH):

Molecular cytogenetics (also known as Fluorescence In Situ Hybridisation) represents a means of increasing the speed, sensitivity and specificity of conventional cytogenetics.

These tests can usually be carried out on the sample supplied for routine cytogenetics. When in doubt, consult the laboratory, as there are many options. Some applications are described below, but please contact the department for specific information, as FISH is a rapidly evolving area.

The technique takes advantage of an intrinsic property of DNA. When similar sequences are rendered single-stranded, they will anneal, or “hybridise” together. By labelling probe sequences of interest with suitable fluorochromes, we can visualise specific sequences on a slide of patient material, using image analysis software.

Microdeletion and microduplication probes:

Used in special cases where the referring clinician suspects a specific syndrome. These are often detectable by conventional cytogenetic methods. Syndromes for which a FISH test is available include: Williams, Prader-Willi, Angelman, Miller-Dieker, Smith-Magenis and Di-George.


SNRPN microdeletion or duplication.


Array and partial karyotype showing duplication of 15q.

Subtelomere studies:

These can be used to FISH samples of blood, amniotic fluid or chorionic villi to detect unbalanced karyotypes resulting from reciprocal translocations.

BACFISH follow-up of Array CGH:

Array CGH findings are confirmed by follow-up FISH on blood samples (lithium-heparin) from the patient and both parents. FISH is done using the specific BAC FISH probes involved in imbalances detected on the patient’s array. The follow-up is used to determine whether the array finding is a coincidental variant or a pathogenic change.



 Duplication detected on array, confirmed by BACFISH interphase left and metaphase right.

Please note: In the case of small imbalances follow-up is done by array CGH on blood samples (EDTA) from both parents, restricted to the chromosome region of interest.


Detection of gene rearrangements in cancer:

Oncogenic “fusion genes” may be created by rearrangement of the genetic material. This is a recognised cause of many cancers and can be highly specific. By designing two-colour FISH probes to both gene partners in a fusion (usually a cancer gene and a promoter gene) the novel sequences may be identified by the close juxtaposition of signals. The haematological malignancies have been the most extensively studied to date.

 Gene fusions currently detectable by FISH include:

  • BCR/ABL in chronic myeloid leukaemia
  • PML/RARA in acute promyelocytic leukaemia
  • TEL/AML in acute lymphoblastic leukaemia
  • t(14;18) in follicular lymphoma



BCR/ABL DF abormal pattern.



MLL BA amplification.


Touch preparations from tumours:

Tumours can be cultured for karyotyping but touch preparations (tumour imprints) can be done from fresh material and directly FISHed for the identification of specific tumour types such as Ewings, rhabdomyosarcoma or synovial sarcoma. Please indicate on the referral form which tumour tests are required.


Tumour touch prep of synovial sarcomocells.


Paraffin Section (PET) FISH

Please provide 4 (m sections on positively charged slides baked overnight at 56(C. It is vital that tissues are not over-fixed in formalin (24-48 hours).


FISH scoring is initially carried out on paraffin sections of NHL (usually DLBCL) using the following break apart (BA) probe panel:





Additional tests can be requested for specific probes such as CCND1 for mantle cell etc. Confirmation can be done using Dual fusion probes. Please contact the lab for information.

Please note: If fresh material is available then this will be FISHed in preference to PET sections as fresh material requires less preparation and is easier to score.

It is very helpful if abnormal regions can be clearly identified either on an accompanying H&E slide or on a diagram on the referral form. These marked areas can then be targeted for screening by FISH.

Please provide a minimum of 6 sections.

IGH BCL2 fusion positive Follicular lymphoma.


Paraffin section FISH can also be done on breast tissues for the detection of Her2 copy number gain compared with a control probe (alpha satellite 17). Ratio=HER2 copy number:Alpha-satellite copy number.

Please note: If ductal carcinoma in-situ (DCIS) is known to be present in a breast tumour it is very important this information is indicated clearly on the referral form, so that the areas of DCIS are not included in the scoring, as this will skew the HER2 ratio. An accompanying H&E slide or diagram marked with the areas of interest is very helpful.

Please provide a minimum of three sections.





Array Comparative Genomic Hybridisation: (enquiries x36735)

Oligonucleotide array comparative genomic hybridisation (aCGH), also known as microarray or CHIP analysis, is the front-line test for constitutional cases where the diagnosis indicates a potential chromosomal imbalance. Referrals may include generalized developmental delay; dysmorphism (especially microcephaly); more severe mental deficit or congenital anomaly.

Basis of aCGH test:

Array comparative genomic hybridisation is performed using a BlueGnome Oligo array. Test DNA is referenced against same sex control DNA and data analysed with BlueFuse Multi software.

The array conforms to the International Standards for Cytogenomic Arrays (ISCA) 8x60k design. The array has 60kb overall median probe spacing, and higher coverage across known disease related regions. A backbone screening resolution of 200kb, encompassing a minimum of three consecutive probes, is applied. Please note aCGH will not detect balanced rearrangements and is limited in detection of mosaicism, and for these reasons there may be cases where a different strategy is more appropriate. Please contact the department if you wish to discuss these tests.



A CGH profile, showing a terminal deletion of 6p.