ChromosOmics - Database

Icon by Leon Liehr              

   - mFISH - whole chromosome paints (wcps) -    
mFISH is also used in detection and necessary for confirmation of chromothripsis -  e.g. :
  • Imaizumi T, Yamamoto-Shimojima K, Yanagishita T, Ondo Y, Yamamoto T (2020) Analyses of breakpoint junctions of complex genomic rearrangements comprising multiple consecutive microdeletions by nanopore sequencing. J Hum Genet 65: 735-741.
  • Rosswog C, Bartenhagen C, Welte A, Kahlert Y, Hemstedt N, Lorenz W, Cartolano M, Ackermann S, Perner S, Vogel W, Altmüller J, Nürnberg P, Hertwig F, Göhring G, Lilienweiss E, Stütz AM, Korbel JO, Thomas RK, Peifer M, Fischer M (2021) Chromothripsis followed by circular recombination drives oncogene amplification in human cancer. Nat Genet 53: 1673-1685.


Some facts


Origin of probes: Whole chromosome painting probes can be derived from flow sorted chromosomes [Speicher et al., 1996; for flow sorting see e.g. Gray et al. 1990 or Carter, 1994] or by chromosome microdissection [Senger et al., 1998; Thalhammer et al., 2004].

COT1-blocking: Normally repetitive sequences have to be blocked in the probe DNA by COT1-DNA. However, also an approach was published [Bolzer et al., 1999] describing the generation of probes depleted of repetitive sequences. By that it is possible to refrain from expensive blocking agent. Dugan et al. 2005 shows another way to use less COT1 DNA.

Number of fluorochromes: Five [Speicher et al., 1996, Schröck et al., 1996, Senger et al., 1998, Padilla-Nash et al., 2006], six [Tanke et al., 1998] or seven [Azofeifa et al., 2000, Saracoglu et al., 2001] different fluorescence dyes are used to achieve the required 24 specific color combinations. The principle of combinatorial labeling as described by others before [Nederlof et al., 1990; Dauwerse et al., 1992, Ried et al., 1992; Wiegant et al., 1992] is used.

Image analysis: As in any case at least one of the used fluorochromes has its emission maximum in the near-infrared spectrum, i.e. is invisible for the human eye, a CCD-camera based image acquisition and a computer based image analysis are required. Automatization is only limited possible [Lee et al., 2001].
Image acquisition can be done based on two different principles: (i) split spectra are acquired via a set of specific filter sets as suggested by Speicher and coworkers (Multiplex-FISH = M-FISH [Speicher et al., 1996]) or (ii) complete emission spectra are acquired by an interferometer-based spectral imaging system as recommended by Schröck and coworkers (spectral karyotyping = SKY [Schröck et al., 1996]). A comparison between both approaches has been done e.g. by Rens et al. (2001). For the classification accuracy of M-FISH see as well Castleman and coworkers (2000) and Rooms et al. (2005), Wang and Castleman (2005) and Wang (2008).

New approaches for image analysing: Blind Spectral Unmixing of M-FISH Images by Non-negative Matrix Factorization [Munoz-Barrutia et al., 2007]; watershed based segmentation method for multispectral chromosome images classification [Karvelis et al., 2006, 2008 and 2009], Maximum-likelihood techniques for joint segmentation-classification [Schwartzkopf et al., 2005]; Wavelet-based compression of M-FISH images [Hua et al., 2005]; Feature normalization via expectation maximization and unsupervised nonparametric classification [Choi et al., 2008]; Color compensation [Choi et al., 2009]; Modeling of clonal expansion from M-FISH experiments [Stolte et al., 2008]; Supervised parametric and non-parametric classification [Sampat et al., 2005]

Combinatorial labeling is most frequently applied for mFISH.

Ratio labeling: The required 24 color combinations can also be achieved using the principle of the combinatorial labeling plus the principle of ratio-labeling [Dauwerse et al. 1992; Nederlof et al., 1992; Morrison et al., 1997] which has been suggested by Tanke and coworkers (COBRA-FISH: COmbined Binary RAtio labelling-FISH [Tanke et al., 1999]). In that approach only 4 fluorochromes are necessary to come to 24 color-combinations or pseudocolors and more than 96 could be easily obtained, according to the authors [Tanke et al., 1999; Szuhai et al., 2000].

Color-changing karyotyping: Another approach to be mentioned in that connection is the recently published mFISH technique named color-changing karyotyping (CCK) [Henegariu et al., 1999]. Using CCK up to 41 different targets can be discriminated by three fluorochromes only. Similar is the approach of Wu et al. (2006) coloring 2 times 12 chromosomes sequentially or Yang et al., 2008.

Counterstaining: Alternative to DAPI is presented by Christian et al. (1998).

Literature to this part
  • Azofeifa J, Fauth C, Kraus J, Maierhofer C, Langer S, Bolzer A, Reichman J, Schuffenhauer S, Speicher MR (2000) An optimized probe set for the detection of small interchromosomal aberrations by use of 24-color FISH. Am J Hum Genet 66: 1684-1688.
  • Bolzer A, Craig JM, Cremer T, Speicher MR (1999) A complete set of repeat-depleted, PCR-amplifiable, human chromosome-specific painting probes. Cytogenet Cell Genet 84: 233-240.
  • Carter N (1993) Cytogenetic analysis by chromosome painting. Cytometry 18: 2-10.
  • Castleman KR, Eils R, Morrison L, Piper J, Saracoglu K, Schulze MA, Speicher MR (2000) Classification accuracy in multiple color fluorescence imaging microscopy. Cytometry 41: 139-147.
  • Choi H, Bovik AC, Castleman KR (2008) Feature normalization via expectation maximization and unsupervised nonparametric classification for M-FISH chromosome images. IEEE Trans Med Imaging 27:1107-1119.
  • Choi H, Castleman KR, Bovik AC (2009) Color compensation of multicolor fish images. IEEE Trans Med Imaging 28:129-136.
  • Christian A, McNiel E, Robinson J, Drabek R, LaRue S, Waldren C, Bedford J (1998) A versatile image analysis approach for simultaneous chromosome identification and localization of FISH probes. Cytogenet Cell Genet 82:172-179.
  • Dauwerse JG, Wiegant J, Raap AK, Breuning MH, van Ommen GJ (1992) Multiple colors by fluorescence in situ hybridization using ratio-labelled DNA probes create a molecular karyotype. Hum Mol Genet 1: 593-598.
  • Dugan LC, Pattee MS, Williams J, Eklund M, Sorensen K, Bedford JS, Christian AT (2005) Polymerase chain reaction-based suppression of repetitive sequences in whole chromosome painting probes for FISH. Chromosome Res 13:27-32
  • Gray JW, Kuo WL, Liang J, Pinkel D, van den Engh G, Trask B, Tkachuk D, Waldman F, Westbrook C (1990) Analytical approaches to detection and characterization of disease-linked chromosome aberrations. Bone Marrow Transplant 6 Suppl 1:14-19.
  • Henegariu O, Heerema NA, Bray-Ward P, Ward DC (1999) Colour-changing karyotyping: an alternative to M-FISH/SKY. Nat Genet 23:263-264.
  • Hua J, Xiong Z, Wu Q, Castleman KR (2005) Wavelet-based compression of M-FISH images. IEEE Trans Biomed Eng 52:890-900
  • Karvelis PS, Fotiadis DI, Georgiou I, Syrrou M (2006) A watershed based segmentation method for multispectral chromosome images classification. Conf Proc IEEE Eng Med Biol Soc 1:3009-3012
  • Karvelis PS, Tzallas AT, Fotiadis DI, Georgiou I (2008) A multichannel watershed-based segmentation method for multispectral chromosome classification. IEEE Trans Med Imaging 27:697-708.
  • Karvelis PS, Fotiadis DI, Tsalikakis DG, Georgiou IA (2009) Enhancement of multichannel chromosome classification using a region-based classifier and vector median filtering. IEEE Trans Inf Technol Biomed 13:561-570.
  • Kearney L (2006) Multiplex-FISH (M-FISH): technique, developments and applications. Cytogenet Genome Res 114:189-198.
  • Lee C, Gisselsson D, Jin C, Nordgren A, Ferguson DO, Blennow E, Fletcher JA, Morton CC (2001) Limitations of chromosome classification by multicolor karyotyping. Am J Hum Genet 68:1043-1047.
  • Munoz-Barrutia A, Garcia-Munoz J, Ucar B, Fernandez-Garcia I, Ortiz-de-Solorzano C (2007) Blind Spectral Unmixing of M-FISH Images by Non-negative Matrix Factorization. Conf Proc IEEE Eng Med Biol Soc 1:6247-6250
  • Nederlof PM, van der Flier S, Wiegant J, Raap AK, Tanke HJ, Ploem JS, van der Ploeg M (1990) Multiple fluorescence in situ hybridization. Cytometry 11: 126-131.
  • Nederlof PM, van der Flier S, Vrolijk J, Tanke HJ, Raap AK (1992) Fluorescence ratio measurements of double-labeled probes for multiple in situ hybridization by digital imaging microscopy. Cytometry 13: 839-845.
  • Morrison LE, Legator MS (1997) Two-color ratio-coding of chromosome targets in fluorescence in situ hybridization: quantitative analysis and reproducibility. Cytometry. 27: 314-326.
  • Raap AK, Tanke HJ (2006) COmbined Binary RAtio fluorescence in situ hybridiziation (COBRA-FISH): development and applications. Cytogenet Genome Res 114:222-226.
  • Rens W, Yang F, O'Brien PC, Solanky N, Ferguson-Smith MA (2001) A classification efficiency test of spectral karyotyping and multiplex fluorescence in situ hybridization: Identification of chromosome homologies between Homo sapiens and Hylobates leucogenys. Genes Chr Cancer 31: 65-74.
  • Ried T, Landes G, Dackowski W, Klinger K, Ward DC (1992) Multicolor fluorescence in situ hybridization for the simultaneous detection of probe sets for chromosomes 13, 18, 21, X and Y in uncultured amniotic fluid cells. Hum Mol Genet 1: 307-313.
  • Rooms L, Reyniers E, Kooy RF (2005) Subtelomeric rearrangements in the mentally retarded: a comparison of detection methods. Hum Mutat 25:513-524.
  • Padilla-Nash HM, Barenboim-Stapleton L, Difilippantonio MJ, Ried T (2006) Spectral karyotyping analysis of human and mouse chromosomes. Nat Protoc 1:3129-3142
  • Sampat MP, Bovik AC, Aggarwal JK, Castleman KR (2005) . Supervised parametric and non-parametric classification of chromosome images. Pattern Recognit 38:1209-1223.
  • Saracoglu K, Brown J, Kearney L, Uhrig S, Azofeifa J, Fauth C, Speicher MR, Eils R (2001) New concepts to improve resolution and sensitivity of molecular cytogenetic diagnostics by multicolor fluorescence in situ hybridization. Cytometry 44: 7-15.
  • Schröck E, du Manoir S, Veldman T, Schoell B, Wienberg J, Ferguson-Smith MA, Ning Y, Ledbetter DH, Bar-Am I, Soenksen D, Garini Y, Ried T. (1996) Multicolor spectral karyotyping of human chromosomes. Science 273: 494-497.
  • Schröck E, Zschieschang P, O'Brien P, Helmrich A, Hardt T, Matthaei A, Stout-Weider K (2006) Spectral karyotyping of human, mouse, rat and ape chromosomes--applications for genetic diagnostics and research. Cytogenet Genome Res 114:199-221.
  • Schwartzkopf WC, Bovik AC, Evans BL (2005) Maximum-likelihood techniques for joint segmentation-classification of multispectral chromosome images. IEEE Trans Med Imaging 24:1593-1610.
  • Senger G, Chudoba I, Plesch A (1998) Multicolor-FISH - the identification of chromosome aberrations by 24 colors. BIOforum 9: 499-503.
  • Speicher MR, Gwyn Ballard S, Ward DC (1996) Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat Genet 12: 368-375.
  • Stolte T, Hösel V, Müller J, Speicher M (2008) Modeling clonal expansion from M-FISH experiments. J Comput Biol 15:221-230.
  • Szuhai K, Bezrookove V, Wiegant J, Vrolijk J, Dirks RW, Rosenberg C, Raap AK, Tanke HJ (2000) Simultaneous molecular karyotyping and mapping of viral DNA integration sites by 25-color COBRA-FISH. Genes Chr Cancer 28: 92-97.
  • Tanke HJ, De Haas RR, Sagner G, Ganser M, van Gijlswijk RP (1998) Use of platinum coproporphyrin and delayed luminescence imaging to extend the number of targets FISH karyotyping. Cytometry 33: 453-459.
  • Tanke HJ, Wiegant J, van Gijlswijk RP, Bezrookove V, Pattenier H, Heetebrij RJ, Talman EG, Raap AK, Vrolijk J (1999) New strategy for multi-colour fluorescence in situ hybridisation: COBRA: COmbined Binary RAtio labelling. Eur J Hum Genet 7: 2-11.
  • Thalhammer S, Langer S, Speicher MR, Heckl WM, Geigl JB (2004) Generation of chromosome painting probes from single chromosomes by laser microdissection and linker-adaptor PCR. Chromosome Res 12: 337-343.
  • Wiegant J, Kalle W, Mullenders L, Brookes S, Hoovers JM, Dauwerse JG, van Ommen GJ, Raap AK (1992) High-resolution in situ hybridization using DNA halo preparations. Hum Mol Genet 1: 587-591.
  •  Wallenborn M, Petters O, Rudolf D, Hantmann H, Richter M, Ahnert P, Rohani L, Smink JJ, Bulwin GC, Krupp W, Schulz RM, Holland H (2018) Comprehensive high-resolution genomic profiling and cytogenetics of human chondrocyte cultures by GTG-banding, locus-specific FISH, SKY and SNP array. Eur Cell Mater 35:225-241.
  • Wang YP. (2008) Detection of chromosomal abnormalities with multi-color fluorescence in situ hybridization (M-FISH) imaging and multi-spectral wavelet analysis. Conf Proc IEEE Eng Med Biol Soc. 2008:1222-1225.
  • Wang YP, Castleman KR (2005) Normalization of multicolor fluorescence in situ hybridization (M-FISH) images for improving color karyotyping. Cytometry A 64:101-109.
  • Wu YP, Yang YL, Yang GZ, Wang XY, Luo ML, Zhang Y, Feng YB, Xu X, Han YL, Cai Y, Zhan QM, Wu M, Dong JT, Wang MR (2006) Identification of chromosome aberrations in esophageal cancer cell line KYSE180 by multicolor fluorescence in situ hybridization. Cancer Genet Cytogenet 170:102-107.
  • Xue YB, Song X (2008) [Progresses on the methods of tumor chromosome aberration analysis.] Yi Chuan 30:1529-1535. Chinese.
  • Yang Y, Chu J, Wu Y, Luo M, Xu X, Han Y, Cai Y, Zhan Q, Wang M (2008) Chromosome analysis of esophageal squamous cell carcinoma cell line KYSE 410-4 by repetitive multicolor fluorescence in situ hybridization. J Genet Genomics 35:11-16.