• ABOUT
  • THE GROUP
  • NEWS & MEDIA
  • EVENTS
  • CAREER
  • CONTACT US
  • 0
Spectral karyotyping

Spectral karyotyping

BY Ms. Nikita Kawli 30th December 2020

Spectral karyotyping (SKY) is a novel method for chromosome examination that has been created dependent on the methodology of the fluorescence in situ hybridization. Owing to its technology for painting each of the 24 human chromosomes with various colors, spectral karyotyping allows it possible to diagnose a range of disease [1].

Chromosomal analysis plays an important role in human leukemia diagnosis, treatment and prognosis [2-6]. In assessing the risk for phenotypic abnormalities, particularly in prenatal circumstances, the identification of marker chromosomes is significant. For the provision of information to couples about the possible phenotypic and/or developmental impact of a de novo rearrangement, the ability to determine the origin of additional genetic materials is very significant. Similarly, the detection of derivative chromosomal content will shed light on the mechanism of infertility when evaluating infertility [7-9]. In constitutional research, spectral karyotyping is an invaluable diagnostic tool for identifying marker chromosomes and chromosome exchanges that are not completely described by traditional cytogenetic methods and in identifying new subgroups of leukemia.

In this technology,chromosomes are painted with 24 chromosome-specific painting probes. This is called Combinatorial chromosome painting techniques. Using three fluorochromes and two haptens, the probes are labelled by degenerated oligonucleotide-primed PCR. Each probe is labeled differentially with one, two, three or four fluorescent dyes resulting in each chromosome having a special spectral signature. With similar principle of labelling the chromosomes,there emerged two techniques -Multiplex fluorescence in situ hybridization (mFISH) and Spectral Karyotyping (SKY), both follows similar basic fluorescence in situ hybridization (FISH) procedures, however the imaging methods are different [10].  

 Schematic representation of SKY Detection.
Fig 1. Schematic representation of SKY Detection.

The differentiation of the chromosomes based on their spectral properties is defined by Spectral karyotyping (Fig1), while mFISH identifies the differentiation of the chromosomes based on the presence or absence of the fluorochrome when visualized with specific filters(Fig 2).

Schematic representation of M-FISH Detection.
Fig 2. Schematic representation of M-FISH Detection.

In SKY technology. using a traditional fluorescence light microscope fitted with a custom-designed triple-bandpass filter and the SpectraCube, a spectral image is acquired after in situ hybridization and immunodetection, which is able to retrieve spectral information for each pixel in a digital CCD image. The 24-colour display and chromosome classification are based on the chromosomes’ unique emission spectra. A detailed description of chromosomal aberrations is provided along with chromosome banding data from an inverted DAPI or a G-banded metaphase [11-12]. 

Optical Design and Analysis Flow
Fig 3. Optical Design and Analysis Flow
The simultaneous measurement of the spectral contents for each pixel in the image
Fig 4. The simultaneous measurement of the spectral   contents for each pixel in the image

The center of the platform for SKY is a  Sagnac  Interferometer, installed  on a rotatable disk to which a CCD camera is attached. The ray of light entering the triangle interferometer is divided into two beams. (a transmitted beam and a reflected beam), traveling in opposite directions, but in space on the same path. They’re hitting the CCD with Optical Path Difference (OPD) and shape a pattern of interference that is influenced in the image, by the spectral content at each point. It captures a sequence of images, at each  different OPD, to form an interferogram  at each pixel. A Fourier-analysis approach is applied to extract the sample’s hyperspectral image.

In MFISH, using specialized single band-pass filter sets and dedicated M-FISH software, microscopic visualization and digital acquisition of each fluorophore is obtained. These acquired images are then superimposed, allowing the classification of individual chromosomes in their group [13].

The main deficiency with mFISH is Cross talk between the filters. The emission spectrum of only two adjacent dyes is shown in the following example(Fig 5). Comparable filter-based measurements may provide fair results as long as the amplitude of those signals is comparable. However to compensate for its weak signal, if the green dye is faint, one will need to increase its exposure time. The cross talk factor will then be even more dominant than the green signal from other dyes (which spread all over due to 10-20 percent non-specific staining). This will cause large misclassifications. 

Cross talk between filters
Fig 5: Cross talk between filters

GenASIs HyperSpectral platform is based on a cutting-edge, dual-mode optical device, which allows both interferometer-based image capture for hyperspectral imaging and direct view mode for high-resolution CCD image capture. The HyperSpectral data reveals the spectrum of every pixel in the image, and provides advanced analysis tools to extract quantitative and hidden information from within a sample. In Direct View mode, the system records image details under extremely low intensities and provides a finely detailed high resolution and high-definition image. SKY is hyper precise, accurate and hypersensitive.

References:

  1. Imataka, G. and Arisaka, O., 2012. Chromosome analysis using spectral karyotyping (SKY). Cell biochemistry and biophysics62(1), pp.13-17.
  2. Guo, B., Han, X., Wu, Z., Da, W. and Zhu, H., 2014. Spectral karyotyping: an unique technique for the detection of complex genomic rearrangements in leukemia. Translational pediatrics3(2), p.135.
  3. Mohr, B., Bornhäuser, M., Thiede, C., Schäkel, U., Schaich, M., Illmer, T., Pascheberg, U. and Ehninger, G., 2000. Comparison of spectral karyotyping and conventional cytogenetics in 39 patients with acute myeloid leukemia and myelodysplastic syndrome. Leukemia14(6), pp.1031-1038.
  4. Lu, X.Y., Harris, C.P., Cooley, L., Margolin, J., Steuber, P.C., Sheldon, M., Rao, P.H. and Lau, C.C., 2002. The utility of spectral karyotyping in the cytogenetic analysis of newly diagnosed pediatric acute lymphoblastic leukemia. Leukemia16(11), pp.2222-2227.
  5. Mrózek, K., 2008, August. Cytogenetic, molecular genetic, and clinical characteristics of acute myeloid leukemia with a complex karyotype. In Seminars in oncology (Vol. 35, No. 4, pp. 365-377). WB Saunders.
  6. Fadl‐elmula, I., Kytölä, S., Pan, Y., Lui, W.O., Derienzo, G., Forsberg, L., Mandahl, N., Gorunova, L., Bergerheim, U.S., Heim, S. and Larsson, C., 2001. Characterization of chromosomal abnormalities in uroepithelial carcinomas by G‐banding, spectral karyotyping and FISH analysis. International journal of cancer92(6), pp.824-831.
  7. Anguiano, A., Wang, B.T., Wang, S.R., Boyar, F.Z., Mahon, L.W., El Naggar, M.M., Kohn, P.H., Haddadin, M.H., Sulcova, V., Sbeiti, A.H. and Ayad, M.S., 2012. Spectral Karyotyping for identification of constitutional chromosomal abnormalities at a national reference laboratory. Molecular Cytogenetics5(1), p.3.
  8. Chen, C.P., Lin, C.C., Su, Y.N., Tsai, F.J., Chern, S.R., Lee, C.C., Chen, W.L., Chen, L.F., Wu, P.C. and Wang, W., 2010. Prenatal diagnosis and molecular cytogenetic characterization of a small supernumerary marker chromosome derived from chromosome 22. Taiwanese Journal of Obstetrics and Gynecology49(3), pp.381-384.
  9. Chen, C.P., Chen, M., Wu, C.H., Lin, C.J., Chern, S.R., Wu, P.S., Chen, Y.N., Chen, S.W., Chang, S.P., Chen, L.F. and Wang, W., 2017. Prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome derived from chromosome 21q11. 2-q21. 1 and a literature review. Taiwanese Journal of Obstetrics and Gynecology56(4), pp.554-557.
  10. Loucas, B.D., 2019. Analysis of Radiation-Induced Chromosome Exchanges Using Combinatorial Chromosome Painting. In Radiation Cytogenetics (pp. 123-135). Humana, New York, NY.
  11. Macville, M., Veldman, T., Padilla-Nash, H., Wangsa, D., O’Brien, P., Schröck, E. and Ried, T., 1997. Spectral karyotyping, a 24-colour FISH technique for the identification of chromosomal rearrangements. Histochemistry and cell biology108(4-5), pp.299-305.
  12. Padilla-Nash, H.M., Barenboim-Stapleton, L., Difilippantonio, M.J. and Ried, T., 2006. Spectral karyotyping analysis of human and mouse chromosomes. Nature protocols1(6), p.3129.
  13. Anderson, R., 2010. Multiplex fluorescence in situ hybridization (M-FISH). In Fluorescence in situ Hybridization (FISH) (pp. 83-97). Humana Press, Totowa, NJ.

Ms. Nikita Kawli

Deputy Manager, Applications


Nikita is Application Specialist for Micro-Imaging Division at DSS Imagetech Pvt Ltd. She is currently responsible for pre-sales and post-sales activities for Applied Spectral Imaging (ASI) products in Cytogenetics and Pathology. Her portfolio includes handling interactive and automated systems for Karyotyping, FISH, Spectral Karyotyping , IHC and Digital Imaging. She also holds an experience in the Diagnostic sector on Molecular genetics. Her academic profile possesses Masters in Applied Genetics , PG- Diploma in Medical Lab Technology , PG- Diploma in Bionanotechnology and Advanced Diploma in Software Engineering.

Latest Articles

Understanding Immunofluorescence and Its Diagnostic Applications
BY DSS Imagetech Pvt Ltd 25th March 2025

Immunofluorescence testing ranks among medicine’s most reliable diagnostic tools, especially when detecting autoimmune diseases. The sensitivity rates reach 100% for certain conditions. Medical professionals have managed to keep this technique at the heart of diagnostics since 1942. It provides exceptional accuracy in identifying diseases in multiple organ systems. The immunofluorescence staining process is a great […]
Read more

Liquid Biopsy: A Non-Invasive Approach to Cancer Detection
BY DSS Imagetech Pvt Ltd 25th March 2025

Cancer is the second leading cause of death worldwide. This grim statistic highlights the need to save lives through better detection methods. We have a long way to go, but we can build on this progress in cancer diagnostics, where liquid biopsy stands at the vanguard of medical advancement. Liquid biopsy revolutionizes cancer detection through […]
Read more

Enhance Genetic Testing with Advanced FISH Instruments from DSS Imagetech
BY DSS Imagetech Pvt Ltd 7th March 2025

One of the most revolutionary techniques of modern molecular biology and genetics, fluorescence in situ hybridization is a precision-based technique known widely for use in genetic diagnostics and research, helping to determine chromosomal anomalies, gene mutation, and many more genetic markers. DSS Imagetech is a trusted company dealing with biotechnology in Delhi, India, and delivers […]
Read more

Unlocking the Mysteries of the Immune System With Clonality Assays
BY Dr. Hitarth Patel 22nd January 2025

The immune system is an extraordinary defense mechanism, safeguarding our bodies against a multitude of threats. Its capacity to discriminate between self and non-self is essential for preserving health in the face of infections and cancer. When the balance within the immune system is disturbed, it can lead to various dysfunctions with serious implications. In […]
Read more

Advancements in Clonality Testing
BY Ms. Megha Dhumal 17th January 2025

The immune system, one of the most intricate networks in the human body, tirelessly protects us from pathogens such as bacteria, viruses, fungi, and more. While a healthy immune system ensures defense and health, any compromise can lead to diseases, autoimmune conditions, allergies, or even cancer. Clonality testing, a crucial advancement in hematologic oncology, has […]
Read more

The Role of Artificial Intelligence in Oncology Diagnostics: Enhancing Accuracy and Efficiency
BY DSS Imagetech Pvt Ltd 30th December 2024

The blending of artificial intelligence (AI) with medical research has paved the path for unprecedented improvements in cancer diagnoses. AI is revolutionizing the science of cancer detection by processing enormous volumes of data, seeing patterns, and making precise predictions. AI-powered technologies are giving physicians crucial tools to diagnose cancer early, customize treatment programs, and enhance […]
Read more