12. The concentration of trypsin working solution is 0.25 %.
13. Time for pretreatment with trypsin should be adjusted for each case according to the spreading technique used, age of slide, degree of contraction of chromosomes, and chromosome morphology. Too long or too short pretreatment time will result in poor banding resolution. Sometimes pretreatment time may be as long as 1 min.
14. The pH of the Earle’s solution must be 6.8; otherwise, the quality of R- banding will be poor.
15. For R-banding, the temperature of water bath must be accurately adjusted to 87.5 °C.
16. Each laboratory should explore the temperature according to its own conditions to obtain ideal R-banding.
17. The time for denaturing in alkaline solution depends on the age of slide. A temperature gradient must be established based on experience.
References
1. Gonon-Demoulian R, Goldman JM, Nicolini FE (2014) History of chronic myeloid leuke- mia: a paradigm in the treatment of cancer.
Bull Cancer 101(1):56–67
2. Wan TS (2014) Cancer cytogenetics: method- ology revisited. Ann Lab Med 34(6):
413–425
3. Sudoyo AW, Hardi F (2011) Cytogenetics in solid tumors: lessons from the Philadelphia Chromosome. Acta Med Indones 43(1):
68–73
4. Bernheim A (2010) Cytogenomics of cancer from chromosome to sequence. Mol Oncol 4(4):309–322
5. Swansbury J (2003) Introduction. In:
Swansbury J (ed) Cancer cytogenetics: meth- ods & protocols. Humana, Clifton, NJ 6. Ushiki T, Hoshi O, Iwai K et al (2002) The
structure of human metaphase chromosomes:
its histological perspective and new horizons by atomic force microscopy. Arch Histol Cytol 65(5):377–390
7. Craig JM, Bickmore WA (1993) Chromosome bands-fl avours to savour. BioEssays 15:
349–354
8. Saitoh Y, LaemmLi UK (1994) Metaphase chromosome structure: bands arise from a
differential folding path of the highly AT-rich scaffold. Cell 76(4):609–622
9. Francke U (1994) Digitized and differentially shaded human chromosome ideograms for genomic applications. Cytogenet Cell Genet 65(3):206–218
10. McGowan-Jordan J, Simons A, Schmid M (eds) (2016) ISCN 2016: An International System for Human Cytogenomic Nomenclature. S. Karger, Basel. [Reprint of Cytogenet and Genome Res 149(1–2)]
11. Simons A, Shaffer LG, Hastings RJ (2013) Cytogenetic nomenclature: changes in the ISCN 2013 compared to the 2009 edition.
Cytogenet Genome Res 141(1):1–6
12. Yunis JJ (1981) Mid-prophase human chromo- somes: the attainment of 2000 bands. Hum Genet 56:293–298
13. Imai HT (1991) Mutability of constitutive het- erochromatin (C-bands) during eukaryotic chromosomal evolution and their cytological meaning. Jpn J Genet 66(5):635–661
14. Wijayanto H, Hirai Y, Kamanaka Y et al (2005) Patterns of C-heterochromatin and telomeric DNA in two representative groups of small apes, the genera Hylobates and Symphalangus.
Chromosome Res 13(7):717–724
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Thomas S.K. Wan (ed.), Cancer Cytogenetics: Methods and Protocols, Methods in Molecular Biology, vol. 1541, DOI 10.1007/978-1-4939-6703-2_7, © Springer Science+Business Media LLC 2017
Chapter 7
Chromosome Recognition
Thomas S. K. Wan , Eleanor K. C. Hui , and Margaret H. L. Ng
Abstract
Chromosomal analysis of human cells serves to characterize aberrations of chromosome number and structure. Individual chromosome can be identifi ed precisely by recognition of its morphological charac- teristics and staining patterns according to specifi c landmarks, regions, and bands as described in the ideo- gram. Since the quality of metaphases obtained from malignant cells is generally poor for karyotyping, a practical and accurate chromosome recognition training guide is mandatory for a trainee or newly employed cytogenetic technologist in a cancer cytogenetics laboratory. The most distinguishable bands for each chromosome are described in detail in this chapter with an aim to facilitate quick and accurate karyotyping in cancers. This is an indispensable chromosome recognition guide used in a cancer cytogenetics laboratory.
Key words Chromosomal analysis , Chromosome pattern , Chromosome recognition , G-banded karyotyping
1 Introduction
The technique of autoradiography had been used in an attempt to improve on the identifi cation of individual chromosomes in the early 60s. However, neither chromosome morphology nor autora- diography provided unequivocal identifi cation. At that time, these non-banded chromosomes can only be arranged into seven readily distinguishable groups (A–G groups) in descending order based on the characteristics of size and centromere location [ 1 ]. The major breakthrough in chromosomal identifi cation came only after the demonstration by Casperson et al. [ 2 ] that each chromosome has its own unique anatomy by virtue of its banding pattern in 1970. The Fourth International Congress on Human Genetics held in Paris in 1971 was a critical developmental milestone in chromosome recognition [ 3 ]. In this landmark achievement, an internationally agreed system for describing the banding pattern of chromosomes was established, by which each homologous chromosome pair can be identifi ed precisely by specifi c landmarks, regions, and bands as described in the ideogram .
The chromosomes are arranged in a karyotype based on the respective centromere position, band pattern, and length of the chromosome arms. Chromosome numbers are designated corre- sponding to decreasing size, except for chromosome 21 that is smaller than 22. The location of the centromere is a key feature described in the chromosome morphology: (1) metacentric chro- mosome refers to a centromeric location near the middle of the chromosome with a ratio of short arm to long arm of 1:1 to 1:1.3;
(2) submetacentric chromosome refers to a centromeric location closer to one end of the chromosome than the other with a ratio of short arm to long arm of 1:1.3–1:7; (3) acrocentric chromosome refers to a centromeric location near the end of the chromosome with a ratio of short arm to long arm >1:7, also a secondary con- striction, or stalk, may separate satellites from the proximal short arm [ 4 ]. Regions and bands are numbered consecutively from the centromere outward along each chromosome arm. The symbols p and q are used to designate the short and long arms of each chromosome respectively [ 5 ].
Since the morphology of metaphases obtained from malignant cells is generally ambiguous and complex, it poses an added chal- lenge to cancer cytogeneticists in chromosome recognition, partic- ularly when the metaphase quality is inferior. The chromosome morphology, length, and banding resolution of cancer cells show a high diversity. It is important to analyze a wide spectrum of meta- phase cells of varying chromosomal qualities to avoid missing detec- tion of the abnormal clone or subclones as normal metaphase cells with better chromosomal resolution may coexist. It often takes a new cytogenetic technologist several weeks to months to become competent and confi dent in recognizing normal and specifi c abnor- mal patterns of chromosomes. The most distinguishable bands for each normal human chromosome are described and highlighted on the partial chromosome and the corresponding ideogram in this chapter. This is an indispensable practical training guide for trainees or newly employed cytogenetic technologists.
2 Chromosome Identifi cation
Group A consists of chromosomes 1-3 and they are usually identifi ed by their size and centromeric index of chromosome (Fig. 1 ).