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Metaphase Harvest and Cytogenetic Analysis of Solid Tumor Cultures

Jonathan Fletcher¹

¹Harvard Medical School, Boston, Massachusetts

Publication Name:

Current Protocols in Human Genetics

Unit Number:

Unit 10.3

DOI:

10.1002/0471142905.hg1003s02

Online Posting Date:

May, 2001

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Abstract

Cytogenetic preparations of solid tumor specimens often demonstrate clonal chromosome aberrations. These clonal aberrations are useful in confirming the neoplastic nature of a given tumor and might also suggest a specific diagnosis or prognosis. Most solid tumors must be cultured for several days before metaphase cells can be obtained for cytogenetic analysis. The Basic Protocol describes harvesting of metaphase cells from cultures prepared from solid tumor specimens. The cells are fixed and then stained using a trypsin-Giemsa (GTG) method to produce characteristic banding patterns for cytogenetic analysis. The first Support Protocol 1 describes disaggregation and short-term tissue culture of solid tumors. The second Support Protocol 2 describes analysis of cells from lymph nodes infiltrated by lymphoma cells and the third Support Protocol 3 describes the direct analysis of cells from malignant effusion (fluid) samples.

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Unit Introduction
Basic Protocol: Cytogenetic Analysis of Metaphase Cells from Solid Tumor, Lymphoma, or Effusion Samples
Support Protocol 1: Disaggregation and Culture of Solid Tumors
Support Protocol 2: Culture of Lymph Node (Lymphoma) Specimens for Cytogenetic Analysis
Support Protocol 3: Preparation of Effusion (Fluid) Specimens for Cytogenetic Analysis
Reagents and Solutions
Commentary
Bibliography
Tables

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Materials

Basic Protocol: Cytogenetic Analysis of Metaphase Cells from Solid Tumor, Lymphoma, or Effusion Samples

Materials

For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock solutions, see appendix 2A; for suppliers, see suppliers appendix.

Actively growing tumor cells (Support Protocol 1)
10 µg/ml Colcemid (GIBCO/BRL)
1× trypsin/EDTA (GIBCO/BRL)
0.067 M KCl
Fixative: 3:1 (v/v) methanol/glacial acetic acid, prepared fresh

25-cm² tissue culture flasks
Centrifuge: Fisher Marathon 21K, Becton Dickinson Primary Care Dynac II, or equivalent

Additional reagents and equipment for chromosome slide preparation (unit 4.1) and GTG-banding staining of chromosomes (unit 4.2)

NOTE: All incubations are performed in a humidified 37°C, 5% CO₂ incubator unless otherwise specified.

NOTE: Quantities specified in this protocol are for cells cultured in 5 ml medium in 25-cm² flasks (lying on their sides). Adjust volumes appropriately when using smaller or larger culture vessels.

Support Protocol 1: Disaggregation and Culture of Solid Tumors

Materials

For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock solutions, see appendix 2A; for suppliers, see suppliers appendix.

Tumor specimen (any solid carcinoma, sarcoma, germ cell, or neural tumor)
Tumor transport solution, room temperature (see recipe)
Complete RPMI/15% FBS (appendix 3G) supplemented with 2.5 µg/ml amphotericin, with and without 1 mg/ml collagenase (see recipe), 37°C (store medium £1 month at 4°C)
Centrifuge: Fisher Marathon 21K, Becton Dickinson Primary Care Dynac II, or equivalent

Additional reagents and equipment for tissue culture (appendix 3G)

NOTE: All incubations are performed in a humidified 37°C, 5% CO₂ incubator unless otherwise specified.

Support Protocol 2: Culture of Lymph Node (Lymphoma) Specimens for Cytogenetic Analysis

Additional Materials

For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock solutions, see appendix 2A; for suppliers, see suppliers appendix.

Lymph nodes
Tumor transport solution, ice cold (see recipe)
Complete RPMI/15% FBS (appendix 3G) supplemented with 2.5 µg/ml amphotericin, with and without 1 mg/ml collagenase (see recipe), 37°C (store medium £1 month at 4°C)
Ficoll (Sigma)
Hanks balanced salt solution (HBSS; appendix 2D)
25-cm² tissue culture flasks or 60-mm-diameter petri dish

NOTE: All incubations are performed in a humidified 37°C, 5% CO₂ incubator unless otherwise specified.

Support Protocol 3: Preparation of Effusion (Fluid) Specimens for Cytogenetic Analysis

Additional Materials

For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock solutions, see appendix 2A; for suppliers, see suppliers appendix.

Malignant effusion sample
10 µg/ml vinblastine (Lilly)
10 mg/ml ethidium bromide solution (appendix 2D)
Complete RPMI/15% FBS (appendix 3G) supplemented with 2.5 µg/ml amphotericin, 37°C
0.85% (w/v) ammonium chloride

NOTE: All incubations are performed in a humidified 37°C, 5% CO₂ incubator unless otherwise specified.

Looking for materials? Suppliers Guide

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Literature Cited

Literature Cited
	Brodeur, G.M., Azar, C., Brother, M., Hiemstra, J., Kaufman, B., Marshall, H., Moley, J., Nakagawara, A., Saylors, R., Scavarda, N., Schneider, S., Wasson, J., White, P., Seeger, R., Look, T., and Castleberry, R. 1992. Neuroblastoma: Effect of genetic factors on prognosis and treatment. Cancer 70:1685-1694.
	Fletcher, J.A., Pinkus, G.S., Weidner, N., and Morton, C.C. 1991a. Lineage-restricted clonality in biphasic solid tumors. Am. J. Path. 138:1199-1207.
	Fletcher, J.A., Kozakewich, H.P., Hoffer, F.A., Lage, J.M., Weidner, N., Tepper, T., Pinkus, G.S., Morton, C.C., and Corson, J.M. 1991b. Diagnostic relevance of clonal cytogenetic aberrations in malignant soft-tissue tumors. New Engl. J. Med. 324:436-443.
	Freshney, R.I. (ed.) 1986. Animal Cell Culture: A Practical Approach. IRL Press, Oxford.
	Harrison, C.J. 1992. The lymphomas and chronic lymphoproliferative disorders. In Human Cytogenetics: A Practical Approach (D.E. Rooney and B.H. Czepulkowski, eds.) pp. 97-120. IRL Press, Oxford.
	Heim, S. and Mitelman, F. 1987. Solid tumors. In Cancer Cytogenetics, pp. 227-264. Wiley-Liss, New York.
	Mandahl, N. 1992. Methods in solid tumor cytogenetics. In Human Cytogenetics: A Practical Approach (D.E. Rooney and B.H. Czepulkowski, eds.) pp. 155-188. IRL Press, Oxford.
	Mitelman, F. 1991. Catalog of Chromosome Aberrations in Cancer. Wiley-Liss, New York.
	Pandis, N., Heim, S., Bardi, G., Limon, J., Mandahl, N., and Mitelman, F. 1992. Improved technique for short-term culture and cytogenetic analysis of human breast cancer. Genes Chrom. & Canc. 5:14-20.
	Sandberg, A.A. 1990. The Chromosomes in Human Cancer and Leukemia. Elsevier, New York.
	Sandberg, A.A., Turc-Carel, C., and Gemmill, R.M. 1988. Chromosomes in solid tumors and beyond. Cancer Res. 48:1049-1059.
	Trent, J.M. 1991. Clinical correlations of chromosome change in human solid tumors: The tip of the iceberg J. Nat. Cancer Inst. 81:1852-1853.
Key References
	Harrison 1992. See above.
Includes several protocols for stimulation of lymphoma cell growth using various mitogens.
	Heim and Mitelman 1987. See above.
Very succinct, well-organized overview of typical cytogenetic aberrations in benign and malignant neoplasms [1st (1987) ed. is already somewhat out-of-date].
	Mandahl 1992. See above.
Exceptional overview of solid tumor cytogenetic methodology, with specific recommendations on growth factors and culture conditions that can be adjusted to facilitate analysis of different tumors.
	Mitelman 1991. See above.
Comprehensive listing of published karyotypes for leukemias, lymphomas, and solid tumors (most karyotypes published in the last decade).
	Sandberg 1988. See above.
Concise review providing an exceptional introduction to the field of solid tumor cytogenetics.