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Metaphase Harvest and Cytogenetic Analysis of Malignant Hematological Specimens

Paola Dal Cin1

1Brigham and Women's Hospital, Boston, Massachusetts

Publication Name: 
Current Protocols in Human Genetics
Unit Number: 
Unit 10.2
DOI: 
10.1002/0471142905.hg1002s36
Online Posting Date: 
May, 2003
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Abstract

Cytogenetic analysis of malignant hematological disease is an important methodology used by clinicians and researchers, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance. The Basic Protocol describes the culture, harvest, and preparation of chromosome spreads from bone marrow aspirates. Five alternate protocols describe adaptations of the Basic Protocol for preparation of specimens from chronic lymphocytic leukemia (CLL) bone marrow, leukemic peripheral blood, CLL peripheral blood, post-bone marrow transplant (post-BMT) peripheral blood, and aplastic anemia bone marrow and peripheral blood. The modifications involve slightly different culture and harvest methods that are necessary to assure optimal results.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Preparation of Chromosome Spreads from Bone Marrow and Leukemic Blood Specimens
  • Alternate Protocol 1: Preparation of Chromosome Spreads from CLL Bone Marrow and Peripheral Blood Specimens
  • Alternate Protocol 2: Preparation of Chromosome Spreads from MM Bone Marrow and Peripheral Blood Specimens
  • Alternate Protocol 3: Preparation of Chromosome Spreads from Lymph Node Specimens
  • Alternate Protocol 4: Preparation of Chromosome Spreads from Spleen
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Tables
     
 
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Materials

Basic Protocol: Preparation of Chromosome Spreads from Bone Marrow and Leukemic Blood Specimens

 Materials
  • Complete culture media, A and B (see recipes)
  • Bone marrow aspirate or leukemic blood, collected in a syringe or Vacutainer tube (Becton Dickinson) containing preservative-free heparin
  • 500 µg/ml ethidium bromide in HBSS (Sigma)
  • 10 µg/ml Colcemid (Irving Scientific)
  • 0.075 M KCl solution, prepared fresh and prewarmed to 37°C
  • 3:1 (v/v) methanol/glacial acetic acid fixative, prepared fresh
  • 50-ml tissue-culture flasks (Falcon)
  • 15-ml culture tubes
  • 37°C, 5% CO2 incubator
  • 15-ml centrifuge tubes (Corning), sterile
  • Tabletop centrifuge (e.g., IEC HN-SII)
  • Pasteur pipets
  • Precleaned microscope slides (Gold Seal; Becton Dickinson; see recipe)
  • Phase-contrast microscope
  • 85° to 90°C oven
  • Additional reagents and equipment for chromosome banding (unit 4.2); karyotyping (appendix 4A), and interpretation of metaphase spreads (unit 8.1)

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

Alternate Protocol 1: Preparation of Chromosome Spreads from CLL Bone Marrow and Peripheral Blood Specimens

 Additional Materials (also see Basic Protocol)
  • Pokeweed mitogen (PWM; see recipe)

Alternate Protocol 2: Preparation of Chromosome Spreads from MM Bone Marrow and Peripheral Blood Specimens

 Additional Materials (also see Basic Protocol)
  • Interleukin 4 (IL-4; see recipe)

Alternate Protocol 3: Preparation of Chromosome Spreads from Lymph Node Specimens

 Additional Materials (also see Basic Protocol)
  • Collagenase solution (see recipe)
  • Bone marrow medium (e.g., GIBCO/BRL or Sigma, or see recipes for complete culture medium A and B)
  • 5-ml petri dish, 60 × 15–mm (Falcon)
  • Disposable scalpels

Alternate Protocol 4: Preparation of Chromosome Spreads from Spleen

 Additional Materials (also see Basic Protocol)
  • Collagenase solution (see recipe)
  • Complete culture medium A and B
  • 5-ml petri dish, 60 × 15–mm (Falcon)
  • Disposable scalpels
Looking for materials?  Suppliers Guide
     
 
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Literature Cited

 Literature Cited
    Boveri, T. 1929. The origin of Malignant Tumors. Baliere, Tindall & Cox, London.
    Crossen, P.E. 1997. Genes and chromosomes in chronic B-cell leukemia. Cancer Genet. Cytogenet. 94:94-44.
    Dal Cin, P. and Morton, C.C. 2003. Cytogenetics for the hematologist. In Blood: Principles and Practice of Hematology (R.I. Handin, S.E. Lux, and T.P. Stossel, eds.) pp. 96-123. Lippincott-Raven, Philadelphia.
    Druker, B.J., Sawyers, C.L., Kantarjian, H., Resta, D.J., Reese, S.F., Ford, J.M., Capdeville, R., and Talpaz, M. 2001. Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N. Engl. J. Med. 344:1038-1042.
    Grimwade, D., Walker, H., Oliver, F., Wheatley, K., Harrison, C., Harrison, G., Rees, J., Hann, I., Stevens, R., Burnett, A., and Goldstone, A. 1998. The importance of diagnostic cytogenetics on outcome in AML: Analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children's Leukaemia Working Parties. Blood 92:2322-2333.
    Heim, S. and Mitelman, F. 1995. Cancer Cytogenetics, 2nd ed. John Wiley & Sons, New York.
    Hernandez, J.M., Gutierrez, N.C., Almeida, J., Garcia, J.L., Sanchez, M.A., Mateo, G., Rios, A., and San Miguel, J.F. 1998. IL-4 improves the detection of cytogenetic abnormalities in multiple myeloma and increases the proportion of clonally abnormal metaphases. Br. J. Haematol. 103:163-167.
    Ikeuchi, T. 1984. Inhibitory effect of ethidium bromide on mitotic chromosome condensation and its application to high resolution chromosome banding. Cytogenet. Cell Genet. 38:56-61.
    Jaffe, E.S., Harris, N.L., Stein, H., and Vardiman, J.W. 2001. World Health Organization Classification of Tumors. Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. IARC Press, Washington D.C.
    Juliusson, G. and Merup, M. 1998. Cytogenetics in chronic lymphocytic leukemia. Semin. Oncol. 25:19-26.
    ISCN. 1991. Guidelines for cancer cytogenetics, Supplement to An International System for Human Cytogenetic Nomenclature (F. Mitelman, ed.). S. Karger, Basel.
    ISCN. 1995. An International System for Human Cytogenetic Nomenclature (F. Mitelman, ed.). S. Karger, Basel.
    Nowell, P.C. and Hungerford, D.A. 1960. A minute chromosome in human chronic granulocytic leukemia. Science 132:1497.
    Rowley, J.D. 1973. A new consistent chromosomal abnormality in chronic myelogenous leukemia identified by quinacrine fluorescence and Giemsa staining. Nature 243:290-293.
    Sandberg, A. 1990. The Chromosomes in Human Cancer and Leukemia, 2nd ed. Elsevier, New York.
 Key References
    Sandberg, 1990. See above.
    Heim, S. and Mitelam, F. 1995. See above
    Barch, M.J., Knutsen, T., and Spurbeck, J.L. The ACT Cytogenetics Laboratory Manual 3rd ed. Lippincott-Raven, Philadelphia.

All of the above provide comprehensive reviews of current knowledge of methodology and chromosome changes in neoplasia.

 Internet Resources
    http://cgap.nci.nih.gov/Chromosomes/Mitelman

F. Mitelman, B. Johansson, and F. Mertens. 2002. Mitelmam Database of Chromosome Aberrations in Cancer.

     
 
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