Metaphase Harvest and Cytogenetic Analysis of Malignant Hematological Specimens

Paola Dal Cin1, Cynthia McLaughlin1

1 CAMD, Cytogenetics Laboratory, Brigham and Women's Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 10.2
DOI:  10.1002/0471142905.hg1002s73
Online Posting Date:  April, 2012
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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 culture, harvest, and preparation of chromosome spreads from bone marrow aspirates. Three alternate protocols describe adaptations of the Basic Protocol for preparation of specimens from chronic lymphocytic leukemia (CLL) blood/bone marrow, plasma cell disorders, mainly bone marrow multiple myeloma (MM), and “solid” hematological samples including lymph nodes and splenic tissue. The modifications involve slightly different culture and harvest methods that are necessary to assure optimal results. Curr. Protoc. Hum. Genet. 73:10.2.1‐10.2.15. © 2012 by John Wiley & Sons, Inc.

Keywords: lymph node; B cell cocktail stimulation; chromosome spreads; mitogen; bone marrow; unstimulated culture

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Chromosome Spreads from Unstimulated Bone Marrow and Leukemic Blood Specimens
  • Alternate Protocol 1: Preparation of Chromosome Spreads from Chronic Lymphocytic Leukemia Bone Marrow or Peripheral Blood Specimens
  • Alternate Protocol 2: Preparation of Chromosome Spreads from Bone Marrow Multiple Myeloma Specimens
  • Alternate Protocol 3: Preparation of Chromosome Spreads from Lymph Node and Spleen Specimens
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Chromosome Spreads from Unstimulated Bone Marrow and Leukemic Blood Specimens

  Materials
  • Complete culture media, A and B (see reciperecipes)
  • 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)
  • Hypotonic solution: 0.075 M KCl solution
  • 3:1 (v/v) methanol/glacial acetic acid fixative, prepared fresh
  • 50‐ml tissue‐culture flasks (Falcon)
  • 37°C, 5% CO 2 incubator
  • 15‐ml centrifuge tubes (Corning), sterile
  • Benchtop centrifuge (e.g., IEC HN‐SII)
  • Vortex mixer
  • Pasteur pipets
  • Precleaned microscope slides (Gold Seal; Becton Dickinson)
  • 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% CO 2 incubator unless otherwise specified.

Alternate Protocol 1: Preparation of Chromosome Spreads from Chronic Lymphocytic Leukemia Bone Marrow or Peripheral Blood Specimens

  • Pokeweed mitogen (PWM; see recipe)
  • GNKG685 (CpG) is a custom oligonucleotide
  • Phorbol 12‐myristate 13‐acetate (PMA)

Alternate Protocol 2: Preparation of Chromosome Spreads from Bone Marrow Multiple Myeloma Specimens

  • Interleukin 4 (IL‐4; see recipe)

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

  • Collagenase solution (see recipe)
  • Bone marrow medium (see reciperecipes for complete culture medium)
  • 5‐ml petri dish, 60 × 15–mm (Falcon)
  • Disposable scalpels
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Figures

Videos

Literature Cited

   Boveri, T. 1929. The Origin of Malignant Tumors. Baliere, Tindall & Cox, London.
   Chen, Z., Issa, B., Huang, S., Aston, E., Xu, J., Yu, M., Brothman, A.R., and Glenn, M. 2005. A practical approach to the detection of prognostically significant genomic aberrations in multiple myeloma. J. Mol. Diagn. 7:560‐565.
   Crossen, P.E. 1997. Genes and chromosomes in chronic B‐cell leukemia. Cancer Genet. Cytogenet. 94:44‐51.
   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.
   Dicker, F., Schnittger, S., Haferlach, T., Kern, W., and Schoch, C. 2006. Immunostimulatory oligonucleotide‐induced metaphase cytogenetics detect chromosomal aberrations in 80% of CLL patients: A study of 132 CLL cases with correlation to FISH, IgVH status, and CD38 expression. Blood 108:3152‐3160.
   Döhner, H., Stilgenbauer, S., Benner, A., Leupolt, E., Krober, A., Bullinger, L., Dohner, K., Bentz, M., and Lichter, P. 2000. Genomic aberrations and survival in chronic lymphocytic leukemia. N. Engl. J. Med. 343:1910‐1916.
   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.
   Heerema, N.A., Byrd, J.C., Dal Cin, P., Dell' Aquila, M.L., Koduru, P., Aviram, A., Smoley, S., Rassenti, L.Z., Greaves, A.W., Brown, J.R., Rai, K.R., Kipps, T.J., Kay, N.E., and Van Dyke, D. 2010. Stimulation of chronic lymphocytic leukemia (CLL) cells with CpG oligodeoxynucleotide (ODN) gives consistent karyotypic results among laboratories: A CLL research consortium (CRC) study. Cancer Genet. Cytogenet. 203:134‐140.
   Heim, S. and Mitelman, F. 2009. Cancer Cytogenetics, 3rd 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.
   ISCN. 1985. An International System for Human Cytogenetic Nomenclature (D.G. Harden and H.P. Klinger, eds.). S. Karger, Basel.
   ISCN. 1991. Guidelines for cancer cytogenetics, Supplement to An International System for Human Cytogenetic Nomenclature (F. Mitelman, ed.). S. Karger, Basel.
   ISCN. 2009. An International System for Human Cytogenetic Nomenclature (L.G. Shaffer, M.L. Slovak, and J.C. Campbell, eds.). S. Karger, Basel.
   Juliusson, G. and Merup, M. 1998. Cytogenetics in chronic lymphocytic leukemia. Semin. Oncol. 25:19‐26.
   Mayr, C., Speicher, M.R., Kofler, D.M., Buhmann, R., Strehl, J., Busch, R., Hallek, M., and Wendtner, C.M. 2006. Chromosomal translocations are associated with poor prognosis in chronic lymphocytic leukemia. Blood 107:742‐751.
   Nowell, P.C. and Hungerford, D.A. 1960. A minute chromosome in human chronic granulocytic leukemia. Science 132:1497.
   Put, N., Konings, P., Rack, K., Jamar, M., Van Roy, N., Libouton, J.M., Vannuffel, P., Sartenaer, D., Ameye, G., Speleman, F., Herens, C., Poirel, H.A., Moreau, Y., Hagemeijer, A., Vandenberghe, P., and Michaux, L. 2009. Improved detection of chromosomal abnormalities in chronic lymphocytic leukemia by conventional cytogenetics using CpG oligonucleotide and interleukin‐2 stimulation: A Belgian multicentric study. Genes Chromosomes Cancer 48:843‐853.
   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.
   Slovak, M.L., Bedell, V., Pagel, K., Cang, K.L., Smith, D., and Somlo, G. 2005. Targeting plasma cells improves detection of cytogenetic aberrations in multiple myeloma: phenotype/genotype fluorescent in situ hybridization. Cancer Genet. Cytogenet. 158:99‐109.
   Swerdlow, S.H., Campo, E., Harris, N.L., Jaffe, E.S., Pileri, Sa., Stain, H., Thiele, J., and Vardiman, J.W. (eds.) 2008. WHO classification of tumors and haematopoietic and lymphoid tissues. Lyon.
Key Reference
   Sandberg, 1990. See above.
  Provides comprehensive reviews of current knowledge of methodology and chromosome changes in neoplasia.
   ISCN. 2009. See above.
Internet Resources
   http://atlasgeneticsoncology.org/
  Atlas of Genetics and Cytogenetics in Oncology and Haematology: Editor: Huret J‐L.
   http://cgap.nci.nih.gov/Chromosomes/Mitelman
  F. Mitelman, B. Johansson, and F. Mertens. Mitelmam Database of Chromosome Aberrations in Cancer.
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