Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Correspondence
  • Published:

Clonal chromosomal abnormalities identified by interphase fluorescence in situ hybridization on collagen cultured hematopoietic stem cells

Leukemia volume 14, pages 949–950 (2000)Cite this article

Myelodysplastic syndromes (MDS) are stem cells disorders which associate a mix of both normal cells and clonal malignant cells in various proportions. Recurrent chromosomal abnormalities are frequently associated with these diseases1 and allow clear distinction between the two cell populations. However, cytogenetic preparations of cells from MDS patients cannot provide information concerning the relative proportion of normal vs clonal cells able to maintain a proliferative potential. To obtain such information, a good way consists of cloning hematopoietic progenitors in semi-solid culture conditions, then in analyzing the chromosomal contents of the colonies thus obtained with interphase fluorescence in situ hybridization (FISH). Several semi-solid media have been used to culture clonogenic hematopoietic progenitors, including agar, plasma clot and methylcellulose. However, further analysis of the colonies which requires the use of dried preparations were often impaired by various drawbacks: (1) Agar gels can be dried but erythroid cell growth efficiency is very poor in this medium. Moreover, dried agar is very dense and does not allow FISH probes to efficiently reach the nucleus. (2) Plasma clots could constitute an alternative method for such studies, but this matrix seems less suitable and convenient than collagen or methylcellulose for good reproducibility of cultures growth and quality of slide preparations.2 (3) Methylcellulose cultures cannot be dried out without losing the clonal organization of the cells. In a previous report,3 we demonstrated that FISH could be performed on individual colonies picked from methylcellulose cultures. However, this procedure: (1) is very time-consuming as each clone must be pippeted individually, washed to remove methylcellulose and cytocentrifuged on a glass slide; (2) allows analysis of just a small proportion of the clones. This selection being operator-dependent, a bias can arise at this step in the choice of the colonies to be analyzed since the majority of small colonies or clusters cannot be efficiently processed by this method; (3) requires the analysis of high numbers of slides to obtain interpretable results. This point represents the main contributing factor to excessive cost of such experiments due to the use of large amounts of expansive FISH probes.

Collagen has been shown to be a suitable matrix for hematopoietic progenitor cloning and to allow standard staining or immunocytochemistry labelling of these cells.45 Moreover, dehydration of the gel on a standard glass slide allows complete analysis of all colonies, even the smallest ones, thus avoiding one of the major drawbacks of the methylcellulose-based technique. Since no report concerning FISH analysis of collagen cultures has been published, we established a new method which allows such a study.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1

References

  1. Fenaux P, Morel P, Lai JL . Cytogenetics of myelodysplastic syndromes Semin Hematol 1996 33: 127–138

    CAS  PubMed  Google Scholar 

  2. Mossuz P, Dobo I, Genevay MC, Allegraud A, Dautel M, Niaussat AE, Cousin F, Praloran V, Boccaccio C, Hermouet S . Use of collagen for standardization of PBSC graft quality evaluation: a multicenter comparative analysis of commercial collagen-based and methylcellulose-based colony-forming unit (CFU) assay kits J Hematother 1998 7: 351–359

    Article  CAS  Google Scholar 

  3. Dupont J-M, Fontenay Roupie M, Dupuy O, Lebarr A, Le Tessier D, Auvinet P, Rabineau D, Fichelson S . Fluorescence in situ hybridization on methylcellulose cultured hematopoietic stem cells from myelodysplastic syndromes Cancer Genet Cytogenet 1998 101: 12–15

    Article  CAS  Google Scholar 

  4. Dobo I, Allegraud A, Navenot JM, Boasson M, Bidet JM, Praloran V . Collagen matrix: an attractive alternative to agar and methylcellulose for the culture of hematopoietic progenitors in autologous transplantation products J Hematother 1995 4: 281–287

    Article  CAS  Google Scholar 

  5. Lanotte M . Terminal differentiation of hemopoietic cell clones cultured in tridimensional collagen matrix: in situ cell morphology and enzyme histochemistry analysis Biol Cell 1984 50: 107–120

    Article  CAS  Google Scholar 

  6. Soenen V, Fenaux P, Flactif M, Lepelley P, Lai JL, Cosson A, Preudhomme C . Combined immunophenotyping and in situ hybridization (FICTION): a rapid method to study cell lineage involvement in myelodysplastic syndromes Br J Haematol 1995 90: 701–706

    Article  CAS  Google Scholar 

  7. List AF, Brasfield F, Heaton R, Glinsmann-Gibson B, Crook L, Teatle R, Capizzi R . Stimulation of hematopoiesis by amifostine in patients with myelodysplastic syndrome Blood 1997 90: 3364–3369

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratoire d'Histologie Embryologie Cytogénétique, CHU, Cochin, AP-HP-Université Paris 5, Paris, France

    D Le Tessier, JM Dupont, A Lebbar & D Rabineau

  2. Laboratoire d'Hématopoïèse, Site Transfusionnel, CHU, Cochin, AP-HP-Université Paris 5, Paris, France

    S Fichelson

  3. Laboratoire d'Hématologie, CHU, Cochin, AP-HP-Université Paris 5, Paris, France

    D Bouscary & F Dreyfus

Authors
  1. D Le Tessier
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. JM Dupont
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S Fichelson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. D Bouscary
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. A Lebbar
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. F Dreyfus
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. D Rabineau
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Le Tessier, D., Dupont, J., Fichelson, S. et al. Clonal chromosomal abnormalities identified by interphase fluorescence in situ hybridization on collagen cultured hematopoietic stem cells. Leukemia 14, 949–950 (2000). https://doi.org/10.1038/sj.leu.2401739

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.leu.2401739

Search

Advanced search

Quick links