Advertisement

Human lymphocyte sorting by gravitational field-flow fractionation

  • Barbara RodaEmail author
  • Pierluigi Reschiglian
  • Andrea Zattoni
  • Pier Luigi Tazzari
  • Marina Buzzi
  • Francesca Ricci
  • Andrea Bontadini
Original Paper

Abstract

Interest in biological studies on various cell types for many biomedical applications, from research to patient treatments, is constantly increasing. The ability to discriminate (sort) and/or quantify distinct subpopulations of cells has become increasingly important. For instance, not only detection but also the highest depletion of neoplastic cells from normal cells is an important requisite in the autologous transplantation of lymphocytes for blood cancer treatments. In this work, gravitational field-flow fractionation (GrFFF) is shown to be effective for sorting a heterogeneous mixture of human, living lymphocytes constituted of neoplastic B cells from a Burkitt lymphoma cell line and healthy T and B lymphocytes from blood samples. GrFFF does not require the use of fluorescent immunotags for sorting cells, and the sorted cells can be collected for their further characterization. Flow cytometry was used to assess the viability of the cells collected, and to evaluate the cell fractionation achieved. A low amount of neoplastic B lymphocytes (less than 2%) was found in a specific fraction obtained by GrFFF. The high depletion from neoplastic cells (more than 98%) was confirmed by a clonogenicity test.

Keywords

Gravitational field-flow fractionation Cell sorting Lymphocytes 

Notes

Acknowledgement

This work was supported by the Ministero Italiano dell’Istruzione, dell’Università e della Ricerca.

References

  1. 1.
    Gnjatic S, Nishikawa H, Jungbluth A, Güre AO, Ritter G, Jäger E, Knuth A, Chen YT, Old LJ (2006) Adv Cancer Res 95:1–30CrossRefGoogle Scholar
  2. 2.
    Salzman HM, Singham SB, Johnston RG, Bohren CF (1989) In: Melamed M, Lindmo T, Mendelshon ML (eds) Flow cytometry and sorting, 2nd edn. Wiley, New York, p 81Google Scholar
  3. 3.
    Hoffman RM, Yang M (2006) Nat Protoc 1:775–782CrossRefGoogle Scholar
  4. 4.
    Giddings JC (1993) Science 260:1456–1465CrossRefGoogle Scholar
  5. 5.
    Ratanathanawongs-Williams SK (2000) In: Schimpf ME, Caldwell KC, Giddings JC (eds) Field-flow fractionation handbook, chap 17. Wiley-Interscience, New YorkGoogle Scholar
  6. 6.
    Lucas A, Lepage F, Cardot PJP (2000) In: Schimpf ME, Caldwell KC, Giddings JC (eds) Field-flow fractionation handbook, chap 29. Wiley-Interscience, New YorkGoogle Scholar
  7. 7.
    Reschiglian P, Zattoni A, Roda B, Michelini E, Roda A (2005) Trends Biotechnol 23:475–483CrossRefGoogle Scholar
  8. 8.
    Caldwell KD, Cheng ZQ, Hradecky P, Giddings JC (1984) Cell Biophys 6:233–251Google Scholar
  9. 9.
    Battu S, Elyaman W, Hugon J, Cardot PJP (2001) Biochim Biophys Acta 1528:89–96Google Scholar
  10. 10.
    Corbiere C, Battu S, Liagre B, Cardot PJP, Beneytout JL (2004) J Chromatogr B 808:255–262CrossRefGoogle Scholar
  11. 11.
    Lautrette C, Cardot PJP, Vermut-Desroches C, Wijdenes J, Jauberteau MM, Battu S (2003) J Chromatogr B 791:149–160CrossRefGoogle Scholar
  12. 12.
    Guglielmi L, Battu S, Le Bert M, Faucher JL, Cardot PJP, Denizot Y (2004) Anal Chem 76:1580–1585CrossRefGoogle Scholar
  13. 13.
    Parsons R, Yue V, Tong X, Cardot PJP, Bernard A, Andreux JP, Caldwell K (1996) J Chromatogr B 686:177–187CrossRefGoogle Scholar
  14. 14.
    Metreau JM, Gallet S, Cardot PJP, LeMaire V, Dumas F, Hernvann A, Loric S (1997) Anal Biochem 251:178–186CrossRefGoogle Scholar
  15. 15.
    Bernard A, Bories C, Loiseau PM, Cardot PJP (1995) J Chromatogr B 664:444–448CrossRefGoogle Scholar
  16. 16.
    Reschiglian P, Zattoni A, Roda B, Casolari S, Moon MH, Lee J, Jung J, Rodmålm K, Cenacchi G (2002) Anal Chem 74:4895–4904CrossRefGoogle Scholar
  17. 17.
    Merino A, Bories C, Gantier JC, Cardot PJP (1991) J Chromatogr 572:291–301CrossRefGoogle Scholar
  18. 18.
    Sanz R, Puignou L, Reschiglian P, Galceran MT (2001) J Chromatogr A 919:339–347CrossRefGoogle Scholar
  19. 19.
    Urbankova E, Vacek A, Chmelík J (1996) J Chromatogr B 687:449–452CrossRefGoogle Scholar
  20. 20.
    Merino-Dugay A, Cardot PJP, Czok M, Guernet M, Andreux JP (1992) J Chromatogr 579:73–83CrossRefGoogle Scholar
  21. 21.
    Benincasa M-A, Moore L, Williams PS, Poptic E, Carpino F, Zborowski M (2005) Anal Chem 77:5294–5301CrossRefGoogle Scholar
  22. 22.
    Bigelow JC, Giddings JC, Nabeshima Y, Tsuruta T, Kataoka K, Okano T, Yui N, Sakurai Y (1989) J Immunol Methods 117:289–293CrossRefGoogle Scholar
  23. 23.
    Wang X-B, Yang J, Huang Y, Vykoukal J, Becker FF, Gascoyne PRC (2000) Anal Chem 72:832–839CrossRefGoogle Scholar
  24. 24.
    Leone F, Cavalloni G, Gunetti M, Pignochino Y, Piacibello W, Aglietta M (2006) Biol Blood Marrow Transplant 12:68–74CrossRefGoogle Scholar
  25. 25.
    Biddison WE (2001) Curr Protoc Cell Biol Chap 2:Unit 2.2Google Scholar
  26. 26.
    Loken MR, Brosnan JM, Bach BA, Ault KA (1990) Cytometry 11:453–459CrossRefGoogle Scholar
  27. 27.
    Temmink JHM, Collard JG, Roosien J, Van den Bosch JF (1976) J Cell Sci 21:563–578Google Scholar
  28. 28.
    Melucci D, Roda B, Zattoni A, Casolari S, Reschiglian P, Roda A (2004) J Chromatogr A 1056:229–236Google Scholar
  29. 29.
    Roda B, Cioffi N, Ditaranto N, Zattoni A, Casolari S, Melucci D, Reschiglian P, Sabbatini L, Valentini A, Zambonin PG (2005) Anal Bioanal Chem 381:639–646CrossRefGoogle Scholar
  30. 30.
    Battu S, Roux A, Delebasee S, Bosgiraud C, Cardot PJP (2001) J Chromatogr B 751:131–141CrossRefGoogle Scholar
  31. 31.
    Moriyama Y, Goto T, Hashimoto S, Furukawa T, Kishi K, Takahashi M, Shibata A (1992) Ann Hematol 4:266–269CrossRefGoogle Scholar
  32. 32.
    Polliack A, Lampen N, Clarkson BD, De Harven E, Bentwich Z, Siegal FP, Kunkel HG (1973) J Exp Med 38:607–624CrossRefGoogle Scholar
  33. 33.
    Epstein MA, Achong BG, Barr YM, Zajac B, Henle G, Henle W (1967) J Natl Cancer Inst 37:547–559Google Scholar
  34. 34.
    Reschiglian P, Melucci D, Torsi G (1996) J Chromatogr A 740:245–252CrossRefGoogle Scholar
  35. 35.
    Chmelik J (1999) J Chromatogr A 845:285–291CrossRefGoogle Scholar
  36. 36.
    Plocek J, Konecny P, Chmelík J (1994) J Chromatogr B 656:427–431CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Barbara Roda
    • 1
    • 2
    Email author
  • Pierluigi Reschiglian
    • 1
    • 2
  • Andrea Zattoni
    • 1
    • 2
  • Pier Luigi Tazzari
    • 3
  • Marina Buzzi
    • 3
  • Francesca Ricci
    • 3
  • Andrea Bontadini
    • 3
  1. 1.Department of Chemistry “G. Ciamician”University of BolognaBolognaItaly
  2. 2.National Institute of Biostructure and BiosystemsInteruniversity ConsortiumRomeItaly
  3. 3.Service of Transfusion MedicineS. Orsola-Malpighi HospitalBolognaItaly

Personalised recommendations