Skip to main content
SpringerLink
Log in

Laser-Based Microdissection of Single Cells from Tissue Sections and PCR Analysis of Rearranged Immunoglobulin Genes from Isolated Normal and Malignant Human B Cells

  • Protocol
  • First Online:
Lymphoma

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1956))

Abstract

Normal and malignant B cells carry rearranged immunoglobulin (Ig) variable region genes, which due to their practically limitless diversity represent ideal clonal markers for these cells. We describe here an approach to isolate single cells from frozen tissue sections by microdissection using a laser-based method. From the isolated cells, rearranged IgH and Igκ genes are amplified in a semi-nested PCR approach, using a collection of V gene subgroup-specific primers recognizing nearly all V genes together with primers for the J genes. By sequence analysis of V region genes from distinct cells, the clonal relationship of the B lineage cells can unequivocally be determined and related to the histological distribution of the cells. The approach is also useful to determine V, D, and J gene usage. Moreover, the presence and pattern of somatic Ig V gene mutations give valuable insight into the stage of differentiation of the B cells.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Cook GP, Tomlinson IM (1995) The human immunoglobulin VH repertoire. Immunol Today 16:237–242

    Article  CAS  Google Scholar 

  2. Corbett SJ, Tomlinson IM, Sonnhammer EL et al (1997) Sequence of the human immunoglobulin diversity (D) segment locus: a systematic analysis provides no evidence for the use of DIR segments, inverted D segments, “minor” D segments or D-D recombination. J Mol Biol 270:587–597

    Article  CAS  Google Scholar 

  3. Ravetch JV, Siebenlist U, Korsmeyer S et al (1981) Structure of the human immunoglobulin mu locus: characterization of embryonic and rearranged J and D genes. Cell 27:583–591

    Article  CAS  Google Scholar 

  4. Hieter PA, Maizel JV Jr, Leder P (1982) Evolution of human immunoglobulin kappa J region genes. J Biol Chem 257:1516–1522

    CAS  PubMed  Google Scholar 

  5. Kawasaki K, Minoshima S, Nakato E et al (1997) One-megabase sequence analysis of the human immunoglobulin lambda gene locus. Genome Res 7:250–261

    Article  CAS  Google Scholar 

  6. Schäble KF, Zachau HG (1993) The variable genes of the human immunoglobulin kappa locus. Biol Chem Hoppe Seyler 374:1001–1022

    Article  Google Scholar 

  7. Vasicek TJ, Leder P (1990) Structure and expression of the human immunoglobulin lambda genes. J Exp Med 172:609–620

    Article  CAS  Google Scholar 

  8. Küppers R, Zhao M, Hansmann ML et al (1993) Tracing B cell development in human germinal centres by molecular analysis of single cells picked from histological sections. EMBO J 12:4955–4967

    Article  Google Scholar 

  9. Rajewsky K (1996) Clonal selection and learning in the antibody system. Nature 381:751–758

    Article  CAS  Google Scholar 

  10. Kanzler H, Küppers R, Hansmann ML et al (1996) Hodgkin and Reed-Sternberg cells in Hodgkin’s disease represent the outgrowth of a dominant tumor clone derived from (crippled) germinal center B cells. J Exp Med 184:1495–1505

    Article  CAS  Google Scholar 

  11. Küppers R, Rajewsky K, Zhao M et al (1994) Hodgkin disease: Hodgkin and Reed-Sternberg cells picked from histological sections show clonal immunoglobulin gene rearrangements and appear to be derived from B cells at various stages of development. Proc Natl Acad Sci U S A 91:10962–10966

    Article  Google Scholar 

  12. Küppers R, Zhao M, Rajewsky K et al (1993) Detection of clonal B cell populations in paraffin-embedded tissues by polymerase chain reaction. Am J Pathol 143:230–239

    PubMed  PubMed Central  Google Scholar 

  13. McCarthy KP, Sloane JP, Wiedemann LM (1990) Rapid method for distinguishing clonal from polyclonal B cell populations in surgical biopsy specimens. J Clin Pathol 43:429–432

    Article  CAS  Google Scholar 

  14. Trainor KJ, Brisco MJ, Wan JH et al (1991) Gene rearrangement in B- and T-lymphoproliferative disease detected by the polymerase chain reaction. Blood 78:192–196

    CAS  PubMed  Google Scholar 

  15. Goossens T, Klein U, Küppers R (1998) Frequent occurrence of deletions and duplications during somatic hypermutation: implications for oncogene translocations and heavy chain disease. Proc Natl Acad Sci U S A 95:2463–2468

    Article  CAS  Google Scholar 

  16. Klein U, Rajewsky K, Küppers R (1998) Human immunoglobulin (Ig)M+IgD+ peripheral blood B cells expressing the CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells. J Exp Med 188:1679–1689

    Article  CAS  Google Scholar 

  17. Goossens T, Bräuninger A, Klein U et al (2001) Receptor revision plays no major role in shaping the receptor repertoire of human memory B cells after the onset of somatic hypermutation. Eur J Immunol 31:3638–3648

    Article  CAS  Google Scholar 

  18. Kurth J, Spieker T, Wustrow J et al (2000) EBV-infected B cells in infectious mononucleosis: viral strategies for spreading in the B cell compartment and establishing latency. Immunity 13:485–495

    Article  CAS  Google Scholar 

  19. Bräuninger A, Küppers R, Spieker T et al (1999) Molecular analysis of single B cells from T cell-rich B-cell lymphoma shows the derivation of the tumor cells from mutating germinal center B cells and exemplifies means by which immunoglobulin genes are modified in germinal center B cells. Blood 93:2679–2687

    PubMed  Google Scholar 

  20. Braeuninger A, Küppers R, Strickler JG et al (1997) Hodgkin and Reed-Sternberg cells in lymphocyte predominant Hodgkin disease represent clonal populations of germinal center-derived tumor B cells. Proc Natl Acad Sci U S A 94:9337–9342

    Article  CAS  Google Scholar 

  21. Müschen M, Küppers R, Spieker T et al (2001) Molecular single-cell analysis of Hodgkin- and Reed-Sternberg cells harboring unmutated immunoglobulin variable region genes. Lab Investig 81:289–295

    Article  Google Scholar 

  22. Küppers R, Bräuninger A, Müschen M et al (2001) Evidence that Hodgkin and Reed-Sternberg cells in Hodgkin disease do not represent cell fusions. Blood 97:818–821

    Article  Google Scholar 

  23. Bräuninger A, Goossens T, Rajewsky K et al (2001) Regulation of immunoglobulin light chain gene rearrangements during early B cell development in the human. Eur J Immunol 31:3631–3637

    Article  Google Scholar 

  24. Hieter PA, Korsmeyer SJ, Waldmann TA et al (1981) Human immunoglobulin kappa light-chain genes are deleted or rearranged in lambda-producing B cells. Nature 290:368–372

    Article  CAS  Google Scholar 

  25. Korsmeyer SJ, Hieter PA, Sharrow SO et al (1982) Normal human B cells display ordered light chain gene rearrangements and deletions. J Exp Med 156:975–985

    Article  CAS  Google Scholar 

  26. Zhang L, Cui X, Schmitt K et al (1992) Whole genome amplification from a single cell: implications for genetic analysis. Proc Natl Acad Sci U S A 89:5847–5851

    Article  CAS  Google Scholar 

  27. Kanzler H, Küppers R, Helmes S et al (2000) Hodgkin and Reed-Sternberg-like cells in B-cell chronic lymphocytic leukemia represent the outgrowth of single germinal-center B-cell-derived clones: potential precursors of Hodgkin and Reed-Sternberg cells in Hodgkin’s disease. Blood 95:1023–1031

    CAS  PubMed  Google Scholar 

  28. Müschen M, Re D, Bräuninger A et al (2000) Somatic mutations of the CD95 gene in Hodgkin and Reed-Sternberg cells. Cancer Res 60:5640–5643

    PubMed  Google Scholar 

  29. Brezinschek HP, Brezinschek RI, Lipsky PE (1995) Analysis of the heavy chain repertoire of human peripheral B cells using single-cell polymerase chain reaction. J Immunol 155:190–202

    CAS  PubMed  Google Scholar 

  30. Brezinschek HP, Foster SJ, Dörner T et al (1998) Pairing of variable heavy and variable kappa chains in individual naive and memory B cells. J Immunol 160:4762–4767

    CAS  PubMed  Google Scholar 

  31. Küppers R (1997) Ongoing somatic mutation in mantle cell lymphomas questioned. Br J Haematol 97:932–934

    PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft and the Deutsche Krebshilfe. We are grateful to all present and previous members of the group and coworkers that were involved in the establishment of the protocols described here.

Author information

Authors and Affiliations

  1. Institute of Cell Biology (Cancer Research), Medical School, University of Duisburg-Essen, Nordrhein-Westfalen, Essen, Germany

    Ralf Küppers

  2. Senckenberg Institute of Pathology, University of Frankfurt, Frankfurt am Main, Germany

    Markus Schneider & Martin-Leo Hansmann

  3. Frankfurt Institute of Advanced Studies, Frankfurt am Main, Germany

    Martin-Leo Hansmann

Authors
  1. Ralf Küppers
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Markus Schneider
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Martin-Leo Hansmann
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ralf Küppers .

Editor information

Editors and Affiliations

  1. Institute of Cell Biology (Cancer Research), Medical School, University of Duisburg-Essen, Nordrhein-Westfalen, Essen, Germany

    Ralf Küppers

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Küppers, R., Schneider, M., Hansmann, ML. (2019). Laser-Based Microdissection of Single Cells from Tissue Sections and PCR Analysis of Rearranged Immunoglobulin Genes from Isolated Normal and Malignant Human B Cells. In: Küppers, R. (eds) Lymphoma. Methods in Molecular Biology, vol 1956. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9151-8_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9151-8_3

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9150-1

  • Online ISBN: 978-1-4939-9151-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation