Skip to main content

PCR-Based Analysis of Rearranged Immunoglobulin or T-Cell Receptor Genes by GeneScan Analysis or Heteroduplex Analysis for Clonality Assessment in Lymphoma Diagnostics

  • Protocol
  • First Online:
Lymphoma

Part of the book series: Methods in Molecular Biology ((MIMB,volume 971))

Abstract

The assessment of the presence of clonal lymphoproliferations via polymerase chain reaction (PCR)-based analysis of rearranged immunoglobulin (Ig) or T-cell receptor (TCR) genes is a valuable technique in the diagnosis of suspect lymphoproliferative disorders. Furthermore this technique is more and more used to evaluate dissemination of non-Hodgkin lymphoma and/or the presence of (minimal) residual disease. In this chapter we describe an integrated approach to assess clonality via analysis of Ig heavy chain (IGH), Ig kappa (IGK), TCR beta (TCRB), and TCR gamma (TCRG) gene rearrangements. The described PCR protocol is based on the standardized multiplex PCRs as developed by the European BIOMED-2 collaborative study (Concerted Action BMH4-CT98-3936). Furthermore it also includes the pre-analytical DNA isolation step from various tissues (formalin fixed paraffin-embedded tissue, fresh tissues, body fluids, peripheral blood and bone marrow), GeneScan analysis of labeled PCR products on a genetic analyzer, heteroduplex analysis of unlabeled PCR products, and post-analytical guidelines for the interpretation of the obtained “molecular morphology” patterns.

On behalf of the EuroClonality/BIOMED-2 Collaborative Study Group

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

Access this chapter

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
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

Similar content being viewed by others

References

  1. Tonegawa S (1983) Somatic generation of antibody diversity. Nature 302:575–581

    Article  CAS  PubMed  Google Scholar 

  2. Davis MM, Bjorkman PJ (1988) T-cell antigen receptor genes and T-cell recognition. Nature 334:395–402

    Article  CAS  PubMed  Google Scholar 

  3. Van Dongen JJM, Langerak AW, Brüggemann M, Evans PA, Hummel M, Lavender FL, Delabesse E, Davi F, Schuuring E, García-Sanz R, Van Krieken JHJM, Droese J, González D, Bastard C, White HE, Spaargaren M, González M, Parreira A, Smith JL, Morgan GJ, Kneba M, Macintyre EA (2003) Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 17:2257–2317

    Article  PubMed  Google Scholar 

  4. Van Zelm MC, Van der Burg M, De Ridder D, Barendregt BH, De Haas EF, Reinders MJ, Lankester AC, Révész T, Staal FJT, Van Dongen JJM (2005) Ig gene rearrangement steps are initiated in early human precursor B cell subsets and correlate with specific transcription factor expression. J Immunol 175:5912–5922

    PubMed  Google Scholar 

  5. Dik WA, Pike-Overzet K, Weerkamp F, De Ridder D, De Haas EF, Baert MRM, Van der Spek P, Koster EEL, Reinders MJT, Van Dongen JJM, Langerak AW, Staal FJT (2005) New insights on human T-cell development by quantitative T-cell receptor gene rearrangement studies and gene expression profiling. J Exp Med 201:1715–1723

    Article  CAS  PubMed  Google Scholar 

  6. Evans PA, Pott C, Groenen PJ, Salles G, Davi F, Berger F, Garcia JF, Van Krieken JHJM, Pals ST, Kluin PM, Schuuring E, Spaargaren M, Boone E, González D, Martinez B, Villuendas R, Gameiro P, Diss TC, Mills K, Morgan GJ, Carter GI, Milner BJ, Pearson D, Hummel M, Jung W, Ott M, Canioni D, Beldjord K, Bastard C, Delfau-Larue MH, Van Dongen JJM, Molina TJ, Cabeçadas J (2007) Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets. Report of the BIOMED-2 Concerted Action BHM4-CT98-3936. Leukemia 21:207–214

    Article  CAS  PubMed  Google Scholar 

  7. Brüggemann M, White H, Gaulard P, Garcia-Sanz R, Gameiro P, Oeschger S, Jasani B, Ott M, Delsol G, Orfao A, Tiemann M, Herbst H, Langerak AW, Spaargaren M, Moreau E, Groenen PJTA, Sambade C, Foroni L, Carter GI, Hummel M, Bastard C, Davi F, Delfau MH, Kneba M, Van Dongen JJM, Macintyre EA, Molina TJ (2007) Powerful strategy for PCR-based clonality assessment in T-cell malignancies. Report of the BIOMED-2 Concerted Action BHM4-CT98-3936. Leukemia 21:215–221

    Article  PubMed  Google Scholar 

  8. Langerak AW, Molina TJ, Lavender FL, Pearson D, Flohr T, Sambade C, Schuuring E, Al ST, Van Dongen JJM, Van Krieken JHJM (2007) PCR-based clonality testing in tissue samples with reactive lymphoproliferations: usefulness and pitfalls. A study from the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 21:222–229

    Article  CAS  PubMed  Google Scholar 

  9. Diss TC, Molina TJ, Cabeçadas J, Langerak AW (2012) Molecular diagnostics in lymphoma: why, when and how to apply. Diagn Histopathol 18:53–63

    Article  Google Scholar 

  10. Van Krieken JHJM, Langerak AW, Macintyre EA, Kneba M, Smith JL, Garcia SR, Morgan GJ, Parreira A, Molina T, Cabeçadas J, Gaulard P, Jasani B, Garcia JF, Ott M, Hannsmann ML, Berger F, Hummel M, Davi F, Brüggemann M, Lavender FL, Schuuring EMD, Evans PAS, White H, Salles G, Groenen PJTA, Gameiro P, Pott C, Van Dongen JJM (2007) Improved reliability of lymphoma diagnostics via PCR-based clonality testing. Report of the BIOMED-2 Concerted Action BHM4-CT98-3936. Leukemia 21:201–206

    Article  PubMed  Google Scholar 

  11. Rothberg PG, Langerak AW, Verhaaf B, Van Dongen JJM, Burack WR, Johnson MD, Slate D, Laughlin TS, Payne K, Figueiredo L, Bandoh BN, Yan Q, Bacon CM, Wright P, Bench A, Du MQ, Liu H (2012) Clonal antigen receptor gene PCR products outside the expected size range. J Hematopathol 5:57–67

    Article  Google Scholar 

  12. Langerak AW, Van Dongen JJM (2012) Multiple clonal Ig/TCR products: implications for interpretation of clonality findings. J Hematopathol 5:35–43

    Article  Google Scholar 

  13. Langerak AW, Groenen PJTA, Brüggemann M, Beldjord K, Bellan C, Bonello L, Boone E, Carter I, Catherwood M, Davi F, Delfau-Larue MH, Diss T, Evans PAS, Gameiro P, Garcia Sanz R, Gonzalez D, Grand D, Håkansson A, Hummel M, Liu H, Lombardia L, Macintyre EA, Milner B, Montes-Moreno S, Schuuring E, Spaargaren M, Hodges E, Van Dongen JJM (2012) EuroClonality / BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations. Leukemia 26:2159–2172

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Anton W. Langerak .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Boone, E., Verhaaf, B., Langerak, A.W. (2013). PCR-Based Analysis of Rearranged Immunoglobulin or T-Cell Receptor Genes by GeneScan Analysis or Heteroduplex Analysis for Clonality Assessment in Lymphoma Diagnostics. In: Küppers, R. (eds) Lymphoma. Methods in Molecular Biology, vol 971. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-269-8_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-269-8_4

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-268-1

  • Online ISBN: 978-1-62703-269-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics