Increased sensitivity of B-cell clonality analysis in formalin-fixed and paraffin-embedded B-cell lymphoma samples using an enzyme blend with both 5′→3′ DNA polymerase and 3′→5′ exonuclease activity

Timea P. Gurbity, Eniko Bagdi, Nicole A. Groen, Leo M. Budel, Mustaffa Abbou, Laszlo Krenacs, Winand N.M. Dinjens

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Polymerase chain reaction (PCR)-based detection of immunoglobulin heavy chain (IgH) gene rear-rangement for determination of B-cell clonality needs to be simple but optimally sensitive. Efficient IgH PCR analysis can be hampered by sequence variability in the template DNA, despite of the use of degenerative primers. To improve sensitivity of the B-cell clonality analysis in formalin-fixed and paraffin-embedded (FFPE) tissues, we have performed framework three-area (FR3)/joining gene (JH) IgH PCR utilizing an enzyme blend (rTth DNA Polymerase, XL) providing both 5′→3′ polymerase and 3′→5′ exonuclease activities. The DNA samples were extracted from FFPE biopsies of 43 mature B-cell lymphoma cases of so-called germinal center and post-germinal center origin, including 6 nodal follicular lymphomas (FL), 15 gastric mucosa-associated lymphoid tissue (MALT) lymphomas, and 22 gastric diffuse large B-cell lymphomas (DLBCL). Of the cases, 31 (17 DLBCL and 14 MALT lymphoma) represented small endoscopic biopsies. Serial dilutions of target DNA were applied to avoid inconsistent bands that may be seen when the input amount of template is too low, which can be the case when DNA is extracted from FFPE endoscopic gastric biopsies. Using conventional Taq polymerase, consistent monoclonal product was found in 53% (23/ 43) of the cases (FL: 67%; MALT lymphoma: 47%; DLBCL: 55%). The rTth polymerase showed reproducible monoclonal pattern in 72% (31/43) of the cases (FL: 67%; MALT lymphoma: 73%; DLBCL: 73%); the sensitivity is compatible with one that can be detected with conventional FR3/JH PCR in fresh/frozen tissues. In conclusion, the rTth DNA polymerase greatly improves sensitivity of FR3/JH PCR in FFPE biopsies of mature B-cell lymphomas, most probably by increasing the primer matches during PCR amplification.

Original languageEnglish
Pages (from-to)643-648
Number of pages6
JournalVirchows Archiv
Volume443
Issue number5
DOIs
Publication statusPublished - Nov 1 2003

Keywords

  • B-cell lymphoma
  • Clonality
  • FR3
  • PCR
  • Paraffin

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Molecular Biology
  • Cell Biology

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