title: Laboratory strategies for efficient handling of paraffin-embedded tissues for molecular detection of clonality in non-hodgkin lymphomas.
abstract: We herein present a technical strategy to optimize DNA isolation from paraffin-embedded tissue (PET). This includes the choice of adequate buffers for proteinase K digestion and multiplex PCR amplifications for assessing the appropriateness of DNA extracts for subsequent PCR assays for detecting clonality. We found that the association of proteinase K digestion in nonionic buffer and subsequent extract dilutions accounted for 79% of successful amplifications. A final efficiency of 88% was achieved by additional organic extractions and/or re-extractions. Comparisons were carried out with control DNA extracts from fresh samples to assess the efficiency of each clonality assay. Immunoglobulin CDRIII rearranged region amplification was more efficient for pregerminal center B-cell lymphomas in contrast to CDRII rearrangement detection, which was more effective for germinal and postgerminal lymphomas. T-cell clonality detection by TCRgamma PCR was less efficient in PET samples than in fresh tissues showing that DNA integrity is more critical for TCR than for IGH amplification. Two inconclusive cases without phenotypic markers and two other atypical lymphoproliferations masked by reactive T cells were diagnosed as plasmablastic lymphomas and as monoclonal B-proliferations, respectively, due to IGH rearrangements.
authors.affiliation: Centro de Transplante de Medula Ossea (CEMO) Instituto Nacional de Câncer (INCa), Rio de Janeiro, Brasil.
authors.forename: Claudio Gustavo
authors.initials: CG
authors.lastname: Stefanoff
authors.forename: Rocío
authors.initials: R
authors.lastname: Hassan
authors.forename: Ana Carolina
authors.initials: AC
authors.lastname: Gonzalez
authors.forename: Luiz Antônio B
authors.initials: LA
authors.lastname: Andrade
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Tabak
authors.forename: Sérgio
authors.initials: S
authors.lastname: Romano
authors.forename: Ilana R
authors.initials: IR
authors.lastname: Zalcberg
mesh_terms: D002999:Clone Cells / Q000473:pathology; D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm / Q000302:isolation & purification*; D019286:Endopeptidase K / Q000378:metabolism; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D015334:Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor; D006801:Humans; D016393:Lymphoma, B-Cell / Q000235:genetics* / Q000473:pathology; D025202:Molecular Diagnostic Techniques; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; D004273:DNA, Neoplasm; D019286:Endopeptidase K