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Laboratory Strategies for Efficient Handling of Paraffin-Embedded Tissues for Molecular Detection of Clonality in Non-Hodgkin Lymphomas

Stefanoff, Claudio Gustavo M.Sc.; Hassan, Rocío M.Sc.; Gonzalez, Ana Carolina; Andrade, Luiz Antônio B. Ph.D.; Tabak, Daniel G. M.D.; Romano, Sérgio M.D.; Zalcberg, Ilana R. M.D., Ph.D.

Diagnostic Molecular Pathology: June 2003 - Volume 12 - Issue 2 - pp 79-87

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 TCRγ 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.

Since the application of polymerase chain reaction (PCR)-based techniques in diagnosis, archival formalin-fixed, paraffin-embedded tissue (PET) has become a valuable source of material for molecular studies (1). In non-Hodgkin lymphomas (NHL), conventional morphologic and immunohistochemical approaches account for approximately 90% of diagnoses. However, a small but significant number of cases exhibit microscopic and clinical characteristics similar to other pathologies (2,3). Some nonlymphoid malignancies and immune benign hyperplasias can be mistaken with lymphomas, and this problem is more conspicuous in the subgroup of small-cell lymphomas (4). Differential diagnosis of follicular lymphomas or peripheral T-cell lymphomas from reactive hyperplasias requires molecular confirmation of clonality (5,6).

Moreover, even in the microarray era, as not all pathology centers can afford tissue banks providing fresh material for reassessing cases of uncertain diagnosis archival PET samples become a valuable source of data. However, extraction of nucleic acids of adequate quality from PET could be problematic because tissue preparation for pathology analysis might adversely affect nucleic acids both physically and chemically (7). Thus, the choice of extraction procedures from PET samples will be crucial for providing adequate amounts of DNA suitable for subsequent PCR amplifications (8–10).

We herein present a technical strategy to optimize DNA isolation from PET. This strategy was developed experimentally and includes different protocols of digestion and purification steps followed by the sequential amplification of constitutive genes and analysis of antigen receptor (AR) gene rearrangements. This method allowed amplification of 88% of all PET-DNA samples, while overall clonality was detected in 78% and 50% of B-cell and T-cell disorders, respectively. Moreover, evidence of molecular recombination of immunoglobulin and T-cell receptor genes was the only diagnostic tool in 19 cases of atypical lymphoid proliferations previously classified as uncertain on histopathologic criteria.

From the Centro de Transplante de Medula Òssea (CEMO) Instituto Nacional de Câncer (INCa), Rio de Janeiro (C.G.S., R.H., A.C.G., D.T., I.R.Z.); Instituto de Biologia, Universidade Federal Fluminense (UFF), Niterói C.G.S., L.A.B.A.); Departmento Genética, Universidade Federal de Rio de Janeiro (UFRJ), Rio de Janeiro (R.H.); and Serviço Patologia, Instituto Nacional de Câncer (INCa), Rio de Janeiro (S.R.), Brasil.

Work supported by PRO-VITA Association, Rotary International, FAPERJ, and INCa.

Address correspondence and reprint requests to Dr. Ilana R. Zalcberg, Instituto Nacional de Câncer, INCa–CEMO, Praça da Cruz Vermelha 23, 7°Andar, 20230-130, Rio de Janeiro, RJ, Brasil (E-mail:

© 2003 Lippincott Williams & Wilkins, Inc.