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.

Prevalencia y características de mutaciones somáticas del gen KRAS en pacientes chilenos con cáncer colorectal

“…Nuestros resultados muestran semejanzas con otros estudios 6,19 , siendo G12D, G12V y G13D las mutaciones más frecuentes con 36%, 25% y 17%, respectivamente. Mutaciones en estos codones son las principales responsables de la activación constitutiva de la proteína, y de la independización de la vía del EGFR por su ligando 3,25 .…”

“…Se extrajo el ADN tumoral de cortes seriados de 8 µm de espesor no teñidos mediante el sistema QIAmp DNA FFPE Tissue extraction kit (QIA-GEN, Hilden, Alemania), según las instrucciones del fabricante. La integridad del ADN se analizó mediante la amplificación por PCR multiplex de 2 amplicones de GAPDH, 1 amplicón de β-globina y 1 amplicón de β-actina 19 .…”

Diagnosing lymphoma in a setting with a high burden of infection: a pediatric case of Epstein-Barr virus-associated aggressive B-cell lymphoma with t(8;14) (q23;q32) and extensive necrosis mimicking tuberculosis

“…applied to DNA that has been extracted from various sources, including FFPE specimens (1) (2) . Of note, PCR results that are relevant to the diagnosis of lymphoma should be interpreted in the context of both clinical and pathologic fi ndings.…”

“…Molecular biology tools are required to confi rm the diagnosis of a small but signifi cant group of NHLs (1) . In this group, molecular clonality analyses are relevant for differentiating reactive conditions from lymphoma, especially when the latter must be distinguished from infection-associated reactive processes (1) (2) .…”

Detección de ADN de parvovirus B19 en donantes de sangre de tres hospitales en Santiago, Chile

“…Se ha demostrado la presencia de ADN viral en 1 de 5.950 donantes de sangre en Francia en un período de dos años, valor que se incrementó hasta 1 en 1.420 en período epidémico 8 . Por otra parte, se ha documentado la presencia de genoma viral en 27 de 16.859 donantes de sangre en Bélgica (1 en 625) 17 . En forma similar, en un estudio realizado en 9.568 unidades de sangre en Estados Unidos de Norteamérica, se demostró la presencia de ADN viral en 11 de ellas (1 en 870) 10 , al igual que en un estudio realizado en Japón, en el cual se detectó la presencia del virus en 6 de 1.000 donantes de sangre (1 en 167) 11 .…”