The effect of fixation on detection of B-cell clonality by polymerase chain reaction

It has been suggested that neutral buffered formalin (NBF)-fixed, paraffin-embedded, or fresh specimens might provide satisfactory DNA templates for polymerase chain reaction (PCR) assays used in establishing the clonality and presumptive B-cell lineage of lymphoma. The suitability of other fixatives used by hematopathologists, such as B5, is still undetermined. Thirty cases were identified from the files of the Cleveland Clinic Foundation, Cleveland Ohio, that showed abnormal immunoglobulin heavy chain (IgH) rearrangement by Southern blot analysis (SBA). Corresponding paraffin-embedded tissue samples fixed in NBF (21 cases), B5 (18 cases), Hollande's fixative (17 cases), zinc formalin (ZF) (5 cases), and Bouin's fixative (3 cases) were studied. With use of consensus primers against the framework 3 (FR3) and FR2 regions of the VH gene, paired against JH primer(s), PCR analysis was performed. bcl-2/IgH translocation was also studied. Ten reactive lymphoid samples were used as controls, and 40 cases were evaluated. Successful amplification of a clonal proliferation was manifested as one or two discrete narrow bands in the appropriate size range. The sensitivity of detecting clonality was 95, 94, 67, 80, and 0% for NBF, Hollande's fixative, B5, ZF, and Bouin's fixative, respectively. Although NBF and Hollande's fixative were 100% specific, consistent false-positive results were a major problem with B5-fixed tissue. Paraffin-embedded tissue, fixed in NBF, Hollande's fixative, and ZF solutions, may be used for DNA extraction and PCR assays for establishing B-cell clonality. The precipitating fixative B5 and Bouin's solution should not be used for this purpose until the issue of false-positive results is resolved.