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 p olymerase chain reaction (PCR) assays used in establishing the clonality an d presumptive B-cell lineage of lymphoma. The suitability of other fixative s used by hematopathologists, such as B5, is still undetermined. Thirty cas es were identified from the files of the Cleveland Clinic Foundation, Cleve land Ohio, that showed abnormal immunoglobulin heavy chain (IgH) rearrangem ent by Southern blot analysis (SBA). Corresponding paraffin-embedded tissue samples fixed in NBF (21 cases), B5 (18 cases), Hollande's fixative (17 ca ses), zinc formalin (ZF) (5 cases), and Bouin's fixative (3 cases) were stu died, With use of consensus primers against the framework 3 (FR3) and FR2 r egions of the VH gene, paired against JH primer(s), PCR analysis was perfor med. bcl-2/IgH translocation was also studied, Ten reactive lymphoid sample s were used as controls, and 40 cases were evaluated, Successful amplificat ion 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% s pecific, consistent false-positive results were a major problem with B5-fix ed tissue. Paraffin-embedded tissue, fixed in NBF, Hollande's fixative, and ZF solutions, may be used for DNA extraction and PCR assays for establishi ng B-cell clonality. The precipitating fixative B5 and Bouin's solution sho uld not be used for this purpose until the issue of false-positive results is resolved.