RCL2, a potential formalin substitute for tissue fixation in routine pathological specimens.

AIMS: To investigate RCL2 as a fixative for tissue fixation in routine histopathological examination and to assess tissue suitability for ancillary investigations. METHODS AND RESULTS: Forty-nine samples from 36 fresh specimens were cut into three equal pieces and fixed in RCL2 diluted in 100% ethanol, RCL2 in 95% ethanol, or neutral buffered formalin as control. Suitability for microtomy, quality of histomorphology, histochemistry, immunohistochemistry, fluorescent and silver in-situ hybridization analysis and extracted genomic DNA were assessed. Microtomy was straightforward in most tissue blocks, but there was difficulty in cutting in approximately a quarter of samples, which required careful handling by an experienced technician. There were no significant differences in tissue morphology between RCL2- and formalin-fixed tissues (P=0.08). Generally, the quality of histochemical staining, immunohistochemistry and in-situ hybridization were comparable to that of formalin-fixed tissues. Inconsistent immunoreactivity was noted, however, with antibodies against pan-cytokeratin and progesterone receptor. Genomic DNA concentration was higher in RCL2-fixed tissues. Using RCL2 diluted in 95% ethanol did not affect fixation quality. CONCLUSION: RCL2 is a potential formalin substitute suitable as a fixative for use in routine histopathological examination; however, difficulty in microtomy and occasional discrepancies in immunohistochemical reactivity require further optimization of the methodology.

Variation in BCL2 protein expression in follicular lymphomas without t(14;18) chromosomal translocations.

AIM: The hallmark of follicular lymphoma is the t(14;18)(q32;q21) chromosomal translocations that lead to deregulation of BCL2 expression in tumour cells. However, not all cases of follicular lymphoma express BCL2, nor is the t(14;18) translocation always present. Follicular lymphomas lacking the BCL2 rearrangement are less well studied with regards to their immunohistochemical and molecular features. This study aims to investigate the BCL2 protein expression pattern in t(14;18) negative follicular lymphomas. METHODS: BCL2 protein expression pattern was analysed in 26 cases of t(14;18) negative follicular lymphoma [determined by fluorescence in situ hybridisation (FISH)], using antibodies against two-different epitopes, i.e., the widely-used antibody BCL2/124 and an alternative antibody E17. RESULTS: Two of the t(14;18) negative cases showed evidence of BCL2 amplification and trisomy 18. A total of 13 cases (50%) lacked BCL2 expression. In 10 cases (38%) the expression was heterogeneous and in only three cases (12%) the BCL2 expression was strongly positive. These cases could thus be subdivided into three subgroups: Group I, normal BCL2 genes (i.e., no evidence of translocation or amplification), and BCL2 protein negative; Group II, normal BCL2 genes but BCL2 protein positive; and Group III, presence of other genetic alterations, i.e., BCL2 amplification and trisomy 18, and BCL2 protein positive. CONCLUSIONS: This study suggests that it may be possible on the basis of staining to predict that the t(14;18) translocation is absent if a case is either negative for BCL2 protein with different antibodies or has heterogeneous BCL2 expression, possibly acquired through a physiological process of differentiation.