Vortex disaggregation for flow cytometry allows direct histologic correlation: a novel approach for small biopsies and inaspirable bone marrows.

Abstract

BACKGROUND: Many approaches to obtaining single cells from tissue for flow cytometric immunophenotyping are used; however, these methods result in tissue that is too disrupted for subsequent histologic examination. We introduce a new technique for cell dissociation of hematopoietic malignancies that preserves tissue for histology. This is especially important with small specimens for which this type of correlation is critical. METHODS: Fresh tissue from lymph node, gastrointestinal (GI) tract, skin, and other soft tissue biopsies, in addition to cores of inaspirable bone marrows, were briefly vortexed until the RPMI cell culture medium became cloudy. Larger specimens such as lymph nodes were sectioned before disaggregating, whereas smaller ones were vortexed in toto. Resultant flow cytometric analyses were compared with the histology and, in some cases, the immunohistochemistry (IHC) to determine whether the data were concordant. Cell suspensions of 104 specimens-composed of 48 lymph nodes, 19 bone marrow cores (BMCs), 11 GI biopsies, 11 skin/soft tissue biopsies, and 15 miscellaneous specimens-were prepared via vortex disaggregation. RESULTS: Flow cytometric analysis of 96 specimens (92.3%) showed adequacy of material and diagnostic correlation with the histology and IHC. Of the eight cases (7.7%) that were discordant, seven were attributable to significant specimen fibrosis or necrosis. With respect to tissue type, this method produced diagnostic cell suspensions for most lymph nodes (95.8%), GI biopsies (90.9%), and BMCs (89.5%); however, it was less useful for skin/soft tissue samples (81.8%). CONCLUSIONS: Disaggregation of tissue for flow cytometric analysis by vortexing appears to provide adequate and representative cellular material. This technique is ideal for inaspirable bone marrows and small biopsies where tissue preservation for histology is paramount.