Evaluation of a method allowing preservation of fresh lymph nodes for flow cytometric immunophenotyping

Abstract

Background: Flow cytometric immunophenotyping (FCI) of lymph nodes (LN) requires fresh unfixed tissue, with analysis being carried out within few hours post surgery. This study evaluated a novel method for fresh LN preservation, in order to allow histomorphology-based FCI. Methods: This study was carried out prospectively on 30 LN with suspected involvement by haematolymphoid neoplasms (HLN). FCI was performed on each fresh and post cryopreserved LN cell suspension. Percentage positivities (PP) and mean fluorescent intensities (MFI) were calculated on both preparations for a combination of T and B-lymphoid antigens together with viability. Results: The cryopreservation method applied in this study did not affect significantly PP and had minor impact on MFI of the tested antigens. Overall, there was minimal decrease in PP and MFI on the cryopreserved cells when compared with fresh cells, for most antigens with only a mild increase in apoptotic cells. However, these changes were not diagnostically significant, since both reactive processes and HLN present within analyzed LN could be identified and differentiated. Viability was more than 75% for all cryopreserved LN composed of haematolymphoid cells. Conclusion: The method presented in this study confers the possibility of storing fresh LN biopsies for later FCI, thus allowing a morphology-based immunophenotypic approach. This would allow a more sensitive, specific, and cost-effective management of LN specimens, whilst maintaining the important benefits provided by FCI.