Efficacy of CytoLyt® Hemolytic Action on ThinPrep® LBC Using Cultured Osteosarcoma Cell Line LM8

Abstract

Objective: The removal of blood components is necessary to improve the quality of the liquid-based cytology (LBC) preparations. In ThinPrep® (TP) samples a cell suspension in a methanol-based fixative undergoes a vacuum filtration method, whereas in SurePath™ (SP) samples a cell suspension in an ethanol-based fixative is processed through a density gradient centrifugation system prior to gravity deposition of the specimen onto a glass slide. We compared the cyto-architectural features for the cytologic diagnosis of endometrial adenocarcinoma using parallel TP and SP preparations in a previous publication. Study Design: We performed our study on LM8 cells (a cultured osteosarcoma cell line). LM8 cells at a concentration of 1.25 × 10³ cell/cm² were seeded on a 35-mm plate in culture medium, which contained 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μ/ml streptomycin in Dulbecco's modified Eagle's medium (DMEM), and aliquots of the cell suspension obtained in this way were compared after the addition of a hemolytic agent, i.e. Cytolyt® (CyL). LBC preparations were then obtained on cell suspensions treated with CyL after different time intervals of hemolysis. Results: Treatment with CyL did not alter the cellularity of the preparation, but reduction of the nuclear area and a tendency towards nuclear chromatin condensation with a subsequent higher brightness were found. Because CyL is a 25% methanol-buffered solution, its alcoholic concentration is low; it was our impression that, while its fixative effect was weak, its hemolytic effect was high. Water influx or efflux through the cell membrane is controlled by osmotic pressure changes induced by the buffer solution in the CyL solution. While CyL was not shown to alter the cell shape, nuclear shrinkage was thought to be probably due to the increasing cell dehydration caused by longer exposure intervals to methanol. Conclusion: This study has allowed us to make significant observations on the hemolytic properties of CyL, and on its combined effects with PreservCyt on the cytomorphology of cells suspensions.