Cell cycle analysis is a recognized and important application of flow cytometry and, more recently, mass cytometry (MCM). Both technologies have been utilized for analysis of the cell cycle state of ex vivo samples from patients with hematologic malignancies. Clinical samples are frequently stored for hours at room temperature or cryogenically frozen before processing and analysis; however, how these processing methods alter cell cycle state is not well described. To understand how storage time and temperature affect the analysis of cell cycle distribution by MCM, two leukemia cell lines, HL-60 and MOLM13, and primary human cells from three human bone marrow aspirates were stored and frozen under a variety of conditions that are likely to be encountered in a clinical setting. Our findings indicate that short delays in sample processing (less than 1 h), have little to no effect on cell cycle distribution, while longer delays or cryopreservation cause significant disruptions to the cell cycle fraction characterized by consistent reductions in IdU incorporation and variable alterations in other cell cycle phases. Analysis of the recovery of cryopreserved leukemia cell lines and marrow cells demonstrated that cell cycle alterations persist for at least 48 h after thawing. Our findings demonstrate that accurate cell cycle analysis requires that samples be processed rapidly after collection, and that cryopreservation significantly alters cell cycle fractions. Measurement of IdU incorporation was the most sensitive to both delays in processing and cryopreservation, while estimation of the total cycling cell fraction using Ki-67 or phosphorylated retinoblastoma protein were least altered by the conditions tested. These findings provide guidance for the ideal approach to collection of samples for cell cycle analysis and can aid interpretation of cell cycle data from samples that cannot be collected under ideal circumstances. © 2018 International Society for Advancement of Cytometry.
Keywords: cell cycle; hematopoiesis; iodo-deoxyuridine; mass cytometry.
© 2018 International Society for Advancement of Cytometry.