Single-Cell Transcriptome Analysis of Acute Myeloid  Leukemia Cells Using Methanol Fixation and Cryopreservation
Introduction: The application of single-cell RNA sequencing has greatly improved our under-standing of various cellular and molecular mechanisms involved in physiological and pathophysi-ological processes. However, obtaining living cells for this technique can be difficult under certain conditions. To solve this problem, the methanol fixation method appeared as a promising alternative for routine clinical use. Materials and Methods: In this study, we selected two AML samples that had been fixed in methanol for 12–18 months. Once the cells were rehydrated, these samples were subjected to single-cell RNA sequencing. We then compared the results obtained from these samples with those obtained from the same samples cryopreserved in DMSO. Results: We used a previously validated methanol fixation protocol to perform scRNA-seq on DMSO cryopreserved cells and cells fixed in methanol for more than one year. Preliminary results show that methanol fixation induces some genetic and transcriptional modification compared with DMSO cryopreservation but remains a valuable method for single-cell analysis of primary human leukemia cells. Conclusions: The initial findings from this study highlight certain resemblances in methanol fixation over a 12-month period and cryopreservation with DMSO, along with associated transcriptional level modifications. However, we observed genetic degradation in the fixation condition when extending beyond one year. Despite certain study limitations, it is evident that short-term methanol fixation can be effec-tively used for leukemia blast samples. Its ease of implementation holds the potential to simplify the integration of this technique into routine clinical practice. Overall design: PBMC (Peripheral Blood Mononuclear Cells) were obtained from the peripheral blood of samples UPN73 and UPN78. After centrifugation to separate them from plasma and other peripheral blood components, a ficoll-Hystopaque density gradient centrifugation method was used. The aim was to conduct a comparative study on the effect of methanol fixation of PBMC from patients with acute myeloid leukemia (AML) versus DMSO cryopreservation. This study was carried out using single cell RNA-seq technology. To carry out this study, we fixed the UPN73 and UPN78 samples in methanol for a period of 12 and 18 months respectively. At the same time, we also cryopreserved the same samples using DMSO before subjecting them to single-cell RNA-seq analysis.