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To achieve high-sensitivity cell measurements (<1 in 10 5  cells) by flow cytometry (FCM), the minimum number of acquired cells must be considered and conventional immunophenotyping protocols fall short of these numbers. The bulk lysis (BL) assay is a standardized erythrocyte lysing approach that allows the analysis of the millions of cells required for high-sensitivity measurable residual disease (MRD) detection. However, this approach has been associated with significant cell loss, along with potential over or underestimates of rare cells when using this method. The aim of this study was to evaluate bulk lysis protocols and compare them with minimal sample perturbation (MSP) protocols, which are reported to better preserve the native cellular state and avoid significant cell loss due to washing steps. To achieve this purpose, we first generated an MRD model by spiking fresh peripheral blood with K562 cells, stably expressing EGFP, at known percentages of EGFP positive cells to leukocytes. Samples were then prepared with BL and MSP protocols and analyzed using FCM. For all percentages of K562 cells established and evaluated, a significant decrease of this population was detected in BL samples compared with MSP samples, even at low K562 cell percentages. Significant decreases for non-necrotic cells were also observed in BL samples relative to MSP samples. In conclusion, the evaluation of the potential effects of BL protocols in obtaining the final count is of great interest, especially for over- or under-estimation of target cells, as in the case of measurable residual disease. Since conventional flow cytometry or minimal sample perturbation assays fall short in obtaining the minimum numbers required to reach high sensitivity measurements, significant efforts may be needed to improve bulk lysis solution reagents., (© 2024 International Society for Advancement of Cytometry.)

Type 1 diabetes (T1D) results from a breakdown in immunological tolerance, with pivotal involvement of antigen-presenting cells. In this context, antigen-specific immunotherapies have been developed to arrest autoimmunity, such as phosphatidylserine (PS)-liposomes. However, the role of certain antigen-presenting cells in immunotherapy, particularly human macrophages (Mφ) in T1D remains elusive. The aim of this study was to determine the role of Mφ in antigen-specific immune tolerance and T1D. To that end, we evaluated Mφ ability to capture apoptotic-body mimicking PS-liposomes in mice and conducted a phenotypic and functional characterisation of four human monocyte-derived Mφ (MoMφ) subpopulations (M0, M1, M2a and M2c) after PS-liposomes uptake. Our findings in mice identified Mφ as the most phagocytic cell subset in the spleen and liver. In humans, while phagocytosis rates were comparable between T1D and control individuals, PS-liposome capture dynamics differed among Mφ subtypes, favouring inflammatory (M1) and deactivated (M2c) Mφ. Notably, high nanoparticle concentrations did not affect macrophage viability. PS-liposome uptake by Mφ induced alterations in membrane molecule expression related to immunoregulation, reduced secretion of IL-6 and IL-12, and diminished autologous T-cell proliferation in the context of autoantigen stimulation. These results underscore the tolerogenic effects of PS-liposomes and emphasize their potential to target human Mφ, providing valuable insights into the mechanism of action of this preclinical immunotherapy., Competing Interests: Declaration of competing interest MV-P and MD are co-founders of Ahead Therapeutics S.L., which aims at the clinical translation of PS-liposome immunotherapy for autoimmune diseases. LA-F, MV, DG, MS, MM-A, SR-V, and BB-Z are employees of this company. MWO works for Sartorius Stedim North America, Inc., which is in the business of selling live cell analysers and reagents. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

While the single-platform flow cytometric CD34+ cell counting method is the preferred choice to predict the yield of mobilized peripheral blood stem cells, most flow cytometers lack the ability of hematology counter analyzers to perform volumetric counting. However, one of the problems using reference microbeads is the vanishing counting bead phenomenon. This phenomenon results in a drop in microbeads concentration and reduces the total and relative number of beads in calibration procedures. In the last years, flow cytometers including a volumetric system to quantify cells have been developed and may represent a promising alternative to enumerate CD34+ cells avoiding the use of beads. In this study we have used a direct true volumetric counting of CD34+ cells under continuous flow pump to overcome potential drawbacks with impact in rare cell analysis. To confirm this hypothesis, we have compared the results of CD34+ cell enumeration using non-volumetric vs. volumetric systems with FC500 (Beckman Coulter) and Attune NxT (ThermoFisher) flow cytometers, respectively, in mobilized peripheral blood samples. No statistically significant differences were observed between measurements of CD34+ cells using beads, when the FC500 and Attune NxT absolute counting values were compared, or when CD34+ counts were compared on the Attune NxT, either using or not using beads. Linear regressions to study the relationship between volumetric and non-volumetric CD34+ counts confirmed the accuracy of each method. Bland-Altman test showed agreement between both methods. Our data showed that CD34+ cell enumeration using a volumetric system is comparable with current counting systems. This method represents an alternative with the advantage of the simplification of sample preparation and the reduction of the analysis subjectivity., Competing Interests: Declaration of competing interest M.D.W. and J.A.B. work for Thermo Fisher Scientific, which is in the business of selling flow cytometers and flow cytometry reagents. The rest of authors declare no potential conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Leukemic stem cells (LSCs) possess similar characteristics to normal hematopoietic stem cells, including self-renewal capacity, quiescence, ability to initiate leukemia, and drug resistance. These cells play a significant role in leukemia relapse, persisting even after apparent remission. LSCs were first described in 1994 by Lapidot et al. Although they have been extensively studied in acute leukemia, more LSC research is still needed in chronic lymphocytic leukemia (CLL) to understand if reduced apoptosis in mature cells should still be considered as the major cause of this disease. Here, we provide new evidence suggesting the existence of stem-like cell populations in CLL, which may help to understand the disease as well as to develop effective treatments. In this study, we identified a potential leukemic stem cell subpopulation using the tetraploid CLL cell line I83. This subpopulation is characterized by diploid cells that were capable of generating the I83 tetraploid population. Furthermore, we adapted a novel flow cytometry analysis protocol to detect CLL subpopulations with stem cell properties in peripheral blood samples and primary cultures from CLL patients. These cells were identified by their co-expression of CD19 and CD5, characteristic markers of CLL cells. As previously described, increased alkaline phosphatase (ALP) activity is indicative of stemness and pluripotency. Moreover, we used this method to investigate the potential synergistic effect of curcumin in combination with fludarabine and ibrutinib to deplete this subpopulation. Our results confirmed the effectiveness of this ALP-based analysis protocol in detecting and monitoring leukemic stem-like cells in CLL. This analysis also identified limitations in eradicating these populations using in vitro testing. Furthermore, our findings demonstrated that curcumin significantly enhanced the effects of fludarabine and ibrutinib on the leukemic fraction, exhibiting synergistic effects (combination drug index, CDI 0.97 and 0.37, respectively). Our results lend support to the existence of potential stem-like populations in CLL cell lines, and to the idea that curcumin could serve as an effective adjuvant in therapies aimed at eliminating these populations and improving treatment efficacy.