This protocol provides instructions to improve flow cytometry analysis of marrow/peripheral blood cells by avoiding erythrolytic solutions, density gradients, and washing steps. We describe two basic approaches for identifying cell surface antigens with minimal sample perturbation, which have been successfully used to identify healthy and pathologically rare cells. The greatest advantage of these approaches is that they minimize the unwanted effect caused by sample preparation, allowing for improved study of live cells at the point of analysis.

Nanoplastics (NPs) and microplastics (MPs) are an emerging threat to global health. They negatively impact ecosystems and many physiological processes, causing alterations in xenobiotic metabolism, nutrient uptake, energy metabolism, or cytotoxicity. In humans, we are beginning to analyze these plastics for the mechanisms by which they enter the organism, accumulate, and diffuse as well as for their pathogenic potential. NP accumulation has been demonstrated in human tissues, such as blood or placenta, while in others it remains largely unstudied. In this work, we detected NP accumulation in bronchoalveolar lavage fluids (BALFs), cerebrospinal fluids (CSFs), lymph nodes (LNs), urine, pleural fluids (PFs), ascitic fluids (AFs), and peripheral blood (PB) by combining fluorescence and nanocytometry techniques. NP analysis was compared with two strains of mice, and the results support that inhalation is the main route of NP accumulation.

Even though anti-PD-1/PD-L1 immune checkpoint inhibitors (ICIs) in non-small cell lung cancer (NSCLC) have improved survival, a high percentage of patients still do not respond to ICIs. Myeloid-derived suppressor cells (MDSCs) are circulating cells that express PD-L1 and can infiltrate and proliferate in the tumor microenvironment, inducing immunosuppression. By evaluating changes in PD-L1 expression of live peripheral blood MDSCs, we are able to define a new PD-L1 index, useful in predicting ICI escape in NSCLC patients before initiating anti-PD-1/PD-L1 immunotherapy. In this study, a cohort of 37 NSCLC patients was prospectively analyzed, obtaining independent PD-L1 indexes. In patients with a PD-L1 index > 5.88, progressive disease occurred in 58.33% of patients [median progression-free survival (PFS) = 5.73 months; 95%CI = 2.67–20.53], showing significant differences when compared with patients with a PD-L1 index ≤ 5.88, in whom 7.69% progressed and median PFS was not reached (NR); p-value = 0.0042. Overall survival (OS) was significantly worse in patients with a high vs. low PD-L1 index (41.67% vs. 76.92%; median OS = 18.03 months, 95%CI = 6.77–25.23 vs. NR, 95%CI = 1.87-NR; p-value = 0.035). The PD-L1 index can be applied to stratify NSCLC patients according to their probability of response to ICIs at baseline. In addition to quantifying tumoral expression, this index could be used to compare nonresponse to treatment.

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.

The widespread presence of nanoplastics (NPs) in the environment creates a significant and growing concern for global health, with ingestion, inhalation, and dermal contact identified as primary exposure pathways. Despite their documented presence in various environmental matrices and human tissues, robust quantitative data on NP levels remains scarce. This study addresses this critical gap by employing a novel and rapid flow cytometry technique to quantify nanoplastic concentrations in commercial waters, common beverages and infant food, with special focus in packaging influence. Pyrogen-free water was analyzed to establish the negative control for NP concentration, yielding 5.24 ± 2.02 events/µL. Ten commercial waters from natural springs in Spain and France showed NP levels ranging from 1.75 NP/µL to 67.94 NP/µL (mean: 19.90 ± 14.53 NP/µL), where three of those brands showed significantly higher NP numbers than the pyrogen-free water control. Compared to pyrogen-free water, infant formula and cereal porridge showed very low NP concentrations, with values of 10.27 ± 6.85 and 6.78 ± 2.27 events/µL, respectively, following triplicate analyses of six samples. Additional analyses comparing three similar soft drinks across different packaging (can, plastic bottle, or glass bottle) found no significant differences in NP concentration attributable to the container type. NPs, as ubiquitous contaminants, can be ingested by organisms through food and drink. Potential NP contamination in commercial water may be due to factors such as source water contamination, filtration and packaging. The presence of very low concentrations of NPs in infant foods suggests rigorous and effective quality control. Finally, the presence of NP in soft drinks was not affected by the type of packaging. Although soft drinks have higher NP levels than water, the type of packaging had no effect on the presence of NP in these soft drinks. Despite all plastic bottles being made of polyethylene terephthalate, variation in NP accumulation implies that material quality, storage condition, and substantially, water treatment and filtering processes contribute to NP contamination. This research gives evidence for widespread nanoplastic accumulation in bottled water, common beverages and infant formula and sets the stage for demanding research to further investigate sources, health effects, and development into effective quality control and preventive measures for public health.