True volumetric counting of CD34+ cells using flow cytometry.

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.

Persistence of Chronic Lymphocytic Leukemia Stem-like Populations under Simultaneous In Vitro Treatment with Curcumin, Fludarabine, and Ibrutinib: Implications for Therapy Resistance.

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.

Functional Flow Cytometry to Predict PD-L1 Conformational Changes.

The programmed cell death protein 1/programmed cell death protein ligand 1 (PD-1/PD-L1) axis is one of the most widely recognized targets for cancer immunotherapy. Importantly, PD-L1 conformational changes can hinder target binding when living cells are used. Antibody affinity, equilibrium binding, association and dissociation rates, and other affinity-related constants are fundamental to ensure target saturation. Here, PD-L1 changes in conformation and their potential impact on PD-L1 function and mutation are explored. Specifically, we present detailed flow cytometry procedures to analyze PD-L1 reactivity in myeloid-derived suppressor cells (MDSCs). This approach can also be used to study the contribution of protein conformational changes in living cells. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Sample preparation for PD-L1+ myeloid-derived suppressor cells detection by flow cytometry Basic Protocol 2: Protocol preparation, sample acquisition, and gating strategy for flow cytometric screening of PD-L1+ myeloid-derived suppressor cells in patients with lung cancer Support Protocol 1: Bioinformatic tools for the analysis of flow cytometric data.

Fast-screening flow cytometry method for detecting nanoplastics in human peripheral blood.

Plastic pollution is a global problem. Animals and humans can ingest and inhale plastic particles, with uncertain health consequences. Nanoplastics (NPs) are particles ranging from 1 nm to 1000 nm that result from the erosion or breakage of larger plastic debris, and can be highly polydisperse in physical properties and heterogeneous in composition. Potential effects of NPs exposure may be associated with alterations in the xenobiotic metabolism, nutrients absorption, energy metabolism, cytotoxicity, and behavior. In humans, no data on NPs absorptions has been reported previously. Given that their detection relies significantly on environmental exposure, we have prospectively studied the presence of NPs in human peripheral blood (PB). Specifically, we have used fluorescence techniques and nanocytometry, together with the staining of the lipophilic dye Nile Red (NR), to demonstrate that NPs can be accurately detected using flow cytometry.•Potential effects of nanoplastics exposure.•Fluorescence techniques and nanocytometry.•Accurate detection using flow cytometry.

Erythrocyte lysing solutions have a detrimental effect in flow cytometric dendritic cell detection.

Flow cytometry (FCM) enumeration of peripheral blood dendritic cells (PBDCs) is a minimally invasive procedure extremely useful for immunological studies. Numbers of PBDCs vary depending on age, lifestyle, or in pathologies like cancer, leukemia or immunodeficiencies. Conventional methods for PBDC identification by FCM involve red blood cell lysis using either formaldehyde or ammonium chloride-based solutions. This specific procedure has been widely reported to cause a detrimental effect as well as an artifactual detection of target populations. Alternatively, minimal sample perturbation assays that avoid the use of erythrolytic solutions with centrifugation steps and preserve the native cellular state are simpler and more robust than conventional methods. In this study, we aimed to evaluate how conventional FCM assays can alter dendritic cell (DC) counting when compared with minimal sample perturbation protocols, in terms of absolute cell counting, percentage and stain index (SI) of PBDC subsets. We evaluated the use of three different erythrolytic solutions (CyLyse, OptiLyse C, and Pharm Lyse) on a series of n = 20 peripheral blood specimens for conventional and plasmacytoid DCs detection as well as for leukocyte and basophil detection. Our results showed a significant reduction of leukocytes and specifically, of DCs and basophils in terms of absolute number when using erythrolytic solutions. In conclusion, our study shows that PBDC counting is heavily affected when lysing solutions are used, indicating that these stellate-shaped populations appear to be more labile.

Impact of red blood cell lysing on rare event analysis.

The challenges associated with analyzing rare cells are dependent on a series of factors, which usually require large numbers of cells per sample for successful resolution. Among these is determining the minimum number of total events needed to be acquired as defined by the expected frequency of the target cell population. The choice of markers that identify the target population, as well as the event rate and the number of aborted events/second, will also determine the statistically significant detection of rare cell events. Sample preparation is another important but often overlooked factor in rare cell analysis, and in this study we examine Poisson theory and methods to determine the effect of sample manipulation on rare cell detection. After verifying the applicability of this theory, we have evaluated the potential impact of red cell lysis on rare cell analysis, and how cell rarity can be underestimated or overestimated based on erythrolytic sensitivity or resistance of healthy leukocytes and pathological rare cells.

Accurate identification of cell doublet profiles: Comparison of light scattering with fluorescence measurement techniques.

Doublet discrimination is usually based on pulse analysis of light scatter parameters. A combination of two pulse parameters (Area, A; Height, H; or Width, W) can be used to discriminate a pulse originated in a single cell from a pulse originated from cells stuck together. Fluorescence signals can be also used to discriminate aggregates, being essential to identify cells in the G2/M phase from doublets in the G0/G1 phase in cell cycle/DNA applications. The most used method combines FSC-A versus FSC-H, whereas other strategies combine FSC-H versus FSC-W, SSC-H versus SSC-A and SSC-H versus SSC-W. However, when studying activated or proliferating cells, scatter discrimination can be difficult. In this study, we have compared the use of light scattering with fluorescence measurement techniques for successful doublet discrimination for single cells. Effective use of FSC and SSC height, area and width are commonly used to eliminate aggregates. However, fluorescence-based methods using viable DNA stains provide a good compromise between performance and accurate manual gating methods, especially for highly concentrated cell products and pathological specimens. Viable DNA dyes, such as Vybrant™ DyeCycle™ Violet stain or Hoechst 33342, can be used to detect nucleated cells in blood and in bone marrow, or to discriminate cell aggregates and debris based on no-lyse no-wash assays, where scatter degradation is a dominant component of the measured data, which increases with event rate.

Flow-cytometry-based protocols for human blood/marrow immunophenotyping with minimal sample perturbation.

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. For complete details on the use and execution of this protocol, please refer to Petriz et al. (2018).

Flow Cytometric Quantification of Cytotoxic Activity in Whole Blood Samples.

Current methods for the determination of cell-mediated cytotoxic activity in blood samples usually isolate peripheral blood mononuclear cells by density gradient centrifugation or alternatively use erythrocyte lysis. Both centrifugation and red cell lysis can cause cellular depletion and cell dysfunction, resulting in erroneous measurements. To address limitations of current assays, we developed an improved strategy to determine cellular cytotoxicity using flow cytometry. Viable nucleic acid stains are used to identify live nucleated cells and discriminate them from non-nucleated erythrocytes, platelets, and debris while avoiding lysing and washing steps to maintain cell functionality. To detect target cells, we have used two different labeling approaches. In the first approach, EGFP-labeled K562 human chronic myelogenous leukemia cells provide a "ready-to-use" target without the need of additional for labeling or staining. For the second approach, we perform parallel cytotoxicity assays in the presence of wild-type K562 cells previously loaded with a fluorescent dye that has spectral properties similar to those of EGFP. Given the importance of cytotoxic assays and the deleterious effects of current sample preparation methods, the aim of this study was to adapt this "untouched cells" flow cytometry method to study cytotoxic activity using unlysed whole blood samples and fluorescent target cells. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Sample preparation for cell-mediated cytotoxic activity determination in unlysed whole blood Basic Protocol 2: Protocol preparation, sample acquisition, and gating strategy for flow cytometric identification of cell-mediated cytotoxic activity using unlysed whole blood samples Support Protocol 1: Optimization of the performance of target cell labeling approaches Support Protocol 2: Assessment of the linearity and reproducibility of cytotoxicity assays.

A Novel Flow Cytometric Method to Study Cytotoxic Activity in Whole Blood Samples.

For several decades, cell-mediated cytotoxicity has been measured using the 51 Cr release assay. This assay, however, has several drawbacks and flow cytometry has been used as an alternative to measure cytotoxic activity. Here, we present a quantitative method for cell-mediated cytotoxicity studies, preserving cellular function with minimal sample manipulation. Cytotoxic activity is simply and reproducibly measured as the ability of cytotoxic cells to lyse K562 target cells previously loaded with Calcein-AM vital stain. After spiking a known number of fluorescent viable K562 target cells into whole blood, cell mixtures are incubated for 2 h in a cell incubator and the remaining spiked cells are counted by flow cytometry. In order to discriminate nucleated cells, erythrocytes, and debris, unlysed whole blood is stained with a cell permeable DNA vital fluorescent dye. Cell-mediated lysis is measured by comparing target counts for different effector-to-target ratios. Since the cytotoxicity of these dyes is relatively low, this method can be broadly applied to studies of innate immune response to tumors and infections, especially where target-killing activity might be compromised by small volume samples or low frequency of cytotoxic cells. © 2020 International Society for Advancement of Cytometry.

Flow Cytometric Quantification of Granulocytic Alkaline Phosphatase Activity in Unlysed Whole Blood.

Translational research has improved the diagnosis and follow-up of hematological diseases and malignancies. However, some classical diagnostics used for research and clinical practice that have remain practically unchanged for decades may be better addressed through advances in flow cytometry technology, whereby more precise measurements may be implemented in a straightforward manner. The current method for semiquantitative analysis of granulocytic alkaline phosphatase (GAP) activity is still based on observer-dependent color-intensity classification. Here, we describe a novel strategy for flow cytometric quantification of GAP activity in which staining and analytical flow cytometry facilitate the detection and quantification of subpopulations of leukocytes with different GAP activities. Our experiments demonstrate the potential of flow cytometry as a simple and highly sensitive approach for measuring GAP activity in unlysed whole blood. Notably, a comparison of flow cytometry and enzyme cytochemistry techniques showed that enzyme activity scores were not similar, indicating that results needs to be interpreted with caution, given that the enzyme-substrate binding affinities may differ, as well as the subjective evaluation of the intensity of the precipitated dye. © 2020 Wiley Periodicals LLC. Basic Protocol: Protocol preparation, sample acquisition, and gating strategy for flow cytometric identification of alkaline phosphatase activity in granulocytes from whole blood samples Support Protocol 1: Sample preparation for granulocyte alkaline phosphatase determination by flow cytometry using no-lyse no-wash methods Support Protocol 2: Data analysis and formula to calculate the GAP score.

Flow cytometric significance of cellular alkaline phosphatase activity in acute myeloid leukemia.

In this prospective hospital-based cohort study that included 43 newly diagnosed patients with acute myeloid leukemia, flow cytometric cellular alkaline phosphatase (ALP) activity within primitive leukemic cells allowed us to identify two groups of patients at diagnosis according to the numbers of leukemic blasts expressing ≥ 12% of ALP+ cells (27 patients, Group A) and less than 12% of ALP+ cells (16 patients, Group B). Differences in outcome for complete response, relapse or treatment resistance, and exitus were statistically analyzed and were significant, when comparing the two groups. The overall survival (OS) and event-free survival (EFS) differences between Group A and B were statistically significant. The survival of Group A patients was significantly shorter than those for Group B. No significant relationship was detected in outcome when comparing ELN prognostic-risk group based on cytogenetic and molecular profile (patients in the favorable, intermediate, and adverse risk groups). Flow cytometric cellular ALP activity at diagnosis may be used to estimate relapses and disease persistence more accurately. The limitations of our study include the small number of patients enrolled and a short follow-up, due to its prospective nature.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

Acoustophoretic Orientation of Red Blood Cells for Diagnosis of Red Cell Health and Pathology.

Distortions of the normal bi-concave disc shape for red blood cells (RBCs) appear in a number of pathologies resulting from defects in cell membrane skeletal architecture, erythrocyte ageing, and mechanical damage. We present here the potential of acoustic cytometry for developing new approaches to light-scattering based evaluation of red blood cell disorders and of the effects of storage and ageing on changes or damage to RBCs membranes. These approaches could be used to immediately evaluate the quality of erythrocytes prior to blood donation and following transfusion. They could also be applied to studying RBC health in diseases and other pathologies, such as artificial heart valve hemolysis, thermal damage or osmotic fragility. Abnormal distributions of erythrocytes can typically be detected after just 30 to 45 seconds of acquisition time using 1-2 µL starting blood volumes.

No lyse no wash flow cytometry for maximizing minimal sample preparation.

Red blood cell lysis is an integral part of many flow cytometry protocols. It's potential to cause artifacts has been known for decades, but lysis free sample preparation has failed to replace lysis in most applications. Studies of various lysing protocols on cell losses and effects on phenotypic markers and cell function began early in the history of immunophenotyping and continue to this day. Opportunities to combine live cell response and functional assessment with phenotyping have sparked increasing interest in no lyse no wash protocols, with minimizing sample preparation effects on the cell biology as the primary goal. No lyse no wash protocols reduce sample handling and are procedurally less complex than lysis protocols, but the impact of keeping intact red blood cells that grossly outnumber the target white blood cells, must be understood to fully take advantage of this simplicity. Presented here are theories and methods for executing and interpreting no lyse no wash assays in whole blood. Methods for distinguishing white blood cells and platelets from red blood cells and improving scatter data by combining 405 nm and 488 nm side scatter are shown. Methods for assessing white blood cell light scattering profiles for individual instruments and sample treatments are discussed within the context of example profiles for no lysis and hypotonic and ammonium chloride lysis treatments. The utility of overcoming no lyse no wash scatter and fluorescence background limitations using alternate scatter and fluorescence thresholding strategies is also discussed in the context of application examples.

Click Chemistry for Analysis of Cell Proliferation in Flow Cytometry.

The measurement of cellular proliferation is fundamental to the assessment of cellular health, genotoxicity, and the evaluation of drug efficacy. Labeling, detection, and quantification of cells in the synthesis phase of cell cycle progression are not only important for characterizing basic biology, but also in defining cellular responses to drug treatments. Changes in DNA replication during S-phase can provide valuable insights into mechanisms of cell growth, cell cycle kinetics, and cytotoxicity. A common method for detection of cell proliferation is the incorporation of a thymidine analog during DNA synthesis. This chapter presents a pulse labeling method using the thymidine analog, 5-ethynyl-2'-deoxyuridine (EdU), with subsequent detection by click chemistry. EdU detection using click chemistry is bio-orthogonal to most living systems and does not non-specifically label other biomolecules. Live cells are first pulsed with EdU. After antibody labeling cell surface markers, fixation, and permeabilization, the incorporated EdU is covalently labeled using click chemistry thereby identifying proliferating cells. Improvements in click chemistry allow for labeling in the presence of fluorescent proteins and phycobiliproteins without quenching due to copper. Measuring DNA replication during cell cycle progression has cell health applications in flow cytometry, fluorescence microscopy, and high content imaging. This protocol has been developed and optimized for research use only and is not suitable for use in diagnostic procedures. © 2017 by John Wiley & Sons, Inc.

Panel development for multicolor flow-cytometry testing of proliferation and immunophenotype in hMSCs.

Adult human mesenchymal stem cells (hMSC) are rare fibroblast-like cells capable of differentiation into a variety of cell tissues which include bone, cartilage, muscle, ligament, tendon, and adipose. Normal adult bone marrow and adipose tissue are the most common sources of these cells. The International Society for Cellular Therapy (ISCT) has proposed a set of standards to define hMSC for laboratory investigations and preclinical studies: adherence to plastic in standard culture conditions; in vitro differentiation into osteoblasts, adipocytes, and chondroblasts; and specific surface antigen expression. Direct measurement of proliferation combined with simultaneous detection of the ISCT-consensus immunophenotypic profile provides data that is used to determine the differentiation status and health of the cells. Flow cytometry provides a powerful technology that is routinely used to simultaneously and rapidly measure multiple parameters in a single sample. This chapter describes a flow cytometric panel for the simultaneous detection of immunophenotypic profile, proliferative capacity, and DNA content measurement in hMSC. Because a relatively small number of cells are needed with this approach, measurements can be made with minimal impact on expansion potential. The ability to assess antigen expression and proliferative status enables the investigator to make informed decisions on expansion and harvesting.

Dual-pulse labeling using 5-ethynyl-2'-deoxyuridine (EdU) and 5-bromo-2'-deoxyuridine (BrdU) in flow cytometry.

Changes in DNA replication during S-phase can give insights into mechanisms of cell growth, cell cycle kinetics, and cytotoxicity. A common method for detection of cell proliferation utilizes the incorporation of a thymidine analog during DNA synthesis. Incorporation of multiple analogs at different time points can further define cell cycle kinetics. Traditionally, the dual-pulse method has been done by combining 5-bromo-2'-deoxyuridine (BrdU) with iododeoxyuridine or chlorodeoxyuridine, with detection using multiple cross-reacting BrdU antibodies. This unit presents a dual-pulse method using the thymidine analog 5-ethyl-2'-deoxyuridine (EdU), detected by click chemistry, combined with BrdU labeling and detection. No cross reactivity with incorporated EdU is observed using the BrdU antibody clone MoBU-1. EdU detection using click chemistry does not cross-react with incorporated BrdU. Cells are first pulsed with EdU, and then pulsed with BrdU; sequential pulses of EdU, followed by BrdU, are done without removing or washing out EdU.

Cell type specific applicability of 5-ethynyl-2'-deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry.

Using the nucleoside analogue EdU (5-ethynyl-2'-deoxyuridine) for thymidine substitution instead of BrdU (5-bromo-2'-deoxyuridine) in cell proliferation assays has recently been proposed. However, the effect of EdU on cell viability, DNA synthesis, and cell cycle progression and consequently its usability for dynamic cell proliferation analysis in vitro has not been explored. We compared the effect of EdU and BrdU incorporation into SK-BR-3 and BT474 breast cancer cells and the impact on cell cycle kinetics, cell viability, and DNA damage. We found that EdU can be used not only for pulse but also for continuous cell labeling and henceforth in high resolution EdU/Hoechst quenching assays. BrdU and EdU proliferation assays based on click chemistry revealed comparable results. However, cell viability of SK-BR-3 breast cancer cells was highly affected by long term exposure to EdU. Both SK-BR-3 as well as BT474 cells show cell cycle arrests upon long term EdU treatment whereas only SK-BR-3 cells were driven into necrotic cell death by long term exposure to EdU. In contrast BT474 cells appeared essentially unharmed by EdU treatment in terms of viability. Consequently using EdU enables highly sensitive and quantitative detection of proliferating cells and facilitates even continuous cell cycle assessment. Nevertheless, potential cellular susceptibility needs to be individually evaluated.