Prognostic Significance of PD-L1 Expression on Circulating Myeloid-Derived Suppressor Cells in NSCLC Patients Treated with Anti-PD-1/PD-L1 Checkpoint Inhibitors.

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.

Bulk lysis procedures alter target cell population counts.

To achieve high-sensitivity cell measurements (<1 in 10 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.

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.

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.

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.

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.

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.

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.

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.

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.

Unmasking the expression of PD-L1 in Myeloid Derived Suppressor Cells: A case study in lung cancer to discover new drugs with specific on-target efficacy.

• PD-L1 displays variation of spatial conformation in response to phorbol ester stimulation, which may confer a critical enhancement in binding affinity. • PD-L1 conformational change may be associated with an immunoregulatory mechanism that affects therapies targeting the PD-1/PD-L1 checkpoint. • Eliminating MDSCs by promoting PD-L1 stabilized unfolded states on both PMN- and M-MDSCs could improve immunotherapy efficacy.

Diagnostic performance of the ClearLLab 10C B cell tube.

Introduction: Pre-analytical and analytical errors can threaten the reliability of flow cytometry (FC) results. A potential solution to some of these is the use of dry, pre-mixed antibodies, such as the ClearLLab 10C system. The purpose of the present study was to compare the diagnostic performance of the ClearLLab 10C B cell tube with that of our standard laboratory practice.

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

For several decades, cell-mediated cytotoxicity has been measured using the 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.

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.

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.

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.

Correction: Is alkaline phosphatase the smoking gun for highly refractory primitive leukemic cells?

[This corrects the article DOI: 10.18632/oncotarget.12497.

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.

Is alkaline phosphatase the smoking gun for highly refractory primitive leukemic cells?

With the aim to detect candidate malignant primitive progenitor populations, we modified an original alkaline phosphatase (ALP) stem cell detection method based on the identification of alkaline phosphatase fluorescent cells in combination with flow cytometry immunophenotyping. Over a period of one year, we have been using this technique to study its activity in patients with leukemia and lymphoma, showing that changes in the alkaline phosphatase levels can be used to detect rare populations of highly refractory malignant cells. By screening different blood cancers, we have observed that this activity is not always restricted to CD34+ leukemic cells, and can be overexpressed in CD34 negative leukemia.

Vybrant DyeCycle Violet Stain Discriminates Two Different Subsets of CD34+ Cells.

Introduction: Studies are needed to understand the role of CD34 expressing cells with regard to efficient engraftment, especially in the adjuvant treatment of cancer.

Materials And Methods: In this study we have used a modified method in our laboratory for routinely counting CD34+ cells. Unlysed whole blood samples were stained with the DNA-selective and cell membrane-permeant Vibrant DyeCycle Violet stain.

Effects of intermittent hypoxia and light aerobic exercise on circulating stem cells and side population, after strenuous eccentric exercise in trained rats.

Our goal was to address if intermittent hypobaric hypoxia (IHH) exposure can help to increase the number of peripheral blood circulating progenitor cells and side population (SP) stem cells, in order to establish the usefulness of this intervention for skeletal muscle repair, because these cells play a role in tissue regeneration. Male Sprague-Dawley rats were studied in two basal states: untrained and trained and compared with 1, 3, 7 and 14 days stages of damage recovery of trained rats that had suffered skeletal muscle injury. Three experimental groups were studied: rats with passive recovery (CTRL); rats exposed to IHH after muscle damage (HYP); and, trained rats that, in addition to IHH, performed light aerobic exercise sessions (EHYP).

Accuracy and reproducibility of stem cell side population measurements on clinically relevant products.

Introduction: In 1996, Goodell et al. first described a rare subpopulation of bone marrow stem cells termed the Side Population (SP). SP cells are known to be CD34 negative and to have a high repopulating capability.