Side Scatter Research Articles

Mass Cytometry – Based Cytotoxicity Assay for Profiling Natural Killer Cells

Phenotypic and functional assessment of Natural Killer (NK) cell activity against cancer cells in a clinical setting requires the integration of labor-intensive multicolor flow cytometry, chromium (Cr) release assays, and CD107a degranulation assays. To merge these into a single high-throughput assay, we developed a method for profiling NK-cancer cell interactions and simultaneous target killing using mass cytometry. In this study, a multiparameter 43-marker mass cytometry panel was applied to a co-culture of immune cells from healthy donors with diverse target cancer cell lines. DNA content, combined with classical CD45 surface staining, were used as gating parameters for co-cultures of immune cells (CD45high/DNAlow) with hematological (CD45low/DNAhigh) and solid cancer cell lines (CD45neg /DNAhigh), recapitulating flow cytometry forward and side scatter. This allows for discrimination of cancer cells from immune populations based on relative “size” and simultaneous assessment of phenotypical changes in both populations. In addition, the sensitivity of mass cytometry allows for the simultaneous detection of changes in NK cell phenotype and their degranulation upon target recognition, and for the analysis of target cells for cytotoxic protein granzyme B content, and cell death. These findings have broad applicability in clinical settings with the aim to phenotype and assess functional changes following NK-cancer cell interactions.

An optomicrofluidic device for the detection and isolation of drop-encapsulated target cells in single-cell format.

Single-cell analysis has emerged as a powerful method for genomics, transcriptomics, proteomics, and metabolomics characterisation at the individual cell level. Here, we demonstrate a technique for the detection and selective isolation of target cells encapsulated in microdroplets in single-cell format. A sample containing a mixed population of cells with fluorescently labelled target cells can be focused using a sheath fluid to direct cells in single file toward a droplet junction, wherein the cells are encapsulated inside droplets. The droplets containing the cells migrate toward the centre of the channel owing to non-inertial lift force. The cells present in the droplets are studied and characterised based on forward scatter (FSC), side scatter (SSC), and fluorescence (FL) signals. The FL signals from the target cells can be used to activate a selective isolation module based on electro-coalescence, using suitable electronics and a program to sort droplets containing the target cells in single-cell format from droplets containing background cells. We demonstrated the detection and isolation of target cells (cancer cells: HeLa and DU145) from mixed populations of cells, peripheral blood mononuclear cells (PBMC) + cervical cancer cells (HeLa) and PBMC + human prostate cancer cells (DU145), at a concentration range of 104-106 ml-1 at 300 cells per s. The performance of the device is characterised in terms of sorting efficiency (>97%), enrichment (>1800×), purity (>98%), and recovery (>95%). The sorted target cells were found to be viable (>95% viability) and showed good proliferation when cultured, showing the potential of the proposed sorting technique for downstream analysis.

In vitro genotoxicity study of silver nanoparticles and titanium dioxide nanoparticles.

Nanoparticles are widely used in cosmetic, pharmaceutical, and food industries, but their safety and genetic toxicity are still unclear. In this study, the genotoxicity of silver nanoparticles (AgNPs) and titanium dioxide nanoparticles (titanium dioxide nanoparticles) were evaluated by in vitro comet assay and PIG-A assay in TK6 cells. We exposed TK6 cells to two types of nanoparticles at the highest concentration of 200 μmol/L for 4 h and conducted the in vitro comet assay. We examined the mutation results of PIG-A gene in vitro after 4 h, 24 ho and 10 days of exposure, respectively. We also examined the endocytosis of nanoparticles in TK6 cells exposed to nanoparticles for 24 h. In the endocytosis assay, with the increase of nano-material concentration, the side scatter (SSC) of TK6 cells in flow cytometry showed a concentration-dependent and time-dependent increase, indicating that TK6 cells could uptake both types of nanoparticles. In the comet assay, AgNPs could induce a concentration-dependent increase in DNA tail intensity. However, titanium dioxide NPs could not induce the concentration-dependent increase of DNA fluorescence intensity of comet tail. In the PIG-A assay, both AgNPs and TiO2NPs did not induce PIG-A gene mutation frequency in TK6 cells. The results showed that AgNPs could induce DNA damage in TK6 cells, but could not induce increase of PIG-A gene mutation frequency. TiO2NPs neither induce DNA damage in TK6 cells nor increase PIG-A mutation frequency. Further tests are needed to determine whether TiO2NPs are genotoxic.

Early Phases of COVID-19 Are Characterized by a Reduction in Lymphocyte Populations and the Presence of Atypical Monocytes.

BackgroundSevere acute respiratory syndrome coronavirus 2 is a recently discovered pathogen responsible of coronavirus disease 2019 (COVID-19). The immunological changes associated with this infection are largely unknown.MethodsWe evaluated the peripheral blood mononuclear cells profile of 63 patients with COVID-19 at diagnosis. We also assessed the presence of association with inflammatory biomarkers and the 28-day mortality.ResultsLymphocytopenia was present in 51 of 63 (80.9%) patients, with a median value of 720 lymphocytes/µl (IQR 520-1,135). This reduction was mirrored also on CD8+ (128 cells/µl, IQR 55-215), natural killer (67 cells/µl, IQR 35–158) and natural killer T (31 cells/µl, IQR 11–78) cells. Monocytes were preserved in total number but displayed among them a subpopulation with a higher forward and side scatter properties, composed mainly of cells with a reduced expression of both CD14 and HLA-DR. Patients who died in the 28 days from admission (N=10, 15.9%), when compared to those who did not, displayed lower mean values of CD3+ (337.4 cells/µl vs 585.9 cells/µl; p=0.028) and CD4+ cells (232.2 cells/µl vs 381.1 cells/µl; p=0.042) and an higher percentage of CD8+/CD38+/HLA-DR+ lymphocytes (13.5% vs 7.6%; p=0.026).DiscussionThe early phases of COVID-19 are characterized by lymphocytopenia, predominance of Th2-like lymphocytes and monocytes with altered immune profile, which include atypical mononuclear cells.

Intracellular Quantification and Localization of Label-Free Iron Oxide Nanoparticles by Holotomographic Microscopy.

BackgroundThe limitations of optical microscopy to determine the cellular localization of label-free nanoparticles prevent a solid prediction of the cellular effect of particles intended for medical applications. To avoid the strong physicochemical changes associated with fluorescent labelling, which often result in differences in cellular uptake, efficiency and toxicity of particles, novel detection techniques are required.MethodsIn the present study, we determined the intracellular content of unlabeled SPIONs by analyzing refractive index (RI)-based images from holotomographic three-dimensional (3D) microscopy and side scatter data measured by flow cytometry. The results were compared with the actual cellular SPION amount as quantified by atomic emission spectroscopy (AES).ResultsLive cell imaging by 3D holotomographic microscopy demonstrated cell-specific differences in intracellular nanoparticle uptake in different pancreatic cell lines. Thus, treatment of PANC-1SMAD4 (1−4) and PANC-1SMAD4 (2−6) with SPIONs resulted in a significant increase in number of areas with higher RI, whereas in PANC-1, SUIT-2 and PaCa DD183, only a minimal increase of spots with high RI was observed. The increase in areas with high RI was in accordance with the SPION content determined by quantitative iron measurements using AES. In contrast, determination of the SPION amount by flow cytometry was strongly cell type-dependent and did not allow the discrimination between intracellular and membrane-bound SPIONs. However, flow cytometry is a very rapid and reliable method to assess the cellular toxicity and allows an estimation of the cell-associated SPION content.ConclusionHolotomographic 3D microscopy is a useful method to distinguish between intracellular and membrane-associated particles. Thus, it provides a valuable tool for scientists to evaluate the cellular localization and the particle load, which facilitates prediction of potential toxicity and efficiency of nanoparticles for medical applications.

Large Extracellular Vesicles Can be Characterised by Multiplex Labelling Using Imaging Flow Cytometry.

Extracellular vesicles (EVs) are heterogeneous in size (30 nm–10 µm), content (lipid, RNA, DNA, protein), and potential function(s). Many isolation techniques routinely discard the large EVs at the early stages of small EV or exosome isolation protocols. We describe here a standardised method to isolate large EVs from medulloblastoma cells and examine EV marker expression and diameter using imaging flow cytometry. Our approach permits the characterisation of each large EVs as an individual event, decorated with multiple fluorescently conjugated markers with the added advantage of visualising each event to ensure robust gating strategies are applied. Methods: We describe step-wise isolation and characterisation of a subset of large EVs from the medulloblastoma cell line UW228-2 assessed by fluorescent light microscopy, transmission electron microscopy (TEM) and tunable resistance pulse sensing (TRPS). Viability of parent cells was assessed by Annexin V exposure by flow cytometry. Imaging flow cytometry (Imagestream Mark II) identified EVs by direct fluorescent membrane labelling with Cell Mask Orange (CMO) in conjunction with EV markers. A stringent gating algorithm based on side scatter and fluorescence intensity was applied and expression of EV markers CD63, CD9 and LAMP 1 assessed. Results: UW228-2 cells prolifically release EVs of up to 6 µm. We show that the Imagestream Mark II imaging flow cytometer allows robust and reproducible analysis of large EVs, including assessment of diameter. We also demonstrate a correlation between increasing EV size and co-expression of markers screened. Conclusions: We have developed a labelling and stringent gating strategy which is able to explore EV marker expression (CD63, CD9, and LAMP1) on individual EVs within a widely heterogeneous population. Taken together, data presented here strongly support the value of exploring large EVs in clinical samples for potential biomarkers, useful in diagnostic screening and disease monitoring.

Abstract 13285: Short-term Low-saturated Fat Diet Compared to High-saturated Fat Diet Improves Monocyte Phenotypes in Subjects With Hypertriglyceridemia

Introduction: Clinical trials suggest that low-saturated fat diet (LSFD) may reduce the risk of atherosclerotic cardiovascular disease (ASCVD) in subjects with hypertriglyceridemia (HTG). Monocytes play crucial roles in atherogenesis. Hypothesis: LSFD vs high-saturated fat diet (HSFD) improves monocyte phenotypes, thereby reducing ASCVD risk, in subjects with HTG. Methods: Subjects with HTG and metabolic syndrome (MetS, n=19) received isocaloric LSFD (~25% of calories from fat, 5% from saturated fat) and HSFD (~52% of calories from fat, 25% from saturated fat) in randomized order for 4 days (days 1-4) plus a breakfast on day 5, separated by a 4- to 6-week washout period. Blood was drawn on day 1 fasting before the diets and 3 times on day 5 (fasting before the breakfast and 4 and 6 hours postprandial) for measurement of lipid profile and analyses of monocyte phenotypes by flow cytometry and monocyte adhesion by a lab-on-a-chip microfluidic assay. Results: On day 5, LSFD, compared to HSFD, induced lower plasma levels of postprandial total triglyceride and LDL-triglyceride and fasting and postprandial total cholesterol, LDL-cholesterol, and small dense LDL-cholesterol. Compared to HSFD, LSFD reduced fasting and postprandial intracellular lipid accumulation in classical and intermediate monocytes examined by nile red staining and indicated by side scatter value of flow cytometric analysis. LSFD versus HSFD also reduced ex vivo uptake of oxidized LDL by classical monocytes at 4 hours postprandially and by intermediate monocytes in fasting state. Surface levels of molecules involved in monocyte adhesion/migration, including CD11c, CD81, and CCR2, were lower on monocytes with LSFD than with HSFD. Consistently, LSFD compared to HSFD reduced monocyte adhesion to VCAM-1. Intracellular levels of cytokines such as IL-1β, TNFα, and IL-6 in monocytes showed no difference between the two diets. Conclusions: In subjects with HTG and MetS, short-term LSFD compared to HSFD reduces monocyte intracellular lipid accumulation and improves monocyte phenotypes with reductions in monocyte adhesion and oxidized LDL uptake. These findings highlight the importance of diet composition in monocyte phenotypes and possibly atherosclerosis risks in patients with HTG and MetS.

Quantification of Light Scattering Detection Efficiency and Background in Flow Cytometry.

Knowledge of the sensitivity of a flow cytometer is essential for data interpretation and comparison. However, light scatter sensitivity currently cannot be quantified due to a lack of a standardized unit. Analogous to the QB approach for fluorescence, we show that it is possible to derive the detection efficiency Q and background B for light scatter detectors, using the scattering cross section (σs) in nm2 as a standardized unit. Side scatter of a mixture of polystyrene beads with known diameter and refractive index was measured at different illumination powers on a customized BD FACSCanto. Poisson statistics was used to derive the number of statistical photoelectrons from the measured coefficient of variation of the bead populations. The resulting light scatter sensitivity was expressed as Q in photoelectrons/nm2, B in nm2, and a resolution limit (R) in nm2. As expected, Q scales linearly with the illumination power (R2 = 0.999), and B is constant over the illumination power (difference < 15%). The resulting R decreased from 1.75·105 nm2 at 20 mW, to 4.03·104 nm2 at 200 mW, which resembles the light scattering of a 450 nm and a 314 nm polystyrene bead, respectively. Comparison of R with the side scatter histograms confirmed that R describes the lower limit at which light scatter signals can be fully discriminated from the background noise. In conclusion, as a proof of principle, we derived Q, B, and R for a scatter detector of a flow cytometer where photon noise associated with the light scattering signal originating from the particle is the dominant source of the measured variation. To quantify the sensitivity of scatter detectors with nanoparticle (<100 nm) sensitivity, our approach requires monodisperse (variation coefficient < 2%) nanoparticles that scatter light isotropically. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.

Novel in vitro assays to detect circulating permeability factor(s) in idiopathic focal segmental glomerulosclerosis.

Many patients with idiopathic focal segmental glomerulosclerosis (FSGS) develop recurrence of proteinuria after kidney transplantation (TX). Although several circulating permeability factors (CPFs) responsible for recurrence have been suggested, there is no consensus. To facilitate CPF identification and predict recurrence after TX, there is a need for robust methods that demonstrate the presence of CPFs. Cultured human podocytes (hPods) and human and mouse glomerular endothelial cells (ciGEnC, mGEnC) were exposed to plasmas of FSGS patients with presumed CPFs, and of (disease) controls. A visual scoring assay and flow cytometry analysis of side scatter were used to measured changes in cellular granularity after exposure to plasma. Nine out of 13 active disease plasmas of 10 FSGS patients with presumed CPFs induced granularity in hPod in a dose- and time-dependent manner. Corresponding remission plasmas induced no or less granularity in hPod. Similar results were obtained with ciGEnC and mGEnC, although induced granularity was less compared with hPod. Notably, foetal calf serum, healthy plasma and a remission plasma partially blocked FSGS plasma-induced hPod granularity. We developed a novel assay in which active disease, presumably CPF-containing, FSGS plasmas induced granularity in cultured hPod. Our results may indicate the presence of CPF inhibitor(s) in healthy and remission plasma. We suggest the presence of a delicate balance between CPF and a CPF inhibitory factor, which is disturbed in patients with active disease. Our novel assays can be applied in future research to identify CPF and CPF inhibitors, and possibly to predict recurrence after TX.

The Utility of Leucocyte Cell Population Data and Scattergram in Rapid Identification of Acute Promyelocytic Leukemia

Introduction: Acute promyelocytic leukemia (APL) is one of the hematological emergencies requiring early detection and treatment. The hematology laboratory plays a pivotal role in management. The correct morphological identification of APL is made even before the confirmation by the cytogenetic or molecular analysis. Automated hematology analyzers are the backbone of the hematology laboratories. Other than providing routine counts and differentials, they are also capable of producing sufficient information in the form of leucocyte cell population data (CPD) scatter plots depicting the leucocyte subpopulation. These data are primarily underutilized, particularly in assessing the lineage of various hematopoietic neoplasm. Here we analyzed the utility of leucocyte CPD and the pattern of scatter plot in Sysmex XN 1000 analyzer for the detection of acute promyelocytic leukemia. Materials and Results: We included 100 controls and 100 AML cases. Leucocyte CPD parameters were compared along with scatter plots from the WDF channel from Sysmex XN1000 analyzer were recorded and analyzed. Immunophenotyping and molecular analysis confirmed the diagnosis in all but two cases, where the patients expired within a few hours of admission in the emergency. Out of a total of 100 cases, 22 were labeled as APL, 63 as AML, and 15 as AML M4/M5. Among the APL cases, in 19 cases, immune-phenotyping was done on peripheral blood, and the remaining three bone marrow aspirate samples were used. The scatter-plot analysis of APL cases showed a characteristic pattern which differentiated this entity from other acute myeloid leukemias. In the case of APL, on Side Fluorescence Light Scatter (SFL) Vs. Side scatter (SSC), the abnormal promyelocytes occupy the space above neutrophils and to monocytes' right, where the immature precursors of myeloid are usually found. In cases where the total leukocyte count was within the normal range, a hiatus between these abnormal promyelocytes and neutrophils are seen. This finding was consistent in all cases irrespective of the morphological variation. On the other hand, other variants of acute myeloid leukemias along with monoblastic leukemias, the scatter plots show the blasts in the monocytic region with extension to the ceiling. On comparison, the myeloblast and monoblasts lie more towards the y-axis than abnormal promyelocytes. All the CPD parameters were compared against the control group and the AML group. The cell population data showed a significant difference in all the parameters representing the side fluorescence, size and granulations except LY-WX (width of dispersion of side scatter) when compared to normal healthy controls. Amidst all the AML cases, APL significantly differed from other cases in NE-SFL, LY-WY, and MO-WZ. AML M4/M5 and APL also varied in LY-Y, NE-WY, NE-WZ, and MO-WY. AMLs excluding AML M4/M5 showed additional variation in MO-Y and MO-Z. Discussion: APL cases have a characteristic scatter plot patter on SFL Vs. SSC. The scatter-plot shows a tear-drop like a collection of cells. These cells have high side scatter and high lateral fluorescence owing to the large size of the cells, apple-core like the nucleus, and dense granulations. The differences in the CPD parameters among APL cases and controls and other acute myeloid leukemia can be attributed to these morphological features of the abnormal promyelocytes. Haider et al. found a significant difference between NE-SFL and other myeloid leukemia. [1] This was in corroboration with our findings. Another research done by Park et al. showed that NE-SFL and MO-WX had the highest sensitivity and specificity in differentiating APL from other AML. [2] Conclusion: We conclude that the new age automated cell counters provide numerous data that remains unexplored and can be utilized further along with the information from the scatter plots to make a rapid diagnosis of Acute Promyelocytic Leukemias.

Predictors of Survival and Time to Progression to Myeloid Neoplasm in Patients with Clonal Cytopenias

Background: Clonal cytopenias of Undetermined Significance (CCUS) have a higher risk of progression to myeloid neoplasm (MN) compared to idiopathic cytopenias of undetermined significance (ICUS) (Malcovati et al., Blood, 2017). The implementation of flow cytometric (FC) immune-phenotyping and next generation sequencing (NGS) in clinical practice has improved the diagnosis of these entities but the clinical significance and interaction of these abnormal results are still unknown. In this study we investigated predictive factors that play role in survival and progression to MN in patients with ICUS/CCUS. Methods: Patients (Pts) who had undergone evaluation for unexplained cytopenia and had undergone FC and bone marrow morphological assessment between 3/2015-3/2020 at Mayo Clinic Rochester were included. Pts were excluded if a malignant hematological disorder was diagnosed prior to the time of FC. FC included evaluation of the following parameters: Abnormal expression of HLA-DR/CD13, CD2, CD7, CD45, and CD56 on blasts; and abnormal CD13/CD16 expression and side scatter on granulocytic cells. NGS panel up to 2018 included 34 genes, ASXL1, BCOR, BRAF, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, MYD88, NOTCH1, NPM1, NRAS, PHF6, PTPN11, RUNX1, SETBP1, SF3B1, TERT, TET2, TP53, U2AF1, WT1, and ZRSR2; in 2018, BRAF was omitted and 12 genes were added, ANRD26, DDX41, ELANE, ETNK1, KDM6A, MYD88, NOTCH1, RAD21, SH2B3, SRP72, SMC3, and STAG2. Results: Characteristics: Of 490 consecutive pts, 238 (median age 69 years (range 19-92), 66% males) met our inclusion criteria as ICUS. 86 (36%) pts had CCUS (abnormal cytogenetics and/or tested positive for pathogenic mutations on NGS). After a median follow-up of 28 months (95% CI: 20 to 31 months), 21 (25%) patients developed MN and 23 (27%) died during follow-up. Comparing ICUS vs CCUS: upon comparing CCUS pts to ICUS, several factors were found to be significantly different: CCUS pts were older (p= .02); majority male (p= .04), had more abnormal HLA-DR/CD13 (p &amp;lt;.0001), more side-scattered light by FC (p &amp;lt;.0001), more pancytopenia on follow up (p= .02) and more morphologic atypia (&amp;lt;.0001). Overall survival (OS) outcomes in CCUS: Several covariates were significant in univariate models, and model selection was used to generate a risk score based on abnormal CD7, abnormal CD13/CD16, abnormal HLA-DR/CD13, splenomegaly and history of prior chemotherapy or radiotherapy (range: 0 - 4; HR=2.58, [95 CI: 1.69 - 3.94], p&amp;lt;.0001). Risk scores were grouped as low risk (score=0), intermediate risk (score=1), and high risk (score=2+), with estimated 2-yr-OS of 95%, 84%, 40%, respectively (Figure 1). An extended risk score model including NGS factors added ASXL1 and IDH1 mutation status to the previous model (range: 0 - 6; HR=2.72, [95 CI 1.82 - 4.06], p&amp;lt;0.0001). MN-free survival (MNFS) in CCUS: 37 pts either progressed to MN and/or died with a median follow up time for MNFS of 22.4 months. Similar for OS, model selection approaches yielded a composite risk score: splenomegaly, BCOR mutation status, mutation in RAS signaling pathway, abnormal expression of flow markers CD13/CD16, HLA-DR/CD13 or CD7 (HR=3.23, [95 CI: 1.90 - 5.49], p=&amp;lt;0.0001). Risk scores were grouped as low risk (score=0), intermediate risk (score=1), and high risk (score ≥ 2), with estimated 1-yr MNFS of 84%, 74%, and 31%, respectively (Figure 2). Conclusion: CCUS has unique features compared to ICUS. Several factors, including clinical characteristics, immune-phenotyping by FC, and somatic mutations have important impact on risk of progression to MN and on overall survival. Our findings serve as proof-of-concept that such integrated models could help identify CCUS patients at higher risks for progression to MN or death. They can guide treatment accordingly and should be evaluated further. Disclosures Shah: Dren Bio: Consultancy.

Sars-Cov-2 Spike Protein Induces Cellular Changes in Primitive and Mature Hematopoietic Cells

The devastating effects of SARS-CoV-2 infection (which causes COVID-19) highlight the need for a deeper understanding of the virus, host response, and the tissues affected by infection, including the hematopoietic stem cells (HSC), hematopoietic progenitors (HPC), and more mature blood cells including immune cells. While other coronavirus infections have been associated with hematopoietic or immune cell complications (Chu et al., J Infect Dis 2016), the effects of SARS-CoV-2 infection on HSC/HPC and mature immune cells are not yet understood. We performed studies to examine the potential susceptibility of HSC/HPC subpopulations and immune cells to infection and functional studies to explore if exposure to SARS-CoV-2 proteins impacts these cells. The most well-studied receptor for SARS-CoV-2 is ACE2 (Hoffmann et al., Cell 2020), a cell surface protein to which SARS-CoV-2 spike protein (S protein) binds to facilitate viral entry to host cells. We explored expression of ACE2 in primitive and mature blood cells. We demonstrated using RT-qPCR as well as western blotting that ACE2 is expressed at both the mRNA and protein levels in pooled low-density peripheral blood cells (PB) from healthy donors (n=5); pooled CD34+ cells from cord blood (CB) enriched for HSC/HPC (n=7); lineage+ (lin+) low density CB cells (n=7), and high density polymorphonuclear leukocytes (PMN) from CB (n=3). We then determined ACE2 expression on specific subpopulations of HSC/HPC and immune cells. Flow cytometry analysis (FACS) demonstrated that ACE2 is expressed on the cell surface of small subpopulations of PB immune cells, including 1-2% of T-cells, 2-4% of B-cells, and <1% of NK cells and monocytes. However, larger populations of HSC/HPC exhibited ACE2 cell surface expression. The percent ACE2+ HSC, multipotent progenitor cells (MPP), and multipotent lymphoid progenitor cells (MLP) were respectively 15-60%, 5-50%, and 5-15% of cells (n=5). We next determined if exposure to viral S protein affects these cell populations. We first tested if SARS-CoV-2 S protein induces responses in HSC/HPC. We isolated CD34+ cells from low density CB and expanded them in the presence of serum and growth factors (100ng/mL TPO, FLT3L, and SCF) in the presence or absence of recombinant full-length S protein. After 7 days expansion, cells were analyzed for immunophenotype using FACS or were plated in semi-solid methylcellulose in an HPC colony forming unit (CFU) assay and grown for 12 days before scoring for functional CFU-Granulocyte/Erythroid/Macrophage/Megakaryocyte (CFU-GEMM) and CFU-Granulocyte/Macrophage (CFU-GM) progenitor cells. CB CD34+ cells exhibit significantly less expansion when exposed to SARS-CoV-2 S protein, showing 33% less expansion of CD34+ cells. The most significant delay in expansion of phenotyped cells is evident in granulocyte-monocyte progenitors (GMP, 38% less expansion), with a more modest 15-30% decrease in expansion for common myeloid progenitors/megakaryocyte-erythroid progenitors (CMP/MEP), HSC, MPP, and MLP (n=4). S protein lessens functional HPC colony forming cell expansion compared to control, with 12-fold and 2.1-fold expansions compared to 30.8-fold and 6-fold expansions respectively for CFU-GM and CFU-GEMM. These data indicate that exposure to SARS-CoV-2 S protein impacts hematopoiesis and myeloid differentiation in vitro. We next tested effects of S protein exposure on PB by isolating low density blood cells from healthy donors and culturing them ex vivo for different time periods in the presence or absence of recombinant full-length S protein. After 2hrs incubation, we observed by FACS a modest but consistent 1.2-fold increase in CD14 positive monocytes in cells exposed to S protein compared vehicle control (n=4). After 18hrs incubation with the S protein, the PB monocytes exhibited consistent changes in morphology, becoming smaller and more granular, determined by changes in forward scatter and side scatter by FACS (n=5). This suggests that exposure to SARS-CoV-2 protein may have a physiologic impact on PB cells ex vivo, despite their low levels of ACE2 expression. Our data thus has important implications for hematopoiesis and immune response in COVID-19 patients. It demonstrates the need to determine whether CB potentially exposed to SARS-CoV-2 should be banked, as the effects we observed may affect the efficacy of CB transplantations.DisclosuresNo relevant conflicts of interest to declare.

Studying Immunophenotypic Modulation at Different Time Points of Chemotherapy : A Walk through the Disease Course in 121 Pediatric B Lymphoblastic Leukemia Cases; Providing Implications for MRD Detection

Introduction Disease monitoring in management of acute leukemia is essentially important in terms of risk stratification and assessing response to chemotherapy throughout the disease course for which minimal residual disease (MRD) testing is the most reliable tool. Measurement of MRD by flow cytometry is based on the principle of studying difference in antigenic expression of the leukemic cells, hematogones and residual normal B cells. Modulation of antigen expression is an established fact that occurs on leukemic blasts during treatment under the influence of chemotherapeutic drugs, which may cause difficulty in analysis and detection of MRD [H.Burnusuzov et al. Folia Medica 2016;58(1):28-35]. Literature has reported antigen expression modulation, particularly during the glucocorticoid phase of induction therapy [Dworzak et al. Cytometry Part B 2010; 78B: 147-153]. For MRD assessment, such phenotypic shifts pose a particular challenge since they can cause misinterpretations and false results. However to our knowledge there hasn't been a large cohort studied to map antigenic modulation and analyze similar antigenic trends in comparison between leukemic blasts and non-leukemic B cells which may coexist during different phases of chemotherapy. Key words: B-lymphoblastic leukemia, Flowcytometry, Antigen modulation, Minimal residual disease, Immunophenotype Methods: The study is conducted at the Hematology department of The Indus Hospital from April 2018 to March 2020. Among 445 pediatric B-lymphoblastic leukemia patients (1 month-18 years); we studied 121 patients who were MRD positive in bone marrow throughout the evaluation period. All patients received Berlin-Frankfurt-Munster (BFM) chemotherapy protocol as per National Cancer Institute (NCI) risk stratification criteria. To observe immunophenotypic modulation (IM) in antigen expression of Tdt, CD34, CD10, CD20, CD45, CD13, CD33 and CD66, compared from the time of diagnosis (day 0), post induction chemotherapy (day 35), post consolidation chemotherapy (day 52) and during maintenance chemotherapy (day78). Using eight color Flowcytometry (BD FACS CANTO-II; DIVA software version 8.0.2), the IM was assessed by comparative analyses of the changes in the mean flourescence intensity (MFI) of leukemic and non-leukemic B cells. The Wilcoxon signed rank test is used to compare median of MFI values at diagnosis and subsequent time points of MRD. Independent sample T-test/Mann-Whitney U test was applied as appropriate to determine significant differences in expression of antigens. A p-value of &amp;lt; 0.05 was considered as significant. Box and whisker plots and run charts will be used for descriptive purposes. Results: Mean age of the patients was 5.7years ±3.7 with male to female 1.7:1.We analyzed forward scatter and side scatter properties and antigen expression of the leukemic and normal residual B-cells. We observed statistically significant differences in MFI levels of CD10, CD20, CD19, CD45, CD66 and Tdt of leukemic cells showing trends at different phases. Down modulation of CD10 was noticed from day 0-day 35 however up modulation was observed in subsequent phases. CD20 and TdT both showed significant and stable up modulation from day 0-day78. A steep down modulation was observed in CD45 and CD19 from day 0 to day 52 and thereafter remained stable. CD34 expression was variable and showed up modulation at day 35 followed by down modulation and then again a reincrease at day 78. CD66, CD33 and CD13 showed similar varying trends exhibiting up modulation at day 35 and then down modulation at day 52 and day 78. Residual normal B cells showed substantial decrease in antigenic expression for CD19, CD20 and CD45 till day 52 followed by an ascent at day 78. Conclusion: Immunophenotypic modulation occurred to different extents in all analyzed samples. Our results confirm the presence of transient immunophenotypic changes for CD10, CD34, CD13, CD33 and CD66 at induction phase of chemotherapy whereas CD20 and TdT showed irreversible gain in expression that is exploitable for MRD detection. The MFI of the different antigens expressed by the leukemic blasts should be taken into consideration and cautiously analyzed for MRD detection. Figure 1 Disclosures No relevant conflicts of interest to declare.

Implementation of Flow Cytometric Monocyte Repartition Assay for Assessment of Chronic Myelomonocytic Leukaemia (CMML) in Bone Marrow

Background: CMML is a myelodysplastic/myeloproliferative overlap syndrome associated with persistent monocytosis. Previous studies have shown a characteristic phenotype of peripheral blood (PB) monocytes in CMML that may have diagnostic utility. There is expansion of the classical monocyte (Mo1) population (CD14+CD16-) with concomitant reduction in the intermediate (Mo2; CD14+16+) and non-classical (Mo3; CD14-16+) populations. It is unclear whether an increased Mo1 population or a reduced Mo2/Mo3 population is a better discriminator in detecting CMML by flow cytometry. Diagnostic thresholds for these populations have most often been established by comparison with normal donors using PB samples. The objective of this study was to determine the optimal cut-off limits for flow cytometric monocyte repartition (FMR) in bone marrow (BM) samples and to assess the diagnostic accuracy of this method in patients undergoing BM evaluation for other causes of monocytosis using a novel gating strategy. Patients/Methods: 71 BM aspirates were analysed comprising 35 confirmed CMML (21 proliferative CMML , 14 dysplastic CMML), 13 other myeloid neoplasm (MN) disease controls and 23 normal bone marrow aspirates. The MN cohort was identified through local samples with a known peripheral blood monocytosis of ≥1.0x109 and peripheral blast count of &amp;lt;20% not fulfilling criteria for CMML. The MN cohort comprised of 4 cases of chronic myeloid leukaemia (CML), 2 cases of myelodysplasia/myeloproliferative neoplasm unspecified (MDS/MPN-U), two cases of atypical CML (aCML), 2 cases of primary myelofibrosis, 1 case of juvenile myelomonocytic leukaemia (JMML), 1 case of chronic neutrophilic leukaemia (CNL) and 1 case of therapy-related acute myeloid leukaemia (T-AML). The median age of CMML patients within our cohort was 74 years (range 44- 89 years) and 77% were male. Stored flow cytometric data were reanalysed to assess monocyte subsets. After initial removal of doublets and non-cellular events, sequential gating selected a CD45+CD33bright population with low side scatter to select monocytes and exclude granulocytes. The lymphocyte gate as determined on the CD45 vs CD33 plot was used to establish a threshold for CD16 positivity. Immature double negative monocytes (CD14-CD16-) were excluded as in published gating strategies. Results: Linear regression analysis of the percentage of both the Mo1 and Mo2 populations demonstrated a significant difference between the CMML, MN, and normal cohorts (global P&amp;lt;0.0001). Post hoc analysis demonstrated a significant increase in the Mo1% fraction (mean 92.5%; 95% Confidence Interval [CI]: 88.1%, 96.9%) in CMML cases when compared to the normal BM cohort (mean 63.5%; 95% CI: 58.1%, 69.0%; comparison P &amp;lt;0.0001) and the MN cohort (mean 79.9%; 95% CI: 72.7%,87.2%; comparison P = 0.004). Conversely, the Mo2% was significantly decreased (mean 6.8% 95% CI: 2.7%, 10.9%) in CMML cases when compared to the normal cohort (mean 33.0%; 95% CI: 27.9%; 38.1%; comparison P value &amp;lt;0.0001) and the MN cohort (mean 17.6%; 95% CI 10.8%; 24.3%; comparison P value 0.008). Receiver Operator Curve (ROC) curve analysis of the Mo1 population demonstrated an area under the curve (AUC) of 0.8738 (95% CI: 0.7897, 0.9579; P&amp;lt;0.0001) with a marginally lower AUC for Mo2% (Figure 1A). Priority was given to selecting a cut-off value with higher specificity, considering that the BM examination is not a screening test. A cut-off value for Mo1% of &amp;gt;88.5% (Figure 1B) gave a sensitivity of 77.1% (95% CI: 60.98%, 87.93%) and specificity of 88.9% (95% CI: 74.69%, 95.59%) (Figure 1A). A Mo2% cut-off value of &amp;lt;12.25% (Figure 1C) gave a sensitivity of 78.8% (95% CI: 62.25%, 89.32%) with a specificity of 80.6% (95% CI: 64.97%, 90.25%) (Figure 1A). Eight patients with CMML were misclassified by the Mo1/Mo2 criteria. Conclusion: Our study confirms that analysis of monocyte subsets on BM samples provides good discrimination of CMML cases when compared to normal controls. There was significant overlap with other MN samples. FMR may be a useful adjunct to BM morphology in cases with subtle dysplasia. Further study is required to understand the overlap in monocyte phenotype between CMML and other myeloid neoplasms. Disclosures Allen: Amgen: Other: Travel Support; Roche: Other: Travel Support; Novartis: Other: Travel Support. Hahn:Roche: Honoraria; Astra Zeneca: Honoraria.

The Use of Monocyte Subset Repartitioning By Flow Cytometry for Diagnosis of Chronic Myelomonocytic Leukemia

Introduction Patients with chronic myelo-monocytic leukaemia (CMML) have been reported to have a relative predominance of classical or MO1 monocytes (CD14+/CD16-) at the expense of MO2 (CD14low/CD16+) and MO3 (CD14-/CD16+) monocytes (Selimoglu-Buet. Blood 2015). These authors suggested that an MO1 percentage cut-off of &amp;gt;94% could predict the diagnosis of CMML with high sensitivity and specificity (both &amp;gt;90%) from other causes of monocytosis. Since then, several independent groups have attempted to reproduce their protocol with variable results. Most recently, the Mayo Clinic (Pophali. Blood Cancer J. 2019) found that an MO1 cut off of &amp;gt; 94% in peripheral blood identified CMML with a sensitivity of 75% and a specificity of 95.4%. These figures were lower than previously reported - calling into question, the utility of flow cytometry to distinguish the aetiology of monocytosis in a real-world setting. Objective Our retrospective audit aimed to establish if monocyte subset repartitioning could be used to reliably diagnose CMML in a real-world sample of patients with monocytosis. Methods In this study, we assessed peripheral blood samples from 35 patients presenting with a monocytosis (absolute monocyte count &amp;gt; 1 x109/L) in a tertiary referral hospital in Brisbane, Australia between June 2015 and Sep 2019. The patients' final clinical diagnosis was extracted from the medical record by two clinicians (AM, PM) blinded to the results of the flow cytometry. Peripheral blood samples were subjected to MFC at a median of &amp;lt;24 hours (range, &amp;lt; 24 hours to 160 hours) after collection. Flow cytometry was performed using the BD FACS Canto II flow cytometer. Monocyte subsets were identified using Kaluza software (Beckman Coulter, USA). A CD45/ side scatter gate was set to locate the monocyte population and specific antibody combinations were used to identify and exclude other lineages; these were - CD24 to exclude granulocytes and B cells, CD16 to exclude neutrophils, CD2 to exclude T cells and CD56 to exclude NK cells. Based on the CD14 and CD16 expression, the monocytes were then divided into: MO1 (CD14+/CD16-), MO2 (CD14low/CD16+) and MO3 (CD14-/CD16+). Results are reported descriptively and Fishers Exact Test (IBM® SPSS® Statistics Version 26) was used to establish if there was any correlation between the percentage of classical monocytes and the diagnosis of CMML. Results Of the 35 patients included, 13 patients had CMML and four patients were diagnosed with another underlying myeloid neoplasm: one patient with myelodysplastic syndrome (MDS-RCMD), one patient with myeloproliferative neoplasm - not otherwise specified (MPN-NOS), one patient with multiple myeloma and one patient with acute myeloid leukaemia with myelodysplasia related changes (AML-MRC). Eighteen cases had non-clonal monocytosis. Six patients had a reactive monocytosis in the setting of autoimmune disease including: granulomatosis with polyangiitis (n=2), rheumatoid arthritis (n=1), IgG4 disease (n=1), mixed connective tissue disease (n=1) and polyarticular gout (n=1). Twelve cases of reactive monocytosis were due to infective and inflammatory causes. Among our 13 patients with CMML, seven cases (53.85%) had an MO1 percentage &amp;gt; 94%. In comparison, only four (18.18%) among the 22 cases of non-CMML were identified to have an MO1 percentage &amp;gt; 94%. There was no correlation between an MO1 percentage cut off of &amp;gt; 94% and a diagnosis of CMML (p = 0.057). Our study also examined the utility of an MO3 percentage cut off of &amp;lt; 1.13%, established by Hudson et al (Am J Clin Pathol 2018). Among our 13 cases of CMML, six patients (46.15%) were noted to have an MO3 percentage &amp;lt; 1.13%. Meanwhile, only eight, out of 22 non-CMML patients (36.36%) had MO3 percentage &amp;lt; 1.13%. There was no association between MO3 percentage and the diagnosis of CMML (p = 0.724). Conclusion Using the MO1 and MO3 percentage cut offs previously established, we were unable to reliably diagnose CMML. Given these findings, we suggest that more research with larger sample sizes is required before monocyte subset analysis can be applied in the clinical laboratory to discriminate reactive monocytosis from CMML. Disclosures Mollee: Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Caelum: Membership on an entity's Board of Directors or advisory committees.

Cryopreserved Platelets Retain Agonist Responsiveness and Support for Factor VIII Function

Platelet activation supports procoagulant activity through phosphatidylserine exposure, secretion of procoagulant factors, and receptor conformational change. For example, thrombin-stimulated platelets bind factor VIII (fVIII) via a macromolecular complex including oligomeric fibrin and the active αIIbβ3 receptor (Phillips et al, JTH 2004; Gilbert et al, Blood 2015). Thus, coagulation assays in which phospholipid vesicles are substituted for platelets do not fully emulate modulators of fVIII activity. Indeed, inhibition of platelet-supported fVIII activity by a panel of mAbs against the C2 domain was not correlated to inhibition of vesicle-dependent activity (Chatterjee et al, JTH 2020). An obstacle to adoption of a platelet-based assay for fVIII is the need for fresh platelets. Therefore we asked whether cryopreserved platelets might support fVIII activity similarly to fresh platelets. Apheresis platelets were mixed with cryopreservatives with or without calcium chelators, in various aliquot sizes, and frozen on various cooling media. Cryopreserved platelets were compared to non-preserved apheresis platelets with regard to agonist response and support of procoagulant activity. Cryopreservation resulted in an increase in subcellular debris and an unresponsive fraction of platelets with decreased forward scatter judged by flow cytometry. Optimized results were obtained when platelet rich plasma with 5% DMSO, in 1 mL aliquots was frozen on powdered dry ice, and stored at -150C. Purification of thawed platelets using a density gradient removed debris and decreased unresponsive platelets resulting in a forward and side scatter profile comparable to fresh platelets. We refer to these as cryopreserved platelets (CryoPlts). CryoPlts were compared to control and outdated apheresis platelets. As with fresh platelets, procoagulant activity of CryoPlts increased with thrombin receptor agonist peptides (TRAP) 1 &amp; 4 and supported a log-linear relationship between time to initial fibrin strand formation and fVIII activity over a range of 0.0001 - 1 u/mL (Fig 1). Further, the degree of inhibition of fVIII activity by mAbs ESH4 and G99 against the fVIII C2 domain, was the same on control and CryoPlts, but markedly different from inhibition in an aPTT-based inhibitor assay. In contrast, outdated apheresis platelets had increased procoagulant activity, minimal agonist response and a shallow curve with varying fVIII concentration. Flow cytometry studies with lactadherin-FITC indicated that 33 ± 14% of CryoPlts had high PS exposure, and the size of this population was minimally affected by TRAP 1+4. In contrast, the main platelet population had a small, uniform, increment in PS exposure comparable to control platelets. Surprisingly, the PS-rich platelets did not significantly affect the time to fibrin formation, confirming that the viable platelets, with limited PS exposure, provide much of the support for fVIII-related procoagulant activity. Flow cytometry indicated αIIbβ3 activation (PAC1-FITC) and α-granule release (anti-P-selectin-PE) were qualitatively intact on CryoPlts, although staining was decreased 70% for PAC1 and 57% for Psel. We also tested whether CryoPlts may be utilized for evaluating response to anti-PF4-heparin antibodies, relevant to heparin-induced thrombocytopenia (HIT). We evaluated platelet response to platelet factor 4 (PF4) and a platelet-activating anti-PF4 antibody (KKO), a combination that induces activation similar to authentic autoimmune antibodies for HIT. The non-activating PF4 antibody RTO served as a negative control. Geometric mean response, corrected for background, was normalized to response to thrombin activation. Both fresh and CryoPlts responded with increases in PAC1 (Fig 2A) and anti-Psel (Fig 2B) binding in response to KKO/PF4 compared to RTO/PF4 . This data demonstrates that the qualitative αIIbβ3 and P-selectin response to HIT-like antibodies is intact. Our results demonstrate a refined cryopreservation protocol of apheresis platelets. These platelets maintain qualitative agonist responsiveness with near-normal support for factor VIII activity, suggesting that they could be used for other platelet-based laboratory or diagnostic assays. Further, our results suggest that major procoagulant activity is provided by platelets with very limited PS exposure, an area for further investigation. Disclosures No relevant conflicts of interest to declare.

Issue Highlights - November 2020.

Issue Highlights - November 2020.

Acute myeloid leukemia with mutated NPM1 mimics acute promyelocytic leukemia presentation.

To investigate clinicopathological and molecular features of NPM1-mutated acute myeloid leukemia that presented with infrequent acute promyelocytic leukemia (APL)-like phenotype and clinical presentation. Cases with both de novo or secondary Acute Myeloid Leukemia (AML) were retrieved. Data from flow cytometry immunophenotyping, cytogenetics, molecular studies, and clinical presentation were analyzed. Cases presented with abnormal coagulation parameters and low platelets count; four of them showed a DIC index compatible with overt DIC. Two cases showed Auer rods. In all cases, immunophenotypes mimicked APL: blasts expressed CD33, CD13, and cytoplasmic MPO but did not express CD34, HLA-DR, or CD11b. Notably, CD4 expression was observed in all cases. Neither t(15;17) nor PML/RARα gene rearrangement was detected. NPM1 gene mutation was identified in all cases. In four cases, TET2 or IDH2 co-mutations were identified. Our findings provide additional evidence of association between NPM1-mutated AML with TET2 or IDH2 co-mutations and the APL-like immunophenotype. This AML subset was found to exist in both de novo and secondary AML. High WBC count and blasts with low to moderate side scatter and significant expression of CD4 are observed features that could assist in the differential diagnosis with APL. The occurrence of significant elevated D-dimer levels, or even overt DIC observed at diagnosis in these cases could be relevant for this AML subtype.

Diameters and Fluorescence Calibration for Extracellular Vesicle Analyses by Flow Cytometry.

Extracellular vesicles (EVs) play a crucial role in the intercellular crosstalk. Mesenchymal stem cell-derived EVs (MSC-EVs), displaying promising therapeutic roles, contribute to the strong rationale for developing EVs as an alternative therapeutic option. EV analysis still represents one of the major issues to be solved in order to translate the use of MSC-EV detection in clinical settings. Even if flow cytometry (FC) has been largely applied for EV studies, the lack of consensus on protocols for FC detection of EVs generated controversy. Standard FC procedures, based on scatter measurements, only allows the detection of the “tip of the iceberg” of all EVs. We applied an alternative FC approach based on the use of a trigger threshold on a fluorescence channel. The EV numbers obtained by the application of the fluorescence triggering resulted significantly higher in respect to them obtained from the same samples acquired by placing the threshold on the side scatter (SSC) channel. The analysis of EV concentrations carried out by three different standardized flow cytometers allowed us to achieve a high level of reproducibility (CV < 20%). By applying the here-reported method highly reproducible results in terms of EV analysis and concentration measurements were obtained.

Differential effects of peritoneal and hemodialysis on circulating regulatory T cells one month post initiation of renal replacement therapy.

Backgroundː Chronic kidney disease stage G5 (CKD G5) patients show an activated but impaired immune system. One function of the FOXP3+ regulatory T (Treg) cells is to preserve tolerance to self while maintaining the ability to fight infectious agents. The aim of this pilot study is to evaluate longitudinal changes in Treg cells before and 1 month after the first dialysis treatment. Materials and methodsː CKD G5 patients not yet on dialysis were enrolled and started on hemodialysis (HD) or peritoneal dialysis (PD). Tregs were analyzed by flow cytometry at two time points: T0 (before the first dialysis treatment) and T1 (1 month after the first dialysis session). Wilcoxon test for dependent samples was used to compare the mean percentage difference between T0 and T1: Δ% = 100 × [(T1 – T0) / T0]. Resultsː 21 patients were enrolled: 8 on HD and 13 on PD. The proportion of total lymphocytes (low side scatter lymphocyte gate) and T lymphocytes (in the CD3+CD4+ gate) did not change significantly 1 month after the start of dialysis in both groups. Treg cells (as CD25+FOXP3+, FOXP3+, or CD25+CD127–), analyzed as percentage of the lymphocyte gate, showed a significant increase post PD (CD25+FOXP3+: median = 35.92; p = 0.0425; FOXP3+: median = 30.85; p = 0.0479 and CD25+CD127–: median = 23.71; p = 0.0215). The same populations, did not change 1 month after the first dialysis session. Conclusionː Our study is the first to evaluate longitudinal effects of dialysis on Treg cells in uremia and suggests that PD was more effective in increasing Treg levels 1 month post initiation of dialysis and may contribute to improvement of inflammatory status. Thus, PD may contribute to better outcomes for patients with renal dysfunction, also maintaining homeostasis of peritoneal and renal tissues.