Abstract: Bone marrow (BM) cavities are the primary sites of continuous and highly regulated blood cell production and reservoirs for long-term immune memory. Within BM tissues, a rare population of self-renewing and mulitpotent hematopoietic stem cells (HSCs) continuously differentiates into progenitors, which in turn generate mature blood derivatives to compensate for the rapid turnover of these cells in the organism. Beyond the different hematopoietic subsets and terminally differentiated immune cells, the BM contains a complex and heterogeneous stromal compartment made of cells of vascular, mesenchymal or neural origin, which crucially participate in the regulation of hematopoiesis and HSC maintenance. In the last decades, this unparalleled complexity of the BM at compositional and functional levels has been dissected through the use of flow cytometry. Nonetheless, flow cytometric techniques do not allow to capture fundamental spatial and morphological information, and miss the vast majority of stromal cells, which are lost during the mechanical dissociation of BM tissues.
To generate spatial maps of BM components, gain information on functional interactions between key cell types and obtain accurate quantifications of stromal cell types, we have developed 3D quantitative microscopy (3D-QM) of hematopoietic tissues. In this workflow we combine advanced tissue processing and clearing protocols for the generation of 3D reconstructions of entire BM cavities with cellular and subcellular resolution, with deep learning-assisted computational tools for the automatic detection of various anatomical landmarks in 3D image data sets. Through the use of 3D-QM, we currently investigate the distribution of HSCs in specific locations to unravel their preferential interactions in specific niches, we describe the homeostatic topology of stromal networks and we study the profound functional and structural remodeling of the complex BM tissue landscape during pathological conditions such as chronic infections, myeloablative insults and hematological neoplasias.
Abstract: Cancer immunotherapeutic approaches based on the redirection of T cell activity toward Tumor-Associated Antigens are actively being investigated. The impressive clinical success of the continuously intravenously infused bispecific T cell-engaging (TCE) antibodies, and of engineered T cells expressing chimeric antigen receptors (CAR-T cells) in hematological malignancies, has led to renewed interest in a novel cancer immunotherapy strategy that combines features of antibody- and cell-based therapies. This emerging approach is based on the endogenous secretion of small-sized Fc-free TCEs by gene-modified T cells (STAb-T cells). Adoptive transfer of genetically modified STAb-T cells secreting anti-CD19 TCEs (STAb-T19) or anti-CD1a TCEs (STAb-T1a) have demonstrated potent anti-tumor activity in hematologic preclinical xenograft models. Our findings suggest that the continued antibody secretion, allows an efficient recruitment of the endogenous T cell pool, resulting in fast and effective elimination of cancer cells that may prevent leukemia frequently associated with CAR-T therapies.
Abstract: The number of parameters that can be measured with cytometry techniques has increased a lot. Manually evaluating all markers and marker combinations is time-consuming and often infeasible and that is where the field of computational cytometry comes in. The default analysis pipeline consists of three main steps: pre-processing and quality control, clustering, and downstream (statistical) analysis. The quality control (i.e. removing low quality events and detecting and solving batch effects) is especially important in a computational setting, because of the garbage in, garbage out principle. The overall pipeline will be discussed and some computational tools like PeacoQC for quality control, CytoNorm for batch effect removal and FlowSOM for clustering will be highlighted.
Abstract: In recent years, the treatment of leukemia and lymphoma has undergone significant changes that have led to greater response rates and prolonged patient survival both in the settings of high-dose chemotherapy (HDT) followed by stem cell transplantation (ASCT) and through the use of new targeted drugs and immune therapy. Because of such improvements, conventional criteria used to assess complete response (CR) have progressively become insufficient to evaluate the quality of response in a significant fraction of the patients, highlighting the need for more sensitive techniques.
In the past, both conventional (4-10 colour) flow cytometry and ASOqPCR have shown to be relatively sensitive approaches to monitor the effect of therapy in leukaemia and lymphoma through the detection of low to minimal levels of measurable (residual) disease (MRD) at the 10-4 to 10-5 level. Subsequent technological advances in the speed of digital flow cytometers have enable the advent of high-sensitive next-generation flow cytometry (NGF) MRD assays, capable of identifying down to 10-6 residual neoplastic cells among millions of bone marrow cells from patients with acute and chronic leukaemia and multiple myeloma based on objective and automated analytical approaches. Such increased sensitivity proved to allow the novel NGF-MRD assays to be broader applicable in BM and blood samples with very important practical implications. More recently, the introduction of antibody- and CAR-T targeted immune therapies, together with the exponential growth of the multicolour capabilities of spectral flow cytometers has fostered the development of more comprehensive antibody monitoring panels and assays which go beyond MRD detection, to the simultaneous identification of MRD and potential targets for e.g., antibody and/or CART-cell mediated immune therapies on residual leukaemia/lymphoma cells, the identification of in vivo expanded blood circulating CART cells and their detail sub-setting, and monitoring of the effect of therapy on the residual innate and adaptative immune cells.
Altogether, this has progressively brought flow cytometry as a key tool for treatment monitoring and treatment-decision making processes in leukaemia and lymphoma patients both in the settings of clinical trials and real-world clinical practice.
Abstract: Occurrence of insertion mutations of the nucleophosmin gene (NPM1mut) is among the most frequent genetic events described in acute myeloid leukemia (AML) and is overall associated with a relatively favourable prognosis. However, clonal evolution of NPM1mut leukemic cells and disease outcome is importantly modulated by comutation patterns of concurrent competing leukemic cell clones. Over years, an increasing amount of phenotype-genotype associations are being unravelled in AML which may contribute to early diagnosis and risk-adapted therapy for these patients. However, leukemia cells from NPM1mut AML patients present at diagnosis with heterogeneous patterns of lineage commitment, including early myeloid maturation arrests and/or more mature traits of myelomonocytic differentiation. More in detail, this translates into a broad spectrum of NPM1mut associated phenotypes, from immature CD117+HLA-DR+ leukemia cells showing minimal differentiation features to more differentiated (e.g. CD34-) monocytic (e.g. CD11b+CD14+ and/or CD64+) and/or neutrophil lineage (e.g. CyMPO+ HLA-DR-) phenotypes, these latter cases (partially) resembling the phenotypic profile of acute monoblastic/monocytic subtypes (e.g. AML with KMT2A-rearranged or CBFB-MYH11) and acute promyelocytic leukemia with PML-RARA (APL), respectively. Such heterogeneity has hampered the identification of baseline immunophenotypic patterns that might accurately predict for NPM1mut AML.
Herewith, we characterized the distribution and immunophenotype of different subsets of bone marrow (BM) leukemia cells studied at diagnosis of a large series of 377 AML patients, including 201 NPM1mut AML cases. Our major goal was to identify reliable phenotypic profiles that could be used for fast screening for the NPM1mut and/or FLT3-ITD in newly diagnosed AML patients. In addition, novel next-generation flow cytometry approaches for automated immunophenotypic discrimination of NPM1mut AML vs. potentially overlapping AML categories (e.g. APL) will be introduced.
Background: B cell maturation antigen (BCMA) is expressed in all normal and malignant plasma cells (PCs). BCMA and its proliferation-inducing ligand (APRIL) play an important role in the long-term the survival of these cells. The enzyme γ-secretase is responsible for the removal of BCMA from PCs. For this reason, it is possible to find soluble BCMA (sBCMA) in peripheral blood (PB).
Aims: This study measured the amount of sBCMA in the PB of patients with multiple myeloma (MM) in order to explore its value for monitoring this disease.
Methods: PB of 5 healthy donors and PB and BM aspirates from 19 patients (12 newly-diagnosed and 7 with relapse/refractory disease) with MM were studied. The concentration of sBCMA and APRIL was evaluated by the ELISA technique in PB and BM. The percentage of infiltration of PCs in BM was evaluated by flow cytometry.
Results & Conclusion: MM patients showed a higher concentration of sBCMA in PB than healthy donors (mean 610.5 ng/mL vs 16.01 ng/mL, p<0.0003). Reganding APRIL, differences were also found between patients with MM and healthy donors, being higher in the control group (mean 138.3 pg/mL vs. 373 pg/mL, p<0.03). In MM patients, mean value of sBCMA detected in BM was 474.1 ng/mL and showed a strong correlation with sBCMA PB levels (R2=0.86 p<0.0001). Finally, a strong correlation between PCs infiltration in BM and the sBCMA detected in PB from MM patients was demonstrated too (R2=0.69, p=0.002). In summary, PB sBCMA can predict the percentage of PCs infiltration in BM. This new biomarker it would be useful for monitoring to disease including non-secretory MM patients. In addition, sBCMA could influence the selection of the appropriate dose of anti-BCMA therapies such as monoclonal antibodies or CART cells currently on the rise. Further translational studies and evaluation are warranted.
Background: Systemic mastocytosis (SM) includes a heterogeneous group of disorders, resulting from an expansion of clonal mast cells (MC) associated with gain-of-function KIT-mutations that lead to MC activation and the release of MC mediators. These might alter the tumour microenvironment with potential consequences on adaptative immune cells, such as T- and NK-cells.
Aims: To investigate the distribution of NK-cells, T-cells and their subsets in blood of SM patients with distinct disease subtypes, and its relationship with other disease features.
Methods: We studied 111 SM patients - 37 bone marrow mastocytosis (BMM), 65 indolent SM (ISM), 9 aggressive SM (ASM)- and 83 age--matched healthy donors (HD). The distribution of blood NK-cells, T-cells and their subsets was investigated by spectral flow cytometry and the EuroFlow Immunemonitoring panel, and correlated with multilineage KITD816V, alpha-tryptasemia genotypes (HAT) and other disease features.
Results & Conclusion: SM patients showed decreased (vs HD) median blood counts (cells/μl) of NK-cells (212 vs 393; p<0.001), cytotoxic T-cells (517 vs 621; p=0.002) and several functional subsets of TCD4+ cells such as Th1 (112 vs 134; p=0.006), Th2-effector-memory (4.1 vs 4.4; p=0.012) and TH22-terminal-effector (0.3 vs 0.4; p=0.001) cells, whereas T-follicular-helper (163 vs 133; p=0.009) and Th1/Th17-like T regulatory cells (0.9 vs 0.7; p=0.003) showed increased values (vs. HD, respectively). Interestingly, different immune profiles were found among distinct diagnostic subtypes of SM, where e.g., ASM patients displayed decreased numbers of total and several maturation-associated and functional subsets of TCD4+ cells. In turn, ISM patients with multilineal KIT had decreased Th2-cell counts (51 vs 65 cells/μl vs MC-restricted cases; p=0.018), whereas HAT+ cases displayed increased Th1/Th17-cell counts (120 vs 77 vs HD; p=0.023) in blood. These results suggest an altered distribution of blood NK and T-lymphocytes in SM, which relates to disease subtype, the pattern of involvement of hematopoiesis by KITD816V and a HAT genotype.
Background: In B-lymphoblastic leukemia (B-ALL), the lineage change of aberrant B precursors to a myeloid lineage during treatment is an extremely rare event, which may cause doubts about the initial diagnosis and the continuation of the established treatment. In recent years, a subgroup of B-ALL with CD2 expression at diagnosis has been described. After initiation of treatment, these patients present a higher probability of transdifferentiation towards monocytoid lineage cells.
Aims: We present a clinical case with the most relevant immunophenotypic findings and explore the possible reasons that justify this phenotypic change.
Methods: Flow cytometry analysis of bone marrow (BM) aspirates were performed using a FACSCantoII cytometer at diagnosis, 15 and 30 days post-treatment. At day +15, a cell culture was performed in presence or absence of methylpredinisolone, cells were extracted at days +2 and +5 and the cell subset percentages, cell death and proliferation were evaluated.
Results & Conclusion: Diagnosis of common B-ALL according to EGIL classification was established, with aberrant CD2 expression. Measurable residual disease (MRD) study on day +15 revealed 28% of aberrant precursors, with a clear differentiation of the original lymphoblasts towards monocytoid cells, being able to establish up to 3 populations. The results obtained in cell culture showed that the presence of methylprednisolone did not modify the percentages of the different subpopulations. The results showed greater proliferation and less cell death in cells with monocytoid differentiation, without appreciating differences due to the presence of methylpredinosolone. On day, +30 aberrant blasts of any type of lineage were not observed. This case demonstrates that despite the clear transdifferentiation of the original blasts, neither the initial diagnosis nor its treatment should be changed. In vitro study revealed that during treatment, cells with monocytoid differentiation showed greater proliferative capacity and resistance to cell death, which explains the behavior of this leukemia.
Sponsored by Beckman Coulter
Abstract: In the last years, extracellular vesicles (EVs), membrane-delimited nanoparticles (30-to-1000 nm) released by all functional cells into the extracellular milieu, have become an attractive target for scientific community. EVs play an essential role as messengers in cell-to-cell communication, delivering their bioactive cargo (proteins, lipids and RNAs) from donor to recipient cell for triggering a specific response in them. EVs has been isolated from all body fluids, where different EV-subsets can be found, making them a challenge to study. The lack of accurate methodologies for isolating/characterizing EV-subsets lead to a poor definition of their composition/function. In the last years, advances in engineering have allowed to develop high-resolution flow cytometers allowing to multiparametric analysis of EVs. Flow cytometry-based method enables distinguishing between EVs vs non-EV particles, giving valuable information about number, size, and phenotype of EVs at single level. Recently, our research group has performed a phenotypical characterization of EV-subsets isolated from pig seminal plasma by flow cytometry, following the International Society of Extracellular Vesicles guidelines. Seminal plasma is a complex fluid secreted mainly by male accessory sex that accompanies sperm during and after ejaculation. This fluid contains a large and heterogeneous population of EVs that can clustered in different subsets, which one will be involved on modulating different sperm physiological processes. In this context, flow cytometric analysis is offered as essential tool for identifying seminal EV-subsets allowing to reach useful information about phenotypic parameters and putative specific function of each EV-subset.
Abstract: Flow cytometry is a highly sensitive tool to study plastic nanoparticles, with advantages over other techniques, such as Raman spectroscopy, which can resolve generally 1-2 µm particle sizes. One direct method to analyze the nanoplastics (NPs) that the human body can accumulate consists of measuring the NPs levels in peripheral blood. We have used Nile Red (NR), a red phenoxazone dye that binds to the surface of plastics and neutral lipids, was used to determine the presence in blood of the most common plastics: low density polyethylene, polystyrene, polyethylene terephthalate, and polyamide.Previous work assessing the levels of MPs or NPs in human peripheral blood has been conducted by Fourier Transform Infrared (FT-IR) spectrometry, by Scanning Electron Microscopy coupled with an Energy Dispersion Detector (SEM-EDX), and by double shot pyrolysis - gas chromatography/mass spectrometry. The flow virometric assay can be used as a potential method to detect RNA viruses. It allows the rapid detection of viral particles and shows high sensitivity and specificity, and can be used complementary with any other technique for virus detection, such as RT-qPCR and RAD methods. The combination with fluorescent monoclonal antibodies against SARS-CoV-2 spike glycoprotein help to confirm the specific detection of the virus. Furthermore, the implementation of this flow virometric method will allow early detection of infection by running rapid high throughput analysis of a large scale of population groups, necessary to detect the presence of asymptomatic individuals. We will discuss the conceptual basis for quantitative fluorescence measurements using flow cytometry, cross-calibration results of different sub-micron beads and instrument models as well as guidelines for flow cytometry data presentation.
Abstract: Chronic inflammatory diseases are often associated with changes in the microbiota, generally termed dysbiosis. We have developed an analysis method, which allows phenotyping of microbiota on the single-cell level by multi-parameter flow cytometry. We determine phenotypic properties of bacteria, such as coating with host immunoglobulins and the expression of surface sugars, to capture the immunological context of their recognition by the host and to reflect metabolic conditions, adhesion ability and cell-crosstalk, respectively. By machine-learning, phenotypic signatures can be delineated which are specific for different chronic inflammatory diseases independently of taxonomic alterations. Thus, microbial phenotyping on the single cell level reveals that intestinal bacteria can serve as biosensors allowing disease classification with the potential for disease monitoring and patient stratification but also for identification of bacterial populations or microbiota conditions potentially relevant in disease pathogenesis.
Background: Nanoplastics (NPs) are plastic particles between 1-1000nm. The accumulation of NPs has been demonstrated in human tissues, such as blood or placenta, while in others it remains unstudied.
Aims: To analyze the accumulation of NPs in human bronchoalveolar lavage (BAL), cerebrospinal fluid (CSF), lymph nodes (LN), biological fluids (BL) and peripheral blood (PB), as well as PB from mice living under SPF confinement.
Methods: Organic matter was degraded in KOH (1%) at 60ºC in a dry block for 10 days. 20µl of the digested sample was diluted in deionized water and stained with 2µl Nile Red for 15min at room temperature. Submicron microspheres were used for calibration. All samples were analyzed on the Attune™ NxT flow cytometer, with accumulation of NPs measured in triplicate.
Results & Conclusion: NPs were detected in all tissues analyzed. Specimens with highest accumulation of NPs were LN and PB, with a median of 7 million NPs per mg of tissue (range: LN 1.02- 17.57 million NPs/mg; PB, 5.11-13.19 million NPs/mg). BAL showed lower accumulation (5 million NPs/mg), and high dispersion between samples (0.55-36.92 million NPs/mg). Pleural liquid, seroma, and CSF, showed lower accumulation of NP, with values of 2 million NPs/mg (range 0.14-0.44 million NPs/mg) in CSF. NP levels in the blood of mice were significantly lower than in humans (0.6 vs. 7 million NPs/mg; mice range: 0.27- 1.08 million; p-value<0.0001). NPs can cross biological barriers, as in the case of CSF accumulation. NPs can enter living organisms through inhalation, as confirmed by detection in BAL. In mice, the lower accumulation of NPs could be related to the air quality in facilities equipped with HEPA filters that clear larger particles. Further studies will be needed to understand the impact of NPs in living organisms as well as their entry, accumulation and elimination pathways.
Background: In Iberia, Mycobacterium bovis, causing animal tuberculosis (TB), circulates in a multi-host system including cattle, wild boar, and red deer. Transmission occurs via direct or indirect contact with infected animals shedding bacteria through several routes. Current knowledge postulates interspecific transmission is favoured by animal contact with natural substrates (soil, water, etc.) contaminated with infected droplets and fluids. However, methodological constraints have hampered the assessment of M. bovis environmental contamination and the validation of this hypothesis.
Aims: With a new real-time monitoring tool, we assessed the burden and viability of M. bovis in natural substrates from a TB hotspot area and assessed the phylogenomic relatedness of environmental and animal isolates.
Methods: Sixty-five natural substrates were collected nearby the International Tagus Natural Park region. These included sediments, sludge, water, and food deployed at unfenced feeding stations. The tripartite workflow included quantification and sorting of different M. bovis cell populations: total, viable, and quiescent. This integrated methodology is based on flow cytometry applying 5-carboxyfluorescein diacetate acetoxymethyl ester to differentiate metabolically active from inactive cells, fluorescence in situ hybridization with an IS6110-specific probe, and fluorescent-activated cell sorting. Taxonomically labelled sorted cells were sequenced after whole-genome enrichment for M. bovis. The phylogenomic analysis explored the signatures of environmental and animal (> 200) M. bovis isolates recovered from the study area.
Results & Conclusion: Thirty-eight percent of samples were positive for M. bovis (mean 3.4x10^4 cells/g or /L), with only 12% showing metabolically active cells (8.9x10^3 cells/g or /L). After a resuscitation step, that number raised to 54%. Environmental isolates were assigned to the two major M. bovis lineages circulating in Portugal, being phylogenetically related to animal isolates. This pioneer multi-tool framework highlights the importance of environmental contamination in indirect transmission and offers additional resources for surveillance, potentially guiding the implementation of biosecurity and control measures.
Background: Phytoplankton are a diverse group of mostly unicellular photosynthetic organisms critical for the global carbon cycle. In fundamental research, it is possible to use and manipulate phytoplankton strains isolated from oceanic waters and maintained in culture collections to investigate a number of biological questions.
Aims: In our flow cytometry facility, we aimed to establish a method to isolate by cell sorting different species of marine phytoplankton that have been co-cultured under different conditions to support research projects.
Methods: We have been focusing, in particular, on Phaeodactylum tricornutum, Nannochloropsis granulata, Dunaliella tertiolecta, Amphidinium carterae and Tisochrysis lutea. Although the different species had distinct morphologies and the light scatter properties were not identical, in most cases there was a significant overlap that prevented their resolution using those parameters alone. Using full spectrum flow cytometry, we characterized the spectral signatures of each phytoplankton species cultured individually. These signatures were based on the endogenous autofluorescence, which is conferred by the differential chlorophyl content, as well as other pigments and metabolites. As expected, similarity indices were very high, but, with the exception of Phaeodactylum, spectral signatures allowed the resolution of all species. Based on the differences in maximal and minimal fluorescence emission in the spectral signatures, we prepared a customized optical configuration for the conventional cell sorter to discriminate the different species. After testing different nozzle sizes and numbers of sorted cells, and without replacing the sheath fluid by sea water, we established a gating strategy that enabled a highly pure isolation from a heterogeneous suspension of four species that could successfully expand in culture after sorting.
Results & Conclusion: In conclusion, our methodology will enable to specifically isolate with a high speed and degree of purity distinct phytoplankton species from co-cultures for further downstream characterization. This opens new opportunities to address important questions in ecology and evolution.
Abstract: Erythropoiesis involves proliferation and differentiation of small populations of hematopoietic stem cells resident in the bone marrow into mature red blood cells. The determination of the cellular composition of the blood is a valuable tool in the diagnosis of different diseases and monitoring of therapy. Different flow cytometry immunophenotyping techniques can be routinely used to characterize the heterogeneous cell populations present in the blood and the hematopoietic cell differentiation and maturation pathways of the bone marrow. In this session, we will discuss the role of flow cytometry in the study of erythropoiesis and classical red blood cell disorders including reticulocyte analysis; quantitative methods for detection of Fetal–Maternal Haemorrhage (FMH); Sickle Cell Disease (SCD); Hereditary Spherocytosis (HS) and related disorders; Paroxysmal Nocturnal Hemoglobinuria (PNH); and red cell survival.
Abstract: En los últimos años, la "antigua" hemoglobinuria nocturna paroxística (HPN) ha logrado nuevos avances en términos de comprensión de su fisiopatología, del enfoque diagnóstico, la optimización de la terapia y el desarrollo dinámico de nuevos agentes terapéuticos.
En esta revisión se describe en donde estamos en el momento actual en cuanto a la fisiopatología y el proceso de diagnóstico de la HPN. El despistaje de HPN se recomienda en pacientes con hemólisis intravascular, síndromes de insuficiencia medular adquirida y en casos de trombosis con localizaciones inusuales. A pesar de la disponibilidad de directrices de consenso para el diagnóstico y monitorización de la HPN, aún se encuentran discrepancias sobre cómo se llevan a cabo sus pruebas diagnósticas, dando lugar a variaciones técnicas que pueden conducir a un diagnóstico incorrecto.
También se describen los determinantes del tratamiento moderno, como las estrategias de inhibidores del C5 del complemento frente al alotrasplante de células madres hematopoyéticas.
Durante los últimos 20 años, la terapia de la HPN se ha basado en la inhibición del complemento terminal mediante anticuerpos bloqueantes, pero recientemente una gran cantidad de nuevos medicamentos que interfieren con la cascada del complemento proximal y terminal, están en desarrollo. Así se presentan los primeros inhibidores aprobados de la vía del complemento proximal que previenen principalmente la hemólisis extravascular y se destacan sus potenciales beneficios.
Abstract: Platelets are essential for the onset and regulation of hemostasis, coagulation, endothelial-cell support and wound repair. On the other hand, platelets are involved in pathological conditions, including thrombosis, inflammation, infection and cancer. Platelets sense their environment through activating or inhibiting cell-surface receptors connected to fast-response transduction pathways. Platelet activation leads to sequential processes of shape change, aggregation, secretion of intracellular factors and shedding of membrane microparticles. These changes contribute locally to form and stabilize the hemostatic plug, while ensuring coagulation, attracting circulating platelets and leukocytes, and promoting repair of vessel lessions. Interestingly, the same platelet functions that elicit their protective action may lead to life-threatening conditions when improperly activated or disregulated. Moreover, platelet function may be affected by congenital or acquired conditions, including hemato-oncological disorders and therapy side-effects. These facts have promoted the study of platelet function in many clinical areas. Flow cytometry (FCM) has paid attention to platelets as early as the 1980s, and novel FCM platelet assays refflect the technical improvements in Cytomics. Currently, FCM is the choice tool to undertake platelet studies in near-physiological conditions. FCM assays of platelets in whole blood use small amount of sample, minimize artifactual activation of platelets and maintain the cellular heterogeneity of circulating blood elements. In this way, FCM allows to follow platelet maturation and destruction, to detect congenital defects of platelets and to provide risk factors related to platelet hypo- or hyper-reactivity in vivo. Such risk factors are based on the activation responses of individual platelets, their interactions with other blood cells and the secretion/shedding of extracellular molecules and microparticles. Moreover, functional ex-vivo real-time assays reveal fast responses of platelets when challenged with agonists. The repertoir of FCM platelet assays allows also to monitor therapy at the patient level, as well as to control the quality of platelet hemoderivates.
Background: Fetomaternal hemorrhage (FMH) refers to the entry of fetal blood into the maternal circulation before or during delivery. This pathological condition presents wide clinical heterogeneity, including devastating consequences for the fetus such as neurologic injury, stillbirth, or neonatal death.
Aims: In 2012, a clinical protocol for flow cytometry-based detection of fetal erythrocytes in maternal samples was established in cases of fetal/neonatal anemia (FA) and unexplained fetal loss (uFL) in order to quantify FMH and guide clinical decisions.
Methods: The Fetal Cell Count Kit (IQP Products, Rozenburglaan, The Netherlands) including anti-carbonic anhydrase (CA) and anti-fetal hemoglobin (HbF) was used to discriminate the different RBC populations in maternal blood. Calculation of FHM volume was performed according to Mollison BMJ 1972. Significant bleed was defined as any bleed exceeding 2mL of fetal erythrocytes according to BCSH guidelines.
Results & Conclusion: 69 cases corresponded to uFL, while 14 corresponded to fetal/neonatal anemia. In 8/83 cases, a significant bleeding was detected. The median FMH volume was 75mL (range 2.4 to 132mL). 28% of FA showed a significant bleeding (range 2.4 to 122mL). In two cases with significant bleeding (one corresponding to uFL and one to FA) the mother was D negative and the fetus/baby was D positive. FMH volume was 103 and 122mL respectively, therefore supplementary doses of anti-D immunoglobulin for the prevention of haemolytic disease of the newborn (HDN) had to be administered and clearance of fetal erythrocytes was subsequently confirmed. Correct quantification of FMH using flow cytometry is feasible and provides valuable clinical information on the origin of uFL and FA. In cases with D antigen discrepancy, communication with the obstetric team allows the calculation of anti-D immunoglobulin dose required to cover the reported bleeding and advice regarding follow-up samples to check for clearance of fetal cells.
Background: Malaria is a vector-borne disease caused by parasites from the genus Plasmodium and transmitted to human by the bite of an Anopheles mosquito. This disease that affects over 200 million people every year. P. falciparum, responsible of the majority of the deaths, infects almost exclusively human erythrocytes.
Aims: The use of humanized mouse models to study this disease have been crucial in the past years to find new drugs to fight malaria. GSK Pf-Hu (Plasmodium falciparum-Humanized) mice model and Flow Cytometry analysis described in this work has been used for preclinical studies of different assets that are currently on clinical phases.
Methods: Parasite growth in Pf-Hu mice blood is reproducible and shows all erythrocytic stages of parasite (rings, trophozoites and schizonts). In mice peripheral blood, parasite is detected by flow cytometry, using a combination of DNA dye (YOYO-1) and a monoclonal antibody specific for mouse erythrocytes (TER119-PE). Parasite is shown as the population YOYO-1+/TER119-PE- in a bidimensional FL1/FL2 dot plot, tacking advantage autofluorescence we are able to discriminate the different erythrocytes stages.
Results & Conclusion: Flow cytometry analysis allows the discrimination of parasite population sensible to the drug treatment when Pf-Hu mice model is used for the screening of new antimalarial compounds. Different standard antimalarial drugs have been used for the setting up of this analysis strategy and it has been demonstrated that a simple bidimensional flow cytometry analysis allows to obtain valuable information to decipher the mechanism of action when little is known about the drugs tested. “The human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents” “All animal studies were ethically reviewed and carried out in accordance with European Directive 2010/63/EU and the GSK Policy on the Care, Welfare and Treatment of Animals.”
Background: Human pyruvate kinase deficiency (PKD), an autosomal recessive disorder produced by mutations in the PKLR gene, is the most common cause of chronic non-spherocytic hemolytic anemia. This deficiency alters the erythrodifferentiation and gives rise to anaemic phenotypes. We have previously shown that the anaemic phenotype has an impact on bone marrow niche by altering cellular subsets. An imbalance in the most immature compartment has been demonstrated but it has not been studied how this alteration could affect physiological processes, such as mobilization.
Aims: Our aim has been to study cellular kinetics after pharmacological induced Mobilization of peripheral blood progenitor cells in a anemic PKD mice model.
Methods: We have designed a 13 colour panel that includes both stem and more mature committed hematopoietic markers such as CD3, CD45R/B220, Ly-6A/E (Sca 1), Ly-6G, Ly-6C, CD11b (Mac-1), CD16/32, CD34, CD71, CD117(c-Kit), TER-119, CD135, F4/80 antigen and viability marker. The samples were acquired in an 4L BD LSRFortessa X-20™ with a total of 20 detectors. The panel was applied at different time points (0, 30´, 90´, 120´, 180´and 24h). Blood samples were also analyzed in a haematological counter(Sysmex XN-1000V).
Results & Conclusion: Results shown an altered kinetic of recovery of white blood cell populations after mobilization in mutant animals. Surprisingly, the results have also shown that the severity of the erythroid phenotype improves after mobilization with Plerixafor at least at the mature level. The number of cells expressing cKit in mutant´s blood at 90´ was also increased after pharmacological treatment compared to healthy control. Whether these kinetic differences are due to intrinsic defects to the progenitor/mature fractions themselves and/or to disturbed niche signals by the constant erytrhoid bias towards the erythroid path remains unclear.
Abstract: We have analyzed BNT162b2 vaccine-induced immune responses in naive subjects and individuals recovered from coronavirus disease 2019 (COVID-19), both soon after (14 days) and later after (almost 8 months) vaccination. We design a 40-color panel to obtain a fully complete screening of the immune system in order to check the profile pre and after vaccination using a Cytek Aurora spectral system. Plasma spike (S)-specific immunoglobulins peak after one vaccine shot in individuals recovered from COVID-19, while a second dose is needed in naive subjects, although the latter group shows reduced levels all along the analyzed period. Despite how the neutralization capacity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mirrors this behavior early after vaccination, both groups show comparable neutralizing antibodies and S-specific B cell levels late post-vaccination. When studying cellular responses, naive individuals exhibit higher SARS-CoV-2-specific cytokine production, CD4+ T cell activation, and proliferation than do individuals recovered from COVID-19, with patent inverse correlations between humoral and cellular variables early post-vaccination. However, almost 8 months post-vaccination, SARS-CoV-2-specific responses are comparable between both groups. Our data indicate that a previous history of COVID-19 differentially determines the functional T and B cell-mediated responses to BNT162b2 vaccination over time.
Abstract: Long COVID can be developed by individuals after an infection with SARS-CoV-2 as described by the WHO. Although this condition is more commonly described in adults, it can occur in children and adolescents with a wide range of estimated prevalence of 1-25%. Little is known about the role of the immune system in longCOVID. The objective of this study is to compare immune cells populations in paediatric populations with and without longCOVID.
Background: T cells are essential for protection against infection by natural- and vaccine-induced immunity. Monitoring T-cell functions is crucial to follow-up COVID-19 vaccination in immunosuppressed patients. Flow cytometry (FCM) has revealed multiple immunological features and consequences of COVID-19 disease.
Aims: To apply FCM for assessing the efficiency of COVID-19 vaccines in immunosuppressed patients, based on circulating antigen-specific T cells, memory subpopulations and cytokine production by T cells upon in vitro activation.
Methods: Twenty-two immunosuppressed patients and 21 controls were studied at months one, three, six and twelve along mRNA vaccination. Circulating CD8 T cells, specific for a Sars-Cov-2 Spike epitope were detected with APC-conjugated dextramer (Immudex). Appropriate DuraClone panels (Beckman Coulter) were used for immunophenotyping, enumeration of T cell-memory subpopulations and quantitation of intracellular cytokines. Assays were run on Gallios flow cytometer (Beckman Coulter) and analyzed with Kaluza software (Beckman Coulter). For statistical analysis, Mann-Whitney test was applied.
Results & Conclusion: Patients presented lower and heterogeneous levels of antigen-specific CD8 cells. Patients showed persistent decrease of CD4 lymphocytes and total lymphocytes and T cells were decreased at 3 months and 6 months. CD28-CD27+ CD4 cells increased, while CD28+CD27+ CD8 cells decreased at 3 and 6 months. There was a marked increase of TEMRA CD4 cells. These findings suggest impaired T cell co-stimulatory function. CD4 cells from patients showed lower production of IL-2 and lower production of cytokines by IL-2+ cells, but greater production of IFNγ and TNFα without IL-2 expression. CD8 lymphocytes from patients showed lower IL-2 expression but were more efficient in producing TNF-α and/or IFN-γ in the absence of IL-2. Our data on immunosuppressed individuals show heterogeneous antigen-specific CD8 memory and lower T-cell activation responses. Our FCM approach may be applied for personalized follow-up of high-risk patients. Sponsored by Fundación Mutua Madrileña (IRAS-VAC Project).
Background: Clinical symptoms following resolution of Sars-CoV-2 infection are known as long-COVID. The incidence and prevalence of chronic symptoms after acute viral infections have gained strong interest over the course of the COVID-19 pandemic. Acute post-infection sequelae have also been observed in other respiratory viral infections, including influenza virus.
Aims: Here, we performed a deep-immune characterization of a cohort of convalescent COVID-19 (post-COVID) and post-influenza (post-FLU) patients in order to describe the immune landscape after acute infection.
Methods: We collected peripheral blood samples from patients who recovered from COVID-19, 3 months after they were discharged from hospital (n = 83), and from post-FLU patients. Immune phenotyping on PBMC was performed using 40-plex mass cytometry, and the levels of 45 circulating cytokines were quantified by Luminex. We also included 17 pre-pandemic healthy donors. Principal component analysis (PCA) and K-means clustering were performed.
Results & Conclusion: PCA revealed notable differences in the immunological profile, especially related to migration, among healthy donors, post-FLU and post-COVID patients, with the expression of chemokine receptors CCR4, CCR6, CXCR3 and CXCR5 on lymphoid and myeloid cells being the most contributing features to the PCA. A logistic regression model was able to classify all individuals according to their diagnosis with good accuracy. Using the 50 features with the highest contribution to PCA, K-means revealed four clusters of individuals. Two of these clusters corresponded to post-COVID patients, which differed in age of individuals but did not correlate with post-COVID symptoms or acute severity. Our results showed immune abnormalities in post-COVID patients three months after hospital discharge, and to a lesser extent in post-FLUindividuals, compared to healthy donors. We were able to divide post-COVID individuals into two immunological groups. The immune alterations in post-COVID were independent of acute or post-syndrome symptoms.
Background: Patients with mature B-cell neoplasms are at risk of severe infections and poor vaccine immunogenicity. The cell-specific extent of immune dysfunction of these patients prior and during vaccination is poorly characterized.
Aims: Characterize the immune response to vaccination in patients with mature B-cell neoplasms using COVID-19 vaccines as a case study.
Methods: This study included 82 patients with mature B-cell neoplasms (7 CLL, 46 lymphoma, 29 MGUS/myeloma) and 96 age-matched health care practitioners (HCP). A total of 1104 blood and serum samples were collected before and at days 7 and 14 after the first dose, as well as at days 7 and 62 after the second. Also before and 17 days after a booster dose. Immune profiling was performed using multidimensional and computational flow cytometry that systematically analyzed 56 cell-types per sample. Serum levels of IgM, IgG and IgA against four viral-antigens were quantified using a multiplex-microsphere-based flow assay. SARS-CoV-2-specific CD8 T cells were quantified using a dextramer panel of spike, membrane, ORF3 and nucleocapsid proteins.
Results & Conclusion: Using HCP as a reference of immune response to COVID-19 vaccination, we observed significant longitudinal deviations in 2/3 granulocytic, 6/6 antigen-presenting cell, 14/18 CD4 and 10/12 CD8 T-cell, and 17/17 B-cell subsets. Accordingly, anti-RBD IgM, IgG and IgA indexes were significantly lower in patients vs HCP. An immune dysregulation longitudinal cumulative score was associated with impaired anti-RBD IgG levels after the second dose. While SARS-CoV-2-specific CD8 T cell percentage after the second dose was similar between patients and HCP, there was an enrichment of TEMRA cells in the former. Furthermore, the booster failed to increase virus-specific CD8 T-cell and antibody levels in patients. This study uncovers the cell-specific extent of immune dysfunction in patients with mature B-cell neoplasms and how it affects the efficacy of vaccination strategies such as for COVID-19.
Abstract: In the aquatic environments, phytoplankton account for approximately half the production of organic matter on Earth, due to their metabolic capacity to fix CO2, producing O2. Some studies have concluded that global phytoplankton concentration has declined over the past century.
Eukaryotic microalgae are part of this phytoplankton and their cellular characteristics make them suitable to be analyzed by flow cytometry. Our studies based on functional flow cytometry showed an additional concern, since growth of this kind of cells is affected by chemical contamination of water ecosystems, but not only. Energy producing metabolism (mainly photosynthesis related parameters), cellular homeostasis (intracellular pH, membrane potential) cell signalling (intracellular calcium, ROS level) or apoptosis-related processes were altered when microalgae were exposed to persistent (metals, pesticides) and emerging pollutants, such as pharmaceuticals or personal care products.
These alterations will need to be considered at a global scale, since geochemical cycling or fisheries depends on these microorganisms.
Abstract: Flow cytometry is considered the state-of-the-art technique for nuclear DNA content estimation. In an era where genomes are sequenced each time faster and cheaper, genome size estimation should precede genome sequencing which often is not the case, leading sometimes to the use of inadequate sequencing strategies. Fungal genome sizes vary over 1600x, ranging from 2.2 (Encephalitozoon romaleae) to 3706 Mbp (Jafnea semitosta), but the patterns of such variations differ across fungal phylogeny, with some phylogenetic groups exhibiting little genome size variations and others encompassing major shifts. In several fungal clades, genome size variation seems to accompany evolution and to relate to fungal life-styles: the genome sizes of saprophytic fungi and those of fungi that interact with animals tend to be smaller than those of fungi that interact with plants, either in mutualism or in parasitism. Fungal genomes are in average smaller than those of plants or animals and the use of flow cytometry for fungal genome size measurement and nuclear cycle analysis is challenging, requiring optimization of isolation procedures, DNA standards and stains. This communication will thus focus on applications and challenges of flow cytometry DNA analyses on fungal biology and plant pathology, aiming to promote the use of this technique in mycology and phytopathology as a fast, low-cost and reliable approach to help solving research questions.
Abstract: Flow cytometry has become one of the basic tools in aquatic microbial ecology, allowing the rapid and efficient analysis of large numbers of individual cells in large numbers of samples. Determining the size, DNA content and natural fluorescence properties of microbial cells provides information on the identity, abundance and even the physiological state of some of the key components of marine food webs. Using flow cytometry allows the routine detection of the most abundant microbial cells in natural waters like viruses, heterotrophic bacteria, cyanobacteria and even small eukaryotes. This makes flow cytometry a valuable tool for understanding the complex interactions and processes within microbial communities in aquatic environments.
In this presentation you will find some examples of how we are using flow cytometry to characterize microbial populations in different marine environments from surface to deep waters and their response to different natural phenomena like a) hydrothermal emissions from a submarine volcano, b) lava and ash inputs from a subaerial volcano into the coastal ocean and c) counting the number of microbes in airborne desert dust and in air over the open ocean.