Flow Cytometry Evaluation Research Articles

Flow cytometry evaluation of phagocytosis and respiratory burst in silver catfish (Rhamdia quelen) leukocytes exposed in vitro to atrazi

Flow cytometry evaluation of phagocytosis and respiratory burst in silver catfish (Rhamdia quelen) leukocytes exposed in vitro to atrazi

Establishing Immunocompetent Leukemia Models to Investigate the Impact of CAR T Cells on the Immune Microenvironment and Bone Marrow Niche

Background Chimeric antigen receptor (CAR) T cells need to overcome limited expansion and persistence within the highly immunosuppressive tumor microenvironment (TME) to successfully eliminate acute myeloid leukemia (AML) in the clinic. CD123 has been identified as a promising target for AML because of its high expression on leukemia stem cells and AML blasts, with a clear increased differential expression when compared to healthy hematopoietic progenitors (HPCs). To date, preclinical evaluation of CD123-CAR T cells have used human xenograft models, which lack a functional immune system. This cross-species evaluation of CAR T cells inadequately addresses interactions between CAR T cells and the immune microenvironment, a limitation of many CAR T cell therapy studies. We have generated immunocompetent murine leukemia models to evaluate murine CD123 (mCD123)-specific CAR T cells and thus investigate the impact of the AML TME on CAR T cell dysfunction. Methods and Results We used biopanning of a murine phage display library on recombinant mCD123 to select a panel of mCD123-specific single chain variable fragments (scFvs). 4 scFvs, encoding 'top binders', were subcloned into a MSGV1 retroviral vector encoding a CD28.z-CAR to generate 4 mCD123-CARs. Second-generation mCD123-CAR T cells were generated by retroviral transduction of activated splenocytes. Only one mCD123-CAR construct was expressed on the cell surface as judged by flow cytometric analysis using F(ab')2 (55.8±11.5% N=7) or recombinant mCD123 (59.0±12.1% N=6) for detection, and T cells expressing this CAR were selected for further analysis. To test the antigen specificity of mCD123-CAR T cells, we expressed full-length mCD123 on 2 murine leukemia cell lines: C1498, a spontaneous myelomonocytic AML cell line, and a Arf-null, BCR-ABL1 preB-ALL. In addition, we analyzed a panel of virally induced NUP98::KDM5A AML generated by retroviral transduction of murine HPCs and determined all samples naturally expressed mCD123 antigen at different densities (range 38-77% N=4). We co-cultured mCD123-CAR in the presence of mCD123+ leukemia cell lines, with mB7-H3-CAR, SP6-CAR, or non-transduced T cells serving as controls. mCD123-CAR T cells recognized CD123+ targets, as evidenced by significantly increased cytokine secretion and cytolytic activity in comparison to controls (N=6, p<0.01). Antitumor activity was maintained in repeat stimulation assays for up to 5 stimulations. To determine in vivo toxicity of mCD123-CAR T cells, non-tumor bearing C57BL/6 mice were given 200 mg/kg cyclophosphamide (Cy) and one effector T cell infusion generated from transgenic luciferase mice (N=5) and monitored by bioluminescence imaging for 10 days. Bone marrow and spleen were harvested 10 days after injection for flow cytometry evaluation and colony formation unit (CFU) readouts. We detected no significant differences in toxicities between mCD123-CAR and control T cells. We next evaluated the anti-leukemia activity of mCD123-CAR T cells. C57BL/6 mice were injected with preB-ALLArf-/CD123+ cells on day 0 and received 100 mg/kg of Cy on day 2. On day 4, they were injected with 10x106 mCD123-CAR T cells. Mice receiving tumor only, tumor + Cy, or SP6-CAR served as controls. mCD123-CAR T cells had significant anti-leukemia activity resulting in a prolonged survival advantage in comparison to controls (N=5 per group, p<0.001). We focused on delineating the TME in our established models and preliminary analysis of comprehensive flow cytometry and single cell transcriptomic data on bone marrow. Our findings revealed that the TME of our established immune competent leukemia CAR T cell therapy models recapitulate human disease, having an increase in dying tumor cells within the bone marrow of mice treated with mCD123-CAR, with a number of other populations (e.g. dendritic cells, regulatory T cells) approximating the tumor-only microenvironment profile. Conclusions We have developed functional mCD123-CAR T cells targeting AML and ALL and have established immunocompetent models to test them. mCD123-CAR T cells demonstrated potent anti-leukemic activity in vitro and have anti-leukemia activity with minimal to no toxicity in vivo. These findings will provide insight into the impact of the AML TME on CAR T cell functionality, and inform future strategies to improve CAR T cells in the immunosuppressive TME.

POCT urine dipstick versus central laboratory analyses: Diagnostic performance and logistics in the medical emergency department

ObjectivesThe aim of this study was to evaluate the logistics and diagnostic performances of dipstick analyses compared to their counterpart central laboratory analyses for detection of bacteriuria, proteinuria, hyperglycemia, ketosis and hematuria. Design and methodsUrine dipstick results, urine culture results, flow cytometric cell counts, U-albumin-to-creatinine ratio, P-glucose and P-beta-hydroxybutyrate were retrospectively reviewed in a cohort of consecutive patients admitted to the medical emergency departments of two Danish hospitals. Sensitivity, specificity and predictive values of traditional dipstick analysis were estimated and dipstick was compared to flow cytometry for detection of significant bacteriuria using logistic regression. Turn-around-time for central laboratory analyses were assessed. ResultsFor each comparison, 1,997 patients or more were included. Traditional dipstick analyses for proteinuria, bacteriuria and ketosis reached sensitivities of up to 90%, while sensitivity for hyperglycemia was 59%. Flow cytometry outperformed traditional dipstick analysis for detection of bacteriuria with a difference in the area under the ROC-curve of 0.07. Turn-around-times for 95% delivery of central laboratory analysis results ranged from approximately 1½ to 2 h. ConclusionsFor the detection of bacteriuria and albuminuria, central laboratory analyses reach better performance than dipstick analysis while achieving acceptable turn-around-times and are thus viable alternatives to dipstick analysis. For detection of ketosis and hyperglycemia, dipstick analysis does not perform adequately, but as very short turn-around-time is often required, these conditions may be best diagnosed by point-of-care blood test rather than dipstick or central laboratory analyses. Dipstick hemoglobin analysis, flow cytometry and microscopic evaluation may serve each their distinct purposes, and thus are relevant in the emergency department.

THE ACUTE LYMPHOBLASTIC LEUKEMIA OF DOWN SYNDROME

Down syndrome (DS) is a genetic disorder caused by the presence of a third copy of chromosome 21.It is usually associated with physical growth delays, mild to moderate intellectual disability, and characteristic facial features .Children with DS are at an elevated risk of leukemia, especially myeloid leukemia. On the other hand, children with DS are at a 20-fold increased risk for acute lymphoblastic leukemia (ALL).In our case, we presented a patient with DS who was diagnosed with ALL. 19-year-old male was admitted to the emergency department due to abdominal pain.On his physical examination,splenomegaly was detected.In laboratory examinations; kidney and liver function tests were normal,lactate dehydrogenase:372 U/L,uric acid: 5.4 mg/dl,white blood cell:25000 × 106/L,lymphocyte: 15780 × 106/L, neutrophil:1140 × 106/L,hemoglobin:10 gr/dl, thrombocyte:12000 × 106/L,coagulation tests were normal and in peripheral blood smear evaluation,90% blast cells were detected. Peripheral blood flow cytometry evaluation was compatible with B-ALL(TdT,CD19,CD10,CD34,cCD79a,CD58,CD9,CD38,CD123,CD20,CD81,CD22 positivity in atypical cells).Bone marrow biopsy was hypercellular.There was diffuse blastic cell infiltration,which stained extensively with TDT,CD79a.Chromosomal analysis is 47XY,+21 and t(12,21) (p13.2;q22.12) (ETV6/RUNX1) (FISH) and 14q32.33 (IGH) FISH were positive,t(9;22) P190 -p210,t(4;11), t(1;19),11q23 were negative.The risk classification was standard risk. AUGMENTED BFM induction chemotherapy protocol was started.Pancreatitis was developed after peg-asparaginase and chemotherapy-related hepatotoxicity(grade 1) was developed.Central nervous system prophylaxis(intrathecal methotrexate) was applied.The control bone marrow biopsy performed after induction was normocellular,the blast rate was <5%.BFM standard risk first consolidation chemotherapy protocol was started.He died of septic shock on the eighteenth day of the first consolidation treatment. Cases of DS-ALL cases are at greater risk for serious side effects from chemotherapeutics,mortality and recurrence than non DS-ALL.Because children with DS have a higher incidence of treatment-related toxicity,survival rates are lower than non-DS children.During ALL induction chemotherapy life-threatening side effects are tumor lysis syndrome,thrombosis,bleeding and infection.In the UKALL 2003 study,DS associated with a significantly increased risk of death from sepsis during chemotherapy.

PSII-A-2 Correlations between Reactive Oxygen Species and Functional Measurements of Spermatozoa from Beef Bulls

Abstract Reactive oxygen species (ROS) have effects on characteristics of spermatozoa, including mitochondrial membrane potential, acrosomal integrity, and structural abnormalities that can influence spermatozoa function. The objective was to determine correlations between sperm response to ROS and functional sperm measurements using ejaculates collected as part of bull breeding soundness exams (BSE). Semen samples were collected from Angus and Charolais bulls (403 ± 11 d of age; n=46) over three days during yearling BSEs. A veterinarian evaluated spermatozoa for percent motility and normal morphology, and percentage progressive motility was measured using an iSperm® analyzer. If ejaculates met minimum thresholds for passing a BSE, they were diluted to 70 million cells/mL using BoviFree® and sent overnight for flow cytometry evaluation. Flow cytometry assays including acrosome and cell membrane integrity, mitochondrial energy potential, and oxidation status. Data were analyzed using Pearson’s correlation coefficients in SAS. Percentage live spermatozoa with positive ROS status was correlated (r = 0.53; P &amp;lt; 0.001) with percentage progressive motility. Percentage live spermatozoa with negative ROS status was moderately correlated with percentage spermatozoa exhibiting secondary abnormalities (r = 0.33; P = 0.02) and tended to be lowly correlated (r = 0.28; P = 0.06) with percentage spermatozoa exhibiting primary abnormalities. Percentage live spermatozoa that had disrupted acrosomes was strongly correlated (r = 0.66; P &amp;lt; 0.001) with percentage live spermatozoa with negative ROS and also moderately negatively correlated (r = -0.31; P = 0.04) with percentage live spermatozoa with positive ROS. Percentage live spermatozoa with positive ROS status was correlated (r = 0.58; P &amp;lt; 0.001) with percentage of spermatozoa with active mitochondrial membranes. Live spermatozoa with positive ROS were strongly correlated (P&amp;lt; 0.001) with live spermatozoa (r=0.94) and live spermatozoa with intact acrosome (r=0.92). These data confirm previous research that shows the detrimental effects of ROS on spermatozoa function.

Intraoperative Flow Cytometry for the Characterization of Gynecological Malignancies.

Simple SummaryAneuploidy and high proliferative potential are distinct features of neoplastic cells. Based on the established role of intraoperative flow cytometry in various types of cancer, the aim of the present study was to investigate its role in cancer cell identification during surgery for gynecological malignancies. The analysis time was 5–6 min per sample. A large percentage of tumors were characterized as aneuploid, while all tumor samples had a significantly high proliferation. Flow cytometry was performed in accordance with pathological evaluation, and the method had high sensitivity and specificity. Our results verify the value of intraoperative flow cytometry in gynecological malignancies, and warrant further investigation in multicenter studies.Cell-cycle analysis has shown the presence of aneuploidy to be associated with poor prognosis. We developed an innovative rapid cell-cycle analysis protocol (the Ioannina protocol) that permitted the intraoperative identification of neoplastic cells in a plethora of malignancies. Herein, we aimed to investigate the potential role of cell-cycle analysis in the intraoperative characterization of gynecological malignancies. Women who underwent surgery for gynecological malignancies in our institution over a three-year period were included in this study. Permanent section pathology evaluation was used as the gold standard for malignancy evaluation. Total accordance was observed between flow cytometry and pathology evaluation. In total, 21 aneuploid cancers were detected following DNA index calculation. Of these, 20 were hyperploid and 1 was hypoploid. In addition, tumor samples were characterized by a significantly lower percentage of cells in G0/G1, as well as an induced tumor index. The response time for flow cytometry to obtain results was 5–6 min per sample. It seems that flow cytometry analyses for intraoperative tumor evaluation can be safely expanded to gynecological malignancies. This is a novel practical approach that has been proven valuable in several tumor types to date, and also seems to be reliable for gynecological malignancies. Intraoperative flow cytometry is expected to be crucial in decisions of lymph node dissection in endometrial cancers, due to its rapid response regarding the tumor invasion of part or all of the myometrial thickness. In this way, the surgeon can quickly modify the plane of dissection. Our results warrant the further investigation of applying iFC in larger, multicenter studies.

Platelet-Derived Procoagulant Microvesicles Are Elevated in Patients with Retinal Vein Occlusion (RVO).

The etiopathogenesis of retinal vein occlusion (RVO) is multifactorial, and the contribution of platelets to RVO development has not been fully elucidated. We aimed to analyze platelet function in RVO patients (n = 35) and controls (n = 35). We found a higher (p < 0.05) level of soluble P-selectin in RVO group vs. controls. Additionally, in RVO patients, the concentration of platelet-derived microvesicles was higher (p < 0.05), and the difference between groups was deeper for the fraction of platelet-derived microvesicles with the procoagulant phenotype (p < 0.0001) and for overall procoagulant microvesicles level (p < 0.0001). The results were similar for the total RVO group and for both RVO types (central- and branched-retinal vein occlusion). We did not find differences in simple platelet parameters (platelet count, mean platelet volume, platelet distribution width, platecrit, reticulated platelets) and inflammatory markers (platelet-lymphocyte ratio, neutrophil-lymphocyte ratio). Similarly, no differences were found for platelet aggregation-stimulated byadenosine diphosphate; collagen; arachidonic acid; and in multiparametric flow cytometry evaluation of P-selectin, PAC-1, and fibrinogen binding for both unstimulated and adenosine diphosphate-, collagen-, and thrombin receptor activating peptide-stimulated platelets. Our results suggest that platelets can contribute to developing RVO by enhancing procoagulant activity through providing a procoagulation surface via platelet-derived microvesicles. The direct role of platelets’ hyperreactivity in developing RVO is less apparent, which is consistent with the complexity and multifactorial background of this disorder.

Now you see me, now you don't: Isolated central nervous system recurrence of CD19-negative diffuse large B-cell lymphoma following CD19-directed CAR T-cell treatment.

The authors discuss a case of CD19-negative diffuse large B-cell lymphoma with central nervous system relapse following CD19-directed CAR T-cell treatment. Absence of CD19 expression by the tumour cells presented a challenge for flow cytometry evaluation.

P362: MINIMAL-LOSS-CYTOMETRY FOR EVALUATION OF CEREBROSPINAL FLUID IN PEDIATRIC ACUTE LYMPHOBLASTIC LEUKEMIA. SINGLE CENTER STUDY.

Background: Central nervous system (CNS) involvement at diagnosis of acute lymphoblastic leukemia (ALL) represents a risk factor for CNS relapse. Currently, examination of the cerebrospinal fluid (CSF) is performed and evaluated quantitatively using a counting chamber and qualitatively using cytomorphological analysis of cytospin. Recently, flow cytometry (FC) evaluation of CSF was shown to provide more reliable results and to predict relapse risk (de Haas et al., 2021, Thastrup et al., 2020). Independently of these studies, we applied a lyse-no wash technique aiming at minimizing cell loss during preparation and at improved cell concentration estimate. Aims: Our aim was to compare our findings with morphological criteria and published data and correlate them with clinical outcome in pediatric patients with ALL. Methods: We included pediatric patients with newly diagnosed ALL (04/2014 – 01/2022), whose CSF examination was performed in CLIP laboratory (n=208). Patients were classified as B cell precursor ALL (BCP ALL; 85%) and T ALL (15%) and treated according to following protocols: AIEOP-BFM ALL 2009 (n=118), AIEOP-BFM ALL 2017 (n=71), EsPhALL(n=8), Interfant06 (n=5), off-protocol (n=6). During follow up, 14 patients relapsed (5 CNS incl. combined, 9 non-CNS). CSF (drawn into tube with no fixative agent and processed within 2 hours) was concentrated by centrifugation and incubated with combination of antibodies (CD20/CD10/CD45/CD34/CD19/CD3/Syto41 in BCP ALL and CD4/CD99/CD5/CD3/CD7/CD16 + 56/CD8/CD45/Syto41 in T ALL). Residual erythrocytes were lysed with NH4Cl solution, followed by immediate acquisition on BD FACS Lyric or BD LSRII cytometers. Blasts were quantified to initial CSF volume. Cluster of 20 events was required to assign samples as FC positive (FC+). In parallel, CNS status was concluded according to morphological criteria, being classified as CNS1 (no blasts), CNS2a/b (≤5 WBC/μL with blasts), CNS2c/CNS3 (≥5 WBC/μL with blasts) according to treatment protocol guidelines. Continuous variables were compared with the Mann–Whitney test, survival data were analyzed using Mantel-Cox test. P values<0.05 were considered as significant. Results: Using FC, we identified atypical blasts in 50 out of the 208 analyzed samples (24%). Median of analyzed CSF volume was 367µL (range 143-1033µL), which enabled median sensitivity 0.054 events/µL (range 0.02-0.14ev/µL). Mean blast concentration was 0.47ev/µL (range 0-25ev/µL) in all samples and 2 ev/µL (range 0.01-25ev/µL, median 0.53ev/µL) in FC+ samples. We observed significantly higher blast concentration in patients with T ALL, in patients with CNS2 and CNS3 and those who subsequently relapsed. Patients with FC+ had significantly lower relapse-free survival than FC- patients (55% vs 94%), which provided better separation than CNS status (68% vs.85% for CNS2/3 vs. CNS1, respectively). Image:Summary/Conclusion: Using fresh CSF sample with lyse-no wash protocol enabled us to detect approximately 10x higher blast count (0.24ev/µL in FC+CNS1 and 1.25ev/µL in FC+CNS2/3) than was recently published (0.015ev/µL in FC+CNS1 and 0.073ev/µL in FC+CNS2/3 in Thastrup et al., 2020), and thus it better correlates to cytomorphological data. Interestingly, the proportion of FC+ patients in our cohort is comparable to (24% vs 25%), showing that although in our method we lose fewer cells, we identify the same proportion of FC+ patients. The presented study shows a higher impact of FC positivity on relapse risk than described previously. The disadvantage of our method is a need for a fresh sample, which is investigated locally. Supported by UNCE/MED/015, NU20J-07-00028.

Abstract 278: Aerobic exercise suppresses melanoma tumor growth via upregulating ERK5 S496 phosphorylation

Abstract Hypoxia, immune cell infiltration, and drug delivery are key elements of therapeutic efficacy in solid tumors. Each are strongly influenced by the tumor microenvironment. Identification of novel methods to change the microenvironment is needed to improve the response of solid tumors, including melanoma, to therapies like immune checkpoint blockade. We and others have demonstrated that aerobic exercise remodels tumor microenvironment in multiple tumor types. Here, we present data to suggest that this exercise-induced remodeling of the tumor microenvironment is partially dependent on modulation of ERK5 in both tumor endothelium and infiltrating immune cells. The depletion of ERK5 in tumor-associated macrophages inhibits the growth of melanoma and lung carcinoma in mouse models, and the depletion of ERK5 in keratocytes prevents tumorigenesis promoted by inflammation. Multiple reports have shown the potential therapeutic approach of both ERK5 knockdown and pharmacological kinase inhibition in regulating inflammation and tumorigenesis. Here we identify the role of ERK5 S496 phosphorylation, known to promote inflammatory signaling, as a novel mediator of exercise induced tumor microenvironment alterations. Utilizing two melanoma models, we found that aerobic exercise suppresses the growth of YUMMER 1.7 tumors but not B16F10 in mice. Consistent with this, single cell RNA sequencing revealed reductions in myeloid derived suppressor cells and a shift in T cell populations favoring a non-exhausted phenotype in YUMMER 1.7. Flow cytometry evaluation demonstrated significantly more CD8+ T cells in YUMMER 1.7, but not in B16F10, tumors from exercised mice. Interestingly, we found increased phosphorylation of ERK5 at the S496 residue when ECs were treated with serum from exercised mice ex vivo. We also found the crucial role of ERK5 S496 phosphorylation in promoting both inflammation and proliferation in ERK5 TEY motif phosphorylation (kinase activity) and transactivation-independent manner in both ECs and macrophages. We generated ERK5 S496A knock-in mice, and found that the ability of exercise to suppress YUMMER 1.7 tumor growth was completely lost in ERK5 S496A knock-in mice, suggesting that ERK5 S496 phosphorylation is a key in exercise-induced tumor growth suppression. We are currently evaluating immune cell infiltration into tumors with or without exercise in the ERK5 S496A knock-in model relative to wild type mice. Our data suggest that ERK5 S496 phosphorylation is a critical mediator of the tumor microenvironment. The often neglected role of ERK5 S496 signaling should be carefully considered in the interpretation of prior reports of ERK5 knockdown and pharmacological kinase inhibition relative to tumorigenesis. Citation Format: Hannah Savage, Sumedha Pareek, Jonghae Lee, Riccardo Ballaro, Venkatasubrahman Samanthapudi, Kyung Ae Ko, Masaki Imanishi, Sivareddy Kotla, Jun-ichi Abe, Keri Schadler. Aerobic exercise suppresses melanoma tumor growth via upregulating ERK5 S496 phosphorylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 278.

Abstract LB025: Malat1-Serpinb6b signaling blocks T cells-mediated pyroptosis and governs cancer dormancy metastatic reactivation

Abstract Metastatic relapse is the major causes of mortality in patients with cancer and occur due to metastatic reactivation of dormant tumor cells. Early dissemination of tumor cells undergoing a protected period of dormancy in the target organs potentially explains this prevalent clinical behavior. Long non-coding RNAs (lncRNAs) are involved in various biological processes and diseases. Malat1 is one of the most abundant and highly conserved nuclear lncRNAs and have shown the associated with metastasis and serving as a predictive marker for various tumor progression. However, the correlation of tumor intrinsic lncRNAs in regulation of tumor dormancy and immune evasion are largely unknown. Using an in vivo screening platform for the isolation of genetic entities involved in either dormancy or reactivation of breast cancer tumor cells, we have identified Malat1 as a positive mediator of metastatic reactivation. To dissect the functional role of Malat1 in metastatic reactivation, we developed a clean Malat1 knockout (KO) model using paired gRNA CRISPR-Cas9 in metastatic murine syngeneic breast cancer. As a proof of concept, we also used inducible knockdown system under in vivo models. To delineate the immune microenvironment, we used single cell RNA-seq, ChIRP-seq, multicolor flowcytometry, RNA-FISH, and coculture experiments. Our data revealed that deletion of Malat1 induces dormancy and attenuated the metastatic colonization resulting in long-term survival of syngeneic mice model. In contrast, overexpression of Malat1 leads to metastatic reactivation of dormant breast cancer cells. Interestingly, 4T1-Malat1 KO dormant breast cancer cells exhibit metastatic outgrowth in T cells defective mice. Our single-cell RNA-seq and multicolor flowcytometry evaluation reveal enhanced T cells and reduced neutrophils proportions in mice with Malat1 KO cells. This indicates a critical role of immune microenvironment via Malat1-dependent immune evasion. Additionally, Malat1 KO inhibits cancer stemness properties. Similarly, RNA-seq and ChIRP-seq data suggest that KO of Malat1 hampers immune evasion and downregulates metastasis associated genes including Serpins and Wnts. Additionally, our data strongly suggests that Malat1 KO cells persists as non-proliferative dormant cells in lung due to CD8+ T cell-umpired immune activity. Our mechanistic studies showed that Malat1 regulated Sepinb6b blocks the CstG enzymatic activity and suppress cleavage of GsdmD and T cells induced pyroptosis. Interestingly, rescue experiments suggest that Malat1 or Serpinb6b protects T cell-induced cell death and induces dormancy re-awakening thereby rescue the metastatic potential of 4T1 Malat1 KO cells. Combination of Malat1 ASO with double immune checkpoint inhibitors greatly affects the metastatic outgrowth in breast cancer. Taken together, our studies demonstrate that tumor intrinsic Malat1 regulates Serpinb6b that suppresses CstG-GsdmD mediated pyroptosis which eventually promotes immune evasion and breast cancer dormancy metastatic reactivation. Citation Format: Dhiraj Kumar, Filippo Giancotti. Malat1-Serpinb6b signaling blocks T cells-mediated pyroptosis and governs cancer dormancy metastatic reactivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB025.

Real-time PCR using atpE, conventional PCR targeting different regions of difference, and flow cytometry for confirmation of Mycobacterium bovis in buffaloes and cattle from the Delta area of Egypt

BackgroundMycobacterium bovis notoriously causes detrimental infections in bovines and humans. In this study, 1500 buffaloes and 2200 cattle were tested by single intradermal comparative cervical tuberculin test and compared with the detection rates of M. bovis isolation, real-time and simplex PCR, and flow Cytometry.ResultsThe tuberculin test is the reference test in Egypt, the positive rate was 54/3700 (1.5%) composed of 18/1500 (1.2%) buffaloes and 36/2200 (1.6%) cattle which were mandatorily slaughtered under the Egyptian legislation, after postmortem examination the non-visible-lesion proportion was 39/54 (72.2%) which surpassed the visible-lesion rate 15/54 (27.8%) with (p < 0.0001). The samples from each case were pooled into one sample representing the case, and the isolation rate of M. bovis was 25/54 (46.3%). Real-time PCR using atpE was positive for mycobacteria on the genus level in 18/18 (100%) and 5/5 (100%) of tissue samples and isolates, respectively; simplex PCR detected M. bovis in 44/54 (81.5%) and 25/25 (100%) of tissue samples and isolates, respectively. Flow Cytometry evaluation of the CD4+, CD8+, WC1+δγ, and CD2+ cell phenotypes showed increased counts in the tuberculin-positive cases compared with negative cases (p < 0.0001), and these phenotypes in the tuberculin-positive cases increased after antigen stimulation than in the negative cases (p < 0.0001). Detection rates of PCR techniques and flow Cytometry exceeded that of bacterial isolation (p < 0.0001) and exhibited a strong correlation.ConclusionsThe skin test suffers from interference from non-tuberculous mycobacteria able to cause false-positive reactions in cattle and other species. Real-time PCR using atpE, conventional PCR targeting RDs, and flow Cytometry are rapid and accurate methods that correlate with the isolation and can be promising for detection and confirmation of infected live and slaughtered cases.

IL-17 Producing T cells as Predictors of Primary Immunodeficiencies in Patients with Candida Infections

Background: IL-17 producing T cells are a distinct subset of CD4+ T cells, which are recognized to have an essential role in protection against certain fungi and extracellular pathogens. Objectives: This study aims to evaluate the number of IL-17 producing T cells as a predictor of primary immunodeficiency disorders in patients with Candida infections and low numbers of IL-17 producing T cells. Methods: Seven newly diagnosed patients with documented Candida infections aged between 4 and 35 years were included in this study. Before establishing the diagnosis of chronic mucocutaneous candidiasis, a thorough immunodeficiency workup, including complete blood count, serum Ig levels and antibody responses, flow-cytometry evaluation, and LTT, was completed for all the patients, and other immunodeficiency disorders, including combined and phagocytic deficiencies, were ruled out. Then, IL-17 producing T cells were detected using antiCD3 and anti-IL-17 antibodies through flow-cytometry evaluation. Results: The IL-17 producing T cells significantly decreased in the peripheral blood of patients with PID and candida infections. A P-value of less than 0.05 was considered statistically significant. Conclusions: To conclude, in cases of recurrent candida infections, initial assessment of IL-17 producing T cells may act as a predictor of an underlying primary immunodeficiency. In patients with low counts of IL-17 producing T cells, selecting a targeted panel of genetic tests may become more helpful in the detection of certain immunodeficiency disorders than performing whole-exome sequence analysis.

Distinct CD8 T Cell Populations with Differential Exhaustion Profiles Associate with Secondary Complications in Common Variable Immunodeficiency

PurposeCommon variable immunodeficiency (CVID) is the most frequent symptomatic primary immunodeficiency, with heterogeneous clinical presentation. Our goal was to analyze CD8 T cell homeostasis in patients with infection only CVID, compared to those additionally affected by dysregulatory and autoimmune phenomena.MethodsWe used flow and mass cytometry evaluation of peripheral blood of 40 patients with CVID and 17 healthy donors.ResultsCD8 T cells are skewed in patients with CVID, with loss of naïve and increase of effector memory stages, expansion of cell clusters with high functional exhaustion scores, and a highly activated population of cells with immunoregulatory features, producing IL-10. These findings correlate to clinically widely used B cell-based EURO classification. Features of exhaustion, including loss of CD127 and CD28, and expression of TIGIT and PD-1 in CD8 T cells are strongly associated with interstitial lung disease and autoimmune cytopenias, whereas CD8 T cell activation with elevated HLA-DR and CD38 expression predict non-infectious diarrhea.ConclusionWe demonstrate features of advanced differentiation, exhaustion, activation, and immunoregulatory capabilities within CD8 T cells of CVID patients. Assessment of CD8 T cell phenotype may allow risk assessment of CVID patients and provide new insights into CVID pathogenesis, including a better understanding of mechanisms underlying T cell exhaustion and regulation.

Myeloperoxidase deficiency: a single center experience

Aim: Myeloperoxidase (MPO) deficiency is the most common inherited defect of phagocytes. In this article, we aimed to reveal clinical characteristics of our patients with primary MPO deficiency.&#x0D; Material and Method: In our study, patients aged 0-18 years, who were consulted to Ankara City Hospital Pediatric Hematology Department between 1 October 2019 and 1 December 2021 due to neutropenia, were retrospectively examined. If a patient had neutropenia in the complete blood count and inconsistently normal neutrophil count in the peripheral blood smear formula it was accepted as pseudoneutropenia. Patients with pseuduneutropenia were included in the study.&#x0D; Results: Fifteen patients diagnosed with MPO deficiency were analyzed in the study. Nine of the patients were female, 6 were male, median age of the patients was 7 (0 – 17.5) years. The mean white blood cell (WBC) count of the patients was reported as 8219±2879/mm3, and the mean neutrophil count and percentage in the complete blood count printout was 33.30±15.88/mm3 and 0,74%±0, 94% respectively. The mean neutrophil count and percentage counted in the peripheral blood smear were 5186±1710 and 63.8%±10.59%, respectively. The mean LUC value on the complete blood count printout was 54.35%±19.47% (Normal range, 0-4%). In the flow cytometry evaluation of peripheral blood samples of the patients, it was observed that neutrophils were stained with CD33, CD13, CD16, CD11b monoclonal antibodies but not with MPO.&#x0D; Conclusion: Peripheral smear evaluation is important when investigating the etiology of neutropenia. Many hematology analyzers using the MPO staining technique are indicative of MPO deficiency by identifying large unstained cells that do not stain with MPO. In patients who present with recurrent infections and MPO deficiency, other reasons that may predispose to infections should be investigated.

Evaluation of Flow Cytometry for Cell Count and Detection of Bacteria in Biological Fluids.

ABSTRACTThe analysis of biological fluids is crucial for the diagnosis and monitoring of diseases causing effusions and helps in the diagnosis of infectious diseases. The gold standard method for cell count in biological fluids is the manual method using counting chambers. The microbiological routine procedures consist of Direct Gram staining and culture on solid or liquid media. We evaluate the analytical performance of SYSMEX UF4000 (Sysmex, Kobe, Japan) and Sysmex XN10 (Sysmex, Kobe, Japan) in comparison with cytological and microbiological routine procedures. A total of 526 biological fluid samples were included in this study (42 ascitic, 31 pleural, 31 peritoneal, 125 cerebrospinal, 281 synovial, and 16 peritoneal dialysis fluids). All samples were analyzed by flow cytometry and subsequently processed following cytological and/or microbiological routine procedures. With regard to cell counts, UF4000 (Sysmex, Kobe, Japan) showed a performance that was at least equivalent to those of the reference methods and superior to those of XN10 (Sysmex, Kobe, Japan). Moreover, the bacterial count obtained with UF4000 (Sysmex, Kobe, Japan) was significantly higher among culture or Direct Gram stain positive samples. We established three optimal cutoff points to predict Direct Gram stain positive samples for peritoneal (465.0 bacteria/μL), synovial (1200.0 bacteria/μL), and cerebrospinal fluids (17.2 bacteria/μL) with maximum sensitivity and negative predictive values. Cell count and detection of bacteria by flow cytometry could be used upstream cytological and microbiological routine procedures to improve and accelerate the diagnosis of infection of biological fluid samples.IMPORTANCE The analysis of biological fluids is crucial for the diagnosis and monitoring of diseases causing effusions and helps in the diagnosis of infectious diseases. The possibility of carrying out cytological and microbiological analyses of biological fluid samples on the same automated machine would simplify the sample circuit (addressing the sample in a single laboratory, 24/7). It would also minimize the quantity of sample required. The performance of cytological and microbiological analysis by the flow cytometer UF 4000 (Sysmex, Kobe, Japan) has never been evaluated yet. This study has shown that bacterial count by flow cytometry using UF4000 (Sysmex, Kobe, Japan) could be used upstream of microbiological routine procedures to improve and to accelerate the diagnosis of infection of biological fluid samples.

Mo-MDSCs are pivotal players in colorectal cancer and may be associated with tumor recurrence after surgery

Colorectal cancer (CRC) is the third most common malignancy. Its development and progression is associated with natural immunosuppression related, among others, to myeloid derived suppressor cells (MDSCs).Overall, 54 patients in different stage of CRC, before any treatment were recruited into the study. The analysis included flow cytometry evaluation of blood MDSCs subsets, correlation their level with the tumor stage and T cell subsets. In the case of 11 patients, MDSCs level was evaluated before and 3 days after surgery, and these patients were monitored for cancer recurrence over 5 years.The results showed that frequency of circulating MDSCs subsets is increased significantly in CRC patients, with highest level detected in most advanced tumor stages. Moreover, only monocytic MDSCs (Mo-MDSCs) positively correlate with regulatory Treg, and negatively with tumor Her2/neu specific CD8+ T cells. Circulating MDSCs, in contrast to tumor resident (mostly Mo-MDSCs), are negative for PD-L1 expression. Additionally, after surgery the blood level of Mo-MDSCs increases significantly, and this is associated with tumor recurrence during a 5-year follow-up.In conclusion, Mo-MDSCs are pivotal players in CRC-related immunosuppression and may be associated with the risk of tumor recurrence after surgery.

Evaluation of Volume-Based Flow Cytometry as a Potential Primary Method for Quantification of Bacterial Reference Material

Evaluation of Volume-Based Flow Cytometry as a Potential Primary Method for Quantification of Bacterial Reference Material

Implementation of TREC/KREC detection protocol for newborn SCID screening in Bulgaria: a pilot study

Neonatal screening for inborn errors of immunity (IEI), based on quantification of T-cell-receptor- excision circles (TRECs) and kappa-deleting recombination-excision circles (KRECs) from dried blood spots (DBS), allows early diagnosis and improved outcomes for the affected children. Determination of TREC/KREC levels from prospectively collected newborns' Guthrie cards and from DBS samples of patients with confirmed IEI was done using a commercial kit. Retrospective assessment of flow cytometry evaluation of TREC/KREC correspondence with lymphocyte subpopulations and evaluation of the correlations between TREC and KREC with immune cells, based on the data from patients with suspected or confirmed immune disorders, were conducted. 2,228 Guthrie cards were tested, 1276 for TREC only and 952 for both TREC and KREC. Eight newborns (0.36%) were TREC positive and 10 (1.05%) had KREC below the cut-off. The re-testing rate was 1.88%. Retrospective analysis demonstrated that the TREC/KREC assay identifies 100% of severe combined immune deficiencies (SCID) cases when DBS were collected at birth. Correlation analysis showed moderate significant correlations between TREC and the absolute numbers of CD4 cells (r = 0.634, p < 0.01) and total T cells (r = 0.536, p < 0.01). The ability of KREC levels to predict abnormal absolute (AUC of 0.772) and relative (AUC 0.731) levels of B cells was demonstrated.

Multiparametric Flow Cytometry Evaluation of CD200L/CD200R- LSC/NK Synapse Including Leukemia Stem Cell (LSC) Fraction As a Potential Therapeutic Target and Marker of NK Cell Exhaustion in Pediatric AML-Conect-AML French Collaborative Network

BACKGROUNDNK cells play a crucial role in the immune surveillance of malignant hemopathies. They undergo fine regulation by the microenvironment and by integrating activating and inhibiting signals trough several receptor/ligand couple interactions, hereafter referred to as “NK synapse”. The ligands are expressed by a variety of cell types in the hematopoietic niche, including most immature leukemic stem cells CD34+CD38-. High expression of inhibiting ligands on AML (acute myeloid leukemia) blasts was associated with adverse clinical outcome . This observation highlights the relevance of identifying new ligand/receptor (L/R) pairs that could be targeted to prevent inhibiting interactions at the NK synapse. Relevant interactions to be blocked would display both ligand and receptor expressions on the leukemic cells and NK cells respectively. PATIENTS AND METHODS23 pediatric AML patients from the pediatric MyeChild01 protocol including in CONECT-AML French national collaborative network project diagnosed between 2018 and 2019 were included in this study. Reference bone marrows used were regenerative (4) or healthy bone marrows (5) . Multicolour flowcytometry protocole used fresh EDTA bone marrow at AML diagnosis and immunostaining with fluorochrome-coupled antibodies using 14 colour panel of L/R couples (Figure 1). Data was acquired on the FORTESSA Becton Dickinson with the Diva software and analysis using script R-PCA and FlowJo . RESULTSWe studied 5 inhibiting NK synapses (iinhibitory ligand/receptor pairs) . Four out of five inhibiting synapses (TIGIT/CD155; PD1-1/PD-L1; CD94/HLA-E and KIR2DL/HLA-A-B-C), showed not significant expression of ligand associated with the corresponding receptor expression. The CD200/CD200R synapse was the only one in which high ligand expression in blasts was significantly associated with high receptor expression on NK cells (Figure 2). This synapse could thus be of interest to develop targeting therapies for CD200-positive pediatric AML, with the strong advantage that patient eligibility could be easily identified at diagnosis. We then realized a principal component analysis, using the R software (PCA), integrating the MFIs of the 5 inhibiting NK synapses and 6 activating NK synapses (Figure 1) for the pediatric AML cohort (ID #1 to #23 ) together with reference bone marrows (healthy donors (n=5; ID #24 to #28) and regenerative bone marrows (n=4; ID #29 to #32)) . The CD200/CD200R synapse was identified as the main variable, explaining the distribution of patients and healthy donors as both CD200 and CD200R expressions happened to be among the most contributive to PCA axes. Interestingly, healthy donors clustered together, close to regenerative bone marrows. Pediatric AML patients distributed heterogeneously (Figure 3). In parallel, we evaluated whether CD200 expression on bulk leukemia blasts including most immature CD34+CD38- LSC was associated with exhaustion markers on NK cells. We found that patients with high and intermediate expression of CD200 on blasts (MFI > 3 rd quartile and comprised between 2 nd and 3 rd quartile, respectively) displayed strong PD-1 and TIGIT expressions on NK cells. Reciprocally, patients with low CD200 expression (MFI< 2 nd quartile) displayed a moderate PD-1 expression on NK cells, and TIGIT expression was more heterogeneous among individuals (Figure 4). CONCLUSIONSHere, we identified CD200 expression in AML blasts including LSC as a marker that could be associated with NK cell exhaustion. at diagnosis. A PCA strategy allowed to observe that this marker differentiated pediatric AML patients NK synapse profiles from healthy donors and regenerative bone marrows sugesting a potential deregulation of bone marrow niche including NK-LSC escape. This suggests that CD200 expression assessment on blasts at diagnosis could be a tool to evaluate NK cell antitumor potential. Indeed, direct NK cell assessment by flow cytometry can be challenging because of blast invasion in the bone marrow. Nevertheless, it remains to be elucidated whether this clustering and exhaustion markers on NK cells correlated with patient clinical outcomes and MRD kinetics including CD34+CD38- LSC flow frequency evaluation that should be useful in most clinical trials to overcome chemoresistance of LSC. These results should be confirmed in a prospectively larger cohort of patients in future clinical trials. [Display omitted] DisclosuresRenard: Jazz Pharmaceuticals: Research Funding.