γδ T-Cell Immunophenotype for the Study of Human Aging.

Flow cytometry serves as a crucial tool in immunology, allowing for the detailed analysis of immune cell populations. γδ T cells, a subset of T cells, play pivotal roles in immune surveillance and immune aging. Assessing the phenotype and functional capabilities of γδ T cells isolated from whole blood or tissue within the context of human aging yields invaluable insights into the dynamic changes affecting immune function, tissue homeostasis, susceptibility to infections, and inflammatory responses.

In Vitro Interaction Between Yeast Extracellular Vesicles and Human Monocyte-Derived Dendritic Cells.

Extracellular vesicles (EVs) are lipid-bound particles produced by a wide variety of cells from different biological species. EVs can carry molecules, such as nucleic acids and metabolites, and are involved in cell functioning, communication, and signaling. Recent literature reported that pathogenic or commensal yeast strains can produce EVs targeting the host's immune system and exerting immunomodulatory actions. In humans, yeast EVs can be endocytosed by dendritic cells (DCs), characterized by phagocyting and migrating capabilities with the role of capturing antigens to present to T lymphocytes, triggering the immune response. Physiological or disease-associated immunosenescence impairs both DC functionality and gut microbiota; thus investigating the interaction between commensal microorganisms and the host's immune system would help elucidate the impact of aging on the immune system-microbiota interplay. We hereby present a protocol for the incubation of in vitro-generated human monocyte-derived DCs with EVs purified from different yeast strains isolated from fermented milk. The protocol includes flow cytometry analysis on DC activation markers and endocytosis assay.

Phenotypic Characterization of B-Lymphocyte Subpopulations in Human Peripheral Blood: A Cost-Effective Seven-Color One-Tube Protocol.

B cells are crucial components of the immune system, responsible for producing specific antibodies in response to infections and vaccines. Despite their uniform appearance, B cells display diverse surface molecules and functional properties, which are not yet fully understood. Apart from antibody production, B cells also play roles in antigen presentation and cytokine secretion, essential for initiating T-cell immune responses. Their significance as disease biomarkers and therapeutic targets has led to increased research focus. However, the lack of standardized protocols for B-cell identification and the variability in defining B-lymphocyte subpopulations pose some challenges. This paper proposes a B-cell identification panel throughout the evaluation of previous cytometry panels and nomenclature heterogeneity for B-cell subpopulations. Major subpopulations recognized in human peripheral blood include transitional, naive, switched memory, unswitched memory, double negative, and plasmablasts, characterized based on their functional and phenotypic features. We present a standardized flow cytometry protocol utilizing surface phenotypic markers (CD3, CD19, IgD, CD27, CD38, and CD24) to differentiate and analyze B-cell subpopulations. This practical and cost-effective panel can be used in various research and laboratory settings. The challenges of standardizing names and markers for classifying B-lymphocyte subpopulations are discussed, along with protocols utilizing multiple markers and gating strategies, allied with the importance of considering viability markers. In summary, this standardized protocol and panel provide a comprehensive approach to identifying B-cell subpopulations to enhance the reproducibility and comparability of B-cell subpopulation studies.

Characterization of Age-Dependent Changes in Skeletal Muscle Repair and Regeneration Using a Mouse Model of Acute Muscle Injury.

Acute skeletal muscle injury initiates a process of necrosis, debris clearance, and ultimately tissue regeneration via myogenesis. While skeletal muscle stem cells (MuSCs) are responsible for populating the proliferative myogenic progenitor pool to fuel muscle repair, recruited and resident immune cells have a central role in the regulation of muscle regeneration via the execution of phagocytosis and release of soluble factors that act directly on MuSCs to regulate myogenic differentiation. Therefore, the timing of MuSC proliferation and differentiation is closely linked to the populations and behaviors of immune cells present within skeletal muscle. This has important implications for aging and muscle repair, as systemic changes in immune system function contribute to a decline in muscle regenerative capacity. Here, we present adapted protocols for the isolation of mononuclear cells from skeletal muscles for the quantification of immune cell populations using flow cytometry. We also describe a cardiotoxin skeletal muscle injury protocol and detail the expected outcomes including immune cell infiltration to the injured sites and formation of new myocytes. As immune cell function is substantially influenced by aging, we extend these approaches and outcomes to aged mice.

Assessment of Viability of Listeria monocytogenes by Flow Cytometry.

In food industry, Listeria monocytogenes contamination can occur accidentally despite the quality control of raw materials and factory. Decontamination processes or inhibitory effects of ingredients/additives in food products are set up to ensure compliance with hygiene and microbiological criteria. These actions represent stresses for the pathogenic agent, causing fluctuations in its physiological states. Moreover, during these environmental stresses, Listeria monocytogenes can enter in a viable but nonculturable (VBNC) state which is not detected by plate counting but by flow cytometry. This technique coupled with cell staining by fluorescent dyes offers the possibility to assess different physiological states based on different cellular parameters: enzymatic activity, transmembrane integrity, membrane potential, and respiratory activity. In this chapter, we present a method to assess the viability of foodborne pathogens using a double-staining principle based on the assessment of membrane integrity and intracellular esterase activity.

Flow cytometry in acute myeloid leukemia and detection of minimal residual disease.

Acute myeloid leukemia (AML) is a common type of acute leukemia (AL), belonging to malignant tumors of the hematopoietic system with the characteristics of rapid disease development, control with extreme difficulties, easy recurrence, poor prognosis, and incidence rate increasing with age. The traditionally diagnostic standard of French American British (FAB), being based on the morphological examination with high human subjectivity, can no longer meet the demand of clinical diagnosis and treatment of AML. Requirements of objective accuracy and low-dose sample, have become the indispensable method for AML diagnosis and monitoring prognosis. Flow cytometry is a modern technology that can quickly and accurately detect the series, antigen distribution, differentiation stage of AML cells, minimal residual lesions after AML therapy, so as to provide the great significance in guiding clinical diagnosis, hierarchical treatment, and prognosis judgement. This article will systematically elaborate on the application of flow cytometry in the diagnosis and classification of AML, and the detection of minimal residual lesions, thereby providing reference significance for dynamic monitoring and prognostic observation of AML with different immune subtypes of FAB.

Transcription Factor Analysis to Investigate Immunosenescence in Rheumatoid Arthritis Patients.

Rheumatoid arthritis (RA) is linked to various signs of advanced aging, such as premature immunosenescence which occurs due to decline in regenerative ability of T cells. RA T cells develop a unique aggressive inflammatory senescent phenotype with an imbalance of Th17/T regulatory (Treg) cell homeostasis and presence of CD28- T cells. The phenotypic analysis and characterization of T cell subsets become necessary to ascertain if any functional deficiencies exist within with the help of transcription factor (TF) analysis. These subset-specific TFs dictate the functional characteristics of T-cell populations, leading to the production of distinct effector cytokines and functions. Examining the expression, activity, regulation, and genetic sequence of TFs not only aids researchers in determining their importance in disease processes but also aids in immunological monitoring of patients enrolled in clinical trials, particularly in evaluating various T-cell subsets [Th17 (CD3+CD4+IL17+RORγt+) cells and T regulatory (Treg) (CD3+CD4+CD25+CD127-FOXP3+) cells], markers of T-cell aging [aged Th17 cells (CD3+CD4+IL17+RORγt+CD28-), and aged Treg cells (CD3+CD4+CD25+CD127-FOXP3+CD28-)]. In this context, we propose and outline the protocols for assessing the expression of TFs in aged Th17 and Treg cells, highlighting the crucial aspects of this cytometric approach.

Factors to consider before choosing EV labeling method for fluorescence-based techniques.

A well-designed fluorescence-based analysis of extracellular vesicles (EV) can provide insights into the size, morphology, and biological function of EVs, which can be used in medical applications. Fluorescent nanoparticle tracking analysis with appropriate controls can provide reliable data for size and concentration measurements, while nanoscale flow cytometry is the most appropriate tool for characterizing molecular cargoes. Label selection is a crucial element in all fluorescence methods. The most comprehensive data can be obtained if several labeling approaches for a given marker are used, as they would provide complementary information about EV populations and interactions with the cells. In all EV-related experiments, the influence of lipoproteins and protein corona on the results should be considered. By reviewing and considering all the factors affecting EV labeling methods used in fluorescence-based techniques, we can assert that the data will provide as accurate as possible information about true EV biology and offer precise, clinically applicable information for future EV-based diagnostic or therapeutic applications.

Differential lipid signaling from CD4+ and CD8+ T cells contributes to type 1 diabetes development.

Introduction: We reported that Ca2+-independent phospholipase A2ß (iPLA2ß)-derived lipids (iDLs) contribute to type 1 diabetes (T1D) onset. As CD4+ and CD8+ T cells are critical in promoting ß-cell death, we tested the hypothesis that iDL signaling from these cells participates in T1D development. Methods: CD4+ and CD8+ T cells from wild-type non-obese diabetic (NOD) and NOD.iPLA2ß+/- (NOD.HET) mice were administered in different combinations to immunodeficient NOD.scid. Results: In mice receiving only NOD T cells, T1D onset was rapid (5 weeks), incidence 100% by 20 weeks, and islets absent. In contrast, onset was delayed 1 week and incidence reduced 40%-50% in mice receiving combinations that included NOD.HET T cells. Consistently, islets from these non-diabetic mice were devoid of infiltrate and contained insulin-positive ß-cells. Reduced iPLA2ß led to decreased production of proinflammatory lipids from CD4+ T cells including prostaglandins and dihydroxyeicosatrienoic acids (DHETs), products of soluble epoxide hydrolase (sEH), and inhibition of their signaling decreased (by 82%) IFNγ+CD4+ cells abundance. However, only DHETs production was reduced from CD8+ T cells and was accompanied by decreases in sEH and granzyme B. Discussion: These findings suggest that differential select iDL signaling in CD4+ and CD8+ T cells contributes to T1D development, and that therapeutics targeting such signaling might be considered to counter T1D.

Early B lymphocyte subsets in blood predict prognosis in sepsis.

Background: B lymphocytes play a key role in immunosuppression. This study investigated the prognostic value of B cell subsets in sepsis. Methods: Flow cytometry was used to assess peripheral B cell subsets from patients with sepsis on the first and seventh days following admission, as well as 111 healthy controls. The patients were divided into survivors and non-survivors, based on 28-day prognosis. Results: The analysis showed abnormal distribution and selective depletion of B cells and its subsets in the early stages of sepsis. On day 1, compared with survivors, non-survivors showed significant decreases in the proportion and absolute count of transitional (Tr) B cells, reductions in the proportion of CD5+ B cells, and increases in the proportion of double-negative (DN) B cells. On day 7, the proportions and absolute counts of Tr and CD5+ B cells significantly decreased whereas the proportion of DN B cells significantly increased in non-survivors. Ninety-four survivors and 15 non-survivors were included in our paired-sample rank-sum test. Compared to day 1, only the survivors showed significant increases in absolute B, Tr B, and CD5+ B cell counts by day 7. Multivariate Cox regression analysis showed that the proportion of DN B cells on day 1 (hazard ratio = 1.092 [95% confidence interval: 1.035-1.152], P = 0.001) was a risk factor for mortality, and Kaplan-Meier survival curve analysis showed that patients with proportions of DN B cells > 11.81% on day 1 had poorer prognoses. Receiver operating characteristic curve analysis showed that B cell subset parameters could predict mortality (area under the receiver operating characteristic curve [AUC], 0.741) and enhanced the prognostic value of the Acute Physiology and Chronic Health Evaluation II score (AUC, 0.840). Conclusion: Our study revealed that deficiencies of B, Tr B, and CD5+ B cells, as well as a persistent increase in the proportion of DN B cells, were associated with poor prognosis-and that B cell subsets showed predictive value to mortality. These results provide new insights into the roles of B cell subsets in sepsis, as well as ways to better manage its progression and predict its course.

Glucose-6-phosphate dehydrogenase deficiency detection using fluorocytometric assay: Evaluation after 1 year of clinical implementation.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common enzymopathy that affects red blood cells (RBCs) and renders them susceptible to oxidative stress. G6PD deficiency can cause hemolytic anemia, especially after exposure to certain drugs or infections. The diagnosis of G6PD deficiency is usually based on spectrophotometric measurement of enzyme activity, but this method has limitations in heterozygous females and in patients with other hematological disorders. In this study, we evaluated the use of flow cytometry as an alternative method for detecting G6PD deficiency in 514 samples (265 females and 249 males) from a clinical laboratory. We compared the results of flow cytometry with those of spectrophotometry and molecular analysis, and assessed the performance of flow cytometry in different subgroups of patients. We found that flow cytometry was able to identify G6PD deficiency in most cases, with high sensitivity and specificity. Flow cytometry also allowed the quantification of the percentage of G6PD-deficient RBCs, which varied among heterozygous females due to X-chromosome inactivation. Moreover, flow cytometry detected several cases of G6PD deficiency that were missed by spectrophotometry, especially in heterozygous females with normal or subnormal enzyme activity. However, flow cytometry also showed some false negative results, mainly in patients with sickle cell disease. Therefore, flow cytometry is a reliable and efficient tool for screening G6PD deficiency, but some precautions should be taken in interpreting the results in patients with other hematological conditions.

SCIP: A scalable, reproducible, and open-source pipeline for morphological profiling image cytometry and microscopy data.

Imaging flow cytometry (IFC) provides single-cell imaging data at a high acquisition rate. It is increasingly used in image-based profiling experiments consisting of hundreds of thousands of multi-channel images of cells. Currently available software solutions for processing microscopy data can provide good results in downstream analysis, but are limited in efficiency and scalability, and often ill-adapted to IFC data. In this work, we propose Scalable Cytometry Image Processing (SCIP), a Python software that efficiently processes images from IFC and standard microscopy datasets. We also propose a file format for efficiently storing IFC data. We showcase our contributions on two large-scale microscopy and one IFC datasets, all of which are publicly available. Our results show that SCIP can extract the same kind of information as other tools, in a much shorter time and in a more scalable manner.

Flow cytometric-based detection of CD80 is a useful diagnostic marker of acute myeloid leukemia in dogs.

Introduction: CD80, a co-stimulatory molecule required for optimal T cell activation, is expressed on antigen-presenting cells, including monocytes and dendritic cells, in dogs and humans. We hypothesized that CD80 would be expressed on tumor cells in dogs from acute myeloid leukemia (AML) but not dogs with lymphoid neoplasms. Methods and results: We first evaluated the cellular staining pattern of a hamster anti-murine CD80 antibody (clone 16-10A1, ThermoFisher Scientific Cat# 17-0801-82, RRID: AB_469417) in blood and bone marrow aspirates from healthy dogs. Using flow cytometric analysis and examination of modified Wright's-stained cytologic smears of unsorted and flow cytometric or immunomagnetic bead-sorted leukocytes, we show that the antibody binds to mature and immature neutrophils and monocytes, but not lymphocytes or eosinophils, in blood and bone marrow. We then added the antibody to routine flow cytometric panels for immunophenotyping hematopoietic neoplasms in dogs. We found that the antibody labeled tumor cells in 72% of 39 dogs with AML and 36% of 11 dogs with acute leukemia expressing lymphoid and myeloid markers ("mixed lineage") but none of the dogs with B (n = 37) or T (n = 35) lymphoid neoplasms. A higher proportion of tumor cells in dogs with AML were labeled with the anti-CD80 antibody vs antibodies against other myeloid-associated antigens, including CD4 (36%, p = 0.003), CD11b (44%), CD11c (46%), CD14 (38%, p = 0.006) and CD18 (59%, clone YFC118). In contrast, antibodies against CD11b and CD11c bound to tumor cells in 8-32% of the lymphoid neoplasms. Discussion: We show that CD80, as detected by antibody clone 16-10A1, is a sensitive and specific marker for AML and would be useful to include in flow cytometric immunophenotyping panels in dogs.

Platelet changes and bleeding symptoms in children, adolescents, and adults with 22q11.2 deletion syndrome.

BACKGROUND: The deletion region of 22q11.2 deletion syndrome (22q11.2DS) contains a gene encoding glycoprotein Ibß (GPIbß), which is required to express the GPIb/IX/V complex on the platelet surface. Therefore, patients with 22q11.2DS may have congenital platelet disorders. However, information is limited on platelets and bleeding symptoms. In this study, we investigated clinical information, including bleeding symptoms, platelet counts, and GPIb expression levels in children and adolescents/adults with 22q11.2DS. PROCEDURE: Thirty-two patients with 22q11.2DS were enrolled in a prospective cohort study between 2022 and 2023 at outpatient clinics within our institute. RESULTS: The median platelet counts in adolescents/adults with 22q11.2DS were significantly lower than those in children (p < .0001). A gradual decrease was found along with increasing age (p = .0006). Values of median GPIb expression on platelet surfaces (66% in children and 70% in adolescents/adults) were significantly lower than those in healthy controls (p < .0001 and p = .0002). Bleeding symptoms included surgery-related bleeding (52%), purpura (31%), and epistaxis (22%); most of them were minor. The median International Society on Thrombosis and Hemostasis bleeding assessment tool score was not significantly different between children and adolescents/adults (p = .2311). CONCLUSION: Although there was an age-related decrease in platelet count and a disease-related decrease in GPIb expression, no difference in bleeding symptoms was found between children and adolescents/adults. 22q11.2DS overall had minor bleeding symptoms in daily life, and the disease had little effect on spontaneous bleeding. However, some patients had major bleeding events; further accumulation of data on hemostasis during surgery and trauma is required.

Breadth and polyfunctionality of T cell responses to human cytomegalovirus in men who have sex with men: relationship with HIV infection and frailty.

Cytomegalovirus (CMV)-seropositive adults have large T cell responses to a wide range of CMV proteins; these responses have been associated with chronic inflammation and frailty in people with or without HIV infection. We analyzed the relationships between chronic HIV infection, frailty, and the breadth and polyfunctionality of CD4 and CD8 T cell responses to CMV. Peripheral blood mononuclear cells from 42 men (20 without HIV and 22 with virologically suppressed HIV) in the Multicenter AIDS Cohort Study (MACS) were stimulated with peptide pools spanning 19 CMV open reading frames (ORFs). As measured by flow cytometry and intracellular cytokine staining for IFN-γ, TNF-α, and IL-2, CD8 T cells from men with HIV responded to significantly more CMV ORFs than those from men without HIV. This was primarily due to a broader response to ORFs that are expressed during the late phase of CMV replication. The number of ORFs to which a participant's T cells responded was positively correlated with the sum of all that individual's T cell responses; these correlations were weaker in men with than without HIV. Polyfunctional CMV-specific CD4 responses (production of more than one cytokine) were significantly lower in men with than without HIV. Frailty status did not substantially affect the breadth or magnitude of the CMV-specific T cell responses. These results suggest that immune control of CMV infection is affected more by chronic HIV infection than by frailty. The differences between men with and without HIV were similar to those reported between young and older adults without HIV. IMPORTANCE: T cell responses to chronic cytomegalovirus (CMV) infection have significant biological and clinical implications in HIV infection and aging. Here, we systematically analyzed the breadth, magnitude, and polyfunctionality of T cell responses to multiple CMV antigens in men with and without HIV in the Multicenter AIDS Cohort Study (MACS), a longstanding study of the natural and treated history of HIV-1 infection in men who have sex with men. We found that the breadth and polyfunctionality of T cell responses to CMV were different between men with chronic, treated HIV and those without HIV. The reason for these differences is unknown, but these findings suggest that people with treated HIV may have more frequent CMV reactivation than people without HIV. Differences between people with and without HIV also resembled differences reported between young and older adults without HIV, supporting a role for the immune responses to CMV in the aging process.

Ecotoxicological effects of suspended sediments on marine microalgae using flow cytometry and pulse-amplitude modulation (PAM) fluorometry.

Microalgal bioassays were conducted to evaluate the ecotoxicological effects of suspended sediments (SS) collected from coastal environments. Growth inhibition was assessed for six microalgal species, and multiple endpoints were measured using flow cytometry (FCM) and pulse-amplitude modulation (PAM) fluorometry for three species (Dunaliella tertiolecta, Isochrysis galbana, and Phaeodactylum tricornutum). Among these, the EC50 for growth inhibition of D. tertiolecta (6700 mg L-1) was notably lower compared to the other species, and among several endpoints, esterase activity was the most inhibited. Species-specific responses to SS exposure were identified, with D. tertiolecta exhibiting greater susceptibility across most endpoints. Meanwhile, measurements of Fo', Fm', and Y(NPQ) in P. tricornutum using PAM fluorometry revealed greater sensitivity. Based on the results of this study and review, the tentative predicted no-effect concentration was calculated as 12.1 mg L-1. Overall, this study provides novel insights into SS ecotoxicity, establishing a crucial baseline for future investigations.

Cytotoxic properties, glycolytic effects and high-resolution respirometry mitochondrial activities of Eriocephalus racemosus against MDA-MB 231 triple-negative breast cancer.

INTRODUCTION: Triple-negative breast cancer (TNBC) represents a significant global health crisis due to its resistance to conventional therapies and lack of specific molecular targets. This study explored the potential of Eriocephalus racemosus (E. racemosus) as an alternative treatment for TNBC. The cytotoxic properties and high-resolution respirometry mitochondrial activities of E. racemosus against the MDA-MB 231 TNBC cell line were evaluated. METHODS: Hexane solvent and bioactive fraction extractions of E. racemosus were performed, while mass spectrometry-based metabolite profiling was used to identify the phytochemical constituents of the extracts. The extracts were further tested against MDA-MB 231 TNBC cells to determine their cytotoxicity. The mode of cell death was determined using flow cytometry. The activities of caspases 3, 8, and 9 were assessed using a multiplex activity assay kit. Glycolytic activity and High-resolution respirometry measurements of mitochondrial function in the MDA-MB 231 cell line were conducted using the Seahorse XFp and Oroboros O2K. RESULTS: Metabolite profiling of E. racemosus plant crude extracts identified the presence of coumarins, flavonoids, sesquiterpenoids, triterpenoids, and unknown compounds. The extracts demonstrated promising cytotoxic activities, with a half maximal inhibitory concentration (IC50) of 12.84 µg/mL for the crude hexane extract and 15.49 µg/mL for the bioactive fraction. Further, the crude hexane and bioactive fraction extracts induced apoptosis in the MDA-MB-231 TNBC cells, like the reference drug cisplatin (17.44%, 17.26% and 20.25%, respectively) compared to untreated cells. Caspase 3 activities confirmed the induction of apoptosis in both cisplatin and the plant crude extracts, while caspase 8 and 9 activities confirmed the activation of both the intrinsic and extrinsic apoptosis pathways. Increased levels of glycolytic activity were observed in the hexane crude extract. High-resolution respiratory measurements showed elevated mitochondrial activities in all mitochondrial states except for complex-IV activity. CONCLUSION: These findings support further exploration of E. racemosus as a potential therapeutic agent for TNBC, offering a promising avenue for the development of targeted treatments with minimal adverse effects.

CD146 Molecule Expression in B Cells Acute Lymphoblastic Leukemia (B-ALLs): A Flow-Cytometric Marker for an Accurate Diagnostic Workup.

Background: B-lineage acute lymphoblastic leukemias (B-ALL) harboring the t(9;22)(q34;q11)/BCR::ABL1 rearrangement represent a category with previously dismal prognosis whose management and outcome dramatically changed thanks to the use of tyrosine kinase inhibitors (TKIs) usage and more recently full chemo-free approaches. The prompt identification of these cases represents an important clinical need. Objectives: We sought to identify an optimized cytofluorimetric diagnostic panel to predict the presence of Philadelphia chromosome (Ph) in B-ALL cases by the introduction of CD146 in our multiparametric flow cytometry (MFC) panels. Methods: We prospectively evaluated a total of 245 cases of newly diagnosed B-ALLs with a CD146 positivity threshold >10% referred to the Division of Hematology of 'Sapienza' University of Rome. We compared the results of CD146 expression percentage and its mean fluorescence intensity (MFI) between Ph+ ALLs, Ph-like ALLs, and molecularly negative ALLs. Results: Seventy-nine of the 245 B-ALL cases (32%) did not present mutations at molecular testing, with 144/245 (59%) resulting in Ph+ ALL and 19/245 (8%) Ph-like ALLs. Comparing the 3 groups, we found that Ph+ B-ALLs were characterized by higher expression percentage of myeloid markers such as CD13, CD33, and CD66c and low expression of CD38; Ph+ B-ALL showed a higher CD146 expression percentage and MFI when compared with both molecular negative B-ALL and Ph-like ALLs; neither the mean percentage of CD146 expression neither CD146 MFI were statically different between molecular negative B-ALL and Ph-like ALLs. Conclusions: Our data demonstrate the association between CD146 expression and Ph+ ALLs. CD146, along with myeloid markers, may help to identify a distinctive immunophenotypic pattern, useful for rapid identification in the diagnostic routine of this subtype of B-ALLs that benefits from a specific therapeutic approach.

Evaluation of flow cytometry-based fluorescent antibody to membrane antigen test to measure the humoral immunity against the varicella-zoster virus.

The fluorescent antibody to membrane antigen (FAMA) test is the gold standard for measuring the immunity induced by varicella vaccines with high sensitivity and specificity. However, certain aspects of the FAMA test, such as time consumption, non-automation, and subjective interpretation by observers using fluorescence microscopy, are obstacles to handling large amounts of samples. To overcome these hurdles, flow cytometry was adopted to analyze and compare the flow FAMA titer with the classic FAMA titer. In addition, to save time in FAMA antigen preparation and reduce lot-to-lot variation, the stability of the FAMA antigen stored in liquid nitrogen was investigated. The FAMA test was performed on sera from 229 children, and antibody titers were analyzed using fluorescence microscopy (classic FAMA) and flow cytometry (flow FAMA). For comparison, glycoprotein enzyme immunoassay (gpEIA) titer was also measured. A strong correlation was found between the flow and classic FAMA titers, and the flow FAMA and gpEIA titers, with Pearson's r of 0.9316 and 0.8588, respectively. Between the classic FAMA and gpEIA titers, the Pearson's r value was 0.8156. The positive percent agreement, negative percent agreement, and area under the curve of the flow FAMA against classic FAMA were 95.0 %, 86.8 %, and 0.909, respectively. And those of the flow FAMA against gpEIA were 80.0 %, 87.6 %, and 0.838, respectively. The FAMA antigen stored in liquid nitrogen was stable for up to 12 months. Based on the above data, flow FAMA has the potential to be used as an alternative to classic FAMA. Moreover, pre-made FAMA antigen may reduce the preparation time and lot-to-lot variation of FAMA test.

Comparative Analysis of Canonical Inflammasome Activation by Flow Cytometry, Imaging Flow Cytometry and High-Content Imaging.

Inflammasome activation occurs in various diseases, including rare diseases that require multicenter studies for investigation. Flow cytometric analysis of ASC speck+ cells in patient samples can be used to detect cell type-specific inflammasome activation. However, this requires standardized sample processing and the ability to compare data from different flow cytometers. To address this issue, we analyzed stimulated and unstimulated PBMCs from healthy donors using seven different flow cytometers. Additionally, human PBMCs were analyzed by fluorescence microscopy, imaging flow cytometry and high-content imaging (HCI). Flow cytometers differed significantly in their ability to detect ASC speck+ cells. Aria III, Astrios EQ, and Canto II performed best in separating ASC speck+ from diffuse ASC cells. Imaging flow cytometry and HCI provided additional insight into ASC speck formation based on image-based parameters. For optimal results, the ability to separate cells with diffuse ASC from ASC speck+ cells is decisive. Image-based parameters can also differentiate cells with diffuse ASC from ASC speck+ cells. For the first time, we analyzed ASC speck detection by HCI in PBMCs and demonstrated advantages of this technique, such as high-throughput, algorithm-driven image quantification and 3D-rendering. Thus, inflammasome activation by ASC speck formation can be detected by various technical methods. However, the results may vary depending on the device used.