Search

Abstract As Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) are easily accessible, easy to isolate, and ethically acceptable, they represent a promising source of MSCs for use in regenerative medicine. Considering decisions on WJ-MSCs collection requires extensive knowledge of the factors that impact their yield. This study's aim was to evaluate the influence of parameters related to mothers and newborns on the WJ-MSCs yield. The WJ-MSCs were isolated and expanded after being isolated from 79 umbilical cord (UC) samples. Population doubling time and cell proliferation were assessed. By flow cytometry analysis, WJ-MSCs were identified by positivity of CD105, CD90, and CD73 and negativity of CD45 and CD34. There was a statistically significant negative correlation between UC width and P1 doubling time. Maternal age and WJ-MSC yield were shown to be negatively correlated. Birth weight and gestational age showed a significant positive correlation between WJ-MSCs yield and neonatal variables. No significant correlations were detected between the WJ-MSCs and the mother parity, nor the neonatal sex, fetal presentation, or head circumference. The WJ-MSCs yield increases with younger maternal age, higher gestational age, and increased neonatal birth weight. Hence, consideration should be given to these factors when selecting the ideal donors.

Abstract Our research focuses on semen collection from 42 African penguin males, grouped by age, habituation levels, and reproductive season timing. We assess behavioral and physiological responses to dorso-abdominal massage, evaluating species-specific seminal traits using conventional and advanced methods. Positive behavioral responses corresponded with successful semen collection. Young and human-reared males exhibited more favorable behaviors, and samples containing spermatozoa were more likely collected during the reproductive season. Age did not influence sample collectability or spermatozoa presence, though mature males produced higher semen volumes. Young males exhibited more sperm morphological abnormalities, especially outside the breeding season. Sperm concentration and number per ejaculate showed no significant differences across age or seasonal groups. Young males had higher motile and progressive sperm percentages, while mature males had more static sperm. Additionally, percentages of live sperm and intact acrosomes were higher in mature males. Young males and samples from non-reproductive season presented more dead sperm with damaged acrosomes. Plasma membrane integrity positively correlated with age for live sperm and negatively for dying sperm. This research demonstrates the feasibility of semen collection from African penguins regardless of habituation level, fills the knowledge gap by describing sperm morphological abnormalities, and provides insights into using flow cytometry in Sphenisciformes.

Abstract The blue bottle genus Physalia is one of the well-known siphonophore belonging to the Cnidaria, Hydrozoa. Physalia is also known as a ferocious predator, occasionally stinging and fatally wounding humans, but key details of its life cycle and reproductive biology are unclear. Physalia have separate sexes, and sexual reproduction occurs through the release of complex structures called gonodendra that contain many gonophores that will release either eggs or sperm. It is not known how mature the gonophores are when the gonodendra are released. In this study, we aim to characterize germ cell maturation by conducting histological, cytological, and gene expression analyses of the gonodendron of Physalia utriculus from Japan. We found a layered structure of the gonophore, consistent with other studies; however, gametes were not found even in gonophores that were within the released gonodendra. Moreover, haploid cells were not detected by flow cytometry. Analysis of the expression of putative germ cell marker and meiosis related genes showed high expression in the gonophore. These results strongly suggest that germ cells do not mature until after gonodendra are released. These findings provide valuable insights into the reproductive ecology and life cycle of Physalia.

Abstract The study of microorganism interactions is important for understanding the organization and functioning of microbial consortia. Additionally, the interaction between yeast and bacteria is of interest in the field of health and nutrition area for the development of probiotics. To investigate these microbial interactions at the cellular and molecular levels, a simple, reliable, and quantitative method is proposed. We demonstrated that flow cytometry enables the measurement of interactions at a single-cell level by detecting and counting yeast cells with bound fluorescent bacteria. Imaging flow cytometry revealed that the number of bacteria attached to yeast followed a Gaussian distribution whose maximum reached 14 bacterial cells using a clinical Escherichia coli strain E22 and the laboratory yeast strain BY4741. We found that the dynamics of adhesion resemble a Langmuir adsorption model, albeit it is a rapid and almost irreversible process. This adhesion is dependent on the mannose-specific type 1 fimbriae, as E. coli mutants lacking these appendages no longer adhere to yeast. However, this type 1 fimbriae-dependent adhesion could involve additional yeast cell wall factors, since the interaction between bacteria and yeast mutants with altered mannan content remained comparable to that of wild-type yeast. In summary, flow cytometry is an appropriate method for studying bacteria-yeast adhesion, as well as for the high-throughput screening of candidate molecules likely to promote or counteract this interaction.

ABSTRACTBackground Diagnosing spontaneous bacterial peritonitis (SBP) requires a high clinical index of suspicion because the clinical presentation varies widely. Early detection and treatment of SBP are crucial for improving survival rates. Ascitic fluid neutrophil count ≥ 250 cells/mm3 is the only required criterion to diagnose SBP, regardless of culture results. The most frequently used manual ascitic neutrophil counting is difficult and time-consuming, and it often leads to frequent delays in obtaining results and a possible under- or overestimation of the neutrophilic count. The flow cytometric counting provides results in 30 minutes and is more accurate, with sensitivity and specificity approaching 100%.Objective This study aimed to assess the diagnostic efficacy of manual counting of neutrophils in comparison to flow cytometry for detecting SBP.Patients and methods In a cross-sectional study, we included 140 cirrhosis-related ascites patients who were clinically suspected to have SBP and admitted to the Alexandria Main University Hospital in the Department of Internal Medicine. Participants with ≥ 250 neutrophil cells/mm3 in ascitic fluid by flow cytometry were defined as SBP patients. In contrast, those with fewer than 250 neutrophil cells/mm3 in ascitic fluid using the flow cytometric method were defined as patients without SBP.Results At a cutoff value of 250 cells/mm3, the ROC curve showed that the manual ascitic fluid method had lower sensitivity, specificity, positive and negative predictive values (67.19%, 86.84%, 81.13%, and 75.86%, respectively, with AUC = 0.945, 95% CI, 0.91–0.97, p

Introduction: The use of functionalized metal nanoparticles against cancer cells has gained assiduous attention. This study aimed to assess the inhibitory activity of iron oxide nanoparticles coated with glucose and conjugated with coumarin (Fe3O4@Glu-Coumarin) on breast cancer cell line, as well as the expression of the caspase-8 and caspase-9 genes. Material & Methods: The physical and chemical properties of nanoparticles were evaluated using FT-IR, XRD, EDS-map, and electron microscope imaging. The toxicity of the synthesized nanoparticles was determined using the MTT assay, and the 50% inhibitory dose (IC50) was calculated. Moreover, the effect of nanoparticles on apoptosis induction was investigated by measuring the expression level of the caspase 8 and 9 genes and flow cytometry analysis. Statistical analyses were performed in SPSS software. One-way analysis of variance (ANOVA) was employed to assess significant differences between nanoparticle-treated and control groups. A p-value of less than 0.05 was regarded as statistically significant. Results: The synthesis of Fe3O4@Glu-Coumarin nanoparticles was confirmed by physicochemical tests, including FT-IR, XRD, EDS-mapping, and electron microscope imaging. The nanoparticles had dimensions of 25 to 50 nm and contained Fe, O, and C elements. The treatment of MCF-7 cells with nanoparticles caused a significant decrease in the survival of cancer cells, and the IC50 was 93μg/ml. The exposure of cells to the nanoparticles caused a marked increase in the expression of caspases 8 and 9 by 2.6 and 2.9 folds, respectively, compared to the control group. In addition, the frequency of apoptotic cells after treatment with the nanoparticles increased from 2.21% to 84%. Discussion & Conclusion: The results of this study pointed out that Fe3O4@Glu-Coumarin increased the expression of the caspases 8 and 9 genes in breast cancer cells and, as a result, can activate the intrinsic and extrinsic apoptotic pathways, thereby inhibiting the proliferation of cancer cells.

Background and Aim: Urinalysis is one of the most commonly performed screening tests in the clinical laboratory to diagnose and monitor various urological as well as systemic conditions. Newly developed automated urine analyzers are expected to routinely screen urine in less time compared to conventional routine microscopy and provide clinicians with prompt clinical information with a lower false-negative rate. The study aimed to evaluate the diagnostic performance of the two automated urine analyzers, URiSCAN Super + YD Diagnostics and Sysmex UC-3500–UF-5000 urine analyzer and compare them with microscopy. Materials and Methods: This was a prospective study performed on 124 randomly selected samples of patients coming to the clinical pathology laboratory at a tertiary care hospital for 3 months between March 1, 2022 and May 30, 2022. Each sample was taken for both automated analyzer testing and microscopic evaluation. The results of urine examination consisting of cells, formed elements, and microorganisms were compared between both the automated urine analyzers and microscopy, and the degree of concordance was calculated utilizing Cohen’s kappa (κ) analysis to see the concordance between all three values. The statistical analyses were performed using SPSS version 25, developed by IBM Co, and P < 0.05 was considered statistically significant. Results: The degree of concordance of red blood cell (RBC), white blood cell (WBC), and epithelial cells (ECs) was good between the two automated analyzers, URiSCAN Super + YD Diagnostics and Sysmex UC-3500–UF-5000 urine analyzer and microscopy with P < 0.05. The concordance between the Sysmex UC-3500–UF-5000 urine analyzer and manual microscopy was better than between manual microscopy and URiSCAN Super + YD Diagnostics. There was no concordance between any of these methods for either crystals, cast, bacteria, and fungi with P > 0.05. Conclusion: The results from the automated analyzers for RBCs, WBCs, and ECs were similar to the result of manual microscopy, and the analyzers can be relied on for testing and can help in reducing turnaround time of routine urine examination. However, bacteria, fungi, dysmorphic red cells, casts, and crystals need to be analyzed by microscopic examination before giving a final diagnosis.

Abstract In the human and veterinary fields, oral vaccines generate considerable interest. In dogs, these vaccines are newly developed, and understanding their mechanisms is crucial. Mesenteric lymph nodes (MLNs) and Peyer's patches (PPs) are important sites for gastrointestinal mucosal induction, yet canine MLNs lack sufficient information. To address this, we collected MLN samples from healthy dogs, performed flow cytometry to characterize immune cells, and conducted single-cell RNA sequencing (scRNA-seq) to explore subpopulations, particularly B and T lymphocytes. This effort enabled the characterization of canine MLN's main cell populations and the construction of a predictive atlas, as well as the identification of particularities of this area.

Abstract Background Tumor cells release extracellular vesicles (EVs) that contribute to the polarization of macrophages towards tumor-associated macrophages (TAMs). High expression levels of the RNA binding protein IGF2BP2/IMP2 are correlated with increased tumor cell proliferation, invasion, and poor prognosis in the clinic. However, there is a lack of understanding of whether IMP2 affects the cargo of cancer cell-derived EVs, thereby modulating macrophage polarization. Methods EVs were isolated from IMP2-expressing HCT116 parental cells (WT) and CRISPR/Cas9 IMP2 knockout (KO) cells. EVs were characterized according to MISEV guidelines, microRNA cargo was assessed by microRNA-Seq, and the protein cargo was analyzed by proteomics. Primary human monocyte-derived macrophages (HMDMs) were polarized by EVs, and the expression of genes and surface markers was assessed using qPCR and flow cytometry, respectively. Morphological changes of macrophages, as well as the migratory potential of cancer cells, were assessed by the Incucyte® system and macrophage matrix degradation potential by zymography. Changes in the metabolic activity of macrophages were quantified using a Seahorse® analyzer. For in vivo studies, EVs were injected into the yolk sac of zebrafish larvae, and macrophages were isolated by fluorescence-activated cell sorting. Results EVs from WT and KO cells had a similar size and concentration and were positive for 25 vesicle markers. The expression of tumor-promoting genes was higher in macrophages polarized with WT EVs than KO EVs, while the expression of TNF and IL6 was reduced. A similar pattern was observed in macrophages from zebrafish larvae treated in vivo. WT EV-polarized macrophages showed a higher abundance of TAM-like surface markers, higher matrix degrading activity, as well as a higher promotion of cancer cell migration. MicroRNA-Seq revealed a significant difference in the microRNA composition of WT and KO EVs, particularly a high abundance of miR-181a-5p in WT EVs, which was absent in KO EVs. Inhibitors of macropinocytosis and phagocytosis antagonized the delivery of miR-181a-5p into macrophages and the downregulation of the miR-181a-5p target DUSP6. Proteomics data showed differences in protein cargo in KO vs. WT EVs, with the differentially abundant proteins mainly involved in metabolic pathways. WT EV-treated macrophages exhibited a higher basal oxygen consumption rate and a lower extracellular acidification rate than KO EV-treated cells. Conclusion Our results show that IMP2 determines the cargo of EVs released by cancer cells, thereby modulating the EVs' actions on macrophages. Expression of IMP2 is linked to the secretion of EVs that polarize macrophages towards a tumor-promoting phenotype.

Acute Myeloid Leukaemia (AML) is a malignant disorder of the bone marrow in which there is maturational arrest in blood cell progenitors, resulting in the failure of normal haematopoiesis. Acute Promyelocytic Leukaemia (APML) is a subtype of AML with a defined clinical course and a biology that is distinct from other forms of AML. Morphologically, the most common form of APML shows the presence of heavily granulated cells with folded and twisted nuclei in the bone marrow. Biologically, the cytogenetic changes define the syndrome, and molecular changes in the chromosomes play a critical role in leukaemogenesis. The occurrence of APML in the paediatric population is very rare, accounting for

Abstract At present, liquid storage is the most efficient method for pig semen preservation. This approach relies upon reducing sperm metabolism, allowing for the maintenance of cell lifespan. In this context, the study of proteins that could protect sperm during liquid storage is of high relevance. The 70 kDa Heat Shock Protein (HSP70) is an anti-apoptotic protein that has been reported to be relevant to sperm survival. Thus, we explored the role of HSP70 during prolonged storage of pig semen at 17 °C. Six semen pools were incubated with YM-1 (0, 0.05, 0.1 and 0.2 μM), an HSP70 inhibitor, and stored at 17 °C for 21 days. On days 0, 4, 10, 14 and 21, sperm quality and function were evaluated through flow cytometry and Computer-Assisted Sperm Analysis (CASA), and HSP70 activity and chromatin condensation were also determined. While inhibition of HSP70 increased progressive motility, Ca2+ and Reactive Oxygen Species (ROS) levels, and mitochondrial activity during the first 10 days of storage, it had a detrimental effect on sperm motility after 14 and 21 days. In spite of this, sperm viability was not altered. We can conclude that HSP70 contributes to the liquid storage of pig semen because it keeps mitochondrial activity low, which is needed for the maintenance of sperm function.

Abstract Sirtuin 3 (SIRT3) belongs to the Sirtuin protein family, which consists of NAD+-dependent lysine deacylase, involved in the regulation of various cellular activities. Dysregulation of SIRT3 activity has been linked to several types of cancer, including breast cancer. Because of its ability to stimulate adaptive metabolic pathways, it can aid in the survival and proliferation of breast cancer cells. Finding new chemical compounds targeted towards SIRT3 was the primary goal of the current investigation. Virtual screening of ~ 800 compounds using molecular docking techniques yielded 8 active hits with favorable binding affinities and poses. Docking studies verified that the final eight compounds formed stable contacts with the catalytic domain of SIRT3. Those compounds have good pharmacokinetic/dynamic properties and gastrointestinal absorption. Based on excellent pharmacokinetic and pharmacodynamic properties, two compounds (MI-44 and MI-217) were subjected to MD simulation. Upon drug interaction, molecular dynamics simulations demonstrate mild alterations in the structure of proteins and stability. Binding free energy calculations revealed that compounds MI-44 (− 45.61 ± 0.064 kcal/mol) and MI-217 (− 41.65 ± 0.089 kcal/mol) showed the maximum energy, suggesting an intense preference for the SIRT3 catalytic site for attachment. The in-vitro MTT assay on breast cancer cell line (MDA-MB-231) and an apoptotic assay for these potential compounds (MI-44/MI-217) was also performed, with flow cytometry to determine the compound’s ability to cause apoptosis in breast cancer cells. The percentage of apoptotic cells (including early and late apoptotic cells) increased from 1.94% in control to 79.37% for MI-44 and 85.37% for MI-217 at 15 μM. Apoptotic cell death was effectively induced by these two compounds in a flow cytometry assay indicating them as a good inhibitor of human SIRT3. Based on our findings, MI-44 and MI-217 merit additional investigation as possible breast cancer therapeutics.

Abstract Fluorescent labels have strongly contributed to many advancements in bioanalysis, molecular biology, molecular imaging, and medical diagnostics. Despite a large toolbox of molecular and nanoscale fluorophores to choose from, there is still a need for brighter labels, e.g., for flow cytometry and fluorescence microscopy, that are preferably of molecular nature. This requires versatile concepts for fluorophore multimerization, which involves the shielding of dyes from other chromophores and possible quenchers in their neighborhood. In addition, to increase the number of readout parameters for fluorescence microscopy and eventually also flow cytometry, control and tuning of the labels’ fluorescence lifetimes is desired. Searching for bright multi-chromophoric or multimeric labels, we developed PEGylated dyes bearing functional groups for their bioconjugation and explored their spectroscopic properties and photostability in comparison to those of the respective monomeric dyes for two exemplarily chosen fluorophores excitable at 488 nm. Subsequently, these dyes were conjugated with anti-CD4 and anti-CD8 immunoglobulins to obtain fluorescent conjugates suitable for the labeling of cells and beads. Finally, the suitability of these novel labels for fluorescence lifetime imaging and target discrimination based upon lifetime measurements was assessed. Based upon the results of our spectroscopic studies including measurements of fluorescence quantum yields (QY) and fluorescence decay kinetics we could demonstrate the absence of significant dye-dye interactions and self-quenching in these multimeric labels. Moreover, in a first fluorescence lifetime imaging (FLIM) study, we could show the future potential of this multimerization concept for lifetime discrimination and multiplexing.

Abstract The use of magnetic metal nanoparticles has been considered in cancer treatment studies. In this study, BiFe2O4@Ag nanoparticles were synthesized biologically by Scenedesmus obliquus for the first time and their anticancer mechanism in a gastric cancer cell line was characterized. The physicochemical properties of the nanoparticles were evaluated by fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic Light Scattering (DLS), and zeta potential analyses. Cell viability and nuclear damage were investigated by the MTT and Hoechst staining assays, respectively. Flow cytometry analysis was performed to determine the frequency of the necrotic and apoptotic cells as well as cell cycle analysis of the nanoparticles-treated cells. Physicochemical characterization showed that the synthesized particles were spherical, without impurities, in a size range of 38–83 nm, with DLS size and zeta potential of 295.7 nm and -27.7 mV, respectively. BiFe2O4@Ag nanoparticles were considerably more toxic for the gastric cancer cells (AGS cell line) than HEK293 normal cells with IC50 of 67 and 117 µg/ml, respectively. Treatment of AGS cells with the nanoparticles led to a remarkable increase in the percentage of late apoptosis (38.5 folds) and cell necrosis (13.4 folds) and caused cell cycle arrest, mainly at the S phase. Also, nuclear fragmentation and apoptotic bodies were observed in the gastric cancer cells treated with the nanoparticles. This study represents BiFe2O4@Ag as a novel anticancer candidate against gastric cancer that can induce cell apoptosis through DNA damage and inhibition of cell cycle progression.

Synchronous diagnosis of two different haematological malignancies of a non-Hodgkin lymphoma and multiple myeloma (MM) is very rare. We report the case of the coexistence of low-grade B-cell lymphoma and MM. The patient was a 79-year-old male who presented with severe transfusion-dependent anaemia and had all CRAB features. Bone marrow flow cytometry revealed lymphocytes and plasma cells which both expressed different light chain restrictions with B-cell showing kappa light chain and plasma cells showing lambda light chain. Serum protein electrophoresis revealed an immunoglobulin G lambda band of 1.42 g/dl. The patient was evaluated extensively to rule out lymphoplasmacytic lymphoma and other plasmacytic lymphomas with positron emission tomography-computed tomography and molecular workup. He was started on therapy with bortezomib–dexamethasone–rituximab. The patient unfortunately expired due to sepsis. The treatment of such an unusual presentation remains difficult. Few case reports of MM with synchronous non-Hodgkins lymphoma are present. We present this case due to the rarity of such a presentation.

The flow cytometry method could support physicians’ decisions in the diagnosis and treatment monitoring of immunodeficient patients. Most clinical recommendations are focused on the search for alterations in T- and B-lymphocyte subsets, less commonly natural killer (NK) cells and granulocytes. While reference values for clinically meaningful lymphocyte subsets have been published ubiquitously among numerous countries, we have not found significant data for a population of adult Polish habitats; thus we determined reference values for T, B, and NK subsets according to sex and age. The female group showed a higher percentage of lymphocytes (CD45 ++ ), T helper lymphocytes with a higher absolute count, as well as CD4/CD8 ratio, marginal zone-like B cells, class-switched B cells, and CD21 low B cells than the male group. The male group was found to have elevated percentages of naïve B lymphocytes, transitional B cells, and plasmablasts. A weak positive correlation with age was found among double positive T lymphocytes, natural killer T cells (NKT) lymphocytes, and CD21 low B cells. A negative correlation with age for double negative T lymphocytes, marginal zone-like B cells, and plasmablasts was noted. The results indicated the importance of creating distinct reference ranges regarding sex and age concerning immunophenotype.

Objective To examine the expression levels of Toll-like receptor 5 (TLR5) on peripheral blood monocytes in patients with rheumatoid arthritis (RA), and to explore its clinical significance. Methods A total of 140 RA patients, along with 70 healthy controls (normal control group) received in Suining Central Hospital from January 2017 to August 2022 were selected. The RA patients were divided into high disease activity group (n=70) and moderate-to-low disease activity group (n=70) based on the disease activity score 28-erythrocyte sedimentation rate (DAS28-ESR). Flow cytometry was used to determine the positive rate of TLR5 on peripheral blood monocytes among three groups. The predictive value of TLR5 expression on monocytes surface for RA was analyzed using receiver operating characteristic (ROC) curves. Results The positive rates of TLR5 on the surface of monocytes in normal control group, moderate-to-low disease activity group and high disease activity group were (46.99±5.79)%, (50.80±5.96)% and (58.86±6.20)%, respectively, and there was a statistically significant difference among the three groups (F=71.550, P＜0.01). The area under the ROC curve for TLR5 in diagnosing RA was 0.79, with a cut-off value of 49.50%, sensitivity of 72.86%, and specificity of 73.57%. Conclusion The TLR5 expressed on peripheral blood monocytes is involved in the pathogenesis and maintenance of disease activity and has predictive value for RA.

Abstract Extracellular vesicles (EVs) have crucial roles in hemostasis and coagulation. They sustain coagulation by exposing phosphatidylserine and initiate clotting by surface expression of tissue factor (TF) under inflammatory conditions. As their relevance as biomarkers of coagulopathy is increasingly recognized, there is a need for the sensitive and reliable detection of TF+ EVs, but their flow cytometric analysis is challenging and has yielded controversial findings for TF expression on EVs in the vascular system. We investigated the effect of different fluorochrome-to-protein (F/P) ratios of anti-TF-fluorochrome conjugates on the flow cytometric detection of TF+ EVs from activated monocytes, mesenchymal stem cells (MSCs), and in COVID-19 plasma. Using a FITC-labeled anti-TF antibody (clone VD8), we show that the percentage of TF+ EVs declined with decreasing F/P ratios. TF was detected on 7.6%, 5.4%, and 1.1% of all EVs derived from activated monocytes at F/P ratios of 7.7:1, 6.6:1, and 5.2:1. A similar decline was observed for EVs from MSCs and for EVs in plasma, whereas the detection of TF on cells remained unaffected by different F/P ratios. We provide clear evidence that next to the antibody clone, the F/P ratio affects the flow cytometric detection of TF+ EVs and should be carefully controlled.

Abstract Background The aim of the study was to investigate whether the expression of CD27−CD38+ in interferon (IFN)-γ+CD4+ T cells stimulated by the specific antigen early secreted antigenic target-6 (ESAT-6)/culture filter protein-10 (CFP-10) could be a potential new therapeutic evaluation indicator for anti-tuberculosis (TB) treatment. Methods Newly diagnosed active pulmonary TB patients, latent TB infection (LTBI) and healthy controls were enrolled from January 2021 to December 2021. PTB patients were treated by standard anti-TB regimen 2HREZ/4HR (2 months of isoniazid (H), rifampin (R), ethambutol (E), and pyrazinamide (Z) followed by 4 months of isoniazid (H) and rifampin (R)). The difference of CD27−CD38+ expression in IFN-γ+CD4+ T cells before treatment, 2 months after treatment, and 6 months after treatment were compared. Results Total 45 PTB patients, 38 LTBI cases and 43 healthy controls were enrolled. The expression of CD27−CD38+ decreased significantly after anti-TB treatment and was comparable with that in LTBI and healthy controls when the 6-month anti-TB treatment course was completed. The decline rate of CD27−CD38+ between 6 months after treatment and baseline was positively correlated with erythrocyte sedimentation rate (r = 0.766, P

ABSTRACTThe diagnosis of acute lymphoblastic leukemia (ALL), which is the most common type of cancer in children, has become more accurate with the use of flow cytometry. Here, this technology was used to immunophenotype leukemic cells in peripheral blood samples from Libyan pediatric ALL patients. We recruited 152 newly diagnosed patients at Tripoli Medical Center (Tripoli, Libya) by morphological examination of blood and bone marrow. Twenty-three surface and cytoplasmic antigen markers were used to characterize B and T cells in circulating blood cells by four-color flow cytometry. Six children (3.9%) turned out to have biphenotypic acute leukemia, 88 (57.9%) had B ALL, and 58 (38.1%) had T ALL. There were 68 cases of pro-B ALL CD10-positive (44.7%), 8 cases of pro-B ALL CD10-negative (5.2%), 6 cases of pre-B ALL (3.9%), and 6 of mature-B ALL (3.9%). CD13 was the most commonly expressed myeloid antigen in ALL. We present immunophenotypic data for the first time describing ALL cases in Libya. The reported results indicate that the most common subtype was pro-B ALL, and the frequency of T-ALL subtype was higher compared to previous studies. Six cases were positive for both myeloid and B lymphoid markers. Our findings may provide the basis for future studies to correlate immunophenotypic profile and genetic characteristics with treatment response among ALL patients.

Abstract The high mortality rate of colon cancer indicates the insufficient efficacy of current chemotherapy. Thus, the discussion on engineered metal nanoparticles in the treatment of the disease has been considered. In this study, silver nanoparticles were functionalized with glutamine and conjugated with thiosemiccarbazide. Then, anticancer mechanism of Ag@Gln-TSC NPs in a colon cancer cell line (SW480) was investigated. Characterizing Ag@Gln-TSC NPs by FT-IR, XRD, EDS-mapping, DLS, zeta potential, and SEM and TEM microscopy revealed that the Ag@Gln-TSC NPs were correctly synthesized, the particles were spherical, with surface charge of − 27.3 mV, high thermal stability and low agglomeration level. Using MTT assay we found that Ag@Gln-TSC NPs were significantly more toxic for colon cancer cells than normal fibroblast cells with IC50 of 88 and 186 µg/mL, respectively. Flow cytometry analysis showed that treating colon cancer cells with Ag@Gln-TSC NPs leads to a considerable increase in the frequency of apoptotic cells (85.9% of the cells) and increased cell cycle arrest at the S phase. Also, several apoptotic features, including hyperactivity of caspase-3 (5.15 folds), increased expression of CASP8 gene (3.8 folds), and apoptotic nuclear alterations were noticed in the nanoparticle treated cells. Furthermore, treating colon cancer cells with Ag@Gln-TSC NPs caused significant down-regulation of the HULC Lnc-RNA and PPFIA4 oncogene by 0.3 and 0.6 folds, respectively. Overall, this work showed that Ag@Gln-TSC NPs can effectively inhibit colon cancer cells through the activation of apoptotic pathways, a feature that can be considered more in studies in the field of colon cancer treatment.

Introduction: Acute myeloid leukaemia (AML) is a tumour of hematopoietic progenitors caused by acquired oncogenic mutations that impede differentiation, leading to the accumulation of immature myeloid blasts in the marrow. Aberrant phenotype is a phenomenon in which lymphoid-associated and other myeloid lineage markers are expressed in myeloblasts or myeloid-associated markers are expressed in lymphoblasts. Materials and Methods: Diagnosed cases of AML were included in this study to study the aberrant expression using multiparametric flow cytometry. Results: Out of a sample size of 50, 30 cases expressed aberrant CD markers. Male: Female ratio was 0.76. Majority of cases belonged to the age group >60 years of age. CD 7 was overall the most common aberrant CD marker. Conclusion: Immunophenotyping has a significant role in diagnosis and predicting prognosis of hematopoietic malignancies in the absence of more advanced diagnostic tools like cytogenetics.

Abstract Background Porcine seminal plasma (SP) is endowed with a heterogeneous population of extracellular vesicles (sEVs). This study evaluated the immunophenotypic profile by high-sensitivity flow cytometry of eight sEV subpopulations isolated according to their size (small [S-sEVs] and large [L-sEVs]) from four different SP sources, namely three ejaculate fractions (the first 10 mL of the sperm rich fraction [SRF-P1], the remaining SRF [SRF-P2], and the post-SRF [PSRF]) and entire ejaculate (EE). Methods Seminal EVs were isolated using a size exclusion chromatography-based protocol from six SP pools (five ejaculates/pool) of each SP source and characterized using complementary approaches including total protein (BCA™assay), particle size distribution (dynamic light scattering), morphology (transmission electron microscopy), and purity (albumin by Western blot). Expression of CD9, CD63, CD81, CD44 and HSP90β was analyzed in all sEV subpopulations by high-sensitivity flow cytometry according to MIFlowCyt-EV guidelines, including an accurate calibration, controls, and discrimination by CFSE-labelling. Results Each sEV subpopulation exhibited a specific immunophenotypic profile. The percentage of sEVs positive for CD9, CD63, CD81 and HSP90β differed between S- and L-sEVs (P

The diagnosis of Multiple Myeloma (MM) is made by demonstration clonal plasma cells in Bone Marrow (BM) aspiration/biopsy, in addition to assessing serum biochemical parameters, conducting radiological examinations, and considering the clinical presentation. In most cases, predominantly mature plasma cells are observed, along with scattered immature forms in the BM. Several morphological variants of plasma cells have been reported, including Auer rod-like inclusions, small lymphocyte-like cells, hairy cell-like cells, anaplastic variants, promonocyte-like cells, crystal-storing histiocytes, Burkitt-like cells, and blastoid cells. In this series of five cases, most showed a typical clinical presentation and laboratory findings suggestive of plasma cell dyscrasia. However, the morphology of each case exhibited unusual morphological variants, posing diagnostic challenges. These variants included Auer rod-like inclusions, small lymphocytes/lymph-plasmacytoid cells, hairy-like cells, multilobulated nuclei, and anaplastic variants mimicking dysplastic megakaryocytes, leading to various differential diagnoses. The age range of these cases was 57-76 years. Most of the cases presented with generalised dull aching body pain. Imaging studies revealed lytic lesions involving various parts of the bone, including the skull, ribs, vertebrae, and femur. Biochemical assays suggested the possibility of plasma cell dyscrasia. Two of the cases had primary Plasma Cell Leukaemia (PCL), which is a rare and highly aggressive plasma cell neoplasm. The anaplastic variant is associated with a poor prognosis, aiding in predicting treatment responses. However, due to the unusual morphological presentation, the diagnosis of MM or PCL was made after conducting ancillary studies such as Serum Protein Electrophoresis (SPEP), serum free light chain assay, Immunofixation Electrophoresis (IFE), and immunophenotyping through Immunohistochemistry (IHC) or flow cytometry.

Background and objective: Chronic lymphocytic leukemia (CLL) is a clonal, mature B-cell neoplasm, its clinical features, cell morphology, and immunophenotyping are used to establish the diagnosis. Immunophenotyping by flow cytometry is the most accurate procedure for diagnosing CLL, there is no single marker exclusively expressed in CLL, however, a complex of immunophenotypic markers that incorporates several B-cell markers and assists in differentiating CLL from other mature B-cell neoplasms, the expression of these immunophenotypic markers is considered in the specific CLL scoring system (Moreue scoring system).this study aimed to assess the clinico – hematological features and immunophenotypic characteristics of CLL patients and to assess the role of immunophenotyping in the differential diagnosis of CLL. Methods: an observational prospective and retrospective study was carried out at Nanakaly hospital for blood diseases and oncology in Erbil city, Iraq. That was conducted on 100 patients newly diagnosed with mature B cell neoplasm which included 68 cases of CLL and 32 cases with other mature B cell neoplasm (MBN), usinga convenience sampling method. The study period was from the1st of September 2021 to the end of April 2022. An interview questionnaire was used to collect the study data from the patients. Results: The mean of age ± standard deviation of the CLL patients (58.85 ± 10.69) years and (66.2%) was male, The most frequent clinical presentations for CLL cases were fatigue (50%), lymphadenopathy (41.2%), and splenomegaly (41.2%), (50%) of CLL patients had anemia, thrombocytopenia was seen in (32.4%) and leukocytosis was seen (98.5%), all patients had lymphocytosis and (97%) had an absolute B lymphocyte count of more than 5 x109 /L. all the studied CLL patients expressed both CD45 and CD19 markers, the expression of the immunophenotypic markers was as following: CD5 (98.5%), CD23 (97.1%) and CD200 (97.1%), Half of the patients (50%) showed expression of monoclonal lambda light chain, sIgM (4.4%), CD20 (88.2%) and CD43 (54.4%) and CD38 (26.5%). The expression of the following marker had a significant role in the differentiation between CLL and other MBN: CD5, CD23, CD79b, FMC7, sIgM, CD200, and CD43. Conclusion: Our results have shown that the clinical presentations and hematological profile of Iraqi CLL patients were not significantly different from that of previous local and global studies, the distinct immunophenotyping, as well as it has been found that immunophenotyping is a promising method for supporting the clinical and morphological characterization of CLL.

Scientific relevance. The number of live bacteria is a quality parameter controlled at all stages of live plague vaccine production. Currently, live microbial cell counting uses a bacteriological method. However, flow cytometry has the potential to increase analytical accuracy and reduce testing time.Aim. This study aimed at testing the applicability of flow cytometry to assessing the quality of live plague vaccines.Materials and methods. The study quantified live microbial cells in 5 experimental batches of live plague vaccine as part of their quality control using the bacteriological method according to the State Pharmacopoeia of the Russian Federation (FS.3.3.1.0022.15). Cytofluorometry of the samples used the SynaptoGreen fluorescent dye.Results. The study quantified live microbial cells in live plague vaccine samples using the bacteriological method and flow cytometry. The results obtained by the bacteriological method ranged from 27.8±2.2 to 56.5±3.1% with an average of 39.8±5.4%. The results obtained by flow cytometry ranged from 29.2±1.2 to 59.1±2.1% with an average of 41.7±5.5%. The statistical analysis showed no significant difference between the results of vaccine quality control by both methods, as well as a high coefficient of determination.Conclusions. The results show that flow cytometry is an appropriate method for the quantification of live microbial cells as part of the quality control of plague vaccines. Being quick, easy, and highly informative, flow cytometry is preferable to traditional methods.

ABSTRACT Objective To evaluate the in vitro and in vivo toxicities of polyethylene glycol-coated gold nanoparticles synthesized using a one-step process. Methods Gold nanoparticles were prepared via a co-precipitation method using polyethylene glycol, and the synthesis product was characterized. For the in vitro evaluation, a flow cytometry analysis with Annexin V and iodide propidium staining was used to assess cytotoxicity in MG-63 cells labeled with 10, 50, and 100µg/mL of nanoparticle concentration. For the in vivo evaluation, nanoparticles were administered intraperitoneally at a dose of 10mg/kg dose in 10-week-old mice. Toxicity was assessed 24 hours and 7 days after administration via histopathological analysis of various tissues, as well as through renal, hepatic, and hematopoietic evaluations. Results Synthesized nanoparticles exhibited different hydrodynamic sizes depending on the medium: 51.27±1.62nm in water and 268.12±28.45nm (0 hour) in culture medium. They demonstrated a maximum absorbance at 520nm and a zeta potential of -8.419mV. Cellular viability exceeded 90%, with less than 3% early apoptosis, 6% late apoptosis, and 1% necrosis across all labeling conditions, indicating minimal cytotoxicity differences. Histopathological analysis highlighted the accumulation of nanoparticles in the mesentery; however, no lesions or visible agglomeration was observed in the remaining tissues. Renal, hepatic, and hematopoietic analyses showed no significant differences at any time point. Conclusion Polyethylene glycol-coated gold nanoparticles exhibit extremely low toxicity and high biocompatibility, showing promise for future studies.

Background: The 2016 WHO classification of T cell acute lymphoblastic leukemia suggest that cases of pre and pro T cell Acute Lymphoblastic Leukaemia (T-ALL) now fall under Early T-cell precursor ALL (ETP-ALL) or Near ETP ALL. Accurate subtyping is essential as the subsets are characterized by distinct molecular profiles and identifying molecular aberrations allow patients to receive novel treatment agents. Aim and Objectives: To study the immunophenotypic characteristics in T-ALL cases and assess the diagnostic difficulties encountered in its subtyping. Material and Methods: Thirty-seven cases of T-ALL were analysed on flow cytometry using a 6/8 colour panel of monoclonal antibodies, including B cell, T-cell, myeloid and stem cell markers. The cases were categorized as ETP-ALL, near ETP-ALL, cortical T-ALL and medullary T-ALL. Clinical details were retrieved from patient case files. Results: Patient age ranged from 1 to 64 years. Male to female ratio was 2.1: 1 and 35.1% cases were documented to have mediastinal mass. Mean haemoglobin level was 8.6 g/dl, and median total WBC count was 118.3 × 10 /µl. Blast percentage ranged from 39-98%, with mean of 84.8%. On flow cytometry analysis, all cases expressed CD7 and cytoplasmic CD3. CALLA+ (CD10+) were 32.4% cases. The aberrant expression of B-cell marker (CD79a) was observed in 5.4% cases. Majority of the cases were classified as medullary T-ALL, constituting 37.8% cases. ETP ALL were 21.6% cases, 13.5% cases were near ETP-ALL and 24.3% cases were cortical T-ALL. One case could not be definitely assigned to a specific category. Conclusion: This study reflects the difficulties encountered in subtyping of T-ALL cases due to antigenic overlap and/or due to lack of an extended panel of secondary markers.

Abstract Objective Differentiation of immortalized Mesenchymal Stromal Cells (iMSCs) into PDGFRα-positive cells under controlled growth conditions has several vital implications in functional studies concerned with the pathogenesis of Diabetic Gastroparesis (DGP). A study published previously by our research group demonstrated the importance of these cells as a novel, in-vitro model for investigating the functional role of neuronal nitric oxide synthase. The currently available methods require fresh differentiation of PDGFRα-positive cells for each round of experimentation. This leads to longer delays, higher usage of reagents, and inconsistency in reproducibility of experiments frequently. We thus aimed to establish through validation that cryopreserving and maintaining the iMSC-derived PDGFRα-positive cells for functional investigations help us to overcome these challenges. Results We demonstrated for the first time that the differentiated PDGFRα-positive cells from iMSCs can be cryopreserved and thawed to be used as per the experimental requirements with prolonged preservation of their characteristics. We assessed the viability of differentiated PDGFRα-positive cells pre- and post-freezing with the subsequent validation of their functional features using flow cytometry, qRT-PCR, and western blotting. We have been successful in demonstrating for the first time that the cryopreservation of previously differentiated PDGFRα-positive cells can be used as a feasible and cost-effective model for experimental reproducibility in functional studies of Diabetes Gastroparesis. Graphical Abstract

Abstract Background Extracellular vesicles (EVs) originating from the central nervous system (CNS) can enter the blood stream and carry molecules characteristic of disease states. Therefore, circulating CNS-derived EVs have the potential to serve as liquid-biopsy markers for early diagnosis and follow-up of neurodegenerative diseases and brain tumors. Monitoring and profiling of CNS-derived EVs using multiparametric analysis would be a major advance for biomarker as well as basic research. Here, we explored the performance of a multiplex bead-based flow-cytometry assay (EV Neuro) for semi-quantitative detection of CNS-derived EVs in body fluids. Methods EVs were separated from culture of glioblastoma cell lines (LN18, LN229, NCH82) and primary human astrocytes and measured at different input amounts in the MACSPlex EV Kit Neuro, human. In addition, EVs were separated from blood samples of small cohorts of glioblastoma (GB), multiple sclerosis (MS) and Alzheimer’s disease patients as well as healthy controls (HC) and subjected to the EV Neuro assay. To determine statistically significant differences between relative marker signal intensities, an unpaired samples t-test or Wilcoxon rank sum test were computed. Data were subjected to tSNE, heatmap clustering, and correlation analysis to further explore the relationships between disease state and EV Neuro data. Results Glioblastoma cell lines and primary human astrocytes showed distinct EV profiles. Signal intensities were increasing with higher EV input. Data normalization improved identification of markers that deviate from a common profile. Overall, patient blood-derived EV marker profiles were constant, but individual EV populations were significantly increased in disease compared to healthy controls, e.g. CD36+EVs in glioblastoma and GALC+EVs in multiple sclerosis. tSNE and heatmap clustering analysis separated GB patients from HC, but not MS patients from HC. Correlation analysis revealed a potential association of CD107a+EVs with neurofilament levels in blood of MS patients and HC. Conclusions The semi-quantitative EV Neuro assay demonstrated its utility for EV profiling in complex samples. However, reliable statistical results in biomarker studies require large sample cohorts and high effect sizes. Nonetheless, this exploratory trial confirmed the feasibility of discovering EV-associated biomarkers and monitoring circulating EV profiles in CNS diseases using the EV Neuro assay. Video Abstract

Amaç: Bu çalışmanın amacı nörobiyoloji çalışmalarında sıkça kullanılmakta olan HT22 fare hipokampal hücre hattının nöronal farklılaşma modeli olarak kullanılabilirliğinin anlaşılması için standart bir farklandırma besiyerine farklı sürelerde verdiği apoptotik cevabın sınanmasıdır. Gereç ve Yöntemler: HT22 hücrelerinin ekilmesi ve farklandırma dışında kültüre edilmesi için HG-DMEM farklandırma için ise B27+ katkılı NB+ medyumu kullanılmıştır. Kontrol grubu da dâhil olmak üzere toplam 9 farklı grup standart olarak kültüre edilmiştir. Deney sonunda AnnexinV/PI işaretlemesiyle erken/geç apoptozis ve nekrozis oranları akım sitometrik olarak belirlenmiştir. Verilerin normal dağılıma uyup uymadığı Shapiro-Wilks testi ile sınandıktan sonra en uygun testle istatistiksel analiz gerçekleştirilmiştir. Bulgular: Gruplar arasındaki karşılaştırmada erken apoptozis (P

The case report presents a patient with chronic lymphocytic leukaemia that was diagnosed in 2006 in Rotterdam, the Netherlands. In September 2010, the patient was admitted to the Department of Haematology in Poland due to progression of the underlying disease. The clinical problem during treatment was the suspicion of Richter’s transformation into another, more aggressive non-Hodgkin lymphoma. The diagnosis was based on the peripheral blood immunophenotype. The patient was diagnosed with an immunoglobulin deficiency. Unfortunately, repeated examinations did not confirm the transformation hypothesis, despite the increasing symptoms. The patient was treated with various therapeutic regimens until May 2021, when an increased number of NK cells was diagnosed in the peripheral blood. NK-cell lymphoproliferative disease was finally diagnosed de novo. Nevertheless, it was found that the patient had active Epstein-Barr virus (EBV) and Cytomegalovirus (CMV) infection. The suspected NK-cell lymphoma/leukaemia was most likely a complication of the active EBV infection and severe immunodeficiency state.

Time-domain measurement of the fluorescence lifetime in flow cytometry offers a complementary method to traditional flow cytometry, especially for the analysis of cells and biomolecules. Short measurement times due to high throughputs (cells/s) make a suited detector mandatory for a sufficient detection of the fluorescence signal and therefore the application of this method. We summarize the requirements for a suited detector for time resolved flow cytometry (TRFC) and present a new developed SPAD array detector with 100 pixels and 340 ns time between measurement windows, allowing a high-throughput of up to 60,000 cells/s. The functionality of the detector is shown with the measurement of a 22.2 ns laser pulse, making it applicable for an improved TRFC.

Background Immunophenotyping and enumeration of plasma cells (PCs) by flow cytometry are deemed to be prognostically significant. However, PCs enumeration by flow cytometry is challenging owing to discrepancy with morphology and PCs loss during sample processing. Enumeration and differentiation of abnormal plasma cells (APCs) and normal plasma cells (NPCs) is difficult because abnormal antigen expression can be seen in subsets of NPCs. This is particularly true when a limited panel of antibodies are relied upon.

Clinical, pathological, and imaging evidence in multiple sclerosis (MS) shows that inflammation starts early and progresses with age. B cells play a central role in this process, contributing to cytokine production, defective regulatory functions, and abnormal immunoglobulin production, even in the central nervous system. Anti-CD20 (aCD20) therapies, which deplete CD20+ B cells, are largely used in the treatment of both relapsing remitting (RR) and progressive (PR) forms of MS. Although effective against MS symptoms and lesions detectable by magnetic resonance imaging, aCD20 therapies can reduce the immune response to COVID-19 vaccination. By using high-parameter flow cytometry, we examined the antigen-specific (Ag+) immune response six months post-third COVID-19 mRNA vaccination in MS patients with RR and PR forms on aCD20 therapy. Despite lower Ag+ B cell responses and lower levels of anti-SARS-CoV2, both total and neutralizing antibodies, RR and PR patients developed strong Ag+ T cell responses. We observed similar percentages and numbers of Ag+ CD4+ T cells and a high proportion of Ag+ CD8+ T cells, with slight differences in T cell phenotype and functionality; this, however, suggested the presence of differences in immune responses driven by age and disease severity.

Introduction The function of cytotoxic cells may be impacted by CD39 expression on CD8-positive T lymphocytes, leading to an imbalance in tumor immunity. Material and methods We analyzed the correlation between the expression of CD39 on CD8-positive T lymphocytes and clinical information in 203 patients with lung adenocarcinoma. Results The expression of CD39 on CD8-positive T lymphocytes was related to T stage, lymph node metastasis and distant metastasis. Furthermore, the CD39 expression reduced the secretion of antitumor cytokines, promoted PD-1 expression and decreased CD28 expression on CD8-positive T lymphocytes. Conclusions This research demonstrated the potential value of CD39 on CD8-positive T lymphocytes as a negative regulator in cancer immunotherapy.

Introduction:Blast percentage (BP) of bone marrow (BM) is crucial for diagnosis, classification, and prognosis in patients with myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML). Cytomorphology (CM) is considered the gold standard for BM blast counts. Immunohistochemistry (IHC) and flow cytometry immunophenotyping (FCI) methods are used as additional tools. This study aims to compare the overall accuracy of CM, FCI, and IHC in determining BM BP and to evaluate the accuracy of diagnostic and prognostic BP groups determined by these methods in MDS and AML cases.Methods:CM, CD34-IHC, and FCI were performed on BM biopsy and aspiration samples from MDS and AML patients diagnosed between 9/2019 and 6/2021. Based on BP, cases were divided into four groups:

Background and objective: Worldwide immunophenotyping by flow cytometry (FCM) in acute leukemia (AL) is the golden step in the diagnosis. It’s very common for acute leukemias to aberrantly express antigens or cluster of differentiation (CD) markers which are usually expressed in other lineages of the disease hence this study aimed at determining the prevalence of aberrancy in AL and to find out the frequency of each aberrant CD marker and their association with the clinic-hematological profile of the cases. Methods: Following history and clinical examination of enrolled patients, blood and/or bone marrow aspirate was drawn for morphological examination and immunophenotyping by FCM from 86 newly diagnosed acute leukemia cases then multiple steps procedure was applied followed by interpretation of the results. Results: The prevalence of aberrant phenotype was 46.5%. The proportional frequency of aberrant phenotype in acute myeloid leukemia (AML) was 41%, in B-acute lymphoblastic leukemia (B-ALL) was 48.8% and in T-acute lymphoblastic leukemia (T-ALL) was 66.6%. The commonest aberrant CD markers in AML were CD22 and CD2, in B-ALL were CD66c and CD13 while in T-ALL were CD13 and CD33. The aberrant phenotype harbored lower white blood cell (WBC) count and blast percentage in PB, also splenomegaly was more frequent in lymphoid positive (Ly+) AML and myeloid positive (My+) T-ALL while in B-ALL, splenomegaly was more frequent in myeloid negative (My-) B-ALL. Conclusion: Aberrant phenotype prevalence in our study sample was comparable to other studies, considerable frequency of aberrant markers is present in cases of AL and some variations exist regarding the clinical and hematological profile of the aberrant group.

Objective To explore the distribution of T lymphocyte subsets in peripheral blood of healthy adults from Chashan Town of Dongguan in Guangdong Province. Methods The venous whole blood samples were collected from 827 healthy adults. The percentage of CD3+ T cells， CD4+ T cells， CD8+ T cells， CD4+CD8+ double positive （DP） T cells， and regulatory T（Treg） cells and CD4+/CD8+ ratio were determined by flow cytometry. Results The percentage of CD3+ T cells， CD4+ T cells， CD8+ T cells， CD4+/CD8+ ratio， DP T cells and Treg cells were determined as 40.5%-75.2%， 35.4%-68.9%， 27.4%-61.8%， 0.6-2.4， 0.2%-2.7% and 2.4%-6.0%， respectively. Significant differences were observed in the distribution of lymphocyte subsets regarding age and gender （all P < 0.05）. Conclusion The distribution of T lymphocyte subsets in healthy adults from Chashan Town of Dongguan in Guangdong Province was successfully analyzed， providing valuable reference for clinicians in this region.

Introduction: Over the past few years, the introduction of chimeric antigen receptor (CAR) T-cell therapy by the FDA has shown remarkable success in treating various hematologic malignancies. However, the limited response and resistance observed in some patients have hindered its broader application.

Background: In today’s generation of targeted therapy, traditional reporting of acute leukemia by morphology using the FAB system of classification has limited uses due to a lack of standardization and use in risk stratification. Immunophenotyping by flow cytometry has gained popularity due to its influence on the treatment and prognosis of the disease. The present study evaluates the judicious application of flow cytometry as an adjunct to morphocytochemical assessment in all newly diagnosed cases of acute leukemia in Southern Odisha. Materials and Methods: A total of 61 cases were subjected to complete blood count analysis, peripheral smear examination, bone marrow study, cytochemistry, and those suspected of leukemia underwent flow cytometry analysis using BD FACS CANTO II as per manufacturer’s instructions. Results: Out of 61 cases of acute leukemia diagnosed, 35 were acute lymphoblastic leukemia, and 24 were acute myeloid leukemia. There was 96.7% correlation between diagnosis by cytomorphology and flow cytometry. Seven cases showed aberrant expression of various antigens. Two cases of acute leukemia on cytomorphology were diagnosed as hematogones and the other as reactive lymphocytosis on flow cytometry. Conclusion: Although morphologic FAB diagnosis is still widely used to classify acute leukemia, flow cytometry has more specificity and should be used as an adjunct to morphology. Along with subclassification of leukemia, flow cytometry can also detect the possibility of an associated genetic abnormality.

Despite their B cell origin, Reed-Berezovsky-Sternberg tumor cells (RBS) in classic Hodgkin's lymphoma (cHL) demonstrate an absolutely unique phenotype. Immunohistochemistry of RBS cells is positive for CD15 antigen in most of cases, CD30, PAX-5; they do not express the T cell antigen CD3, В cell CD19, and in most cases are negative for the B cell antigen CD20, as well as for common leukocyte antigen CD45. Taking into account such unequivocal immunophenotype, RBS cells can be identified by multiparameter flow cytometry. Thus, J.R. Fromm et al. (2006, 2014) have convincingly shown the possibility to identify RBS cells in a puncture and/or biopsy sample of lymphatic nodes in cHL and were of the fair opinion that such rather simple and reproducible technique as flow cytometry could be an additional diagnostic instrument in cHL. We have tested the technique proposed by J.R. Fromm et al. for the assessment of lymphatic node involvement in cHL and used 8 to 10-parameter flow cytometry for detection RBS cells in cHL in 8 biopsy samples of a lymphatic node, and confirmed the feasibility to identify RBS cells by high performance flow cytometry. We also performed morphological and immunohistochemical assessment of the biopsy samples of lymphatic nodes from patients with suspected cHL. The study included clinical cases with immunohistochemically confirmed cHL (n = 8), and the control samples were from those with other diagnoses than Hodgkin's lymphoma. In all cases of cHL we found RBS cells. In future we plan to analyze larger case samples by flow cytometry.

Aims. This study compared the results obtained by basic immunophenotyping of cerebrospinal fluid (CSF) cells by flow cytometry (FC) to the results of conventional cytology and evaluated the possibility of detailed analyses of CSF B-cell subpopulations. Methods. Samples from 42 patients were examined by conventional cytology (native and/or pre-centrifuged CSF) and FC. The results from 15 patients without evidence of organic neurological disease were used to estimate reference ranges. Results. Pre-centrifugated CSF had significantly higher cell yield on the cytologic slide, but cell subpopulation percentages were altered; the percentage of lymphocytes was significantly higher and monocytes significantly lower compared to both native CSF slides and FC. The percentage of granulocytes was higher in FC compared to cytology. For leukocyte count, the following reference ranges were estimated for Fuchs-Rosenthal chamber (FR) counting and FC, respectively: leukocytes ≤4.7/μL and ≤2.5/μL, lymphocytes ≤4.1/μL and ≤1.8/μL, monocytes ≤1.2/μL and ≤0.9/μL, and granulocytes 0/μL and ≤0.2/μL. The following reference ranges were estimated for basic subpopulations: T-lymphocytes 84.1-100%, B lymphocytes 0.0-1.5%, NK cells 0.0-6.3%, NKT cells 0-9.5%, and CD3+CD4+/CD3+CD8+ 0.8-4.9. Using a volume of 1.2-2.4 mL, the number of B lymphocytes was too low (

Objective B-lymphocyte progenitors, namely the hematogones (HGs), may pose problems in morphological assessment of bone marrow, not only during the diagnostic workup but also while evaluating bone marrow for remission status following chemotherapy. Here, we describe a series of 12 cases of acute lymphoblastic leukemia (ALL) that included both B-ALL and T-ALL cases, which were evaluated for remission status and revealed blast-like mononuclear cells in bone marrow in the range of 6 to 26%, which on immunophenotypic analysis turned out to be HGs. Materials and Methods This is a case series of 12 ALL cases who were undergoing treatment at the Army Hospital (Referral and Research), New Delhi. All these cases were under workup for post-induction status (day 28) and to check for suspected ALL relapse. Bone marrow aspirate (BMA), biopsy, and immunophenotyping were performed. Multicolored flow cytometry was performed using CD10, CD20, CD22, CD34, CD19, and CD38 antibodies panel. Results BMA assessment of 12 cases revealed a maximum of 26% blastoid cells and a minimum of up to 6%, raising the suspicion of hematological relapse. However, on clinical assessment, these patients were well preserved, with preserved peripheral counts. Hence, marrow aspirates were subjected to flow cytometry using the CD markers panel, as discussed above, which revealed HGs. These cases were followed by minimal residual disease (MRD) analysis that revealed MRD-negative status, further confirming our findings. Conclusion This case series highlights the importance of morphology and bone marrow immunophenotyping in unveiling the diagnostic dilemma in post-induction ALL patients.

Objective Sepsis is a major global health issue due to its high death and morbidity rates. To avoid the negative effects of sepsis and decrease mortality, it is vital to diagnose and treat it as soon as possible. Blood cultures can take up to 2 days to give result, and they are not always reliable. According to recent studies, neutrophil CD64 expression might be a sensitive and specific option for assessing sepsis. This study aimed to evaluate the diagnostic performance of a flow cytometry analysis for the expression of neutrophil CD64 in sepsis and its comparison with other standard tests in a tertiary care center. Materials and Methods Prospective analysis on 40 blood samples from suspected sepsis patients admitted to intensive care units with criteria for the systemic inflammatory response syndrome on presentation was performed for expression of neutrophil CD64, C-reactive protein, procalcitonin, and complete blood count. Ten healthy volunteers were also enrolled in this prospective study. The laboratory results were compared in different groups. Results The neutrophil CD64 had the highest diagnostic value to differentiate between patients of sepsis and nonsepsis groups with a sensitivity of 100% (95% confidence interval [CI]: 77.19–100%) and 100% (95% CI: 55.32–86.83%); specificity of 90.00% (95% CI: 59.58–99.49%) and 87.24% (95% CI: 66.69–99.61%); and likelihood ratio of 10.00 and 7.84, respectively. Conclusion The neutrophil CD64 expression provides a more sensitive, specific, and novel marker for the early detection of sepsis in critically ill patients.

Objective To compare the performance of conventional and spectral flow cytometers in detecting auto-fluorescence samples. Methods Single cell samples with auto-fluorescence were prepared and stained by annexin V(AV)binding and propidium iodure (PI) to detect apoptosis. The same auto-fluorescence sample was run on conventional and spectral flow cytometers side by side. Results By avoid using FITC in conventional cytometers, the better result was gotten in detecting auto-fluorescence samples. Full spectrum cytometry presented better resolution even there was spectral overlap. It was easy to discriminate the signals derived from auto-fluorescence and FITC. The protocol of annexinV binding and PI staining to analyze apoptosis was optimized. Conclusions Spectral flow cytometry shows relatively excellent performance under certain conditions in detecting of apoptosis derived from auto-fluorescence samples.

Abstract Background Nivolumab is an anti-PD1 antibody that has dramatically improved metastatic melanoma patients’ outcomes. Nevertheless, many patients are resistant to PD-1 inhibition, occasionally experiencing severe off-target immune toxicity. In addition, no robust and reproducible biomarkers have yet been validated to identify the correct selection of patients who will benefit from anti-PD-1 treatment avoiding unwanted side effects. However, the strength of CD26 expression on CD4+ T lymphocytes permits the characterization of three subtypes with variable degrees of responsiveness to tumors, suggesting that the presence of CD26-expressing T cells in patients might be a marker of responsiveness to PD-1-based therapies. Methods The frequency distribution of peripheral blood CD26-expressing cells was investigated employing multi-parametric flow cytometry in 69 metastatic melanoma patients along with clinical characteristics and blood count parameters at baseline (W0) and compared to 20 age- and sex-matched healthy controls. Percentages of baseline CD4+CD26high T cells were correlated with the outcome after nivolumab treatment. In addition, the frequency of CD4+CD26high T cells at W0 was compared with those obtained after 12 weeks (W1) of therapy in a sub-cohort of 33 patients. Results Circulating CD4+CD26high T cells were significantly reduced in melanoma patients compared to healthy subjects (p = 0.001). In addition, a significant association was observed between a low baseline percentage of CD4+CD26high T cells (

Background The diagnostic gold standard for myelodysplastic syndrome (MDS) is based on bone marrow morphology, progenitor cell count and cytogenetics. But the dysplasia is not specific for MDS. In the context of poor quality of bone marrow smear, very mildmorbid hematopoiesis, insignificant increase in blasts, especially a normal karyotype, the diagnosis of MDS is often challenging. Multiparameter flow cytometry has become a key tool for the diagnosis of MDS. Some studies have shown that integrated flow cytometric score (iFS) is the best scoring system for MDS diagnosis, but its use has not been reported in Chinese population. Objective To assess diagnostic and prognostic values of iFS in MDS to demine whether it is an appropriate flow cytometry scoring system for clinical diagnosis and prognosise valuation of MDS. Methods The immunophenotype data of 83 patients with MDS and 77 patients with non-MDS treated in Department of Hematology, the Affiliated People's Hospital of Ningbo University between January 2019 and April 2022 were analyzed retrospectively. Low-grade MDS was defined as less than 5% blasts in bone marrow, including MDS with single lineage dysplasia, MDS with multilineage dysplasia, MDS with ring sideroblasts, MDS, unclassifiable, and MDS with isolated del (5q). The results of chromosome karyotype and bone marrow morphology of MDS patients were collected. The probability of phenotypeabnormalities in each cell lineinterpreted according to the iFS was compared between MDS and non-MDS patients. ROC analysis was conducted to assess the performance of iFS and Ogata score in diagnosing MDS, with sensitivity, specificity and the area under the ROC curve (AUC) calculated. In the MDS group, the Spearman's rank correlation was adopted to analyze the correlation of iFS grade with cytogenetic risk category and Revised International Prognostic Scoring System (IPSS-R). The event-free survival curve was plotted by the Kaplan-Meier method and compared between MDS patients diagnosed by the iFS and the other MDS patients by Log-rank test. Results MDS group had higher prevalence of phenotype abnormalities of myeloid progenitors (71.1% vs 1.3%), granulocytes/monocytes (73.5% vs 18.8%) and erythrocytes (60.2% vs 14.2%) than non-MDS group (P