cytometry

摘要： The aim of this study was to find a way to efficiently separate neuronal cells from the cerebral cortex of adult rats,providing a reference method for rapid acquisition of neuronal cells from the adult rat brain.Fifteen SD rats were randomly divided into three groups,with five SD rats in each group.Then,neuron cells were isolated from the adult rat cerebral cortex by the grinding method,the trypsin method,and the collagenase II method,respectively.The expression of anti-NeuN in the neurons of each group was analyzed by flow cytometry.The acquisition rates and morphology of neurons of each group were observed by immunofluorescence staining.The grinding or collagenase II method is more suitable for rapid acquisition of neuronal cells from an adult rat’s cerebral cortex.The number of neuron cells obtained by the trypsin method were very few,so it is not convenient for later experiments.

摘要： Myelodysplastic syndromes(MDS)are highly heterogeneous myeloid neoplasms,and a large number of patients are difficult to diagnose and classify by blood and bone marrow examination.As a surface marker of granulocyte,studies have shown CD10 can be used to define the degree of granulocyte maturation in MDS patients.However,whether it can be used for differential diagnosis of MDS and other hematological diseases remains inconclusive.To explore the value of CD10 for differential diagnosis of MDS,60 newly diagnosed MDS,20 aplastic anemia(AA)patients,and 35 iron-deficient anemia(IDA)patients were selected for this study.Bone marrow(BM)specimens were processed for surface marker analysis and labeled with pre-conjugated monoclonal antibodies.Stained cells were detected by flow cytometry.Our results indicated that CD10-positive granulocytes were significantly decreased in BM of MDS patients than AA and IDA patients,and the level of CD10-positive mature granulocytes was not associated with the clinical stages of malignancy.Receiver operating characteristic(ROC)areas under the curve(AUC)of CD10-positive granulocytes was 0.86 and 0.85,respectively,in MDS patients than the IDA group and AA group with good specificity and sensitivity.Further,CD10-positive granulocytes were increased after effective treatment.In conclusion,we found the decrease in CD10-positive granulocytes has a differential diagnostic value of MDS.

摘要： Cyanobacterial harmful algal blooms (CHAB), caused by eutrophication, are known to threaten both wildlife and human health. Due to urbanization and land use changes, an increase of CHAB’s at a more frequent rate within Barnegat Bay has been observed. In order to detect possible CHAB causing cyanobacteria, water samples were collected from 12 different locations within Barnegat Bay. Each sample was filtered through a 30- and 0.4-μm polycarbonate filter sequentially. Flow cytometry was carried out for the filtrate collected between 0.4- and 30-μm. Chelex DNA extraction, polymerase chain reaction (PCR), and gel electrophoresis were then performed for all sites using four primer sets (27F/785R, PSF/UR, CYA359F/CYA781R and MSF/MSR) designed to detect cyanobacteria. Flow cytometric results indicated the water samples contained a wide range of cyanobacteria, including M. aeruginosa, Cylindrospermum spp., Anabaena spp., and Synechococcus sp. IU 625 ranges from 3.16 to 8.17 × 107 cells·L-1. PCR-based assays suggest that general cyanobacteria as well as phytospecific species were present for all sites, but no toxin-producing Microcystis aeruginosa was detected. Plaque assays demonstrated the presence of cyanophages for S. IU 625, Anabaena spp., and M. aeruginosa at all sites, up to 105 PFU·ml-1.

摘要： Background: Mature red blood cells lack protein synthesis and are unable to restore inactivated enzymes, damaged cytoskeleton and membrane proteins. An oxidation breakdown of band 3 is probably part of the mechanism leading to the generation of a senescent cell antigen. This specific signal serves for the clearance of RBCs by inducing the binding of autologous IgG and C3, leading to phagocytosis. In addition, phosphatidilserin molecules appear in the outer membrane and the CD47 expression diminishes. Methods: Erythrocytes of different ages from whole blood were studied by flow cytometry analysing light scatter proprieties, binding of autologous IgG, C3 complement deposits, externalization of phosphatidylserine and CD47 expression. Dot-plot analysis based on forward scatter versus side scatter parameters showed two RBCs populations of different sizes and density. RBCs were further incubated with Alexa 488 IgG, APC-anti-C3, PE-annexin-V and PE-CD47. The comparison of the values obtained for the different variables studied in SeRBC and YRBC populations was carried out by the Student t-test for matched samples or by the Wilcoxon test (after verification of the normality assumption). Results: The percentage of IgG and C3 positive cells was significantly higher in senescent red blood cells population. The fraction of annexin-V positive RBCs was also larger in SeRBCs while the CD47 expression was lower in this population. Conclusions: These results indicate that flow cytometry allow differenciation of erythrocytes populations of different ages, turning this tool into an useful alternative option to study erythrocyte aging process. These findings will contribute to a better understanding of the process and mechanisms involved in erythrocyte senescence process.

摘要： Circulating tumor cells are cells that detach from the primary tumor site and migrate to the bone marrow or other tissues where they can initiate a metastatic site. Liquid biopsies are an emerging tool in the past decades that enables us to detect Circulating Tumor Cells in patients’ blood. Flow cytometry is a powerful tool used in liquid biopsy diagnostics. This aims to prove the sensitivity and specificity of a flow cytometric panel for the detection of CTCs in breast cancer patients using healthy individuals’ samples as controls. The study was blinded to the data analyzing researcher. Statistical analysis followed and results show 86.9% area under the curve which indicates that the particular method can be very promising for diagnosing breast cancer.

摘要： Colorectal cancer(CRC) is a heterogeneous disease, with a diverse and plastic immune cell infiltrate. These immune cells play an important role in regulating tumour growth-progression or elimination. Some populations of cells have a strong correlation with disease-free survival, making them useful prognostic markers. In particular, the infiltrate of CD3^+ and CD8^+ T cells into CRC tumours has been validated worldwide as a valuable indicator of patient prognosis. However, the heterogeneity of the immune response, both between patients with tumours of different molecular subtypes, and within the tumour itself, necessitates the use of multiparametric analysis in the investigation of tumour-specific immune responses. This review will outline the multiparametric analysis techniques that have been developed and applied to studying the role of immune cells in the tumour, with a focus on colorectal cancer. Because much of the data in this disease relates to T cell subsets and heterogeneity, we have used T cell populations as examples throughout. Flow and mass cytometry give a detailed representation of the cells within the tumour in a single-cell suspension on a per-cell basis. Imaging technologies, such as imaging mass cytometry, are used to investigate increasing numbers of markers whilst retaining the spatial and structural information of the tumour section and the infiltrating immune cells. Together, the analyses of multiple immune parameters can provide valuable information to guide clinical decision-making in CRC.

摘要： Renal transplantation remains the best option for patients suffering from end stage renal disease(ESRD).Given the worldwide shortage of organs and growing population of patients with ESRD,those waitlisted for a transplant is ever expanding.Contemporary crossmatch methods and human leukocyte antigen(HLA) typing play a pivotal role in improving organ allocation and afford better matches to recipients.Understanding crossmatch as well as HLA typing for renal transplantation and applying it in clinical practice is the key step to achieve a successful outcome.Interpretation of crossmatch results can be quite challenging where clinicians have not had formal training in applied transplant immunology.This review aims to provide a worked example using a clinical vignette.Furthermore,each technique is discussed in detail with its pros and cons.The index case is that of a young male with ESRD secondary to Lupus nephritis.He is offered a deceased donor kidney with a 1-0-0 mismatch.His complement dependent cytotoxicity(CDC) crossmatch reported positive for B lymphocyte,but flow cytometry crossmatch(FCXM) was reported negative for both B and T lymphocytes.Luminex-SAB(single antigen bead) did not identify any donor specific antibodies(DSA).He never had a blood transfusion.The positive CDCcrossmatch result is not concordant with DSA status.These implausible results are due to underlying lupus erythematosus,leading to false-positive B-lymphocyte crossmatch as a result of binding immune complexes to Fc-receptors.False positive report of CDC crossmatch can be caused by the underlying autoimmune diseases such as lupus erythematosus,that may lead to inadvertent refusal of adequate kidney grafts.Detailed study of DSA by molecular technique would prevent wrong exclusion of such donors.Based on these investigations this patient is deemed to have "standard immunological risk" for renal transplantation.

摘要： Acute-on-chronic liver failure (Acute-on-chronic liver failure, ACLF) is acute liver function decompensation on the basis of chronic liver disease. The progression of ACLF develops from advanced phase, plateau phase to remission phase. The pathophysiological basis of ACLF in different phases is various. In advanced phase, immune imbalance and systemic inflammatory reaction plays key roles. In this study, we try to assess the association between expression of NK cells and its receptors and prognosis of patients with ACLF in advanced phase. A total of 35 inpatients with HBV acute-on-chronic liver failure in advanced phase were recruited. They were divided into case group (n = 18) and control group (n = 17) according to whether the patients was dead in the 12 weeks. PBMC were detected for the frequency and expression of NK cell receptors by flow cytometric analysis. Our results demonstrated that patients who died had lower expression of NK cells and inhibitory receptor KIR3DL1, higher levels of FASL. During 12-week follow-up in those case alive, we found that NK cells increased, while expression of FASL decreased. High short-term mortality of ALCF was associated with NK cell, especially related to KIR3DL1 and FASL (PNK = 0.036, PKIR3DL1 = 0.0265, PFasL = 0.0008).

摘要： Objective: There are two monocyte populations in human blood: CD14+CD16- classical monocytes and CD14+CD16+ inflammatory monocytes. CD14+CD16+ inflammatory monocytes, account for approximately 10% of the total monocytes, may be expanded in various types of inflammatory conditions. The purpose of this study was to investigate whether the expansion of the CD14+CD16+ monocyte population represents a risk factor of aseptic loosening (AL). Methods: Peripheral monocytes subsets were measured in revision patients with AL (n = 35) and in patients with stable implants (SI, n = 56). The gene profiles of TNFα, IL-1β, CD16, CD68 and TRAP5B from collected loosening periprosthetic tissues were analyzed. Results: There were no significant differences in the CD14+CD16+ monocyte populations between the SI and AL patients. The CD14+CD16+ monocytes were marginally higher in revision patients with osteolysis (n = 30), compared to patients without osteolysis (n = 5) though no statistically difference was found. There was an association between the CD14+CD16+ monocyte subpopulation and the tissue gene profiles, including IL-1β (p = 0.063), CD68 (p = 0.036), and TRAP5B (p = 0.073). Conclusion: It was demonstrated that the expansion of CD14+CD16+ monocytes reflects, to some extent, the inflammatory status of the loosening periprosthetic tissues. It is unclear if some of those SI patients (no pain and negative radiograph) who have a higher frequency of CD14+CD16+ monocytes may be at the early stage of AL. Further evaluation of CD14+CD16+ monocyte population, independently or combined with other factors, will be useful to design a risk profile for AL incidence and progression.

摘要： Monosomic lines of Nicotiana tabacum are helpful to confirm the location of genes on specific chromosomes. In the cross N. nudicaulis and N. tabacum, hybrid seedlings express lethal symptoms, which are controlled by the S subgenome of N. tabacum. To identify the responsible chromosome, we needed to produce chromosome lacking lines (CLLs) of N. tabacum L. “Red Russian” and use them to cross with N. nudicaulis. From a cross of (N. tabacum × N. tomentosiformis) × N. tabacum, 380 BC1 individuals were obtained. Using a Haplo-Q line (a monosomic line lacking the single linkage group 11) and N. tabacum, we found that qPCR is a simple and reliable screening method for CLLs of N. tabacum. The marker PT30342 is located on linkage group 11, and the -Ct value (Ct Actin - Ct PT30342) was 2.0 for a disomic line and was 1.097 for a Haplo-Q line. By the use of flow cytometry, qPCR and chromosome counting together as a screening method, we identified 6 CLLs lacking 2 to 6 chromosomes. Compared with conventional methods, our method is a rapid technique for making and screening CLLs ofthe S or S/T subgenome of N. tabacum. Further, these CLLs will be useful to identify the location of two or more factors on chromosomes controlling a variety of genetic problems affecting breeding. Here, we only made CLLs of the S or S/T subgenome of N. tabacum. We will use the method established in this study to produce CLLs of the T subgenome of N. tabacum, and gather a full set of CLLs of N. tabacum. qPCR could also be applied to the identification of chromosome aberrations in other plants.