Accurate characterization of nanoparticles is of paramount importance for advancing our understanding of life sciences, improving disease diagnosis and treatment, and fostering the development of nanotechnology. Due to the high degree of individual variation and heterogeneity among nanoparticles, there is an urgent need for the development of rapid detection techniques at the single-particle level. This review article introduces the development of nano-flow cytometry (nFCM), a technology based on Rayleigh scattering and sheath-flow single-molecule fluorescence detection. nFCM can achieve highly sensitive, selective, and high-throughput multi-parameter analysis of the size distribution, particle concentration, and biochemical characteristics of synthetic nanoparticles (7 ~ 500 nm), as well as naturally occurring biological nanoparticles such as extracellular vesicles and viruses, at a rate of up to 10 000 particles per minute. The article discusses the significance, challenges, progress, and industrial transformation of the nFCM development, reviews its applications in nanoparticle research, and discusses its future application prospects.
Flow cytometry (FCM) is a widely used single-cell analysis technique in biological research due to its ability to simultaneously and quantitatively analyze and sort cells or biological particles with multiple parameters in a fast linear flow state. Conventional FCM based on fluorescence has been developed for over 50 years and is a mature technology. However, it requires invasive blood sampling, which disrupts the organism’s physiological environment and limits real-time detection. The in vivo flow cytometry (IVFC) is an emerging technology that enables non-invasive detection of cell samples within living organisms. Through the principles of fluorescence excitation, photoacoustic effect, and photothermal effect, various IVFC techniques have been developed. The basic principles and development of different types of IVFC techniques are summarized and compared with the basic parameters, and the research findings on circulating tumor cells and the current research results of novel label-free assays are reviewed with the aim to show the current development trends and application prospects of IVFC.
Human immunodeficiency virus (HIV)-associated diffuse large B-cell lymphoma (DLBCL) is a rare disease with high malignancy, complex treatment, poor prognosis, and high mortality rate. Currently, the international prognostic index (IPI) is widely used to assess the prognosis of DLBCL patients. However, due to the uniqueness of HIV infection, HIV-associated DLBCL patients have different clinical characteristics compared to HIV-negative DLBCL patients, and the IPI cannot accurately distinguish HIV-associated DLBCL patients with high risks and poor prognosis. In response to the challenges faced in the prognosis evaluation of HIV-associated DLBCL patients, this review summarizes the research progress of biomarkers for prognosis evaluation of HIV-associated DLBCL patients, providing reference for improving the accuracy of prognosis evaluation of patients with HIV-associated DLBCL.
Copy number variations (CNVs) are genomic rearrangements characterized by the increase or decrease in copy numbers of genomic segments that are typically longer than a few thousand base pairs. They are submicroscopic deletions and duplications, and segmental duplications. CNVs are associated with various diseases such as human birth defects, developmental disorders, and cancer, representing one of the significant mechanisms underlying these conditions. A comprehensive understanding of CNVs in the genome is essential for better comprehension of the relationship between genes and diseases, genetic-environmental interactions, and the correlation between genomic variations and species evolution. Therefore, research on diseases related to CNVs has emerged as a critical area in medical genetics. This review summarizes the molecular mechanisms, detection methods, and recent advances in the pathogenicity of submicroscopic CNVs.
Objective: To investigate the immunophenotypic characteristics of nodal follicular helper T-cell lymphoma, angioimmunoblastic-type (nTFHL-A), and to assess the diagnostic value of flow cytometry in nTFHL-A. Methods: A total of 227 patients were included in our study. They were initially diagnosed with nTFHL-A in the Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, China, from January 2010 to August 2023. Results: The lack and decreased expression of surface CD3 and the CD7 deficiency were the most common immunophenotypeic abnormalities of pan-T cell markers in nTFHL-A, and CD10 expression was the most specific for nTFHL-A. Almost all neoplastic T cells had bright PD-1 expression and CD45RO expression. Flow cytometry may present false negative results in patients with CD3-positive or slightly decreased expression and meantime with CD7-positive or partial expression (tissue 11.3%, bone marrow 10.3%). Bright PD-1 expression can improve nTFHL-A detection and also help to differentiate it from other peripheral T-cell lymphomas. Conclusions: Flow cytometry is an effective tool to screen, identify or differentiate nTFHL-A.
Objective: To investigate the clinical and laboratory features of chronic myelogenous leukemia with marked thrombocytosis (CML-T), and analyze the key points of differentiation from essential thrombocythemia (ET). Methods: By reviewing 15 patients with CML-T admitted to our hospital from January 2013 to January 2023 and 15 randomly selected patients with ET during the same period, we analyzed and compared their clinical manifestations and related laboratory results. Results: The CML-T group presented significantly elevated platelet counts up to 2 387 × 109/L. Meanwhile, the BCR∷ABL1 fusion gene was detected in all CML-T patients, and all of them were of the p210 type. Compared with the ET group, the CML-T group had a higher white blood cell (WBC) count, basophil percentage and mean platelet volume (MPV), as well as a higher prevalence of immature granulocytes in the PB smear, with all P values < 0.05. On bone marrow aspiration examination, the CML-T group presented a higher degree of hyperplasia, a higher myeloid-erythroid ratio, a higher number of megakaryocytes, a smaller mean diameter of megakaryocytes, and a more severe myelofibrosis grading, with all P values <0.05. Conclusions: CML-T has an insidious onset, with only elevated PLT count, normal or mildly elevated WBC count, occasional immature granulocytes in the PB, and rare splenomegaly, making it easy to be misdiagnosed as ET. When the PLT count is significantly higher with an increased percentage of basophils, increased MPV and the presence of immature granulocytes in PB, CML-T should be screened and BCR∷ABL1 fusion gene detection should be suggested in time. In addition, the degree of bone marrow hypercellularity, high myeloid-erythroid ratio, increased megakaryocyte count, and characteristic small megakaryocyte morphology can provide important diagnostic clues to help avoid misdiagnosis and achieve early diagnosis and treatment.
Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of lymphomas derived from neoplastic transformation of mature T lymphocytes. The value of CD3-/dimCD4+ abnormal cell population detected in the bone marrow and peripheral blood is unclear in the diagnosis of PTCL. In this study, 67 patients with CD3-/dimCD4+ abnormal T-cell populations were grouped according to the final pathological and clinical diagnosis. The patients' data including peripheral blood count, biochemistry indices, immunoglobulin, inflammatory factors, and residual normal T-cell subpopulations were used to explore whether this abnormal lymphocyte population is significant for the identification of various types of PTCL. The results indicate that CD3-/dimCD4+ cells found in the bone marrow are associated with T cell disorders, and the angioimmunoblastic T-cell lymphoma (AITL) is the most common type of PTCL. However, the immunophenotyping and laboratory indices of different PTCL groups overlap each other, which should be carefully differentially diagnosed by combining the clinical features and pathological results.
Objective: To investigate the expression of CD27 antigen in myeloma cells of multiple myeloma (MM) patients and its clinical diagnostic value. An analysis of the correlation between CD27 expression and cytogenetic abnormality, as well as the immune cell population will be provided. Methods: Immunophenotypes of myeloma cells in 124 MM patients were detected to analyze the expression of CD27 on malignant plasma cells, and the expression of T subsets, B subsets and Th1/Th2/Th17 subsets in peripheral blood. The presence of cytogenetic abnormalities, such as t (14; 16), p53 deletion, del (13q14.3), IGH rearrangement, t (4; 14), t (11; 14) and del (13q14) were analyzed. Clinical data of 124 patients were collected to compare whether there were significant differences between CD27+MM patients and CD27-MM patients. A total of 57 patients who underwent 4 courses of chemotherapy in our hospital were screened for efficacy evaluation, and to find the difference in efficacy evaluation between CD27+MM patients and CD27-MM patients. All patients who received treatment in our hospital were followed up for survival analysis. Results: The CD27+ rate of abnormal plasma cells was 29.03% (36/124) in 124 MM patients. By analyzing the clinical baseline data of CD27+MM and CD27-MM patients, statistically significant differences were found in lymphocyte count, serum creatinine, lactate dehydrogenase, and β2-microglobulin levels between two groups. Meanwhile, the cytogenetic and FISH results showed that the incidence of IGH rearrangement was lower in CD27+MM patients (31.58%) than in CD27-MM patients (53.41%). The R-ISS stratification in CD27+MM patients was lower than that in CD27-MM patients. The levels of CD19+B cells in CD27+MM patients were lower than those in CD27-MM patients, while the levels of TNF-α in Th1/Th2/Th17 subsets and the CD3+CD4+ T cell subsets in CD27+MM patients were higher than those in CD27-MM patients. After 4 courses of chemotherapy, the CR+VGPR rate was higher in CD27+MM patients than in CD27-MM patients. Conclusions: The low expression of CD27 on malignant plasma cells in MM patients may be related to the disease progression and poor prognosis.
Objective: To investigate the clinical significance of the expression level of programmed death molecule 1 ligand 1 (PD-L1) in the occurrence, transformation, and treatment of acute myeloid leukemia (AML). Methods: First, using GEO software and GEO2R calculation method, with P<0.05 and | logFC |>1 as conditions, we analyzed the expression of common immune monitoring points in public databases. The data were analyzed using TCGA-LAML queue data and survival R packets for high expression PD-L1 patients. Second, we selected newly diagnosed AML patients in our department from October 2017 to March 2023 to verify our hypothesis. Flow cytometry was used to detect the expression levels of PD-L1 in the bone marrow T lymphocyte programmed cell death receptor 1 (PD-1) and CD34+progenitor cells before treatment. The correlation between PD-1 and PD-L1 expression levels and patient clinical characteristics, efficacy, and survival was analyzed. Results: The expression of lymphocyte activation gene 3 (LAG3), PD-1, PD-L1, and T cell immunoglobulin domain and mucin domain-3 (TIM3) at immune checkpoint in the initially diagnosed AML patients was higher than that in normal individuals (P<0.05), and the survival rate was higher in the group with low PD-L1 expression at initial diagnosis. Recurrent and refractory AML and secondary AML have higher levels of PD-L1 expression at initial diagnosis. The high expression of PD-L1 in newly diagnosed AML patients tends to have complex chromosomal karyotypes, poorer prognosis, and shorter survival. Conclusions: The PD-1/PD-L1 signaling pathway may be involved in the immune escape mechanism of myeloid tumor cells, and its expression level is positively correlated with the malignancy and progression of the disease, while negatively correlated with treatment efficacy and survival.
Objective: To explore the clinical significance of flow cytometry (FCM) immunophenotype analysis in the diagnosis and treatment of multiple myeloma (MM). Methods: A total of 117 newly diagnosed MM patients admitted to the Hematology Department of Ningxia Medical University General Hospital from January 2019 to June 2023 were selected for immunophenotypic analysis using flow cytometry with polychromatic fluorescent labeled antibodies. At the same time, bone marrow cell morphology, immunofixation electrophoresis (IFE), and quantitative immunoglobulin detection results were collected and analyzed. Results: 117 MM patients underwent gate analysis using CD45/SSC and CD45/CD38 dot maps. Abnormal cell populations were observed in areas with CD45 negative or weakly positive, CD38 strongly positive, and SSC larger than those with nuclear red blood cells, accounting for approximately 0.1% to 91.5% of nuclear cells. CD38 was strongly expressed in all 117 cases, CD138 in 111 cases, CD56 in 78 cases, CD229 in 67 cases, CD27 in 48 cases, CD200 in 44 cases, CD117 in 40 cases, CD269 in 30 cases, CD28 in 25 cases, CD20 in 19 cases, CD81 in 9 cases, CD13 in 7 cases, and CD19 in 4 cases and the positive expression rates were 100%, 94.87%, 66.67%, 57.26%, 41.03%, 37.61%, 34.19%, 25.64%, 21.37%, 16.24%, 7.69%, 5.98%, and 3.42%, respectively. All 117 cases were restrictedly expressed with cytoplasmic light chains (100%), of which 64 cases were restrictedly expressed with cKappa light chains (54.70%) and 53 cases with cLambda light chains (45.30%). Morphological examination of bone marrow cells revealed myeloma cells in 111 cases (94.87%) under microscope, and M protein was detected in 114 cases using IFE, with a detection rate of 97.44%. Conclusions: FCM immunophenotyping can accurately, quickly, and objectively distinguish between benign and malignant plasma cells, reveal the clonal expression of cytoplasmic light chain and the expression characteristics of CD molecules in myeloma cells, and provide objective basis for the early diagnosis, classification, prognosis evaluation, and treatment of MM. It has good clinical application value.
Objective: This study intends to investigate the levels of bone marrow tumor necrosis factor-alpha (TNF-α) in adult acute B-lymphoblastic leukemia (B-ALL) patients and the role of TNF-α in the efficacy of chemotherapy and tumor recurrence. Methods: Peripheral blood and bone marrow plasma samples were prospectively collected from 107 patients with B-ALL (disease group), 25 healthy donors (healthy controls), and 77 patients with other types of hematologic neoplasms (disease controls). Plasma TNF-α concentration was determined by chemiluminescence immunoassay. Gal-9 expression in leukemia cells from remission and relapse patients was determined by flow cytometry and TNF-α was co-cultured in vitro with primary cells from the patients to detect the changes in the expression of Gal-9. Results: Bone marrow TNF-α levels were elevated in B-ALL patients and TNF-α levels correlate with whether or not they are in remission from the first chemotherapy treatment. Bone marrow TNF-α concentrations at the time of initial diagnosis were lower in the first-time chemotherapy complete remission patients compared to those in non-complete remission patients. The leukemia cell Gal-9 was significantly up-regulated in relapsed patients and TNF-α induced Gal-9 up-regulation in vitro. Conclusions: This study reveals that bone marrow and peripheral blood TNF-α levels are elevated in adult B-ALL patients and induce up-regulation of Gal-9 in leukemia cells to promote disease progression and tumor relapse.
Purpose: The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours defines new diagnostic criteria for chronic myelomonocytic leukemia (CMML) and myelodysplastic neoplasms (MDS). This study aims to explore the clinical and molecular characteristics of CMML and MDS patients belonging to these categories. Method: A thorough review of clinical records of 52 patients with increased peripheral blood absolute monocyte count (AMC) and bone marrow pathological hematopoiesis was performed and their diagnoses were revised according to the new criteria. These patients were compared with 48 non-AMC increased MDS (non MDS-Mo) patients. A retrospective analysis of clinical characteristics and prognostic differences of patients with different diagnoses was conducted. Results: Among 52 patients with increased AMC and pathological hematopoiesis, 35 cases were previously diagnosed with CMML, of which 3 cases (8.6%) were revised to acute myeloid leukemia (AML) due to the presence of NPM1 mutation; 17 cases with increased AMC were previously diagnosed with MDS, of which 4 cases (23.52%) were revised to CMML. There was no difference in clinical characteristics and prognosis between CMML patients with AMC ≥ 1.0 × 109/L (32 cases) and AMC<1.0 ×109/L (4 cases). The MDS with monocytosis group (MDS-Mo) showed a difference in the frequency of driving gene mutations from CMML (AMC<1.0×109/L). Compared to the non MDS-Mo group, the MDS-Mo group showed more TP53 mutations and chromosomal karyotypes of -5/5q -, -7/7q -, with a significantly shorter median survival time (3.85 years vs. 19.43 years). Conclusions: Adhering to the 2022 World Health Organization (WHO) standards can better distinguish between CMML and AML, as well as MDS. MDS-Mo patients have characteristic molecular mutations and genetic characteristics, and their prognosis is worse than that of patients with CMML and non MDS-Mo. Therefore, it is recommended to consider MDS-Mo as an independent subtype of MDS.
Objective: To analyze the expression of TRBC1 and TCR Vβ repertoire in different subsets of T lymphocytes in healthy donors and compare two flow cytometry methods for detecting T cell clones and their judgment criteria. Methods: Peripheral blood was collected in 40 healthy individuals, in which five subsets were divided, including CD7+CD5+(T1), CD7+CD5dim+(T2), CD7+CD5dim2+(T3), CD7+CD5-(T4) and CD7-CD5+(T5) gated on total CD3+ cells and CD3+TCR γδ- T cells, respectively. The expression of CD4, CD8, TCR Vβ repertoire and TRBC1 were analyzed in these subsets. Results: Thirty-nine healthy donors were included for statistical analysis. The average proportions of T1, T2, T3, T4, and T5 in CD3+cells were 57.29%, 27.74%, 5.15%, 1.43%, and 5.99%, respectively. T1 and T5 are mainly composed of CD4+cells, while T2, T3, and T4 are mainly composed of CD8+cells. The TRBC1+% in T2, T3, and T4 subsets of CD3+ is significantly lower than the corresponding value of TCR γδ- within T cells of CD3+ (P<0.01). Abnormal TRBC1+% (<15% or >85%) was found in 22 subsets in 21 healthy subjects, mainly in the T3 and T4 subsets. Partial subsets show a single dominant Vβ class, with dominant Vβ class proportion ≥60% or Vβ sum value of <15% used as the criterion for determining abnormal clones. After revising the TRBC1 clonal diagnostic criteria to TRBC1+%<12% or >82%, the consistency between the two methods for determining clonality was 96.77% (180/186). After the effect of TCR γδ+ cells is removed, the distribution trend remains unchanged. The final confirmed incidence of monoclonal T cells was 38.46% (15/39), and 86.67% (13 cases) had a proportion of clonal T cells in lymphocytes below 5%, indicating physiological clonal proliferation. The other two cases accounted for 6.76% and 33.24%, respectively, which may be T-cell clones of uncertain significance (T-CUS) and T lymphocyte proliferative diseases. Conclusions: Many physiologic T-cell clones were found frequently in CD5dim+ or CD5- subsets after gated by CD7/CD5 pattern in healthy people. The significance and interpretation of clonal T cells must be closely integrated with clinical practice.
Objective: The objective of the research is to establish a logistic regression model for the differential diagnosis of iron deficiency anemia (IDA) and thalassemia (TT) and to explore its application value. Methods: The research analyzed medical records of 121 patients diagnosed with both IDA and TT at the First Affiliated Hospital of Kunming Medical University between 2021 and 2022. A total of 100 specimens were arbitrarily chosen from each cohort to constitute the experimental groups, while the remaining 21 were designated for validation reasons. After performing a univariate comparison to establish statistical significance, the subsequent action was to identify variables with significant differences for the multi-factor analysis. Based on the analysis, four variables - gender, hemoglobin, mean corpuscular hemoglobin concentration, and red cell distribution width - were identified as independent factors that influence the outcome. These four variables established a logistic regression model, which was evaluated for its differential diagnosis effect by analyzing sensitivity, specificity, and other indicators on a subject’s working characteristics curve. Results: The results of the univariate comparison illustrate that the IDA group has lower values of red blood cells, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration in their peripheral blood compared to the TT group. Conversely, higher values of peripheral blood red cell distribution width-standard deviation are observed in the IDA group in comparison to the TT group, and the difference is statistically significant with a P-value less than 0.05. Gender, hemoglobin (Hb), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and red cell distribution width (RDW-SD) were analyzed via binary non-conditional logistic regression. The study showed that higher RDW-SD values were linked to an increased risk of IDA, while more significant reductions in Hb and MCHC were associated with a higher risk of IDA at a significance level of P<0.05. The logistic regression model is presented as:Logit(P)=-19.288-1.685X1+0.035X2+0.066X3-0.076X4. In the experimental group, the area under the receiver operating characteristic (ROC) curve of the logistic model is 0.947, the sensitivity is 90%, and the specificity is 91%; the sensitivity and specificity of the verification group are 100% and 72.4%, respectively. Conclusions: Logistic regression models have certain application value in the differential diagnosis of IDA and TT.
Objective: To investigate the diagnostic value of total protein and cytokine levels in cerebrospinal fluid (CSF) on central nervous system lymphoma (CNSL) and its impact on prognosis. Methods: A total of 43 patients who underwent cerebrospinal fluid testing at Chongqing University Cancer Hospital between October 2021 and February 2023 were enrolled into our study. Eighteen patients diagnosed with CNSL were in the CNSL group, and 25 patients with clinical high-risk factors for central invasion of lymphoma but without central nervous infiltration of lymphoma were in the control group. The basic clinical information, CSF biochemical indexes and CSF cytokine were collected. Mann-Whitney U test and t test were used to compare the differences of CSF related indexes between the two groups. The receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of CSF related indicators for CNSL patients, and the Kaplan-Meier curve was used to analyze the effect of CSF related indexes on the prognosis of patients. Results: The CNSL group had significantly higher levels of CSF total protein and CSF TNF-α, TNF-β, IL-17A, IL-6, and IL-10 than the control group (P< 0.05). The area under the curve (AUC) of CSF total protein combined with cytokines TNF-α, TNF-β, IL-17A, IL-6 and IL-10 in the diagnosis of CNSL was 0.957, with 94.1% sensitivity, and 95.5% specificity, which was significantly higher than the diagnostic value of CSF total protein and CSF cytokines alone (P< 0.05). The 2-year overall survival (OS) rates of the CNSL and the control groups were 29.1% and 56.0%, and the 2-year progression-free survival (PFS) rates were 23.1% and 68.0%, respectively. Patients in the CNSL group had shorter median PFS (P= 0.03), and there was no significant difference in median OS between the two groups (P= 0.11). Patients with high level of CSF total protein level, high expression of CSF TNF-α, IL-6, and IL-10 and low expression of CSF IL-17A and TNF-β had shorter PFS and OS, but the differences were not statistically significant (P> 0.05). Conclusions: The prognosis of CNSL patients is worse than that of patients with high risk of central nervous infiltration of lymphoma. The expression levels of CSF total protein combined with CSF cytokines TNF-α, TNF-β, IL-17A, IL-6 and IL-10 can assist in the diagnosis of CNSL and have a certain impact on the prognosis of patients with CNSL.
Objective: To investigate the changes and clinical significance of the absolute count of peripheral blood immune cells in children with immune thrombocytopenia (ITP). Methods: A total of 41 newly diagnosed ITP children admitted to the Department of Pediatrics, General Hospital of Ningxia Medical University from January 2022 to August 2023 were selected as the case group, while 30 healthy children were selected as the control group. Flow cytometry was used to detect and compare the absolute count and percentage levels of CD3+, CD3+CD4+, CD3+CD8+T cells, CD3-CD19+B cells and CD3-CD16+CD56+NK cells, as well as the CD4+/CD8+ratio in peripheral blood between the two groups. Results: The absolute count levels of CD3+ and CD3+CD4+T cells, as well as the absolute count and percentage levels of CD3-CD19+B cells in the case group were significantly increased compared to the control group (P<0.05). The percentage of CD3-CD16+CD56+NK cells significantly decreased (P<0.05). There was no statistically significant difference between two groups in the absolute count of CD8+T cells,the percentage of CD3+,CD4+,CD8+T cells, the ratio of CD4+/CD8+ and the absolute count of CD3-CD16+CD56+NK cells (P>0.05). Conclusions: There is a disorder in the immune cells of peripheral blood in children with ITP. The absolute count level can objectively and truly reflect the changes of immune cells in children with ITP. Judging abnormal changes in immune cell levels should be based on the absolute count level to analyze the percentage level changes, so as to provide better assistance for clinical disease diagnosis and treatment.
Objective: To study the differential gene expression in senescent endothelial cells after IL-17 induction and to explore possible mechanisms of action of IL-17-induced endothelial cell senescence. Methods: Unstimulated human umbilical vein epithelial cells (HUVEC) and IL-17-stimulated HUVEC were subjected to senescence index assay and RNA-seq sequencing, and the differential genes were analyzed by KEGG enrichment, and the differential genes were screened for qRT-PCR validation. Results: Cellular senescence occurred in IL-17-stimulated HUVEC compared to unstimulated HUVEC. After cluster analysis, SMAD6 was selected for qRT-PCR validation. The results of the analysis were consistent with the trend of the sequencing results. Conclusions: IL-17 can activate or inhibit a number of senescence-related genes and pathways that lead to endothelial cell senescence.
Objective: To establish a droplet digital PCR (ddPCR) method for detecting the low-level JAK2V617F mutation and to explore its application value in the diagnosis of myeloproliferative neoplasm (MPN). Methods: Site-specific TaqMan-MGB probes were used to establish the ddPCR method for detecting the JAK2V617F mutation in genomic DNA. JAK2V617F mutations were detected by ddPCR, real-time quantitative PCR and next-generation sequencing (NGS). The consistency of the detection results of different methods was compared. Results: The ddPCR method for detecting the JAK2V617F mutation based on specific TaqMan-MGB probes was successfully established, and the detection sensitivity was at least 0.05%. The ddPCR method and qPCR method were used to detect the JAK2V617F mutation in 82 subjects with MPN and 8 healthy controls. The consistency rate was 96.7%. For the 52 samples with positive results by both methods, the correlation coefficient of quantitative data was 0.971 4 (P<0.000 1). Eleven samples were verified by NGS and the correlation coefficient between the two quantitative results was 0.983 9 (P<0.000 1). Conclusions: The ddPCR with the specific TaqMan-MGB probe method can detect the JAK2V617F mutation conveniently and accurately, which may have potential application value in the screening and dynamic monitoring of the JAK2V617F mutation.
Objective: To explore the changes of T cell subsets and macrophage subtypes in severe aplastic anemia (SAA) mouse models. Methods: To induce the SAA mouse models, the B6D2F1 mice first received a 5 5 Gy total body irradiation (TBI) and then were infused with C57BL/6 lymph node cells at 4×106/mouse via a tail vein injection, while the B6D2F1 mice receiving TBI alone were used as the control group. The mice were euthanized on day 12-13, and their peripheral blood, lymph nodes, spleen, and bone marrow were collected for analysis. T cell subsets and phenotypic differentiation of CD4+ and CD8+ cells were detected by flow cytometry, and the proportion and polarization of macrophages in bone marrow were detected by flow cytometry and immunohistochemistry. Results: Relative to control mice that received 5 Gy TBI without lymph node cells infusion, SAA mouse models exhibited severe bone marrow failure characterized by a significant reduction in the hematopoietic area of the sternum and decreased levels of white blood cells, hemoglobin, and platelets in peripheral blood 12-13 days post-radiation. In the SAA group, the frequencies of CD8+ T cells increased and the ratio of CD4+/CD8+ lymphocytes decreased significantly in lymph nodes, spleen, and peripheral blood. In CD4+ and CD8+ T cells of SAA mouse models, the percentage of CD44+CD62L- effector memory T cells increased, while the proportion of Na?ve T cells and CD44+CD62L+ central memory T cells decreased significantly. CD11b+F4/80+ macrophages increased in the bone marrow of SAA mouse models, and most macrophages were CD86- cCD206+ and iNOS- cCD206+ M2 macrophages. Conclusions: In the SAA mouse models, the ratio of CD4+/CD8+ lymphocytes is significantly decreased, and the lymphocytes are mainly CD44+CD62L- effector memory T cells. Bone marrow macrophages increased and were mainly polarized towards M2 macrophages.
With the development of flow cytometry instruments, the accuracy and stability of flow cytometry are continuously improved. An increasing number of studies suggest the clinical value of quantitative detection of protein molecules expressed on cells. The demand for quantitative flow cytometry is increasing, but the traditional method cannot meet the needs of polychromatic antibody quantification due to its own limitations that antibody binding quantification is still limited to using of a single fluorescent labeled antibody. Quantum simply cellular (QSC) beads could be used for antibody binding quantification, but the quantification method using QSC beads was not widely adopted because of the huge difference between the results using this method and using the classical methods. Considering that the reason for the failure of the QSC method was the poor specificity of the QSC beads, this paper proposes a new method that has improved the staining technique by using a protein free microsphere staining buffer and is combined with a suitable specific capture microsphere (the anti-mouse IgG k beads for human cell analysis) as a calibrate for the value of QSC standards. In the test of CD3, CD4, CD8, CD25, CD27, CD28, CD45RA, CD127 and CD197 on the surface of human T cells, reasonable results were obtained. Although the accuracy of this technique still needs more tests, it has many possible uses, including calculating the absolute number of antibody binding, as a reference for polychromatic fluorescence compensation, visualizing the compensated spillover spread, calculating the amount of fluorescence produced by a single antibody under specific detection conditions, indicating the quality of antibody, and tracking the instrument performance. The calibrated fluorescence value of antibody-bound microspheres can be references to design a multicolor panel and help to achieve a comparable detection system among different platforms. The quantification of polychromatic flow cytometry is of great significance for single cell multi-omics technology. Our study suggests that a specific binding antibody light chain of a capture beads series can be used to achieve accurate and simple multiple quantification of cell surface proteins.
Objective: To construct the SKM-1 cell line with stable knockdown of the TET2 gene, and to infect SKM-1 cell line with the packaged H_TET2-shRNA lentivirus to obtain the H_TET2-shRNA SKM-1 stable strain. Methods: The SKM-1 cell line with stable knockdown of TET2 gene was verified by qPCR and Western blot (WB) experiments. The proliferative activity and apoptosis of SKM-1 cells before and after transfection were detected using the cell counting kit-8 (CCK-8) cell viability assay and flow cytometry, respectively. Results: qPCR verified that the TET2 gene expression was significantly reduced in SKM-1 cells with H_TET2-shRNA. Western blot showed that the TET2 protein expression was also significantly reduced in SKM-1 cells with H_TET2-shRNA and the CCK-8 cell viability assay confirmed that stable knockdown of the TET2 gene did not affect cell viability. The cell cycle assay revealed that S-phase cells accounted for 48.1% of the total cell number after knockdown of TET2, which was higher than that of the SKM-1 group (42.3%) and the control empty plasmid group. The apoptosis assay revealed that the apoptosis rate of SKM-1 cells in the TET2 knockdown group was 0.6% lower than that of the empty plasmid group (4.4%). Conclusions: SKM-1 cell line with stable low expression of the TET2 gene was constructed by lentivirus-mediated transfection, and the constructed cell line was verified to be successful by using qPCR and WB experiments. It was confirmed that the stable knockdown of TET2 did not affect the viability of the cells by using the CCK-8 cell viability assay. The cell cycle assay of flow cytometry found that the knockdown of TET2 affected the function of cellular DNA replication, which led to the abnormalities of cellular interphase division and affected cell growth and self-repair. The flow cytometry apoptosis assay found that knockdown of TET2 would inhibit apoptosis, which may be related to the fact that TET2 is an anti-oncogene.
In order to investigate the difference of cytokines between immune infertility patients and healthy women, and the relationship between cytokine levels and treatment outcomes in immune infertility patients, 96 women with female immune infertility and 57 healthy women were enrolled as the experimental and control groups, respectively. The levels of 7 cytokines were measured by flow cytometry, and fertility status was followed for 2 years. The results showed that there were significant differences in the serum levels of 6 cytokines including IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ, between the experimental group and the control group (P< 0.05). The levels of IL-2, IL-6, TNF-α, IFN-γ, and IL-17A in the non-pregnant group with poor treatment effect in the experimental group were significantly higher than those in the pregnant group (P< 0.05). Based on cytokine data and reproductive outcomes of patients, a new prognostic prediction model for immune infertility was successfully constructed with the PyCaret library. The bagging quadratic discriminant analysis (Bagging QDA) model performed best on the training set, with a sensitivity of 72.73%, specificity of 81.25%, and accuracy of 72.41% on the test set. In summary, immune infertility is associated with Th1/Th2 cytokine abnormalities, while the Bagging QDA model has high accuracy in predicting the prognosis of immune infertility.
