Objective This study investigated the regulatory effect of high mobility group protein B1 (HMGB1) in the peripheral blood of patients with primary immune thrombocytopenia (ITP) on myeloid dendritic cells (mDC) and Th17/regulatory T cells (Treg) balance. Methods The study enrolled 30 newly diagnosed ITP patients and 30 healthy controls.Flow cytometry was used to measure the proportion of mDC, Th17, and Treg cells in the peripheral blood of ITP patients and healthy controls. ELISA was conducted to quantify the serum levels of HMGB1, interleukin 6 (IL-6), IL-23, IL-17, and transforming growth factor β(TGF-β). The mRNA levels of retinoic acid-related orphan receptor γt(RORγt) and forehead box P3(FOXP3) were detected by real-time PCR. The correlation between the abovementioned cells, cytokines, and platelet count was assessed using Pearson linear correlation analysis. Results The proportion of Th17 cells and the expression levels of HMGB1, IL-6, IL-23, IL-17 and the level of RORγt mRNA in the peripheral blood of ITP patients were higher than those in healthy controls. However, the Treg cell proportion and TGF-β level were lower in ITP patients than those in healthy controls. In patients with ITP, the proportion of mDC and the level of FOXP3 mRNA did not show significant changes. The proportion of mDC cells was significantly correlated with the expression of IL-6 and IL-23. Moreover, the expression of HMGB1 showed a significant correlation with the expression of mDC, IL-6, IL-23, RORγt mRNA, and IL-17. Notably, both the proportion of mDC cells and the expression of HMGB1 were negatively correlated with platelet count. Conclusion The high expression of HMGB1 in peripheral blood of ITP patients may induce Th17/Treg imbalance by promoting the maturation of mDC and affecting the secretion of cytokines, thereby potentially playing a role in the immunological mechanism of ITP.
Humans ; Th17 Cells/cytology* ; HMGB1 Protein/genetics* ; T-Lymphocytes, Regulatory/cytology* ; Female ; Male ; Dendritic Cells/metabolism* ; Adult ; Middle Aged ; Purpura, Thrombocytopenic, Idiopathic/genetics* ; Nuclear Receptor Subfamily 1, Group F, Member 3/genetics* ; Young Adult ; Interleukin-23/blood* ; Interleukin-17/blood* ; Interleukin-6/blood* ; Forkhead Transcription Factors/genetics* ; Myeloid Cells/cytology* ; Aged

OBJECTIVES: To investigate the clinical and immunological features of children with primary immune thrombocytopenia (pITP) or connective tissue disease (CTD) with thrombocytopenia as the initial manifestation at initial diagnosis, and to provide a basis for early differentiation. METHODS: A retrospective study was performed on 236 children with pITP (pITP group) or CTD with thrombocytopenia as the initial manifestation (CTD-TP group) who were admitted from January 2019 to August 2024. Clinical and immunological indicators were compared between the two groups to identify potential influencing factors for early differentiation and their discriminative validity. RESULTS: Compared with the pITP group, the CTD-TP group had a significantly older age of onset and significantly lower leukocyte count, eosinophil count, lymphocyte count, and complement C4 level (P<0.05), as well as significantly higher levels of C-reactive protein, IgE, and IgM (P<0.05). The logistic regression analysis showed that age, IgE, IgM, total B cells, and complement C4 were predictive factors for early differentiation between pITP and CTD-TP (P<0.05). The receiver operating characteristic curve analysis showed that a combination of these five factors had a good discriminative validity, with an area under the curve of 0.944. The correlation analysis showed a negative correlation between IgG and platelet count in the pITP group (r_s=-0.363, P<0.05) and a positive correlation between NK cells and platelet count in the CTD-TP group (r_s=0.713, P<0.05). CONCLUSIONS There is heterogeneity in the clinical and immunological indicators between children with pITP and CTD-TP at initial diagnosis, and these research findings can help with the early differentiation between the two diseases.
Purpura, Thrombocytopenic, Idiopathic/immunology* ; Diagnosis, Differential ; Connective Tissue Diseases/immunology* ; Retrospective Studies ; Early Diagnosis ; Age of Onset ; Leukocyte Count ; Complement C4/immunology* ; C-Reactive Protein/immunology* ; Immunoglobulin E/immunology* ; Immunoglobulin M/immunology* ; Humans ; Male ; Female ; Infant ; Child, Preschool ; Child ; Adolescent ; Biomarkers/blood*

OBJECTIVE: To investigate the expression of CSF-1 and CSF-1R in the peripheral blood of children with immune thrombocytopenia (ITP) and its clinical significance. METHODS: Forty-four children with ITP treated in our hospital from February 2023 to January 2024 were selected as the observation group, and 40 healthy children were selected as the control group during the same period, and relevant clinical data were collected. Peripheral blood mononuclear cells (PBMC) of children with ITP and healthy children were separated, and the plasma levels of M1 macrophage-associated cytokines (TNF-α, IL-6), M2 macrophage-associated cytokines (IL-10, TGF-β), and CSF-1 were detected by ELISA in the children of both groups. The mRNA levels of M1 macrophage surface markers (CD86, iNOS), M2 macrophage surface markers (CD206, Arg-1) and CSF-1R were detected by RT-PCR in PBMC of children in both groups. Western blot was used to detect the expression of CSF-1R protein in PBMC of the two groups of children. The correlation between platelet count and CSF-1R mRNA expression in PBMC, TNF-α, IL-6, IL-10, TGF-β and CSF-1 in plasma was analyzed. RESULTS: Compared with the control group, the levels of IL-10, TGF-β, CSF-1 and platelet count in plasma of children with ITP were significantly decreased (P < 0.01), and the levels of TNF-α and IL-6 were significantly increased (P < 0.01); the mRNA levels of the M1 macrophage surface markers (CD86, iNOS) in PBMC of children with ITP were significantly increased (P < 0.05), mRNA levels of M2 macrophage surface marker CD206 in PBMC of children with ITP were decreased compared with controls but the difference was not statistically significant ( P >0.05), mRNA levels of Arg-1 were decreased, the difference was statistically significant (P < 0.05). The mRNA and protein levels of CSF-1R in PBMC of ITP children were higher than that in controls. CSF-1R expression in PBMC of ITP was positively correlated with platelet count, IL-10, CSF-1 were positively correlated (r =0.822,0.481,0.405). CONCLUSION CSF-1 is significantly reduced in the plasma of ITP, and CSF-1R mRNA and protein expression is significantly elevated in PBMC of ITP, which are involved in the regulation of macrophage M1/M2 imbalance, and could serve as a potential therapeutic target for ITP.
Humans ; Purpura, Thrombocytopenic, Idiopathic/blood* ; Macrophage Colony-Stimulating Factor/metabolism* ; Leukocytes, Mononuclear/metabolism* ; Child ; Interleukin-10/blood* ; Macrophages/metabolism* ; Tumor Necrosis Factor-alpha/blood* ; Interleukin-6/blood* ; Male ; Female ; Transforming Growth Factor beta/blood* ; Receptor, Macrophage Colony-Stimulating Factor/metabolism* ; Clinical Relevance

Objective: To report the clinical manifestations and laboratory features of five patients with congenital thrombotic thrombocytopenic purpura (cTTP) and explore its standardized clinical diagnosis and treatment along with a review of literature. Methods: Clinical data of patients, such as age of onset, disease manifestation, personal history, family history, and misdiagnosed disease, were collected. Treatment outcomes, therapeutic effects of plasma infusion, and organ function evaluation were observed. The relationship among the clinical manifestations, treatment outcomes, and ADAMTS13 gene mutation of patients with cTTP was analyzed. Additionally, detection of ADAMTS13 activity and analysis of ADAMTS13 gene mutation were explored. Results: The age of onset of cTTP was either in childhood or adulthood except in one case, which was at the age of 1. The primary manifestations were obvious thrombocytopenia, anemia, and different degrees of nervous system involvement. Most of the patients were initially suspected of having immune thrombocytopenia. Acute cTTP was induced by pregnancy and infection in two and one case, respectively. ADAMTS13 gene mutation was detected in all cases, and there was an inherent relationship between the mutation site, clinical manifestations, and degree of organ injury. Therapeutic or prophylactic plasma transfusion was effective for treating cTTP. Conclusions: The clinical manifestations of cTTP vary among individuals, resulting in frequent misdiagnosis that delays treatment. ADAMTS13 activity detection in plasma and ADAMTS13 gene mutation analysis are important bases to diagnose cTTP. Prophylactic plasma transfusion is vital to prevent the onset of the disease.
Female ; Pregnancy ; Humans ; Adult ; Blood Component Transfusion ; Plasma ; Purpura, Thrombotic Thrombocytopenic/therapy* ; Mutation ; Purpura, Thrombocytopenic, Idiopathic ; ADAMTS13 Protein/therapeutic use*

OBJECTIVE: To investigate the effect of enhanced autophagy in megakaryocyte to proplatelet formation in children with immune thrombocytopenia(ITP). METHODS: Giemsa staining and immunofluorescence staining were used to observe megakaryocyte morphology and proplatelet formation, Western blot was used to determine the expression of cytoskeleton protein and autophagy related protein. Autophagr regulation drugs Rap or 3-MA was used to regulate autophagy of megakaryocytes. RESULTS: Some vacuole-like structures was found in ITP megakaryocytes of the children, the expression of LC3II/I (ITP 1.32±0.18； Ctrl 0.49±0.16，P<0.05) and Atg5-Atg12 (ITP 0.69±0.17； Ctrl 0.12±0.08，P<0.05) was significantly higher in ITP children as compared with those in control group. The immu- nofluorescence staining showed that the cytoskeleton arrangement in megakaryocytes of ITP children was abnormal, and the phosphorylation of myosin light chain was also increased(ITP 0.74±0.09, Ctrl 0.05±0.02，P<0.05). In vitro, inducer or inhibitor of autophagy could regulate the production of proplatelet and the expression of cell cycle related protein, including CyclinD1（Veh 1.08±0.12; Rap 0.46±0.04; Rap+3-MA 0.70±0.03）, CyclinD2（Veh 0.47±0.04; Rap 0.27±0.04; Rap+3-MA 0.41±0.03）, P21（Veh 0.15±0.01; Rap 0.04±0.01; Rap+3-MA 0.05±0.01）. CONCLUSION Enhanced autophagy is the key factor of poor proplatelet formation in megakaryocytes of ITP children.
Autophagy ; Blood Platelets ; Humans ; Megakaryocytes ; Purpura, Thrombocytopenic, Idiopathic ; Thrombocytopenia

OBJECTIVE: To compare the effect of Sheng-Xue-Xiao-Ban Capsule (SXXBC) and indirubin to the peripheral platelets of the Idiopathic thrombocytopenic purpura (ITP) model mouse. METHODS: The ITP mouse model was established by the method of passive immunization. SXXBC and indirubin were used for intervention treatment. Then the hemorrhagic phenomena of ITP mice were observed and the numbers of peripheral platelets, hemoglobin and white blood cells, bone marrow megakaryocytes and their classification and coagulation function were detected and compared. RESULTS: The improvement rate of hemorrhage in SXXBC group was 40% for small dose, 60% for medium dose and 80% for high dose, while the improvement rate of hemorrhage in indirubin group was 30% for small dose, 50% for medium dose and 60% for high dose. There was no statistically significant difference in the improvement rate of hemorrhage between the two groups (P＞0.05). Compared with the model control group, PLT and Hb increased in different doses of SXXBC and indirubin group 4th-8th day after drug intervention (P＜0.05, 0.01). However, there was no significant difference between the different doses of SXXBC group and indirubin group (P＞0.05). Compared with the model control group, the WBC in each group was significantly lower (P＜0.05, 0.01) on the 4th-8th day after drug intervention; However, there was no statistical significance between the two groups of SXXBC and indirubin (P＞0.05). Compared with the model control group, the total number of megakaryocytes in each treatment group were decreased (P＜0.05, P＜0.01), in which the number of primary megakaryocytes in the large and medium dose groups of SXXBC and indirubin were decreased (P＜0.05, 0.01), and the number of juvenile megakaryocytes in the large dose group of SXXBC and indirubin were also decreased (P＜0.05). The number of granular megakaryocytes were decreased in each intervention groups (P＜0.05, 0.01), and the number of thromocytogenic megakaryocyte was increased in the high and medium dose groups of SXXBC and indirubin (P＜0.01). The time of prothrombin was shortened in the high and medium dose groups of SXXBC and indirubin (P＜0.05), and the fibrinogen (FIB) content in the high and medium dose groups of SXXBC was close to that of the normal control group. CONCLUSION Both of the SXXBC and the indirubin standard all show good hemostatic effects. Indirubin shows a positive effect on increasing the peripheral platelet and hemoglobin in ITP model mice, regulating the immune response, reducing the total number of bone marrow megakaryocytes, increasing the thromocytogenic megakaryocyte, and increasing coagulation function.
Animals ; Blood Platelets ; Capsules ; Indoles ; Megakaryocytes ; Mice ; Purpura, Thrombocytopenic, Idiopathic/drug therapy*

OBJECTIVE: To establish a flow cytometric immunobead array assay (FCIA) to quantify platelet antibodies and to explore its application in the diagnosis and treatment of ITP. METHODS: The guantitative standard curve was established by binding the human IgG of known concentration on antibody-coated microbeads; at the same time, the platelet-specific antigen and antibody complex was captured and levels of platelet antibodies were detected using the microbeads coated by 5 kinds of antibodies against platelets suca as GPIX (SZ1), GPⅠb (SZ2), GpⅢa (SZ21), GPⅡb (SZ22) and p-selection (SZ51). The fluorescence signal detected by flow cytometry were transformed into the conentration of platelet antibodies in samples through the quantitative standard curve, thereby establishing the method for quantititive detection of platelet-specific antibodies in plasm samples (FCIA), moreover the property, efficiency and clinical application of establishod FCIA method were evaluated. RESULTS: The FCIA could detect 5 kinds of antibodies against GPIX, GPⅠb, GpⅢa, GPⅡb and β-selection within a broad range of 33.29-1280 ng/ml, 45.17-1280 ng/ml, 42.07-1280 ng/ml, 46.40-1280 ng/ml, 42.48-1280 ng/ml and 42.48-1280 ng/ml respectively, and their recovery rates were 115.23%, 112.58%, 117.47%, 107.64% and 112.67% respectively. The intra-assay coefficient of variation (CV) for anti- GPIX, -GPⅠb, -GpⅢa, -GPⅡb and p-selection antibodies was 3.54%, 3.63%, 4.66%, 6.43% and 6.67% respectively, and the inter-assay CV for above mentioned antibodies were 10.89%, 7.57%, 10.34%, 6.95% and 10.72% respectively. The detection showed that the levels of 5 kinds of platelet-specific antibodies in ITP group all were higher than those in non-ITP and healthy control groups (P＜0.01). The sensitivity, specificity and accuracy of quantitatively detecting 5 kinds of antibodies for diagnosis of ITP by FCIA were 68.29%, 84.98% and 78.95% respectively, while the sensitivity, specificity and accuracy of detecting 5 kinds of antibodies by modified indirect MAIPA were 41.46%, 90.41% and 72.81% respectively. CONCLUSION The established quantitative FCIA for detection of antibodies provides a powerful tool for diaghosis and evaluation of therapeutic efficacy and prognosis of ITP patients.
Antibodies ; Autoantibodies ; Blood Platelets ; Flow Cytometry ; Humans ; Purpura, Thrombocytopenic, Idiopathic

OBJECTIVE: To investigate the changes of mean platelet volume (MPV), platelet distribution width (PDW) and platelet associated antibodies (PAIg) in children with acute immune thrombocytopenic purpura (aITP), and to explore the diagnostic value of MPV, PDW, PAIg and their combination for megakaryocyte dysmaturity in aITP children. METHODS: Plt count, MPV and PDW of 36 aITP children were measured by using Sysmex XN automatic blood cell analyzer, and 33 children with acquired thrombocytopenic purpura (ATP) without megakaryocyte dysmaturity. The expression of PAIg was detected by flow cytometry, and the number and classification of megakaryocytes in the bone marrow were performed by marrow cytology. The diagnostic significances of MPV, PDW, PAIg and their combination as well as the sensitivity and specificity for megakaryocytes dysmaturity in aITP were assessed through calculating the area under ROC curve (AUC), after determining the influence of each parameters on the megakaryocyte dysmaturity by Logistic regression. RESULTS: MPV, PDW and PAIg of aITP children were significantly higher than those of the ATP children (P＜0.05), while the Plt count and number of thromocytogenic megakaryocytes per area (1.5 cm×3 cm) were less than those of the controls (P＜0.05). Count of RBC and WBC, percentages of neutrophil granulocytes and lymphocydes in aITP were similar to those in the controls（P＞0.05）. The results of Logistic regression showed that Plt count, MPV, PDW and PAIg were the factors influencing megakaryocyte dysmaturity in aITP children, and the regression model has a high statistical significance (χ=65.491，P=0.001) and r square (R=0.713). The AUC of the combined detection of Plt count, MPV, PDW and PAIg was 0.863, which was much higher than that of Plt count, MPV, PDW, PAIg individually or in pairs. The sensitivity and specificity of the combined detection were 79.167% and 89.697%, which were higher than those of Plt count, MPV, PDW, PAIg individually or in pairs. CONCLUSION The diagnostic significance of MPV and PDW for megakaryocyte dysmaturity in aITP are insufficient, but the diagnostic efficacy can be improved by combined examination with PAIg.
Antibodies ; Blood Platelets ; Child ; Humans ; Mean Platelet Volume ; Megakaryocytes ; Platelet Count ; Purpura, Thrombocytopenic, Idiopathic ; diagnosis

Abstract　Immune thrombocytopenia (ITP) is a complex autoimmune disease characterized by less than 100×10/L platelet count in peripheral blood. The pathogenesis of ITP is complex and has not been fully elucidated. Currently, researches on the pathogenesis of ITP mainly focus on the disorders of humoral immunity and cellular immunity. In recent years, some new progress has been made in the study of this pathogenesis, including the platelet clearance pathway that is not dependent on Fc γ R mediation, the metalloproteinase (ADAM) 10 that can regulate T and B cells, and the abnormal expression of micro RNA in genetic factors. Under the joint action of multiple factors, the imbalance of the immune system in the body leads to the occurrence of ITP. This article reviews the research progress on humoral immunity, cellular immunity and other possible new pathogenesis of ITP in recent years.
ADAM10 Protein ; Blood Platelets ; Humans ; Platelet Count ; Purpura, Thrombocytopenic, Idiopathic

OBJECTIVE: To explore the clinical significance of platelet membrane glycoprotein (GPIIb/IIIa) detection by flow cytometry (FCM) combined with enzyme-linked immunosorbent assay (ELISA) in diagnosis and treatment of immune thrombocytopenia(ITP). METHODS: Fifty-two patients with immunological thrombocytopenia admitted to the Department of Hematology in our hospital during October 2014-October 2018 were enrolled in ITP group. Thirty healthy people from the physical examination center were enrolled in control group. The positive expression rate of platelet membrane glycoprotein was measured by FCM, and the peripheral platelet count was detected by automatic analyzer, and the plasma membrane glycoprotein level was measured by ELISA. The difference between the two groups was compared. The platelet membrane glycoprotein levels in ITP patients before and after treatment were compared. RESULTS: The positive expression rate of GPIIb/IIIa (i,e CD41/CD61) and plasma GPIIb/IIIa levels in ITP patients were significantly lower than those in the healthy controls. The increased expression of platelet GPIIb/IIIa was associated with the response to therapy. The positive expression rate and level of GPIIb/IIIa postively correlated with its platelet count. The sensitivity of platelet GPIIb/IIIa combined with platelet count for diagnosis of ITP was 90.38%, the specificity was 93.33%, the positive likelihood ratio was 13.57, and the positive predictive value was 95.92%. CONCLUSION The platelet membrane glycoprotein detection as a preliminary screening method used for diagnosis of immune thrombocytopenia is simple, convenient, sensitive and rapid, thus may be considered as a new method for clinical diagnosis.
Autoantibodies ; Blood Platelets ; Humans ; Platelet Glycoprotein GPIIb-IIIa Complex ; Purpura, Thrombocytopenic, Idiopathic