The Study On The Pathogenesis And Intervention Mechanism Of Immune Thrombocytopenia

Immune thrombocytopenia(ITP)is an acquired,heterogeneous,autoimmune disease.It is mainly characterized by isolated thrombocytopenia and the most common bleeding disease in clinical practice,accounting for about a third of all bleeding diseases.No "gold standard" test exists to reliably establish the diagnosis.Although the new 2019 consensus has been published,its main focus is on treatment,and ITP has so far been a diagnosis of exclusion.ITP is mainly divided into primary and secondaryary.The former refers to the exclusion of all known causes of secondary ITP,while secondary ITP is mainly triggered by genetic or acquired susceptibility diseases,such as:autoimmune disease(systemic lupus erythematosus,antiphospholipid antibody syndrome,etc.),thyroid disease,drug-induced thrombocytopenia,allogeneic thrombocytopenia,proliferative disease of the lymphatic system,abnormal myelodysplasia(aplastic anemia and myelodysplastic syndrome),hematological malignancies,chronic liver disease,hypersplenism,consumptive thrombocytopenia,pregnancy thrombocytopenia,acute and chronic infections,pseudothrombocytopenia,and congenital thrombocytopenia.The pathogenesis of ITP is complex and is the result of the combined effects of multiple mechanisms,of which the immune intolerance to autoantigens is the most basic mechanism.The classical pathogenesis of ITP is specific platelet autoantibodies and cytotoxic T cells directly kill platelets,resulting in increased platelet destruction.At the same time,cytotoxic T cells and platelet autoantibodies can directly affect megakaryocytes of the bone marrow,leading to their maturation disorders and affecting platelet generation from the bone marrow level.In addition,in recent years,platelets are desialylated and lead to their destruction in the liver,which is an important pathway for platelet clearance that is independent of the c-terminal receptor of the Fc portion of the immunoglobulin(Fc receptor).The treatment of ITP follows the principle of individualization,and factors such as patient age,bleeding degree,comorbidities,and combined medication need to be weighed.Its treatment strategy is aimed at restoring platelet counts that are compatible with hemostatic effects rather than returning platelet counts to normal values.First-line therapy focuses on inhibiting the production of autoantibodies and platelet degradation.Corticosteroids are the first choice for ITP first-line therapy,which can broadly condition autoimmune disorders.It mainly includes traditional doses of prednisone and high-dose dexamethasone.Given that traditional doses of prednisone need to be gradually reduced and have a longer course of treatment,high-dose dexamethasone has gradually replaced traditional doses of prednisone due to the advantages of fast onset,shortening course of treatment,and greatly reduced side effects of corticosteroids.Although corticosteroids are effective and economical,about one-third of the patients in the clinic are still ineffective and need to be transferred to secondary-line treatment.Secondary-line treatment is the main method for long-term treatment of ITP patients,including anti-CD20 monoclonal antibody rituximab,recombinant human thrombopoietin receptor agonist(TPO-RA),splenectomy,and immunosuppression agent.With the continuous emergence of new therapeutic drugs and the risks associated with surgery-related infections,thrombosis and mortality,splenectomy rates have gradually decreased.It is reported in the literature that secondaryary ITP(HCV-related),that is,secondary ITP(HCV-associated)patients,has a higher incidence and severity of bleeding than patients with primary ITP,which severely affects the patients'surgery and clinically invasive procedures,increased platelet transfusion costs,blood transfusion-related risks,and impact on the application of antiviral drugs.These have become difficult problems for clinical doctors,and new mechanisms need to be discovered urgently and may provide a basis for improving platelet therapy.As a human-mouse chimeric monoclonal antibody antibody,rituximab,can bind to CD20,a specific transmembrane receptor on the surface of B cells,and thus cause the B cell's punching effect.Possible mechanisms include complement-dependent cytotoxicity.(CDC),antibody-dependent cell-mediated cytotoxicity(ADCC),etc.,eventually lead to B-cell clearance.The total effective rate is 60%,and the long-term effective rate is 20 to 25%.Because equivalent to"drug resection",it is now attracting much more attention in the treatment of primary ITP.However,its price is relatively expensive,and there is still a lack of clinical indicators for predicting its response to treatment.In this study,the average fluorescence intensity of fluorescein-labeled ECL and RCA-I antibodies and real-time quantitative PCR are used to detect platelet desialylation and Neul expression levels for the first time in the secondary ITP(HCV-associated),which has a greater risk of hemorrhage.The pathogenesis of platelet clearance in patients provides new insights and provides a strong theoretical basis for improving platelet therapy.At the same time,the anti-nuclear antibody(ANA)test is used to evaluate the short-term and long-term treatment effects of rituximab in the treatment of patients with primary ITP,and the possible interference effects of other detection indicators are compared,which is useful for clinical prediction of ITP.Rituximab treatment response provides effective laboratory indicators,filling up domestic and overseas blank.Part I:The Platelet Desialylation:A Novel Pathogenesis and Intervention Mechanism of Secondary Immune Thrombocytopenia(Hepatitis C Virus-associated)Objective:In this study,the platelet desialylation levels of patients with secondaryary ITP(HCV-related)and normal controls were analyzed,also involved the correlation with platelet destruction.The patients with secondaryary ITP(HCV-related)were tested for anti-HCV.The levels of platelet desialylation before and after virus treatment were collected to analyze its relationship with curative effect.The platelet desialylation and the expression of neuraminidase 1(Neu1)were detected after treatment.Therefore,it was clear whether there was abnormality of platelet desialylation in patients with secondaryary ITP(HCV-related),and changes in platelet desialylation during the course of HCV and after treatment.The aim was to explain a new pathogenesis of secondary ITP(HCV-associated),so as to provide new interventions for the treatment of secondary ITP(HCV-associated).Methods:Forty-five patients with secondary ITP(HCV-associated)were enrolled and received direct antiviral drug(DAA)treatment.Peripheral venous blood samples were collected before treatment.Clinical efficacy was evaluated twelve weeks after the start of treatment.At the same time,twenty-one healthy volunteers were enrolled as normal controls.And platelet counts were measured by routine blood tests.1.Flow cytometry was used to detect the level of ?-galactose(?Gals)exposed to platelet asialization,that is,platelets were labeled with the PE-Cy5-CD41a monoclonal antibody,and co-expressed with FITC-ECL,FITC-RCA-I After incubation,the average fluorescence intensity of the latter two were used to reflect the level of asialization of platelets.2.RT-PCR was used to detect the copy number of Neu1 in patients with secondary ITP(HCV-associated)and normal controls before and after treatment.Results:1.The average fluorescence intensity of platelet ECL and RCA-I in secondary ITP(HCV-associated)patients was significantly higher than that in normal controls.2.Patients with secondary ITP(HCV-associated)had sustained virological response for 12 weeks(SVR12)and no response(NR)rates were 91.4%and 8.6%,respectively.The average fluorescence intensity of ECL and RCA-I in SVR12 patients after treatment were significantly higher than before treatment Decreased,and the average fluorescence intensity of ECL and RCA-I before and after treatment in NR patients was not statistically different.3.Compared with healthy controls,secondary ITP(HCV-associated)patients had higher Neul copy numbers.Conclusion:1.Secondary ITP(HCV-associated)patients in the experimental group had significantly higher levels of platelet desialylation than healthy controls.2.After DAA treatment,the levels of platelet desialylation decreased and platelet counts increased in patients in the redipavir/sofibvir(Harvoni)and vepartavir/sofibvir(Epclusa)groups.Therefore,it was clarified that the increase of platelet asialization level may be closely related to the occurrence of secondary ITP(HCV-associated),which was a new mechanism for secondary ITP(HCV-associated),and it was expected to provide new treatment for patients with secondary ITP(HCV-associated)strategy.3.There was no significant difference in the mean fluorescence intensity of platelet ECL and RCA-I and the copy number of Neu1 mRNA between the two treatment groups.It was suggested that the level of thrombocytopenia in secondary ITP(HCV-associated)patients had no obvious correlation with this type of antiviral therapy,which indirectly proved the important role of platelet desialylation in secondary ITP(HCV-associated).Part II:Correlation between antinuclear antibodies and rituximab intervention in patients with primary immune thrombocytopeniaObjective:To detect peripheral blood ANA levels in patients with primary ITP before rituximab treatment,compare early efficacy of ANA-positive and ANA-negative ITP patients,including effectiveness and time to onset,also evaluate gender,age,and initial platelet count,IgG level,megakaryocyte count in bone marrow,and possible effects of different doses of rituximab between the two groups.Compare the long-term efficacy of the two groups,including a follow-up rate of 6 months,1 year,and 2 years of continuous remission and evaluation of survival curve.ANA screening could be a useful test for predicting rituximab response in ITP.Methods:1.Using the method of continuous cohort study,retrospectively analyzed the clinical records of a total number of adult primary ITP(2012.01-2018.12)from three hospitals.All included patients had their ANAs tested before rituximab treatment.2.ANAs were determined by the immune fluorescent technique on Hep-2 cell substrate.3.According to the ANA titer in patients before treatment,they were divided into 98 cases of ANA positive group and 189 cases of negative group.4.Simultaneously collect the patienst' age,gender,disease duration,baseline platelet count,serum immunoglobulin(1g)G level,bone marrow megakaryocyte count,and bleeding score.5.All patients were followed up for at least 2 years after rituximab treatment,and the early and long-term effectiveness of the ANA positive and negative groups were evaluated.The evaluation of the early effective rate was based on the latest ITP diagnosis and treatment guidelines at home and abroad.The long-term effective rate referred to the time that the peripheral platelet count of the patient continues to reach more than 30 ×109/L after the early remission.Results:1.There were no significant differences between the ANA positive and negative groups in gender,age,median platelet count,median IgG level,ITP duration,bleeding score,and median count of bone marrow megakaryocytes.2.Compared with the ANA-negative group,the early effective rate was significantly higher in the ANA-positive group and there was a significant statistical difference.3.In the ANA-positive group,the total effective rate and complete response(CR)rate decreased linearly at 6 months of follow-up,while the total effective rate and CR rate of ANA-negative patients did not decrease significantly.4.The total effective rate of the ANA positive group was less than 5%after 1 year of follow-up,and the effective rate was close to 0 after 2 years of follow-up.In the ANA-negative group,the total effective rate was still 35.4%and the remission rate was 19%after 1 year of follow-up.The total effective rate was still higher than 20%after 2 years of follow-up.Conclusion:1.Compared with the ANA-negative group after rituximab treatment,ITP patients in the ANA-positive group had higher early total effective rate and complete response rate,but shorter long-term effective rate.2.The long-term effectiveness of the ANA-negative group is comparable to other studies reported in rituximab treatment.3.ANA screening may be an effective predictor of rituximab treatment response in ITP.