儿童原发性免疫性血小板减少症颅内出血的多中心临床研究

目的:探讨我国儿童免疫性血小板减少症(ITP)颅内出血的发生率、相关临床因素及预后。方法:对四大儿童血液病中心2001-2011年收治的儿童ITP并发颅内出血者17例进行回顾性分析。结果:①ITP患儿颅内出血的发生率为0.46%。②颅内出血高危因素为急性患儿起病1个月内,慢性患儿病程1年后;并发感染、剧烈运动和外伤;血小板计数小于10×109/L。③并发颅内出血者病死率为11.7%。④积极治疗后预后良好。结论:ITP患儿的颅内出血是一种可以通过积极治疗而达到有效治愈的疾病,幸存下来的患儿多数无严重的神经系统后遗症。     

小儿特发性血小板减少性紫裥抗血小板抗体亚类的研究

采用鼠抗人IgG亚类单克隆抗体酶联免疫吸附法对急、慢性ITP患儿进行血小板表面相关IgG(PAIgG)亚类及血清IgG亚类测定。结果表明:ITP患儿抗血小板抗体有IgG亚类限制性,主要以PAIgG_1和PAIgG_3增高为主。其原因可能与某些感染及免疫调节异常有关。血清IgG亚类缺陷可能是ITP病因之一。PAIgG亚类在诊断和判断疗效中起重要作用。     

特发性血小板减少性紫癜患者血小板相关抗体水平与预后的关系

目的:探讨特发性血小板减少性紫癜(ITP)与血小板相关抗体之间的相互关系,为临床诊断和治疗提供帮助。方法:回顾性分析了2000年1月～2007年4月应用流式细胞术检测140例临床确诊的ITP患者初诊时及治疗前后不同血小板相关抗体——血小板膜糖蛋白抗体(PAIg)的表达。结果:ITP患者初诊时PAIgG、PAIgA及PAIgM抗体表达水平均高于正常对照组;PAIgG、PAIgM、PAIgA表达水平与血小板数之间均呈负相关,相关系数分别为γ=-0.72、γ=-0.83和γ=-0.67。在确诊ITP后予激素等免疫治疗无效转变为慢性ITP的患者时,PAIgM抗体水平要明显高于激素治疗有效的急性ITP患者,并且PAIgM异常增高或联合PAIgG增高的比例要显著多于急性ITP患者(均P<0.05)。35例PAIgG和PAIgM同时升高的ITP患者,经治疗血小板升至正常后,PAIgG、PAIgM抗体表达水平均有显著下降。结论:ITP的发生与血小板抗体密切相关,初诊ITP患者PAIgM单独或联合PAIgG异常增高常提示预后不良,易转变为慢性ITP。     

CMV和EBV感染与儿童ITP的关系

目的 :探讨巨细胞病毒 (CMV)和Epstein Barr病毒 (EBV)感染与儿童特发性血小板减少性紫癜 (ITP)的关系。方法 :采用基础PCR方法检测了 44例ITP患儿外周血白细胞中CMV和EBV的感染情况 ,并结合实验室及临床特征进行分析。结果 :①ITP患儿CMV感染阳性率较高 ,达 6 1.4% ;在慢性反复发作的患儿中 ,CMV阳性率更高 (71.9% ) ,显著高于急性ITP患儿 (33.3% ) (χ2 =5 .47,P <0 .0 5 )。CMV阳性患儿血小板自身抗体阳性率较高 ,血小板及巨核细胞数均较低 ,出血症状明显 ,激素治疗效果较差。② 5 0 %的ITP患儿外周血EBVDNA阳性 ,急性患儿较多见 ,但与慢性患儿比差异无显著性意义。EBV阳性患儿血小板自身抗体阳性率也较高 ,以抗GPⅡb/Ⅲa升高最明显 ,显著高于EBV阴性患儿 (χ2 =4.96 ,P <0 .0 5 )。与CMV阳性患儿相比 ,EBV阳性患儿血小板减少多为轻、中度 ,骨髓中巨核细胞数正常或增多 ,出血症状少见 ,预后较好。结论 :CMV和EBV感染与部分儿童ITP的发病有关 ,不同的病毒感染有不同的发病机制和临床特征。     

流式细胞术研究特发性血小板减少性紫癜患者血小板相关抗体

目的：探讨特发性血小板减少性紫癜（ITP）患者血小板表面相关抗体（PAIg）在诊断及预后的价值。方法：应用流式细胞术（FCM）检测84例ITP患者及20例正常人PAIgG、PAIgM、PAIgA。结果：初发ITP和复发ITP患者组与正常对照组比较，PAIgG、PAIgA差异有统计学意义（P〈0．01），PAIgM差异无统计学意义（P〉0．05）。PAIgM与PAIgA之间有显著相关性，r=0．451（P〈0．01）。结论：①PAIg增高可作为诊断初发ITP的重要指标之一；②FCM检测ITP患者血小板表面PAIg敏感性好、特异性高，适用于临床，对ITP的诊断及预后评价有较好的实用价值；③初发型ITP患者以PAIgG和PAIgA增高为主，预后较好；复发型以PAIgM增高为主，预后较差，易复发。     

人微小病毒B19感染与小儿ITP的关系

采用PCR检测41例特发性血小板减少性紫瘢（ITP）患儿和22例健康儿童的血清人微小病毒B19（HPVB19）DNA，并用酶联免疫法（ELISA）检测血小板相关抗体。结果ITP组患儿血清中HPVB19DNA阳性率39．0％（16／41）显著高于对照组；ITP组中急性型HPVB19DNA阳性率50．0％（14／28）显著高于慢性型15．4％（2／13）；病毒感染阳性患儿的血小板相关抗体PAIgG、PAIgA明显高于病毒感染阴性患儿。ITP患儿血清中HPVB19DNA阳性率高，病毒感染可导致血小板相关抗体升高而致血小板减少。     

慢性特发性血小板减少性紫癜患者PAIgG、ACAIgG的测定及意义

应用ELISA法对 41例慢性特发性血小板减少性紫癜 (ITP)患者和 2 5例健康对照者进行血浆和 (或 )血清血小板相关抗体 (PAIgG)、抗心磷脂抗体 (ACAIgG)测定 ,并常规血小板计数。发现ITP患者治疗有效组治疗后血小板计数明显升高、PAIgG含量显著降低 ,治疗前较治疗后及对照组均有显著性差异 (P <0 .0 1 ) ,且血小板计数与PAIgG之间呈显著性负相关 (r =- 0 .738,P <0 .0 0 1 )。而治疗无效组血小板计数、PAIgG均无明显变化。ACAIgG治疗前后无明显变化 ,且未发现与血小板及PAIgG相关。PAIgG、ACAIgG含量增高提示ITP患者针对血小板磷脂及其他糖类蛋白等不同抗原产生了自身免疫反应 ,为免疫抑制剂的应用提供了理论依据     

CD28/CTLA-4:B7在特发性血小板减少性紫癜患者外周血淋巴细胞中的表达及意义

目的:探讨特发性血小板减少性紫癜(ITP)患者外周血淋巴细胞CD28、CTLA-4(CD152)、B7-1(CD80)及B7-2(CD86)的表达及意义。方法:采用免疫荧光标记和流式细胞术检测41例ITP患者和40例健康对照者外周血CD3+CD28+细胞、CD3+CD152+细胞、CD80+CD19+细胞和CD86+CD19+细胞分别占淋巴细胞的比例及血小板表面相关抗体水平,进行2组对比、分析。结果:与正常对照组相比,急性ITP患者外周血CD3+CD28+细胞和CD3+CD152+细胞差异无统计学意义(P0.05),CD80+CD19+细胞增多(P0.05),CD86+CD19+细胞显著增多(P0.01),慢性ITP患者CD86+CD19+细胞增多(P0.05);急性ITP患者外周血CD86+CD19+细胞较慢性ITP患者增多(P0.05);与正常对照组相比,急性ITP患者PAIg's、PAIgG和PAIgM水平显著增高,慢性ITP患者PAIgG水平增高;CD80、CD86表达与PAIgG水平之间存在显著的相关性(均P0.01)。结论:ITP患者外周血B淋巴细胞上CD86和CD80表达均异常,可能与其发病相关。     

炔羟雄烯唑对免疫性血小板减少性紫癜的作用

免疫性血小板减少性紫癜(ITP)通常是由 IgG 型扰血小板抗体引起的。ITP 患者血小板表面的 IgG 一般是增高的,其水平随血小板数目变化而变化。许多慢性 ITP 使用炔羟雄烯唑(danazol)和长春花碱治疗     

蛋白A柱免疫吸附疗法对血小板减少病人的作用

免疫性血小板减少性紫癜(ITP)是一种伴有抗血小板抗体形成的疾病。最近 Bertram 等证实,用葡萄球菌蛋白 A 柱对人类免疫缺陷病毒感染伴发 ITP 的病人进行免疫吸附治疗是有效的。本文报道用蛋白 A 柱对其它类型血小板减少的病人进行免疫吸附治疗的结果。病人 10例血小板减少患者,其中急性 ITP 2例,先前未接受过任何治疗的慢性 ITP 1例,曾用多种方法治疗无效的慢性 ITP 3例,再生障碍性贫血2例,慢性粒细胞白血病急性变2例。后4例由于同种免疫对输注血小板无反应。所有病人在进入本研究时血小板均<20 000/μi。方法免疫吸附采用葡萄球菌蛋白 A 硅柱(Pros-orba columns),采集200～300cc 血浆,通过此柱后再以10ml/min 的速率回输给患者,每周进行1～2     

Investigation of megakaryocyte apoptosis in children with acute and chronic idiopathic thrombocytopenic purpura

OBJECTIVE: Although the platelet destruction shows a primary role in the thrombocytopenia of idiopathic thrombocytopenic purpura (ITP), it has been demonstrated that impaired platelet production may also contribute to the severity of thrombocytopenia in ITP. The present study examined megakaryocyte apoptosis in bone marrow aspirates of children with acute and chronic ITP and investigated the role of megakaryocyte apoptosis in ITP pathophysiology. METHODS: Thirteen children diagnosed with acute ITP and eight children diagnosed with chronic ITP comprised the study group. Ten children, who were hospitalized for scoliosis operation but healthy otherwise, comprised the control group. In all children, megakaryocytes were isolated from the same amount of bone marrow aspirate samples using MACS CD61 MicroBeads (Miltenyl Biotec, Auburn, CA, USA). Megakaryocyte apoptosis was studied with transferase-mediated d-UTP-bitin nick end-labeling method. RESULTS: Isolated megakaryocyte counts did not differ significantly between acute ITP, chronic ITP and control groups. The percentage of apoptotic megakaryocytes did not differ significantly between acute ITP group and control group and between chronic ITP group and control group. The percentage of apoptotic megakaryocytes in patients with chronic ITP was significantly lower than the patients with acute ITP. There was no correlation between the percentage of apoptotic megakaryocytes and platelet counts of the cases. CONCLUSIONS: Increased megakaryocytic apoptosis does not play a role in the pathogenesis of dysmegakaryopoiesis and impaired platelet production in children with ITP. Decreased megakaryocyte apoptosis in cases with chronic ITP may be due to suppression of megakaryocyte maturation, as the terminal phase of the megakaryocyte lifespan is characterized by the onset of apoptosis.     

HLA antigens, blood groups and immunoglobulin levels in idiopathic thrombocytopenic purpura

Thirty-three patients with idiopathic thrombocytopenic purpura (ITP) were tested for HLA-A, B and C antigens, platelet antibodies, immunoglobulin levels and ABO blood groups. With one exception, ITP proved not to be significantly associated with the HLA antigens studied; an increased frequency of HLA-A28 was found in chronic ITP patients (50 vs. 18.7% in the control population). An increased incidence of blood group A was found in ITP patients (64 vs. 37.98% in the control population), especially in those with acute ITP (84.7%). A significant reduction of IgG levels was noted in patients with chronic ITP, while below-normal levels of IgA were found in both chronic and acute ITP patients. There was no difference in levels of IgM. Circulating platelet isoantibodies were demonstrated in 67.6% of the ITP patients. No correlation was demonstrated between the presence of platelet antibodies, immunoglobulin levels of HLA antigens.     

Micromethod for demonstrating increased platelet surface immunoglobulin G: findings in acute, chronic, and human immunodeficiency virus-1-related immunologic thrombocytopenias

A method has been developed for the demonstration of increased platelet surface IgG that uses 1 ml of blood regardless of the platelet count. Platelets are gel filtered to remove plasma and contaminating lymphocytes. They are then reacted with fluorescein-conjugated antihuman IgG and analysed by flow cytometry. Percent positive staining cells vary from 10% to 80% of total cells examined. A platelet antibody index is derived from the product of percent positive staining cells X mean fluorescence intensity of positive staining cells. All patients studied with chronic idiopathic thrombocytopenia purpura (ITP) or human immunodeficiency virus-1 (HIV-1)-related thrombocytopenia had increased platelet surface IgG. Twelve acute children and 11 chronic children had indices averaging 3.5- and 8.9-fold greater than 12 normal children, respectively. Five of 12 children with acute ITP had normal platelet IgG. There was no linear correlation between the platelet antibody index and platelet count. Platelets of patients with acute, chronic, or HIV-1-related ITP displayed autofluorescence. In chronic ITP, the percentage of platelets displaying autofluorescence had a significant negative correlation with the platelet count. This technique will be a valuable diagnostic tool in the pediatric population.     

Platelet-associated IgG in immune thrombocytopenic purpura

A method for the measurement of immunoglobulin G associated with gel- filtered platelets is described and finding in 70 control subjects and 37 patients with immune thrombocytopenic purpura (ITP) are reported. Control platelet-associated IgG (PAIgG) levels (nanograms IgG per 10(9) platelets) averaged (+/-SD) 1231+/-424; samples studied after 24 and 48 hr remained within the control range. PAIgG values of 19 adult and 12 childhood patients with chronic ITP averaged 4711+/-3025 and 4923+/- 3955, respectively, and differed significantly from controls (p less than 0.001). There was an inverse correlation between PAIgG values and the chronic ITP patient's platelet count. Six patients with childhood acute ITP had PAIgG levels ranging from 5588 to 56,250 and appeared to represent a different statistical population from those with chronic ITP. In chronic ITP patients responding to splenectomy, there was an immediate normalization of PAIgG levels; however, a certain percentage of patients studied several months after splenectomy evidenced elevated PAIgG levels in association with normal platelet counts. These data showed that the direct measurement of platelet associated antibody is a useful technique in the diagnosis and follow-up of patients with chronic ITP. Preliminary studies in patients with acute ITP have suggested that this method may be useful in differentiating acute and chronic childhood ITP.     

Characterization of autoantibodies against the platelet glycoprotein antigens llb/llla in childhood idiopathic thrombocytopenia purpura

The majority of children with idiopathic thrombocytopenia (ITP) have an acute self-limiting course and no diagnostic test has been identified which will predict the course of thrombocytopenia and detect those with the chronic autoimmune form. The detection of autoantibodies directed against the platelet glycoprotein complex llB/llla, may identify patients with chronic ITP. Serum anti-GP llb/llla antibodies were assessed by the indirect MAIPA assay in 54 children with immune thrompocytopenla at initial presentation along with an additional 7 children previously diagnosed with chronic ITP, to determine if there was a difference in antibody positivity between acute and chronic ITP patients, and whether the identification of antibodies could be used as a predictive test at diagnosis. There was no significant difference in the percentage of antibodies detected in children classified with acute ITP (27/40–68%) compared to children with chronic ITP (13/21-62%, P > 0.05). Patients with acute ITP had significantly lower mean platelet counts at diagnosis compared to the chronic ITP group (16,225/mm³ vs. 32,250/mm³, P < 0.05), though there was no significant difference in the bleeding manifestations between the acute and chronic ITP groups. Serum anti-GP llb/llla antibodies are detected in a high percentage of children with ITP and autoantibodies appear to be involved in the pathogenesis of both acute and chronic ITP. The detection of anti-GP llb/llla antibodies at diagnosis, however, does not appear to be a useful prognostic test in childhood ITP. © 1995 Wiley-Liss, Inc.     

Intravenous Gammaglobulin Therapy in Idiopathic Thrombocytopenic Purpura

The effect of high-dose intravenous gammaglobulin (IVG) therapy with a CLB preparation was studied in 42 patients: 8 patients had acute and 26 patients had chronic idiopathic thrombocytopenic purpura (ITP); 5 patients had thrombocytopenia accompanied by various diseases such as systemic lupus erythematosus, auto-immune haemolytic anaemia and neutropenia; 3 patients had hypoplastic anaemia and 1 patient had neutropenia and rheumatoid arthritis. After treatment, a rise in platelet count occurred in about 75% of the patients with ITP, although there was no sustained response in any of the patients. There was no correlation between the strength of platelet antibodies as detected by the direct immunofluorescence test before infusion and the pattern of response to the infusion. In most cases of ITP, no immune complexes, as measured by Clq-binding assay, were observed. Furthermore, we found no relationship between the amount of Clq-binding activity of patients' sera and the reaction pattern after infusion of IVG. Splenectomy of the patient had no influence on the outcome of IVG therapy.     

Circulating thrombopoietin level in chronic immune thrombocytopenic purpura

The circulating thrombopoietin (TPO) level in 43 patients with chronic immune thrombocytopenic purpura (ITP) was examined by an ELISA system. The TPO level (mean±SD) in ITP patients was mildly elevated (1.86±1.17 fmol/ml) compared to that in normal subjects (0.76±0.21), and was within the normal range in 30% of ITP patients. In contrast, the TPO level in patients with aplastic anaemia was very high, 12.35±6.42 fmol/ml. There was no correlation between TPO level and platelet count in ITP patients. Splenectomy was performed in two ITP patients, after which platelet counts increased to normal levels and TPO levels showed a transient increase. These data suggest that reactive TPO production against thrombocytopenia in ITP is small when compared to that in aplastic anaemia. Relative endogenous TPO deficiency may play some role in the pathophysiology of thrombocytopenia in ITP patients.     

Idiopathic Thrombocytopenic Purpura in Children; Detection of Platelet Autoantibodies by Immunofluorescence

There are 3 varieties of idiopathic thrombocytopenic purpura (ITP) in children: the acute, the chronic and the recurrent forms. We have tested the platelets from 80 children with these forms of ITP by an immunofluorescence test. Platelet-bound immunoglobulins were detected in 19/22 acute, 41/48 chronic and 5/10 recurrent cases of ITP, i.e. in 65 of a total of 80 cases. A significance differences between acute and chronic ITP was found in the observation that only IgM was bound to the platelets of 7/16 patients with acute ITP. In all cases of chronic ITP with a positive direct immunofluorescence test, IgG was present on the platelet membrane. It is assumed because some patients with chronic ITP do not have elavated platelet-bound immunoglobulins, and show refractoriness to immunosuppressive therapy, that in these patients the disorder is not of a humoral autoimmune nature.     

Functional role of CD4+CD25+ regulatory T cells and transforming growth factor-beta1 in childhood immune thrombocytopenic purpura

CD4+CD25+ regulatory T cells (Tregs) are critical in maintaining self tolerance and preventing organ specific autoimmune diseases. Their role in childhood immune thrombocytopenic purpura (ITP), an immune disorder in which the production of platelet autoantibodies might be caused by cytokine network dysregulation, has not been clearly defined. Transforming Growth Factor-beta1 (TGF-beta1) has been suggested to mediate Tregs suppressive function. The aim of this study was to assess cell populations of CD4+CD25+ T regulatory cells as well as mRNA expression of TGF-beta1 level in peripheral blood of children with ITP and evaluate their possible role in prediction of disease severity and response to therapy. Thirty-three children with ITP (13 acute and 20 chronic cases) and 10 healthy controls were studied. CD4+CD25+ regulatory T cells were assessed in peripheral blood by flowcytometry. Expression of TGF-beta1 mRNA was examined by real-time quantitative polymerase chain reaction assay. The frequency of CD4+CD25+ Tregs was significantly decreased in acute and chronic ITP patients as compared to controls. Acute ITP patients had significantly lower percentage of Tregs compared with chronic ITP patients. Higher frequency of CD4+CD25+ Tregs was detected in chronic ITP patients with platelet count >100 x 10(9)/L compared with patients with platelet count <100 x 10(9)/L and in steroid responsive patients compared with steroid resistant patients. The expression of mRNA TGF-beta1 level was significantly lower in acute and chronic ITP patients compared with controls and in chronic ITP patients with pltc <100 x 10(9)/L in comparison with patients with pltc > 100 x l0(9)/L. A significant positive correlation was found between percentage of CD4+CD25+ Tregs and mRNA expression of TGF-beta1 in chronic ITP patients. In conclusion, immune regulation of TGF-beta1 by Tregs may play a fundamental role in the pathogenesis of childhood immune thrombocytopenic purpura. This might form a base for future specific immunomodulatory therapies for ITP.     

An FcγRIIb transmembrane polymorphism in Chinese ITP patients

Immune thrombocytopenic purpura (ITP) is putatively associated with self-antibodies against platelet. FcγRIIb is a key regulator of B cell responses. To explore the relationship between the polymorphism of FcγRIIb transmembrane portion and ITP, a cohort control study was carried out. Two hundred and eighty ITP patients and 243 healthy volunteers were enrolled in this study. Most of the ITP patients were followed up for at least 6 months following diagnosis to allow classification of chronic or acute ITP. The concentrations of IgG/IgA/IgM antiplatelet antibodies (PAIgG/IgA/IgM) were determined by a competitive micro-ELISA method. Genomic DNA was isolated and a single nucleotide polymorphism (SNP) of the FcγRIIb transmembrane exon located at position 695 was detected by real-time florescent PCR. The presence of 695T > C polymorphism was detected by the pattern of melting curve peak. The distribution of FcγRIIb genotypes was not significantly different between ITP patients and healthy controls. The homozygous 695C/C proportion in child-onset ITP patients was lower than that in the healthy control group, but had no statistical significance. FcγRIIb transmembrane polymorphism had no relationship with chronic ITP or acute ITP when compared with healthy controls. The FcγRIIb 695C allele carrying had no influence on the levels of platelet antibodies such as IgG, IgA or IgM. However, the PAIgA/IgM levels associated with the clinical experience of developing chronic ITP. Here we concluded one hot-spot polymorphism in FcγRIIb transmembrane sequences was not associated with the development of ITP.