血小板自身抗体的流式细胞仪检测与意义

目的：应用流式细胞术（ＦＣＭ）测定血小板自身抗体ＰＡＩｇＧ,研究其对待发性血小板减少性紫癜（ＩＴＰ）诊断的意义。方法：应用ＦＣＭ检测２０例ＩＴＰ患者血小板膜表面及血浆中的抗血小板抗体,并与ＥＬＩＳＡ所测结果进行比较。结果：ＦＣＭ测得１４例（７０％ＩＴＰ患者血小板膜自身抗体ＰＡＩｇＧ阳性,９例（４５％）血浆抗血小板抗体阳性。ＥＬＩＳＡ显示１２例（６０％）患者ＰＡＩｇＧ阳性,７例（３５％）血浆抗血小板     

慢性特发性血小板减少性紫癜自身抗体克隆性分析

目的 了解慢性特发性血小板减少性紫癜(ITP)自身抗体克隆性生成特性。方法 采用改良的单克隆抗体免疫固定特异血小板抗原(MAIPA)法检测43例慢性ITP患者血清血小板膜糖蛋白(GP)特异性IgG抗体及其重链亚型和轻链表型,采用PCR技术分析患者淋巴细胞免疫球蛋白重链基因重排。结果 43例中16例(38％)血清中至少存在一种抗GP(GPⅡb／Ⅲa、GPⅠb、GPⅠa、GPⅣ、GPⅤ)IgG抗体。73％(8／11)的血清糖蛋白特异性自身抗体表现重链限制性,仅表达一种重链亚型;80％(16／20)的糖蛋白特异性抗体仅表达一种轻链表型;6例患者的糖蛋白特异性抗体既表现为轻链限制性又表现为重链限制性。PCR分析显示,3例存在淋巴细胞重链基因重排。结论 部分慢性ITP患者GP特异性自身抗体源于寡克隆B淋巴细胞增生。     

特发性血小板减少性紫癜患者血小板相关抗体及网织血小板检测的临床意义

目的探讨血小板相关抗体(PAIgG)和网织血小板(RP)检测在特发性血小板减少性紫癜(ITP)治疗中的临床意义。方法收集常州市第一人民医院2003-06～2004-10住院的ITP患者48例,根据疗效分为两组,其中临床有效组40例,临床无效组8例,以50例健康人为正常对照。应用流式细胞仪(FCM)测定其治疗前后血浆和(或)血清PAIgG及RP%,并常规血小板计数。结果ITP患者临床有效组治疗后较治疗前血小板计数明显升高、PAIgG和RP%显著降低(P<0.01)。临床无效组治疗前后血小板计数、PAIgG和RP均无明显变化(P>0.05)。两组治疗前3项指标与对照组比较差异有显著性意义(P<0.01)。结论PAIgG和RP动态检测将是ITP患者血小板治疗效果的预测指标之一。     

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。     

评价血小板相关抗体定量测定法对特发性血小板减少性紫癜的诊断价值

目的 :评价血小板相关抗体 (PAIg)定量测定法对诊断特发性血小板减少性紫癜 (ITP)的价值。 方法 :应用酶联免疫竞争抑制试验 (ELISA)定量测定血小板相关抗体 ,计算试验的灵敏度、特异度、准确度、阳性预测值 ,绘制受试者ROC曲线进行诊断价值的评价。结果 :根据常用判断PAIg阳性的临界值 ,血小板相关抗体试验诊断ITP的灵敏度较高 (95 % ) ,但特异度低 (48.8% ) ,准确度低 (5 7.5 % ) ,阳性预测值低 (30 .2 % )。用ROC曲线寻找诊断ITP的最佳临界值 ,用新的临界值 (PAIgG >16 0ng/10 7血小板或PAIgA >2 5ng/10 7或PAIgM >35ng/10 7)时 ,可提高诊断试验的特异度 ,从 4 8.8%提高到 6 2 .8% ,可确诊更多的患者 ;但准确度和阳性预测值仍低 ,分别为 6 7.9%和 36 .0 %。结论 :PAIg试验诊断ITP的特异度、准确度、阳性预测值较低 ,帮助诊断ITP的临床价值有限 ,即使用新的临界值提高了试验的特异度 ,但诊断ITP的价值仍不大。     

网织血小板检测在特发性血小板减少性紫癜中的临床价值

特发性血小板减少性紫癜(ITP)是一种因为产生抗自身血小板抗体促使血小板减少而使机体器官受损的特异性自身免疫性疾病。血小板减少引起的出血是ITP较常见的表现之一。血小板减少的主要原因大致为两类：血小板生成减少和血小板破坏加速。网织血小板(RP)是新释放入血液循环的血小板，是富含RNA类似于网织红细胞(Ret)的最年轻的血小板。RP的数目反映了人体血小板的更新速度、血小板增生情况。2002年11月～2003年4月我们检测了50例健康人、48例ITP患者、35例急性白血病、25例再生障碍性贫血外周血的RP，旨在探讨RP在特发性血小板减少性紫癜诊断和治疗中的临床价值，以便更好为临床上诊治ITP提供一种新的手段。     

流式细胞术检测血小板相关IgG的临床应用

目的：研究流式细胞式（ＦＣＭ）检测血小板相关免疫球蛋白Ｇ（ＰＡＩｇＧ）的特点及在特发性血小板减少性紫癜（ＩＴＰ）诊断中的意义。方法：用ＦＣＭ检测了４７例ＩＴＰ、１３例非免疫性血小板减少、１０例自身免疫溶血性贫血、１８例其它免疫系统疾病患者以及３１例健康志愿者的ＰＡＩｇＧ,并与ＥＬＩＳＡ竞争法检测结果比较。结果：ＦＣＭ检测ＩＴＰ患者ＰＡＩｇＧ的平均荧光强度和阳性血小板百分率均明显高于正常对照组（Ｐ〈０．０１）,１１例有峰形异常。ＩＴＰ患者ＦＣＭ检测的阳性率（８７．２％）比ＥＬＩＳＡ法（８３．０％）稍高,两种方法的符合率为８５．１％。但两种方法都存在特异性不高的问题。结论：ＦＣＭ检测ＰＡＩｇＧ具有快速、简便和灵敏等特点,可作为免疫性血小板减少实验诊断的新方法。     

人源化抗血小板膜糖蛋白Ⅱb/Ⅲa抗体库的构建及临床价值

目的筛选出抑制血小板聚集的血小板膜糖蛋白(GP)Ⅱb/Ⅲa自身抗体,用噬菌体表面展示技术构建人源化抗血小板GPⅡb/Ⅲa单链噬菌体抗体(ScFv)库。方法用单克隆抗体特异性俘获血小板抗原(MAIPA)技术和血小板聚集试验筛选出血浆中含有抑制血小板聚集的血小板GPⅡb/Ⅲa自身抗体的特发性血小板减少性紫癜(ITP)患者。从筛选出患者的外周血淋巴细胞中提取mRNA,用RT PCR扩增出人免疫球蛋白的重链可变区(VH)和轻链可变区(VL)基因片断,用DNA linker将VH和VL连接成ScFv基因片断。用限制性内切酶SfiⅠ/NotⅠ酶切ScFv后克隆到噬菌体载体pHEN2,然后转化大肠杆菌TG1。用辅助噬菌体M13K07援救转化后的TG1,产生ScFv。结果95例慢性ITP患者中41例(43.2%)血浆中抗GPⅡb/Ⅲa自身抗体阳性,强阳性患者5例(5.3%)。2例(2.1%)明显抑制血小板聚集功能。扩增出380～400bp大小的VH和VL基因,用连接肽(Gly4Ser)3成功地连接成约780bp大小的ScFv片断。ScFv克隆到pHEN2并转化大肠杆菌TG1后,形成2.1×107个克隆。用辅助噬菌体M13K07援救TG1后产生的噬菌体抗体库滴度为1.62×1010cfu/ml。结论少数抗GPⅡb/Ⅲa自身抗体可抑制血小板聚集功能。用噬菌体表面展示技术构建了ScFv库,可用来筛选人源化抗血小板GPⅡb/ⅢaScFv。     

特发性血小板减少性紫癜患者抗心磷脂抗体的意义

自 1 98 3年抗磷脂抗体 (APL )检测方法建立以来 ,促进了人们对 APL及与 APL相关疾病的研究。 APL可引起组织损伤 ,致多种临床症状 ,称为抗磷脂综合征 (APS)。 APL也可出现于多种疾病中。关于 APL在特发性血小板减少性紫癜 (ITP)的作用的研究 ,文献报道较少见。本文对 68例 ITP患者血清 Ig G型抗心磷脂抗体 (Ig G- ACLA)进行检测 ,以探讨其在 ITP患者发病中的意义。1　材料与方法1 .1 　 病例选择ITP组 68例为我院门诊及住院的 ITP患者 ,均符合 1 995年第五届血栓与止血学术会议修订的ITP诊断标准。其中男 2 0例 ,女 48…     

特发性血小板减少性紫癜患者血小板抗体等四项参数测定的价值

目的　探讨特发性血小板减少性紫癜 (ITP)患者血小板抗体 (PAIgG)、血小板膜蛋白(CD62P)、网织血小板 (RP)及淋巴细胞亚群变化及意义。方法　应用流式细胞术检测 5 8例ITP组及 2 0例正常对照组外周PAIgG、血CD62P、RP、淋巴细胞亚群的表达。结果　ITP组的血小板数明显低于正常对照组 (P <0 .0 1) ,PAIgG、CD62P、RP均明显高于正常对照组 (P <0 .0 1)。在淋巴细胞亚群中 ,ITP组CD3、CD4、CD4/CD8比值明显低于正常对照组 (P <0 .0 1) ,CD8、CD19细胞则显著高于正常对照组 ,而CD16+ 5 6与正常对照组无明显差异。结论　PAIgG、CD62P、RP及淋巴细胞亚群的变化可较好地反映ITP这一病理过程 ,对提高诊断水平及指导临床有一定实用价值。     

Analysis of outcome of laparoscopic splenectomy for idiopathic thrombocytopenic purpura by platelet count

Laparoscopic splenectomy (LS) is now performed routinely in patients with idiopathic thrombocytopenic purpura (ITP) refractory to the medical treatment. Low preoperative platelet count was deemed to be a contraindication for a laparoscopic approach; however, there is no data reporting the outcome in those patients. We aimed to evaluate the influence of the preoperative platelet count on the operative and postoperative course and complication rate. Retrospective cohort study that was conducted in tertiary care university-affiliated medical center and included 110 consecutive patients who underwent LS. All patients were divided into three groups by their preoperative platelet counts: 50 x 10(9)/L (n = 80). The outcome and the influence of preoperative factors predictive of complications, blood transfusion, and length of stay were compared between the groups. Patients with a platelet count of 20 x 10(9)/L before surgery. Patients with counts >20 x 10(9)/L can safely undergo LS.     

Relationship between platelet volume and anti-platelet autoantibodies in idiopathic thrombocytopenic purpura

We used flow cytometry to explore the relationship between platelet volume and anti-platelet autoantibodies in 71 patients with idiopathic thrombocytopenic purpura (ITP). An increase in platelet volume was found more frequently in patients with a platelet count of less than 20,000/microliters. Platelet volume was larger in patients without anti-GPIIb/IIIa autoantibodies than in patients with these autoantibodies. Furthermore, the platelet count was significantly lower in patients without anti-GIIb/IIIa autoantibodies than in the patients with these autoantibodies. There was a positive correlation between a large platelet volume in patients with a platelet count of less than 30,000/microliters and high platelet-associated IgM levels. These results suggest that the platelet volume is related to the severity of thrombocytopenia in ITP.     

Discrepancy between impedance and immunofluorescence platelet counting has implications for clinical decision making in patients with idiopathic thrombocytopenia purpura

This study investigated whether differences occur between the impedance and immunofluorescence methods for platelet quantification in idiopathic thrombocytopenia purpura (ITP). Immunofluorescence gave a platelet count >50% higher than the impedance test in 9/35 (26%) patients, of which 4/35 (11%) were >100% higher. The clinical severity of thrombocytopenia was changed as a result of the immunofluorescence test in 14/35 (40%) patients. Neither mean platelet volume nor platelet distribution width predicted impedance/immunofluorescence method discrepancy. It is suggested that immunofluorescence platelet counts should be performed on all ITP patients when the implementation of a therapeutic or diagnostic intervention is being considered.     

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.     

The IgG subclasses of platelet-associated autoantibodies directed against platelet glycoproteins IIb/IIIa in patients with idiopathic thrombocytopenic purpura

The majority of patients with idiopathic thrombocytopenic purpura (ITP) have antiplatelet autoantibodies that are most frequently directed against platelet glycoproteins IIb/IIIa or Ib/IX/V. However, there is some debate whether the immune response is oligoclonal or polyclonal in nature. We investigated the subclass distribution of anti-IIb/IIIa IgG autoantibodies in 59 prospectively studied patients with ITP. We also tested patients with a variety of thrombocytopenic disorders (n=31) and healthy controls (n=30). Platelet lysates were tested for IgG anti-IIb/IIIa autoantibodies, and the specific IgG subclass distribution was measured using antigen capture assays. All testing was done blinded to diagnosis and other assay results. After unblinding, we found that 43 of the 59 ITP patients had anti-IIb/IIIa autoantibodies (sensitivity=73%). Anti-IIb/IIIa autoantibodies were also detected in five of the 31 non-ITP patients, but in none of the 30 healthy controls (specificity=91%). The IgG subclass assay was positive in 39 of the 43 ITP patients with anti-IIb/IIIa antibodies (sensitivity=92%) and in 12 samples that had no detectable anti-IIb/IIIa antibodies including two ITP patients (specificity=83%). The most common subclass in the ITP patient samples was IgG1 (77%), either alone (n=14) or with other IgG subclass antibodies (n=19). However, there were also patients with only IgG2 (n=2), IgG3 (n=3) or IgG4 (n=3) antibodies. Our results are consistent with the hypothesis that ITP is a heterogeneous disorder and that some patients have evidence of oligoclonality, whereas other patients have polyclonal autoantibodies.     

Pulsed high-dose dexamethasone in refractory chronic idiopathic thrombocytopenic purpura: a report on 10 cases

Summary. We treated 10 patients who had chronic refractory idiopathic thrombocytopenic purpura (ITP) with high-dose dexamethasone (DXM, 40mg/d for 4 sequential days every month). The interval from diagnosis ranged from 49 to 300 months, and patients had previously received a median of 5.5 treatments (including splenectomy in nine cases). Median platelet count was 14 × 10⁹/1 (range 6-26 × 10⁹/1) at the onset of DXM and eight patients had bleeding symptoms. Eight patients received at least three cycles of DXM.
Five patients had a response (i.e. platelet count at least doubled and increased by >20 × 10⁹/1), including one almost complete remission and four minor responses (MR).
Of the MR, one was probably due to concurrent IVIg administration, and all four MR were transient, in spite of further cycles of DXM. In three patients DXM was a failure after three or four cycles. In two patients DXM had to be stopped after one course because of major side-effects (systemic hypertension with stroke and insulin-dependent diabetes, respectively).
In our experience, high-dose DXM had a relatively limited effect in chronic refractory ITP and was associated with severe side-effects in some cases.     

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.     

Effect of Helicobacter pylori eradication on platelet recovery in Japanese patients with chronic idiopathic thrombocytopenic purpura and secondary autoimmune thrombocytopenic purpura

The prevalence of Helicobacter pylori infection and the effect of its eradication on platelet count in 48 Japanese patients with autoimmune thrombocytopenic purpura (AITP), including 40 chronic idiopathic thrombocytopenic purpura (ITP) and eight secondary AITP, were investigated. H. pylori infection was found in 25 ITP patients (62.5%) and in two secondary AITP (25%). H.pylori eradication was obtained in 19 of 19 infected ITP patients (100%), who were not in remission (platelets < 100 x 109/l) at the time of infection assessment. During follow-up (median 14.8 months), 12 of 19 H. pylori-eradicated patients (63.2%) showed a significant increase in platelet count accompanied by a significant decrease of platelet-associated immunoglobulin G (IgG). This response was maintained in all responding patients throughout the follow-up period. However, two infected patients with secondary AITP did not show platelet increase after eradication. The assessment of H. pylori infection and its eradication should be attempted in ITP as this approach could be an effective strategy, at least for some of these patients.     

Effects of IgG and its F(ab')2 fragments of some patients with idiopathic thrombocytopenic purpura on platelet aggregation

OBJECTIVES: To make humanized monoclonal antibodies by phage surface display technology, we screened out the specific anti-platelet glycoproteins (GPs) IgG antibody from patients with chronic idiopathic thrombocytopenic purpura (ITP), which can inhibit platelet aggregation. METHODS: We studied plasmas from 68 patients with ITP for the presence of IgG antibodies specific for GPIIb/IIIa and/or GPIb/IX using modified monoclonal antibody immobilization of platelet antigen assays. The IgG antibody and its F(ab')(2) fragments of the positive plasmas which could inhibit platelet aggregation function were prepared and purified. Their immunoreactivity to platelet GPs and effects on platelet function were further analyzed. RESULTS: GPIIb/IIIa- and GPIb/IX-specific antibodies were found in 21 and 19 patients, respectively. Six of them had antibodies against both GP complexes. Among the 34 positive plasmas, four with positive anti-GPIIb/IIIa autoantibody showed significant inhibition of platelet aggregation induced by adenosine diphosphate (ADP), whereas one with GPIb/IX-specific antibody inhibited ristocetin-induced platelet aggregation. The purified IgG and its F(ab')(2) fragments from two patients not only retained the ability to bind to platelet GPs but also impaired the in vitro ADP-induced platelet aggregation. CONCLUSIONS: F(ab')(2) portion of the IgG is a functional fragment, which is responsible for the autoantibody interaction with platelet GPs in ITP, and some of them also affect platelet function, which can be used to develop completely humanized anti-GPIIb/IIIa small molecular phage antibody.