ITP血小板特异性抗体和T淋巴细胞亚群及NK细胞变化的意义探讨

目的：检测特发性血小板减少性紫癜（ITP）患者抗血小板膜糖蛋白（GPⅡb／Ⅲa、GPⅠb／Ⅸ）特异性抗体表达、T淋巴细胞亚群及NK细胞的变化,探讨相关因素在ITP发病机制中的作用。方法：应用改良血小板抗原单克隆抗体固相化检测技术（MAIPA）、流式细胞术分别检测52例ITP和24例正常对照组抗血小板膜糖蛋白（GPⅡb／Ⅲa、GPⅠb／Ⅸ）特异性抗体表达、T淋巴细胞亚群及NK细胞变化。结果：ITP组的血小板计数明显低于正常对照组（P〈0．05）;抗GPⅡb／Ⅲa及GPⅠb／Ⅸ抗体A值均高于正常对照组（P〈0．05）;相关分析表明ITP组血小板计数与两种特异性抗体水平均呈负相关关系;在T淋巴细胞亚群变化中,ITP组CD3^＋T淋巴细胞百分比、CD4^＋T淋巴细胞百分比及CD4^＋／CD8^＋的比值均明显低于正常对照组（P〈0．05）,CD8^＋T淋巴细胞百分比则显著高于正常对照组（P〈0．05）;NK细胞百分比明显低于正常对照组（P〈0．05）。结论：血小板特异性抗体及T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的实用价值。     

流式微球技术检测血小板膜糖蛋白自身抗体的方法建立及临床价值

目的:建立流式微球技术检测血小板膜表面糖蛋白(GPⅡb/Ⅲa和GPⅠb/IX)特异性自身抗体的方法,并评价其对特发性血小板减少性紫癜(ITP)的诊断价值及临床意义。方法:应用抗人血小板GPⅡb/Ⅲa(CD41a)、GPⅠb/IX(CD42b)单抗包被微球,将血小板从待测血液样本中分离并裂解,血小板裂解后与包被好的微球共同孵育,加入PE标记的羊抗人IgG多克隆抗体,上机检测,流式细胞仪分析ITP组和非免疫性血小板减少组患者自身抗体表达情况。将所测样本的平均荧光强度值(MFI)与正常对照(3个不同正常人血样)MFI的均值进行比较,计算其比率。结果:ITP组该比率均值及范围:GPⅡb/Ⅲa:6.52(0.22～30.20),GPⅠb/IX:6.13(0.56～22.00);非免疫性血小板减少组为:GPⅡb/Ⅲa:1.20(0.32～3.04),GPⅠb/IX:1.302(0.33～3.56);正常对照组比率均值及范围为:GPⅡb/Ⅲa:0.96(0.23～2.28),GPⅠb/IX:0.97(0.22～2.13)。ITP组2种自身抗体荧光强度比率明显高于非免疫性血小板减少组和正常对照组(均P<0.01)。以正常对照...     

改良MAIPA与网织血小板检测对免疫性血小板减少症的诊断价值

目的:探讨血小板膜特异性抗体及网织血小板(RP)检测在免疫性血小板减少症(ITP)诊断中的意义及2者的相关性。方法:通过改良的单克隆抗体特异性俘获血小板抗原实验(MAIPA)检测90例ITP患者与20例非免疫性血小板减少患者(NITP)血浆中抗血小板膜糖蛋白(GP)Ⅱb/Ⅲa和Ⅰb/Ⅸ的特异性抗体;应用流式细胞术(FCM)检测以上患者RP%值。结果:①ITP患者抗GPⅡb/Ⅲa、抗GPⅠb/Ⅸ抗体共阳性率为33.30%,单一抗GPⅡb/Ⅲa抗体为8.89%,抗GPⅠb/Ⅸ抗体为12.22%;ITP初发患者血小板膜特异性抗体阳性率为58.73%,复发患者为44.44%,2者差异无统计学意义(P0.05);糖皮质激素应用前的MAIPA实验阳性率为58.46%,高于用药后的阳性率44.00%;PLT≤30×109/L的ITP患者MAIPA的阳性率58.18%高于PLT30×109/L患者的阳性率48.75%,但2者差异无统计学意义(P0.05)。②ITP患者的RP%(5.45±5.78)与正常人(0.81±0.41)比较差异有统计学意义(P0.05),NITP患者RP%(0.89±0.06)与正常人比较差异无统计学意义(P0.05);应用糖皮质激素前92.30%ITP患者高于对照,用药后为72.00%,用药前后RP%比较差异有统计学意义(P0.05)。结论:检测血小板膜特异性抗体的MAIPA实验对ITP的诊断特异性高,单一抗体检测阳性率低,通过2种特异性抗体联合检测可以提高MAIPA的敏感性。糖皮质激素的应用对抗GPⅡb/Ⅲa、抗GPⅠb/Ⅸ抗体的阳性率有影响。RP的检测对于ITP的诊断具有重要意义,该实验也可用于协助激素治疗效果的判断。     

络泰注射液对慢性肺源性心脏病病人血小板活化功能的影响

目的观察络泰注射液对慢性肺源性心脏病活化血小板的影响。方法应用洛秦注射液:应用流式细胞仪(FCM)对62例慢性肺源性心脏病人和60名健康体检者进行血小板活化功能的检测,测定血小板活化依赖性颗粒膜蛋白(CD62p)、血小板糖蛋白GPⅡb/GPⅢa复合物的数值。对照组CD62p,GPⅡb/GPⅢa复合物的变化以及应用洛秦注射液;治疗组用药前后CD62p,GPⅡb/GPⅢa复合物的变化。结果慢性肺源性心脏病病人CD62p较对照组升高(P<0.05);治疗组治疗后其CD62p的均值(2.51±0.68)较对照组(2.89±0.92)下降;治疗组治疗后GPⅡb/GPⅢb的均值(92.72±7.15)较对照组的(98.81±8.53)下降,治疗后治疗组CD62p、GPⅡb/GPⅢb均较治疗前明显下降(P<0.05)。结论肺心病病人存在血小板功能活化;络泰注射液对肺心病病人血小板的活化具有抑制作用。     

TIGIT、CD226、CD96和CD155在血小板糖蛋白自身抗体阳性的原发性免疫性血小板减少症的表达及临床意义

目的:探讨TIGIT、CD226、CD96和CD155在血小板糖蛋白自身抗体(platelet glycoprotein autoantibody, GPs)阳性的原发性免疫性血小板减少症(immune thrombocytopenia, ITP)患者外周血CD8⁺T细胞和树突状细胞(dendritic cell, DC)膜表面的表达,并分析其临床意义。方法:选取41例GPs阳性的ITP患者和20例健康对照者,采用流式细胞术检测CD8⁺T细胞表面TIGIT、CD226和CD96表达比例,并检测ITP患者外周血DC细胞的亚群变化及浆细胞样DC(plasma DC,pDC)上CD155的表达比例。结果:GPs阳性ITP组患者的pDC和CD155⁺pDC比例均明显高于对照组,且GPⅠb/Ⅸ⁺/Ⅱb/Ⅲa^+/-组均明显高于GPⅠb/Ⅸ^-/Ⅱb/Ⅲa⁺组(P<0.05,F=40.83;P<0.01,F=59.50)。GPs阳性ITP...     

抗血小板抗体在系统性红斑狼疮血小板减少患者中临床意义的研究

目的 明确抗血小板抗体在系统性红斑狼疮(SLE)血小板减少患者中的临床意义.方法 采用改良抗原捕获酶联免疫吸附试验(ELISA)法检测抗血小板抗体(抗GPⅡb/Ⅲa、GPⅠb/Ⅸ、GPⅠa/Ⅱa、GPⅣ抗体),分别比较治疗前SLE血小板减少与SLE非血小板减少患者抗血小板抗体的阳性率、SLE血小板减少患者治疗前后抗血小板抗体的阳性率、SLE血小板减少治疗前患者病情与抗血小板抗体的关联性.统计方法采用秩和检验和x2检验.结果 治疗前SLE血小板减少组抗GPⅡb/Ⅲa抗体、抗GP Ⅰb,Ⅸ抗体阳性率分别为50%、67%.非血小板减少组阳性率分别为11%、28%,2组间阳性率差异有统计学意义(P＜0.05);治疗后SLE血小板减少组抗GPⅡb/Ⅲa抗体、抗GP Ⅰb/Ⅸ抗体阳性率分别为6%、28%,较治疗前显著降低(P＜0.05);SLE血小板减少组治疗前抗GPⅡb/Ⅲa抗体、抗GP Ⅰb/Ⅸ抗体之间显著关联,该2种抗体均与SLEDAI评分有显著关联性,与抗核抗体、抗双链DNA(dsDNA)抗体、抗中性粒细胞胞质抗体(ANCA)则无显著关联;各组间未检测出抗GPⅣ和GPⅠa/Ⅱa抗体.结论 抗GPⅡb/Ⅲa、GPⅠb/Ⅸ抗体在病情活动SLE血小板减少患者中表达显著升高,与SLE血小板减少病情发生发展和转归相关.

Abstract：

Objective To evaluate the clinical significance of antiplatelet antibody in patients with systemic lupus erythematosus complicated with thrombocytopenia.Methods Antiplatelet antibody (anti-GP Ⅱb/Ⅲa antibody, anti-GP Ⅰb/Ⅸ antibody, anti-GP Ⅰa/Ⅱ a antibody, anti-GP Ⅳ antibody) were detected by modified antigen capture ELISA. The positive rate of antiplatelet antibody between SLE complicated with thrombocytopenia group and without thrombocytopenia group before therapy were compared,and the positive rate of antiplatelet antibody before therapy and after therapy in SLE complicated with thrombocytopenia were compared,and the relevance between antiplatelet antibody and conditions in SLE complicated with thrombocytopenia were analyzed. Rank test and Chi square test were used for statistical analysis. Results The positive rate of anti-GP Ⅱb/Ⅲa antibody and anti-GP Ⅰb/Ⅸ antibody in SLE complicated with thrombocytopenia group before therapy was 50% and 67% respectively, however,the positive rate in SLE without thrombocytopenia group before therapy was 11% and 28% respectively,there was significant difference between the two groups (P＜0.05) and the positive rate of anti-GP Ⅱb/Ⅲa antibody and anti-GP Ⅰb/Ⅸ antibody in SLE complicated with thrombocytopenia group after therapy was 6% and 28% respectively, which was significantly lower than those before therapy (P＜0.05). In SLE complicated with thrombocytopenia group before therapy, there was significant relevance between anti-GP Ⅱb/Ⅲ a antibody and anti-GP[b/Ⅸ antibody, and there was significant relevance between these two antibodies and SLEDAI score,but no significant relevance between these two antibodies and ANA,dsDNA, ANCA. Neither anti-GPⅣ antibody nor anti-GP Ⅰ a/Ⅱ a antibody was detected in patients of this study. Conclusion The positive rate of antiplatelet antibody (anti-GP Ⅱb/Ⅲ a antibody, anti-GP Ⅰb/Ⅸ antibody) is significantly higher in patients with active systemic lupus erythematosus complicated with thrombocytopenia,and these two antibodies are significantly associated with clinical outcomes.     

利妥昔单抗治疗糖皮质激素无效的特发性血小板减少性紫癜疗效观察

目的 探讨利妥昔单抗治疗特发性血小板减少性紫癜(ITP)的疗效、安全性及治疗前后B细胞、血小板膜糖蛋白(GP)特异性自身抗体的变化.方法 利妥昔单抗(375 mg/m2,每周1次,连用4周)治疗12例糖皮质激素治疗无效的ITP患者,监测治疗前后的血常规、血清免疫球蛋白定量(IgG、IgM、IgA)、血小板GPⅡb/Ⅲa和(或)GP Ⅰ b/Ⅸ特异性自身抗体、CD+3、CD+4、CD+8、CD+19、CD+20细胞数.结果 4例完全有效,3例部分有效,2例微效,3例无效.随访中位时间5(0.5～12)个月,疗效均维持较好.有效患者治疗后血小板自身抗体均转阴.治疗前后血清IgG、IgM、IgA无明显变化,CD+3、CD+4、CD+8细胞数无明显变化.治疗后CD+19/CD+20细胞数(4.1±2.2)×106/L与治疗前(295.0±86.4)×106/L比较明显下降(P＜0.01).无严重不良反应.结论 利妥昔单抗治疗糖皮质激素无效的ITP患者安全、有效.     

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。     

冠心病患者阿司匹林抵抗与血小板膜糖蛋白CD62p及GPⅡb/Ⅲa相关性的研究

目的:观察冠心病(CHD)患者阿司匹林治疗前后血小板膜糖蛋白CD62p和GPⅡb/Ⅲa的变化,探讨其与阿司匹林抵抗(AR)的关系。方法:采用流式细胞术检测60例CHD患者(CHD组)及20例健康人(对照组)的血小板CD62p、GPⅡb/Ⅲa表达的阳性率。根据血小板聚集率(PAG)将CHD组分为AR亚组和阿司匹林敏感亚组(AS亚组)。60例CHD患者接受阿司匹林(100mg,qd)等治疗,2周后复检CD62p、GPⅡb/Ⅲa,并采用比浊法分别测定由二磷酸腺苷(ADP)、花生四烯酸(AA)诱导的PAG。结果:CHD患者治疗后血小板CD62p、GPⅡb/Ⅲa表达阳性率均比治疗前明显降低(P<0.01),但仍高于对照组(P<0.01)。入选60例CHD患者中有8例(13.3%)存在AR。AR亚组血小板GPⅡb/Ⅲa表达阳性率高于AS亚组(P<0.01)。结论:GPⅡb/Ⅲa可作为CHD患者阿司匹林治疗中早期识别AR的指标之一。     

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。     

免疫性血小板减少性紫癜35例患者外周血免疫细胞亚群的检测及其临床意义

目的 探讨免疫细胞亚群的变化在免疫性血小板减少性紫癜(ITP)发病机制中的作用及其临床意义.方法 应用流式细胞术检测35例ITP患者治疗前、后及20例正常对照者免疫细胞亚群各指标的变化,包括CD3+、CD4+、CD8+、CD56+、CD19+淋巴细胞及CD4+/CD8+比值.结果 ITP患者CD3+ T淋巴细胞百分比(61.58±6.45)%、CD4+ T淋巴细胞百分比(28.38±4.89)%、CD4+/CD8+比值(0.99±0.22)较对照组[(67.85±4.68)%、(38.00±3.37)%、1.54±0.13]均减低(P值均＜0.05),治疗后3项指标[(69.41±5.03)%、(38.17±3.18)%、1.60±0.15]均升高至正常水平;CD8+ T淋巴细胞百分比(29.20±4.50)%及CD19+ B淋巴细胞百分比(17.74±4.14)%较对照组[(24.82±2.93)%、(12.09±3.51)%]升高(P值均＜0.05),治疗后2项指标[(24.06±3.02)%、(10.90±3.55)%]均降至正常水平;ITP患者CD56+细胞百分比治疗前(15.80±2.85)%、治疗后(15.16±2.77)%与对照组(16.36±2.75)%差异无统计学意义(P＞0.05).结论 免疫细胞亚群紊乱参与了ITP的发病,对其检测可作为ITP的辅助诊断,在指导治疗方面可能有一定的意义.

Abstract：

Objective To explore the clinical significance of immunocyte subsets before and after immunosuppressive therapy in the peripheral blood of patients with immune thrombocytopenic purpura (ITP).MethodsThe percentages of immunocyte subsets in the peripheral blood of 35 patients with ITP and 20 healthy controls were detected by flow cytometry,including CD3+,CD4+,CD8+,CD56+,CD19+ lymphocytes and CD4+/CD8+.Results The percentages of CD3+ T lymphocyte (61.58 ± 6.45 ) %,CD4+ T lymphocyte (28.38 ±4.89)% and the ratio of CD4+/CD8+ 0.99 0.22 in patients with ITP were lower than those in healthy controls[( 67.85 ± 4.68 ) %,( 38.00 ± 3.37 ) %,1.54 ± 0.13,all P ＜ 0.05].After immunosuppressive therapy,the percentages of CD3+ T lymphocyte ( 69.41 ± 5.03 ) %,CD4+ T lymphocyte (38.17 ±3.18)% and the ratio of CD4+/CD8+ 1.60 ±0.15 recovered to control levels.The percentages of CD8+ T lymphocyte (29.20 ±4.50)% and CD19+B lymphocyte ( 17.74 ±4.14)% were higher than those in healthy controls[( 24.82 ± 2.93 ) % and ( 12.09 ± 3.51 ) %,all P ＜ 0.05].After the immunosuppressive therapy,the percentages of CD8+ T lymphocyte ( 24.06 ± 3.02 ) % and CD19+ B lymphocyte ( 10.90 ± 3.55 ) %recovered to control levels.There were no significant difference of the percentage of CD56+ lymphocyte among ITP patients ( 15.80 ± 2.85 )%,ITP patients after immunosuppressive therapy ( 15.16 ± 2.77 )% and healthy controls ( 16.36 ± 2.75 ) %.ConclusionThe aberrant immunocyte subsets are involved in the pathogenesis of ITP,and detection of immunocyte subsets might be helpful for the diagnosis and determination of therapeutic outcome of ITP.     

Pathogenesis of chronic immune thrombocytopenia: increased platelet destruction and/or decreased platelet production

Chronic immune thrombocytopenia (ITP) is a haematological disorder in which patients predominantly develop skin and mucosal bleeding. Early studies suggested ITP was primarily due to immune-mediated peripheral platelet destruction. However, increasing evidence indicates that an additional component of this disorder is immune-mediated decreased platelet production that cannot keep pace with platelet destruction. Evidence for increased platelet destruction is thrombocytopenia following ITP plasma infusions in normal subjects, in vitro platelet phagocytosis, and decreased platelet survivals in ITP patients that respond to therapies that prevent in vivo platelet phagocytosis; e.g., intravenous immunoglobulin G, anti-D, corticosteroids, and splenectomy. The cause of platelet destruction in most ITP patients appears to be autoantibody-mediated. However, cytotoxic T lymphocyte-mediated platelet (and possibly megakaryocyte) lysis, may also be important. Studies supporting suppressed platelet production include: reduced platelet turnover in over 80% of ITP patients, morphological evidence of megakaryocyte damage, autoantibody-induced suppression of in vitro megakaryocytopoiesis, and increased platelet counts in most ITP patients following treatment with thrombopoietin receptor agonists. This review summarizes data that indicates that the pathogenesis of chronic ITP may be due to both immune-mediated platelet destruction and/or suppressed platelet production. The relative importance of these two mechanisms undoubtedly varies among patients.     

Evaluation of platelet kinetics in patients with liver cirrhosis: similarity to idiopathic thrombocytopenic purpura

BACKGROUND: Thrombocytopenia is a common manifestation of liver cirrhosis (LC), but its underlying mechanism is not fully understood. The purpose of the present paper was to evaluate the platelet kinetics in LC patients by examining several non-invasive convenient markers. METHODS: Fifty-seven LC patients, 32 patients with idiopathic thrombocytopenic purpura (ITP), 12 with aplastic anemia (AA), and 29 healthy individuals were studied. Plasma thrombopoietin was measured by enzyme-linked immunosorbent assay. Absolute reticulated platelet (RP) count and plasma glycocalicin were used as indices for thrombopoiesis, and the indices for platelet turnover were the RP proportion and the plasma glycocalicin normalized to the individual platelet count (GCI). RESULTS: There was no difference in thrombopoietin levels between LC patients and healthy controls. The RP proportion and GCI were significantly higher and the absolute RP count and glycocalicin significantly lower in LC patients than in healthy controls. These markers in ITP and LC patients were comparable, but significantly different from those in AA patients. The bone marrow megakaryocyte density in LC and ITP patients was similar, and significantly higher than in AA patients. CONCLUSIONS: Cirrhotic thrombocytopenia is a multifactorial condition involving accelerated platelet turnover and moderately impaired thrombopoiesis. Thrombopoietin deficiency is unlikely to be the primary contributor to cirrhotic thrombocytopenia.     

系统性红斑狼疮患者外周血CD4+CD25highFoxp3+调节性T细胞数量和Foxp3mRNA的表达水平与疾病活动性的相关性

目的 研究系统性红斑狼疮(SEE)患者CD4+CD25highFoxp3+调节性T细胞的数量及其功能基因Foxp3 mRNA的表达水平与SLE疾病活动性和肾脏损伤的相关性.方法 采用四色流式细胞术以Foxp3-异硫氰酸荧光素(FITC )/CD25-藻红蛋白/CD4-多甲藻叶绿素蛋白(PerCP)/CD3-藻蓝蛋白7抗体组合检测40名健康对照者及42例SLE患者外周血CD4+CD25highFoxp3+调节性T细胞的数量,实时荧光定量聚合酶链反应(PCR)检测特异性转录因子Foxp3 mRNA的表达水平,并分析其与SLE患者疾病活动指数(SLEDAI)、补体C3及血清抗双链DNA(dsDNA)抗体的关系.统计学方法采用t检验和Spearman相关分析.结果 活动期SLE患者外周血CD4+CD25highFoxp3+调节性T细胞数量显著低于健康对照组[(4±3)％与(7±4)％,P＜0.05],稳定期与健康对照组差异无统计学意义(P＞0.05);活动期SLE患者外周血CD4+CD25highFoxp3+调节性T细胞数量及CD4+CD25highFoxp3+调节性T细胞/CD4+比值显著低于稳定期患者[(4±3)％,(9±6)％与(5±4)％,(10±6)％,P均＜0.05];活动期SLE患者外周血Foxp3 mRNA的表达水平明显低于稳定期和对照组(P＜0.01,P＜0.05);SLE患者并发肾病组外周血CD4+CD25highFoxp3+调节性T细胞数量及CD4+CD25highFoxp3+调节性T细胞/CD4+比值显著低于SLE非肾病组(P＜0.05).相关分析显示,SLE患者外周血CD4+CD25highFoxp3+调节性T细胞数量与SLEDAI呈负相关(r=-0.5782,P＜0.05);CD4+CD25highFoxp3+调节性T细胞/CD4+比值与SLEDAI呈负相关(r=-0.4913,P＜0.05),与补体C3呈正相关(r=0.3687,P＜0.05);SLE患者外周血CD4+CD25highFoxp3+调节性T细胞数量与Foxp3 mRNA的表达水平呈正相关(r=0.6142,P＜0.0l).结论 SLE患者外周血CD4+CD25highFoxp3+调节性T细胞和Foxp3 mRNA的变化可能是导致SLE疾病发生和发展的关键因素之一,与疾病的活动性有密切关系.     

Suppression of in vitro megakaryocyte production by antiplatelet autoantibodies from adult patients with chronic ITP

Chronic immune thrombocytopenic purpura (ITP) is manifested by autoantibody-induced platelet destruction. Platelet turnover studies suggest that autoantibody may also affect platelet production. To evaluate this, we studied the effect of plasma from adult patients with chronic ITP on in vitro megakaryocyte production. CD34(+) cells, obtained from healthy donors, were cultured in medium containing PEG-rHuMGDF and 10% plasma from either ITP patients or healthy subjects. Cultures containing plasma from 12 of 18 ITP patients showed a significant decrease (26%-95%) in megakaryocyte production when compared with control cultures. Positive ITP plasmas not only reduced the total number of megakaryocytes produced during the culture period but also inhibited megakaryocyte maturation, resulting in fewer 4N, 8N, and 16N cells. The role of antibody in this suppression is supported by 2 factors: (1) immunoglobulin G (IgG) from ITP patients inhibited megakaryocyte production when compared with control IgG; and (2) adsorption of autoantibody, using immobilized antigen, resulted in significantly less inhibition of megakaryocyte production when compared with unadsorbed plasma. These results show that plasma autoantibody from some adult patients with ITP inhibits in vitro megakaryocyte production, suggesting that a similar effect may occur in vivo.     

Rapid flow cytometric quantitation of reticulated platelets in whole blood

Young or reticulated platelets contain some residual mRNA, which is rapidly degraded after platelet release into the circulation. In order to minimize platelet activation and possible loss of large platelets during sample handling a whole blood method has been developed utilising the RNA fluorochrome thiazole orange (TO) in combination with an antibody to anti-glycoprotein Ib (GpIb) directly conjugated to phycoerythrin (PE), to specifically stain reticulated platelets via flow cytometric analysis. In this study whole blood analysis of platelet mRNA was undertaken in healthy normal subjects and a variety of patients with haematological abnormalities. The percentage of Gp Ib positive platelets containing mRNA in normals (n = 22) was 11.61% with a two SD range of 3.19-20.01%. The percentage of reticulated platelets was significantly increased (mean mRNA content ± one SD) in sickle cell disorders (n = 22) 38.12% ± 18.42 (P < 0.001); thalassaemia (major, intermedia and trait) (n = 24) 29.76 ± 19.15 (P < 0.001); ITP (N = 20) 23.53% ± 13.04 (P < 0.02) and essentialthrombocythemia (N = 32) 37.12 ± 19.84 (P < 0.001). Platelets from patients with reactive thrombocytosis (N = 15) were only 12.23% positive (±6.95) and not significantly different from the normal range (P = 0.95). This method offers a rapid and simple procedure for assessment of reticulated platelets in whole blood and suggests that there may be an increased platelet turnover in certain haemoglobinopathies.     

The clinical significance of circulating B cells secreting anti-glycoprotein IIb/IIIa antibody and platelet glycoprotein IIb/IIIa in patients with primary immune thrombocytopenia

Abstract

Objectives

The objective of the study is to evaluate the possible roles of the detection of circulating B cells secreting anti-glycoprotein IIb/IIIa antibody, platelet glycoprotein IIb/IIIa, and anti-glycoprotein IIb/IIIa antibody in the diagnosis of primary immune thrombocytopenia (ITP) patients.

Methods

Circulating B cells secreting anti-glycoprotein IIb/IIIa antibody, platelet glycoprotein IIb/IIIa and anti-glycoprotein IIb/IIIa antibody in 64 patients with ITP, 33 non-ITP patients, and 32 controls were measured with enzyme-linked immunospot assay (ELISPOT), monoclonal antibody immobilization of platelet antigens assay (MAIPA) and flow cytometic analysis (FCM), respectively.

Results

Compared with the controls and non-ITP patients, the frequency of circulating B cells secreting anti-glycoprotein IIb/IIIa antibody was significantly increased, whereas the positive rate of platelet glycoprotein IIb/IIIa was significantly decreased (P < 0.05) in ITP patients, respectively. The sensitivities for the diagnosis of ITP of ELISPOT and FCM were 68.8% and 57.8%, and the specificities of 90.9% and 90.9%, respectively. The sensitivities of ELISPOT and FCM were higher than MAIPA's sensitivity (39.1%) (P < 0.05). However, there was no apparent difference of the sensitivities of ELISPOT and FCM and the specificities of those three detections (MAIPA's specificity was 81.8%) (P > 0.05).

Discussion

ELISPOT and FCM for detecting the circulating B cells secreting anti-glycoprotein IIb/IIIa antibody and the platelet glycoprotein IIb/IIIa were as specific as that of MAIPA for assay of anti-glycoprotein IIb/IIIa antibody, but ELISPOT and FCM had higher sensitivities. So ELISPOT and FCM were sensitive and specific for identifying patients with autoantibody-mediated thrombocytopenia and these should be used as diagnostic tests in clinic.     

Indirect study of thrombopoiesis (TPO, reticulated platelets, glycocalicin) in patients with hereditary macrothrombocytopenia

Chronic isolated hereditary macrothrombocytopenia (CHMT) is a congenital form of macrothrombocytopenia that seems to be due to defective production secondary to a disturbance in megakaryocyte fragmentation. To better understand the pathogenesis of thrombopoiesis in this hereditary thrombocytopenic disorder, we determined the percentage of reticulated platelets (RP), plasma glycocalicin (GC) and thrombopoietin (TPO) levels in 29 patients with CHMT, 23 patients with immune thrombocytopenic purpura (ITP), and 17 patients with thrombocytopenia secondary to decreased bone marrow megakaryocytes (hypoplasia). The % RP was similar in CHMT (2.27 +/- 1.33) and hypoplasia (1.98 +/- 1.35) patients and markedly lower than that in ITP patients (8.80 +/- 7.97; p <0.001), suggesting that the production of new platelets is reduced in CHMT. Plasma GC was within the normal range (0.84 +/- 0.16 microg/mL) both in patients with CHMT (0.63 +/- 0.20 microg/mL) and ITP (0.82 +/- 0.90 microg/mL), while it was significantly decreased in patients with hypoplasia (0.16 +/- 0.04 microg/mL; p < 0.001). When the GC value was normalized for platelet count, the GC index was normal in CHMT patients (2.05 +/- 1.1) and in patients with hypoplasia (0.85 +/- 0.10) while it was significantly increased in ITP patients (10.88 +/- 18.00; p<0.001); thus, patients with CHMT seem to have a normal platelet turnover. TPO was significantly increased in CHMT (195 +/- 72 pg/ml) as compared with normal (80 +/- 53 pg/ml; p < 0.002); however, the mean level was not as high as in ITP patients (345 +/- 167 pg/mL; p < 0.001). This finding suggests that CHMT syndrome is not secondary to a defective production of TPO and that megakaryocyte mass is nearly normal.     

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.     

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.