流式微球技术检测糖蛋白Ⅱb/Ⅲ a自身抗体在特发性血小板减少性紫癜诊断中的应用

目的 建立流式微球技术检测血小板膜表面糖蛋白Ⅱ b/Ⅲa(GPⅡb/Ⅲa)特异性自身抗体的方法,比较该方法和改良间接单克隆抗体俘获血小板抗原技术(MAIPA)在特发性血小板减少性紫癜(ITP)与非免疫性血小板减少性紫癜鉴别诊断中的价值.方法 应用抗人血小板GPⅡb/Ⅲa单抗(CD41a)包被微球.分离血小板,将血小板裂解后与包被过的微球孵育,再加入PE标记的羊抗人IgG多克隆抗体,流式细胞术分析.分别用该方法和改良间接MAIPA检测血小板表面和血浆中的糖蛋白特异性自身抗体,并将检测结果进行比较.结果 将样本的平均荧光强度值(MFI)与正常对照MFI的均值比较,计算比率.该比率均值及范围在ITP组为3.36(0.84～22.94),非免疫性血小板减少组为1.16(0.67～5.59),正常对照组为1.08(0.72～1.76).非参数检验得ITP组荧光强度比率与非免疫性血小板减少组及正常对照组存在显著差异(P《0.01).若将正常对照组上限作为界值,比率大于1.76定为阳性,则流式微球技术诊断ITP的敏感性为71.43%,特异性为94.28%,阳性预测值为95.24%,其敏感性高于改良间接MAIPA的测定值(51.79%)(χx2=4.57，P<0.05)。由ROC曲线得流式微球法区分ITP患者与正常人的鉴别效度为0.916。结论 流式微球技术检测血小板膜表面糖蛋白特异性自身抗体，耗时短、重复性好，敏感性高于改良间接MAIPA，对提高ITP的实验诊断水平及指导临床治疗有一定的价值。     

血小板特异性抗体联合检测在免疫性血小板减少症鉴别诊断中的价值

目的通过检测血小板减少症患者外周血B淋巴细胞血小板膜糖蛋白特异性抗体的变化,探讨其表达水平变化对血小板减少症鉴别诊断的临床意义。方法应用流式微球技术检测血小板减少症患者及健康对照者外周血GPⅡb/Ⅲa、GPⅠb/ⅠX的表达水平。结果原发ITP与继发ITP患者血小板特异性抗体GPⅡb/Ⅲa、GPⅠb/ⅠX与对照组比较差异有统计学意义(P0.05);血小板特异性抗体GPⅠb/ⅠX诊断原发ITP的敏感度43%,特异度89%;GPⅡb/Ⅲa诊断原发ITP的敏感度86%,特异度83%。两者联合诊断敏感度90%,特异度83%。结论联合检测两种血小板抗体,提高原发ITP和SLE的诊断阳性率;GPⅠb/ⅠX在ITP中的诊断优势不如GPⅡb/Ⅲa。     

应用流式细胞术检测原发免疫性血小板减少症患者血小板膜糖蛋白Ⅱb/Ⅲa的表达及其临床意义

免疫性血小板减少症(Primary immune thrombocytopenia,ITP)是一类由体液和细胞免疫异常导致血小板破坏增多的疾病[1-2]。发病机制与免疫功能异常引起的自身抗血小板抗体产生关系密切。血小板表面膜糖蛋白(GP)Ⅱb/Ⅲa是血小板及巨核细胞主要的膜糖蛋白,GPⅡb/Ⅲa抗体是ITP患者自身抗体的主要成分之一。目前测定血小板膜糖蛋白特异性抗体的方法主要有放射免疫微球法[3]、单克隆抗体俘获血小板抗原技术( MAIPA)[4],我们用流式细胞术(FCM)检测血小板减少患者及正常人血小板GPⅡb/ⅢRa的变化,探讨FCM检测血小板GPⅡb/Ⅲa在诊断免疫性血小板减少性紫癜中的意义。     

血小板特异性自身抗体检测在特发性血小板减少性紫癜中的意义

目的 检测特发性血小板减少性紫癜(ITP)患者体内的血小板特异性自身抗体与临床严重程度和临床疗效间的关系. 方法 应用改良的单克隆抗体特异性俘获血小板抗原(MAIPA)法检测血小板膜糖蛋白(GPⅡ bⅢa、GPIb)特异性自身抗体. 结果 ITP组(40例),10例为单一抗GPⅡ b Ⅲa抗体阳性.6例为单一抗GPIbα抗体阳性,20例为双抗体阳性,4例为双抗体阴性.非免疫性血小板减少组与正常对照组均为阴性.抗GPⅡbⅢa抗体(b=-0.071,P<0.01)、抗GPlbα抗体(b=-0.092,P<0.01)均与采血时血小板计数呈显著负相关.治疗后,双抗体阳性组8例治疗无效,单抗体阳性组(16例)1例治疗无效(χ2=6.09,P<0.05). 结论 血小板特异性自身抗体检测对鉴别特发性和非免疫性血小板减少症有一定价值.抗体种类与临床疗效有一定关系.     

自身免疫性血小板减少性紫癜相关抗体的研究

目的　探索对自身免疫性血小板减少性紫癜 (AITP)诊断特异和敏感的实验方法。方法　采用单克隆抗体特异性俘获血小板抗原技术 (MAIPA技术 )并加以改进 ,对比检测血小板洗脱液和血浆中血小板膜糖蛋白特异性抗体。结果　AITP患者血浆游离抗血小板膜糖蛋白 (GPⅡb Ⅲa、GPⅠb Ⅸ )抗体总阳性率为 38.89%(5 4例中 2 1例 ) ,洗脱血小板表面抗血小板膜糖蛋白 (GPⅡb Ⅲa、GPⅠb Ⅸ )抗体总阳性率为 6 8.5 2 %(5 4例中 37例 ) ,两者差异有显著性 (校正 χ2 =19.39,P <0 .0 0 5 )。原发AITP组血浆游离及洗脱血小板表面抗血小板糖蛋白 (GPⅡb Ⅲa、GPⅠb Ⅸ )特异性抗体总阳性率与继发性AITP组比较差异无显著性。AITP患者血小板数量与自身抗体滴度呈明显负相关。结论　MAIPA法检测血小板洗脱液中抗血小板糖蛋白抗体在AITP的诊断和治疗中具有高度特异性 ,且敏感性较血浆抗体检测显著提高。     

直接MAIPA对免疫性和非免疫性血小板减少性紫癜的鉴别诊断

目的检测免疫性与非免疫性血小板减少患者血小板膜糖蛋白特异性自身抗体，评价该方法在免疫性和非免疫性血小板减少性紫癜鉴别诊断中的价值。方法应用改良直接单克隆抗体俘获血小板抗原技术(MAIPA)检测血小板膜糖蛋白(GPⅡh／Ⅲa、GP I b和GPI a／Ⅱa)特异性自身抗体。结果免疫性血小板减少患者自身抗体阳性率(76．4％)显著高于非免疫性血小板减少患者(3．6％)(P＜0．05)。直接MAIPA诊断免疫性血小板减少的敏感性为76．4％，特异性为96．4％，阳性预测值为97．1％。GPⅡh／Ⅲa特异性自身抗体阳性的免疫性血小板减少患者血小板计数与自身抗体吸光度比值呈显著负相关(r=-0．338，P＜0．05)。结论直接MAIPA检测血小板膜糖蛋白特异性自身抗体对于鉴别免疫性与非免疫性血小板减少有一定意义。     

血小板膜表面自身抗体表达与大剂量地塞米松治疗ITP疗效的关系

有统计学意义(P<0.05),GPⅠb自身抗体对疗效没有明显影响(P>0.05).结论 血小板膜GPⅡb/Ⅲa特异性自身抗体检测可作为ITP患者口服大剂量地塞米松疗效预测的潜在指标.     

南宁地区成人及儿童ITP患者体内血小板自身抗体特异性分布的研究

目的：研究血小板膜糖蛋白特异性自身抗体在成人及儿童原发免疫性血小板减少症（ITP）患者中分布的异同。方法应用酶联免疫吸附试验（PAKAUTO 试剂盒）检测 ITP 组（83例）及非 ITP 组（58例）患者血小板自身抗体,并分析成人组ITP（46例）,儿童组 ITP（37例）的抗 GP Ⅱ b／Ⅲ a 、抗 GP Ⅰ b／Ⅸ及抗 GP Ⅰ a／Ⅱ a 自身抗体特异性分布规律。结果 ITP 组血小板自身抗体阳性率为66．27％,高于非 ITP 组的6．90％,差异有统计学意义差异有统计学意义（P＜0．05）。成人组女性 ITP 患者占60．87％,儿童组女性 ITP 患儿占64．86％,差异无统计学意义（P ＞0．05）,但两组女性发病率均高于男性,差异有统计学意义（P＜0．05）。成人组 ITP 患者血小板自身抗体阳性率为63．04％,儿童组 ITP 患儿抗体阳性率为70．27％,差异无统计学意义（P＞0．05）;且成人组与儿童组 ITP 患者血小板自身抗体特异性分布差异无统计学意义（P ＞0．05）,均以抗 GP Ⅱ b／Ⅲ a 和抗 GPⅠ b／Ⅸ抗体多见。结论血小板自身抗体检测对 ITP 诊断、鉴别诊断及治疗均有重要的参考价值,GP Ⅰ a／Ⅱ a 抗体介导的 ITP很值得深入研究。     

分泌GPⅡb/Ⅲa抗体B细胞及血小板特异性抗体检测在特发性血小板减少性紫癜诊断中的意义

目的 检测特发性血小板减少性紫癜(ITP)患者及非免疫性血小板减少患者分泌GPⅡb/Ⅲa抗体B细胞、血小板特异性抗体的变化,评价其对诊断ITP及非免疫性血小板减少疾病的作用及临床意义.方法 应用酶联免疫斑点技术(ELISPOT)及改良血小板抗原单克隆抗体固相化检测技术(MAIPA)分别检测58例ITP患者、33例非免疫性血小板减少患者及31名正常对照者分泌GPⅡb/Ⅲa抗体B细胞频数、血小板特异性抗体(抗GPⅡb/Ⅲa抗体)表达的变化.结果 ITP患者分泌GPⅡb/Ⅲa抗体B细胞频数[(6.6±4.2)/105个外周血单个核细胞(PBMNC)]明显高于非免疫性血小板减少患者[(2.2±2.0)/105个PBMNC](P＜0.05)及正常对照组[(1.3±0.5)/105个PBMNC](P＜0.05),而分泌GPⅡb/Ⅲa抗体B细胞频数在非免疫性血小板减少患者及正常对照组间的差异无统计学意义(P＞0.05).通过ELISPOT法检测分泌GPⅡb/Ⅲa抗体B细胞对ITP诊断的敏感性为70.69%,特异性为90.91%,高于改良MAIPA法的敏感性(χ2=7.03,P＜0.05).根据ROC曲线,ELISPOT区分ITP患者和非免疫性血小板减少患者的鉴别效度为0.886.结论 通过检测分泌GPⅡ/bⅢa抗体B细胞及血小板特异性抗体能够较好地反映ITP的发病机制.应用ELISPOT方法检测ITP患者分泌GPⅡb/Ⅲa抗体B细胞具有较高敏感性及特异性,可提高ITP的实验窒诊断水平,对指导治疗有一定的临床意义.     

特发性血小板减少性紫癜患者脾脏CD5+和CD5-B细胞共同参与血小板自身抗体的产生

目的：研究慢性特发性血小板减少性紫癜（ITP）患者脾脏CD5^ B细胞水平的变化及CD5^ 和CD5^-B细胞与血小板膜糖蛋白（GP）特异性自身抗体产生的关系，以识别致病B细胞亚群。方法：应用双色流式细胞仪检测8例慢性ITP患者脾脏CD5^ B细胞水平。选择4例血浆抗GPⅡb/Ⅲa和抗GP Ⅰb/Ⅸ抗体双阳性ITP切脾患者，应用Ficoll密度梯度离心及花环形成分离法分离脾脏B淋巴细胞，继而采用镝产珠分选法分选、纯化CD5^ B细胞和CD5^-B细胞，并分别进行体外培养，应用改良MAIPA法检测血浆和细胞培养上清液的血小板特异性抗体。结果：ITP患者脾脏CD5^ B细胞水平圈晨自身免疫性疾病患者略有增高，二者之间差异无统计学意义。CD5^ B细胞水平与患者血小板计数无相关性。4例血浆抗GPⅡb/Ⅲa抗体和抗GPⅠb/Ⅸ抗体双阳性。另外1例CD5^ B细胞培养液抗GPⅡb/Ⅲa抗体阴性，抗GPⅠb/Ⅸ抗体阳性;CD5^-B细胞培养液抗GPⅡb/Ⅲa抗体和抗GPⅠb/Ⅸ抗体双阳性。结论：脾脏CD5^ 和CD5^-B细胞 均可产生血小板GP特异性自身抗体，抗体产生种类和滴度无明显差异。提示二者共同参与了ITP的发病过程。     

The detection methods for antiplatelet autoantibodies (phase II and phase III assay)

A variety of methods are utilized to detect antiplatelet autoantibodies nowadays, platelet-associated immunoglobulin G(PAIgG) is a phase II assay of limited value in the meaning of its sensitivity and specificity for the diagnosis of idiopathic thrombocytopenic purpura(ITP), although it has been ordered in many occasions. The newer antigen-specific assays(phase III), which can identify autoantibodies against platelet glycoprotein(GP)s, such as GP IIb/IIIa, with greater specificity but lower sensitivity are rarely performed in clinical situations in Japan. Development of novel systems to detect clinically more relevant markers, including specific antiplatelet autoantibodies, is necessary for the diagnosis of ITP.     

A comparison of monoclonal antibody immobilization of platelet antigen (MAIPA) and immunobead methods for detection of GPIIb/IIIa antiplatelet antibodies in immune thrombocytopenic purpura

In this study we have compared two assays for the detection of autoantibodies GpIIb/IIIa, platelet bound and in serum, in immune thrombocytopenic purpura (ITP). Both assays were found to have a similar sensitivity, but the monoclonal antibody immobilization of platelet antigen (MAIPA) assay was more reproducible than the immunobead assay. The MAIPA and immunobead assay demonstrated an 81% concordance of results for serum antibody detection and a 78% concordance for platelet-associated antibody detection, with an 8–12% incidence of false positive or negative results.     

Anti-platelet antibody and platelet function]

The effect of anti-platelet antibodies, including murine monoclonal antibodies, autoantibodies and alloantibodies, on platelet function was analyzed. The target antigen of these antiplatelet antibodies, investigated in the present study, was a glycoprotein IIb/IIIa, which is a receptor of fibrinogen and plays an important role in platelet aggregation. Some of these antibodies inhibited agonist-induced platelet aggregation. The target antigen of one murine monoclonal antibodies, designated OP-G2, was a glycoprotein IIb/IIIa and interestingly, this antibody induced platelet aggregation, which required divalent cation and fibrinogen. We compared the epitope of these antibodies by inhibition assay and found the epitope of these antibodies to be very close. The binding of OP-G2 to the platelets required Ca2+. These data suggest that OP-G2 recognizes an epitope at or in very close proximity to the fibrinogen binding site of GPIIb/IIIa, as compared with other antibodies.     

Idiopathic thrombocytopenic purpura

Idiopathic(autoimmune) thrombocytopenic purpura(ITP) is characterized by thrombocytopenia and normal to increased numbers of megakaryocytes. ITP is a disease caused by circulating autoantibodies that react with the platelet membrane. It is thought that platelet-associated IgG(PAIgG) plays an important role in the mechanism of ITP, since increases in PAIgG are closely related to decreases in platelet count in patients with this disease. However, it is possible that PAIgG includes immune complex and platelet antibodies to human platelet alloantigens(HPA) other than autoantibodies. There have been several recent reports on autoantibodies to glycoprotein IIb/IIIa and Ib/IX. Although the etiology of ITP remains unclear, both genetic and environmental factors appear to be involved in it. With respect to the genetic aspects of ITP, the human leukocyte antigen (HLA) haplotype of patients is considered a potentially important factor in etiology. Various methods have been used for the treatment of chronic ITP. However, splenectomy and corticosteroids are still the mainstays of therapy. Because of the heterogeneity of the disease, however, approximately 20% of cases are refractory to these treatments. New therapy of chronic or refractory ITP with thrombopoietin, antiCD40 ligand antibody, and Helicobactor pylori eradication have recently been reported.     

Significance of detection of anti-GPIIb/IIIa antibody secreting B cells and platelet-specific antibody in patients with idiopathic thrombocytopenic purpura

OBJECTIVE: To detect the frequencies of anti-GPIIb/IIIa antibody secreting B cells and platelet-specific antibody in patients with idiopathic thrombocytopenic purpura (ITP) and non-immune thrombocytopenia, and to evaluate their roles in the diagnosis of ITP and their clinical significance. METHODS: The frequencies of circulating B cells secreting anti-GPIIb/IIIa antibody and platelet-specific antibody in 58 ITP patients, 33 non-ITP patients and 31 healthy controls were tested by Enzyme-linked Immunospot Assay (ELISPOT) and modified monoclonal antibody immobilization of platelet antigens assay (MAIPA) respectively. RESULTS: The frequencies of circulating B cells secreting anti-GPIIb/IIIa antibody in ITP patients \[(6.6 ± 4.2)/10(5) PBMNC\] were significantly increased (P < 0.05) than that of the controls \[(1.3 ± 0.5)/10(5) PBMNC\] and non-immune thrombocytopenic purpura patients \[(2.2 ± 2.0)/10(5) PBMNC\]. However there was no apparent difference between the latter two groups (P > 0.05). ELISPOT had a sensitivity of 70.69%, a specificity of 90.91% for the diagnosis of ITP, the sensitivity being higher than that of modified MAIPA's (43.10%) (χ(2) = 7.03, P < 0.05). The ROC curve showed the discriminative validity of cytometric bead array was 0.886. CONCLUSION: The frequencies of circulating B cells secreting anti-GPIIb/IIIa antibody may reflect the pathogenesis of ITP. ELISPOT assay have high sensitivity and specificity than modified MAIPA for the diagnosis of ITP and the guidance for clinical therapy.     

Detection and differentiation of platelet-specific antibodies by flow cytometry: the bead-mediated platelet assay

BACKGROUND: Platelet-reactive antibodies cause a number of clinical disorders. The detection and differentiation of these antibodies are prerequisites for the adequate treatment of these disorders. The bead- mediated platelet assay described here enables the detection and differentiation of platelet-bound antibodies by the use of flow cytometry. STUDY DESIGN AND METHODS: The bead-mediated platelet assay is based on the isolation of human platelet glycoproteins by using flow cytometric standardization beads after the incubation of typed platelets with human sera. The specificity and sensitivity of this assay were tested with five sera, each containing a known platelet- reactive antibody. The monoclonal antibody-specific immobilization of platelet antigens assay was used as a reference test. RESULTS: The bead- mediated platelet assay was able to determine the glycoprotein specificity of the antibody without cross-reactions in every case. In serial dilution tests, the bead-mediated platelet assay was able to detect the antibodies at higher dilutions than the monoclonal antibody- specific immobilization of platelet antigen assay. Total test time was 3.5 hours. CONCLUSION: The bead-mediated platelet assay is a fast and reliable method for the detection and differentiation of platelet- reactive antibodies.     

Flow cytometry analysis of platelet antibodies

We have devised assays to detect both circulating alloantibodies to platelets (indirect assay) and platelet-association IgG and IgM (direct assay) using a flow cytometric technique. A variety of patients with immune thrombocytopenia were studied. Employment of a confocal lens in the flow cytometer increased the discrimination power of the instrument. Patients with autoimmune thrombocytopenia (idiopathic thrombocytic purpura [ITP], systemic lupus erythematosus (SLE), lymphoma, leukemia, and drug-induced thrombocytopenia showed a significant increase in platelet-associated antibody. Circulating antibodies to platelets (alloantibodies) were demonstrated in cases of platelet refractoriness and neonatal isoimmune purpura. Day-today precision of the assays ranged from 3% to 6% (coefficient of variation). No interference was shown in the presence of hemoglobin (5 g/L), triglycerides (10 g/L), or polyclonal and monoclonal immunoglobulinemia (50 g/L: IgG, IgA, IgM). The sensitivity of the direct assay was 500 attograms of IgG or IgM platelet.     

血小板特异性抗体对特发性血小板减少性紫癜诊断价值的研究

本研究的目的是比较特发性血小板减少性紫癜 (ITP)、慢性再生障碍性贫血 (CAA)、恶性血液病患者及健康志愿者特异性抗体水平 ,以评价血小板特异性抗体在ITP诊断中的价值。用改良单克隆抗体特异性俘获血小板抗原 (MAIPA)技术同时检测血小板GPIb/Ⅸ、GPIIb/Ⅲa、GPⅣ、GPⅤ的特异性抗体。结果表明 :ITP组、CAA组、恶性血液病患者及健康志愿者血小板特异性抗体总阳性率分别为 6 9.99% ,10 % ,2 0 %和 0 %。ITP组与CAA组存在显著性差异 ( χ2 =2 0 .71,P <0 .0 0 5 ) ,ITP组与恶性血液肿瘤化疗组存在显著性差异 ( χ2 =12 .2 2 ,P <0 .0 0 5 )。健康志愿组无 1例阳性。结论 :多种抗体同时检测可提高敏感性 ,血小板特异性抗体对ITP是一种特异性高、敏感性强的实验室诊断指标。