自身免疫性血小、板减少性紫癜的特异性免疫学诊断的研究

目的 检测自身免疫性血小板减少性紫癜（AITP）患者及非免疫性血小板减少症患者的抗血小板特异性抗体,并与血小板相关抗体（PAIgG)相比较,评价其诊断及鉴别诊断价值。方法 用酶联免疫吸附竞争法检测PAIgG,改良单克隆抗体特异性俘获血小板抗原（MAIPA）技术检测抗血小板GPⅡb/Ⅲa、GPIb/Ⅸ的特异性抗体。结果 血小板特异性抗体较PAIgG的敏感性低,但特异性明显增强。结论 抗血小板特异性抗体对鉴别免疫性与非免疫性血小板减少具有重要临床意义。     

一种新的血小板特异性同种抗原Br~a

根据单克隆抗体特异的固定血小板抗原为原理而建立的糖蛋白特异性酶免疫测定试验(MAIPA)清晰地鉴别出一种新的血小板特异性同种抗体,抗-Br~a、本文报道由这种抗体引起的4例新生儿同种免疫性血小板减少性紫癜(NAIT).4个生NAIT患儿后的母亲的血清中均含有一种与以前所发现的所有血小板抗体特异性不同的抗体,称为抗-Br~a。其相应抗原与所有已知血小板同种抗原有明显不同。新抗原的分离类型与已识别的Zw或Bak系统的抗原明显不同,其遗传方式为常染色体共显性遗传,血小板配组分析也认为新抗原与Zw或Bak抗原不同。此外群体调查显示其表现型频率(20%)与计算的基因频率(0.1056)亦与所有其他已知抗原不     

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。     

血小板输注无效患者血小板特异性糖蛋白抗体表达的实验研究

目的探讨血小板输注无效(PTR)患者血小板特异性糖蛋白抗体的表达情况。方法采用ELISA方法检测56名临床上确诊为PTR患者的血小板特异性糖蛋白抗体、应用PCR方法检测其血小板特异性抗原(HPA)1-6,15基因分型。结果 56名PTR患者检出血小板特异性糖蛋白抗体,8例表达阳性(占14.3%),包括血小板特异性糖蛋白GPⅡb/Ⅲa阳性7例,GPⅠa/Ⅱa阳性1例、GPⅠb/Ⅰx阳性1例、GPⅣ阳性1例。结合8例患者的抗体检测反应格局和HPA抗原基因型结果分析抗体的特异性为:4例存在抗-HPA3b、3例存在抗-HPA3a、1例存在抗-HPA5b。结论反复输血的血小板输注无效患者,应选择HPA抗原相匹配的血小板供者,以改善血小板输注效果。     

血小板输注无效患者血小板同种抗体及血小板特异性糖蛋白抗体表达的研究

目的 探讨血小板输注无效与血小板同种抗原或血小板特异性抗原的相关性.方法 选择65例临床确诊血小板输注无效患者作为研究对象,应用酶联免疫吸附实验(ELISA)方法检测血清、血小板洗脱液中血小板特异性抗体;应用HLA抗体特异性检测试剂盒,对组合反应性抗体(PRA)阳性的患者进行HLA抗体特异性分析;用HPA分型试剂盒检测8个血小板同种抗原系统HPA-1、2、3、4、5、6、9、15;用HLA分型试剂盒对HLA-A/B抗原进行基因分型.结果 65例患者HLA-A/B抗原,HPA-1、2、4、5、6、9、15抗原的基因频率分布与健康献血员比较差异无统计学意义.HPA-3a、3b抗原频率分别为0.65、0.35,与健康献血员比较差异有统计学意义(P＜0.05).65例患者中HLA抗体单独阳性24例(36.9%),HLA抗体和血小板特异性糖蛋白抗体共同阳性14例(21.5%);HLA抗体和血小板洗脱液特异性糖蛋白抗体共同阳性6例(9.2%),血小板洗脱液特异性糖蛋白抗体阳性13例(20%),HLA抗体、血小板特异性糖蛋白抗体及血小板洗脱液特异性糖蛋白抗体共同阳性4例(6.2%);HLA-A/B特异性抗体中,HLA-A*9抗体占全部抗体的46.2%,HLA-B*40抗体占33.6%.血清血小板特异性抗体以GPⅡb/Ⅲa为主(26.2%),其次为GP Ⅰa/Ⅱa(21.5%),血小板洗脱液中,血小板特异性抗体以GPⅡb/Ⅲa和GP Ⅰb/Ⅸ为主(41.5%).对2例患者进行了遗传学调查,发现产生的血小板特异性糖蛋白抗体和HLA抗体与父母血小板抗原及HLA抗原不相合呈密切相关.结论 血小板输注无效患者中,HLA抗体占主要地位,其次为血小板特异性糖蛋白抗体.     

血小板谱抗原的建立及其在鉴定血小板特异性抗体中的应用

目的 建立血小板谱抗原,鉴定引起血小板输注无效和新生儿血小板减少性紫癜的血小板特异性抗体,为血小板血型研究和临床治疗提供依据。方法根据中国人群人类血小板同种抗原(HPA)-1-HPA-16等位基因频率分布资料,利用聚合酶链反应-序列特异引物(PCR-SSP)技术对O型血小板供者进行HPA-1-HPA-6、HPA-15分型,筛选合适的供者,组成血小板谱抗原。通过建立的血小板谱抗原,利用简易致敏红细胞血小板血清学技术(SEPSA)鉴定同种免疫反应产生的血小板抗体的特异性。结果从O型血小板供者中筛选出11名供者,建立了血小板特异性抗体鉴定谱抗原。其可鉴定HPA-1-HPA-6,HPA-15抗体的特异性。在所筛检1 120份样本中,有3例患者检出HPA抗体,其中HPA-4b(Penb)抗体1例,HPA-15a(Govb)抗体2例。结论通过血小板谱抗原鉴定血小板抗体的特异性,对提高临床输注血小板的安全性和有效性,以及预防新生儿血小板减少性紫癜有积极的意义.     

简单实用的特异性抗原检测用于血小板抗体试验

背景目前用于鉴定血小板(PLT)自身和同种抗体反应的特异性抗原测定法对大多常规实验室来说太复杂,不实用。本文介绍一新的类似红细胞的特异性抗原微粒检测(ASPA)。研究设计与方法溶解血小板,然后同包被有针对不同血小板糖蛋白复合物的单克隆抗体(MoAbs)的红染聚苯乙烯微粒孵育。这些微粒直接测定包被的自身抗体(n=8)或间接测定血清     

充当免疫原的血小板

血小板同种抗原可以是与其他细胞共有的同种抗原或血小板特异性抗原。在共有性同种抗原之中,最重要的是HLAⅠ类抗原和ABO抗原。目前,已有五个血小板(特异性)抗原体系被确定,其中每个体系各有两种抗原。这些抗原体系已由妊娠患者或受血患者的抗体验明,而且通常和新生儿同种免疫性血小板减少症(NAIT)或输血后紫癜(PTP)相关。对血小板特异性抗原产生免疫应答是异常情况而不是规律。出现频率最高的抗体是抗-PI~(A1),占80～90%的病例,其次是抗—Br~4(10～15%);其他抗体     

抗HPA-3a抗体所致新生儿同种免疫性血小板减少性紫癜一例并文献复习

目的 探讨抗血小板特异性抗原(HPA)-3a抗体所致新生儿同种免疫性血小板减少性紫癜(NAITP)的诊断和治疗.方法 采用多重PCR及基因测序技术检测1例出血伴血小板减少新生儿及其父母HPA-1~21bw系统基因型,采用流式细胞术(FCM)和血小板抗原单克隆抗体特异性免疫固定检测技术(MAIPA)检测患儿及其母亲血清血小板特异性抗体并进行特异性鉴定.结果 患儿出生后2 h出现全身多发皮下出血点、血尿及咖啡色呕吐物.基因分型显示患儿为HPA-3ab、母亲为HPA-3bb、父亲为HPA-3aa;患儿及母亲血清中均含与患儿父亲血小板反应的特异性抗体,经MAIPA技术鉴定为抗HPA-3a抗体.结论 发现1例抗HPA-3a抗体所致NAITP患者,通过临床特征分析为该病的诊断和治疗提供借鉴与参考.     

血小板减少性疾病PAIgG的测定

人类血小板膜上有多种抗原,产生的血小板相关抗体(PAIgG)主要有红细胞血型(ABH)抗体、白细胞(HLA-I类)抗体和血小板特异性抗体(HPA)。PAIgG的测定对于诊断原发性血小板减少性紫癜(ITP)、血小板输注无效、免疫性血小板减少性紫癜、再生障碍性贫血等血小板减少性疾病有重要意义。     

Establishment of a cell line panel as an alternative source of platelet antigens for a screening assay of anti-human platelet antibodies

Background: A panel of platelets expressing various human platelet antigens (HPAs) for a platelet antibody screening assay is difficult to prepare because some antigens are rarely expressed. Therefore, an alternative method without using platelets would be helpful in detecting HPA antibodies. This study describes the establishment of cell lines that stably express specific HPAs and their application for detecting specific antibodies. Methods: Wild‐type β3, HPA‐1b, ‐6b, ‐7b and ‐7 variant cDNA as well as wild‐type αIIb and HPA‐3b cDNA were individually co‐transduced with wild‐type αIIb and β3 cDNA in the K562 cell line. We performed an immunobead monoclonal antibody immobilisation of platelet antigens (MAIPA) assay to evaluate this cell line panel for antibody detection using identified sera containing HPA antibodies, whose specificities had been determined by the mixed passive haemagglutination test. Results and Conclusion: Of the 12 sera containing HPA‐1a (n = 2), HPA‐3a (n = 6), HPA‐6b (n = 3) or HPA‐7 variant (n = 1) antibodies, all antibodies were detected and determined by our new method, except for two HPA‐3a antibodies. One of the two antibodies was also negative for conventional platelet MAIPA, suggesting that the cell line panel might be used as an alternative source of platelet antigens in the MAIPA assay.     

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.     

Complement-Fixing Platelet Iso-Antibodies in Serum of Transfused Persons. Correlation of Antibodies with Platelet Survival in Thrombocytopenic Patients

Sera from 172 patients who had received an average of 34 transfusions per patient were assayed for platelet antibodies using a complement fixation technic. Fifteen patients formed a total of 31 antibodies. Eighteen of these were identified as reacting with six specific platelet antigens which were present in from 3 to 49 per cent of the general population. The antigens are common to leukocytes, but not erythrocytes, and are genetically determined. No antibodies were found in serum from 55 non-transfused persons. The probability of antibody formation increased with repeated antigen exposure. Stored whole blood was at least as effective as platelet concentrates in provoking antibodies. There was a significant relationship between the presence of complement fixing platelet antibodies and the occurrence of "fever-chill" transfusion reactions. In three patients it was possible to correlate the efficacy of platelet transfusion therapy with the antigen content of infused platelets.     

Human platelet alloantigens.

Antibody formation against alloantigens of the human platelet membrane is responsible for clinical syndromes and transfusion related conditions as neonatal alloimmune thrombocytopenia (NAIT), post-transfusion purpura (PTP), platelet transfusion refractoriness (PTR) and passive alloimmune thrombocytopenia. Moreover, rare cases of alloimmune reactions involving platelets have been observed after transplantation of hematopoietic stem cells. Among alloantigens of the platelet membrane shared with other cells (type I alloantigens) are the glycoconjugates of the ABO system and class I human leukocyte antigen (HLA) antigens. Antibodies against these structures are responsible for PTR and for febrile nonhemolytic transfusion reactions. Antibodies against type II antigens (formerly termed "platelet specific antigens") have been observed in NAIT, PTP and passive alloimmune thrombocytopenia. ABH antigens have been identified on intrinsic platelet membrane glycoproteins. Moreover, it is now clear that HLA class I antigens are an integral part of the platelet membrane. The quantity of both HLA and ABH-antigen expression on the platelet membrane varies considerably. Single point mutations account for almost all platelet specific alloantigens, but most antigenic determinants seem to depend upon glycoprotein conformation: generally, platelet specific alloantibodies fail to recognize synthetic peptides encompassing the polymorphic residues. Restriction fragment polymorphism analysis and allele-specific PCR have been implemented for genotyping of platelet alloantigens in many laboratories. Antigen specific assays using monoclonal antibodies (MAIPA, immunobead assay) became de facto standard for diagnosis of platelet antibodies in serum/plasma samples. It can be expected that innovative techniques as human alloantibody fragments produced by phage display technique and the production of recombinant antigens will allow rapid and reliable phenotyping and antibody detection in the future.     

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.     

Analysis of granulocyte-reactive antibodies using an immunoassay based upon monoclonal-antibody-specific immobilization of granulocyte antigens

Summary. To detect human granulocyte-reactive antibodies, a glycoprotein-specific enzyme immunoassay for platelet antibodies was adapted for the use of granulocytes as target cells. Peripheral blood granulocytes were simultaneously incubated with a monoclonal antibody (mAb) and the serum to be investigated. After solubilization, aliquots of the cell lysate were transferred to plastic tubes coated with goat anti-mouse antibodies. Following immobilization of the trimolecular (mAb-glycoprotein-human antibody) complex it was detected by addition of enzyme-labelled goat anti-human antibodies using a luminescence technique. This assay allowed identification of different granulocyte-reactive antibodies present in the same sample without the need for complicated absorption studies. Alloantibodies against HLA and the granulocyte-specific NA antigens as well as isoantibodies against the Fc-gamma-receptor III (FcRIII) were detectable using mAb-specific immobilization of granulocyte antigens (MAIGA). Binding of autoantibodies to the FcRIII and to the CD 11b/CD 18 complex could be shown.     

Frequency and specificity of platelet-specific alloantibodies in HLA-immunized haematologic–oncologic patients

The frequency and specificity of platelet-alloantibodies to human platelet antigens (HPA) -1, -3 and -5 was investigated in 59 multitransfused, HLA-immunized patients. Using the MAIPA test (monoclonal antibody specific immobilization of platelet antigens) platelet alloantibodies could be demonstrated in 10 (17%) patients.   In one patient the antibody was present prior to any transfusions and probably induced by multiple previous pregnancies. This antibody was directed to HPA-5b. The remaining nine antibodies were found in patients ( n  = 36) with HLA-antibodies reacting with over 95% of unselected lymphocytes. In these patients the target antigens were HPA-1b in six, HPA-3a in one and both antigens in two patients.   Our findings demonstrate platelet alloimmunization induced by transfusions to be restricted to patients with high HLA-immunization. 25% of these patients (9/36) show platelet-specific antibodies, primarily HPA-1b.     

Diagnosis and management of neonatal alloimmune thrombocytopenia

Neonatal alloimmune thrombocytopenia (NAT) is a life-threatening bleeding disorder caused by maternal platelet antibodies produced in response to fetal platelet antigens inherited from the father. Antiplatelet antibodies cross the placenta and cause destruction of fetal platelets, leading to severe thrombocytopenia, and potentially bleeding, including fatal intracerebral hemorrhage. Incompatibilities between maternal and fetal platelets for the human platelet antigen 1a (previously called PL(A1)) account for most of the patients with NAT, but other antigens are commonly implicated. Diagnostic testing for NAT involves genotyping of maternal, paternal, and sometimes fetal DNA; platelet antigen phenotyping; and maternal platelet antibody investigations using specialized platelet glycoprotein specific assays. The management of women and infants at risk for NAT remains largely empiric; and mounting evidence points to prohibitive risks of invasive procedures such as fetal blood sampling and intrauterine platelet transfusions, except in rare circumstances. Improvements in our understanding of the pathophysiology of NAT, and of clinical and laboratory predictors of severity, may help develop better treatments and improve our ability to identify mothers at risk.     

The Assessment of Drug-Dependent and Isoimmune Antiplatelet Antibodies by the Use of Platelet Aggregometry

The technique of platelet aggregometry provides a simple, quantitative, and specific method for the detection of drug-dependent and isoimmune antiplatelet antibodies. In the presence of antiquinidine antibody, quinidine causes lysis of normal platelets in platelet-rich plasma. The resulting changes in optical density are readily detected in the aggregometer. The initial rate of lysis is a function of the antibody titer, but is relatively independent of the platelet count. In vitro, quinidine produces platelet swelling and inhibits aggregation of platelets by adenosine diphosphate, epinephrine, and collagen. Isoimmune antibodies cause aggregation of platelets in platelet-rich plasma. In studies of a single family the rate of aggregation is proportional to the number of HL-A antigens present on the normal platelets against which the antibody is directed. The simple technique of platelet aggregometry may be a useful adjunct in the selection of compatible donors for platelet transfusion. Serum derived from patients with idiopathic thromboytopenic purpura did not cause platelet aggregation.