Detection of platelet activation with monoclonal antibodies and flow cytometry. Changes during platelet storage

To investigate whether changes in platelet condition during platelet storage correlate with an altered expression of platelet membrane proteins, the binding of monoclonal antibodies (MoAbs) to fresh platelets was compared with MoAbs' binding to thrombin-activated platelets and to platelets stored as platelet concentrates. The MoAbs included antibodies against the platelet glycoprotein (GP) IIb/IIIa complex and against two activation-dependent antigens, one of which was a component of the internal platelet alpha-granule membrane (GMP 140) and the other of which was a 53-kD protein derived from platelet lysosomes. The binding of MoAbs to platelets fixed with 1 percent paraformaldehyde was measured by flow cytometry. In thrombin-activated platelets, a threefold increase was found in the expression of GP IIb/IIIa over that in fresh platelets. The binding of the activation-dependent MoAbs increased from 2 to 3 percent to 70 to 80 percent of the platelets. Storage of platelet concentrates for 5 days resulted in a 60 percent increase in GP IIb/IIIa expression compared to Day 0 and increased binding of the MoAbs directed against GMP-140 from 3 to 16 percent and against the 53-kD protein from 2 to 8 percent of the platelets, respectively. These changes correlated with modifications in platelet morphology (decrease in swirling), leakage of lactate dehydrogenase, and release of beta-thromboglobulin. These data indicate that platelets become activated and are damaged during the storage of platelet concentrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

1350名患儿血小板抗体检测结果分析

目的分析血小板抗体检测患儿阳性率,探讨其与血小板输注次数及其疾病相关性。方法回顾性分析本院2016年1月—2018年6月1 350例患儿血小板抗体检测结果,根据血小板抗体检测时血小板输注次数分成3组:0次组(675例)、1—3次组(384例)和>3次组(291例),分别统计分析各组血小板抗体检测阳性发生率,并分析抗体阳性患儿的疾病分布情况。结果 1 350例患儿共检出血小板抗体阳性162例(12%)。0次组血小板抗体检测阳性28例(4.15%),1—3次组和>3次组的阳性例数分别为55例(14.32%)和79例(27.15%),血小板抗体阳性率随血小板输注次数增加而升高(z=104.2,P<0.05)。162例阳性患儿疾病分布情况为:血液病142例(18.54%)、实体肿瘤8例(8.99%)、重症感染5例(4.77%)、新生儿血小板减少3例(3.75%)、外科手术患儿3例(1.66%)、其他疾病患儿1例(0.78%),血液病患儿阳性率最高(χ~2=94.321,P<0.05)。结论血小板输注次数与血小板抗体产生密切相关,且疾病本身亦可能导致血小板抗体阳性。     

Detection of plasmapheresis-induced platelet activation using monoclonal antibodies

Twelve volunteer plasma donors were studied so as to determine the extent and duration of in vivo platelet activation caused by automated plasmapheresis. Samples obtained immediately before and after donation were mixed with murine monoclonal antibodies PAC-1 and S12, which bind specifically to activated platelets. Antibody binding on platelets was quantitated by flow cytometry. The change in the mean fluorescence intensity (MFI) (MFI after donation minus MFI before donation) was 61 +/- 23 (confidence interval [CI], 48-75) for PAC-1 and 56 +/- 64 (CI, 19-92) for S12 in plasmapheresis donors, as compared to 0.3 +/- 0.8 (PAC-1; CI, -0.2-0.8) and 0.3 +/- 0.9 (S12: CI, -0.3-0.9) in whole blood donors (p less than 0.05). Additional studies showed circulating activated platelets up to 48 hours after plasmapheresis. In contrast to other data, significant platelet activation was demonstrated following plasmapheresis on an automated machine. None of the donors had clinical complications. Nevertheless, it may be appropriate to delay subsequent plasmapheresis and platelet procurement from such donors until evidence of platelet activation has disappeared.     

Detection of HLA antibodies by platelet crossmatching techniques

Thirty-seven monospecific HLA antibodies directed against all common HLA-A and -B loci and reactive by the microlymphocytotoxicity assay (LCT) were tested against platelets carrying the corresponding antigen by three platelet crossmatch methods, the platelet immunofluorescence test (PIFT), platelet enzyme-linked immunosorbent assay (P-ELISA), and platelet radioimmunoassay (P-RIA). Positive reactions were obtained with the PIFT in 67 percent, the P-ELISA in 41 percent, and the P-RIA in 49 percent of 85 cell-serum pairs. The same cell-serum combinations gave 49 percent positive reactions in the lymphocyte immunofluorescence test. Three multispecific HLA antisera were positive in nine of nine cell-serum combinations by all four methods. Thirteen transfusions were given to eight patients with known HLA antibodies. All donor-recipient pairs were LCT positive, six were PIFT positive, and seven were PIFT negative. Three of seven PIFT-negative and none of six PIFT-positive transfusions were successful. Thus, platelet crossmatching is less sensitive than the LCT for the detection of complement-binding monospecific HLA antibodies. The platelet crossmatch, however, is able to identify some potentially successful HLA-incompatible donors for patients with multispecific HLA antibodies and limited access to HLA-identical donors.     

大血小板检测及临床意义探讨

目的：评价ADVIA120血液分析仪对大血小板的检测及大血小板的临床意义。方法：将富含大血小板标本分别用免疫学法、手工法,ADVIA120和Coulter-jt血液分析仪地血小板,以免疫学法结果为参考值,比较手工法,ADVIA120和Coulter-JT与免疫学法的相关性,临床分析290例大血小板标本。结果：3种方法与免疫学法比较的相关系数分别为0．8121、0．9306和0．7669;妊娠和恶性肿瘤者大血小板分别占17．9％和20．0％。结论：ADVIA120血液分析仪是目前检测血小板较理想的仪器之一;妊娠和恶性肿瘤病人血中会出现较大的大血小板。     

Quantitative and qualitative platelet disorders.

Platelet disorders are common during childhood. Those conditions that are clinically significant are generally diagnosed with ease. Qualitative platelet disorders rarely cause clinical problems. The armamentarium of diagnostic tests, although limited, is usually satisfactory to allow for accurate diagnosis and effective treatment of those children with platelet disorders who are at risk of hemorrhage.     

肾移植后人类白细胞抗原抗体检测的临床意义

目的：了解肾移植受者的抗人类白细胞抗原（HLA）IgG抗体水平及其对肾移植效果的影响。方法：应用莱姆德抗原板和混合抗原板通过微量酶联免疫吸附法检测685例患者的抗HLA特异性IgG 抗体。结果：685例肾移植受者中HLA－IgG抗体阳性者占12％，致敏受者移植后排斥反应发生率为50％，明显高于HLA－IgG抗体阴性受者（P＜0．01），而移植物存活率则显著低于HLA－IgG抗体阴性受者（P＜0．01），移植后HLA－IgG抗体水平升高组，其排斥发生率和移植物丢失率分别为82％和77％，均显著高于HLA－IgG抗体无变化组（P＜0．01），而排斥逆转率则显著低于后者（P＜0．01）。结论：HLA－IgG抗体是预测受者HLA免疫致敏的一个敏感指标；动态监测HLA－IgG抗体水平的变化对临床筛选合适供者，减少排斥反应，提高移植物存活率重要意义。     

Detection of drug-dependent platelet antibodies using immobilized Staphylococcal protein A

Serum samples from eight patients suspected of having drug-induced immunologic thrombocytopenia provoked by a variety of medications were tested for drug-dependent platelet antibodies with a newly developed assay that forms rosettes of antibody-coated platelets around Staphylococcal protein A-Sepharose beads. The same samples were then tested with immunofluorescence and 51Cr-release assays. Seven of eight patients (87.5%) tested positive for drug-dependent antibodies by the rosette assay. In contrast, two of eight (25.0%) and one of eight (12.5%) patients tested positive for drug-dependent antibodies by immunofluorescence and 51Cr release, respectively. These results demonstrate that the new rosette method is significantly more sensitive and specific for the detection of drug-dependent platelet antibodies than either immunofluorescence or 51Cr release.     

Detection of platelet isoantibodies by (3H)serotonin platelet release and its clinical application to the problem of platelet matching.

The detection of platelet isoantibodies by the release of (3H)serotonin from platelets has been evaluated. The conditions for optimal release of (3H)serotonin with platelet isoantibodies using a microtechnique have been defined. A group of cardiac surgery patients were followed pre- and post-transfusions, with 48percent developing a positive serotonin release assay. Of these patients, 16percent also had a platelet complement-fixing and/or lymphocytotoxic isoantibody. There was variation in the degree of correlation between (3H)serotonin release and lymphocytotoxicity using individual National Institutes of Health typing serum. The matching obtained between family members by both techniques showed a close correlation when each technique was evaluated separately using the same NIH typing serum. The detection of iso-antibodies in patients with hematological malignancies correlated with the unresponsiveness to unmatched platelet transfusions in 15 out of 17 cases. The use of the patient's isoantibody to matched platelets of family members by (3H)serotonin release correlated well with the clinical response to transfusion with these platelets. The data suggest that (a) platelet isoantibodies can be detected with increased frequency by (3H)serotonin release; (b) (3H)serotonin release is a specific reaction depending on the surface antigen of the platelet; and (c) the method can be used to match compatible family members for platelet transfusions.     

The platelet factor 3 immunoinjury technique re-evaluated. Development of a rapid test for antiplatelet antibody. Detection in various clinical disorders, including immunologic drug-induced and neonatal thrombocytopenias.

A modified version of the platelet factor 3 immunoinjury technique for the detection of antiplatelet antibody is described in detail; heat-inactivated sera and frozen platelets are employed. The modification allows the results of the test to be available within 4 to 5 hours after drawing the blood sample, in contrast to the 3 days required in the original method. The technical work involved is significantly reduced. The reproducibility of the technique is improved. Results obtained with the two tests correlate well. Data accumulated in 458 cases of clinical disorders associated with antiplatelet antibody are reported. Drug-dependent antiplatelet antibody was demonstrated in 26 cases of thrombocytopenia associated with 19 different drugs. Data are also presented for 23 cases of neonatal thrombocytopenia.     

Diagnostic approach to platelet function disorders.

Platelet disorders are common bleeding disorders, with a variety of congenital and acquired causes. The diagnostic evaluation of platelet disorders challenges both clinicians and clinical laboratories, as testing for these conditions is complex, not well standardized and time consuming. An understanding of normal platelet function has provided insights on the pathogenesis of many platelet function disorders. Knowledge of the key features of platelet disorders aids their diagnostic assessment. Tests for aggregation, secretion and dense granule defects continue to be the most helpful for the evaluation of suspected platelet function disorders.     

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.     

急性白血病患者血清血小板衍生生长因子检测及临床意义

目的探讨血小板衍生生长因子(PDGF)在急性白血病中的表达及意义。方法急性白血病患者(实验组)42例,依据法美英(FAB)诊断标准分为急性淋巴细胞白血病(ALL)组和急性非淋巴细胞白血病(ANLL)组;同时,根据有无髓外浸润再分为髓外浸润组和非髓外浸润组,又根据诊断与治疗分别分为复发和初诊、部分缓解和完全缓解亚组。正常对照者(对照组20例)。应用酶联免疫吸附法(ELISA)检测各组研究对象的血清PDGF水平。结果实验组血清PDGF(406.77±119.35)ng/L明显高于对照组(128.59±15.23)ng/L(P<0.01);ALL血清PDGF(503.32±89.78)ng/L高于ANLL(334.36±81.67)ng/L(P<0.05);但在ALL和ANLL各亚型之间PDGF水平差异无统计学意义;髓外浸润组PDGF(437.11±100.99)ng/L比非髓外浸润组(330.93±131.99)ng/L表达高(P<0.05);复发患者PDGF(442.20±91.26)ng/L、初诊患者(447.70±100.46)ng/L表达均高于部分缓解(280.07±34.64)ng/L、完全缓解组患者(253.27±91.53)ng/L,(P<0.05);急性白血病组患者治疗前血清中PDGF(406.77±119.35)ng/L明显高于治疗后(181.25±52.69)ng/L(P<0.05)。结论 PDGF在急性白血病中高表达,可能与白血病的发生及疗效有关。     

自身免疫性肝病相关抗体检测及其临床意义

自身免疫性肝病是一组由于机体自身抗体存在而引起肝组织持续损伤为特征的慢性肝病,主要包括自身免疫性肝炎(autoimmune hepatitis,AIH)、原发性胆汁性肝硬化(primary biliary cirrhosis,PBC)和原发性硬化性胆管炎(primary sclerosing cholangitis,PSC)[1]。由于我国的肝炎和肝硬化多因肝炎病毒尤其是乙型肝炎病毒所致,长期以来对自身免疫性肝病认识不足,研究不够,以致误诊、漏诊不在少数,而延误了对患者的治疗。自上世纪80年代我国开始实行新生儿肝炎疫苗接种以来,乙型病毒性肝炎发病逐步下降,乙型病毒性肝炎的治疗迅速发展。非病毒性…     

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.