Increased amounts of von Willebrand factor are bound to microparticles after infusion of desmopressin

Effects of desmopressin (DDAVP) in platelet disorders and primary haemostasis cannot be attributed solely to the increase in FVIII/VWF (von Willebrand factor), as VWF/FVIII concentrates have no effect in these circumstances. Microparticles (MP) can support haemostasis by expression of phospholipids, tissue factor and VWF on their surface. We hypothesized that significant amounts of VWF are bound to MP after DDAVP administration and that consequently depletion of MP should influence VWF:Ag and VWF:RCo plasma levels. Platelet‐poor plasma was either obtained well from healthy controls or before and after DDAVP administration from patients with von Willebrand's disease (type 1 or possible type 1) or patients with other bleeding disorders as controls. Concentrations of MP and VWF parameters were determined before and after MP depletion by different methods (magnetic bead selection, plasma microfiltration, ultracentrifugation). Platelet MP and VWF‐bearing MP were significantly increased after DDAVP. MP depletion by magnetic bead selection led to a significant reduction in VWF:Ag (?18.0%) and VWF:RCo (?27.7%) plasma levels without changes in VWF multimer composition. As results were similar for DDAVP control subjects, the amount of VWF bound to circulating microparticles was significantly higher after DDAVP administration compared with healthy controls (reduction ?11.7%). DDAVP leads to a release of microparticles and increases the amount of VWF bound to microparticles which might explain the clinical efficacy of DDAVP in platelet disorders.     

CBA法研究HLA全相合与单倍体相合移植患者血清细胞因子表达的差异性

本研究旨在探讨非清髓异基因造血干细胞HLA全相合移植（HLA-identical nonmyeloablative allogeneichematopoietic stem cell transplantation,iNAHSCT）和单倍体相合移植（HLA-haploidentical nonmyeloablative allogeneichematopoietic stem cell transplantation,hiNAHSCT）患者血清细胞因子表达的差异性,用流式细胞微珠阵列法（flow cy-tometric bead array,CBA）检测20例白血病患者移植前后不同时间点IL-2、IL-4、IL-6、IL-10、TNF-ɑ、γ-IFN、IL-17浓度的变化。结果表明,IL-2分别在iNAHSCT及hiNAHST后1周、2周起表达上调,两类移植各时间点比较无差异（P〉0.05）;γ-IFN都在移植后4周起上调,两类移植后的浓度无差异（P〉0.05）;IL-4在iNAHSCT及hiNAHSCT中分别于移植后2周、移植后1周起增高,iNAHSCT的IL-4浓度高于hiNAHSCT（P〈0.01）;IL-6在iNAHSCT及hi-NAHSCT中分别在移植后1周、移植后2周起上调,移植后4周为峰值,且hiNAHSCT的IL-6浓度高于iNAHSCT（P〈0.02）;IL-10在iNAHSCT、hiNAHSCT中分别于从移植后1、2周起增高,iNAHSCT中IL-10的表达高于hiNAH-SCT（P〈0.05）。TNF-ɑ在hiNAHSCT中从移植后1周、在iNAHSCT中从移植后2周起上调;hiNAHSCT中TNF-ɑ增高的幅度高于iNAHSCT（P〈0.01）。IL-17分别于hiNAHSCT及iNAHSCT后移植后1周、移植后4周时增高,hi-NAHSCT IL-17增高的幅度高于iNAHSCT的（P〈0.05）。结论：两类移植后所检测到的细胞因子水平均明显增高,移植后4周为峰值。IL-6、TNF-ɑ、IL-17在hiNAHSCT中增高明显,而IL-4,IL-10在iNAHSCT中增高显著。     

用免疫磁珠法检测抗白念珠菌烯醇化酶抗体

摘要：目的：建立检测人血清抗白念珠菌烯醇化酶(Eno)IgG类抗体的免疫磁珠法,并评估其在念珠菌血症诊断中的价值。 方法：将Eno重组蛋白耦联至磁性微珠上,以辣根过氧化物酶（HRP）标记的羊抗人IgG为二抗,优化免疫磁珠法的反应条件,考察其精密度和特异性。收集经血培养确诊的念珠菌血症患者（n=113）、念珠菌定植者（n=50）、细菌菌血症患者（n=30）和健康人对照者（n=100）血清,用免疫磁珠法测定抗Eno抗体,并与ELISA法结果进行比较。 结果：免疫磁珠法测定抗Eno抗体的批内、批间变异系数（CV）分别为8.2%和14.2%,重组Eno抗原对相应抗体的阻断率为91.6%。以吸光度（A）值0.29为cut off值时,诊断念珠菌血症的敏感性、特异性、阳性预测值和阴性预测值分别为80.5%、92.3%、90.1%和84.5%。免疫磁珠法的敏感性和特异性与ELISA法比较无统计学差异,但检测流程从37 ℃ 2.5 h缩短至常温1 h。 结论：免疫磁珠法检测抗Eno IgG类抗体简便、快捷、可靠,在侵袭性念珠菌感染研究中具有潜在的应用价值。     

流式免疫微球捕获技术和荧光定量PCR检测BCR-ABL融合基因的方法学比较

目的:对流式微球捕获技术和荧光定量PCR技术在BCR-ABL融合基因中的检测进行比较和评价。方法:选取18例慢性粒细胞性白血病(chronic myeloid leukemia,CML)患者、6例急性髓系白血病(acute myeloblasticleukemia,AML)患者、9例缺铁性贫血(iron deficiency anemia,IDA)患者,分别用流式微球捕获技术和荧光定量PCR技术检测其骨髓BCR-ABL融合基因蛋白和mRNA。结果:6例AML和9例IDA患者均未检出阳性。18例CML患者荧光定量PCR检出16例P210和2例P190,流式微球捕获技术则均检出阳性。流式微球捕获技术的lgMFI(平均荧光强度)和荧光定量PCR法的lgCopies(拷贝数)的相关系数为0.708(P<0.01)。结论:流式免疫微球捕获技术对BCR-ABL各种断裂类型敏感性高,但不能区分断裂类型,适合作为初筛实验。     

用蛋白质组学方法分析先兆子痫孕妇血清差异蛋白表达和急性反应蛋白

目的用蛋白质组学方法寻找先兆子痫患者血清蛋白的差异表达。方法 5例重度先兆子痫患者血清和5例正常妊娠对照血清分别混合。每个混合样本血清用亲水亲脂柱(hydrophilic-lipophilic balance,HLB)和多肽配体库磁珠(peptide ligandlibrary bead,PLLB)方法去除表达丰富蛋白,使低含量蛋白表达,洗脱液用1D胶联合液相色谱质谱串联技术(liquidchromatography tandem mass spectrometry,LC-MS/MS)对蛋白表达进行定量分析;结果 HLB方法共有28种蛋白发生差异表达;PLLB方法共有51种蛋白发生差异表达,包括白介素17F(IL-17F)和铜蓝蛋白(ceruloplasimin,CP)等。结论 PLLB联合1D胶-LC-MS/MS是鉴定血清中差异表达蛋白的一种有效方法;炎性反应因子可能与先兆子痫的发病机制有关。     

免疫磁珠捕获法分离抗酸杆菌的实验研究

目的建立免疫磁珠捕获法,用于分离抗酸杆菌,以提高痰菌阴性肺结核的诊断率。方法选取浸润型肺结核患者19例作为实验组,对照组6例,为肺部普通细菌感染者。受检者留取痰液,分别做直接涂片抗酸染色检查及免疫磁珠分选后抗酸染色检查。结果 19例浸润型肺结核患者中,7例痰涂片抗酸染色检查阳性,免疫磁珠分选后抗酸染色仍阳性;在12例痰涂片阴性肺结核患者中,经免疫磁珠分选后6例抗酸染色阳性。6例普通细菌性肺炎患者经免疫磁珠分选后抗酸染色均为阴性。结论用免疫磁珠捕获法分离抗酸杆菌能显著提高痰菌阴性肺结核的诊断效率。     

A novel assay to measure B cell responses to keyhole limpet haemocyanin vaccination in healthy volunteers and subjects with systemic lupus erythematosus

The aim of the study was to characterize performance of a complementary set of assays to measure antigen-specific immune responses in subjects immunized with a neoantigen. Healthy volunteers (HV) (n = 8) and patients with systemic lupus erythematosus (SLE) (n = 6) were immunized with keyhole limpet haemocyanin (KLH) on days 1 and 29. Serum antibodies were detected using a flow cytometric bead array (CBA) that multiplexed the KLH response alongside pre-existing anti-tetanus antibodies. Peripheral blood mononuclear cells were studied by B cell ELISPOT. These assays were built upon precedent assay development in cynomolgus monkeys, which pointed towards their utility in humans. Primary anti-KLH IgG responses rose to a mean of 65–93-fold above baseline for HV and SLE patients, respectively, and secondary responses rose to a mean of 260-170-fold above baseline. High levels of anti-tetanus IgG were detected in pre-immunization samples and their levels did not change over the course of study. Anti-KLH IgG1-4 subclasses were characterized by a predominant IgG1 response, with no significant differences in subclass magnitude or distribution between HV and SLE subjects. Anti-KLH IgM levels were detectable, although the overall response was lower. IgM was not detected in two SLE subjects whodid generate an IgG response. All subjects responded to KLH by B cell ELISPOT, with no significant differences observed between HV and SLE subjects. The CBA and B cell ELISPOT assays reliably measured anti-KLH B cell responses, supporting use of this approach and these assays to assess the pharmacodynamic and potential safety impact of marketed/investigational immune-therapeutics.     

Immunomagnetic bead-based cell concentration microdevice for dilute pathogen detection

A cell concentration microdevice for immunomagnetic pathogen isolation from a dilute sample is presented. Cells are driven by integrated on-chip pumps through a fluidized bed of immobilized immunomagnetic beads. Off-chip polymerase chain reaction and capillary electrophoretic analysis are used to determine capture efficiencies of E. coli and to optimize the system. Beads are immobilized after each split in a bifurcated channel system to ensure a balanced distribution of beads in all the capture channels. The addition of a pumping flutter step to repeatedly drive sample through the bead bed was found to enhance capture. Capture efficiencies of 70% and a limit of detection of 2 cfu/microL were achieved; specific capture of E. coli at a concentration of 100 cfu/microL in a 100-fold background of S. aureus is shown. This capture/concentration system is an important step in overcoming the macro-to-micro interface challenge in the development of microdevices for pathogen detection.     

社区常见恶性肿瘤筛查研究

目的探寻一种简便的恶性肿瘤初筛自查方法。方法将人体肿瘤分为三类:体表肿瘤约占15％;空腔脏器肿瘤占65％;深层实体脏器肿瘤20％。通过科普宣传教育,让群众学会自查体表部位肿瘤,每年用自查盒助查各空腔脏器有无微量出血,隐血阳性时,去医院进一步精查。北京部分大学选40～70岁居民1万人,分成试验组、对照组各5千人。结果试验四年后:试验组共查出79例癌,年平均癌检出率482.5人/10万,癌死亡率为12.2/10万;对照组癌死亡率206.2人/10万,两组有显著性差异。在癌症高发区,同时作扩大普查试验:广东四会市肿瘤所,在门诊普查1669人,检出25例癌,鼻咽癌占24例。1999～2000年,江苏省食管胃癌高发区,普查近8万人,检出480例癌。加上“九五”以前的普查统计,用秦氏自查盒已筛查431075人,检出1272例癌,癌前病变1万多例。结论试验证明该方案设计合理,在当前是一种最简便的恶性肿瘤初筛自查方法。