Standardized measurement of major immunoglobulin class (IgG,IgA, and IgM) antibodies to β2glycoprotein I in patients with antiphospholipid syndrome

Patients with the antiphospholipid syndrome (APS) have autoantibodies directed against epitopes on β₂ glycoprotein I (β₂GPI). We describe herein the performance characteristics of standardized enzyme-linked immunosorbent assays (ELISAs) for anti-β₂GPI of the three major immunoglobulin classes: IgG, IgA, and IgM. All three assays generated highly linear standard curves (5 points, r ≥ 0.993 for each); precision was excellent both intra-assay and run-to-run, with coefficients of variation (CV) ranging from 2.3% to 6.6%. Values for IgG anti-β₂GPI correlated strongly with those obtained by an earlier method (r = 0.80, P < 0.0001). A study group consisting of 203 healthy subjects was used to generate percentile-based reference intervals for all three classes of anti-β₂GPI. APS subjects' anti-β₂GPI were found to differ significantly (P < 0.0001 for each) for those of the control population. All three assays correlated well with β₂GPI-dependent anticardiolipin antibody (aCL) measurements; IgG: r = 0.94 (P < 0.0001), for IgA: r = 0.82 (P < 0.001) and for IgM: r = 0.84 (P < 0.0001). We suggest that these ELISAs may provide valuable standardized measurements of IgG, IgA, and IgM anti-β₂GPI. J. Clin. Lab. Ana. 12:293–297, 1998. © 1998 Wiley-Liss, Inc.     

Identification of miRNAs as potential modulators of tissue factor expression in patients with systemic lupus erythematosus and antiphospholipid syndrome

Summary. Background: Tissue factor (TF) is the main initiator of the coagulation cascade and elements that may upregulate its expression might provoke thrombotic events. Systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) are autoimmune diseases characterized by a high TF expression in monocytes. Objectives: To examine the role of microRNAs (miRNAs) in TF expression and to evaluate their levels in SLE and APS patients. Methods: An in silico search was performed to find potential putative binding sites of miRNAs in TF mRNA. In vitro validation was performed transfecting cells expressing TF (THP‐1 and MDA‐MB‐231) with oligonucleotide miRNA precursors and inhibitors. Additionally, reporter assays were performed to test for the binding of miR‐20a to TF mRNA. Levels of miRNAs and TF were measured by quantitative (qRT‐PCR) in patients with APS and SLE. Results: Overexpression of miRNA precursors, but not inhibitors, of two of the members of cluster miR‐17～92, for example miR‐19b and miR‐20a, in cells expressing TF decreased TF mRNA, protein levels, and procoagulant activity between 30% and 60%. Reporter assays showed that miR‐20a binds to TF mRNA. Finally, we measured levels of miR‐19b and miR‐20a in monocytes from patients with APS and SLE and observed significantly lower miRNAs levels in comparison with healthy subjects inversely correlated with the levels of TF. Conclusions: Down‐regulation of miR‐19b and miR‐20a observed in patients with SLE and APS could contribute to increased TF expression and thus provoke the hypercoagulable state characteristic of these patients.     

系统性红斑狼疮合并抗磷脂综合征患者的临床分析

目的 研究系统性红斑狼疮合并抗磷脂综合征(SLE-APS)患者的临床特点.方法 对39例SLE-APS患者的临床和实验室资料进行回顾性分析.结果 39例患者中,有31例共发生了48次血栓栓塞事件,以深静脉血栓及脑梗死为主.26例已婚有生育史女性患者中,有12例发生病态妊娠.28例抗心磷脂抗体阳性;16例狼疮抗凝物阳性.24例患者先确诊SLE,平均9.5年后又出现APS样表现;12例先出现反复病态妊娠或血栓事件,平均4.8年后演变为SLE;3例一发病便同时符合SLE和APS的分类标准.有5例在发生血栓事件或病态妊娠时的狼疮活动指数<5分.结论 SLE-APS患者血栓事件及病态妊娠发生率增多.APS可出现于SLE之前、之后或同时.狼疮患者可以在病情稳定期出现APS的表现.详细询问病史、常规检测抗心磷脂抗体,有助于发现SLE-APS的高危因素,积极预防APS的发生.     

BioPlex 2200全自动免疫分析仪检测血清中抗双链DNA抗体的临床应用

目的分析比较BioPlex 2200全自动免疫分析仪和酶联免疫吸附测定(ELISA)检测血清中抗双链DNA(dsDNA)抗体的灵敏度、特异度及其优缺点。方法采用BioPlex 2200和ELISA检测101例纳入研究者的血清抗dsDNA抗体。纳入研究者中49例为临床确诊的系统性红斑狼疮患者,52例作为对照(包括32例其他结缔组织病患者和20例体检健康者)。结果BioPlex 2200检测抗dsDNA抗体的灵敏度为57.14%,特异度为94.23%;ELISA检测抗dsDNA抗体的灵敏度为53.06%,特异度为96.15%。两种方法总体符合率为95.05%,χ2检验分析,差异无统计学意义(χ2=78.69,P>0.05),Kappa值评价一致性较好(Kappa=0.880)。BioPlex 2200检测抗dsDNA抗体,3组抗dsDNA抗体水平比较,差异有统计学意义(F=61.52,P<0.05)。结论BioPlex 2200和ELISA检测血清抗dsDNA抗体结果有较好的一致性,检测迅速,值得临床推广应用。     

两个不同厂家自身抗体定量检测试剂盒的优劣评价

目的：评价四川省新成生物科技有限责任公司（A厂家）研制的赛盟自免棆抗dsDNA抗体检测试剂盒（酶联免疫吸附试验）与已上市销售的同类产品的优劣。方法收集2013年6～8月临床确诊为系统性红斑狼疮（SLE）的患者血清50例设为试验组,收集同期体检健康者血清50例设为对照组,分别采用A厂家和德国某知名B厂家生产的抗dsDNA抗体检测试剂盒进行定量检测,对检测结果进行统计分析。结果A厂家的抗dsDNA抗体检测试剂盒（酶联免疫吸附试验）阳性符合率高于B厂家同类试剂,结果差异具有统计学意义（P＜0．05）,两个厂家的阴性符合率差异无统计学意义（P＞0．05）;线性相关性分析r＝0．9848,转换为定性结果后采用Kappa分析,Kappa系数为0．91。结论A厂家生产的抗dsDNA抗体检测试剂盒（酶联免疫吸附试验）和德国某知名B厂家已上市销售的同类产品具有等效性,符合临床应用要求。     

血小板活化因子在急性肝肾衰竭动物模型中对肾功能的影响

目的:了解血小板活化因子(PAF)在急性肝、肾衰竭动物模型中对肾功能的影响及可能的作用机制。方法:设4组大鼠,分别注射D-氨基半乳糖(D-GaLN)加内毒素(LPS),D-GaLN加PAF,D-GaLN加LPS加PAF受体拮抗剂及生理盐水,诱导急性肝、肾衰竭动物模型,测肝、肾功能指标并观察肝、肾组织病理改变。结果:注射D-GaLN大鼠的血清丙氨酸氨基转移酶、总胆红素比生理盐水组升高10倍以上,差异有显著性,病检有肝细胞坏死。注射PAF或LPS组与PAF受体拮抗剂组或生理盐水组比,肾功能指标差异有显著性。肾组织病检可见部分肾小管坏死。结论:PAF与急性肝、肾衰竭动物模型中的急性肾衰有关;PAF可能参与了内毒素相关的肝肾综合征(HRS)的形成;PAF受体拮抗剂对HRS可能有治疗作用。     

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。     

系统性红斑狼疮中Th17细胞相关细胞因子的变化

目的探讨系统性红斑狼疮（systemic lupus erythematosus,SLE）血浆中T辅助17（Th17）细胞相关的细胞因子白细胞介素（interleukin,IL）-17、IL-6、IL-23、IL-27和转化生长因子β（transforming growth factor-beta,TGF-β）水平的变化。方法 SLE组43例;健康对照组20例。采用酶联免疫吸附测定法测定血浆中IL-17、IL-6、IL-23、IL-27和TGF-β水平,分析它们的相关性。结果 SLE组与健康对照组比较,血浆中IL-17（74.32±22.76）ng/L vs（32.12±5.68）ng/L,IL-6（62.06±9.92）ng/L vs（37.98±15.41）ng/L,IL-23（30.29±12.59）ng/L vs（15.10±12.31）ng/L和IL-27（27.61±11.16）ng/L vs（16.17±8.35）ng/L的浓度都明显升高（P〈0.01）,TGF-β的血浆浓度则明显降低（35.80±11.65）ng/L vs（69.08±28.76）ng/L（P〈0.01）。IL-17与IL-6,IL-23和IL-27之间呈正相关（r=0.542、0.887、0.757,均P〈0.05）,与TGF-β之间则呈负相关（r=-0.268,P〈0.05）。结论 IL-17及其相关的细胞因子在SLE表达异常,IL-17在SLE发病中具有重要作用。     

抗核小体抗体对系统性红斑狼疮诊断价值的探讨

目的：探讨抗核小体抗体对系统性红斑狼疮的诊断和判断疾病活动度的价值。方法：采用酶联免疫吸附试验（ELISA）检测45例系统性红斑狼疮、25例类风湿关节炎、16例原发性干燥综合征、8例多发性肌炎／皮肌炎、5例混合性结缔组织病、18例骨性关节炎和30例健康对照组血清中的抗核小体抗体，将抗核小体抗体与疾病活动度（以SLEDAI评分）、抗双链DNA抗体、抗Sm抗体等指标进行比较。结果：抗核小体抗体在45例系统性红斑狼疮患者34例（75．5％）阳性，其敏感度和特异度分别为75．5％，93，1％；抗核小体抗体与SLEDAI评分、抗双链DNA抗体、肾损害有相关性。结论：抗核小体抗体在系统性红斑狼疮中敏感性较高，是诊断系统性红斑狼疮和了解狼疮活动度的良好指标。     

False-positive tests for heparin-induced thrombocytopenia in patients with antiphospholipid syndrome and systemic lupus erythematosus

Summary.  Background : Heparin-induced thrombocytopenia (HIT) is a severe complication of heparin therapy that can be associated with arterial or venous thrombosis and is caused by antibodies against platelet factor 4 (PF4)–heparin complex. Patients with antiphospholipid syndrome (APS) have been reported with positive tests for PF4–heparin complex antibodies by antigen assay. Whether such patients can be treated with heparin is a dilemma. Objectives : To determine the incidence and nature of the HIT immune reaction in patients with APS and/or systemic lupus erythematosus (SLE). Methods : Antibodies against PF4–heparin complex were assayed by particle gel immunoassay (PaGIA), or enzyme immunoassay (EIA) with or without an excess of heparin. EIA for PF4 alone was also performed. Functional assays for HIT, that is, heparin-induced platelet activation (HIPA) and heparin-induced platelet aggregation, were also performed. Results : In 32 of 42 patients (76.2%) with APS, APS and SLE, SLE, or SLE with antiphospholipid antibodies, EIA IgG or PaGIA for PF4–heparin complex antibodies were positive. Of these 32 samples, 26 (81.3%) tested positive for anti-PF4 antibodies. All 24 samples that were positive for PF4–heparin complex by EIA IgG were also positive for EIA IgG in the presence of heparin excess, and all were negative by the HIPA and heparin-induced platelet aggregation tests. Conclusion : A large proportion of patients with APS and/or SLE give false-positive HIT antigen test results that are presumably related to autoantibodies against PF4, which can be distinguished from true HIT antibodies by EIA for PF4–heparin complexes tested with heparin excess, and by functional assays.     

血小板活化因子在新生儿缺氧缺血性脑病中作用

目的探讨血小板活化因子(PAF)与新生儿缺氧缺血性脑病(HIE)之间的关系。方法应用改良的Henson法测定实验鼠和HIE血清PAF水平,应用拮抗剂银杏提取物(BN52021)治疗实验鼠。结果实验鼠和患儿血清PAF明显高于对照组(P<0.001),对PAF增高的实验鼠,应用BN52021治疗,可以明显降低PAF的水平。结论PAF可做为判断HIE病情轻重程度和预后的重要指标,BN52021能明显抑制HIE小鼠体内PAF的产生,降低PAF在血清中的含量(P<0.001)。     

抗突变型瓜氨酸波形蛋白抗体对类风湿性关节炎的临床诊断价值

目的 探讨抗突变型瓜氨酸波形蛋白抗体(anti-mutated citrullinated vimentin,anti-MCV)对类风湿性关节炎(rheumatoid arthritis,RA)的临床诊断价值. 方法 用酶联免疫吸附试验法测定80例RA患者和35例其他自身免疫性疾病的非RA患者血清抗MCV抗体,用免疫速率散射比浊法同步检测类风湿因子(rheumatoid factor,RF),计算绘制受试者工作特性(ROC)曲线下面积. 结果 抗MCV和RF检测的敏感度分别为85.0%,70.0%;特异度为91.4%,45.7%;一致率分别为87.0%,62.6%,Kappa值分别为0.713,0.164,抗MCV检测均优于RF检测(均P<0.05).抗MCV抗体的ROC曲线下面积为0.923(95%可信区间为0.872～0.973),RF为0.658(95%可信区间为0.559～0.757),抗MCV抗体对RA的临床诊断价值较高. 结论 抗MCV抗体可作为临床诊断RA的一项很好的血清学指标,具有较高的敏感度和特异度,优于RF等其他血清学指标.对于筛查和辅助诊断类风湿性关节炎具有重要意义.     

乙酰胆碱和血小板激活因子含量变化与血管性痴呆发生机制的相关性

目的动态观察乙酰胆碱和血小板激活因子含量变化对血管性痴呆(vasculardementia,VD)发生机制的意义。方法Wistar大鼠共30只,将大鼠随机分为模型组(A组)和对照组(B组)。模型组用0号丝线间歇阻断双侧颈总动脉10min,共阻断双侧颈总动脉3次,每次间隔10min,同时尾静脉放血2ml,对照组仅分离双侧颈总动脉。用比色法和高效薄层色谱法,分别测定血管性痴呆大鼠手术后,第2,3,5天海马乙酰胆碱(acetylcholine,ACh)和血小板激活因子(plateletactivatingfactor,PAF)含量。结果手术后第2,3,5天大鼠海马ACh含量,在模型组与对照组间差异均有显著性意义(F=20.53,P<0.05);而PAF含量两组间则差异均无显著性意义(F=2.98,P>0.05)。结论血管性痴呆大鼠海马ACh含量的持续降低,可能是VD发生。发展的重要原因之一;而PAF则可能主要参与急性脑缺血早期的病理生理过程,对脑缺血后期的病理生理变化意义不大。     

Alterations of cerebral blood flow and antiphospholipid antibodies in patients with systemic lupus erythematosus

Twenty-two patients with systemic lupus erythematosus and 13 healthy controls were included in a cerebral blood flow study and underwent brain-dedicated single-photon emission computed tomography using^99mtechnetium-d, 1-hexamethylpropylene amine oxime together with a brain computed tomography scan. Plasma levels of antiphospholipid antibodies (lupus anticoagulant and anticardiolipin IgM and IgG antibodies) were also determined. Brain computed tomography showed signs of focal cerebral ischemia in 4 patients (18%), whereas cerebral blood flow by single-photon emission computed tomography was abnormal in 13 of 22 patients (59%), who showed bilateral or monolateral hypoperfusion in the temporo-parietal regions. Patients with abnormal cerebral blood flow had a longer duration of disease than those with normal blood flow (8.9±1.9 years vs. 5.3±1.5 years,P<0.05). Plasma antiphospholipid antibodies were present in 15 patients (68%), but the prevalence was similar in those with normal (6/9, 66%), or abnormal (9/13, 69%) cerebral blood flow. No statistically significant difference in lupus anticoagulant or anticardiolipin antibodies was observed between patients with and without cerebral blood flow abnormalities. Our study shows that patients with systemic lupus erythematosus frequently have cerebral blood flow abnormalities, which could precede those observed by computed tomography. Plasma lupus anticoagulant and anticardiolipin titers were not correlated with normal cerebral blood flow.     

Revised classification criteria for antiphospholipid syndrome and the thrombotic risk in patients with autoimmune diseases

BACKGROUND: The classification criteria for antiphospholipid syndrome (APS) were updated in 2006. Objective: The aim of the study was to analyze associations between clinical complications and laboratory test abnormalities typical for APS in a group of patients with autoimmune diseases, based on the recently updated criteria. PATIENTS/METHODS: Three hundred and thirty-six patients were enrolled into the study, with the majority (n = 235) suffering from systemic lupus erythematosus. Laboratory determinations included: lupus anticoagulant (LA), anticardiolipin (aCL) and anti-beta(2)-glycoprotein I (anti-beta(2)GPI) antibodies (ABs) [of both immunoglobulin G (IgG) and IgM class]. RESULTS: A significant association was found between laboratory and clinical features of APS; odds ratios (ORs) for thrombosis associated with the presence of LA, aCL, and anti-beta(2)GPI Abs were 4.04 [95% CI: 2.44-6.68], 3.71 (95% CI 2.32-5.92) and 2.57 (95% CI 1.60-4.1), respectively. Detailed analysis showed marked differences between the risk of clinical complications associated with the presence of an antibody in the IgG class (OR 4.15, 95% CI 2.42-7.12, and OR 4.77, 95% CI 2.37-9.61 for aCL and anti-beta(2)GPI, respectively) and in the IgM class (OR 2.2, 95% CI 1.31-3.70, and OR 1.9, 95% CI 1.15-3.14 for aCL and anti-beta(2)GPI, respectively). The postulated inclusion of anti-beta(2)GPI antibody positivity into the previous laboratory criteria changed only slightly the number of patients diagnosed with APS (from 112 to 117). CONCLUSIONS: The updated APS classification criteria clearly represent a step forward. However, our results argue against the use of overall positivity for aCL or anti-beta(2)GPI, and favor a clear distinction between the IgG and IgM classes of antiphospholipid ABs. Patients with both LA and anti-beta(2)GPI IgG or LA and aCL IgG positivity may represent the subgroups at the highest risk of thrombotic complications.     

血小板活化因子与婴幼儿牛奶蛋白过敏严重程度的相关性

目的 探讨血小板活化因子(PAF)与婴幼儿牛奶蛋白过敏(CMPA)严重程度的相关性.方法 选择2018年7月至2019年12月在中山市博爱医院儿保科和儿童消化科确诊为CMPA的婴幼儿60例,根据过敏症状将其分为轻中度过敏组(n=30)和重度过敏组(n=30),另随机选取无任何过敏病史的30例健康儿作为对照组.比较各组的...     

血浆溶血血小板活化因子测定及在儿童哮喘中应用

建立血浆溶血血小板活化因子（ｌｙｓｏ－ＰＡＦ）的测定方法，探讨在儿童哮喘中的应用。本文建立的方法线性范围２～１０μｇ／Ｌ，最适兔血小板浓度２５０～４００×１０９／Ｌ。用本法检测体检正常儿童２６例，儿童哮喘４３例，儿童肺炎１８例。正常儿童血浆ｌｙｓｏ－ＰＡＦ参考范围５８．６２±４１．７２μｇ／Ｌ，哮喘组ｌｙｓｏ－ＰＡＦ水平较肺炎及正常对照组显著增高（Ｐ＜０．０１）。而肺炎组与对照无明显差别（Ｐ＞０．０５），实验结果表明，血浆ｌｙｓｏ－ＰＡＦ测定可作为儿童哮喘辅助诊断及疗效观察的一项重要指标。     

3种抗双链DNA抗体检测方法比较及其联合检测对系统性红斑狼疮的诊断价值研究

目的分析间接免疫荧光法(IIF)、酶联免疫吸附试验(ELISA)和免疫印迹法(IBT)检测抗双链DNA(dsDNA)抗体的差异,以及联合检测在系统性红斑狼疮(SLE)诊断中的应用价值。方法选取2012年1月至2015年3月确诊的SLE患者50例,以及同期其他自身免疫病(AID)患者100例和体检健康者100例。分别采用3种方法检测其血清抗dsDNA抗体,比较3种方法的灵敏度与特异度,并分析3种方法联合检测的灵敏度与特异度。结果IIF法特异度(99.5%)最高,ELISA法灵敏度(74.0%)最高。IIF与ELISA法、IIF与IBT法、ELISA与IBT法检测SLE患者抗dsDNA抗体的检出率比较,差异均有统计学意义(χ~2值分别为11.435、13.994、4.539,P0.05);且一致性检验的Kappa值(κ)分别为0.411、0.522、0.278。3种方法串联检测特异度提高到99.5%,并联检测的灵敏度提高到82.0%。结论 3种检测抗dsDNA抗体的方法中,IIF特异度最高,ELISA灵敏度最高,联合检测可提高检测灵敏度与特异度。