体外转运红细胞囊泡的GPI锚定蛋白cD59保护阵发性睡眠性血红蛋白尿症红细胞

阵发性睡眠性血红蛋白尿症(PNH)是一种后天获得性克隆溶血疾病,由于血细胞膜上缺乏具有调节蛋白补体的GPI-锚定蛋白如CD59和CD55等,所以对补体敏感,易产生溶血。红细胞存储或ATP耗空时有囊泡释放,经免疫印迹试验证明囊泡中富含CD59。应用免疫亲和层析柱分离PNH CD59~-红细胞,然后与红细胞囊泡保温,保温后分别测定PNH CD59~-细胞表面CD59含量和溶血度。用流式细胞仪测定CD59,结果发现保温后CD59含量增加;用蛇毒因子溶血试验测得溶血度有显著下降。体外实验证明囊泡上的CD59可以转运到PNH CD59~-红细胞上,并具有抑制补体溶血的功能,使PNH CD59~-红细胞在受补体攻击时不易溶血。     

CD55与CD59及其在PNH患者血细胞膜上的表达

CD55与CD59是两种重要的补体调节蛋白,广泛存在于各种细胞膜表面,具有抑制补体系统激活,充当信号,协助T细胞活化等功能。在阵发性睡眠性血红蛋白尿症(PNH)患者血细胞膜上,CD55与CD59表达较正常水平低,籍此不仅可部分解释PNH的病理机制,且有助于该病的诊断和鉴别诊断,乃至未来的基因治疗。     

阵发性睡眠性血红蛋白尿症实验室诊断

阵发性睡眠性血红蛋白尿症(PNH)是一种后天获得 的血细胞膜缺陷的克隆性疾病,临床上可表现血管内溶 血、栓塞、全血细胞减少等,有时可出现骨髓衰竭[1]。现 已证实,大多数PNH患者异常克隆与正常造血并存,使本 病的诊断和发病机制的研究受到限制。PNH患者红细胞 膜表面缺乏加速衰变因子(DAF,CD55)、反应性溶血的膜 抑制物(MIRL,CD59)和C8结合蛋白(或称同种限制因 子,HRF)等,使红细胞对补体敏感而溶血。上述3种蛋 白都经糖基化磷脂酰肌醇(GPl)连接在细胞膜上[2]。近 年来,由于各种检测技术的发展使得PNH发病机制的研 究、PNH…     

CD55和CD59检测对诊断阵发性睡眠性血红蛋白尿的意义

目的：探讨检测患者红细胞和中性粒细胞膜上CD55和CD59表型的缺失对诊断阵发性睡眠性血红蛋白尿（PNH）的意义。方法：应用流式细胞仪间接免疫荧光标记法检测40例正常人（对照组），29例PNH患者（PNH组），16例自身免疫性溶血性贫血患者（AIHA组），12例缺铁性贫血患者（IDA组）的红细胞和中性粒细胞CD55和CD59表型的缺失情况。结果：PNH组红细胞和中性粒细胞CD55^-、CD59^-的百分率与对照组比较有显著性差异（P〈0．01），AIHA组、IDA组与对照组比较无显著性差异（P〉0．05）。结论：利用流式细胞术同时检测患者红细胞和中性粒细胞膜CD55和CD59表型的缺失是目前诊断PNH最可靠、最敏感的方法。     

41例阵发性睡眠性血红蛋白尿症患者血细胞CD55和CD59抗原检测

采用流式细胞术(FCM)和免疫荧光标记技术对阵发性睡眠性血红蛋白尿症(PNH)患者完全或部分缺乏退变加速因子(DAF,CD55)和反应性溶血膜抑制物(MIRL,CD59)的异常红细胞和粒细胞进行分析[1-5],提高了对PNH 的诊断水平,但各实验室之间缺乏统一的判断标准.我们应用Biocytex公司提供的标准化的Redquant CD55/CD59 和Cellquant CD55/CD59试剂盒,可以简便、快速地对PNH细胞表型进行定量分析,现报道如下.     

CD55、CD59与再生障碍性贫血

<正>补体调节蛋白CD55、CD59与阵发性血红蛋白尿症(paroxysmal nocturnal hemoglobinuria,PNH)关系密切,CD55、CD59已被广泛应用于PNH诊断中[1],并将CD55、CD59表达缺乏的细胞称为PNH细胞。Ruiz-Delgado等[2]发现红细胞表面CD55、CD59     

低分子量肝素和地塞米松对PNH患者红细胞体外抑制溶血作用的研究

目的 研究低分子量肝素和地塞米松对阵发性睡眠性血红蛋白尿症（PNH）患者红细胞体外溶血的作用。方法 采集6例典型溶血发作的PNH患者外周血，采用Ham试验、微量补体溶血敏感试验（mCLST)检测单独或联合加入不同剂量的低分子量肝素和地塞米松后对溶血的影响；同时检测各种不同剂量的药物对活化部分凝血活酶时间（APTT）的影响。结果 （1）低分子量肝素、地塞米松体外对PNH患者的红细胞均有抑制溶血的作用，此种作用随剂量的增大而增大。当同时加入上述两种治疗量的药物时，对溶血的抑制作用明显加强。同时，在此种剂量下APTT的延长未超过2倍。（2）Ham试验检测时地塞米松有抑制溶血的作用，mCLST检测时地塞米松随加入方法的不同对溶血的作用不同。低分子量肝素在Ham和mCLST试验中均可发挥抑制溶血的作用。结论 低分子量肝素、地塞米松均有抑制PNH患者红细胞溶血的作用，其作用机制存在差异；二者具有协同作用，治疗剂量对出凝血的影响较小，在PNH患者红细胞溶血发作的治疗中有应用前景。     

CD55/CD59缺陷检测及其在阵发性睡眠性血红蛋白尿诊断中的意义

目的：应用外周血红细胞CD55和CD59的检测并结合相关检验，建立有效的诊断和鉴别诊断阵发性睡眠性血红蛋白尿(PNH)的实验体系。方法：运用流式细胞术检测10例PNH患外周血红细胞和粒细胞的CD55和CD59，并分别对缺陷红细胞作激光散射分析(FS)及FS／CD55和FS／CD59双参数分析，结合溶血性贫血(溶贫)相关其他检验，作出PNH诊断。结果：5例PNH发作期患酸溶血试验呈阳性，CD55和CD59缺陷红细胞在流式细胞术检测中具有特征性的改变；5例PNH缓解期酸溶血试验等无明显改变，患外周血CD55和CD59缺陷红细胞的数量较少。运用流式细胞术分别作红细胞FS／CD55和FS／CD59双参数分析，发现CD59缺陷与红细胞FS具有相关性。正常红细胞与缺陷红细胞的FS分布存在明显差异。部分再生障碍性贫血患外周血也存在PNH样细胞，但FS／CD59检测结果与PNH患具有不同特征。结论：流式细胞术检测外周血红细胞CD55和CD59缺陷结合溶血性贫血的常规检验可对PNH作出较明确的诊断和鉴别诊断。     

中药在抑制补体活性及溶血反应中的作用

补体是免疫系统的重要组成部分,补体过度激活可引起炎症反应、组织损伤及各类免疫性溶血反应。临床上常见的溶血反应多为新生儿溶血及血型不合溶血性输血反应,两者均由补体活化介导红细胞破坏而发生溶血,且危害较大。目前,溶血性输血反应及新生儿溶血病的治疗主要应用免疫抑制剂等药物,虽起效快,但具有一定毒副作用。然而,我国中药资源丰富,应用理论博大精深,若能以中药抑制补体活性,采用中西医结合输血,溶血性疾病的防治将会更加安全有效。因此,本文将对部分已知中药在抑制补体活性及减轻溶血反应中的作用作一综述,以期全面了解中药在补体抑制剂研发及溶血性疾病防治中的潜在应用价值。     

CD59检测在阵发性睡眠性血红蛋白尿症诊断中的意义

目的　探讨CD5 9检测对阵发性睡眠性血红蛋白尿症 (paroxysmalnocturnalhemoglobinuria ,PNH)及再生障碍性贫血 (AA) PNH综合征的临床诊断意义。方法　用FITC标记的CD5 9单抗 ,以流式细胞仪检测 13例PNH、37例AA和 2 0例正常人外周血红细胞和粒细胞膜表面CD5 9的表达情况。结果　正常人红细胞和粒细胞CD5 9表达阳性率均 >98% ;13例PNH患者红细胞及粒细胞CD5 9表达阳性率均 <90 % ;37例再障患者中 2 4例表达正常 ,13例红细胞或粒细胞CD5 9表达阳性率均有不同程度的减低。结论　CD5 9检测是诊断PNH较直接而又特异、敏感的方法 ,特别是对表现不典型的PNH及AA PNH综合征的诊断具有重要意义     

嗜水气单胞菌hec毒素溶血试验诊断阵发性睡眠性血红蛋白 …

目的 研究嗜水气单胞菌ｈｅｃ毒素对红细胞的作用,为阵发性睡眠性血红蛋白尿症（ＰＮＨ）的临床诊断提供一种新方法。方法 从患暴发性传染病的家养鲫鱼分离到嗜水气单胞菌,其培养上清经硫酸铵沉淀获得粗提毒素,经ＤＥＡＥ纤维素层析得提纯毒素。粗提毒素作用于ＰＮＨ和其他贫血患者及正常人的红细胞,采用９６孔板在波长６３０ｎｍ酶标仪比色;同时用ＣＤ５９单抗和ＦＴＴＣ标记的羊抗鼠ＩｇＧ标记红细胞,以流式细胞仪分析毒素     

Mechanisms and clinical implications of thrombosis in paroxysmal nocturnal hemoglobinuria

Summary. Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired disease characterized by a clone of blood cells lacking glycosyl phosphatidylinositol (GPI)‐anchored proteins at the cell membrane. Deficiency of the GPI‐anchored complement inhibitors CD55 and CD59 on erythrocytes leads to intravascular hemolysis upon complement activation. Apart from hemolysis, another prominent feature is a highly increased risk of thrombosis. Thrombosis in PNH results in high morbidity and mortality. Often, thrombosis occurs at unusual locations, with the Budd–Chiari syndrome being the most frequent manifestation. Primary prophylaxis with vitamin K antagonists reduces the risk but does not completely prevent thrombosis. Eculizumab, a mAb against complement factor C5, effectively reduces intravascular hemolysis and also thrombotic risk. Therefore, eculizumab treatment has dramatically improved the prognosis of PNH. The mechanism of thrombosis in PNH is still unknown, but the highly beneficial effect of eculizumab on thrombotic risk suggests a major role for complement activation. Additionally, a deficiency of GPI‐anchored proteins involved in hemostasis may be implicated.     

补体调节蛋白在三种干细胞缺陷性贫血中的表达及其意义的研究

目的 观察糖化磷脂酰肌醇（ＧＰＩ）连接的补体调节蛋白（ＣＲＰ）－ＣＤ５５和ＣＤ５９在阵发性睡眠性血红蛋白尿症（ＰＮＨ）,再生障碍性贫血（再障）和骨髓增生异常综合征（ＭＤＳ）患的外周血血细胞表达的变化并分析其在上述疾病中的临床意义。方法 采用荧光标记抗ＣＤ５５和ＣＤ５９单克隆抗体,流式细胞术测定３０名正常人,２１例ＰＮＨ患,３１例再障患及２１例ＭＤＳ患外周血中性粒细胞,淋巴细胞和红细胞膜抗原     

Paroxysmal nocturnal hemoglobinuria and the transfusion of washed red cells. A myth revisited

Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon, acquired clonal stem cell disorder primarily affecting red cells that have an abnormal sensitivity to complement lysis. Since 1948, the use of saline-washed red cells (WRBCs) has been advocated to minimize hemolysis after transfusion to patients with PNH. Thirty-eight years of experience (1950 through 1987) with patients who had PNH were reviewed. Twenty-three patients with a positive Ham's test had been transfused with 556 blood components, including 431 RBC products: 94 units of whole blood, 208 units of packed RBCs, 80 units of white cell-poor RBCs, 38 units of WRBCs, 5 units of frozen RBCs, and 6 units of intraoperatively salvaged RBCs. Only one documented episode of posttransfusion hemolysis related to the underlying diagnosis of PNH was found, and it was associated with the transfusion of a unit of type O whole blood to an AB-positive individual. This unit contained ABO-incompatible plasma; this case was similar to one in an earlier report from which originated the recommendation for using WRBCs. The posttransfusion increment in hemoglobin concentration in patients receiving ABO-identical packed RBCs was comparable to that in patients receiving frozen or washed RBCs. These findings indicate that the use of WRBCs is unnecessary and that patients with PNH should be transfused with group-specific blood and blood products.     

Association of Blood Group Antigen CD59 with Disease

In 2014, the membrane-bound protein CD59 became a blood group antigen. CD59 has been known for decades as an inhibitor of the complement system, located on erythrocytes and on many other cell types. In paroxysmal nocturnal haemoglobinuria (PNH), a stem cell clone with acquired deficiency to express GPI-anchored molecules, including the complement inhibitor CD59, causes severe and life-threatening disease. The lack of CD59, which is the only membrane-bound inhibitor of the membrane attack complex, contributes a major part of the intravascular haemolysis observed in PNH patients. This crucial effect of CD59 in PNH disease prompted studies to investigate its role in other diseases. In this review, the role of CD59 in inflammation, rheumatic disease, and age-related macular degeneration is investigated. Further, the pivotal role of CD59 in PNH and congenital CD59 deficiency is reviewed.     

阵发性睡眠性血红蛋白尿症临床缓解患者仍可查出异常血细胞

目的观察临床完全缓解的阵发性睡眠性血红蛋白尿症（ＰＮＨ）患者体内是否仍有异常造血细胞并探讨其临床意义。方法用免疫荧光标记及流式细胞术检测患者骨髓有核细胞、外周血红细胞ＣＤ５９抗原表达及骨髓ＣＤ３４＋造血干／祖细胞数。结果２例临床完全缓解的ＰＮＨ患者体内仍呈正常造血细胞与异常克隆并存状态,但与未缓解ＰＮＨ患者相比,完全缓解患者外周血红细胞、骨髓单个核细胞及ＣＤ３４＋细胞中均以正常的ＣＤ５９＋细胞为主,表明体内异常克隆造血优势已经消失。结论临床完全缓解的ＰＮＨ患者仍可有残存的异常克隆;ＰＮＨ达到临床完全缓解并不一定需要体内异常克隆的彻底消灭;ＰＮＨ患者体内异常克隆的造血优势在一定条件下是可以消失的。     

Serum-Red Cell Interactions at Low Ionic Strength: Erythrocyte Complement Coating and Hemolysis of Paroxysmal Nocturnal Hemoglobinuria Cells

Complement coating and hemolysis were observed when erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) were incubated in isotonic sucrose solution in the presence of small amounts of serum. Normal cells were likewise coated with complement components but did not hemolyze. Both normal and PNH erythrocytes reduced the hemolytic complement activity of the serum used in this reaction.Experience with other simple saccharides and related compounds suggests that the low ionic strength of the sucrose solution is the feature that permitted complement coating of red cells and hemolysis of PNH erythrocytes. Isotonic solutions of other sugars or sugar alcohols that do not readily enter human erythrocytes could be substituted for sucrose.The mechanism for these reactions may possibly relate to the agglutination observed with erythrocytes tested in the serum-sucrose system. Even though PNH hemolytic activity could be removed by prior heating of serum or barium sulfate treatment of plasma, the agglutination phenomenon still persisted.The in vitro conditions necessary for optimal sucrose hemolysis of PNH erythrocytes were described and compared with those of the classical acid hemolysis test. The requirement for less serum in the sucrose hemolysis system than needed in the standard acid hemolysis reaction makes certain experiments, especially those using large amounts of autologous PNH serum, much more feasible. Additional advantages of the sucrose hemolysis test are that it can be carried out at room temperature in the presence of oxalate and citrate and that critical pH control is not essential. To date, the sucrose hemolysis test has been a sensitive and specific one for PNH. A modified test used for screening purposes, the "sugar water" test, is very easy to perform.     

Expression of cryptantigen Th on paroxysmal nocturnal hemoglobinuria erythrocytes in association with a hemolytic exacerbation.

Paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes lack complement regulatory membrane proteins and are susceptible to complement. Although the critical role of complement in intravascular hemolysis in PNH is accepted, the precise mechanism of complement activation in vivo is unknown. Accordingly, in a PNH patient who was suffering from a hemolytic precipitation soon after a common cold-like upper respiratory infection, we analyzed the erythrocytes with lectins and by flow cytometry to detect membrane alteration that lead to complement activation. The lectin reactivity of erythrocytes showed the expression of cryptantigen Th. The patient serum at the time of the hemolysis induced the expression of Th on erythrocytes from PNH patients and from healthy volunteers in vitro, whereas neither the patient serum after recovery from the hemolysis nor blood type-matched control serum from healthy donor showed this activity. Moreover, autologous serum selectively hemolyzed Th+ PNH erythrocytes, but not Th- PNH erythrocytes, or Th+ control erythrocytes. Hemolysis was not observed either in complement-inactivated serum or in blood type-matched cord blood serum, which lacks natural antibodies to cryptantigens. These findings indicate that the immunoreaction of infection-induced Th with natural antibody on PNH erythrocytes is a trigger of the complement activation, leading to intravascular hemolysis.