PGE1对血小板的体外激活抑制作用

目的 研究前列腺素E1（PGE1）在血小板冻于前预处理过程中对血小板激活和功能的影响,为血小板冻干前处理过程筛选血小板激活损伤保护剂。方法 测定血小板平均体积（MPV）,采用流式细胞术（FCMs）分析血小板CD62p、PAC-1的表达。以反映血小板状态。测定血小板对凝血酶（thrombin）、二磷酸腺苷（ADP）和瑞斯托菌素（Restocetin）的最大聚集率,以反映血小板功能,研究血小板预处理前后的变化以及前列腺素的保护作用。结果预处理后,血小板平均体积（MPV）未发生明显改变,但血小板CD62p表达率显著增加,达20％。PGE1抑制血小板CD62p表达,这种抑制作用随PGE1浓度增加而递增。预处理过程对Restocetin和ADP诱导的血小板聚集有一定影响,聚集抑制率为10％和23％,对凝血酶诱导的聚集抑制不显著。PGE1不影响血小板对Restocetin的聚集反应,但PGE1≥1μmol／L可抑制血小板对ADP的聚集反应,PGE1≥5μmol／L时,显著抑制凝血酶诱导的血小板聚集。结论 前列腺索E1浓度为1μmol／L,可抑制血小板在预处理过程中的激活损伤,且保留血小板的聚集功能。     

供血者性别对血小板激活标志物含量的影响

目的 探讨浓缩血小板保存过程中血小板激活的性别差异。方法 采用PRP法制备浓缩血小板,并于常规条件下保存5天,间隔取样,分别用RIA、ELISA方法检测血小板表面P-selectin分子数及血浆中可溶性P-selectin(sP-se-lectin)含量。结果 血浆中sP-selectin含量在保存1天后即明显升高,而血小板表面P-selectin分子数在保存3天后明显升高,二者与保存前比较均有显著性差异(P<0.05)。在保存期内,男女供血者的血小板表面P-selectin分子数之间比较无统计学差异(P>0.05),而女性供血者血浆中sP-selectin含量明显高于男性(P<0.05)。结论 在相同刺激因素影响下,女性供血者之血小板更易释放sP-selectin,因此在应用sP-selectin作为浓缩血小板质控指标及临床疾病的诊断时,应考虑供血者性别差异。     

富血小板凝胶制备方法的比较与优选

背景：富血小板凝胶含有多种细胞生长因子,对于局部创伤及骨缺损的修复具有非常重要的作用。富血小板凝胶作为组织工程的支架的研究越来越多,但是对于其最优化的制备方法目前尚无统一标准。 目的：简要综述近年来富血小板凝胶所含生长因子及制备方法的相关研究,总结多种富血小板凝胶的制备方法并进行比较,以此找出较为优化的制备方法。 方法：以＂富血小板血浆,富血小板凝胶,组织工程＂为检索词检索中国期刊全文数据库（1980年1月至2013年1月）,以＂Platelet-rich plasma,Platelet-rich plasma gel,Tissue Engineering＂为检索词检索PubMed数据库（1980年1月至2013年1月）,纳入与富血小板凝胶检测及制备方法相关的研究,排除不相关学科及内容的研究,检索得到322篇相关文献,筛选后最终纳入67篇。 结果与结论：目前富血小板凝胶的多种制备方法都存在各自的优势,不能单纯凭借某一项数据断定何种制备方法最好,需根据不同需要确定使用不同的方法。随着富血小板凝胶研究的不断深入,将会对创伤及骨缺损的临床修复以及组织工程研究起到至关重要的作用,也是未来组织工程支架研究的重要方向。     

富血小板血浆治疗坐骨结节滑囊炎

背景：坐骨结节滑囊炎被认识长久,但其治疗手段仍保留在40年前的封闭及手术等方法。目的：分析富血小板血浆注射治疗坐骨结节滑囊炎的疗效。方法：收集15例坐骨结节滑囊炎患者资料。所有患者双侧同时接受富血小板血浆（n=10例,共20侧）或封闭（n=5例,共10侧）注射治疗。随访6-14个月,采用目测类比评分法、治疗满意度评分及复发率进行评估,比较这两种治疗方法在各观察时间点的评分,评估治疗效果。结果与结论：在目测类比评分方面,富血小板血浆组和封闭组综合各时间点的结果比较差异无显著性意义（F=0.219,P=0.643）;分别在各个时间点两组间比较,富血小板血浆注射的疼痛评分在短期内（治疗后1周内）高于封闭注射,但长期随访的疼痛评分低于封闭组。在总体满意度、疗效和不良反应评分方面,富血小板血浆注射在短期内（尤其是治疗后1周）的评分不如封闭注射,但持久的长期评分却高于封闭注射。在方便性评分方面,两者差异无统计学意义。末次随访富血小板血浆注射治疗的复发率较封闭注射低。分析结果说明对于坐骨结节滑囊炎的治疗,富血小板血浆注射和封闭注射均可缓解疼痛,封闭注射的短期疗效更好,尤其是在治疗后1周内,但其长期疗效不佳,而富血小板血浆注射起效缓和,但疗效持久、复发率更低。     

血小板减少性紫癜激活的富血小板血浆凝固时间实验观察

目的研究激活的富血小板血浆凝固时间（APRPCT）测定对血小板减少性紫癜出血的预示作用及意义,探讨该实验方法与临床实验室常用的血小板计数、血浆凝血酶原时间、活化部分凝血活酶时间、血浆凝血酶时间、血浆纤维蛋白原测定间的相关性。方法制备激活富血小板血浆凝固时间激活试剂,检测健康对照组及血小板减少性紫癜组APRPCT,观察两组间差异。结果硅微粒激活的富血小板血浆凝固时间,健康对照组、血小板减少性紫癜组间差异有统计学意义（P〈0．05）。结论激活的富血小板血浆凝固时间可用于血小板减少性紫癜的出血预示。     

左旋精氨酸和西洛他唑对血小板体外激活的抑制作用

本实验旨在研究左旋精氨酸(L-arginine)和西洛他唑(cilostazol)在体外对血小板激活的抑制和功能保护作用,为可逆性血小板抑制剂在血小板保存中的应用提供依据。采用对血小板CD62p和PAC-1表达及凝血酶激活后再表达率的流式细胞分析(FCMs)、血小板聚集试验以及血小板凝血活性检测等手段,观察血小板激活、血小板功能状态。结果表明,体外处理后血小板CD62p和PAC-1表达率显著增加,西洛他唑和左旋精氨酸对这一过程CD62p和PAC-1表达有较强抑制作用,且随浓度增加而递增。西洛他唑可抑制凝血酶激活后CD62p和PAC-1的表达,左旋经氨酸仅抑制凝血酶激活后的PAC-1表达。左旋精氨酸和西洛他唑对3种诱导剂诱导的血小板聚集呈现不同程度的抑制作用,抑制作用随浓度递增。左旋精氨酸≥15mmol/L时,血小板聚集时间延长甚至不凝集。西洛他唑在浓度1-4mmol/L范围均延长血小板聚集时间。结论:5mmol/L和10mmol/L的左旋精氨酸均可抑制血小板体外处理过程的激活,且血小板的聚集和再表达功能不受影响,5mmol/L作用更佳。1mmol/L浓度西洛他唑抑制血小板体外激活,可保留血小板部分聚集活性和再表达能力。     

特发性血小板减少性紫癜患者激活的富血小板血浆凝固时间测定及临床意义

目的研究激活的富血小板血浆凝固时间（activated platelet rich plasma coagulation time，简称APRPCT）对特发性血小板减少性紫癜（idiopathic thrombocytopenic purpura，ITP）出血的预示作用及意义，探讨该实验方法与临床实验室常用的血小板计数（PLT）、血浆凝血酶原时间测定（PT）、活化部分凝血活酶时间测定（APTT）、血浆凝血酶时间测定（TT）、血浆纤维蛋白原测定（Fib）间的相关性。方法根据APRPCT实验原理，优选浓度为2g／L的硅微粒作为APRPCT激活试剂，制备质控血浆监测APRPCT实验的过程和质量，以2g／L的硅微粒检测正常对照组及ITP组的APRPCT，统计分析两组间的APRPCT有无显著性差异。结果浓度为2g／L的硅微粒测定APRPCT，正常对照组较ITP组明显延长，两组之间差异有统计学意义（P〈0．05）。结论APRPCT能同时反映血小板数量和质量的变化，其预示出血倾向的特异性明显优于血小板计数，可作为ITP患者的出血预示指标。     

脑梗塞患者血小板活性的动态观察

目的：观察脑梗塞患者血小板α－颗粒膜蛋白的变化。方法：用放免法测定４０例脑梗塞患者及２０例健康者对照血小板表面及血浆内α－颗粒膜蛋白值。结果：正常对照血小板Ｇｍｐ－１４０６５０±１８０分子数／血小板,血浆Ｇｍｐ－１４０１１．７±１．９μｇ／ｍｌ,４０例脑梗塞患者Ｇｍｐ－１４０９６３±３０２分子数／血小板,血浆Ｇｍｐ－１４０３９．４±１４．９μｇ／ｍｌ。二者有显著差异。结论：脑梗塞患者血小板活性增高。     

全血(22±2)℃保存24 h对白膜法分离血小板体外激活和血浆凝血因子活性的影响

目的 研究全血20 ～24℃保存24h对白膜法制备的浓缩血小板体外激活和血浆凝血因子活性的影响.方法 对采集的无偿献血者全血(400 mL)分组:对照组(全血室温放置6h)和试验组[全血(22±2)℃保存24h].将全血进行成分分离,得红细胞,浓缩血小板和血浆.用流式细胞仪检测2组血小板激活标志-膜表面糖蛋白分子CD62P表达率;用血凝仪检测血浆的凝血因子(F)Ⅱ,Ⅶ,Ⅴ,Ⅸ,X,Ⅺ及纤维蛋白原的活性.结果 实验组和对照组比较,血小板膜表面糖蛋白分子CD62P的表达率为:CD62P(10.08±0.71)％和(13.08±0.62)％(P＜0.05);凝血因子(F)Ⅱ,Ⅶ,Ⅴ,Ⅸ,X,Ⅺ,纤维蛋白原活性未有统计学差别,(F)Ⅶ因子活性(0.98±0.07) U/mL和(1.17±0.06)U/mL(P ＜0.05).结论 全血(22±2)℃保存24h制备的浓缩血小板外激活程度降低,血浆凝血因子除FⅧ外,其它因子活性未受显著影响.     

腺苷对血小板体外激活的抑制作用

本研究的目的主要是研究腺苷对血小板的体外激活抑制作用,为血小板冻干前处理过程筛选血小板功能保护剂提供依据.采用流式细胞术（FCM）测定血小板膜表面CD62P和PAC-1的表达,应用APACT-2聚集仪测定瑞斯托菌素（restocetin）、凝血酶（thrombin）、二磷酸腺苷（ADP）和没食子酸（propyl gallate）诱导的血小板聚集率.研究结果表明,冻干预处理后血小板膜表面CD62P表达显著增加,腺苷浓度为0.75 mmoL/L时可抑制CD62P表达,且呈剂量效应;腺苷可抑制没食子酸诱导的血小板聚集反应,但对瑞斯托霉素诱导的血小板聚集无抑制作用,腺苷浓度≥1.0 mmol/L对凝血酶诱导的聚集有显著抑制作用.因此,腺苷可抑制体外处理导致的血小板激活,对瑞斯托霉素和凝血酶诱导血小板聚集反应无明显抑制.结论：腺苷可作为血小板体外激活抑制剂及冻干前处理的功能保护剂.     

In vitro platelet activation by an echo contrast agent.

OBJECTIVE: We investigated whether an ultrasonic echo contrast agent containing microbubbles (Levovist [SH U 508A]; Schering AG, Berlin, Germany) could in routine use activate platelets. METHODS: Levovist and its main component, galactose, were mixed with separate samples of whole blood (1.5-75 mg/mL) from 5 healthy volunteers to form a 1-mL suspension sample. After in vitro exposure to ultrasound emitted from a commercial ultrasonic scanner at a pulse frequency of 3.5 MHz with a mechanical index of 1.9 and an exposure duration of 5 minutes, 5 microL of the sample was incubated for 20 minutes with the fluorescein isothiocyanate-labeled CD61 antibody, which is a platelet-specific antigen, and the phycoerythrin-labeled CD62P (P-selectin) antibody, an activation-specific antigen, both on the platelet surface. After more than 30 minutes of fixing in 1% paraformaldehyde, flow cytometric analysis was performed. RESULTS: The percentage of CD62P-expressing platelets increased according to the concentrations of Levovist and galactose, which showed almost equal effects. Ultrasound exposure did not enhance the effect except at the highest concentration of Levovist (75 mg/mL). CONCLUSIONS: In vitro, a galactose-based echo contrast agent could not activate the platelets at its routine concentration.     

超临界流体技术合成的复合型骨替代生物材料:体内外生物学评价

背景:在复合材料内部增加细胞黏附性较好的高分子材料或同骨组织羟基磷灰石成分相似的无机材料,可以改善材料的表面化学结构.过去的十几年,产生和发展了许多合成生物降解支架材料的新技术,虽然超临界流体技术应用在合成支架中时间尚短,但有着其他技术不可比拟的众多优点,目前越发受到学者的重视.目的:以聚乳酸和同种异体骨粉为原材料,通过超临界流体技术合成复合型骨替代生物材料,评价其生物学特性.方法:首先将同种异体皮质骨粉与聚乳酸在超临界二氧化碳作用下合成多孔复合型骨生物替代材料.将材料浸提液与成骨细胞系复合培养,体外观察细胞形态、增殖情况;通过材料浸提液皮内注射实验和材料肌袋内埋入实验,体内观察动物的致敏、组织炎症发生情况.结果与结论:体外大体观察该种复合材料形状、大小、孔隙可控,孔径适中,有较好的硬度,体外实验表明细胞相容性良好,对细胞的增殖无毒副作用;体内实验表明材料植入动物体内未发生致敏反应,组织相容性良好,但无异位成骨作用.说明制备的新型复合骨组织生物材料在细胞学和生物相容性检测上可以满足组织工程支架和骨组织替代的要求,该种合成技术及多孔生物材料有广阔发展前景.     

多孔偏磷酸钙生物材料的体外加速降解实验

背景：偏磷酸钙的降解性能已得到一定证实,但传统降解方法实验周期长,工作量大,影响对偏磷酸钙生物材料的深入研究。目的：与传统实验方法对比,建立流动装置,寻找一种快速研究偏磷酸钙降解性能的方法。设计、时间及地点：对比观察实验,于2007-09/2008-03在广东省人民医院医学研究中心完成。材料：多孔偏磷酸钙材料和基于离心泵的循环装置及反应器由广东省人民医院医学研究中心提供。方法：取偏磷酸钙材料若干,分析天平称重后分成两组。新型流场方法：置于循环装置中,流速分别为0.15,0.30m/s。管路内灌满37℃生理盐水50mL。传统静态方法：直接浸泡在37℃生理盐水50mL中28d。主要观察指标：新型流场方法于运行循环装置2,4,8,16,24h测定偏磷酸钙失重率,传统静态方法于放置2,4,8,16,28d测定偏磷酸钙失重率。同时扫描电镜观察其表面形貌。结果：采用传统静态方法,28d失重率为9.2%;采用新型流场方法,流速分别为0.15,0.30m/s时,24h偏磷酸钙的失重率约为15%和25%。扫描电镜显示,传统静态方法偏磷酸钙表面发生了物质沉积,影响了其失重率,而新型流场方法偏磷酸钙表面未发生物质沉积。结论：采用流动装置反映偏磷酸钙材料的降解规律与传统方法基本相同,流场方法可加快降解实验的进程。     

In vitro assessment of platelet function

With the realization that the skin bleeding time is often an unreliable measure of platelet function, efforts have been made to identify ways to assess qualitative platelet dysfunction. Currently available techniques measure platelet adhesion, platelet aggregation, the ability of platelets to retard or stop flow through filters, and the contribution of platelets to in vitro clot formation. Glass bead adhesion, which continues to be performed in some laboratories, is gradually being replaced by measures of platelet adhesion to filters composed of glass fibers, Dacron fibers, or collagen. In each instance, anticoagulated platelet-rich plasma or whole blood flows through the filter under a regulated pressure gradient. The amount of blood flowing through the filter versus time and/or the time to filter occlusion are measured. Recent developments in platelet aggregation have focused on whole blood and stagnation point flow aggregation techniques. Whole blood aggregation does not require blood sample processing and accommodates blood obtained from citrated vacutainer tubes. Stagnation point flow measures both platelet adhesion and aggregation and may be able to detect pathologically-enhanced platelet function. Global measures of hemostasis attempt to simultaneously evaluate the adequacy of fluid phase coagulation and platelet function. Currently available techniques include Thromboelastography, SonoClot Analyzer, Hemodyne Hemostasis Analyzer, PITT, and Hemostatometry. Although each of these technologies have been shown to provide interesting data in the research setting, the ability of any of these techniques to detect abnormal or clinical inadequate platelet function remains to be established.     

In vitro evaluation of platelet concentrates during storage: Platelet counts and markers of platelet destruction.

BACKGROUND: Differences in platelet counts are observed by use of automated haematology analyzers making interlaboratory comparison difficult. MATERIALS AND METHODS: Twenty-eight single-donor platelet concentrates (PCs) were collected. Platelet concentration and markers of platelet destruction were investigated during storage for 11/12 days. RESULTS: Increasing impedance-immunoplatelet ratio was observed during storage, correlating to platelet fragments, large platelets, platelet density and cell-lysis. High variability was observed for optical-immunoplatelet ratio. CONCLUSION: Immunoplatelet count or correction factor calculated by impedance-immunoplatelet ratio should be used to confirm that platelet unit meets platelet count requirements or to compare results from clinical trials. Optical platelet count is not recommended.     

In vitro evaluation of prestorage pools consisting of mixed A and O platelet concentrates

BACKGROUND: Current FDA regulations allow the prestorage pooling of whole blood-derived platelet concentrates (PCs) of identical ABO type in a recently cleared platelet (PLT) pooling bag (Acrodose, Pall Medical). It is unclear how ABO-mixed PC pools would store if pooled before storage. STUDY DESIGN AND METHODS: Pools consisting of ABO-identical PLTs and mixed A and O PLTs in varying proportions were evaluated on Days 1, 5, and 7 of storage with measures of the PLT storage lesion, lymphocyte activation, and activation of the complement and coagulation system. Data were analyzed by analysis of variance. RESULTS: Pools did not differ on Day 7 in pH (p = 0.63), hypotonic shock response (p = 0.25), extent of shape change (p = 0.26), morphology score (p = 0.18), or white cell count (p = 0.79), but surface P-selectin expression was more evident in the ABO-mixed pools (p = 0.02). Small microscopic clumps of PLTs were observed in all pools, but were more prominent in the ABO-mixed pools (p < 0.01). PLT counts, however, did not differ between pools (p = 0.93), indicating that only a small proportion of PLTs were clumped. Surface A-antigen expression was proportional to the number of A PCs in each pool and did not vary between study days. Anti-A(1) titers were either unchanged or decreased by one dilution. Complement and coagulation activation markers did not differ between pools. CONCLUSION: Pooling A and O PCs is associated with evidence of increased microscopic PLT clumping and activation, but these differences are not exacerbated with 7-day storage. Other major measures of PLT quality do not differ, and there was no evidence of a mixed lymphocyte reaction.     

In vitro and in vivo evaluation of cotton wool filtration of platelet concentrates obtained by automated and manual apheresis

The effect of cotton wool filtration of apheresis platelet concentrates (PCs) on platelet viability and complement activation was evaluated by two laboratories. PCs were prepared by automated (Lab A, n = 5) or manual (Lab B, n = 5) apheresis. After storage for 1 day, the PC was filtered through cotton wool before transfusion on one occasion and, on the other occasion, filtered through a standard screen filter before transfusion to the same donor. Five paired studies were performed by each laboratory. Except for a small, but significant reduction in mean platelet size, from 7.3 +/- 1.1 to 6.6 +/- 0.9 microns 3, after cotton wool filtration, no effect of filtration on various tests of in vitro platelet function and morphologic integrity was found. As demonstrated by autologous radiolabeled studies, no effect of cotton wool filtration on platelet viability was found by Laboratory B, while Laboratory A found a slight increase in the percentage of recovery from 59 +/- 4 to 68 +/- 13 percent, and a small reduction in survival, from 8.2 +/- 0.9 to 7.7 +/- 0.5 days after cotton wool filtration (p less than 0.05). Cotton wool filtration was associated with a slight increase in C3a levels found in manual apheresis PCs. Neither laboratory found any effect of cotton wool filtration per se on the recipients' white cell (WBC) counts or C3a and C5a levels after transfusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro evaluation of metabolic changes and residual platelet responsiveness in photochemical treated and gamma-irradiated single-donor platelet concentrates during long-term storage

BACKGROUND: Photochemical treatment (PCT) prevents replication of pathogens in platelet concentrates (PCs) by cross-linking nucleic acids and thus affects all cells containing DNA or RNA. STUDY DESIGN AND METHODS: Fourteen double-dose single-donor PCs were divided into two study arms. The double-dose PCs were split in two identical units, PCT and conventional control PCs. Study Arm A consisted of seven PCT PCs with corresponding untreated controls, whereas Study Arm B consisted of seven PCT PCs with corresponding gamma-irradiated control. Metabolic changes and agonist-induced platelet (PLT) response were evaluated during storage for up to 12 days. RESULTS: Higher rate of PLT destruction, illustrated by reduced PLT content, increased lactate dehydrogenase levels, and higher CD61+ microparticle formation rate, were observed after PCT. Generally PCT accelerated metabolic changes in PCs and reduced agonist-induced (collagen or thrombin receptor activator peptide [TRAP]) aggregation responses. Flow cytometric analysis of CD62P and CD42b (GPIbalpha) expression showed higher spontaneous PLT activation in PCT PCs from 5 days of storage. Correspondingly, a reduced capacity for up regulation of CD62P expression and down regulation of CD42b was observed in PCT PLTs after stimulation by the agonists ADP or TRAP. CONCLUSION: Generally reduced in vitro PLT quality was observed after PCT during storage for up to 12 days, with marked reduction from 5 days of storage. Compared to conventional PCs, reduced agonist-induced aggregation and glycoprotein expression were observed after PCT during storage, corresponding to significantly higher level of spontaneous PLT activation in PCT PCs. Clinical studies of efficacy and safety of PCT PCs stored for more than 5 days are recommended.     

Ultraviolet irradiation of platelet concentrate abrogates lymphocyte activation without affecting platelet function in vitro

We studied the effect of ultraviolet (UV) radiation on platelet concentrates. Samples irradiated at 310 mm for 30 minutes at a dose of 1782 J per m2 showed no loss of platelet function in vitro as determined by adenosine diphosphate, collagen, or ristocetin-induced aggregation. Lymphocytes isolated from irradiated units were unable to act as responders or stimulators in a mixed-lymphocyte reaction. These data suggest that UV radiation of platelet concentrates may result in a cell suspension that is unable to evoke an immunological response.     

In vitro evaluation of cefoperazone.

The activity of cefoperazone, a new broad-spectrum cephalosporin, was tested in vitro against 670 clinical isolates of gram-negative bacilli and gram-positive cocci. With the exception of Enterobacter spp., it inhibited the majority of all organisms tested at a concentration of 6.25 microgram/ml. Of particular interest is its good activity against Pseudomonas aeruginosa isolates which are usually very resistant to cephalosporins. When compared with other antibiotics, it was more active than any available cephalosporin against the Enterobacteriaceae, and its activity was comparable to the investigational drugs tested. Except against Pseudomonas, cefoperazone was less active than moxalactam (LY127935). No significant decrease in activity was noted in medium and pH variation studies. A considerable decrease in activity resulted when the size of the inoculum was incrased from 10(5) to 10(7) cells/ml. The minimal bactericidal concentrations were within one or two dilution values of the minimal inhibitory concentrations against the majority of isolates tested, except Staphylococcus aureus.