优化流式细胞术测定保存血小板CD62p表达

CD62p阳性率是保存血小板质量监测的重要指标之一。为在静脉全血内血小板CD62p测定方法的基础上优化建立流式细胞术(FCM)测定保存血小板表达CD62p的方法，在血小板检测标本内加入0．1mmol/L潘生丁稳定血小板；对TBS溶液进行了改良，添加了1．1mmol/L的EDTA和100μmol/L潘生丁，用以代替PBS；采用新鲜富含血小板血浆(FPRP)制备的阴、阳性对照；进行方法学评价。结果表明：加入0．1mmol/L潘生丁和1．1mmol/L的EDTA可以有效防止实验过程中的血小板人为激活；采用新鲜富含血小板血浆(FPRP)制备的阴、阳性对照既可优化FCM分析方法，还可用于检验所购荧光抗体的质量，保证实验结果的有效性。采用泛血小板特异性标志物CD61可灵敏特异地识别血小板，提高了实验抗干扰能力。制备保存血小板时采用的是专用大号采血针，抽血流畅，对血小板CD62p表达测定人为影响很小，明显优于用注射器采集患静脉全血的做法。CD62p测定灵敏度可达1％，制备的标本可稳定48小时。结论：利用该流式细胞术可以灵敏而特异地测定保存血小板的CD62P表达率；该方法简便易行，提高了结果的准确性，具有良好的稳定性和重复性。     

深低温保存新鲜富含血小板血浆质量分析

为了评价批量制备的深低温保存富含血小板血浆(cryo-FPRP)的效率和效果,在优化建立的无偿献血者血小板的批量分离和制备cryo-FPRP过程中测定了ACD抗凝的200ml全血、FPRP、含5%DMSO的FPRP(DM-SO-PRP)和-80℃冰箱然冰冻保存后复温的FPRP(Cryo-FPRP)的血小板计数(Plt)、血小板平均体积(MPV)、血小板分布宽度(PDW)、血浆内pH、血浆乳酸(LA)浓度和乳酸脱氢酶(LDH)浓度、细菌培养、血小板CD62p阳性率、PAC-1阳性率及抗GPIb-Ⅸ-Ⅴ荧光强度,也测定了FPRP和cryo-FPRP的促凝血活性.结果表明①血小板的总平均得率为70%;FPRP中残余的红细胞≤1×109/袋;白细胞≤1×107/袋.②血浆pH、LA浓度和PAC-1阳性率在整个制备和保存过程中没有明显变化.③血浆内LDH、血小板CD62p阳性率和血小板抗GPIb-Ⅸ-Ⅴ荧光强度在血小板冰冻保存后出现明显升高.④与FPRP比较,cryo-FPRP诱导的血浆凝固时间明显缩短.结论①本研究选择的离心分离制备FPRP条件可高效分离无偿献血者的血小板,同时不会导致血小板激活率增加.②深低温保存无偿献血者的新鲜富含血小板血浆可有效防止代谢产物的积累和pH下降,并且可以高效预防血小板GPⅡb/Ⅲa的激活.③冰冻保存后部分血小板胞膜受到明显损害.④血小板胞膜表面GpIb-Ⅸ-Ⅴ复合物的数量增加,促凝血活性明显增强.     

血小板保存对P—选择素的影响

我们对浓缩血小板保存过程对血小板膜表面 P选择素 ( P- selectin)分子数及血浆 P-选择素 ( SP-selectin)的含量进行动态观察 ,并对不同性别供血者的血小板激活差异进行比较。材料和方法一、浓缩血小板制备　采用富含血小板血浆( PRP)法 ,并将制备的浓缩血小板按供血者性别进行了分组。所选择的男女供血者在年龄及浓缩血小板中的血小板计数等均无统计学差异。浓缩血小板制备后于血小板保存箱内 2 2℃振荡保存 ,并分别于保存 0 ,1 ,3,5d后取样待检。二、血小板表面 P-选择素分子数测定　采用12 5I标记的单克隆抗体 SZ51 (苏州医学院血栓…     

流式细胞术测定保存血小板再表达CD62p方法的建立及应用

目的　①建立并优化测定血小板再表达膜表面激活标志物CD6 2p能力的流式细胞术 (FCM) ;②测定2 2℃保存液体血小板不同保存期再表达CD6 2p的能力 ,以评价该指标在血小板质量监测以及新的保存方法研究中的应用价值。方法　①采用浓度梯度法优化GPRP浓度条件 ,采用析因设计优化凝血酶浓度和 37℃孵育时间条件 ,寻找最佳阴、阳性对照 ;②将 13份血小板按AABB标准保存 ,并延长保存至 12d ,测定每天血小板CD6 2p再表达率 ,与其相应体内存活期作直线相关性分析。结果　①约 1.5× 10 6个血小板内加入 2 .5mmol/LGPRP可有效防止过量凝血酶诱导的聚集反应 ,孵育条件以 1U/L凝血酶 37℃ 15min然后室温置 15min最佳。采用新鲜富含血小板血浆(FPRP)为阴性对照 ,以凝血酶激活FPRP为阳性对照效果良好。② 2 2℃保存血小板CD6 2p再表达率与其相应体内存活期呈明显正相关 ,单份相关系数 (r) 0 .91～ 0 .99,总体相关系数为 0 .99。结论　①优化建立了一种灵敏简便 ,稳定性和重复性并好的流式细胞术方法用于测定保存血小板CD6 2p再表达率 ;②保存液体血小板CD6 2p再表达率与其相应体内存活期呈明显正相关 ,且相关性良好 ,提示CD6 2p再表达率是监测保存血小板质量的良好指标 ,同时还可能在新的保存方法研究和临床血小板功?     

深低温保存增强血小板促凝血功能的研究

为了探索血小板冰冻保存后膜表面促凝血活性相关分子变化与冰冻血小板体内即刻止凝血功能增强之间的可能联系，用流式细胞术检测了新鲜血小板冰冻保存前后V因子结合能力、血小板膜表面GPIb—Ⅸ-V分子（CD42a）密度，用血凝仪测定激活血小板诱导血浆凝固时间（aPACT）变化，用血细胞计数仪测定血小板计数、NIPV和PDW。研究结果表明，与新鲜血小板比较，深低温保存后血小板激活血小板诱导血浆凝固时间缩短43．9％；结合V因子的荧光强度平均增加117％；结合膜表面GPIb—Ⅸ—V分子的荧光强度增加32％。结论：冰冻血小板体内即可止凝血功能增强，可能与血小板冰冻保存后膜表面促凝血活性分子表达增加或功能增强，促凝血功能明显增强，发挥快速止血功能有关。     

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。     

冰冻保存血小板制备过程中的质量控制

22℃保存的血小板 ,保存期短 ,已不能满足日益增长的临床需求 ,且易受到细菌污染 ,而低温条件下贮存血小板 ,可使血小板得到长时间有效地贮存 ,从根本上解决了临床输注血小板供不应求的难题。冰冻保存血小板具有良好的临床止血效果 ,可有效防止细菌的污染 ,且具有长期大量保存稀有血型血小板的优点 [1] ,目前在临床已被广泛使用 ,但只有正确掌握冰冻血小板的制备和质量控制方法 ,才能提高冰冻血小板的数量和质量。1　制备过程中易出现的质量问题　冰冻保存血小板是由全血制备成富含血小板血浆 ( FPRP) ,在无菌条件下加入防冻剂二甲基亚砜 …     

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.     

白膜法汇集浓缩血小板的研究进展

手工分离制备浓缩血小板有富含血小板血浆法（PRP法）和白膜法（BC法）2种。BC法制备的血小板被欧洲许多国家广泛应用，其优点在于：白细胞含量少、血小板膜表面CD62p的表达率及糖分解率显著降低、pH保持恒定等。目前，白膜法汇集浓缩血小板的辐照、过滤、洗涤、病毒灭活、冰冻保存已成为输血界研究的焦点。血小板膜微粒衍生物的制备研究也已初见成效。     

凝胶过滤血小板的分离制备

本文应用Sepharose2B作为分离介质，建立了凝胶过滤制备血小板的方法。利用Sepharose2B的分子筛效应，可使血小板与血浆成份分离，并且过滤的血小板能较好地保存血小板活性，加入纤维蛋白原及复钙后，5μMADP诱导的血小板聚集曲线良好．与离心洗涤分离血小板的方法相比，凝胶过滤制备血小板具有简便、血小板丢失少、活性保存完整的特点，有助于血小板特性的体外研究．     

冷冻干燥保存血小板的实验研究

本研究探讨冷冻干燥保存血小板的方法,以获得可长期冻干保存的血小板制品,使之能在常温条件下保存、占用空间小、重量轻、便于长距离运输,能够满足突发事件和战伤救治的需要。在冷冻干燥保存过程中添加血小板可逆性激活抑制剂、DMSO和海藻糖等低温保护剂,进行预处理、冷冻、一级干燥、二级干燥,再水化,并同时测定血小板回收率,凝血酶聚集反应,促凝血功能,CD62p表达率和PAC-1表达率等。结果表明:血小板回收率为56.29%,其对凝血酶的聚集反应与对照组无明显差异,对ADP和丙基没食子酸诱导的聚集反应较对照组分别降低49.34%和26.25%,促凝血功能与对照组比较也无统计学差异;冻干血小板CD62p表达率为42.36%,PAC-1表达率为2.12%,凝血酶激活后CD62p再表达率为50.88%,PAC-1再表达率为54.55%。结论:添加血小板可逆性激活抑制剂,海藻糖和DMSO后的冻干血小板,其聚集活性和促凝血功能与新鲜血小板无明显差异,血小板可逆性激活抑制剂降低了冻干血小板的CD62p表达,增强了冻干血小板的生存能力,因而延长了其生存时间,因此可以该冻干方法为基础进一步提高冻干保存血小板的效率。     

Apoptotic activity in stored human platelets

BACKGROUND: Platelets possess some of the machinery required for apoptotic cell death. However, disruption of mitochondria function, implicated in several models of cell death, has not been extensively studied in platelets. Mitochondrial viability and several other measures of apoptotic death in stored and experimentally stressed platelets were evaluated. MATERIALS AND METHODS: Platelet mitochondrial transmembrane potentials (Deltapsim) were studied by staining platelets with JC-1, a dye that fluoresces at different wavelengths based on the state of mitochondrial polarization. Annexin V binding, a measure of phosphatidylserine (PS) exposure, and CD62P expression, an indicator of platelet activation, were determined by flow cytometry. Caspase-3 activity was measured with an enzyme assay and by Western blotting. Experimental platelet stressors included storage for 7 days, azide exposure, calcium ionophore stimulation, and plasma deprivation. RESULTS: As measured by flow cytometry, Deltapsim values were similar in freshly drawn platelets and in platelet concentrates stored for up to 7 days. However, compared to fresh platelets, stored platelet concentrates had significantly increased PS exposure (3.1 vs. 5.1%, p = 0.015), CD62P expression (6.5 vs. 13.5%, p = 0.0067), and caspase-3 activity. Azide exposure, which decreased ATP release 20 to 30 percent, did not affect the Deltapsim. Stressed platelets exhibited higher degrees of mitochondrial depolarization in response to calcium ionophore stimulation than platelets that were not stressed. Plasma deprivation also resulted in significant alterations in Deltapsim, PS exposure, and CD62P expression. CONCLUSIONS: Platelet mitochondria maintain Deltapsim when stored for up to 7 days under standard blood bank storage conditions. Therefore, changes in platelet mitochondria Deltapsim do not correlate with downstream markers of apoptotic death such as caspase activation and PS exposure.     

25Gy γ射线辐照对手工富集人血小板CD62p表达的影响

本研究观察25Gyγ射线照射对22℃保存条件下的手工富集血小板悬液不同保存期CD62p、血小板计数及平均血小板体积(MPV)的影响。将富浆法分离的16袋血小板,每袋平均分为两份,其中1袋经25Gy137铯γ射线辐照,另1份不辐照,然后按美国血库协会(AABB)标准保存72小时。经辐照的血小板作为观察组,未辐照的血小板作为对照组。流式细胞术检测两组血小板辐照前及保存24和72小时后P选择蛋白的表达水平;同时用血细胞计数仪检测血小板数和平均血小板体积(MPV)。结果表明:辐照组与对照组血小板表达CD62p百分率均随保存时间的延长而增高;保存24小时与新鲜血小板比较有显著性差异(p(0.05),保存72小时与保存24小时比较有非常显著性差异(p(0.01)。在保存24、72小时后,两组血小板CD62p表达率、血小板计数无显著差异(p0.05);但两组血小板在保存72小时后MPV与新鲜血小板比较有显著性差异(p(0.05)。结论:γ射线照射不影响血小板的数量和质量,但手工血小板液态储存时间应尽可能缩短。     

冰冻保存血小板新亚群及其新特性体外研究

目的　探讨血小板冰冻保存后血小板新亚群的产生和其获得新特性的分子基础。方法　采用流式细胞术在不同条件下比较分析新鲜血小板和冰冻保存血小板膜表面功能分子表达变化差异。结果　根据表达和再表达CD6 2p能力不同可将冰冻血小板分为三个亚群 ,CD6 2 p再表达率为 5 1 .7%± 7.8% ;新鲜血小板只有一个明显的亚群 ,CD6 2 p再表达率为 98.3%± 0 .6 %。结合磷脂酰丝氨酸 (PS)、CD6 2 p和CD4 2b分子变化可将冰冻血小板分出新的亚群 ,其显著特征包括PS阳性和CD4 2b分子密度显著降低 ;而CD6 2p阴性亚群对钙离子的激活更为敏感。结论　血小板通过冰冻保存后 ,产生了新亚群。新亚群膜表面功能分子的变化及其表达能力改变可能与其体内存活率减低而即刻止血功能增强密切相关     

富含血小板血浆制备过程中血小板CD62p表达

为了探讨大批量制备冰冻保存富含血小板血浆（Cryo-PRP)全过程中影响血小板质量变化的因素，以优化Cryo-PRP制作过程，提高Cryo-PRP质量，采用流式细胞术测定Cryo-PRP全过程中血小板膜表面CD62p表达。冰冻保存血小板全过程包括血液采集，离心制备，添加DMSO，－80℃冰箱保存和38℃水浴解冻，结果表明，在血液采集、离心制备，添加DMSO过程中血小板膜CD62p阳性率有一个突然的升高，其幅度约占整个过程的82％，对血小板的损害较大，结论：在Cryo-PRP的制备过程中，血小板的离心制备和DMSO的添加方式均可以得到良好的优化的优化，而对冰冻富含血小板血浆的质量影响也易于控制，但冰冻保存的损害较大，且不可避免，因此，迫切需要一种新型的血小板冰冻保护液和改进冰冻保存方法，以冰冻损害，进一步提高冰冻血小板质量。     

Annexin V and platelet antigen expression is not altered during storage of platelet concentrates obtained with the AMICUS cell separator.

During storage of platelet concentrate the so-called "storage lesion" occurs. During this time, platelets loose their morphological and functional capacities that are necessary for proper in vivo efficacy following transfusion. Annexin V represents a marker for apoptosis. In this study, Annexin V and additional antigens were analyzed by flow cytometry. Platelet concentrates were obtained with a new cell separator (AMICUS Separator, Fenwal). Following apheresis, platelet units were stored for an experimentally prolonged time of seven days. Daily aliquots of the platelet-rich plasma were obtained to measure Annexin V and platelet antigens CD62p, CD63, CD41a, CD42b, and the binding of fibrinogen. All analyses were performed using flow cytometry. During storage, no significant changes in mean channel fluorescence intensity (MCFI) of CD41a (P = 0.99) and CD42b (P = 0.29), percentage of CD62p+ and CD63+ platelets (P = 0.23 for CD62p; P = 0.52 for CD63), and the binding of fibrinogen to platelets occurred (P = 0.85). Also, the expression of Annexin V remained constant with no significant change (P = 0.36). This study shows that antigens of platelets, obtained with the AMICUS cell separator are well preserved during storage. Regarding Annexin V, no obvious signs of apoptosis can be detected by flow cytometry. These findings demonstrate the high degree of biocompatibility of the apheresis device and storage container.     

Autoapheresis and intraoperative blood salvage in oncologic surgery

Platelet activation occurs during the collection, processing and storage of platelet concentrates. The effect of the platelet activation on the functional state of stored platelets remains however undefined. We employed flow cytometric analysis to evaluate the extent of platelet activation and the physiological response to thrombin stimulation of platelets stored for up to five days under routine blood bank conditions. Platelet surface expression of the activation markers CD62 and CD63 was examined, along with modulation of platelet membrane glycoproteins (GP) Ib and IIbIIIa. Platelet dense granule content was determined using a mepacrine uptake assay and the extent of platelet microparticle generation was quantified. Thirteen random-donor platelet concentrates prepared under routine conditions by a platelet-rich-plasma protocol were examined. Platelets were found to be activated following preparation on day 1. Although a gradual increase was seen with increasing storage time, this was not statistically significant for CD62 or CD63 expression, GPIIbIIIa or GPIb modulation or dense granule release; the generation of platelet microparticles did, however, increase with increasing storage time. The characteristic increase in surface expression of CD62, CD63 and GPIIbIIIa and decrease in GPIb and dense granule content in response to thrombin stimulation was observed with all concentrates, but these measures of platelet functional reserve showed decreasing platelet function with increasing storage time. The results indicate that platelets are activated by day 1, likely as a consequence of manipulation during collection and processing, but are not further progressively activated with increasing storage time; they do, however, become relatively hypofunctional with increasing storage.