超低温保存血小板的制备与临床应用

血小板在成分输血中越来越显得重要,主要是血小板在止血和凝血过程中起到极其重要的作用.单采新鲜血小板于22±2℃条件下保存,容易发生细菌污染而引起输血反应,5 d保存期短很容易过期.不能满足临床输血紧急需求,供求矛盾突显.冰冻保存是延长血小板效期的最佳选择.冰冻保护剂有聚乙烯咯烷酮(PVD)、聚乙二醇(PEG)、Thrombosol,也有甘油、甘露醇、羟乙基淀粉等.目前常用的大多使用5%～6%的二甲基亚砜(DMSO)[1].笔者就当前超低温保存冷冻血小板(下称冷冻血小板)的制备和临床应用情况作简要的综述.     

高血小板浓度在采集和冷冻干浓缩血小板中的影响

采用5％或6％二甲基亚砜(DMSO)冷冻保存血小板是浓缩血小板(PC)长期保存的最佳方法，这个浓度的DMSO对血小板没有毒性。虽然冷冻和融化过程由于血小板膜完整性丧失，引起血小板激活和细胞溶解，但冷冻保存血小板能被安全地输注，其止血功能优于22C保存5天的PC，且回收率相似。     

高血小板浓度在采集和冷冻干浓缩血小板中的影响

采用5％或6％二甲基亚砜(DMSO)冷冻保存血小板是浓缩血小板(PC)长期保存的最佳方法,这个浓度的DMSO对血小板没有毒性。虽然冷冻和融化过程由于血小板膜完整性丧失,引起血小板激活和细胞溶解,但冷冻保存血小板能被安全地输注,其止血功能优于22C保存5天的PC,且回收率相似。     

冷冻血小板的稳定性研究

目的探讨冷冻血小板融化复苏后的稳定性。方法将血小板分别置于-80℃和-85℃～-120℃保存，观察冷冻血小板融化复苏后纤维蛋白及絮状不可逆聚集的情况。结果-80℃保存血小板纤维蛋白及絮状不可逆聚集率为24．0％，～85℃～-120℃保存血小板纤维蛋白及絮状不可逆聚集率为0。血小板保存温度不同，纤维蛋白及絮状不可逆聚集发生率的差异有显著性（Х^2-243．18，P〈0．01），冷冻血小板冷冻速度和保存温度对血小板质量的影响有显著性。结论冷冻血小板于-85℃～-120℃保存的稳定性更好。     

二甲基亚砜与羟乙基淀粉对血小板的冷冻保护作用

目的 观察二甲基亚砜(DMSO)与羟乙基淀粉(HES)联用保护剂冷冻保护血小板的效果,探寻更适合冷冻血小板的保护剂.方法 实验设未冷冻组,DMSO组,HES组,DMSO与HES联用组.各组加血小板混匀后置-80℃冰箱保存,38℃水浴融化,检测血小板回收率,MPV值,观察血小板超微结构,测定CD42b、CD62p的表达水平.结果 HES单独使用对血小板的保护作用不强;DMSO单独使用组与DMSO与HES联用组对血小板的外部形态和内部结构的保护及CD62p的表达水平,MPV增加值,后者优于前者,CD42b表达水平DMSO组优于DMSO与HES连用组.结论 DMSO与HES联用对血小板的冷冻保护有一定协同作用,它与DMSO联用可作为冷冻血小板的保护剂进一步研究.     

血小板保存3天后再冷冻保存的实验研究及临床应用

本研究探讨血小板常温保存3天后再进行-80℃冰箱内冷冻保存及临床应用的可行性。对当天冷冻和保存3天后再冷冻血小板进行了计数，并检测聚集力、黏附力以及CD62p的表达，并通过可对比性病例观察保存3天后再冷冻与当天冷冻血小板，临床应用的可能性。结果表明：在保存期3天之内血小板数量、低渗性休克反应、黏附功能无显著差异性改变（P〉0．05），但聚集功能和CD62p的表达率的变化有显著性差异（P〈0．05）。当天保存并冷冻的血小板与保存3天后再冷冻的血小板各项指标的变化都无显著性差异。CD62p的再表达率（CD62p凝血酶激活后阳性率-CD62p激活前的阳性率）也无显著性差异，分别是51．1±4．5和51．1±4．4（P〉0．05）。临床应用当天冷冻和保存3天后再冷冻血小板的CCI值分别是48％和49％，无显著性差异（P〉0．05）。结论：血小板保存3天后可以再-80℃冷冻保存，其临床应用效果与当天冷冻血小板比较后无明显差异。     

二甲亚砜与海藻糖联用对冷冻保存血小板的保护作用

本研究观察二甲亚砜（DMSO）与海藻糖联用对冷冻保存血小板的保护效果。实验分设空白组、海藻糖组、DMSO组、5％DMs0加海藻糖联用组、2．5％DMSO加海藻糖联用组。各组血小板置-80℃冰箱保存,37℃水浴融化。采用血细胞计数仪测定血小板回收率和MPV值、电子显微镜观察血小板超微结构变化和流式细胞仪测定血小板的CD41、CD42b、CD61及CD62p的表达水平。结果表明：海藻糖单独应用对提高回收率的保护作用不强,但海藻糖处理后冷冻保存的血小板的形态接近正常。DMSO在保证冷冻保存血小板的回收率和血小板整体完好性方面作用较为突出,但其血小板形态偏向于肿胀,仍有部分血小板呈异形性改变。DMSO和海藻糖合用对维持血小扳外部形态和内部结构接近正常稳态方面具有保护作用,同时保证冷冻保存血小板具有理想的回收率和较高的CD41、CD42b、CD61、CD62p表达水平。结论：DMSO和海藻糖联合应用对冷冻保存血小板具有协同保护作用,但DMSO和海藻糖单用或合用都较难抑制冷冻保存血小板的活化,两者合用作为血小板冷冻保护剂有望促使冷冻保存血小板临床输注效果的进一步提高。     

二甲基亚砜(DMSO)对血小板体外聚集功能的影响

目前,DMSO冰冻保存血小板作为22±2℃液体保存血小板的补充产品,在外科手术应用中已取得良好的临床疗效[1].然而冰冻血小板仍缺乏统一的质量标准[2],而且现有体外功能测定并不一定能完全反映其在体内发挥功能的实际情况[3].必须寻求能如实反映血小板体内功能的指标及简便易行的质量控制方法才能控制生产工艺,提供质量可靠的冰冻血小板制品.为此,笔者通过检测花生四烯酸诱导的DMSO终浓度不同的液体血小板体外聚集功能,进一步分析讨论了聚集功能作为DMSO冰冻保存血小板质量控制的指标是否合适?现将结果报告如下.     

人卵巢组织冷冻保存及其评价方式的研究与进展

背景：如何保存有生育需求的患者的卵巢功能使其能获得一定的生育能力,具有重要的意义及广阔的应用前景。目的：综合近年来卵巢组织冷冻的相关研究,对保存及评价方法作以综述,以探讨如何建立最佳的卵巢组织冷冻方案。方法：由第一作者检索1994年1月至2014年1月PubMed数据及万方数据库相关文献。英文检索词为ovariantissue,ovariantissuecryopreservation,freezingfactors;中文检索词为卵巢组织,卵巢组织冷冻,冷冻影响因素。纳入与卵巢组织冷冻技术、影响冷冻化效果的因素、冷冻组织复苏及人卵巢组织移植相关的文献59篇进行分析。结果与结论：目前,卵巢组织冷冻主要分为慢速冷冻、快速冷冻及玻璃化冷冻技术,而不同的患者、组织切片的大小、冷冻保护剂种类、渗透时间及冷冻载体的不同,都会影响到冷冻效果。卵巢组织复苏后,主要通过组织学显微镜下观察卵泡及颗粒细胞的形态并进行计数,观察亚细胞结构的改变评价冻融效果;凋亡信号的检测;免疫组织化学分析检测复苏后增殖及凋亡相关的信息;体外培养检测内分泌水平的变化;基因水平的检测等。近年来,卵巢组织冷冻后的临床应用是将冷冻复苏后的组织进行移植,使患者恢复生殖内分泌功能,或获取成熟的生殖细胞,从而保存女性生育能力。然而,卵巢组织冷冻仍存在一些问题尚需进一步研究。     

亚硝基谷胱甘肽对冷冻血小板膜糖蛋白分子的影响研究

本研究探讨亚硝基谷胱甘肽(S-nitrosoglutathione,GSNO)对冷冻血小板膜糖蛋白分子的影响。用流式细胞术对新鲜血小板、冷冻血小板及加入GSNO后的冷冻血小板的膜糖蛋白分子进行测定并进行比较。结果表明:GSNO明显降低了血小板的聚集率;PAC-1变化不显著;CD42b、CD62P变化在新鲜液态血小板组与冷冻血小板组、加有GSNO的冷冻血小板组差别显著;冷冻血小板组、加有GSNO的冷冻血小板组的血小板膜糖蛋白变化差别不显著。结论:GSNO抑制血小板的聚集,保持血小板的功能,可以用作冷冻保护剂。加入GSNO的冷冻血小板可能存在分子重排、结构上的变化及其它作用机制。     

Correlation of in vivo and in vitro functions of fresh and stored human platelets

BACKGROUND: Long-term storage of human platelets has been hindered by the loss of function of the platelets stored under current protocols. Novel preservation methods have encouraged examination of platelet function of cells preserved by cooling and freezing. The function of the platelets was assessed by using both in vitro assays and an in vivo rabbit bleeding model. STUDY DESIGN AND METHODS: Human platelets were stored in the presence or absence of 2 microM: cytochalasin B and 80 microM: EGTA/AM at 4 degrees C for 14 days or by freezing in the presence or absence of 5 percent DMSO. After the storage period, the platelets were resuspended in normal saline and infused into rabbits. Platelet function was assessed in vivo in a kidney bleeding model and in vitro by platelet-induced clot retraction and by platelet aggregation. RESULTS: Platelets stored at either 4 degrees C or -145 degrees C exhibited shorter survival times in the rabbit circulation than did fresh platelets. Platelets cooled to 4 degrees C, in both the presence or absence of cytochalasin B and EGTA/AM treatment, or frozen in the absence of DMSO were not effective in halting bleeding. However, frozen DMSO-treated platelets were as effective as fresh platelets in stopping bleeding. In vitro assays showed that cooled platelets treated with cytochalasin B and egtazic acid/AM and frozen DMSO-treated platelets retained 30 to 40 percent platelet function, while the cooled and frozen control samples exhibited no platelet-induced clot retraction. With thrombin as the agonist, only frozen DMSO-treated platelets exhibited a tendency to aggregate, although at only 22 percent of the aggregation function of fresh platelets. CONCLUSION: It is possible to freeze platelets and retain in vivo efficacy if the cryopreservative DMSO is included in the preparation. In vitro responses were greatly reduced by all of the storage protocols, but it may not be necessary to retain 100 percent in vitro function to have a platelet substitute or storage product that functions satisfactorily in vivo.     

Dimethylacetamide, a New Cryoprotective Agent for Platelets

Dimethylacetamide (DMAC) was used as cryoprotective agent for the preservation of platelets in the frozen state. The addition of dextrose to the platelet suspension greatly increased the cryoprotective effects of this agent. Rat platelets preserved with 5 per cent DMAC and 5 per cent dextrose showed a higher percentage of morphologically intact cells and higher clot retraction-promoting activity than platelets frozen in other concentrations of these two agents. The infusion of these platelets into thrombocytopenic rats resulted in platelet-count increments greater than those observed after administration of platelets frozen in 5 per cent dimethylsulfoxide (DMSO) and 5 per cent dextrose. At room temperatures, DMAC appeared to be less damaging to platelets than DMSO. Transfusions of human platelets frozen with DMAC and dextrose to thrombocytopenic patients with acute leukemia were associated with temporary increases of their platelet counts.     

Dimethyl sulfoxide: effects on function of fresh platelets and on the viability of platelets in storage

Dimethyl sulfoxide (DMSO) is used as a cryoprotective agent when platelets are frozen. We examined the effect of DMSO (0.1 to 10%) on platelet aggregation, release, and prostaglandin synthesis (as indicated by malondialdehyde formation) in response to thrombin, collagen, arachidonic acid and calcium ionophore. Inhibition was observed at the lowest levels of DMSO, varied with the type of stimulus, and was reversed by washing the platelets. Inhibition of aggregation, release, and malondialdehyde formation were dose-dependent with thrombin or collagen. DMSO did not inhibit malondialdehyde formation stimulated by arachidonic acid, nor did it consistently inhibit any function stimulated by calcium ionophore. When platelets were stored as platelet-rich plasma at 20 to 24 degrees C for 48 hours, with and without 5 percent DMSO, and subsequently washed, the platelets stored with DMSO were more reactive in vitro. These results indicate that platelet function inhibition by DMSO not only is reversible, but protects the platelets during storage. The factor limiting the use of DMSO in platelet storage is potential systemic toxicity, not its effects on platelets.     

Platelet preservation by freezing. Use of dimethylsulfoxide as cryoprotective agent

Variables important in the preservation of platelets by freezing with dimethylsulfoxide (DMSO) as cryoprotective agent were studied in normal volunteers and thrombocytopenic patients. Use of 5 per cent DMSO and a freezing rate of 1-3 degrees C/minute yielded optimal preservation of platelet viability. The addition of 5 per cent Dextrose did not improve results. In vivo yield using 5 per cent DMSO was superior to previous results in which glycerol was used as the cryoprotective agent. Viability after freezing was equivalent when platelets were frozen in small (10 ml) and large (60 ml) volumes of plasma. The larger volume had the advantage that a smaller percentage of the platelets was lost during transfer from one plastic container to another.     

Viability and function of platelets frozen at 2 to 3 C per minute with 4 or 5 per cent DMSO and stored at −80 C for 8 months

Each of 15 healthy male volunteers was treated with 650 mg of aspirin 24 hours before the autologous transfusion of one unit of freeze- preserved platelets. Freeze-thaw-wash recovery values in vitro, viability and function in vivo, and the bleeding time and platelet aggregation response were measured. The platelets were frozen with 4 or 5 per cent dimethylsulfoxide (DMSO) at an overall rate of 2 to 3 C per minute and were stored at −80 C in a mechanical freezer for up to eight months. They were washed by dilution/centrifugation. The mean recovery in vitro of platelets frozen with 4 per cent DMSO was 76+/−16%; the value was 64+/−16% for platelets frozen with 5% DMSO. The mean in vivo 51Cr recovery of autologous platelets frozen with 4% DMSO was 34+/−6%, and for platelets frozen with 5% DMSO it was 33+/−7%. In both groups the platelet lifespan was normal. There was a significant reduction in bleeding time after the transfusion of a single unit of autologous platelets preserved with either 4 or 5% DMSO, but no improvement in the aspirin-induced platelet aggregation pattern.     

Cryopreservation of human platelets with dimethyl sulfoxide: changes in biochemistry and cell function

BACKGROUND: The shelf life of liquid-stored platelet concentrates is limited to 5 days. Therefore, much work has been carried out in an attempt to establish the optimum method for cryopreservation. Among the various cryoprotectants, dimethyl sulfoxide (DMSO) has been shown to be the most effective. However, DMSO-frozen platelets are characterized by a number of cell lesions. This report describes metabolic and functional changes that should give rise to some concern about the functional integrity of these cells. STUDY DESIGN AND METHODS: Single- donor platelet concentrates were frozen in liquid nitrogen by use of DMSO (5%). After thawing, the cells were washed and resuspended in autologous plasma. Before, during, and after the freezing process, samples for analysis of metabolic measures (e.g., pH; calcium, potassium, and lactate dehydrogenase concentrations; plasma complement factors) and functional measures (e.g., aggregometry, in vitro bleeding time, alpha-granule membrane protein-140 expression) were taken. RESULTS: Mean platelet volume increases during the deep-freezing process. Potassium, calcium, and lactate dehydrogenase are released from the intracellular space to the extracellular space. A strong activation of complement, which is mainly due to the addition of DMSO, is observed. Platelets become activated as indicated by the expression of alpha-granule membrane protein-140. Accordingly, decreased platelet function can be observed. CONCLUSION: DMSO-frozen platelets are characterized by several metabolic and functional changes. Although these cells have been shown to exert hemostatic effects in vivo, it is conceivable that those effects could be improved by further development of platelet-freezing techniques.     

Therapeutic transfusions of previously frozen washed human platelets

The platelets used in this study were collected by serial centrifugation, and within four hours of collection were frozen with 5% dimethylsulfoxide (DMSO) at an overall rate of 2 to 3 C per minute by storage in a mechanical refrigerator at −80 C. The frozen platelets were stored for four to ten weeks before thawing and washing. After washing, the units were kept at room temperature for six to eight hours before transfusion. The units were pooled, and an average of eight units was given to each of four patients, with a range of three to 14 units per transfusion. In vitro recovery after washing was about 65 per cent and in vivo recovery of the 51chromium labeled (51Cr) platelets was about 35 per cent. The infusion of these previously frozen washed platelets corrected prolonged bleeding times in patients. The homologous platelets were transfused along with other blood products to treat patients with hematologic disorders. The circulation and function of the donor platelets were influenced by compatibility of the platelets, the quality of platelet preservation and the patient's disease state.     

In vivo platelet function following cardiopulmonary bypass

In order to test the in vivo hemostatic function of platelets exposed to cardiopulmonary bypass, template bleeding times (TBT) were performed prebypass, three hours postbypass and 18 to 20 hours postoperatively in 22 patients undergoing open-heart surgery. The Harker-Slichter formula was applied to platelet counts below 100,000/cu mm to determine the expected TBT if the platelets function normally but were associated with a prolonged bleeding time merely from thrombocytopenia. Half of the patients received only frozen erythrocyte transfusions. Twenty of the 22 patients (91%) had normal or shorter than the expected TBT. Eight of the ten patients with platelet counts under 100,000/cu mm had shorter than predicted TBTs. All of the patients receiving only frozen erythrocytes had normal or shorter than predicted TBTs. It is concluded that in vivo platelet hemostatic function is usually normal postbypass, that routine platelet transfusions are therefore not necessary, and that most open-heart surgical procedures can be performed using frozen erythrocyte transfusions exclusively.     

冰冻血小板制备研究

[目的]探讨冰冻单采血小板制备的可行性,为其临床应用提供可靠依据.[方法]通过认真选择献血员,单采血小板,并对40袋新鲜血小板进行计数等质控,用二甲基亚砜（DMSO）作冰冻保护剂,-80℃深低温保存1年内,解冻后观察外观,分别进行计数、计算回收率、测定PH值、粘附率、无菌试验,并且与新鲜血小板进行比较.[结果]-80℃保存冰冻单采血小板1年内血小板计数、pH值、粘附率与冻前新鲜血小板进行比较差异无显著性（P〉0.05）;血小板平均体积、血小板体积分布宽度冰冻前后差异有显著性（P〈0.01）,保存1年内平均回收率为90.49% , 无菌实验无细菌生长.[结论]-80℃深低温保存冰冻血小板质量达到标准要求,可以应用于临床.     

贮存温度波动与冰冻血小板不可逆聚集发生的相关性研究

目的探讨冰冻血小板保存温度波动与冰冻血小板不可逆聚集情况发生的相关性。方法对2006-2008年共1842袋冰冻血小板在保存期间不同温度波动范围与融化后发生血小板不可逆聚集的情况进行统计分析。结果冰冻血小板保存温度分别在-80-60℃和-80-50℃范围波动,冰冻血小板融化复苏后血小板不可逆聚集发生率分别为6．39％和31．21％,保存温度在-80-70℃范围波动的对照组血小板不可逆聚集发生率为2．25％,前两种保存温度与对照组比较,组间差异均有统计学意义（P〈0．01）;保存温度在-80-50℃范围波动,手采和单采两种冰冻血小板融化复苏后,单采冰冻血小板不可逆聚集的发生率为72．97％,手采冰冻血小板不可逆聚集为18．33％,二者的发生率差异有统计学意义（χ^2=39．33,P〈0．01）;保存温度在-80-60℃范围波动,单采与手采两种冰冻血小板比较,差异无统计学意义（χ^2=0．89,P〉0．05）。结论当冰冻血小板保存温度分别在-80-60℃和-80-50℃波动时,融化复苏后血小板不可逆聚集有较高的发生率,在-80-70℃范围波动时,血小板不可逆聚集发生率较低-80-70℃的温度范围可以作为目前采供血机构保存冰冻血小板选择温度条件的参考。