Cold-stored platelets: A product with function optimized for hemorrhage control

Early administration of blood products following severe trauma is pivotal for establishing hemostasis and achieving successful outcomes. Platelet transfusions, in particular, provide rapid control of hemostasis and help to restore platelet dysfunction induced by trauma. In the U.S. platelets used for therapeutic purposes are stored at room temperature with a limited shelf life of 5-7 days. Issues with room temperature storage of platelets, including an increased risk of bacterial growth and a decline in platelet hemostatic function, have led to a resurgence in interest in cold-stored platelets for therapeutic transfusion. This review presents the current state of cold-stored platelets and cold-stored whole blood as treatment for actively bleeding patients. Usage of cold stored platelets in alternative areas, such as in the field of regenerative medicine, is also discussed.     

Cryopreserved buffy-coat-derived platelets reconstituted in platelet additive solution: A safe and available product with sufficient haemostatic effectiveness

BackgroundPlatelets (PLTs) stored at 20–24 °C have a short shelf life of only 5 days, which can result in their restricted availability. PLT cryopreservation extends the shelf life to 2 years.MethodsWe implemented a method of PLT freezing at ?80 °C in 5–6% dimethyl sulfoxide. Buffy-coat-derived leucodepleted fresh PLTs blood group O (FP) were used for cryopreservation. Cryopreserved pooled leucodepleted PLTs (CPP) were thawed at 37 °C, reconstituted in PLT additive solution SSP + and compared to FP regarding PLT content, PLT concentration, pH, volume, PLT loss, anti-A/B antibody titre, total protein, plasma content, and PLT swirling. Clot properties were evaluated via rotational thromboelastometry. PLT microparticle number and surface receptor phenotype were assessed via flow cytometry.ResultsCPP met the required quality parameters. The mean freeze-thaw PLT loss was 22.24 %. Anti-A/B antibody titre and plasma content were significantly lower in CPP. CPP were characterised by faster clot initiation and form stable PLT clots. The number of PLT microparticles increased 25 times in CPP and there were more particles positive for the activation marker CD62 P compared to FP.ConclusionThawing and reconstitution are easy and fast processes if platelet additive solution is used. Low anti-A/B antibody titre and plasma content make possible the use of CPP of blood group O reconstituted in SSP + as universal ABO products, including clinical situations where washed PLTs are required. Clot properties evaluated via rotational thromboelastometry demonstrated that CPP retain a significant part of their activity compare to FP and are haemostatically effective.     

血小板激活时其胞质内钙离子变化的测定

目的建立测定血小板激活时细胞内钙离子变化的方法。方法摸索荧光染料Ｆｌｕｏ－３染色血小板内钙离子的适宜条件，应用流式细胞仪测定标记了Ｆｌｕｏ－３／ＡＭ的血小板激活后细胞内钙离子的变化。结果Ｆｌｕｏ－３标记血小板时，随着染料浓度增加和孵育时间延长，基线荧光强度和钙离子载体Ａ２３１８７刺激后的最大荧光强度均提高，但比值基本一致，不受染料条件的影响。无论二磷酸腺苷（ＡＤＰ）还是凝血酶作诱导剂时，染色和未染色血小板之间的最大聚集率均无差异。ＡＤＰ可引起血小板内钙离子增高，并随着ＡＤＰ浓度增加，钙离子荧光强度增强。结论建立了流式细胞仪测定血小板激活时细胞内钙离子变化的方法。     

972例次血液病患者输注机采血小板后回顾性分析

目的探讨机采血小板治疗血液病的效果及临床科学、合理应用血小板的情况。方法收集2007年本院血液科输注血小板136例(972次)的资料,比较输注前后24h内病历中记载的血小板检查结果,以血小板增加值判断血小板输注效果。结果972例次中输注后24h内复查血小板的为661例次(68.00%),输注前后24h内均检查血小板的为475例次(48.86%);所有输注血小板患者的血小板数量与输注前比较显著提高(P<0.01),总有效率为75.74%(103/136);但在输注前后24h内检查血小板的475例次中,血小板输注有效率为58.73%。结论机采血小板对血液病具有较好的治疗作用,但科学合理有效使用血小板的意识仍亟待加强。     

血细胞分析仪检测网织血小板对血小板减少性疾病的临床意义

目的：研究血细胞分析仪检测网织血小板（RP）对血小板减少性疾病诊断和治疗的临床意义。方法：用XE-5000血细胞分析仪检测40例ITP,35例AA及60例正常人外周血的网织血小板百分比（RP%）,并计算RP绝对值。结果：ITP组RP百分比为（23.61±7.26）%,明显高于对照组（7.62±2.01）％）（P〈0．01）,其RP绝对值为（8.36±4.01）×109/L,则低于对照组（21.35±7.78）×109/L（P〈0.05）,ITP患者经过治疗好转后RP百分比明显降低（8.58±1.97）％,与治疗前比有统计学意义（P〈0．01）;AA组RP百分比（（4.12±1.81）％）（P〈0.05）和RP绝对值（2.63±1.22）×109/L （P〈0．01）都低于对照组。结论：血细胞分析仪检测RP简便易行,对于血小板减少性疾病的诊断,鉴别诊断以及疗效观察有重要价值。     

一氧化氮供体改善血小板保存质量的初步研究

目的探讨一氧化氮(NO)供体S-亚硝基乙酰青霉胺(SNAP)对常温保存血小板过程中血小板质量的影响。方法离心法制备浓缩血小板共12人份,38~40 ml/份。将相同血型的2袋混合,加入复温后的冰冻血浆至约100 ml,混匀后均分、转移至2个血小板专用保存袋,分别为实验组:保存前加入终浓度10~5mol/L SNAP;对照组:加入等体积的无菌生理盐水。(22±2)℃振荡保存7 d,分别在d1、d3、d5、d7取样检测血小板计数、pH、血小板活化率及抗低渗性休克反应等指标。结果 2组血小板在保存过程中pH均保持在6.8以上;血小板活化率均不断升高,实验组从(5.93±1.43)%升高到(44.22±6.84)%,对照组从(8.22±1.33)%升高到(54.32±5.68)%,d1、d3、d5、d7 2组血小板之间活化率差异有统计学意义(P<0.05);d1、d5、d7实验组血小板抗低渗休克反应分别为(65.98±7.57%)、(53.1±8.44)%、(44.23±0.08)%,对照组为(50.92±4.48)%、(40.06±4.66)%、(35.28±0.04)%,d1、d5、d7实验组抗低渗休克反应能力高于对照组,差异具有统计学意义(P<0.05)。结论在血小板保存过程中加入NO供体SNAP一定程度上可以抑制血小板活化,改善血小板功能。     

植酸钠和百维利肽体外抑制血小板活化的实验研究

目的探讨植酸钠和百维利肽在体外对血小板激活的抑制和功能保护的作用。方法测定血小板对诱导剂的聚集反应、血小板CD62p和PAC-1表达、凝血酶激活后血小板的CD62p和PAC-1再表达以及血小板聚集功能,研究在离心、洗涤和重悬等体外处理过程中,不同浓度的植酸钠和百维利肽对血小板的激活损伤、血小板功能保存和血小板促凝血活性的影响。结果经体外处理后的血小板CD62p和PAC-1表达显著增加;1mmol/L植酸钠可抑制PAC-1表达,但对血小板CD62p和PAC-1再表达无明显抑制作用,血小板对诱聚剂仍有聚集反应性;百维利肽浓度为1μmol/L时,对CD62p和PAC-1表达有较强的抑制作用,CD62p和PAC-1表达率仅为0.78%和0.24%,抑制血小板CD62p和PAC-1再表达,保留血小板除凝血酶之外诱导的聚集反应;植酸钠浓度≤2mmol/L可显著延长血小板发生聚集的时间,≥3mmol/L时,血小板不聚集;百维利肽浓度≤3μmol/L时,血小板聚集时间无明显延长。结论1mmol/L的植酸钠可抑制血小板GPⅡb/Ⅲa构象的改变,并保留血小板的部分凝集和再表达功能;1μmol/L的百维利肽可作用于凝血酶的激活途径,抑制血小板激活,且能部分保留血小板的再表达和聚集功能,适当减少其作用浓度会对血小板有更好的功能保护效果。     

Evaluation of platelets collected by a new portable apheresis device

We studied the efficiency of platelet collection by the Mobile Collection System (MCS) using two types of experimental protocols and evaluated the effect of storage at 22°C on the platelet concentrates (PC). MCS is a new blood cell separator that combines discontinuous flow features with a new computerized operating system and can be used to harvest either full units of apheresis PC (SDP _protocol) or half units of PC together with one to two units of plasma (PLP _protocol). On the average, 1.98 × 10¹¹ ± 0.46 × 10¹¹ (mean ± SD) platelets were obtained by the PLP protocol and 3.01 × 10¹¹ ± 0.70 × 10¹¹ and 4.2 × 10¹¹ ± 1.12 × 10¹¹ by the early and later versions of the SDP protocols, respectively. The mean number of WBC per PC ranged from 3.3 to 4.7 × 10⁸. During the storage period pH stayed above 7.0. On the average, the production of one molecule of lactate corresponded to the consumption of 0.538 molecules of glucose, indicating that less than 8% of glucose was consumed by the oxidative pathway. There were only small increases in LDH and B thromboglobulin concentrations. Furthermore, the ability of platelets to recover from osmotic shock and to aggregate following exposure to dual agonists declined only slightly during storage, indicating that both viability and function of platelets collected by the MCS were preserved during storage. © 1993 Wiley-Liss, Inc.     

网织血小板和血小板参数在急性冠脉综合征中的变化及意义

目的:探讨急性冠脉综合征(ACS)患者网织血小板(RP)和血小板参数的变化及意义。方法:随机选取ST段抬高心肌梗死(STEMI)27例为STEMI组,非ST段抬高心肌梗死(NSTEMI)25例为NSTEMI组,不稳定心绞痛(UA)31例为UA组,健康体检者30例为对照组进行检测。用CD61-PE单抗标记血小板,噻唑橙(TO)为染料,应用富含血小板血浆(PRP)法检测RP;用COULTERJT型全自动血液分析仪测定血小板参数[血小板计数(PLT)和血小板平均体积(MPV)]。结果:STEMI、NSTEMI、UA组3组RP%均显著性高于对照组(P<0.05);STEMI、NSTEMI组的RP%显著高于UA组(P<0.05);STEMI、NSTEMI组的MPV均显著高于对照组(P<0.05)。结论:RP可能成为评估ASC患者血小板活性的新指标,它和MPV的检测可能对ACS有着一定的临床意义。     

冰冻血小板与新鲜血小板的疗效比较

目的比较冰冻血小板和新制备的血小板的在临床上的应用效果。方法选268例新制备的血小板与296例冰冻的血小板输注病例,观察两组输注血小板前后的临床表现并进行血小板的计数。结果在564例病案中,输注新制备血小板后的患者外周血血小板上升的程度和总有效率明显高于输注冰冻血小板的患者,两者之间差异有显著性(P<0.05)。结论输注新鲜血小板或冰冻血小板均能达到控制及预防出血的治疗作用,并且提升机体血小板数值,虽然新鲜血小板的疗效优于冰冻血小板,但冰冻血小板可以在抢救危重患者时代替机采新鲜血小板。     

冻干保存血小板可逆性抑制激活的实验研究

目的探索最优的血小板激活抑制剂组成方案,以建立一套完善的血小板冻干前处理程序。方法选择已基本确定有效作用浓度的6种血小板激活抑制剂如前列腺素E1、腺苷、左旋精氨酸、植酸钠、百维利肽和西洛他唑,结合血小板冷冻干燥保护剂的负载过程,以CD62p、PAC-1、MPV和P lt作为血小板活化指标,CD62p和PAC-1的再表达率作为血小板反应性指标,诱导剂诱导的血小板最大聚集率和SPAT作为血小板聚集和促凝血功能的指标;采用正交实验设计,分8个实验组,研究分析各因素在负载中对血小板状态和功能的影响,选择最优的负载液组成。结果A因素(负载环境)水平Ⅰ在抑制血小板MPV增大、D62p和PAC-1的表达以及再表达率保留、血小板最大聚集率和SPAT影响均优于水平Ⅱ(P均<0.05);B因素(PGE1)C因素(左旋精氨酸)F因素(植酸钠)G因素(百维利肽)的水平Ⅱ则均由于各自的水平Ⅰ。因此,在8个实验条件中,以第3实验组(A1B2C2D1E1F2G2)对血小板功能保护的最好。结论在血小板冻干前保护剂负载过程程序,在血浆负载环境中添加1μmol/L前列腺素E1,5 mmol/L左旋精氨酸,0.5 mmol/L植酸钠和0.5μmol/L百维利肽,可有效抑制血小板激活,使保存的血小板具有表达CD62p和PAC-1活性、聚集反应和促凝血活性。     

The evolving role of platelets in inflammation

Summary.  Platelets are small in size and simple in structure. Nevertheless, these anucleate cytoplasts utilize complex molecular systems to regulate a variety of biological functions. Here we review evolutionary paths, traditional roles, and previously unrecognized biological capacities of platelets that interface thrombosis with inflammation and potentially identify new roles in inflammatory diseases.     

Two subpopulations of thrombin-activated platelets differ in their binding of the components of the intrinsic factor X-activating complex

Binding of fluorescein-labeled coagulation factors IXa, VIII, X, and allophycocyanin-labeled annexin V to thrombin-activated platelets was studied using flow cytometry. Upon activation, two platelet subpopulations were detected, which differed by 1-2 orders of magnitude in the binding of the coagulation factors and by 2-3 orders of magnitude in the binding of annexin V. The percentage of the high-binding platelets increased dose dependently of thrombin concentration. At 100 nm of thrombin, platelets with elevated binding capability constituted approximately 4% of total platelets and were responsible for the binding of approximately 50% of the total bound factor. Binding of factors to the high-binding subpopulation was calcium-dependent and specific as evidenced by experiments in the presence of excess unlabeled factor. The percentage of the high-binding platelets was not affected by echistatin, a potent aggregation inhibitor, confirming that the high-binding platelets were not platelet aggregates. Despite the difference in the coagulation factors binding, the subpopulations were indistinguishable by the expression of general platelet marker CD42b and activation markers PAC1 (an epitope of glycoprotein IIb/IIIa) and CD62P (P-selectin). Dual-labeling binding studies involving coagulation factors (IXa, VIII, or X) and annexin V demonstrated that the high-binding platelet subpopulation was identical for all coagulation factors and for annexin V. The high-binding subpopulation had lower mean forward and side scatters compared with the low-binding subpopulation ( approximately 80% and approximately 60%, respectively). In its turn, the high-binding subpopulation was not homogeneous and included two subpopulations with different scatter values. We conclude that activation by thrombin induces the formation of two distinct subpopulations of platelets different in their binding of the components of the intrinsic fX-activating complex, which may have certain physiological or pathological significance.