GMA保存血小板的实验研究

目的:探讨葡萄糖甘露醇腺嘌呤(GMA)作为添加剂对血小板的保存效果。方法:将GMA替代血浆制备的浓缩血小板(GMA-PC)和以ACD血浆为介质的浓缩血小板(ACD-PC)置22℃水平摇箱内保存,于各时间段取样测定有关指标。结果:两组PC的血小板低渗休克反应和以ADP诱导的血小板聚集率随保存时间的延长平行降低,差异无显著性(P> 0.05),在保存120h 期间,GMA-PC的乳酸脱氢酶释放量低于ACD- PC,前者的形态积分则显著高于后者,提示用GMA 保存的血小板其形态和细胞膜的完整性优于用ACD血浆保存的血小板。结论:初步证实用GMA替代血浆制备和保存浓缩血小板具有可行性     

两种血小板保存箱保存富血小板血浆效果的比较

目的　探讨血小板体外保存指标 ,验证国产血小板保存箱实际保存效果。方法　取新分离的富血小板血浆 (PRP) 12袋 ,每袋平均分成两袋 ,分别放入国产XHZ IA型血小板保存箱和进口FORMA 36 0 6型血小板保存箱中保存 5天 ,每天取样检测血小板聚集功能、低渗休克反应回复功能、CD6 2p表达率等体外保存指标。结果　两种保存箱保存PRP各项体外保存指标无显著差别。结论　XHZ IA血小板保存箱与FORMA血小板保存箱保存PRP的实际保存效果无差别。     

血小板添加液延长血小板保存期的研究

目的探讨血小板浓缩液(PC)加入血小板添加液延时贮存后的质量。方法将新鲜全血(400ml/袋)在4—6h内分离出的白膜层(BC)于22℃静置过夜,取同血型的7袋BCs汇集制备成PC后滤白,用血小板添加液(PAS-ⅢM+血浆)贮存21d,并在存储期内分时间段测定其血小板含量、红细胞和白细胞残留量、最大聚集率、CD41和CD62p阳性表达率、代谢产物和pH值等。结果血小板含量≥2.5×1011/袋,WBC残留量≤3.0×105/袋;贮存至d10,其pH值维持在7.00左右,CD62p阳性表达率45%,ADP和肾上腺素合用诱导的聚集反应率为15%—88%,凝血酶诱导的聚集反应率为89%—99%,HSR恢复率为41%—69%。10d内有可供血小板代谢需要的葡萄糖,乳酸盐浓度随着葡萄糖含量降低而升高,PO2随着PCO2降低而增加。结论加入PAS-ⅢM+血浆的PC贮存10d仍含有可供血小板正常代谢的能量物质,具有较好的聚集功能和抗低渗休克反应的能力。     

单采血小板冷冻保存的质量评价

[目的]研究单采血小板冰冻保存。[方法]在单采血小板中加入二甲基亚砜(DMSO)，终浓度为5％，-120℃保存。观察冰冻一周，一个月后血小板聚集功能和回收率的改变、血小板低渗休克反应回复率的变化。[结果]冻存一周，一个月后单采血小板仍具有较高的回收率，血小板聚集功能和低渗休克反应回复率有一定下降。[结论]单采血小板冰冻保存是可行的，具有较好的疗效。贮存期可以延长。     

高甘油三脂对机采血小板保存期间活化及聚集功能的影响

目的探讨高甘油三脂(TG)对机采血小板保存的影响。方法使用全自动血细胞分离机(MCS+)采集浓缩血小板(PC)24份,用高浓度TG贫血小板同种异体血浆(PPP)制备血小板悬浮于高TG血浆的模型,调整每份PC的TG终浓度分别为正常人群中值(2.3 mmol/L)的1倍、2倍和3倍,用低浓度PPP调整PC的TG使其终浓度为正常人群中值的0.7倍作为对照。样本于22℃保存,并分别于d 0、1、2、3、4、5测定胶原诱导的血小板聚集率和血小板膜CD62P和CD63的阳性表达率。结果随着保存期的延长,各组的血小板聚集率呈下趋势;相同保存时间内,TG含量越高血小板聚集率降低越明显;在保存的d 1—5,高TG组与对照组相比差异有统计学意义。与此同时,CD62P和CD63的阳性表达率呈上升趋势;在保存的d 0—5,高TG组的CD62P和CD63的阳性表达率>对照组。结论悬浮于高甘油三脂血浆的血小板体外保存研究表明,高含量TG不利血小板的保存质量,可增加血小板的活化和降低血小板聚集功能。     

全血或白膜混合过滤制备的血小板浓缩物的体外功能

背景和方法由白膜制备保存5天后的血小板浓缩物(PC) 质量的数据不多,作者用去除白细胞的全血(Terumo WBSP,n =10)或白膜(Pall Autostop,n=10)制备的PCs在血浆中保存 7天,测定血小板参数和血小板激活标志物来评估PCs质量。结果两种类型的PC在保存过程中葡萄糖水平降低,但不是完全消失(>11 mmol／L,保存的第7天)。在保存中乳酸水平增加,一直<20 mmol／L,所有单位在第7天时pH维持>6．8,低渗休克反应计分>47％。第1天血小板激活标志高于WBSP PC,但到     

血小板低温保存的初步研究

血小板输注应用的进展,要求体外长期保存血小板,低温保存是目前公认细胞长期保存的最好方法。本研究用体外回收率、血小板聚集试验、血小板低渗休克试验与透射电镜观察等手段,研究了不同降温方法与冻存条件下血小板的活力。结果发现:(1)用自动控温装置降温效果最好。(2)选用5％DMSO低温保护剂与1℃/min降温率为最好冻存条件,此时70％以上血小板活力得以保存。(3)血小板低温损伤主要发生在降温时融合热释放期(-6℃以上)。(4)-20℃冻存是不可取的。(5)血小板聚集功能在冻存后严重下降,有必要进一步研究解决方法。     

2种血小板保存袋保存效果的比较

目的比较国产和同类进口产品保存血小板保存袋对机采血小板的保存效果。方法机采血小板无菌加入到2种血小板保存袋于22℃保存,分别在0、3、5和7 d取样品测定血小板聚集功能、膜蛋白CD62p、血小板代谢功能、pH、浓度、MPV、PDW、血小板低渗休克反应(HSR)及细菌培养。结果 2种血小板保存袋常温条件下保存的血小板各项检测指标差异无统计学意义。结论产品保存血小板保存袋相比,各项检测指标差异无统计学意义,2种保存袋保存效果基本一致。     

血小板冷冻保存的研究

用终浓度4％二甲基亚砜(DMSO)作为防冻剂,于—30℃和—80℃冷冻保存血小板以开展低温保存血小板的输注。体内外研究结果表明:DMSO对血小板粘附和单一诱聚剂所致的聚集有可逆性抑制作用;洗涤去除DMSO后,粘附、聚集可以恢复。联合诱聚剂所致的聚集不受DMSO影响。冻存后的血小板粘附、聚集和低渗休克反应较冻前有所降低,与保存时间(1和3个月)无关。—80℃保存的血小板质量优于—30℃保存者。输注—80℃保存的血小板可提高患者的血小板数,因而能有效地预防和控制因血小板减少导致的出血。     

机采血小板常温保存过程中代谢及体外功能变化的研究

目的 研究机采血小板常温保存过程中代谢及体外功能的变化规律。方法 通过对15U保存0～9d机采血小板的血气指标、葡萄糖、乳酸、乳酸脱氢酶、血小板膜表面CD62p表达率、凝血酶激活后CD62p再表达水平、血小板低渗休克反应进行系统监测,分析机采血小板体外保存过程中代谢指标与体外功能的关系。结果 血小板在体外保存9d的过程中,乳酸与H^＋逐渐堆积,pH值随之下降;血小板低渗休克反应在7d时开始明显下降;CD62p表达率随保存天数增加而逐渐增高,凝血酶激活后CD62p再表达率逐步下降;血气、葡萄糖、乳酸等代谢指标与血小板低渗休克反应、CD62p的表达率及再表达率具有显著相关性。结论 对代谢指标的监测可以间接反映血小板体外功能的变化,从而对血小板保存质量作出系统评价。     

A new polyvinylchloride blood bag plasticized with less-leachable phthalate ester analogue, di-n-decyl phthalate, for storage of platelets

To compare changes in platelets stored in the new di-n-decyl phthalate (DnDP)-plasticized polyvinyl chloride (PVC) bag with those in a di-(2-ethylhexyl) phthalate (DEHP)-plasticized PVC bag, single-donor apheresis platelet concentrates (PCs), 133 +/- 11 x 10(7) platelets per ml (n = 7), were stored with 94 +/- 3 ml of plasma in a new 1-liter bag with a surface area of 44 +/- 7.1 cm2 per 10(10) platelets. Oxygen and carbon dioxide gas diffusion properties of PVC-DnDP films were respectively, 1.6 and 2 times those of standard PVC-DEHP films. The amounts of DnDP leaked into the plasma of PCs were low at 0.58 +/- 0.06 mg per bag after 5-day storage, which is about one-eightieth the amount of DEHP leaked. The pH of PCs in PVC-DnDP bags amounted to 6.99 +/- 0.03 after 5-day storage, with glycolysis accelerated somewhat in the new bags. However, the platelet oxygen consumption was no different from that in the PVC-DEHP bags. Platelet aggregation and responses to hypotonic shock were significantly better in the new bags at the end of storage. Shape changes of platelets into spherical forms with dendrites were more frequently observed in PVC-DnDP bags than in PVC-DEHP bags. The study indicated that platelets stored in the new DnDP-plasticized PVC bags have retained aggregation and responses to hypotonic shock more than platelets in the PVC-DEHP bags, but spherical forms and anaerobic metabolism increased in the new bags.     

New plasma-reduced synthetic media, Fukushima cocktails, for the storage of platelets for transfusion

BACKGROUND: Donor plasma proteins are associated with non-hemolytic allergic reactions, such as urticaria or laryngeal edema, in platelet-transfusion recipients. Replacement of plasma with synthetic media from platelet concentrates (PCs) is considered to be effective in preventing such reactions. However, platelets preserved in media depleted of less than 10% plasma are reported to have functions inferior to those preserved in plasma. METHODS: Fukushima Cocktails (FCs) contain glycerol (25, 50 or 100mM), sodium acetate, glucose and other components. To test the effect and determine the most suitable concentration of glycerol for platelet preservation, functions of platelets including aggregation, hypotonic shock response and swirling pattern and released biochemicals were measured with platelets preserved in Fukushima Cocktails. The effects of residual plasma on platelet functions were also evaluated. Autologous platelets stored for 3 days in solution containing 50 mM glycerol were transfused into healthy volunteer donors to evaluate their safety and survival. RESULTS: The functions (aggregation and hypotonic shock response) of platelets preserved in Fukushima Cocktails with 10% residual plasma were preserved for 5-7 days as well as plasma controls, whereas platelets stored for 9 days in a medium lacking glycerol became swollen and beta-thromboglobulin and thromboxane B(2) increased. When the residual plasma was more than 5%, platelet functions including aggregation, hypotonic shock response and swirling pattern were well preserved for 7 days. The in vivo platelet survival rates at 24 and 48 h after transfusion of platelets stored for 3 days in Fukushima Cocktail were 77% and 60%, respectively, which were not less than autologous plasma-stored platelets. CONCLUSION: Glycerol at a concentration of around 50 mM has a beneficial effect on platelet preservation for more than 7 days. The results of these experiments indicate that platelets stored in Fukushima Cocktail should be useful clinically.     

2种国产血小板滤器滤除白细胞对体外血小板功能的影响

目的考察2种血小板滤器滤除手工法制备的浓缩血小板中的白细胞后血小板功能的变化情况。方法采用富血小板血浆法(PRP法)以400 ml新鲜全血制备浓缩血小板,将6袋ABO同型的浓缩血小板汇集,并用2种国产血小板型去白细胞滤器过滤,各10例(分别以A、B组代之),测定过滤前后的血小板计数、白细胞计数、pH值、血小板CD62p阳性表达率、血小板聚集和低渗休克等指标。结果血小板去白过滤后,A、B 2种滤器(组)的血小板回收率、剩余白细胞数及pH值分别为(87.01±3.47)%vs(87.88±4.77)%、(0.95±0.90)×106vs(0.45±0.58)×106及(7.13±0.13)vs(6.80±0.26)(P>0.05);血小板过滤前后CD62p阳性表达率、血小板最大聚集率和低渗休克,A组分别为(8.06±4.11)%vs(8.21±4.50)%、(70.55±27.21)%vs(71.63±32.24)%和(68.14±10.13)%vs(69.18±9.38)%,B组分别为(10.34±3.26)%vs(10.47±2.42)%、(56.30±18.43)%vs(59.49±19.15)%和(75.73±5.50)vs(73.74±6.52)%(P>0.05)。结论所考察的2种血小板型去白细胞滤器过滤浓缩血小板未增加血小板的活化,对血小板聚集功能及抗低渗休克能力无明显影响,血小板回收率及剩余白细胞数符合相关标准。     

Extension of platelet concentrate storage by addition of sodium bicarbonate

Viability of platelet concentrate (PC) stored in polyvinylchloride bags in an elliptical rotator at 22 degrees C (standard PC) was assessed by in vitro tests, and an alternate approach to extending the shelf-life of PC by the addition of hypertonic sodium bicarbonate (test PC) was investigated. The fall in the pH which occurred during storage in standard PC was arrested in test PC. Furthermore, platelets stored under these test conditions maintained their morphology better than in standard PC as judged by their mean platelet volume and platelet distribution width. Recovery of stored platelets from hypotonic shock at 37 degrees C following resuspension in fresh plasma was better for test platelets. Results indicated that platelets in standard PC were viable up to day 3 but were not viable at day 7. Platelets store better in PC to which sodium bicarbonate has been added and behave as viable platelets up to 7 days.     

Platelet storage in Fresenius/NPBI polyolefin and BTHC-PVC bags: a direct comparison

SUMMARY: New platelet storage systems, such as changes in the plastic of the storage bags, require validation. In this study, pooled buffy coat platelets stored in Fresenius/NPBI polyolefin bags were compared with those stored in Fresenius/NPBI butyryl-trihexyl citrate (BTHC) plasticized polyvinyl chloride (PVC). The CompoSelect thrombocyte polishing filter system (1000 mL polyolefin bag) and the CompoStop F730 system (1300 mL BTHC-PVC bag) were used to prepare paired, plasma-suspended, buffy coat platelet concentrates. Samples were taken up to day 7 for in vitro analysis. In a separate experiment, 12 units were prepared using the CompoStop F730 system and samples taken after leucofiltration for FXIIa assay. By day 7, platelet concentrates stored in BTHC-PVC demonstrated significantly higher pH levels (7.32 +/- 0.05 vs. 7.26 +/- 0.05) and a greater degree of cell lysis as shown by increased lactate dehydrogenase levels (497 +/- 107 vs. 392 +/- 81 U L(-1)). The supernatants contained higher concentrations of soluble P-selectin and the chemokine 'regulated on activation, normal T-cell expressed and presumably secreted', which are released from the alpha-granules during activation. The ATP concentrations were significantly lower in BTHC-PVC. Platelet counts, mean platelet volume and hypotonic shock response were similar for both bags. FXIIa antigen concentrations were 0.6 +/- 0.2 ng mL(-1) indicating that activation of the contact factor pathway had not occurred. Although the CompoStop F730 leucoreduction filter did not activate the contact system, platelets stored in 100% plasma in BTHC-PVC bags demonstrated different in vitro characteristics from those stored in polyolefin. Further work is required to demonstrate whether these differences will affect in vivo recovery and survival.     

Comparative Studies of the Viability of Human Platelets Stored at 4 C in Plastic and Glass Containers

Studies were done to investigate whether the surface of the container (plastic or glass) would influence the viability of human platelets stored at 4C for short intervals.
Cr⁵¹-labeled platelets prepared as concentrates suspended in plasma were preserved at 4 C for 24, 48 or 72 hours and their capacity to recirculate and survive after infusion into the respective donors ("viability") was determined. In addition, the clot retraction property of the stored platelets was measured.
Platelet viability was sharply reduced after storage at 4 C in all experiments. When the storage period was limited to 24 hours, survival curves of platelets preserved in plastic bags were similar to those of platelets preserved in glass bottles. However, after 48 hours of storage, viability of platelets preserved in plastic containers had values significantly higher than those of platelets preserved in glass containers. After 72 hours of storage, platelet viability was reduced to minimal values but was still greater for platelets stored in plastic bags.
The study of clot retraction confirmed data previously obtained and showed that this platelet property was preserved better by storage in glass bottles rather than in plastic bags. Platelet viability was, however, lost very rapidly during storage at 4 C in either type of container so that the unfavorable effect of plastic on the preservation of clot retraction was thought not to be of practical importance in platelet transfusion therapy.     

Metabolic changes during platelet storage

Platelet concentrates (PC) were stored in plastic bags with continuous shaking at 4, 22, and 37 C. Various metabolic parameters were examined over a 72-hour period. At 22 C, the pH and PO2 declined over 72 hours while the PCO2 and lactate increased. Hypotonic shock declined to 70 per cent. This differed from the small amounts of CO2 and lactate found at 4 C and the marked accumulation of metabolites and almost complete loss of shock response at 37 C. Aggregation was always better maintained with 4 C storage. The toxic effect of the accumulation of metabolites on the platelets was tested by adding lactate to fresh PC at zero time. This was effective in lowering the initial pH, markedly inhibiting the response to aggregation and decreasing the total accumulation of lactate during storage, but did not produce an inhibition of hypotonic shock response. The effect of accumulation of toxic metabolites was further investigated by using 72-hour plasma and platelets and recombining each of them with fresh preparations. Platelets were tested under degassed conditions to outline the requirements for oxygen and gasious exchange. Surprisingly, there was less accumulation of lactate and CO2 and better hypotonic shock response. These experiments have detected various changes in viability markers in platelets that are stored under actual blood bank conditions and indicate that the accumulation of lactate is not totally responsible for the toxic inhibition of platelet performance that is found upon storage at 22 C.     

In vitro function and in vivo viability of stored platelet concentrates. Effect of a secondary plasticizer component of PVC storage bags

Previous studies have shown that CL-2399 (Cutter) and PL-130 (Fenwal) polyvinyl chloride (PVC) plastic bags are unsatisfactory for storage of platelet concentrates (PC) at 22 degrees C. In an effort to explain the effects of plastic bags, the chemical make-up of CL-2399 and PL-130 PVC films was determined and compared with that of P1-146 (Fenwal) PVC, which is satisfactory for PC storage at 22 degrees C. The only significant difference between the three materials was the incorporation of tetrahydrofurfuryl oleate (THFO) as a secondary plasticizer in CL-2399 and PL-130. The response of platelets to aggregating agents, uptake of serotonin, recovery from hyptonic stress, and serotonin release during storage following storage in a modified CL- 2399 plastic prepared without THFO and designated CL-3000 (Cutter) was equivalent to PL-146 and far superior to CL-2399. In vivo studies in two laboratories of platelets stored in CL-3000 bags showed satisfactory recovery (56 +/− 4.2% and 46.7 +/− 2.7%) and survival (6.4 +/− 0.4 days and 7.4 +/− 0.6 days). From these studies we conclude that the THFO secondary plasticizer component of PL-130 and CL-2399 is the cause of the poor platelet viability of platelets stored in these plastic bags. The mechanism of impairment is not known. The causative agent(s) may be degradation products of THFO (formed during manufacture of the PVC film) that are leached from the plastic into PC during storage.     

Platelet storage: an assessment of the requirements for plasma and oxygen

Platelets from several units of plasma were pooled, and then resuspended either as platelet concentrate (PC) (60 ml) or as platelet rich plasma (PRP) (220 ml) and followed during 72 hours of storage at 22 C. Aggregation, pH, and hypotonic shock response are better maintained in the larger volume of plasma. However the decreased pH and function in the PC is not the result of lactate production. While the relative concentration, expressed in mg/dl, is higher in the PC, the absolute production per platelet and, therefore, the total amount of lactate is higher in PRP. Glucose levels are always sufficiently high to permit glucose to be used as a metabolic substrate even after 72 hours. PC maintained in nitrogen have better in vitro function than do platelets stored either in oxygen or CO2. CO2 can easily diffuse through the plastic bag; the same is not true of oxygen. Less than 15 per cent of the volume of oxygen introduced into the bag passed out through the PVC plastic over a 72-hour period. The data suggest that during storage, anaerobic glycolysis is the preferred metabolic route. The relatively poor performance of the PC maybe the result of limitation of some unidentified substrate or cofactor which is present in plasma and which is necessary for maintaining function.