白细胞过滤对血小板保存期间炎性细胞因子水平的影响及临床效果的评估

目的　考察浓缩血小板悬液 (plateletconcentratesuspend ,PCs)在保存期内IL 1β、IL 6、IL 8和TNF α的浓度变化和过滤对其的影响 ,了解在保存前滤除PCs中的白细胞是否能有效地减少这些细胞因子的积累和降低受血者非溶血性发热性输血反应 (febrilenonhemolytictransfusionreactions,FNHTR)发生率。 方法　将 1单位PCs分成两等份 ,分别给予血小板专用白细胞滤器过滤处理和不滤除白细胞处理 ,保存 5d。在 0、3、5d测定IL 1β、IL 6、IL 8和TNF α含量及白细胞计数 ,采用配对t检验进行统计分析 ;临床观察未滤组和过滤组PCs输后FNHTR发生率。结果　PCs中的白细胞计数与保存 5d时IL 1β、IL 6、IL 8和TNF α水平之间呈正相关。未滤组PCs中有较多白细胞混入 [(35 1± 81)× 10 6/袋 ],在保存期间IL 1β、IL 6、IL 8和TNF α水平明显升高 ;过滤组的PCs残余白细胞 <1× 10 6/袋在保存期间诸细胞因子均保持在 0d水平 ;临床观察显示 ,末滤PCs与过滤PCs输注后FNHTR发生率分别为 2 0 .83%和 5 .83% ,P <0 .0 1。结论　保存前用血小板专用去白细胞滤器去除PCs中残留的白细胞能有效地防止细胞因子的积累 ,同时保留 95 %以上的血小板。输注滤除白细胞的PCs能有效地减少FNHTR发生     

四种血小板制品滤除白细胞效果的实验研究

目的观察过滤对血小板的影响,遴选适于过滤的血小板制品。方法对机采血小板、手工浓缩血小板和相应的两种冰冻血小板进行过滤,观察血小板回收率、白细胞去除率和血小板聚集率等多项血小板功能指标。结果机采血小板回收率为86．81％,手工浓缩血小板回收率仅为73．08％;两种冰冻血小板制品损失量达60%～70％;过滤后机采血小板达到去白细胞标准,而手工血小板未达到去白细胞标准;过滤对液态保存血小板制品的功能无明显影响,但手工浓缩血小板功能低于机采血小板。结论冰冻保存的血小板制品不适于去白细胞过滤;手工浓缩血小板制备工艺尚需进一步改进和提高,并研制与之相适应的去白细胞滤器;机采血小板质董拔好．国产滤器可达到去白细胞标准．是当前适于滤除白细胞的血小板制品。     

不同常规保存血小板冰冻保存效果研究

目的了解常规保存不同时间的机采血小板冰冻前后质量指标变化及输注疗效，探讨血小板冰冻处理前常规保存调控的最佳方案。方法对120袋机采血小板随机分成6组，在（22±2）℃平床振荡条件下，分别保存0、1、2、3、4、5d然后制备冰冻血小板，并对血小板冰冻前与复温后分别计数血小板，检测pH值，跟踪调查输注冰冻血小板的患者，计算回收率。结果有效期内（22±2）℃振荡保存的血小板产品中血小板计数无显著性差异，pH值下降明显；冰冻前后血小板计数有显著性差异，pH值无差异。保存3d内的血小板冰冻后血小板的输注回收率无差异，与保存4d、5d的血小板冰冻后的回收率有显著差异。结论（22±2）℃振荡保存3d内的血小板可以制备冰冻血小板，保存4-5d的血小板可以输注但不宜制备冰冻血小板。     

以富血小板血浆法或白膜法制备的浓缩血小板中的白细胞碎片

背景和目标浓缩血小板(PCs)中白细胞碎片可能导致受体发生同种免疫。材料和方法白细胞碎片含量水平因制备方法不同而异,作者比较了两种不同方法制备的PCs,分别是白膜法(BC-PCs)制备的血浆或血小板保存液(Composol)保存的PCs和富血小板血浆(PRP-PCs)。结果过滤后的结果表明,两种     

保存期内不同时间制备去白细胞混合浓缩血小板制剂的质量研究

目的探讨浓缩血小板保存期内不同时间制备的去白细胞混合浓缩血小板制剂的质量。方法采用2种时间点制备去白细胞混合浓缩血小板:方法 1为常规制备方法,将采血后24 h内经白膜法制备的5袋同型浓缩血小板汇集后去除白细胞,并在血小板保存箱内振荡保存7 d;方法 2为改良法,将采血后24 h内经白膜法常规制备的浓缩血小板在血小板保存箱内振荡保存,d5将同型5袋浓缩血小板汇集,用一次性白细胞输血过滤器过滤处理,制备成混合浓缩血小板制剂,再在血小板保存箱内保存2 d。2种方式制备的混合浓缩血小板制剂各15袋,检测保存0d,5 d和7 d时血小板含量、红细胞混入量、血小板代谢情况及炎性因子含量等质量指标。结果 2种方法制备的产品质量在常规的保存5 d均可达到国家标准。改良法血小板保存d7与常规法d7比较,细胞因子IL-6(84.80±13.45vs 3.83±0.44)pg/m L,IL-10(67.90±12.02 vs 14.93±2.98)pg/m L,TNF-!(43.17±5.12 vs 7.58±0.71)pg/m L明显升高,差异具统计学意义(P值0.01),其它指标没有明显差异。结论浓缩血小板在制备后5 d内均可再进行汇集成混合浓缩血小板制剂。     

血小板添加液维持血小板12天保存有很好体外质量

背景如果血小板的体内外变量允许更长的保存，并做细菌筛查，浓缩血小板(PCs)的保存可以延长到超过5天。本研究的目的是检测PCs在不同的保存溶液中的保存特性，如仅血浆，或血浆混合PAS-Ⅱ，PAS-Ⅲ，PAS-ⅢM，及Composol。研究设计及方法5份白膜制备的过滤去白细胞的PCs保存在1．3L丁酰-3-己基-枸橼酸塑料PVC血袋中。首先，比较含     

用于浓缩血小板的去白细胞滤器质量评价

目的考察用于浓缩血小板(PCs)的去白细胞滤器的化学性能及过滤前后血小板质量的变化情况。方法按照中华人民共和国医药行业标准《一次性使用去白细胞滤器》(YY0329-2009)制备检验液,测定还原物质、金属离子、酸碱度、蒸发残渣、紫外吸光度和溶血率,并对ABO血型相匹配的PCs进行手工汇集和过滤,测定过滤前后的血小板数量、pH、白细胞数量、血小板平均体积(MPV)和血小板低渗休克相对变化率。结果滤器的化学性能指标和溶血率均符合我国行业标准要求,血小板回收率为(89.99±5.37)%,剩余白细胞数为(0.93±0.58)×106个,血小板低渗休克相对变化率为(2.75±4.93)%。过滤前后pH值和MPV无明显变化。结论该种用于PCs的去白细胞滤器具有血小板回收率高、白细胞残留少的特点,对血小板功能无显著影响,能安全有效地去除PCs中的白细胞。     

-80℃保存机采浓缩血小板的研究

目的：探讨-80℃冰冻保存机采浓缩血小板的功能及止血效果。方法：随机抽取冰冻保存3个月之内、3～6个月之间、6～9个月之间的冰冻血小板样品于42℃解冻后进行相关实验，比较不同保存时期冰冻血小板的功能及回收率；分血液病组和非血液病组观察患者输注冰冻血小板24h后出血症状的控制及校正血小板增高指数CCI值。结果：冻存3个月内、3～6个月的两组冰冻血小板的黏附功能、聚集功能与新鲜机采血小板相比差异均无显著性（P〉0．05），而冻存6～9个月的冰冻血小板的黏附功能与新鲜机采血小板相比则差异有高度显著性（P〈0．01）。3组冰冻血小板的第Ⅲ因子活性测定与新鲜血小板相比。均在正常范围，平均回收率为85．2％。两组患者在输注冰冻血小板24h后能有效控制出血症状的比率平均为93、3％；而CCI值≥10000／μL的比率，非血液病组显著高于血液病组。结论：5％二甲基亚砜-80℃冰冻保存半年以内的机采浓缩血小板具有良好的止血功能     

采集后6小时与72小时制备的冰冻单采血小板质量研究

目的通过比较采集后6h和72h制备的冰冻单采血小板制品的多项质量参数，为冰冻单采血小板的制备标准提供依据。方法以美国产Trima血细胞分离机配套全密闭7d保存袋采集的单采血小板，分别于采集后6h和72h制备为冰冻血小板制品，1周后复苏留样，分别检测血小板计数（PLT，MPV，PCT，PDW）、血小板粘附性、血小板P选择蛋白、PF3A、PF4、pH值、血块收缩试验（血浆法）。结果采集后6h和72h制备的冰冻单采血小板，两者相比差异无统计学意义（t〈0．01，P〉0．05）。单采血小板在不同保存条件下，PF3A均保持了良好的PF3活性，单采血小板在常规或冰冻保存条件下，PF4及P选择蛋白的表达均明显增加，采集后6h和72h制备的冰冻单采血小板相比，差异无统计学意义（t〈0．01，P〉0．05）；单采血小板在常规保存3d内，粘附功能和血块收缩试验没有明显变化，而冰冻保存的单采血小板这两项指标呈明显减退现象，血小板的活力下降明显。结论以美国产Trima血细胞分离机配套全密闭7d保存袋采集的单采血小板，分别于采集后6h和72h制备的冰冻血小板制品，两者的体外指标分析结果相比差异无显著性，且都能有效的改善和控制急性大出血患者的出血倾向，用于抢救急性大出血患者疗效是可靠的。     

改良血小板添加液低温(10℃)保存血小板的动物实验研究

目的对添加海藻糖的血小板添加液替代70%血浆冷藏的血小板进行动物体内输注效果评价。方法采集兔心脏血,按常规方法制备浓缩血小板,将浓缩血小板分3组保存。血浆常温组:添加100%血浆,22℃保存;添加液低温组:添加70%PAS-ⅢM/30%血浆,10℃保存;冰冻保存组:添加100%血浆,-85℃保存。以新鲜血小板为对照,常温血浆保存组在d 3,添加液低温保存组在d 3、7、9,冰冻保存组在d 20复苏后分别检测血小板缺乏兔模型的血小板24 h回收率和生存率。结果除保存9 d的添加液低温组血小板存活率明显低于新鲜血小板存活率(P<0.05)外,血浆常温组、添加液低温组以及新鲜血小板的24 h回收率和存活率相互比较差异均无显著统计学意义(P>0.05)。冰冻保存组血小板24 h回收率和存活率均低于其他各组(P<0.05)。结论改良的PAS-ⅢM能够替代血浆在低温条件下用于血小板的保存,能维持血小板的体内功能。     

Effect of filtration of platelet concentrates on the accumulation of cytokines and platelet release factors during storage

BACKGROUND: Platelet transfusions are frequently accompanied by febrile nonhemolytic transfusion reactions. These may be due, in part, to the release of cytokines interleukin 1 beta (IL-1 beta), interleukin 6 (IL- 6), interleukin 8 (IL-8), and tumor necrosis factor alpha (TNF-alpha) by white cells (WBCs) into the plasma during storage of platelet concentrates (PCs). Acting as endogenous pyrogens, these agents may induce inflammatory responses. STUDY DESIGN AND METHODS: This study proposed to determine if WBC reduction in PCs by filtration significantly reduced the levels of cytokines normally generated during storage of unfiltered PCs up to 5 days. Serotonin, platelet-derived growth factor (PDGF-AB), and von Willebrand factor levels were also assessed to establish whether or not filtration or storage elicited significant platelet activation and granule release. RESULTS: Filtration significantly reduced total WBC counts by 99.1 percent before storage (p < 0.001) without affecting total platelet counts. Compared to unfiltered PCs, filtration prevented a rise in the levels of each cytokine by Day 3 for IL-1 beta (27.7 vs. 0.6 pg/mL; p < 0.05), IL-6 (114.2 vs. 0.4 pg/mL; p < 0.001), and IL-8 (4.2 vs. 0.02 ng/mL; p < 0.001). By Day 5, further increases in the levels of all cytokines were noted in unfiltered PCs, but Day 0 levels remained in filtered PCs (IL-1 beta: 105.4 vs. 0.4 pg/mL, p < 0.001; TNF-alpha: 42.2 vs. 7.5 pg/mL, p < 0.025; IL-6: 268.8 vs. 0.4 pg/mL, p < 0.001; and IL-8: 7.6 vs. 0.02 ng/mL, p < 0.001). From Day 0 to Day 5, there were significant increases in serotonin (21.3 vs. 6.3 ng/mL, p < 0.05), PDGF-AB (72.6 vs. 25.8 ng/mL, p < 0.001), and von Willebrand factor (4.7 vs. 2.7 IU/mL, p < 0.05) in unfiltered PCs, with similar increased levels being observed in filtered PCs during storage. CONCLUSION: These data indicate that the accumulation of high levels of cytokines in stored PCs could be prevented by WBC-reduction filtration of PCs without the induction of significant platelet activation or granule release. As cytokines have the potential to induce febrile nonhemolytic transfusion reactions in patients, the transfusion of WBC-reduced PCs would be expected to reduce the frequency and severity of such reactions.     

Cytokines in WBC-reduced apheresis PCs during storage: a comparison of two WBC-reduction methods

BACKGROUND: Several studies have suggested that cytokine accumulation during storage of platelet concentrates (PCs) may mediate nonhemolytic febrile transfusion reactions and that a reduction in WBC numbers prevents the generation of cytokines. Despite efforts to minimize WBC contamination in apheresis PCs, high numbers of WBCs and increased cytokine levels may still occur, depending on the quality of the apheresis device employed. STUDY DESIGN AND METHODS: This study was undertaken to investigate whether PCs collected with WBC-reduction devices (Spectra LRS, COBE;or MCS+ LDP, Haemonetics) were sufficiently depleted of WBCs to limit cytokine accumulation during storage. The study evaluated 1) the levels of cytokines of WBC and platelet origin in two types of apheresis PCs during storage and 2) the effects of prestorage filtration on cytokine levels in the Spectra LRS PCs. RESULTS: In the Spectra LRS PCs, low levels of IL-6, IL-8, and monotype chemoattractant protein 1 (MCP-1) were detected in Day 1 PCs, and they remained consistent during the shelf life. RANTES, platelet factor 4 (PF4), beta-thromboglobulin (beta-TG), and transforming growth factor (TGF)-beta1 were also detected in these PCs, and their levels increased significantly on storage. Prestorage filtration of Spectra LRS PCs did not further reduce the levels of IL-6, IL-8, MCP-1, PF4, beta-TG, and TGF-beta1 in the filtered component. In the MCS+ LDP PCs, IL-6 was detected on Day 1, and its level increased significantly on storage, whereas the levels in the Spectra PCs remained steady. IL-8 levels were lower in MCS+ LDP PCs than in Spectra LRS PCs of the same age. MCP-1 levels were similar in both products on Day 1 and marginally increased in stored MCS+ LDP PCs. Substantial amounts of RANTES, PF4, beta-TG, and TGF-beta1 occurred in Day 1 MCS+ LDP PCs, and, on storage, these levels rose significantly. CONCLUSION: Despite a significant reduction in levels of WBC-derived cytokines, platelet-derived cytokines were present in different amounts in the two products.     

White cell apoptosis in platelet concentrates

BACKGROUND: The aim of the present study was the evaluation of the apoptosis in residual white cells (WBCs) contained in platelet concentrates (PCs) and of the relationship of this apoptosis with the concentration of inflammatory cytokines in the medium and with platelet activation. STUDY DESIGN AND METHODS: Three independent methods were used to evaluated apoptosis in WBCs present in 9 PCs, either from single donors by apheresis (SD-PCs) or from pooled buffy coats (BC-PCs). All PCs were divided in two parts, one of which was irradiated. PCs were stored up to 4 days at room temperature, and samples were withdrawn daily for analysis of apoptosis, of platelet activation (surface and soluble CD62P), and of cytokine concentration (interleukin [IL]-1alpha, IL-1beta, IL-6, IL-8, and tumor necrosis factor alpha). RESULTS: Apoptosis was found to occur with storage in both irradiated and nonirradiated units. Platelet activation increased with storage time and was higher in BC-PCs. The amount of released cytokines was rather variable among PC units. Only IL-8 was consistently found to increase with storage time. CONCLUSIONS: Apoptosis of residual WBCs occurred in PC units as a function of storage time. The amount and the time course of apoptosis seem to correlate with IL-8 release rather than with platelet activation or with the occurrence of febrile nonhemolytic transfusion reactions.     

Cytokine generation in stored platelet concentrates

BACKGROUND: Cytokines, because of the nature of their immunoinflammatory actions, are potential mediators of the symptom complex of nonhemolytic transfusion reactions. One possible source of cytokines in the transfusion setting is the stored blood component itself. STUDY DESIGN AND METHODS: To test this possibility, the plasma portion of stored platelet concentrates (PCs) was assayed for the presence of interleukins 1 beta (IL-1 beta), 6 (IL-6), and 8 (IL-8) and tumor necrosis factor alpha (TNF-alpha). Samples were taken from PCs obtained from the inventory of a regional blood center (n = 120; 30 each of 2-, 3-, 4-, and 5-day-old units). RESULTS: Detectable levels of IL-8 were measured in 59 percent of the PCs sampled, ranging from 30 percent of the 2-day-old units to 83 percent of the 5-day-old units. The median IL-8 concentration ranged from undetectable levels in 2-day- old units up to 1100 pg per mL in 5-day-old units. The mean IL-8 concentration in 5-day-old units, 11,600 pg per mL, was 100 times the mean for 2-day-old units, which was 116 pg per mL (p < 0.0001). The highest levels of IL-8, 50,000 to 200,000 pg per mL, in general were found in units with the longest storage times and highest white cell counts. Sequential sampling of 17 individual PCs over 7 days of storage confirmed that IL-8 increases progressively with increasing storage time. Parallel, but smaller, increases in IL-1 beta were observed in those units with high IL-8 concentrations. TNF-alpha was detected in 3 (10%) of 30 five-day-old PCs, but never exceeded 55 pg per mL in any unit tested. IL-6 at levels of 740 and 508 pg per mL was detected in two 5-day-old units with high white cell counts of 9500 and 14,800 per microL, respectively, but not in 21 additional units tested with white cells < or = 9200 per microL or storage time of < or = 2 days. White cell reduction by third-generation filters on Day 1 of platelet storage prevented the generation of IL-8 and IL-1 beta to Day 5 of storage. CONCLUSION: Although IL-8 achieved levels in some units of PCs that appear capable of causing physiologic changes, the potential adverse effect on transfusion recipients of the infusion of cytokines in PCs remains to be investigated.     

Cytokine accumulation in photochemically treated and gamma-irradiated platelet concentrates during storage

BACKGROUND: Photochemical treatment (PCT) for pathogen reduction of platelet concentrates (PCs) affects all cells containing DNA and/or RNA. Soluble mediators, which may cause transfusion reactions, are determined by the balance between secretion and/or cell destruction and binding and/or degradation. STUDY DESIGN AND METHODS: Ten double-dose single-donor leukoreduced PCs were split in two identical units. Two study arms were created: Study Arm A consisting of five PCT PCs with corresponding untreated control PCs and Study Arm B consisting of five PCT PCs with corresponding gamma-irradiated control PCs. PCs that had added PAS-III (Intersol) were treated with amotosalen and ultraviolet A light. Corresponding controls PCs, to which PAS-II (T-sol) were added, received no treatment or were gamma-irradiated before storage. Platelet (PLT)-derived (CCL5/RANTES, CXCL4/PF4, CCL3/MIP-1alpha, transforming growth factor [TGF]-beta, CXCL8/interleukin [IL]-8, IL-1beta) as well as white blood cell (WBC)-associated (IL-6, IL-10, IL-11, IL-12, tumor necrosis factor, interferon-gamma) cytokines were investigated by enzyme-linked immunosorbent assay and cytometric bead array during storage for up to 12 days. RESULTS: Independent of previous treatment we observed that all concentrates showed low levels of WBC-associated cytokines. PLT-derived cytokines were detected at higher levels and showed significant increase during storage. Statistical analysis showed lower PLT content per unit in PCT PCs, higher levels of activation variables in PCT PCs, and higher levels and accumulation rate of CCL5, CXCL4, TGF-beta, and CXCL8 in PCT PCs. CONCLUSION: PLTs are the main source of released cytokines during storage of untreated, gamma-irradiated, and PCT PCs. PCT may affect the level of PLT-derived cytokines in PCs. No additional reduction of WBC-associated cytokines were observed after PCT in prestorage leukoreduced PCs.     

Increased tumor necrosis factor alpha (TNF alpha), interleukin 1, and interleukin 6 (IL-6) levels in the plasma of stored platelet concentrates: relationship between TNF alpha and IL-6 levels and febrile transfusion reactions

Increased interleukin 6 (IL-6) levels were found in 8 of 12 platelet concentrates (PCs) after 3 days of storage and in 10 of 12 PCs after 5 and 7 days of storage. Most of the PCs with an increased IL-6 level also showed increased tumor necrosis factor alpha (TNF alpha) and interleukin 1 beta (IL-1 beta) levels. Levels of IL-6 increased by 3 log10 over the base level during storage. Increased levels were found when the PC white cell count exceeded 3 × 10(9) per L. A linear correlation was found among the levels of TNF alpha, IL-1 beta, IL-1 alpha, and IL-6 in the PCs (r > 0.885). Comparison of the TNF alpha, IL- 1 beta, and IL-6 levels in samples taken at various storage times indicates that the increased levels are the result of an active synthesis and release of interleukins during storage. In a second part of the study, 45 transfusions of white cell-reduced PCs were studied. Six transfusions were complicated by a febrile reaction. These reactions were related to high levels of IL-6 and TNF alpha in the PCs (p < 0.0001). These cytokines are known as endogenous pyrogens. These findings indicate that transfusion reactions might be due to the intravenous administration of plasma with high cytokine levels and might not always result from an antigen-antibody reaction.     

Cytokine accumulation in stored red cell concentrates: effect of buffy-coat removal and leucoreduction

The accumulation of cytokines in stored red blood cell concentrates (RCCs) has been implicated as a potential cause of transfusion reactions associated with the use of such products. At present, it is unclear whether there is any link between residual leukocyte and/or platelet content with cytokine levels in various RCCs. In this study, we have therefore assessed cytokine levels of leukocyte (e.g., IL8) and platelet (e.g., RANTES, TGF-beta1) origin in supernatants of RCCs prepared by the plasma reduced method or by depletion of the buffy coat. We have also assessed whether the Duffy antigen receptor (DARC, a promiscuous receptor for some chemokines) has any role in the diminution of cytokine levels in stored blood components by comparing cytokine levels in stored plasma reduced RCCs derived from both DARC +ve and DARC -ve individuals. In addition, comparison of filtered and non-filtered products of the same origin has also been conducted. Results showed that supernatants from DARC -ve concentrates contained higher levels of IL-8 up to days 14/15 of storage compared with DARC +ve RCCs. However, at later time points, similar levels of IL-8 were observed in RCCs regardless of their Duffy receptor status. For TGF-beta1 and RANTES, no significant difference in the levels of these cytokines was detected between DARC +ve and DARC -ve concentrates. Removal of leukocytes and platelets by conventional leukocyte filtration significantly reduced the accumulation of cytokines. Buffy coat reduced RCCs contained minimal amounts of IL-8 and TGF-beta1 but no RANTES. We conclude therefore, that the levels of cytokines in the supernatants of RCCs stored at 4 degrees C are related mainly to their leucocyte and platelet content.