Electronmicroscopic examination of white cell reduction by four white cell-reduction filters

The mechanisms of white cell (WBC) reduction in 16-hour-old CPDA-1 red cell (RBC) concentrates by filtration on a column filter and on three different flatbed filters were studied by electron microscopy, with special emphasis on cell-to-cell interaction, cell damage, and interaction of blood cells with the material. Generally, lymphocytes were removed by mechanical sieving and monocytes by adherence and mechanical sieving. Granulocyte depletion occurred by mechanical sieving, direct adhesion to the fibers, and indirect adhesion to activated and spread platelets. In the column filter, most granulocytes were captured by adhesion. In the coarse layers of two of the flatbed filters, indirect adhesion was most prominent, whereas direct adhesion was most prominent in the other flatbed filter. For the most part, granulocytes were captured by direct adhesion in the fine layers, but in one flatbed filter, capture apparently occurred by mechanical sieving. The results of this study suggest that the efficiency and the mechanism of WBC reduction depend on the physicochemical characteristics of the non-woven materials in the filters as well as the cellular composition of the RBC concentrates.     

A case of transfusion-associated graft-versus-host disease not prevented by white cell-reduction filters

A case of transfusion-associated graft-versus-host disease (TA-GVHD) in a patient with non-Hodgkin's lymphoma is reported. The patient, a 67-year-old woman, was diagnosed as having diffuse, mixed type non-Hodgkin's lymphoma, at clinical stage IIIA. She was treated with combination chemotherapy and received multiple blood transfusions for anemia and thrombocytopenia. Although white cells (WBCs) were reduced in the transfused components by WBC-reduction filters, the patient developed TA-GVHD that was confirmed by skin biopsy. It is suggested that the WBC reduction attained with these filters does not prevent TA-GVHD in immunocompromised patients. It is recommended that all blood components should be irradiated before transfusion to such patients.     

Experimental 6 log10 white cell-reduction filters for red cells

White cell (WBC) reduction of blood components has been receiving increased attention as a way of reducing transfusion-related complications such as WBC-associated HLA alloimmunization and transmission of cell-associated viral diseases. Currently available filters are limited to removing approximately 3 log10 (99.9%) of WBCs from red cells (RBCs). The performance of two experimental filters that were designed to remove 6 log10 WBCs from fresh RBCs during component preparation was evaluated. Both filters were able to meet this objective in less than 40 minutes with RBC losses of less than 15 percent under nonoptimized conditions. Filtered RBCs showed storage parameters within the normal range over a 42-day period. The use of these filters, if combined with a sterile docking device or if incorporated into a collection set, should provide the means to supply highly WBC-reduced RBCs with a normal shelf life.     

Prestorage removal of Yersinia enterocolitica from red cells with white cell-reduction filters

Prestorage removal of phagocytic white cells (WBCs) may increase the survivability of contaminating bacteria in units of stored red cells. Fourteen units of whole blood were inoculated with 65 colony-forming units per mL of Yersinia enterocolitica (serotype O:3) and processed into AS-3-preserved red cells. Five red cell units were filtered with a prototype third-generation filter and five red cell units with a second generation filter. WBC reduction was performed on the day of collection. Four red cell units were not filtered. Three noninoculated whole blood units served as negative controls; two were filtered (one with each type of WBC-reduction filter) and one remained unfiltered. All red cell units were then stored at 4 degrees C for 42 days. One of the five filtered red cell units (20%) in each filter group supported growth of Y. enterocolitica. In contrast, 4 (100%) of 4 unfiltered inoculated red cell units had growth (p = 0.04). Overall, 2 (20%) of 10 units of WBC-reduced red cells supported the growth of Y. enterocolitica, as compared to 100 percent of unfiltered red cell units after inoculation (p = 0.015). Bacterial contamination was not detected in any of the three noninoculated units. It can be concluded that prestorage WBC filtration significantly reduces the potential for growth of Y. enterocolitica in red cells stored at 4 degrees C for 42 days.     

Comparative analysis of six different white cell-reduction filters for packed red cells

BACKGROUND: The reduction of white cells in blood components before transfusion by filters with at least 3 log10 depletion may prevent adverse transfusion reactions such as HLA alloimmunization, febrile reactions, transmitted infections, and immunomodulation. A new generation of filters with 4 log10 depletion is now available. STUDY DESIGN AND METHODS: The aim of this study is to compare the efficiency of white cell reduction by six commercial filters for packed red cells with 3 and 4 log10 depletion (claimed by manufacturers). The analysis of white cell concentration in the white cell-reduced units was performed by flow cytometry and with a Nageotte chamber. RESULTS: The last generation of filters (BPF4, RC400, R01 Plus) show mean residual white cell numbers of 0.18 +/− 0.14, 0.26 +/− 0.21, and 0.25 +/− 0.15 × 10(6), respectively, by flow cytometric analysis and 0.05 +/− 0.04, 0.18 +/− 0.15, and 0.38 +/− 0.23 × 10(6), respectively, by Nageotte chamber evaluation. The 3 log10 depletion filters (R01, Leucostop-4LT- mono, R200) have mean residual white cell numbers of 1.41 +/− 0.92, 2.4 +/− 1.99, and 1.05 +/− 0.64 × 10(6), respectively, by flow cytometric analysis and 3.56 +/− 1.7, 1.67 +/− 1.3, and 3.21 +/− 4.1 × 10(6), respectively, by Nageotte chamber evaluation. The data show that the BPF4, RC400, and R01 Plus filters are likely to be more efficient by 1 log10 reduction than the R01, Leucostop-4LT-mono, and R200 filters. CONCLUSION: The most recent generation of filters is able to deplete white cells from packed red cells by 4 log10; in particular, with one of the filters, the residual WBC content was less than 0.5 × 10(6) per unit in all experiments, while two other filters reached that level in 9 of 10 experiments.     

Cost-effectiveness of white cell-reduction filters in treatment of adult acute myelogenous leukemia

The objective of this study was to compare the cost and cost- effectiveness of three transfusion strategies in the treatment of acute myelogenous leukemia: 1) the use of unfiltered pooled platelets until alloimmunization developed and of crossmatch-compatible single-donor platelets thereafter; 2) the use of filtered blood components until alloimmunization occurred and of crossmatch-compatible single-donor platelets thereafter; and 3) the use of single-donor platelets from the beginning. The data sources were English language articles on transfusion medicine in acute leukemia and the management of acute leukemia and review of the transfusion experience at the H. Lee Moffitt Cancer Center. The method was decision analysis with a software program for cost-effectiveness, sensitivity analysis, threshold evaluation, and Monte Carlo sensitivity analysis. In the basic models, the total costs of the first, second, and third strategies are, respectively, $12,557.14, $11,406.17, and $13,016.16 without bone marrow transplant and $14,002.72, $12,281.89, and $13,727.48 with bone marrow transplant. The threshold between the first and second strategies in regard to risk of refractoriness to filtered blood components and pooled platelets was 0.30 and 0.27, respectively, without bone marrow transplant and 0.28 and 0.40 with bone marrow transplant. According to a Monte Carlo sensitivity analysis of 500 samples, the second strategy is more cost- effective than the first in 76 percent of cases. It is concluded that the use of filtered blood components is unlikely to increase the cost of treatment.     

不同方法制备的浓缩血小板表面CD62分子的表达

目的　了解不同制备方法对浓缩血小板表面CD6 2表达的影响。方法　采用藻红素标记的抗CD6 2单抗 ,经流式细胞仪分析血小板表面CD6 2表达情况。结果　 (6 .5 5± 3.5 2 ) %的机采血小板表达CD6 2 ,(9.2 6± 5 .4 0 ) %的白膜回浆法 (BC法 )血小板表达CD6 2 ,高达 (5 0 .30± 2 0 .4 2 ) %的富血小板血浆法 (PRP)血小板表达CD6 2。结论　相对于PRP法 ,机采法和BC法更适合于浓缩血小板的制备。     

Quality and clinical response to transfusion of prestorage white cell-reduced apheresis platelets prepared by use of an in-line white cell-reduction system

BACKGROUND: This study evaluated the quality and clinical effectiveness of white cell (WBC)-reduced apheresis platelets collected by the use of a new technology, fluidized particle-bed separation. STUDY DESIGN AND METHODS: In phase 1, six suitable donors underwent two separate plateletpheresis procedures on one occasion, each separated by less than 10 minutes. In random order, a control unit was collected with the COBE Spectra and a test unit with the Spectra Leukocyte-Reduction System (LRS). The quality of apheresis platelet components was assessed by an in vitro test panel, and residual WBCs were counted by Nageotte chamber and flow cytometric methods. For the in vivo studies, the test and control units were randomly labeled with either 51Cr or 111In at the end of storage and transfused simultaneously to the donor. Samples were taken for calculation of platelet survival and recovery. In phase II, 109 thrombocytopenic patients were given platelets collected by use of the Spectra LRS. RESULTS: Test platelets had significantly fewer residual WBCs (median 7.6 x 10(4)) than control platelets (median 3.9 x 10(5)), with equivalent in vitro function values. Test and control platelets had similar recovery and survival. Transfused platelets collected by use of the LRS achieved a mean 1-hour corrected-count increment of 19.3. CONCLUSION: The LRS collects platelet components with significantly lower WBC contamination without adverse effects on the function or in vivo survival of the platelets.     

Removal of soluble biologic response modifiers (complement and chemokines) by a bedside white cell-reduction filter

BACKGROUND: Biologic response modifiers infused with stored platelet concentrates (PCs) are believed to contribute to symptoms seen during transfusion reactions. Although prestorage white cell reduction is known to decrease the production of some biologic response modifiers during storage, the possibility that poststorage (bedside) white cell reduction could reduce the amount of biologic response modifiers already present in stored PCs during bedside filtration has not been well studied. STUDY DESIGN AND METHODS: Individual PCs were pooled on storage Days 2 and 5 and passed through a third-generation white cell- reduction filter. The results from a series of in vitro PC assays were studied, before and immediately after filtration, as were levels of C3a and interleukin 8 (n = 5). Levels of other biologic response modifiers- C5a, interleukin 1 beta, interleukin 6, tumor necrosis factor alpha, and RANTES-were also studied. Removal of interleukin 8 and RANTES was studied further by using serial filtration of units of PC. RESULTS: For the in vitro platelet assays studied, pH was unchanged after filtration from prefiltration values in units of PCs pooled on storage Day 2 or 5. A 4 log10 reduction in white cells was reliably seen after filtration in Day 2 and 5 pooled PCs. Postfiltration platelet loss was 14.8 percent for Day 2 pooled PCs and 9.6 percent for Day 5 pooled PCs. For pools of both Day 2 and Day 5 platelets, postfiltration levels of CD62 (P-selectin, CD62P) were unchanged from prefiltration levels, as were results for morphology scores. Levels of C3a decreased after filtration in both the Day 2 pooled PCs (448 ng/mL before filtration vs. 20 ng/mL after filtration) and the Day 5 pooled PCs (1976 ng/mL before filtration vs. 124 ng/mL after filtration). Levels of interleukin 8 were similarly reduced after filtration in the Day 2 pooled platelets (188 pg/mL before filtration vs. 27 pg/mL after filtration) and the Day 5 pooled platelets (2234 pg/mL before filtration vs. 799 pg/mL after filtration). Levels of interleukin 8 in other components evaluated after filtration declined similarly. However, levels of the proinflammatory cytokines interleukin 1 beta and interleukin 6 did not decline after filtration. Serial filtration studies showed that, although levels of interleukin 8 and RANTES were initially lowered by filtration, they returned to prefiltration values with increases in the volume of filtration. CONCLUSION: The third-generation bedside filter used in this study reliably reduced the level of white cell contamination to 4 log10 white cells per PC. It also lowered the levels of interleukin 8, RANTES, and C3a. The filter did not, however, remove (scavenge) the proinflammatory cytokines interleukin 1 beta and 6. The mechanism of chemokine and C3a removal by the filter is unknown, but it may be related to ionic interactions between these biologic response modifiers and the filter medium.     

Effects of prestorage white cell-reduction of apheresis platelets on platelet glycoprotein Ib and von Willebrand factor

Twenty plateletpheresis components were harvested from 11 healthy donors and stored in polyolefin bags on a horizontal flatbed agitator at 22 degrees C. After 24 hours, white cells were reduced in one aliquot by centrifugation while the other aliquot was stored unaltered. Samples were obtained aseptically from each of these platelets at intervals for up to 10 days, and measurements were made of platelet glycoprotein Ib (GPIb) by both flow cytometry and polyacrylamide gel electrophoresis, of ristocetin-induced platelet aggregation by impedance aggregometry, and of plasma and platelet von Willebrand factor (vWF) by enzyme-linked immunosorbent assay. Storage of platelets under these conditions was associated with only minor decreases in surface GPIb, intraplatelet vWF, and ristocetin-induced platelet aggregation, and no differences were observed between the white cell-reduced and nonreduced aliquots. No benefit of white cell reduction in such components before prolonged storage is evident in the vWF-platelet interaction.     

Prevention of transfusion-associated Chagas' disease: efficacy of white cell-reduction filters in removing Trypanosoma cruzi from infected blood

BACKGROUND : Transfusion-associated Chagas' disease (TA-CD) is a worldwide problem. Measures adopted to prevent TA-CD include the clinical and serologic screening of blood donors and/or the inactivation of Trypanosoma cruzi present in collected blood, using gentian violet as the trypanocidal agent. This study investigated the efficacy of white cell-reduction filters in removing T. cruzi from infected blood. STUDY DESIGN AND METHODS : Human blood was contaminated with 2 or 150 T. cruzi parasites per mL and then left unfiltered or filtered with white cell-reduction filters that provided either 2, 3, or 6 log10 white cell removal. The efficacy of the parasite removal of these filters was evaluated by microscopic enumeration of active forms of T. cruzi both in vivo and in vitro. The in vivo experiments were done in Swiss mice that had been intraperitoneally inoculated with T. cruzi-infected human blood. The in vitro experiments were performed with fresh human blood that had been deliberately contaminated with T. cruzi. RESULTS : The number of parasites seen in mice inoculated with unfiltered blood containing 2 or 150 parasites per mL was significantly higher than the number of parasites seen in mice inoculated with blood from the same sample, but filtered with white cell-reduction filters providing 3 or 6 log10 white cell removal. Fifty to 70 percent of the mice given T. cruzi-infected (2 parasites/mL) filtered blood did not develop T. cruzi infection. In vitro, the use of white cell-reduction filters, providing 2, 3, or 6 log10 white cell removal, significantly reduced the number of parasites seen in culture. CONCLUSION : The present experimental data provide evidence that white cell-reduction filters are effective in reducing the number of parasites in T. cruzi- infected blood and that this efficacy depends, in part, on the concentration of parasites in the artificially infected blood. Properly designed clinical studies of known carriers of T. cruzi must be conducted to determine whether the use of white cell-reduction filters may be an alternative method of reducing the incidence of TA-CD.     

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?     

去白细胞机采血小板膜表面CD62P及血栓弹力图变化分析

目的 掌握去白细胞机采血小板在保存期内的功能变化,为临床输注成分血提供参考依据.方法 选择2010年5月至2014年1月于日照市中心血站无偿献血的30例无偿献血者的血样为研究对象.男性献血者为21例,女性为9例;年龄为19～42岁,平均为25.2岁.所有献血者均符合《献血者健康检查要求》(GB18467-2011).所有血液的采集均获得献血者知情同意,并在知情同意一栏签字后献血.通过白细胞过滤方法制备去白细胞机采血小板,于(22±2)℃血小板恒温振荡保存箱中振荡保存,并分别于保存前以及保存后第1,3,5及7天检测去白细胞机采血小板膜表面CD62P(P选择素)的表达率和血栓弹力图(TEG),进而评价随保存时间延长血小板功能的变化情况.结果 ①去白细胞机采血小板保存前白细胞残余量为(5.9±2.1)×105/袋,保存至第5和7天,白细胞残余量相对于保存前明显减少,且差异均有统计学意义(P＜0.05),白细胞残余量随保存时间的延长而减少.②去白细胞机采血小板保存前血小板膜表面CD62P表达率较低,仅为(10.3±2.3)％,保存后第3,5及7天与保存前比较,血小板膜表面CD62P表达率均显著增加,且差异均有统计学意义(P＜0.05),并随保存时间延长,血小板膜表面CD62P表达率逐渐增加.③去白细胞机采血小板保存后第1,3,5及7天R值较保存前显著增加,差异均有统计学意义(P＜0.05),且R值随保存时间延长呈明显增加趋势.而K值和α角在保存期内与保存前比较,差异均无统计学意义(P＞0.05).保存后第5,7天MA值较保存前显著降低,差异均有统计学意义(P＜0.05).结论 去白细胞机采血小板在保存期内存在明显的血小板损耗,但对其凝血功能影响不大,保存5d内的去白细胞机采血小板仍是比较理想的成分血.     

Inhibition of activated protein C by platelets.

Activated protein C (APC), an anticoagulant that acts by inactivating Factors Va and VIIIa, is dependent on a suitable surface for its action. In this study we examined the ability of human platelets to provide this surface and support APC-mediated anticoagulant effects. The activity of APC was examined in three systems: the Factor Xa recalcification time of Al(OH)3 adsorbed plasma, studies of thrombin generation in recalcified plasma, and assessment of the rate of inactivation of purified Factor Va. In comparison with phospholipid, intact platelets required significantly greater concentrations of APC to achieve a similar degree of anticoagulation. When washed platelet membranes were substituted for intact platelets, adequate support of APC was observed and the anticoagulant effect was similar to that obtained with phospholipid. Platelet releasate obtained by stimulation of platelets with thrombin and epinephrine contained an inhibitor that interfered with the ability of phospholipid and washed platelet membranes to catalyze the anticoagulant effects of APC. A noncompetitive inhibition was suggested by Dixon plot analysis of the interaction between platelet releasate and APC. The activity of the platelet APC inhibitor was immediate and was not enhanced by heparin, distinguishing it from the circulating protein C inhibitor. The presence of this inhibitor in the platelet and its release with platelet stimulation emphasizes the procoagulant role of this cell.