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.     

Effect of filtration and storage of platelet concentrates on the production of the chemotaxins C5a, interleukin 8, tumor necrosis factor alpha, and leukotriene B4

BACKGROUND: The production in platelet concentrates (PCs) of C3 activation products (C3bc), terminal complement complex (TCC), and chemotaxins C5a, interleukin (IL)-8, tumor necrosis factor alpha (TNFalpha), and leukotriene B4 (LTB4) and the proposed reduction in concentration of the chemotaxins by white cell reduction were examined. STUDY DESIGN AND METHODS: Samples were collected from supernatants of PCs produced by apheresis (apheresis PCs) or from buffy coats (BC PCs) immediately after the production, after white cell-reduction filtration on Day 1, and after 5-day storage, and examined by enzyme immunoassays. RESULTS: Complement was activated in all PCs during storage, and the concentration of activation products was not influenced by prestorage filtration. In prestorage white cell-reduced BC PCs, only C3bc levels increased. Levels of IL-8, TNFalpha, and LTB4 increased during storage of apheresis PCs, but not in filtered units, except for LTB4. In contrast, levels of IL-8 decreased after storage of filtered BC PCs. C5a correlated significantly with IL-8, which also correlated with TNFalpha and LTB4. CONCLUSION: Both C5a and TNFalpha generation in apheresis PCs seem to induce white cell IL-8 production, which mediates cellular LTB4 release. Prestorage white cell reduction is recommended for reducing chemotactic cytokine and leukotriene levels in all PCs. Production of BC PCs is recommended to achieve less complement activation, which is not affected by filtration.     

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.     

Effects of filtration and gamma radiation on the accumulation of RANTES and transforming growth factor-β1 in apheresis platelet concentrates during storage

BACKGROUND: Platelet-derived biologic response modifiers (BRMs) including RANTES and transforming growth factor (TGF)-beta1 accumulate in platelet components during storage because of platelet activation, and they may play a causative role in nonhemolytic febrile transfusion reactions. The majority of PCs with high unit values are provided by single donor apheresis in Japan. STUDY DESIGN AND METHODS: RANTES and TGF-beta1 levels in platelet units prepared from single-donor apheresis platelet concentrates (apheresis PCs) and units from whole blood (buffy coat PCs) were investigated. The effects of prestorage and poststorage filtration and gamma radiation on the levels of RANTES and TGF-beta1 in the supernatant of apheresis PCs during storage were also examined. RESULTS: The levels of RANTES and TGF-beta1 increased during storage from Day 0 to Day 5. The levels of RANTES and of TGF-beta1 correlated with the platelet concentration (p<0.01), but not with the residual white cell concentration in apheresis PCs that were not white cell reduced by filtration (p>0.05). In addition, there was a correlation between RANTES and TGF-beta1 levels (p<0.01). In white cell-reduced apheresis PCs using negatively charged filters as well as in gamma-radiated apheresis PCs, the levels of these two BRMs-did not differ at any storage time from those of untreated apheresis PCs. Filtration of apheresis PCs with negatively charged filters after 3 days of storage significantly (p<0.05) reduced the levels of RANTES, but not of TGF-beta1. There was no reduction in the levels of RANTES and TGF-beta1 levels by positively charged filters. The RANTES levels in buffy coat PCs were slightly higher than but not significantly different from those of apheresis PCs during storage, except for the level on Day 1. There were no differences in the TGF-beta1 levels in apheresis and buffy coat PCs during storage. CONCLUSION: Prestorage filtration and gamma radiation had neither preventive effects on the accumulation of RANTES and TGF-beta1 nor adverse effects on platelet activation. Negatively charged filters might be useful for the reducing the levels of RANTES in stored apheresis PCs.     

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.     

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.     

Complement activation in prestorage leucocyte-filtered plasma

Complement activation and generation of pro-inflammatory cytokines occur during storage of blood components. Prestorage leucocyte filtration of platelet concentrates and red cells diminishes the accumulation of leucocyte-derived cytokines during storage, however, transfusion reactions are not eliminated. We investigated inflammatory mediator release during storage of plasma and whole blood and the effect of prestorage leucocyte filtration of plasma. Twenty-four blood units were collected from healthy blood donors and stored for 35 days. Eight units were stored as whole blood, eight units as plasma and eight units as prestorage filtered plasma. Samples were collected weekly for analyses of potassium, leucocytes, free plasma haemoglobin, complement activation (C3a and SC5b-9) and pro-inflammatory cytokines [interleukin (IL)-6, IL-8 and tumor necrosis factor (TNF)-alpha]. Elevated levels of C3a and SC5b-9 were registered in filtered plasma, from the beginning of storage. C3a levels increased during storage. There was a higher rate of change during storage in C3a (P < 0.01) and SC5b-9 (P < 0.05) in plasma compared with filtered plasma. Interleukin (IL)-8 is released in whole blood. The cytokine levels generated in plasma and filtered plasma were low. Complement activation is present in whole blood, plasma and filtered plasma during storage. Prestorage filtration of plasma activates the complement cascade but does not influence cytokine generation.     

Evaluation of pooled platelet concentrates using prestorage versus poststorage WBC reduction: impact of filtration timing

BACKGROUND: Concern for the undesirable consequences of transfusing passenger WBCs is leading to the general use of WBC-reduced platelet concentrates (PCs). However, the impact of prestorage versus poststorage WBC reduction on the quality of platelet products has not been clearly defined. STUDY DESIGN AND METHODS: Pooled PCs were WBC reduced before or after 5-day storage, by use of a WBC filter (PXL-8, Pall Corp.). Samples from pools were taken on days 1 and 5, before and after filtration, and on Day 9 of storage and assessed for cell counts, biochemical values, expression of platelet glycoproteins, thrombin generation, and content of IL-6, IL-8, TNFalpha, transforming growth factor beta1 (TGFbeta1), and anaphylatoxins C3a and C4a. RESULTS: Filtration of fresh and 5-day-stored pooled PCs via a PXL-8 filter was similarly efficient, rendering pools with low WBC counts (<1 x 10(6) cells) and high platelet recovery (>95%). No major changes were found in the metabolic behavior or the expression of platelet GPIb, GPIIb/IIIa, CD62, and CD63 in PCs filtered before or after storage. Filtration, either before or after storage, increased by less than 5 percent the proportion of CD62+ platelets. Moreover, no changes were found in the concentration of prothrombin fragments 1 and 2 and thrombin-antithrombin complexes in the pooled PCs derived from the time of filtration. Finally, prestorage WBC reduction abrogated the accumulation of IL-6 and IL-8, but it did not prevent that of anaphylatoxins C3a and C4a nor of TGFbeta1. However, filtration through a PXL-8 filter significantly reduced (40-90%) the amount of IL-8, C3a, and C4a in the filtrate. CONCLUSIONS: The timing of PXL-8 filtration of PCs has little impact on the efficiency of WBC reduction and on platelet recovery, and it does not seem to affect the quality of platelets or the generation of thrombin in the PCs. As regards the goal of reducing the amount of bioactive products in PCs, it remains uncertain as to whether prestorage WBC reduction fully eliminates the need for poststorage filtration. Prestorage filtration leads to low levels of IL-6 and IL-8 in PCs, but it does not impair the poststorage content of TGFbeta1 or anaphyla-toxins. By contrast, poststorage PXL-8 filtration removes significant amounts of C3a and C4a, and thus it might provide clinical benefits beyond those of prestorage WBC reduction.     

Cytokine generation in stored, white cell-reduced, and bacterially contaminated units of red cells

BACKGROUND: Proinflammatory cytokines were measured in the supernatant portion of stored, bacterially contaminated, and/or white cell (WBC)- reduced units of red cells (RBCs). Previous studies from this laboratory and others have shown that cytokines are generated in platelet concentrates during storage. This earlier work has been expanded to the study of stored RBCs. STUDY DESIGN AND METHODS: Units of AS-1 RBCs (n ? 10 non-WBC-reduced; n ? 10 WBC-reduced) were obtained from a regional blood center, and each was split on Day 3 of storage into three equal portions by sterile techniques. One portion was kept sterile (control), and the other two were inoculated with Yersinia enterocolitica and Staphylococcus aureus, respectively, at 1 to 3 colony-forming units per mL. The RBCs were stored at 1 to 6 degrees C for 42 days. Sequential samples were taken during storage and assayed for interleukin 8 (IL-8), interleukin 1 beta (IL-1 beta), interleukin 6, WBC count, and bacteria count. For the WBC-reduced group (n ? 10), WBC removal was done by filtration on Day 3 of storage, before bacterial inoculation. RESULTS: IL-8 was detected in the supernatant portion of all 42-day-old, non-WBC-reduced (mean WBCs ? 4760 ± 3870/μL) units of AS-1 RBCs at levels ranging from 63 to 1610 pg per mL. By contrast, at 2 to 3 days of storage, lower levels of IL-8 (range, 0-280 pg/mL) were detected in the same units. IL-8 levels increased progressively during storage in most (7/10) units. The highest mean levels of IL-8 were reached by outdate at Day 42. Y. enterocolitica-contaminated units had statistically higher levels of IL- 8, with a range of 170 to 2100 pg per mL, by 42 days of storage. S. aureus grew poorly in stored units of RBCs and failed to further stimulate cytokine production. No WBC-reduced unit (mean WBCs ? 0.5 ± 0.6/μL), even when contaminated with bacteria, had more than 260 pg per mL of IL-8. Although IL-1 beta was not detected in any unit of RBCs at 3 days of storage, it increased to low levels (5-13 pg/mL) in all units tested at 42 days. Interleukin 6 was not detected in any unit at any storage time. CONCLUSION: IL-8 and IL-1 beta accumulated in the supernatants of stored RBCs despite cold storage conditions. IL-8 reached levels > 1000 pg per mL in the supernatants of some RBC units. IL-1 beta increased to significant but low levels (< 13 pg/mL). WBC filtration early in storage prevented the accumulation of IL-8. The physiologic significance to transfusion recipients of IL-8 in RBC supernatants is currently unknown and deserves further investigation.     

Chemokines in stored platelet concentrates

BACKGROUND: Platelets contain several mediators, belonging to a family of proinflammatory cytokines named chemokines, that are stored in the organelles. Release and accumulation of these chemokines during storage of platelet concentrates (PCs) might be responsible for nonhemolytic transfusion reactions. STUDY DESIGN AND METHODS: Analysis was done of pH and the levels of platelet factor 4, beta-thromboglobulin, interleukin 8, RANTES, macrophage-inflammatory protein-1 alpha, lactate dehydrogenase, and serotonin in the supernatant of stored PCs on Days 1, 3, 5, and 8. PCs were prepared by apheresis or from pools of four buffy coats. Buffy coat PCs were filtered before storage. RESULTS: Nonfiltered apheresis PCs, which had a higher white cell contamination (p < 0.01), contained significantly more platelets than did buffy coat PCs (p = 0.02). The pH decreased significantly in apheresis PCs (p = 0.01), whereas there was a significant increase in lactate dehydrogenase (p < 0.001). In buffy coat PCs, pH remained stable and lactate dehydrogenase increased moderately. Concentrations of platelet factor 4 and beta-thromboglobulin increased steadily in both preparations over the storage period (p < 0.001). Macrophage- inflammatory protein-1 alpha was hardly detectable in the supernatant of both PCs, while RANTES levels increased significantly with storage time (p < 0.001). Interleukin 8 was not found in the supernatant of any PCs, with the exception of one apheresis PC with high white cell contamination (> 10(9)/ L). Serotonin levels were higher in apheresis PCs (p = 0.01), but the levels did not correlate with storage time. CONCLUSION: Platelet factor 4, beta-thromboglobulin, and RANTES were released from platelets during storage and accumulated over time in the PCs. These chemokines might play a causative role in nonhemolytic transfusion reactions because of their inflammatory potential, but the clinical effects of the transfusion of PCs with high chemokine contents remain to be investigated.     

Preparation and storage of white blood cell-reduced split apheresis platelet concentrates for pediatric use

BACKGROUND: White blood cell (WBC) reduction and bacterial screening induce unacceptable product loss when platelet (PLT) concentrates (PCs) for pediatric transfusion are prepared from whole blood. The aim was to investigate PCs, WBC reduced and bacterially screened, from single-donor apheresis procedures, divided in 3 or 4 pediatric units and stored up to 5 days. STUDY DESIGN AND METHODS: PCs were collected with an apheresis machine and WBC reduced by in-process filtration. The PCs were sampled for bacterial screening and subsequently divided in 70-mL products. Initially, storage characteristics of split units in 400-mL polyvinylchloride (PVC) bags with 17 split PCs originating from five apheresis donations were studied. When a 600-mL container made of the more gas-permeable polyolefin became available, a paired comparison was performed with 9 split PCs from nine donations and with a higher-yield PLT collection procedure. RESULTS: Split PCs contained 69 x 10(9) +/- 14 x 10(9) PLTs in 69 +/- 1 mL of plasma, and storage in the PVC containers gave a pH value of 6.86 +/- 0.10 on Day 6 (mean +/- SD, n = 17). When comparing the containers, the PVC bag contained 98 x 10(9) +/- 15 x 10(9) PLTs in 72 +/- 4 mL versus 102 x 10(9) +/- 18 x 10(9) PLTs in 74 +/- 8 mL for the polyolefin bag (n = 9, not significant). This gave pH values on Day 6 of 6.12 +/- 0.50 in the PVC container, whereas pH remained acceptable in the polyolefin container: 6.85 +/- 0.10 on Day 6 (p < 0.01). CONCLUSION: PCs for pediatric use from split single-donor apheresis concentrates, WBC reduced and bacterially screened, can be stored for up to 5 days in a 600-mL polyolefin container with maintenance of good in vitro storage variables.     

白细胞过滤对冰冻保存浓缩血小板制剂细胞因子和血小板功能的影响

目的探讨白细胞过滤对浓缩血小板悬液(platelet concentrate suspend,PCs)在冰冻保存前后的一些细胞因子及血小板体外功能的影响。方法取25份浓缩血小板样品(40ml/份),将每份样品再等分为4份,其中2份白细胞过滤(过滤组),另2份不过滤(未过滤组);将过滤组和未过滤组的分别常规保存和冰冻保存。常规保存0、1、3d和冰冻保存3个月解冻后0、1、3d的所有样品均采用ELISA法测定IL-2、IL-6、INFγ-、TNF-α、IL-10的含量;对冰冻保存复溶后当天的样品和新鲜样品分别作血小板聚集功能、粘附功能及血小板第Ⅲ因子活性检测;采用配对t检验作统计分析。结果未过滤组PCs冰冻保存后复溶0d与常规保存0d的PCs细胞因子的水平无明显升高,两种条件保存后1、3d细胞因子的水平均显著升高;过滤组PCs在冰冻保存和常规保存时细胞因子均无显著变化。过滤组和未过滤组PCs经冰冻保存后的血小板体外功能活性均无明显变化。结论PCs中的细胞因子在常规保存及冰冻保存复溶后随时间延长呈显著性增加趋势,白细胞过滤可明显减轻这种效应。白细胞过滤对冰冻保存的血小板体外功能活性无明显影响。     

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.     

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.     

Interleukin 1 beta and tumor necrosis factor levels in stored platelet concentrates and the association with gene polymorphisms

BACKGROUND: Cytokines (IL-1beta and TNF) generated by WBCs during storage of PLT concentrates have been associated with febrile nonhemolytic transfusion reactions. STUDY DESIGN AND METHODS: This study was undertaken to investigate whether there is an association between the polymorphisms of IL1B -511C/T and +3953C/T, IL1RN intron 2 VNTR and TNFA-308G/A genes and the increase of cytokines during the storage of PLT concentrates produced by plasma-rich PLTS (PRP-PC) or apheresis PLTs. RESULTS: Thirty PRP-PCs were studied and a progressive increase of IL-1beta and TNF during storage was revealed. IL1-beta and TNF levels were inversely correlated with the content of PLTs in PRP-PCs detected on Day 3 (p = 0.004) and Day 5 (p = 0.019), but not on Day 7. There was association of IL1B-511T polymorphism and IL-1beta levels (Day 5, p = 0.063, only tendency and on Day 7, p = 0.038, significant). There was no association of the other polymorphisms (IL1B+3953C/T, IL1RN intron 2 and TNFA-308G/A) with their respective cytokines. CONCLUSION: The great variation of cytokine levels in the plasma of PLT concentrates (PCs) during storage may also be caused by cytokine gene polymorphisms, as well as WBC contamination, material that the bags are made of, and storage time, as previously described.     

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.     

Elimination of cytokine production in stored platelet concentrate aliquots by photochemical treatment with psoralen plus ultraviolet A light

BACKGROUND: Cytokines generated in platelet concentrates (PCs) during storage have been implicated as possible mediators of febrile nonhemolytic transfusion reactions. Two potential methods of white cell inactivation were compared for their ability to reduce cytokine synthesis in pooled random-donor PC aliquots: treatment with gamma-radiation and photochemical treatment (PCT) using psoralens and ultraviolet A light. STUDY DESIGN AND METHODS: ABO-matched PC aliquots were pooled and divided into separate aliquots. Aliquots (20 mL) were taken from each pool to serve as an untreated control and to undergo gamma-radiation. Aliquots were treated by using either gamma-radiation (2500 or 5000 cGy) or virucidal PCT. PCT with the psoralens 8-methoxypsoralen (8-MOP), aminomethyltrimethyl psoralen (AMT), and S-59 was investigated. PC aliquots were stored for 7 days and analyzed for levels of interleukin 8 by use of an enzyme-linked immunosorbent assay. Levels of DNA adduct formation were determined by using 3H-labeled psoralens. RESULTS: Levels of interleukin 8 in the untreated random-donor PC aliquots increased with increasing white cell counts, but they were not affected by pooling. The untreated control aliquots and the aliquots treated with gamma-radiation had significant increases in levels of interleukin 8 after 5 to 7 days of storage (p<0.05). PCT with S-59 resulted in a significant reduction in cytokine synthesis (p<0.05). Day 5 to 7 levels of interleukin 8 did not differ significantly from Day 0 levels. Inhibition of interleukin 8 production by PCT increased with increasing levels of DNA modification (S-59 > AMT > 8-MOP). CONCLUSION: PCT that utilizes S-59 has been developed to inactivate potential viral and bacterial pathogens in PC aliquots while maintaining in vitro platelet function. These data demonstrate that PCT of aliquots of pooled PC aliquots before storage also prevents white cell cytokine synthesis during storage. PCT may therefore offer the potential for reducing cytokine-associated febrile nonhemolytic transfusion reactions.     

Improved removal of white cells with minimal platelet loss by filtration of apheresis platelets during collection

BACKGROUND: Filtration of apheresis platelets to remove white cells (WBCs) requires operator intervention after the collection procedure (postcollection filtration), which may cause variable and unsatisfactory filter performance (WBC removal and platelet loss). The MCS+ LN9000 apheresis system filters platelets through a WBC-reduction filter during each collection cycle (continuous filtration) at a flow rate of 15 to 25 mL per minute. Apheresis platelets obtained by continuous filtration were evaluated in terms of platelet loss, WBC removal, and platelet storage properties and then were compared to unfiltered apheresis platelets and to apheresis platelets that underwent postcollection filtration. Two WBC-reduction filters were tested (LRF6 and LRFXL). STUDY DESIGN AND METHODS: In 70 apheresis platelets, postcollection filtration was performed by using the LRF6 at flow rates of 80 mL per minute (n = 30) and 50 mL per minute (n = 30) and the LRFXL at 50 mL per minute (n = 10). One hundred fifty-eight apheresis platelets underwent continuous filtration through the LRF6 (n = 58) or the LRFXL (n = 100). Unfiltered apheresis platelets (controls) (n = 30) were obtained by the same collection protocol. RESULTS: Estimated platelet loss with continuous filtration was 7 percent for the LRFXL and 3 percent for the LRF6. A reduction in the filtration flow rate from 80 to 50 mL per minute with postcollection filtration through the LRF6 resulted in markedly lower WBC levels, with 10 percent versus 57 percent of the apheresis platelets having WBC counts <1 × 10⁵, respectively. Additional improvements in WBC removal were found with continuous filtration; 85 percent of the apheresis platelets filtered with the LRF6 and 100 percent of the apheresis platelets filtered with the LRFXL had WBC counts <1 × 10⁵. CONCLUSIONS: Continuous or postcollection filtration of freshly collected apheresis platelets resulted in minimal platelet loss. Better WBC removal from apheresis platelets was obtained with continuous filtration than with postcollection filtration, likely because of the slower flow rate. Platelet storage quality was not affected by filtration.