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.     

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.     

Generation of kinins during preparation and storage of whole blood-derived platelet concentrates

BACKGROUND: Leukoreduction of platelet (PLT) concentrates (PCs) may be associated with hypotension in recipients, and a role for bradykinin (BK)-related peptides has been proposed for this side effect. STUDY DESIGN AND METHODS: The concentration of BK and one of its vasoactive metabolites, des-arginine(9)-BK (des-Arg(9)-BK), was measured in a large number of PCs as a function of leukoreduction and storage duration with specific enzyme immunoassays and complementary techniques. RESULTS: On Day 0 of storage, kinins were detected in leukoreduced and unfiltered PCs at a concentration lower than 100 pg per mL. During storage, both kinin levels peaked on Day 5 of storage, with a concentration higher than 1 ng per mL in 22 percent of PCs whether filtered on Day 0 or not. Physicochemical and pharmacologic characterizations of immunoreactive kinins confirm their nature. In vitro activation of the contact system of the corresponding PLT-poor plasma showed that a high kinin concentration on Day 5 of the storage corresponded with a low kinin-forming capacity of plasma. On Day 7, BK was no longer elevated presumably due to its degradation and the depletion of kinin-forming capacity of the plasma in stored PCs. The activities of metallopeptidases that metabolize BK-related peptides in plasma from PCs were at levels similar to those recorded in the plasma of a normal reference population and were unaffected by storage. CONCLUSION: Storage of PCs contributes to the hydrolysis of high-molecular-weight kininogen and generation of pharmacologically relevant BK levels that might pose a hazard in susceptible patients.     

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.     

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.     

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.     

Febrile reactions to platelet transfusion: the effect of increased interleukin 6 levels in concentrates prepared by the platelet-rich plasma method

Background: A relation between febrile reactions to platelet transfusion and high cytokine levels in platelet concentrates (PCs) was found previously. The levels of cytokines such as interleukin (IL)-6 are related to the while cell content of the PC during storage. Therefore, early removal of white cells should prevent reactions. Study Design and Methods: This prospective study was set up to compare methods for the preparation of random PCs, the platelet-rich plasma method (PRP-PCs), which results in a high white cell content, and the buffy coat method (BC-PCs), which results in a low white cell content, with regard to the frequency and severity of reactions to platelet transfusion and the IL-6 level of the PC. IL-6 was chosen because it is the major mediator of the acute-phase response. White cells were reduced in all PCs before transfusion. Results: Platelet transfusions (n = 584) in 64 patients were studied. An overall reaction frequency of 7.2 percent was observed. Transfusion reactions were seen predominantly in patients who received PRP-PCs (PRP-PCs: 9.3% vs. BC-PCs: 2.7%, p = 0.007). Allergic reactions were limited to transfusions of PRP-PCs. The following PRP-PC characteristics were significantly correlated with febrile transfusion reactions: IL-6 level (p < 0.0001), initial white cell count (p = 0.001), and storage time (p = 0.02). In this group, reactions were less frequent in patients receiving pretransfusion medication (p < 0.001). In the PRP-PC group, IL-6 content (p = 0.01) and initial white cell count (p = 0.04) were also significantly correlated with allergic reactions, which indicated that these or associated factors might have an effect on the outcome of this type of reaction. Conclusion: Febrile reactions are highly correlated with IL-6 levels in PCs. The low white cell content of BC-PCs is associated with undetectable IL-6 levels and a reduced frequency of febrile as well as allergic reactions in recipients. The BC method is the preferable one for the production of random-donor PCs.     

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.     

Comparison of bacteria growth in single and pooled platelet concentrates after deliberate inoculation and storage

BACKGROUND: The ability to store pools of platelet concentrates (PCs) for extended periods would provide logistical flexibility. However, reports of severe adverse reactions due to the transfusion of contaminated PCs led to an examination of whether the total bacteria levels after storage of pools containing a deliberately inoculated platelet unit would be significantly different than the levels in paired unpooled concentrates. STUDY DESIGN AND METHODS: A single PC was deliberately inoculated on Day 0 with one of three bacterial species (0.1–8.0 colony-forming units/mL). On Day 1, the deliberately inoculated PC was divided into three equal parts and either 1) pooled with 5 half-volume, ABO- and Rh-identical PCs; 2) similarly pooled and white cell reduced; or 3) kept as a control. Sterile connections were used during pooling; modified storage containers were used to ensure the correct surface-to-volume ratio of the single unit. RESULTS: Between Day 2 and Day 5 of storage, in 26 of 36 paired samples, nonfiltered pools containing Escherichia coli had greater total numbers of bacteria than did the paired single PCs. Day 2 pools had total bacteria levels approximately five times higher (colony-forming units/mL × container volume) than those in single units (p < 0.05). There was rapid growth of Staphylococcus aureus by Day 2 in pooled and unpooled PCs; by Day 3, total bacteria levels were approximately five times higher in pools than in single units (p < 0.05). Between Days 3 and 5 of storage, in 23 of 27 paired samples, nonfiltered pools containing S. aureus had greater total bacteria levels than the single PCs. By Day 5, 15 of 16 non-white-cell reduced pools had total levels of Staphylococcus epidermidis bacteria approximately five times those in the paired single PCs. Greater total bacteria levels in pooled units than in single units generally occurred when bacteria in pools reached the stationary phase of growth (when bacteria concentration became constant), and they were well correlated with the sixfold volume of pooled units. White cell reduction did not substantially affect the time required to attain stationary phase. CONCLUSION: The potential during storage for greater total bacteria levels in pools than in single PCs is a consequence of the greater volume of the pool.     

The effect of storage on the expression of platelet membrane phosphatidylserine and the subsequent impacton the coagulant function of stored platelets

BACKGROUND: Platelet concentrates (PCs) derived from whole blood and stored under standard blood bank conditions undergo changes that are referred to as the platelet storage lesion. This study assesses the effect of PC preparation and storage on the distribution of phosphatidylserine (PS) in the platelet membrane and the effect that this distribution may have on the thrombogenic potential of stored PCs. STUDY DESIGN AND METHODS: Fresh platelets and PCs donated by healthy donors were obtained. PCs derived from platelet-rich plasma were studied on Day 1, Day 3, and Day 6 of storage under blood bank conditions. RESULTS: Platelet aggregation after exposure to the platelet agonists ADP and epinephrine singly declined progressively, but, when ADP and epinephrine in combination and collagen and thrombin in combination were used as agonists, the decline in platelet aggregation was less marked. PS expression as measured by Annexin V binding (mean and SD) was 2.02 +/- 0.93 percent in fresh platelet samples and increased to 5.39 +/- 4.2 percent on Day 1, 22. 1 +/- 7.1 percent on Day 3, and 39.5 +/- 12.1 percent on Day 6. Platelet prothrombinase activity (mean +/- SD) as measured by thrombin generation increased from 1.49 +/- 0.7 micro per mL in fresh platelet samples to 3.68 +/- 1.1 micro per mL in Day 1 platelets (p<0.001), 5.15 +/- 2.5 micro per mL in Day 3 platelets (p<0.001), and 4.65 +/- 2.48 micro per mL in Day 6 platelets (p<0. 001). CONCLUSION: These results show that PS expression increases after preparation of PCs from platelet-rich plasma and rises progressively during platelet storage under blood bank conditions. Furthermore, the greater PS expression is associated with increased platelet- dependent thrombin-generating capacity.     

Role of circulating cytokines and chemokines in exertional heatstroke

OBJECTIVE: The interplay between inflammatory and anti-inflammatory cytokines, as well as chemokines, has not been well explored in exertional heatstroke. DESIGN: Prospective, observational study. PATIENTS: Seventeen military recruits who developed exertional heatstroke and 17 exertional controls who did not develop exertional heatstroke during the same training exercises. SETTING: University teaching hospital. MEASUREMENTS AND MAIN RESULTS: The severity of exertional heatstroke was evaluated using a Simplified Acute Physiology Score. Plasma cytokines and chemokines were determined using enzyme-linked immunosorbent assay kits. Body temperatures were 41.2 +/- 1.2 degrees C and 37.6 +/- 0.8 degrees C in exertional heatstroke and exertional controls, respectively. Significantly, plasma cytokines including interleukin (IL)-1beta (3.1 +/- 1.6 vs. 1.2 +/- 0.8 pg/mL; p <.05), tumor necrosis factor alpha (4.9 +/- 4.1 vs. 1.2 +/- 2.4 pg/mL; p <.05), IL-6 (15.8 +/- 3.2 vs. 1.2 +/- 1.2 pg/mL; p <.01), interferon gamma (7.3 +/- 4.9 vs. 2.4 +/- 4.1 pg/mL; p <.01), IL-2 receptor (1568 +/- 643 vs. 610 +/- 214 pg/mL; p <.01), IL-4 (2.5 +/- 1.2 vs. 1.2 +/- 0.8 pg/mL; p <.05), and IL-10 (12.9 +/- 9.4 vs. 2.5 +/- 4.9 pg/mL; p <.01) and serum chemokines IL-8 (84.2 +/- 79.9 vs. 10.4 +/- 3.2 pg/mL; p <.01), monocyte chemoattractant protein 1 (959 +/- 589 vs. 158 +/- 217 pg/mL; p <.01), and RANTES (12464 +/- 10505 vs. 5570 +/- 2894 pg/mL; p <.01) were elevated in exertional heatstroke compared with exertional controls. Among cytokines, IL-6, interferon gamma, and IL-2 receptor were positively correlated with Simplified Acute Physiology Score (r =.573, p <.01; r =.625, p <.01; and r =.56, p <.05, respectively). Among chemokines, only serum monocyte chemoattractant protein 1 was positively correlated with Simplified Acute Physiology Score (r =.78, p <.001). There was no correlation between either cytokines or chemokines and body temperature. CONCLUSIONS: Proinflammatory cytokines IL-1beta, tumor necrosis factor alpha, IL-6; T helper 1 cytokines INF-gamma and IL-2 receptor; and chemokines IL-8, monocyte chemoattractant protein 1, and RANTES are increased in patients with exertional heatstroke. T helper 2 cytokines may play a role as anti-inflammatory cytokines. IL-6, interferon gamma, IL-2 receptor, and monocyte chemoattractant protein 1 may serve as prognostic indicators of disease severity in exertional heatstroke.     

Febrile and allergic transfusion reactions after the transfusion of white cell-poor platelet preparations

BACKGROUND: Nonhemolytic transfusion reactions (NHTRs) frequently occur after platelet transfusions. White cell (WBC)-derived inflammatory cytokines can cause these reactions, but they are rarely found in WBC-poor platelet preparations. Transfusion reactions were investigated with regard to the residual WBC content in the stored platelet concentrate in two consecutive study periods. STUDY DESIGN AND METHODS: In the first study period, platelet concentrates were WBC-reduced by bedside filtration. In the second period, all platelet concentrates were filtered before storage. Recipients who experienced transfusion reactions were examined with regard to their main clinical symptoms during and after transfusion. In the supernatant of the involved platelet concentrates, concentrations of interleukin (IL)-1beta, IL-6, IL-8, tumor necrosis factor (TNF)alpha, macrophage inflammatory protein 1alpha, and RANTES were analyzed. RESULTS: The incidence of transfusion reactions remained steady when the transfusion regimen was changed from bedside filtration to prestorage WBC filtration (1.63% and 1.56%; p = 0.84). In both periods, NHTRs were predominantly of allergic origin. Inflammatory mediators IL-1beta, IL-6, IL-8, and TNFalpha were detectable in only a minority of platelet components involved in NHTRs. Platelet concentrates involved in allergic reactions contained high concentrations of RANTES (668 +/- 223 ng/mL). CONCLUSIONS: Prestorage WBC filtration did not reduce the incidence of these reactions, and inflammatory cytokines were of minor relevance. The proinflammatory platelet-derived chemokine RANTES, which accumulates even in WBC-reduced platelet concentrates, was associated with allergic transfusion reactions. Platelet-derived mediators may be a key to understanding NHTRs.     

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.     

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     

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.     

Improvement of platelet storage conditions by using new polyolefin containers

BACKGROUND : Storage of pooled platelet concentrates (PCs) with yields above 3.0 × 10¹¹ platelets per unit in a 1-L PL-732 polyolefin container for 5 days often results in a drop in pH to below 6.0. Recently, new oxygen-permeable platelet containers (1-L PL-2410, 1-L and 1.5-L Compoflex) have been developed. The maximal platelet storage capacities of the new containers and the PL-732 were compared. STUDY DESIGN AND METHODS : Large platelet pools (n = 27) with platelet concentrations between 1.2 and 1.4 × 10¹¹ per L were made from 3 to 5 PCs prepared from buffy coats. The pools were divided in equal volumes among the PL-732 and the three new platelet containers. Platelet counts in the PCs ranged from 1.0 to 5.0 × 10¹¹ per unit. All PCs were stored on a flatbed shaker at 22 ± 2°C and evaluated on Days 1, 3, 5, and 7 by measuring platelet count, pH, pO₂, pCO₂, HCO₃-, glucose, lactate, platelet swirling, and soluble p-selectin. RESULTS : Day 7 storage of PCs (n = 6) with yields between 3.0 and 4.0 × 10¹¹ platelets in PL-732 showed mean ± SD pH values of 5.93 ± 0.05 and lactate values of 32.3 ± 7.9 mmol per L; in 4 of these 6 PCs, pH was below 6.0. In contrast, storage of these PCs in 1-L PL-2410 and 1.5-L Compoflex containers and of 2 of these 6 PCs in 1-L Compoflex containers showed pH values above 6.8. Lactate values were 15.5 ± 1.3, 15.3 ± 1.8, and 19.5 ± 4.7 mmol per L, respectively (p < 0.001 vs. PL-732). The platelet storage capacity of the new containers with platelet yields between 4.0 and 5.0 × 10¹¹ per unit (n = 6) was evaluated. Day 7 storage of these PCs in the 1.5-L Compoflex showed an average pH value of 6.74 ± 0.20; in 2 of 6 PCs, pH was below 6.8. The average pH value in the PL-2410 was 6.38 ± 0.31, and in all PCs, pH was below 6.8. Average lactate values were 17.8 ± 5.7 and 25.8 ± 5.6 mmol per L (p < 0.05), respectively. Soluble p-selectin values on Day 7 of storage increased approximately twofold in all PCs. CONCLUSION : The new oxygen-permeable containers showed platelet quality comparable to that with the PL-732 and for longer storage periods and at higher platelet counts.     

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.