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.     

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, 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.     

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.     

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.     

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.     

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 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.     

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.     

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.     

Automated blood component collection with the MCS 3p cell separator: evaluation of three protocols for buffy coat-poor and white cell- reduced packed red cells and plasma

BACKGROUND: Automated collection of blood components with a cell separator (MCS 3p, Haemonetics), was performed according to three protocols. STUDY DESIGN AND METHODS: The first protocol provided 2 units of fresh-frozen plasma (FFP); and one buffy coat-poor red cell (RBC) concentrate in additive solution. The second protocol included an additional in-line filtration of the RBC in a closed system after storage at 4 degrees C for 24 hours. In the third protocol, an additional platelet concentrate (PC) was recovered from the buffy coat. Cell counts and biochemical characterization of the RBCs (n=20 each) were determined on Days 0, 1, 14, 28, and 49. RESULTS: The RBC volume was 336 +/? 9 mL (first protocol), 337 +/? 7 mL (second protocol) and 293 +/? 12 mL (third protocol) with a hematocrit of 59 +/? 2, 53 +/? 3, and 61 +/? 5, percent respectively. On Day 49, hemolysis was 0.24 +/? 0.1 percent (first protocol), 0.33 +/? 0.32 percent (second protocol), and 0.38 +/? 0.1 percent (third protocol). The filtered RBC concentrate met the international standards for white cell-reduced RBCs. Filtration resulted in a clinically irrelevant increase of hemolysis. The in vitro RBC values (lactate dehydrogenase, 2-hydroxybutyrate dehydrogenase, hemolysis, potassium, 2,3 DPG, ATP) were at least equal to those in RBCs collected by conventional whole-blood donation. There is a trend toward extended preservation of 2,3 DPG in RBCs collected by apheresis. Two units of FFP could be collected with each donation (first protocol: 420 +/? 55 mL, 5.4 +/? 7 WBCs/microL, 6.5 +/? 5 × 10(3) platelets/microL; second protocol: 440 +/? 33 mL, 3 +/? 5.2 WBCs/microL, 32 +/? 12 × 10(3) platelets/microL; third protocol: 398 +/? 32 mL, 5 +/? 12 WBCs/microL; 3.4 +/? 3.5 × 10(3) platelets/microL). PCs prepared from the buffy coat collected by the third protocol contained 90 +/? 30 × 10(9) platelets in 88 +/? 14 mL of plasma. In vitro test results in these PCs were superior to those in PCs collected by conventional whole-blood donation. The procedure was well tolerated by all donors. No adverse reactions appeared. CONCLUSION: Erythroplasmapheresis with the MCS 3p cell separator is a useful alternative to conventional whole-blood donation and separation.     

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.     

In vitro safety profile of G-CSF-mobilized whole blood after storage for 7 days in an infusable-grade L15 medium

BACKGROUND: G-CSF-mobilized whole blood (WB) is a cost-reducing and simple alternative for peripheral blood progenitor cell transplantation. Recently, it was demonstrated that mobilized WB supplemented with Leibovitz's L15 medium permitted prolonged preservation of clonogenic cells at ambient temperature. In this study, an infusable-grade L15 medium (IG-L15) was developed, and the safety profile of mobilized WB after 7 days of storage was investigated. STUDY DESIGN AND METHODS: IG-L15 was manufactured in a closed system under good manufacturing practice conditions. Proinflammatory cytokine levels and hemolysis in mobilized WB were determined after 7 days of storage in different containers and were compared with current clinical mobilized WB values after 1 to 3 days of storage at 4 degrees C. RESULTS: IG-L15 and L15 maintained clonogenic cells equally. In the samples of mobilized WB that were returned to the patient, cytokine levels were not elevated in comparison with freshly collected mobilized WB. By using IG-L15 in polystyrene-coated cell culture bags, median (range) levels of 9.4 (2.2-69.8) pg per mL (IL-1beta), 31.6 (6.1-146.5) pg per mL (TNF-alpha), 76.9 (15.5-934.9) pg per mL (IL-6), and 7195 (104-205,600) pg per mL (IL-8) were found after 7 days. Higher cytokine levels were found with L15 and different containers. He- molysis was less than 0.5 g per dL in all cases. CONCLUSION: The storage of mobilized WB for 7 days in IG-L15 at ambient temperature is possible with adequate preservation of clonogenic cells, but cytokine levels may require plasma removal before return.     

Filtration through a polyester white cell-reduction filter of plasma- poor platelet concentrates prepared with an acetate-containing additive solution

It is of practical importance to known whether the adsorption of platelets and contaminating white cells (WBCs) by the WBC-reduction filter is altered when platelet concentrates (PCs) are prepared in a plasma-poor condition with an acetate-containing additive solution (Seto sol). Plasma-poor PCs with 11-percent residual plasma were prepared from apheresis platelet-rich plasma by using a sterile docking device with steam-sterilized Seto sol. Seto sol contains 115 mM (115 mmol/L) NaCl, 4 mM (4 mmol/L) KCl, 3 mM (3 mmol/L) MgCl2, 10 mM (10 mmol/L) Na3PO4, 15 mM (15 mmol/L) acetate, 3 mM (3 mmol/L) Na3 citrate, and 10 mM (10 mmol/L) glucose (pH 7.1). The solution was steam- sterilized under nitrogen gas. On Days 1 and 5, pooled Seto sol PCs (2.4 × 10(11) platelets) were filtered with a polyester filter at a flow rate of 10 mL per minute. The WBC-removal rate was over 99.9 percent with a platelet recovery of 88 percent following Day 1 filtration. These values were very similar to those of plasma PCs, and 84-percent recovery was achieved following Day 5 filtration. However, when 1 unit of Seto sol PCs with half the number of platelets was filtered with the polyester filter, platelet recovery was about 16 to 17 percent less than that of plasma PCs. Platelet quality was maintained if pooled Seto sol PCs were filtered on Day 1 and stored for over 4 days. Filtration did not alter platelet function in 1-day-old or 5-day-old Seto sol PCs.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

A comparison of prestorage WBC-reduced whole-blood-derived platelets and bedside-filtered whole-blood-derived platelets in autologous progenitor cell transplant

BACKGROUND: Prestorage WBC-reduced platelet concentrates (PCs) can be manufactured from platelet-rich plasma (PRP) by in-line filtration of PRP. There are few published data on the clinical use of these products, as compared to bedside-filtered pools of standard PCs (S-PCs) manufactured from PRP. STUDY DESIGN AND METHODS: A prospective, randomized trial was conducted in autologous progenitor cell transplant patients requiring platelet transfusions with each patient as his or her own control who was given a pool of 5 units of WBC-reduced PCs and a pool of 6 units of S-PCs within a 3-hour period. The pools were characterized before transfusion for platelet and WBC content, P-selectin expression, and IL-8. The patients were monitored with platelet counts and vital signs and observed for reactions. Data were analyzed using Mann-Whitney U tests. RESULTS: Thirty-three transfusions were administered to 13 patients. Median platelet content in the WBC-reduced PC pools was lower than that in the S-PC pools (3.3 vs. 4.0 x 10(11), p<0.01). Median WBC content was 4 to 5 log less in the WBC-reduced PC pools (2.5 x 10(4) vs. 4.6 x 10(8), p<0.01). Median IL-8 levels (pg/mL) were lower in the WBC-reduced PC pools (2 vs. 36, p<0.01). No differences were observed in CCI, but the median absolute increase after transfusion of the S-PC pools was higher (25 vs. 19 x 10(9)/L, p<0.01), which reflected the larger size of the S-PC pools. No overall differences in vital signs were recorded. Two reactions were observed, both in temporal association with the transfusion of pools of S-PCs. CONCLUSIONS: A pool consisting of 5 units of WBC-reduced PCs gave a median platelet increment of 19 x 10(9) per L in these thrombocytopenic patients and has a median WBC content 1 to 2 log below the accepted threshold for primary alloimmunization or CMV transmission.     

Preparation of white cell-reduced platelet concentrates from whole blood during component preparation

This article introduces a new method of component preparation that is capable of producing white cell (WBC)-reduced platelet concentrates (PCs) from whole blood. Whole blood is separated into packed red cells (RBCs) and platelet-rich plasma (PRP) by centrifugation, and the PRP is expressed through a newly designed WBC removal filter into the platelet storage bag. The filtered PRP is then centrifuged and yields WBC-reduced PCs and plasma for freezing as fresh-frozen plasma (FFP). The method uses standard triple-pack blood bags and centrifugation protocols. Fifteen WBC-reduced PCs prepared with this technique had an average volume of 56.7 mL, an average Day 5 platelet content of 8.6 x 10(10) per unit, and an average Day 5 WBC content of 0.83 +/- 0.7 x 10(4) per unit (0.14 WBCs/microL). This represents WBC removal equal to at least 99.9 percent (3 log10) of the WBCs found in standard PCs prepared in our laboratory by an identical centrifugation protocol. Paired studies documented a 4.5-percent platelet loss by filtration. Filtration had no effect on the plasma prepared for FFP as measured by prothrombin time; activated partial thromboplastin time; factors I, V, VIII:C, and VIII:von Willebrand factor; antithrombin-III; albumin; globulin; or total protein. This method holds promise as a simple and highly effective technique for the production of WBC-reduced PCs by filtration during component preparation.     

Characterization of reactions after transfusion of cellular blood components that are white cell reduced before storage

Background: During the storage of cellular components before transfusion, cytokines that may mediate transfusion reactions are released from white cells (WBCs). Adverse effects of transfused cellular blood components therefore depend not only on the number of residual WBCs in blood components, but also on the timing of WBC reduction. Study Design and Methods: Febrile nonhemolytic transfusion reactions (FNHTRs), allergic reactions, and other reactions were characterized in recipients of 4728 units of red cells (RBCs) and 3405 bags of single-donor apheresis platelets (SDAPs), all of which underwent prestorage WBC reduction. To delineate the impact of prestorage versus poststorage WBC reduction of RBCs on transfusion reactions, these results were compared with reactions occurring after the transfusion to similar recipients of 6447 bags of RBCs that underwent poststorage WBC reduction by bedside filtration and 5197 units of SDAPs that underwent prestorage WBC reduction. The levels of interleukin (IL) 1 beta, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-alpha) were measured in a subset of 20 implicated cellular blood components at the time of transfusion reactions and correlated with the duration of storage before transfusion. Results: The incidence of reactions was greater after transfusions of SDAPs (5.49%) than of RBCs (1.63%). The incidence of FNHTRs after transfusion of RBCs that were WBC reduced before storage (1.1%) was significantly lower (p = 0.0045) than that after transfusion of RBCs that were WBC reduced after storage (2.15%). Although allergic reactions to RBCs that were WBC reduced before storage were also less common (0.41%) than those to RBCs that were WBC reduced after storage (0.51%), the difference was not significant (p = 0.067). At the time of reactions to RBCs and SDAPs that were reduced before storage, the level of IL-6 was negatively correlated (r = -0.54, p = 0.014) with the duration of storage before transfusion, and there was no correlation between the level of either IL-1 beta or IL-8 and the interval before transfusion. TNF-alpha was not detectable in any implicated component. Conclusion: FNHTRs, but not allergic reactions, were less common after transfusion of RBCs that were WBC reduced before storage than after the transfusion of those WBC reduced after storage at the bedside by filtration. The level of IL-6 in implicated cellular blood components that were WBC reduced before storage was inversely correlated with the length of storage before transfusion. Further studies are needed to determine whether the transfusion of cellular blood components that were WBC reduced before storage can both diminish the incidence of adverse reactions and improve outcome.