Cytokines as quality indicators of leucoreduced red cell concentrates.

Different types of filters are currently used for leucodepletion of red cell concentrates. These filters meet the specification for leucoreduction (<5 x 10(6) leucocytes/ATD) but the quality of the final product may differ depending on the performance of the filters for effective removal of both leucocytes, platelets and possibly cytokines which are associated with transfusion reactions. We measured the levels of three representative cytokines: IL-8, RANTES and TGF-beta1 in red cell concentrates prior to and subsequent to the filtration procedure on day 1 and after a storage period of 35 days. Low levels of IL-8 (10-24 pg/ml) in the control unfiltered concentrates on day 1 which increased by approximately twofold on storage. Filtration reduced the levels of IL-8 on day 1 and day 35, in filtered concentrates in comparison with their control unfiltered counterparts. Leucoreduced concentrates produced by three different filters showed similar IL-8 levels on day 1 and day 35. However, concentrates prepared using another type of process showed a twofold increase in IL-8 levels on storage in comparison with day 1. None of the concentrates tested contained any detectable RANTES and TGF-beta1 suggesting a minimal platelet content. These results indicate that a combination of IL-8, RANTES and TGF-beta1 are useful quality indicators for validation of leucoreduced red cell preparations.     

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.     

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.     

Low cytokine contamination in buffy coat-derived platelet concentrates without filtration

BACKGROUND: Cytokines (interleukin [IL]-1 beta, IL-6, and tumor necrosis factor [TNF]) generated by white cells during the storage of platelet concentrates can cause febrile nonhemolytic transfusion reactions. The high rate of febrile reactions reported in other studies was not observed in the patients in the authors' center. This discrepancy prompted the determination of cytokine levels in buffy coat- derived platelet concentrates. STUDY DESIGN AND METHODS: Platelet concentrates were produced from buffy coats by a standard large-scale production process. Buffy coats were separated from the red cell and plasma components, and then platelets were recovered from the buffy coats by a soft-spin procedure. Levels of cytokines (IL-1 beta, IL-6, IL-8, and TNF) were determined with commercial enzyme-linked immunosorbent assays. RESULTS: In platelet concentrates produced by the buffy coat method, IL-1 beta, IL-6, IL-8, and TNF were observed at or below the detection limit of current enzyme-linked immunosorbent assays after 5 days' storage at 22 +/− 2 degrees C. Therefore, prestorage filtration had no measurable effect on cytokine levels. In controls, IL- 1 beta, IL-6, IL-8, and TNF were quantitatively detected after exogenous addition of recombinant cytokines or exposure to lipopolysaccharide. CONCLUSION: Platelet concentrates prepared from buffy coats may be virtually free of cytokines (IL-1 beta, IL-6, IL-8, and TNF) during 5 days of storage. Filtration is not required to reduce the recipient's cytokine exposure via such platelet concentrates.     

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)-1β, IL-6, IL-8, tumor necrosis factor (TNF)α, macrophage inflammatory protein 1α, 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-1β, IL-6, IL-8, and TNFα 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.     

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。     

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.     

Release of potential immunomodulatory factors during platelet storage

BACKGROUND: Blood platelets (PLTs) link the processes of hemostasis and inflammation. Recent studies have demonstrated that PLTs promote immunity and inflammation mainly by means of the CD40/CD40L pathway. Our objective was to describe the accumulation of cytokines in PLT concentrates during storage. STUDY DESIGN AND METHODS: Pools of PLT concentrates were prepared, separated from plasma, and resuspended in clinical-grade storage medium; samples were taken on Days 0, 1, 2, 3, and 5 for analysis, without replacement (i.e., without soluble protein dilution). Interleukin (IL)-6, IL-8, PLT-derived growth factor (PDGF)-AA, soluble CD40 ligand (sCD40L), RANTES, and transforming growth factor-beta production were measured by specific enzyme-linked immunosorbent assays. RESULTS: Over time, the levels of RANTES, IL-8, and IL-6 were stable. In contrast, the levels of PDGF-AA and sCD40L increased. Ex vivo production of sCD40L was quantified at levels sufficient to induce B-cell effects based on previous studies of in vitro induced B-cell activation and differentiation by sCD40L. Cytokine and/or chemokine levels were generally higher in PLT concentrate supernatants and/or PLT lysates in comparison to PLT-free plasma, allowing the determination of which cytokine and/or chemokine was absorbed or secreted by transfusion-grade PLTs over time. CONCLUSION: Our data provide evidence that stored PLTs contain molecules with known immunomodulatory competence and secrete them differentially over time during storage for transfusion purposes.     

单采血小板储存样品中某些细胞因子含量的变化

本研究探明不同血细胞分离机所采集的单个供者血小板（single donor platelets，SDP）在储存期间细胞因子含量的变化。使用MCS^＋、Trima和Amicus3种血细胞分离机采集18份SDP，于血库标准条件下储存，于第1、3、5、7天取样检测储存期内白介素8（IL-8）、RANTES、CD154和肿瘤生长因子β1（TGF-β1）、血管内皮细胞生长因子（VEGF）等细胞因子含量的变化情况。结果显示：MCS^＋、Trima和Amicus机器采集的SDP，在储存期间随着时间的延长细胞因子IL-8、RANTES、CD154、TGF-β1及VEGF的含量逐渐增高，但MCS^＋机器采集SDP的IL-8的含量在保存期的增高水平，与Trima和Amicus法采集的SDP水平相比有显著性差异（P〉0．05）；而其余的细胞因子含量虽有增高，但无显著性差异．结论：单采血小板储存期间IL-8、RANTES、CD154、TGF-β1和VEGF等细胞因子的含量随保存时间的延长有升高的趋势；少白细胞的单采血小板中的IL-8表达相对较少。     

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.     

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.     

Impact of buffy coat storage on the generation of inflammatory cytokines and platelet activation

BACKGROUND: Whole blood-derived buffy coat (BC) has become an alternative source from which to prepare random-donor platelet concentrates. The influence of prolonged storage of BC prior to platelet concentrate preparation is a matter of controversy. The impact of BC storage on cytokine release was evaluated and the platelet activation quantified. STUDY DESIGN AND METHODS: BCs were prepared from whole-blood donations after hard-spin centrifugation. After 1, 3, 6, 12, and 24 hours of storage at 22 degrees C without agitation, samples were withdrawn for cell count and blood gas analysis and measurement of interleukin (IL)-1beta, IL-6, IL-8, tumor necrosis factor alpha and platelet factor 4. Platelet surface markers CD41a, CD42b, CD62P, and CD63 were analyzed by flow cytometry, and the antibody-binding sites were quantified by using microbeads. RESULTS: Inflammatory cytokines IL- 1beta, IL-6, and tumor necrosis factor alpha were hardly detectable in stored BCs but levels of IL-8 increased in 25 percent of BCs after 24 hours. A constant increase in platelet factor 4 was observed, which accelerated after 12 hours of storage. Analysis of platelet surface markers showed an initial decrease of platelet activation, followed by an increase after storage for 12 to 24 hours. CONCLUSION: Storage of BCs for up to 12 hours without agitation showed a good preservation of platelets but storage of BCs for 24 hours resulted in increased platelet activation and significantly higher release of platelet factor 4 and IL-8. Stored BCs might well be suitable for platelet preparation, but their storage time should not greatly exceed 12 hours.     

Transforming growth factor beta is increased in plasma of patients with hematologic malignancies after transfusion of platelet concentrates

BACKGROUND: Transforming growth factor beta 1 (TGF-beta 1) acts as a potent inhibitor of bone marrow proliferation. High concentrations were found in human platelets, which release this cytokine during storage. STUDY DESIGN AND METHODS: TGF-beta 1 levels during a storage period of 5 days were compared in the plasma of platelet concentrates prepared by apheresis or by the buffy coat method. In addition, TGF-beta 1 plasma levels were monitored in patients with hematologic malignancies before and after transfusion. RESULTS: TGF-beta 1 levels in the supernatant of platelet concentrates were found to be 55 times higher than those in the plasma of healthy volunteer donors. During storage, an additional increase was observed. Accordingly, the transfusion of platelet concentrates resulted in a significant increase of plasma TGF-beta 1 levels in patients with hematologic malignancies (before transfusion: 2.2 +/− 0.5 ng/mL; after transfusion: 2.9 +/− 0.6 ng/mL), and these higher levels persisted for at least 4 hours. CONCLUSION: Because TGF- beta 1 reduces the clonogenic capacity of hematopoetic progenitor cells, a myelosuppressive effect of platelet transfusions is suggested.     

Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones

A cytokine synthesis inhibitory factor (CSIF) is secreted by Th2 clones in response to Con A or antigen stimulation, but is absent in supernatants from Con A-induced Th1 clones. CSIF can inhibit the production of IL-2, IL-3, lymphotoxin (LT)/TNF, IFN-gamma, and granulocyte-macrophage CSF (GM-CSF) by Th1 cells responding to antigen and APC, but Th2 cytokine synthesis is not significantly affected. Transforming growth factor beta (TGF-beta) also inhibits IFN-gamma production, although less effectively than CSIF, whereas IL-2 and IL-4 partially antagonize the activity of CSIF. CSIF inhibition of cytokine synthesis is not complete, since early cytokine synthesis (before 8 h) is not significantly affected, whereas later synthesis is strongly inhibited. In the presence of CSIF, IFN-gamma mRNA levels are reduced slightly at 8, and strongly at 12 h after stimulation. Inhibition of cytokine expression by CSIF is not due to a general reduction in Th1 cell viability, since actin mRNA levels were not reduced, and proliferation of antigen-stimulated cells in response to IL-2, was unaffected. Biochemical characterization, mAbs, and recombinant or purified cytokines showed that CSIF is distinct from IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IFN-gamma, GM-CSF, TGF-beta, TNF, LT, and P40. The potential role of CSIF in crossregulation of Th1 and Th2 responses is discussed.     

TNF-alpha mediates chemokine and cytokine expression and renal injury in cisplatin nephrotoxicity

The purpose of these studies was to examine the role of cytokines in the pathogenesis of cisplatin nephrotoxicity. Injection of mice with cisplatin (20 mg/kg) led to severe renal failure. The expression of cytokines, chemokines, and ICAM-1 in kidney was measured by ribonuclease protection assays and RT-PCR. We found significant upregulation of TNF-alpha, TGF-beta, RANTES, MIP-2, MCP-1, TCA3, IL-1beta, and ICAM-1 in kidneys from cisplatin-treated animals. In addition, serum, kidney, and urine levels of TNF-alpha measured by ELISA were increased by cisplatin. Inhibitors of TNF-alpha production (GM6001, pentoxifylline) and TNF-alpha Ab's reduced serum and kidney TNF-alpha protein levels and also blunted the cisplatin-induced increases in TNF-alpha, TGF-beta, RANTES, MIP-2, MCP-1, and IL-1beta, but not ICAM-1, mRNA. In addition, the TNF-alpha inhibitors also ameliorated cisplatin-induced renal dysfunction and reduced cisplatin-induced structural damage. Likewise, TNF-alpha-deficient mice were resistant to cisplatin nephrotoxicity. These results indicate cisplatin nephrotoxicity is characterized by activation of proinflammatory cytokines and chemokines. TNF-alpha appears to play a central role in the activation of this cytokine response and also in the pathogenesis of cisplatin renal injury.     

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.     

Binding of HIV-1 to RBCs involves the Duffy Antigen Receptors for Chemokines (DARC)

The Duffy Antigen Receptor for Chemokines (DARC) belongs to a family of erythrocyte chemokine receptors that bind C-X-C and C-C chemokines such as interleukin 8 (IL-8), monocyte chemoattractant protein 1 (MCP-1) and regulated-on-activation, normal T cell-expressed and -secreted (RANTES). but not macrophage inflammatory protein 1α (MIP-1α) or MIP-1β. DARC has also been identified to a receptor for malaria parasites Plasmodium vivax and Plasmodium knowlesi. In the present study, we show that HIV-1 binds to RBCs from Caucasian individuals via DARC making RBCs able to transmit HIV to peripheral blood mononuclear cells (PBMCs). Furthermore, binding of HIV-1 particles to RBCs is inhibited by treating these cells with recombinant RANTES, but not with recombinant MIP-1α prior to their incubation with HIV-1. This finding suggests that RBCs may function as a reservoir for HIV-1 or as a receptor for the entry of HIV-1 into CD4-cell subsets as well as neurons or endothelial cells.     

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.