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.     

Platelet capacity of various platelet pooling systems for buffy coat-derived platelet concentrates

BACKGROUND: Platelet (PLT) storage lesions might depend on the total PLT count in the storage container and also on the PLT pooling system, especially the storage container, that is used for preparation of PLT concentrates (PCs). In this study, the PLT capacity of four commercially available PLT pooling systems was studied. MATERIALS AND METHODS: Four PCs were prepared in pooling systems of Baxter, Fresenius, Terumo, or Pall. The PCs were pooled and divided with various total PLT counts over the four storage containers (<225 × 10⁹, 225 × 10⁹‐324 × 10⁹, 325 × 10⁹‐424 × 10⁹, and >424 × 10⁹ PLTs). Volumes were kept equal by adding plasma to PCs with less than 425 × 10⁹ PLTs until a same volume as for PCs with more than 424 × 10⁹ PLTs was reached. PCs were stored at room temperature and tested for various in vitro variables on Days 1, 3, 5, 7, and 9. Paired experiments were repeated for each system five times. RESULTS: In vitro variables remained good for 9 days, that is, swirling score of 2 or more, pH value of 6.8 or more, glucose level of 10 mmol per L or more, lactate level of less than 25 mmol per L, and CD62p expression of less than 50 percent, for PCs in Baxter systems with more than 225 × 10⁹ PLTs, for PCs in Fresenius and Terumo systems with 225 × 10⁹ to 424 × 10⁹ PLTs, and for PCs in Pall systems with fewer than 425 × 10⁹ PLTs. CONCLUSION: PLT capacity depended on the PLT pooling systems used. All systems provide acceptable storage conditions. The Baxter system was the only system with capacity for more than 424 × 10⁹ PLTs per PC.     

Overnight or fresh buffy coat-derived platelet concentrates prepared with various platelet pooling systems.

BACKGROUND: For logistic reasons, possibilities to produce both platelet (PLT) concentrates prepared from fresh or overnight-stored whole blood (fresh and o/n PCs, respectively) are convenient. The consequences of both possibilities are not well described. The PLT pooling system used might also influence the condition of PCs. Our aim was to compare fresh and o/n PCs with different PLT pooling systems. STUDY DESIGN AND METHODS: Fresh and o/n PCs were prepared from buffy coats and plasma in PLT pooling systems of Baxter, Fresenius, Terumo, or Pall (n = 5). PCs were stored for 9 days. The in vitro quality was determined by the PLT count, pH, glucose, lactate, pO(2), pCO(2), CD62P expression, and annexin V binding. RESULTS: The o/n PCs showed higher PLT count (approx. 460 x 10(9)/PC vs. approx. 310 x 10(9)/PC), pCO(2), and lactate concentration and lower pH, pO(2), glucose concentration, CD62P expression (until Day 5), and annexin V binding (until Day 7) compared with fresh PCs (p < 0.05). Only for o/n PCs in the Baxter and Fresenius systems did the pH and glucose concentration remain higher, and the lactate concentration and CD62P expression remained lower than that of o/n PCs in the Pall and Terumo systems. The pH for fresh PCs in the Baxter and Fresenius systems was more often greater than 7.4 than for fresh PCs in the Terumo or Pall systems. CONCLUSION: The quality of PCs depended on whether PCs were prepared from fresh or overnight-stored whole blood and on the used PLT pooling system. The main difference between fresh and o/n PCs was the PLT count.     

Elimination and multiplication of bacteria during preparation and storage of buffy coat-derived platelet concentrates

BACKGROUND: The prevalence of bacterial contamination of random-donor platelet concentrates (PCs) is considerably lower than that of blood donations. Which key steps of the preparation procedure contribute to the elimination of bacteria was investigated. STUDY DESIGN AND METHODS: Ten bacteria species were used. Blood donations were spiked with bacteria and stored at 22 degrees C for 8 hours. The buffy coats were kept for 6 hours. PCs were prepared from pools of 4 buffy coats. At each preparation step and during PC storage, bacteria contents were measured. In additional experiments, the titers of spiked blood and buffy coats were determined after storage at 20, 22, or 24 degrees C for 8 and up to 24 hours, respectively. RESULTS: Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, and Yersinia enterocolitica were completely inactivated during storage in blood or buffy coats. Titer reduction was between 3.32 and 4.62 log. Bacillus cereus, Propionibacterium acnes, Staphylococcus aureus, and Staphylococcus epidermidis did not multiply. Compared with their values in spiked blood the titers in the PCs were reduced by 1.7 to 2.8 log. Klebsiella pneumoniae was the only species that grew in blood. With the exception of P. acnes, those species that were not removed by the preparation process multiplied in the PCs. Remarkable donor-to-donor variations of the bactericidal activities of buffy coats were detected when the storage time was prolonged to 24 hours. CONCLUSIONS: Bacteria are significantly eliminated by the preparation procedure for random donor PCs. Also, blood and buffy coats are bactericidal for most species. When buffy-coat storage is prolonged, it cannot, however, be predicted whether specific strains vanish or multiply.     

Markers of platelet activation and apoptosis during storage of apheresis- and buffy coat-derived platelet concentrates for 7 days

BACKGROUND: The different production methods for platelet concentrates (PCs) result in products with variable in vitro quality and in vivo viability. The aim of this study was to compare in vitro variables of PCs produced by apheresis (AP-PC) or the buffy coat (BC-PC) method by applying a number of new and established assays.
STUDY DESIGN AND METHODS: Standard TRIMA Accel (Gambro BCT) AP-PCs (n = 20) and BC-PCs (n = 20) were stored in 100 percent plasma and changes in mitochondrial membrane potential (ΔΨ_m) were assessed using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethlybenzimidazolcarbocyanine iodide (JC-1) dye on Days 1, 3, 5, and 7. The capacity of platelets (PLTs) for oxidative phosphorylation was also monitored by measuring oxygen consumption using a Clark-type electrode. PLT viability was measured using a new assay that utilizes the vital stains calcein-AM and FM4-64. Expression of phosphatidylserine (PS), CD42b, CD47, CD61, and CD62P was also assessed.
RESULTS: Although the JC-1 ratio (FL2/FL1) decreased significantly in both preparations, the percentage of PLTs with depolarized ΔΨ_m increased significantly in BC-PCs but not in AP-PCs. However, no significant change was detected in the PLTs' ability to consume oxygen in both preparations. PLTs in BC-PCs also showed significantly lower GPIb, CD47, and CD61 expression than AP-PCs on Day 1. PLTs in both preparations, however, showed a similar increase in CD62P and PS expression during storage, without significant loss of viability.
CONCLUSIONS: PLTs in AP-PCs and BC-PCs undergo different degrees of deterioration in mitochondrial integrity and thus may undergo different degrees of apoptosis. Interventions that maintain mitochondrial integrity may improve PLT viability in vitro.     

Platelet storage lesion of WBC-reduced, pooled, buffy coat-derived platelet concentrates prepared in three in-process filter/storage bag combinations

BACKGROUND: With the implementation of universal WBC reduction in the United Kingdom, in-process WBC-reduction filters for pooled buffy coat (BC)-derived platelet concentrates (PCs) are used in routine production. The effects of three filter/storage bag combinations on platelet activation and microvesiculation and on the activation of coagulation were investigated. STUDY DESIGN AND METHODS: Using pooled BCs from the same donors, three filter/storage bag combinations (Autostop BC/CLX, Pall Biomedical; Sepacell PLX5/PL2410, Asahi Medical; and Imugard III-PL 4P/Teruflex, Terumo) were compared with unfiltered controls for their effects on microvesiculation and other storage-induced changes in platelets. Process efficiency was measured by platelet yield and residual WBC count. The storage changes were assessed: pH, activation of platelets measured by CD62P on the platelet surface and in supernatant plasma, quantitation of platelet-derived and RBC-derived microvesicles, cellular injury measured by annexin V in the supernatant plasma, and activation of the coagulation system measured by kallikrein-like and thrombin-like activities, prothrombin fragment 1+2, and thrombin-antithrombin complex. RESULTS: All three filters were comparable in terms of platelet recovery and WBC removal, and none induced immediate platelet activation or microvesiculation. With storage, platelet activation or microvesiculation increased in platelets prepared by all three filters and in unfiltered controls, but these effects were significantly less in the Imugard PCs than in controls. These findings were consistent with those for annexin V in the supernatant plasma, which were lower in Imugard PCs than in other products. Sepacell and Imugard filters reduced RBC-derived microvesicles to 50 percent of control levels, but the Autostop filter had no effect. On storage, levels of RBC-derived microvesicles in filtered products remained static, but levels in the unfiltered control doubled. Kallikrein- and thrombin-like activities were generated only by the Autostop filter without any further increment on storage. CONCLUSION: WBC-reduced pooled BC-PCs prepared by various filter/bag combinations were equivalent on Day 1 but differed during storage in terms of platelet activation or microvesiculation.     

医用电解质溶液作添加液制备汇集少白细胞血小板

目的采用医用电解质溶液作添加液(PAS),建立1种白膜法制备添加液汇集少白细胞血小板(PAS汇集BC-PCs)的方法。方法从400ml全血中分离白膜层,容量35—40ml,放(22±2)℃静置过夜,将ABO同型的6袋白膜汇集,加220g添加液(90%复方电解质注射液、8%ACD-A血液保养液和2%含量为50g/L的碳酸氢钠注射液的混合液)稀释白膜,汇集后的白膜在(22±2)℃中,以300×g离心10min,将上层的富含血小板悬液再经白细胞滤除器过滤去除白细胞,并转移到血小板保存袋内,即制备成1个成人治疗量的PAS汇集BC-PCs,制备过程在一个特制的密闭系统内完成。结果共制备30个成人治疗量的PAS汇集BC-PCs,其容量为(270±32)ml、血小板含量为(2.96±0.31)×1011、WBC混入量为(1.3±0.2)×106、RBC混入量为(5.8±1.1)×109、CD62P表达率为(22.5±10.6)%。保存8d后的pH为7.14±0.04、低渗休克反应率(HSR)为(54.0±8.2)%、CD62P表达率为(45.7±13.8)%。结论由复方电解质注射液、ACD-A保养液和碳酸氢钠注射液的混合液为添加液汇集血小板的方法可行。     

Superior integrin activating capacity and higher adhesion to fibrinogen matrix in buffy coat-derived platelet concentrates (PCs) compared to PRP-PCs

Background

Regardless of different sources, methods or devices which are applied for preparation of therapeutic platelets, these products are generally isolated from whole blood by the sedimentation techniques which are based on PRP or buffy coat (BC) separation. As a general fact, platelet preparation and storage are also associated with some deleterious changes that known as platelet storage lesion (PSL). Although these alternations in platelet functional activity are aggravated during storage, whether technical issues within preparation can affect integrin activation and platelet adhesion to fibrinogen were investigated in this study.

Quality of platelet concentrates derived by platelet rich plasma, buffy coat and Apheresis.

Platelets derived from platelet rich plasma, buffy coat and Apheresis technologies differ in terms of in vitro functional activity, aggregation states and storage characteristics, as measured by automated cell counters, and pH assessment. This may be related to either differences in the subpopulation of platelets and leucocytes recovered or the processing/storage induced cellular damage. Some types of methodologies, such as BC derived-PC, appear to have a higher rate of bacterial contamination.     

白膜放置时间对制备手工浓缩血小板质量的影响

目的评价全血当天分离的白膜(BC)在不同时间分离制备的浓缩血小板(PC)的质量,为手工制备PC提供参考。方法将80袋400 mL全血于采集6 h内分离出BC,将5袋同血型BC由无菌接驳机对接合并成1袋(1个治疗量)后,再均分在3个血小板保存袋内,1袋即刻(0 h组)轻离心分离制备PC,另2袋在22℃血小板保存箱分别振摇4 h(4 h组)和16 h(16 h组)后再分离PC。对所有标本留样进行血小板质量检测,包括Plt、血小板回收率、CD62P、聚集率、RBC混入量、WBC混入量、FHb含量。结果 3组PC制剂RBC混入量、Plt、血小板回收率、CD62P、聚集率差异无统计学意义;WBC混入量:0 h(6.76±1.29)和4 h组变化不明显,16 h(3.78±0.45)组降低明显(P<0.05);FHb含量:随BC处理时间延长有增高趋势,16 h(65.62±11.11)与0 h(33.45±6.95)比差异具统计学意义(P<0.05)。结论随BC放置时间延长对制备PC制剂的质量有一定影响。     

In-process cell counts during preparation of platelet concentrates from buffy coats

To ensure the quality of platelet concentrates (PCs), we studied in-process recoveries of blood cell counts in pooled PCs derived from four or five buffy coats (BCs) from Biopack Compoflex Systems in Bern (PC-BC/4 or PC-BC/5) and from five BCs from Optipac (Baxter) in Zurich (PC-BC/5). BCs were pooled employing a sterile connecting device and flushing them with 300 mL of platelet additive solution. The pools were centrifuged for 12 min at 500 g at 20 degrees C and filtered with PALL's Auto-Stop BC-leukocyte removal filter. Automated platelet counting was performed on whole blood donation, on single BC, on pooled BC and in the final product. Four out of 10 PC-BC/4 (= 40%) and 29 out of 30 PC-BC/5 (= 97%) had a total platelet count of > 200 x 10(9) platelets. Average percentage recoveries in PC compared to the pre-centrifugation BC pools were similar with the Biopack Compoflex and the Optipac systems, 62% and 57% respectively, whereby the absolute platelet count per one donation was similar, i.e. 49.5 x 10(9), 55 x 10(9) and 53 x 10(9) in PC-BC/4 and PC-BC/5 from Bern and PC-BC/5 from Zurich. There was a significant positive correlation between the inital number of BCs taken for pooling and the final platelet counts in the PCs. In order to recover a minimal platelet content of 200 x 10(9) platelets per pooled unit, it is safer to start out with five rather than with four donations unless recoveries during the production steps can be improved.     

Freezing human platelets with 6 percent dimethyl sulfoxide with removal of the supernatant solution before freezing and storage at -80 degrees C without postthaw processing

BACKGROUND: Platelets (PLTs) can be frozen with 6 percent dimethyl sulfoxide (DMSO) at -80 degrees C for up to 2 years. This method has been modified by concentrating the PLTs and removing the supernatant before freezing. STUDY DESIGN AND METHODS: High-yield leukoreduced PLTs stored at 22 degrees C for up to 5 days were divided into three equal volumes: one was frozen with 6 percent DMSO at -80 degrees C, thawed, washed, and resuspended in plasma (old method with DMSO); the second was treated with 6 percent DMSO, concentrated to remove the supernatant DMSO, frozen at -80 degrees C, thawed, and diluted with 0.9 percent NaCl (new method with DMSO); and the third was treated with 0.9 percent NaCl without DMSO, concentrated to remove the supernatant solution, frozen at -80 degrees C, thawed, and diluted with 0.9 percent NaCl (new method without DMSO). RESULTS: Freeze-thaw-wash recovery of PLTs frozen by the old method with DMSO was 74 +/- 2 percent with 5 percent PLT microparticles. Freeze-thaw recovery was 94 +/- 2 percent with 7 percent PLT microparticles (new method with DMSO) and 69 +/- 9 percent with 15 percent PLT microparticles (new method without DMSO). Total DMSO in washed PLTs was 400 and 600 mg in PLTs concentrated before freezing. In vivo recovery of PLTs frozen by the new method with DMSO and transfused into normal volunteers was 30 percent and the life span was 7 days. CONCLUSION: Concentrating PLTs before freezing simplified the procedure by eliminating postthaw washing. PLTs frozen by this method had more PLTs with reduced GPIb and increased annexin V binding than those frozen by the old method.     

Leukoreduced buffy coat-derived platelet concentrates photochemically treated with amotosalen HCl and ultraviolet A light stored up to 7 days: assessment of hemostatic function under flow conditions

BACKGROUND: Amotosalen plus ultraviolet A light photochemical treatment (PCT) inactivates high titers of bacteria, and other pathogens, in platelet concentrates (PCs) potentially allowing the storage of platelets (PLTs) for up to 7 days. Adhesion and aggregation of PLTs to injured vascular surfaces are critical aspects of PLT hemostatic function. STUDY DESIGN AND METHODS: Two ABO-identical leukoreduced buffy coat-derived PCs in additive solution were mixed and divided: one-half underwent PCT (PCT-PCs) and the other was kept as a control (C-PCs); both were stored under standard conditions. The total number of paired PCs studied was nine. Samples were taken on Day 1 (before PCT) and after 5 and 7 days of storage. The adhesion and aggregation capacities were evaluated under flow conditions in a ex vivo perfusion model. RESULTS: Compared to control, PCT resulted in a decrease in PLT count of 6.5 percent (p = 0.004) and 10.2 percent (p = 0.008) after 5 and 7 days' storage, respectively (n = 9). PLT interaction with subendothelium was mainly in form of adhesion. The surface covered by PCT PLTs on Day 1 was 26.0 +/- 4.2 percent (mean +/- SEM). On Day 5, PCT-PCs showed a covered surface of 20.9 +/- 2.2 percent, and the C-PCs, 20.6 +/- 1.6 percent. After 7 days, PCT-PCs produced a nonsignificant higher PLT deposition compared to control (27.1 +/- 2.9% vs. 21.2 +/- 2.8%, p = 0.06). CONCLUSION: PCT of PCs and storage up to 7 days was associated with a 10.2 percent decrease in PLT count due to processing losses compared to C-PC. PLT adhesive and aggregating capacities under flow conditions of PCT-PCs were similar to C-PCs and remained well preserved for up to 7 days of storage.     

Overnight or fresh buffy coat–derived platelet concentrates prepared with various platelet pooling systems

BACKGROUND: For logistic reasons, possibilities to produce both platelet (PLT) concentrates prepared from fresh or overnight-stored whole blood (fresh and o/n PCs, respectively) are convenient. The consequences of both possibilities are not well described. The PLT pooling system used might also influence the condition of PCs. Our aim was to compare fresh and o/n PCs with different PLT pooling systems.
STUDY DESIGN AND METHODS: Fresh and o/n PCs were prepared from buffy coats and plasma in PLT pooling systems of Baxter, Fresenius, Terumo, or Pall (n = 5). PCs were stored for 9 days. The in vitro quality was determined by the PLT count, pH, glucose, lactate, pO₂, pCO₂, CD62P expression, and annexin V binding.
RESULTS: The o/n PCs showed higher PLT count (approx. 460 × 10⁹/PC vs. approx. 310 × 10⁹/PC), pCO₂, and lactate concentration and lower pH, pO₂, glucose concentration, CD62P expression (until Day 5), and annexin V binding (until Day 7) compared with fresh PCs (p < 0.05). Only for o/n PCs in the Baxter and Fresenius systems did the pH and glucose concentration remain higher, and the lactate concentration and CD62P expression remained lower than that of o/n PCs in the Pall and Terumo systems. The pH for fresh PCs in the Baxter and Fresenius systems was more often greater than 7.4 than for fresh PCs in the Terumo or Pall systems.
CONCLUSION: The quality of PCs depended on whether PCs were prepared from fresh or overnight-stored whole blood and on the used PLT pooling system. The main difference between fresh and o/n PCs was the PLT count.     

Pathogen reduction treatment of buffy coat platelet concentrates in additive solution induces proapoptotic signaling

BACKGROUND: Pathogen reduction technology (PRT) can potentially reduce the risk of transfusion‐transmitted infections. However, PRT treatment of platelet (PLT) concentrates also results in reduced PLT quality and increased markers of apoptosis during storage. The aim of this study was to investigate changes to the expression and activation of proteins involved in apoptosis signaling. STUDY DESIGN AND METHODS: Samples from riboflavin and ultraviolet light PRT‐treated and untreated (control) buffy coat–derived PCs in 70% SSP+ and 30% plasma were taken on Days 1, 5, and 7 of storage. Phosphatidylserine (PS) exposure, expression of Bcl‐2 family proteins, cytochrome c release, and cleavage of caspase‐3 and caspase‐3 substrates were analyzed using flow cytometry and Western blotting. RESULTS: Compared to untreated controls, markers of apoptosis signaling were increased after PRT and subsequent storage. PS exposure on the PLT outer membrane was significantly higher after PRT on Days 5 and 7 of storage (p < 0.05). Expression of proapoptotic Bak and Bax was higher after PRT and subsequent storage. Cytochrome c release and caspase‐3 cleavage were also greater and occurred earlier in the PRT‐treated PLTs. The cleavage of caspase‐3 substrates gelsolin and ROCK I were also increased after PRT, compared to untreated controls. CONCLUSIONS: This study demonstrated an increase in proapoptotic signaling during PLT storage, which was exacerbated by PRT. Many of these differences emerged outside the current 5‐day storage period. These changes may not currently influence PLT transfusion quality, but will need to be carefully evaluated when considering extending PLT storage beyond 5 days.