Red blood cells as oxygen carrier during normothermic machine perfusion of kidney grafts: Friend or foe?

Renal ex vivo normothermic machine perfusion (NMP) is under development as an assessment tool for high-risk kidney grafts and as a means of achieving more physiologically accurate organ preservation. On-going hemolysis has been reported during NMP, as this technique relies on red blood cells for oxygen delivery. In this study, we confirm the occurrence of progressive hemolysis during 6-hour kidney NMP. NMP-associated erythrostasis in the glomeruli and in peri-glomerular vascular networks points to an interaction between the red blood cells and the graft. Continuous hemolysis resulted in prooxidative changes in the perfusate, which could be quenched by addition of fresh frozen plasma. In a cell-based system, this hemolysis induced redox stress and exhibited toxic effects at high concentrations. These findings highlight the need for a more refined oxygen carrier in the context of renal NMP.

Platelet concentrates in platelet additive solutions generate less complement activation products during storage than platelets stored in plasma.

BACKGROUND: Platelet transfusions can be associated with adverse reactions, such as febrile non-haemolytic transfusion reaction (FNHTR). It has been suggested that damage-associated molecular patterns (DAMP) and complement play a role in FNHTR. This study investigated the nature of DAMPs and complement activation products contained in platelet concentrates during storage, with a specific focus on different platelet storage solutions. MATERIALS AND METHODS: Buffy coats (BC) from healthy donors were pooled (15 BC per pool) and divided into three groups of the same volume. After addition of different storage solutions (plasma, platelet additive solutions [PAS]-C or PAS-E; n=6 for each group), BC pools were processed to platelet concentrates (PC). Leukoreduced PCs were stored on a shaking bed at 20-24°C and sampled on days 1, 2, 6 and 8 after collection for selected quality parameters: platelet activation, DAMPs (High Mobility Group Box 1 [HMGB1], nucleosomes), and complement activation products. RESULTS: During storage, equal levels of free nucleosomes and increasing concentrations of HMGB1 were present in all groups. Complement activation was observed in all PC. However, by day 8, the use of PAS had reduced C3b/c levels by approximately 90% and C4b/c levels by approximately 65%. DISCUSSION: Nucleosomes and HMGB1 were present in PCs prepared in plasma and PAS. Complement was activated during storage of platelets in plasma and in PAS. The use of PAS is associated with a lower amount of complement activation products due to the dilution of plasma by PAS . Therefore, PC in PAS have less complement activation products than platelets stored in plasma. These proinflammatory mediators in PC might induce FNHTR.

Comparison of Washing Efficiency and Recovery of Blood Cells Between Centrifugation, Coarse Filtration and Microfiltration Techniques to Prepare Autologous Blood for Transfusion.

Purpose: Cell salvage is the process by which blood lost in surgery is collected and washed or filtered to produce autologous blood for re-transfusion to the patient. Cell salvage aims to reduce the need for donor blood. Centrifugal cell salvage washing technique is a preferred medical treatment in order to retain lost red blood cells (RBCs) without contaminants. Although this technology very efficiently collects and washes shed blood, it is costly and often impractical or unavailable, especially in middle- or low-income countries. This study assessed two innovative filter devices as an alternative to centrifugal cell salvage technology: a coarse collection filter device (Hemafuse) and a microfiltration device (HemoClear). In contrast to centrifugal technology, both filter devices do not require electricity, nor costly equipment and extensive training. We compared the effectiveness of these filtration technologies to remove plasma constituents and recover and concentrate the cellular components with centrifugal technology (autoLog® device). Methods: Whole blood was processed with each technology according to the device manufacturer's instructions. Before and after processing, the blood products were analyzed for supernatant solutes and cellular composition. Results: The centrifugal technology confirmed its efficacy to remove potentially harmful solutes and capture red blood cells. The microfiltration technology (HemoClear) reached comparable levels of removal of solutes, with a potential advantage over centrifugal technology in the ability to also recover platelets. The coarse filtration technology (Hemafuse) had no washing capacity but, like the microfiltration technology, has the advantage of recovering platelets. Conclusion: Innovative filtration devices represent an alternative to centrifugal technology in the preparation of autologous blood for reinfusion. The HemoClear technology for the first time enables the recovery of washed platelets and red blood cells. Clinical trials will have to be performed to investigate the clinical value of this new autologous blood product.

Clinical and in vitro evaluation of red blood cells collected and stored in a non-DEHP plasticized bag system.

BACKGROUND AND OBJECTIVES: Di-ethyl-hexyl-phthalate (DEHP) is currently the main plasticizer used for whole blood collection systems. However, in Europe, after May 2025, DEHP may no longer be used above 0.1% (w/w) in medical devices. DEHP stabilizes red cell membranes, thereby suppressing haemolysis during storage. Here we compared in vitro quality parameters of red cell concentrates (RCCs) collected and stored in DEHP-, DINCH- or DINCH/BTHC-PVC hybrid blood bags with saline-adenine-glucose-mannitol (SAGM) or phosphate-adenine-glucose-guanosine-saline-mannitol (PAGGSM) storage solution. Last, we performed haemovigilance surveillance for RCC collected in DINCH-PVC and stored in PAGGSM/BTHC-PVC. MATERIALS AND METHODS: In vitro quality parameters of RCC were determined during 42 days of storage. Haemovigilance surveillance was conducted to compare the frequency and type of transfusion reaction. RESULTS: Haemolysis levels were increased in SAGM/BTHC-PVC as compared to SAGM/DEHP-PVC (0.66% ± 0.18% vs. 0.36% ± 0.17%). PAGGSM storage solution was able to adequately suppress haemolysis to levels observed during storage in SAGM/DEHP-PVC, both in BTHC-PVC (0.38% ± 0.12%), and to a slightly lesser extent in DINCH-PVC (0.48% ± 0.17%). A total of 1650 PAGGSM/BTHC-PVC and 5662 SAGM/DEHP-PVC RCC were transfused yielding a transfusion reaction frequency of 0.24% (95% CI 0.0000-0.0048) and 0.44% (95% CI 0.0027-0.0061) respectively. CONCLUSION: The in vitro quality of RCC stored in PAGGSM/BTHC-PVC and SAGM/DEHP-PVC is comparable. There is no indication that transfusion of erythrocytes stored in PAGGSM/BTHC-PVC results in increased transfusion reaction frequency. These initial results provide a basis for further clinical evaluation to narrow down the confidence interval of transfusion reaction frequency.

Recovery of platelet-rich red blood cells and acquisition of convalescent plasma with a novel gravity-driven blood separation device.

OBJECTIVES: Our objectives were to determine the separation characteristics and blood product quality of a gravity-driven microfiltration blood separation system (HemoClear, The Netherlands). BACKGROUND: A range of centrifugal blood separation devices, including intraoperative cell salvage devices (cell savers) and apheresis machines, are available to assist in preparing both allogenic and autologous blood products. These devices are expensive to operate and require extensive training. METHODS AND MATERIALS: Nine whole blood units were collected under standard conditions and analysed for haematological parameters, thromboelastographic properties, platelet morphology and activation, and red blood cell (RBC) deformability and morphology. Three whole blood units were separated by means of the HemoClear device, into a liquid and cellular component. The cellular component was diluted with SAGM and cold stored for 14 days. To simulate cell salvage six whole blood units were diluted with isotonic saline, followed by multiple HemoClear separation rounds. RESULTS: The recovery of both RBCs (100 ± 1.6%) and white blood cells (99 ± 4.5%) after undiluted filtration were very high, while platelet recovery was high (83 ± 3.0%). During the filtration, and cold storage after filtration storage both the non-deformable RBC fraction and the RBC maximum elongation remained stable. Parameters of thromboelastography indicated that platelets remain functional after filtration and after 7 days of cold storage. In the cell salvage simulation the total protein load in the cellular fraction was reduced by 65 ± 4.1% after one washing round and 84 ± 1.9% after two consecutive washing rounds. CONCLUSION: The novel blood filter studied effectively separates whole blood into diluted plasma and platelet-rich RBCs. Moreover, the device effectively washed diluted whole blood, driving over 80% of proteins to the liquid component.

Frozen Blood Reserves.

Frozen blood reserves are an important component in meeting blood needs. The idea behind a frozen blood reserve is twofold: to freeze units of rare blood types for later use by patients with special transfusion needs and for managing special transfusion circumstances. The permeating additive glycerol is used as a cryoprotectant to protect red blood cells (RBCs) from freezing damage. The use of thawed RBCs has been hampered by a 24-h outdating period due to the potential bacterial contamination when a functionally open system is used for addition and removal of the glycerol. The introduction of an automated, functionally closed system for glycerolization and deglycerolization of RBCs improved the operational practice. More importantly, the closed process allowed for extended shelf life of the thawed RBCs. In the current chapter, a cryopreservation procedure for RBCs using a functionally closed processing system is described.

Residual red cells in blood components: A multisite study of fully automated enumeration using a hematology analyzer.

BACKGROUND: Manufacture of platelet concentrates (PCs) and plasma may fail to remove all residual red blood cells (rRBCs). Measuring rRBCs for compliance to guidelines has proven challenging, leading to an absence of a consensus methodology. Sysmex hematology analyzers with the Blood Bank mode (BB mode) analysis option offer the potential for automated rRBC counting. We therefore performed a two-site appraisal of the system. STUDY DESIGN AND METHODS: Performance characteristics were determined using platelet and plasma samples spiked with RBCs. Sample stability (n = 47) and the impact of sample type were also assessed. Components (platelets, n = 1474; plasma, n = 77) prepared using different routine manufacturing methods were tested to assess variation in rRBC concentration. RESULTS: Linearity studies up to 19 000 RBCs/µL demonstrated good correlation between expected and observed results (R2 ≥ 0.9731), and flow cytometric results also correlated well with BB mode (R2 = 0.9400). Precision analysis gave a limit of quantitation of 6 to 7 RBCs/µL, and carryover was 0.03%. Ethylenediaminetetraacetic acid and plain tube results were not significantly different (P ≥ 0.10), and samples were stable up to 24 hours. Apheresis PCs produced at two sites had lower rRBC concentrations (medians, 17 and 13 RBCs/µL) than those produced with the buffy coat method either manually (median, 681 RBCs/µL) or with the automated Terumo Automated Centrifuge and Separator Integration process (median, 81 RBCs/µL). All PCs failing visual inspection as having RBCs ≥4000 RBCs/µL were also detected by the BB mode. CONCLUSION: The BB mode had acceptable performance characteristics and has the potential for integration into a fully automated process control system for rRBC enumeration in plasma and PCs.

Lifestyle behaviours are not associated with haemolysis: results from Donor InSight.

BACKGROUND: Lifestyle behaviours such as physical activity, sedentary behaviour and dietary habits have been shown to influence blood lipid levels, and both lifestyle and blood lipids may be associated with haemolysis during storage of blood products. We aimed to investigate whether lifestyle behaviours are associated with degree of haemolysis in red cell concentrates (RCC), and if such associations are mediated by low-density lipoprotein (LDL) cholesterol and triglyceride levels. MATERIALS AND METHODS: Cross-sectional analyses were performed in data from 760 Dutch blood donors participating in Donor InSight, an observational cohort study. Linear regression analyses were conducted to assess associations of lifestyle behaviours with haemolysis levels in RCC 28 days after blood sampling. Lifestyle behaviours included moderate-to-vigorous physical activity and sedentary behaviour measured by accelerometry, and self-reported intake of a selection of foods potentially related to blood lipids, i.e. consumption of eggs, meat, nuts and fish. Potential mediating roles of both LDL cholesterol and triglyceride levels were investigated separately. All analyses were adjusted for relevant confounders. RESULTS: No statistically significant nor substantial associations of any of the lifestyle behaviours with haemolysis in RCC were found, nor were there any associations between lifestyle behaviours and blood lipids. We did find consistent positive associations of LDL cholesterol and triglyceride levels with haemolysis in RCC during storage. DISCUSSION: In this large cohort, blood lipid levels were consistently associated with haemolysis in RCC. Nonetheless, there was no evidence for an association between lifestyle behaviours and haemolysis in RCC, or for mediating effects by blood lipid levels.

Prevention of red cell storage lesion: a comparison of five different additive solutions.

BACKGROUND: In Europe, red cell concentrates (RCC) are usually stored in SAGM (saline-adenine-glucose-mannitol). During storage, in vitro red cell quality declines, including lowered energy status and increased cell lysis. Recently, several additive solutions (ASs), designed to diminish the decline in in vitro quality during storage, have been developed. These new solutions have mainly been developed to better maintain red blood cell (RBC) 2,3-biphosphoglycerate (2,3 BPG) levels and energy status during storage. High levels of 2,3 BPG allow for better oxygen release while high energy status is necessary for function and survival of RBC in vivo. In a paired study design, RBC ASs were compared for their ability to provide improved in vitro quality during hypothermic storage. MATERIALS AND METHODS: For each experiment, 5 whole blood units held overnight were pooled and split. The whole blood units were processed according to the buffy coat method. RBCs were resuspended in either SAGM, PAGGSM, PAG3M, E-Sol 5 or AS-7 and leucoreduced by filtration. RCCs were stored for eight weeks at 2-6 °C and sampled weekly for analysis of in vitro quality parameters. RESULTS: Red cell concentrates stored in PAG3M, E-Sol 5 and AS-7 showed significantly higher lactate production and higher levels of intracellular adenosine triphosphate (ATP) and total adenylate. 2,3 BPG levels rapidly declined during storage in SAGM and PAGGSM. The decline in 2,3 BPG was inhibited during storage in E-Sol 5 and AS-7, while in PAG3M, 2,3 BPG level increased above the initial level till day 35 and remained detectable till day 56. Haemolysis was comparable for all ASs until day 35, upon prolonged storage, haemolysis in SAGM was higher than with the other ASs. As compared to SAGM, storage in PAGGSM, PAG3M, E-Sol 5 and AS-7 better maintained morphological properties. DISCUSSION: Storage of RBCs in the new generation ASs yield RBCs with more stable metabolite levels and improved overall quality during storage as compared with RBCs stored in SAGM.

The effect of prefreeze rejuvenation on postthaw storage of red blood cells in AS-3 and SAGM.

BACKGROUND: We investigated whether improving the metabolic status of red blood cell concentrates before freezing could extend the postthaw shelf life beyond 14 days while still meeting the requirements for hemolysis (0.8%) and total adenylate (>82% of original values). STUDY DESIGN AND METHODS: At Day 8 after collection, four leukoreduced red blood cell concentrates in saline-adenine-glucose-mannitol (SAGM) were pooled, mixed, and split (n = 4). Of these concentrates, two were rejuvenated in Rejuvesol. In addition, two leukoreduced red blood cell concentrates in phosphate-adenine-glucose-guanosine-gluconate-mannitol (PAGGGM) were pooled, mixed, and split at Day 8 after collection (n = 4). All concentrates were glycerolized, frozen, and stored for at least 2 weeks at -80°C. After thawing and deglycerolization, from each pair, one red blood cell concentrate was resuspended in SAGM, and one was suspended in AS-3. During postthaw storage at 2 to 6°C for 35 days, all concentrates were sampled weekly and analyzed for hematologic, metabolic, and morphologic parameters. RESULTS: Both Rejuvesol and PAGGGM treatment produced increased adenosine triphosphate and total adenylate and 2,3-diphosphoglycerate levels compared with untreated red blood cell concentrates. Regardless of prefreeze Rejuvesol or PAGGGM treatment, postthaw hemolysis remained below 0.8% during 7 days in SAGM and during 35 days in AS-3. At Day 35 of postthaw storage in AS-3, total adenylate in nonrejuvenated red blood cell concentrates had decreased to 72% of the original values; whereas, in prefreeze Rejuvesol-treated and PAGGGM-treated concentrates, adenylate values were still were at 101% and 98%, respectively. CONCLUSION: Based on maximum allowable hemolysis of 0.8% and total adenylate content greater than 82% of the original value, thawed, prefreeze Rejuvesol-treated or PAGGGM-treated red blood cell concentrates can be stored for 35 days at 2 to 6ºC in AS-3.

Cryopreservation of red blood cells.

Cryopreservation of red blood cell concentrates (RBCs) is an important method for maintaining an inventory of rare RBC units and managing special transfusion circumstances. The permeating additive glycerol is used as a cryoprotectant to protect RBCs against freezing damage. The use of thawed RBCs was hampered a 24-h outdating period due to potential bacterial contamination when a functionally open system was used for addition and removal of the glycerol. With the introduction of a functionally closed system for the glycerolization and deglycerolization of RBC units, extended post-thaw storage became possible. Here, we describe the cryopreservation of red blood cells according to the high-glycerol method, using a functionally closed processing system.

In vitro evaluation of the quality of blood products collected and stored in systems completely free of di(2-ethylhexyl)phthalate-plasticized materials.

BACKGROUND: The plasticizer di(2-ethylhexyl)phthalate (DEHP) is a common component in blood bags. DEHP is noncovalently bound to polyvinylchloride (PVC) polymer and can leach into the blood product. There are public concerns that exposure to DEHP might induce developmental and reproductive toxicity in humans. The aim of this study was to evaluate an alternative plasticizer, di(isononyl) cyclohexane-1,2-dicarboxylate (Hexamoll DINCH, BASF SE), for its use in blood bags. STUDY DESIGN AND METHODS: Whole blood (WB) was collected into DEHP-containing and DEHP-free collection systems. After overnight hold, WB was centrifuged and separated in plasma, buffy coat, and red blood cells (RBCs). Buffy coats and plasma were used to make platelet (PLT) concentrates in DEHP-free systems. After addition of additive solution (AS), SAG-M, PAGGS-M, AS-3, or PAGGG-M, RBCs were leukoreduced and analyzed for in vitro characteristics and plasticizer levels during storage. RESULTS: The use of DINCH-based systems had no effect on WB composition, blood processing, and plasma quality. PLT in vitro quality variables were maintained during storage in DEHP-free systems. During storage in SAG-M, hemolysis was significantly higher in DINCH-PVC while potassium leakage and adenosine triphosphate content were comparable. During storage in alternative ASs, hemolysis was reduced compared to storage in SAG-M. CONCLUSIONS: The complete absence of DEHP in the collection system had no effect on WB composition, processing, or plasma and PLT quality. During storage in SAG-M, the absence of DEHP resulted in increased hemolysis. With alternative ASs like PAGGS-M, AS-3, or PAGGG-M, the absence of DEHP had no effect on hemolysis. Leakage of DINCH into the blood product was less pronounced than that of DEHP.

Evaluation of the quality of blood components obtained after automated separation of whole blood by a new multiunit processor.

BACKGROUND: The Reveos system (Terumo BCT) is a fully automated device able to process four whole blood (WB) units simultaneously into a plasma unit, a red blood cell (RBC) unit, and an interim platelet (PLT) unit (IPU). Multiple IPUs can be pooled to form a transfusable PLT product. The aim of our study was to evaluate the quality of components made with the Reveos system from either fresh (2-8 hr) or overnight-held WB. STUDY DESIGN AND METHODS: A prototype of the Reveos system was used to process WB. RBCs were resuspended in SAGM, leukoreduced, and assayed for in vitro quality variables during a 42-day storage period at 2 to 6 °C. Twenty-four-hour in vivo recovery was determined on Day 42. Plasma was assayed for cellular contamination and activation variables. IPUs were pooled with SSP+ additive solution for in vitro quality assessments during a 7-day storage period at room temperature. RESULTS: Reveos-produced RBCs and plasma units met the predefined requirements. RBC recovery was superior to control units. On Day 42, hemolysis was below 0.8% and in vivo recovery was above 75% for all RBCs. Cellular contamination was lower for Reveos-produced plasma. PLT yield was higher with overnight-stored WB. PLT quality was well maintained during storage with no significant differences between the two groups. CONCLUSION: Blood components prepared with the Reveos from fresh or overnight-held WB meet quality criteria without any relevant difference between the two groups. The Reveos system has the potential to increase efficacy and standardization of blood component preparation.

The effects of cryopreservation on red blood cell rheologic properties.

BACKGROUND: In transfusion medicine, frozen red blood cells (RBCs) are an alternative for liquid-stored RBCs. Little is known about the rheologic properties (i.e., aggregability and deformability) of thawed RBCs. In this study the rheologic properties of high-glycerol frozen RBCs and postthaw stored in saline-adenine-glucose-mannitol medium were compared to those of conventionally liquid-stored and fresh RBCs. STUDY DESIGN AND METHODS: Fresh RBCs were obtained from healthy volunteers. Leukoreduced liquid-stored and thawed deglycerolized RBC units were obtained from the Sanquin Blood Bank. RBCs were tested for aggregability (aggregation index [AI]), deformability (elongation index [EI]), and various hematologic variables. RESULTS: The AI of thawed RBCs was reduced, compared to fresh and liquid-stored RBCs (p<0.05). The EI of stored RBCs was significantly enhanced over a shear stress range of 2.0 to 50Pa compared to fresh RBCs (p<0.05). No significant differences in EI between thawed and 21- or 35-day liquid-stored RBCs were observed. The osmotic fragility, hemolysis, mean cell volume, and mean cell hemoglobin concentration of thawed RBCs were markedly altered, compared to fresh and liquid-stored RBCs (p< 0.05). The adenosine triphosphate (ATP) content of thawed RBCs was similar to 3- or 21-day liquid-stored and fresh RBCs. CONCLUSIONS: Thawed RBCs are more fragile than conventionally liquid-stored and fresh RBC. The freeze-thaw-wash process, however, did not adversely affect the aggregability and deformability or the ATP content of thawed RBCs. Based on the rheologic properties, cryopreserved RBCs are a valuable alternative to liquid-stored RBCs.

Photodynamic treatment with mono-phenyl-tri-(N-methyl-4-pyridyl)-porphyrin for pathogen inactivation in cord blood stem cell products.

BACKGROUND: Hematopoietic stem cell transplants and culture of hematopoietic progenitor cells require pathogen-free conditions. The application of a method of pathogen inactivation in red blood cells using photodynamic treatment (PDT) was investigated for the decontamination of cord blood stem cell (CBSC) products. STUDY DESIGN AND METHODS: CBSC products, spiked with Gram-positive and Gram-negative bacteria, were treated with PDT using mono-phenyl-tri-(N-methyl-4-pyridyl)-porphyrin (Tri-P(4)) and red light. After PDT, in vitro and in vivo evaluation of the CBSC functions were performed. RESULTS: PDT of CBSC products resulted in the inactivation of the bacteria, with Staphylococcus aureus being the most resistant. Complete decontamination was achieved when CBSC products were contaminated with low titers of bacteria. PDT had no effect on white blood cell viability, the ex vivo expansion potential of the progenitor cells, and their capacity to differentiate to various hematopoietic cell lineages. However, PDT reduced the engraftment of human CBSCs in NOD/SCID mice, particularly affecting the B-cell lineage engraftment. CONCLUSION: Pathogen inactivation of CBSC with Tri-P(4)-mediated PDT is feasible at contamination level up to 10 to 20 colony-forming units per mL and can be considered when ex vivo expansion culture is anticipated. However, this treatment is not recommended for transplantation purposes at this time. Further investigations may elucidate why engraftment is diminished.

Improved postthaw viability and in vitro functionality of peripheral blood hematopoietic progenitor cells after cryopreservation with a theoretically optimized freezing curve.

BACKGROUND: The freezing curve currently used for the cryopreservation of peripheral blood stem cell transplants (PBSCTs) has been determined empirically. Although the use of cryopreserved PBSCTs is successful and usually leads to rapid hematopoietic recovery, the freeze-thawing process is known to induce a significant degree of cell death. Furthermore, the infusion of dimethyl sulfoxide (DMSO), used to protect the cells against damage induced by freezing, can cause morbidity. Therefore, optimizing the current cryopreservation protocol (with 10% DMSO and a slow linear cooling curve) with theoretically optimized freezing curves and a lower DMSO concentration might improve the recovery after transplantation. STUDY DESIGN AND METHODS: A theoretical model was used to predict optimal freezing curves for 5 and 10 percent DMSO. CD34+-selected and -unselected PBSCs were cryopreserved with the current or the new freezing curves. Postthaw quality was evaluated by cell viability, colony formation, and megakaryocyte outgrowth. RESULTS: With 10 percent DMSO, the use of the predicted optimal freezing curve resulted in increased postthaw viability of CD34+ cells, colony formation, and megakaryocyte outgrowth. Lowering the DMSO concentration to 5 percent resulted in improved postthaw viability and functionality, which was not further improved by use of the theoretically optimized freezing curve. CONCLUSIONS: Our results indicate that the current cryopreservation method for PBSCTs can be improved by either lowering the DMSO concentration to 5 percent or by using the theoretically optimized freezing curve. Infusion of less DMSO and more viable cells might improve the outcome of PBSCT.

Altered processing of thawed red cells to improve the in vitro quality during postthaw storage at 4 degrees C.

BACKGROUND: The use of a functionally closed system (ACP215, Haemonetics) for the glycerolization and deglycerolization of red blood cell (RBC) units allows for prolonged postthaw storage. In this study, the postthaw quality of previously frozen, deglycerolized RBCs resuspended in saline-adenine-glucose-mannitol (SAGM) or additive solution AS-3 was investigated. STUDY DESIGN AND METHODS: Leukoreduced RBC units were frozen with 40 percent glycerol and stored at -80 degrees C for at least 14 days. The thawed units were deglycerolized with the ACP215, resuspended in SAGM or AS-3, and stored at 2 to 6 degrees C for up to 21 days. RESULTS: The mean +/- standard deviation in vitro freeze-thaw-wash recovery was 81 +/- 5 percent. During storage, hemolysis of deglycerolized cells remained below 0.8 percent for 2 days in SAGM and for 14 days in AS-3. This difference was explained by the protective effect of citrate, which is present in AS-3. Cells stored in AS-3 showed a lower glycolytic activity and a faster decline in adenosine 5'-triphosphate (ATP) than cells in SAGM. Increasing the internal pH of cells before storage in AS-3 by use of phosphate-buffered saline (PBS) in the deglycerolization procedure resulted in elevated lactate production and better maintenance of intracellular ATP content. After 3 weeks of storage, the ATP content of PBS-washed cells amounted to 2.5 +/- 0.5 micromol per g of hemoglobin (Hb), whereas for saline/glucose-washed cells this value was decreased to 1.0 +/- 0.3 micromol per g of Hb. CONCLUSIONS: Leukoreduced, deglycerolized RBCs can be stored for 48 hours in SAGM. Improved ATP levels during refrigerated storage can be observed with thawed cells, resuspended in AS-3, when PBS is used as a washing solution.

Effect of albumin on the photodynamic inactivation of microorganisms by a cationic porphyrin.

BACKGROUND: Photodynamic inactivation (PDI) employs visible light and a photosensitizer to inactivate cells. The technique is currently clinically used for the treatment of several malignancies. However, the PDI of microorganisms still remains in the research phase. PURPOSE: To study the effect of human blood plasma and human serum albumin (HSA) on the PDI of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. METHODS: PDI experiments were performed using white light (30 mW cm-2) and the cationic 5-phenyl-10,15,20-tris(N-methyl-4-pyridyl)porphyrin chloride (TriP[4]) as photosensitizer. RESULTS: The microorganisms could be successfully photoinactivated by TriP[4] when suspended in phosphate buffered saline (PBS). In this medium, P. aeruginosa was the most resistant microorganism. Changing the suspending medium from PBS to human blood plasma reduced the PDI of all three microorganisms. In human blood plasma C. albicans was the most resistant microorganism. The same results were obtained with 4.5% and 7% HSA/PBS suspensions. CONCLUSIONS: Albumin inhibits the PDI of S. aureus, P. aeruginosa and C. albicans in a dose dependent manner. However, our results are encouraging towards the potential future application of PDI for the treatment of superficial wound infections caused by S. aureus, P. aeruginosa and C. albicans.

Positively charged porphyrins: a new series of photosensitizers for sterilization of RBCs.

BACKGROUND: Photodynamic treatment could be a way to inactivate pathogens in RBCs. The objective of this study was to characterize the virucidal activity and RBC-damaging activity of a series of cationic porphyrins. Using the most efficacious photosensitizer, various in-vitro human RBC quality variables and in-vivo RBC survival in Rhesus monkeys were evaluated. STUDY DESIGN AND METHODS: RBCs, spiked with 5 log of extracellular VSV, were treated with porphyrins (25 micro mol/L) and red light (100 W/m2) and essayed for virucidal activity. In-vitro RBC quality variables were assessed during 5 weeks of storage in various ASs. In-vivo survival was investigated with autologous RBCs in Rhesus monkeys. RESULTS: Tri-P(4) was by far the best sensitizer of a series tested, giving the least hemolysis under conditions that resulted in 5 log-kill of extracellular VSV. Under our experimental conditions, the percentage hemolysis in treated cells was 5.1 +/- 1.1 percent after 5 weeks of storage in SAG-M compared to 1.9 +/- 1.1 percent in the untreated control. Storage in AS-3 resulted hemolysis of 2.3 +/- 1.9 percent. With the exception of IgG binding and potassium leakage, RBC quality variables remained unchanged after photodynamic treatment. Addition of reduced glutathione (GSH) during treatment reduced IgG binding. The 24-hour recovery and T50 of treated RBCs in Rhesus monkeys were satisfactory. CONCLUSION: Porphyrin Tri-P(4) may be a suitable photosensitizer for sterilization of RBCs. However, further exploration to optimize the method is necessary to reach clinically acceptable goals.