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.

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.

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.

Effect of monovalent and divalent cations on the photoinactivation of bacteria with meso-substituted cationic porphyrins.

It is well established that for successful photoinactivation (PI) of gram-negative bacteria a cationic photosensitizer is required. This requirement suggests a charge-dependent interaction between the photosensitizer and the gram-negative bacterium, which may be influenced by the presence of ions in the suspending medium. The aim of the present study was to investigate the effect of cations Na+ and Ca2+ on the efficacy of the PI of the gram-negative Pseudomonas aeruginosa and the gram-positive Staphylococcus aureus. The bacteria were suspended in buffer containing either meso-tetra(N-methyl-4-pyridyl)-porphyrin or meso-mono-phenyl-tri(N-methyl-4-pyridyl)-porphyrin as photosensitizer and various concentrations of Na+ or Ca2+. The cell suspensions were exposed to a broadband light dose of 9 J/cm2. In buffer without added cations, P. aeruginosa and S. aureus were equally sensitive to PI. Addition of cations strongly decreased the sensitivity of both bacteria to PI, with the PI of P. aeruginosa being much more decreased than that of S. aureus, and Ca2+ being more effective than Na+. The decreased sensitivity was accompanied by a reduced binding of the photosensitizers to the bacteria.

Stability after thawing of RBCs frozen with the high- and low-glycerol method.

BACKGROUND: RBCs can be frozen with either the high-glycerol method (HGM) or the low-glycerol method (LGM). To date, the use of frozen RBCs is hampered by a 24-hour outdating period after thawing. A closed washing system (ACP 215) may solve this problem. STUDY DESIGN AND METHODS: We compared the effects of high- (40%) and low-glycerol (19%) concentration, with and without freezing (at -80 degrees C for HGM, -196 degrees C for LGM) on the in vitro quality of RBCs after deglycerolization with the closed washing system and during storage at 4 degrees C in SAGM after thawing. RESULTS: Glycerol treatment by itself induced hemolysis during processing, which was more pronounced in HGM cells. The freeze-thaw-wash process decreased the stability of RBCs, particularly in LGM cells during storage after thawing. In contrast to LGM cells, in HGM cells no additional effect of freeze or thaw on stability of washed cells was seen during the first week of storage after thawing. Changes in osmotic resistance and cellular metabolism could not explain the observed differences in RBC stability. CONCLUSION: The closed washing system is able to process both high- and low-glycerol-treated RBCs. Stability after washing during cold storage in SAGM, as measured by hemolysis, is better for HGM cells as compared to LGM cells.

Relation between K+ leakage and damage to band 3 in photodynamically treated red cells.

Potassium leakage is one of the first events that appear after photosensitization of red blood cells. This event may subsequently lead to colloid osmotic hemolysis. The aim of our study was to determine which photodynamically induced damage is responsible for increased membrane cation permeability. This was done by studying the effect of dimethylmethylene blue (DMMB)-mediated photodynamic treatment (PDT) on different membrane transport systems. Inhibition of band 3 activity (anion transport) showed a comparable light dose dependency as PDT-induced potassium leakage, whereas glycerol transport activity was inhibited only at higher light doses. Dipyridamole (DIP), an inhibitor of anion transport, protects band 3 against DMMB-induced damage, and prevents the increase in cation permeability of the membrane. Damage to glycerol transport was partially reduced when PDT was performed in the presence of DIP. Because DIP has no affinity for the glycerol transporter, this protection might result from the reduced photodamage to band 3. These results support the hypothesis that band 3 might be involved in glycerol transport. Glucose transport was not affected by DMMB-mediated PDT. The present results are the first to show a causal relationship between DMMB-mediated photodamage to band 3 and increased cation permeability of red blood cells.