Generation of singlet oxygen induces phospholipid scrambling in human erythrocytes.

Maintenance of phospholipid asymmetry of the plasma membrane is essential for cells to prevent phagocytic removal or acceleration of coagulation. Photodynamic treatment (PDT), which relies on the generation of reactive oxygen species to achieve inactivation of pathogens, might be a promising approach in the future for decontamination of red blood cell concentrates. To investigate whether PDT affects phospholipid asymmetry, erythrocytes were illuminated in the presence of 1,9-dimethyl-methylene blue (DMMB) as photosensitizer and subsequently labeled with FITC-labeled annexin V. This treatment resulted in about 10% annexin V positive cells, indicating exposure of phosphatidylserine (PS). Treatment of erythrocytes with N-ethylmaleimide (NEM) prior to illumination, to inhibit inward translocation of PS via the aminophospholipid translocase, resulted in enhanced PS exposure, while treatment with H(2)O(2) (previously shown to inhibit phospholipid scrambling) greatly diminished PS exposure, indicating the induction of phospholipid scrambling by PDT. Only erythrocytes illuminated in the presence of DMMB showed translocation of NBD-phosphatidylcholine (NBD-PC), confirming scrambling induction. Double label experiments indicated that PS exposure does not occur without concurrent scrambling activity. Induction of scrambling was only moderately affected by Ca(2+) depletion of the cells. In contrast, scavengers of singlet oxygen were found to prevent phospholipid scrambling induced by PDT. The results of this study show that phospholipid scrambling is induced in human erythrocytes by exposure to singlet oxygen.

Composition of the additive solution affects red blood cell integrity after photodynamic treatment.

BACKGROUND AND OBJECTIVES: Photodynamic treatment is a promising technique for pathogen inactivation of red blood cell concentrates. For protocol optimization, the influence of the composition of the storage solution on the integrity of phototreated red cells was studied. MATERIALS AND METHODS: Red blood cells were resuspended in the storage solutions SAG-M or AS-3 to a haematocrit (Hct) of 30%. After addition of the photosensitizer, 1,9-dimethylmethylene blue (DMMB) (25 microm), the suspensions were illuminated with red light, and potassium leakage and delayed haemolysis were determined. In some experiments, the cells were washed after illumination and resuspended in modified storage solutions. RESULTS: Illumination of red cells in the presence of DMMB resulted in an immediate, light-dose-dependent increase in potassium leakage. The illumination conditions used induced no detectable haemolysis immediately after photodynamic treatment. Potassium leakage was higher when the illumination was performed in AS-3. In contrast, delayed haemolysis, measured after overnight storage, was considerably lower when cells were stored in AS-3. This protection was mainly a result of the presence of citrate in AS-3. In addition, other impermeant solutes protected against haemolysis. CONCLUSIONS: The additive solution strongly influences the integrity of red cells after photodynamic treatment. Whereas the solution in which the cells are illuminated has a small effect on red cell integrity, the main influence of the additive solution is during post-treatment storage. Red cell integrity is best maintained when illumination is performed in SAG-M followed by storage in AS-3. The presence of non-permeant solutes, such as citrate, in the solution used for storage, prevents haemolysis of the phototreated, cation-permeable cells by counterbalancing the osmotic activity of haemoglobin.

Additive effects of dipyridamole and Trolox in protecting human red cells during photodynamic treatment.

BACKGROUND AND OBJECTIVES: Photodynamic treatment (PDT) of red blood cell (RBC) suspensions has been reported to result in virus inactivation, but also in deterioration of cell quality. Recently, we have demonstrated the potential usefulness of the reactive oxygen species scavenger dipyridamole in selectively protecting RBCs against the harmful side-effects of PDT. Unfortunately, dipyridamole-conferred protection against long-term photohaemolysis was incomplete. In the present study, dipyridamole was applied in combination with Trolox (a hydrophilic vitamin E analogue) in order to augment RBC protection. MATERIALS AND METHODS: Leucodepleted RBC suspensions (30% haematocrit) were treated with 1,9-dimethylmethylene blue (DMMB) and red light, and the effect of inclusion of dipyridamole and Trolox was assessed on potassium leakage as well as on short-term and long-term photohaemolysis. Possible interference of the scavenger cocktail with virus inactivation was examined using extracellular pseudorabies virus (PRV). RESULTS: Treatment of RBC with DMMB and red light resulted in enhanced potassium leakage and both short- and long-term haemolysis. Dipyridamole and Trolox showed additive protective effects against induction of potassium leakage and photohaemolysis, suggesting different protection mechanisms for the two scavengers. Combined inclusion of dipyridamole and Trolox did not interfere with efficacy of PRV inactivation. CONCLUSIONS: Combined inclusion of dipyridamole and Trolox results in substantially improved selectivity of photodynamic treatment of RBC suspensions.