Vassilis L. Tzounakas

Effects of pre-storage leukoreduction on stored red blood cells signaling: a time-course evaluation from shape to proteome.

The introduction of pre-storage leukoreduction in the preparation of standard RBCs intended for transfusion provided significant improvement in the quality of labile products and their post transfusion viability and effects, although the literature data are controversial. To elucidate the issue of the probable leukoreduction effects on RBCs storage lesion, we evaluated various storage quality measures in RBCs stored in either leukoreduced (L) or non-leukoreduced (N) units, with emphasis to senescence and oxidative stress associated modifications. Our data suggest that the residual leukocytes/platelets of the labile products represent a stressful storage factor, countering the structural and functional integrity of stored RBCs. Hemolysis, irreversible echinocytosis, microvesiculation, removal signaling, ROS/calcium accumulation, band 3-related senescence modifications, membrane proteome stress biomarkers as well as emergence of a senescence phenotype in young RBCs that is disproportionate to their age, are all encountered more or mostly in N-RBCs compared to the L-RBCs, either for a part or for the whole of the storage period. The partial, yet significant, alleviation of so many storage-related manifestations in the L-RBCs compared to the N-RBCs, is presented for the first time and provides a rational mechanistic interpretation of the improved storage quality and transfusions observed by the introduction of pre-storage leukoreduction. This article is part of a Special Issue entitled: Integrated omics.

Donor-variation effect on red blood cell storage lesion: A close relationship emerges.

Although the molecular pathways leading to the progressive deterioration of stored red blood cells (RBC storage lesion) and the clinical relevance of storage‐induced changes remain uncertain, substantial donor‐specific variability in RBC performance during storage, and posttransfusion has been established (“donor‐variation effect”). In‐bag hemolysis and numerous properties of the RBC units that may affect transfusion efficacy have proved to be strongly donor‐specific. Donor‐variation effect may lead to the production of highly unequal blood labile products even when similar storage strategy and duration are applied. Genetic, undiagnosed/subclinical medical conditions and lifestyle factors that affect RBC characteristics at baseline, including RBC lifespan, energy metabolism, and sensitivity to oxidative stress, are all likely to influence the storage capacity of individual donors’ cells, although not evident by the donors health or hematological status at blood donation. Consequently, baseline characteristics of the donors, such as membrane peroxiredoxin‐2 and serum uric acid concentration, have been proposed as candidate biomarkers of storage quality. This review article focuses on specific factors that might contribute to the donor‐variation effect and emphasizes the emerging need for using omics‐based technologies in association with in vitro and in vivo transfusion models and clinical trials to discover biomarkers of storage quality and posttransfusion recovery in donor blood.

Donor variation effect on red blood cell storage lesion: a multivariable, yet consistent, story.

Previous studies have shown that baseline hematologic characteristics concerning or influencing red blood cell (RBC) properties might affect storage lesion development in individual donors. This study was conducted to evaluate whether variation in hemolysis, microparticle accumulation, phosphatidylserine (PS) exposure, and other storage lesion–associated variables might be a function of the prestorage hematologic and biologic profiles of the donor.

Uric acid variation among regular blood donors is indicative of red blood cell susceptibility to storage lesion markers: A new hypothesis tested.

Oxidative stress orchestrates a significant part of the red blood cell (RBC) storage lesion. Considering the tremendous interdonor variability observed in the “storability,” namely, the capacity of RBCs to sustain the storage lesion, this study aimed at the elucidation of donor‐specific factors that affect the redox homeostasis during the storage of RBCs in standard systems.

Glucose 6-phosphate dehydrogenase deficient subjects may be better "storers" than donors of red blood cells.

Storage of packed red blood cells (RBCs) is associated with progressive accumulation of lesions, mostly triggered by energy and oxidative stresses, which potentially compromise the effectiveness of the transfusion therapy. Concerns arise as to whether glucose 6-phosphate dehydrogenase deficient subjects (G6PD(-)), ~5% of the population in the Mediterranean area, should be accepted as routine donors in the light of the increased oxidative stress their RBCs suffer from. To address this question, we first performed morphology (scanning electron microscopy), physiology and omics (proteomics and metabolomics) analyses on stored RBCs from healthy or G6PD(-) donors. We then used an in vitro model of transfusion to simulate transfusion outcomes involving G6PD(-) donors or recipients, by reconstituting G6PD(-) stored or fresh blood with fresh or stored blood from healthy volunteers, respectively, at body temperature. We found that G6PD(-) cells store well in relation to energy, calcium and morphology related parameters, though at the expenses of a compromised anti-oxidant system. Additional stimuli, mimicking post-transfusion conditions (37°C, reconstitution with fresh healthy blood, incubation with oxidants) promoted hemolysis and oxidative lesions in stored G6PD(-) cells in comparison to controls. On the other hand, stored healthy RBC units showed better oxidative parameters and lower removal signaling when reconstituted with G6PD(-) fresh blood compared to control. Although the measured parameters of stored RBCs from the G6PD deficient donors appeared to be acceptable, the results from the in vitro model of transfusion suggest that G6PD(-) RBCs could be more susceptible to hemolysis and oxidative stresses post-transfusion. On the other hand, their chronic exposure to oxidative stress might make them good recipients, as they better tolerate exposure to oxidatively damaged long stored healthy RBCs.

Data on how several physiological parameters of stored red blood cells are similar in glucose 6-phosphate dehydrogenase deficient and sufficient donors

This article contains data on the variation in several physiological parameters of red blood cells (RBCs) donated by eligible glucose-6-phosphate dehydrogenase (G6PD) deficient donors during storage in standard blood bank conditions compared to control, G6PD sufficient (G6PD+) cells. Intracellular reactive oxygen species (ROS) generation, cell fragility and membrane exovesiculation were measured in RBCs throughout the storage period, with or without stimulation by oxidants, supplementation of N-acetylcysteine and energy depletion, following incubation of stored cells for 24 h at 37 °C. Apart from cell characteristics, the total or uric acid-dependent antioxidant capacity of the supernatant in addition to extracellular potassium concentration was determined in RBC units. Finally, procoagulant activity and protein carbonylation levels were measured in the microparticles population. Further information can be found in “Glucose 6-phosphate dehydrogenase deficient subjects may be better “storers” than donors of red blood cells” [1].

Metabolic Linkage and Correlations to Storage Capacity in Erythrocytes from Glucose 6-Phosphate Dehydrogenase-Deficient Donors

Objective In glucose 6-phosphate dehydrogenase (G6PD) deficiency, decreased NADPH regeneration in the pentose phosphate pathway and subnormal levels of reduced glutathione result in insufficient antioxidant defense, increased susceptibility of red blood cells (RBCs) to oxidative stress, and acute hemolysis following exposure to pro-oxidant drugs and infections. Despite the fact that redox disequilibrium is a prominent feature of RBC storage lesion, it has been reported that the G6PD-deficient RBCs store well, at least in respect to energy metabolism, but their overall metabolic phenotypes and molecular linkages to the storability profile are scarcely investigated. Methods We performed UHPLC-MS metabolomics analyses of weekly sampled RBC concentrates from G6PD sufficient and deficient donors, stored in citrate phosphate dextrose/saline adenine glucose mannitol from day 0 to storage day 42, followed by statistical and bioinformatics integration of the data. Results Other than previously reported alterations in glycolysis, metabolomics analyses revealed bioactive lipids, free fatty acids, bile acids, amino acids, and purines as top variables discriminating RBC concentrates for G6PD-deficient donors. Two-way ANOVA showed significant changes in the storage-dependent variation in fumarate, one-carbon, and sulfur metabolism, glutathione homeostasis, and antioxidant defense (including urate) components in G6PD-deficient vs. sufficient donors. The levels of free fatty acids and their oxidized derivatives, as well as those of membrane-associated plasticizers were significantly lower in G6PD-deficient units in comparison to controls. By using the strongest correlations between in vivo and ex vivo metabolic and physiological parameters, consecutively present throughout the storage period, several interactomes were produced that revealed an interesting interplay between redox, energy, and hemolysis variables, which may be further associated with donor-specific differences in the post-transfusion performance of G6PD-deficient RBCs. Conclusion The metabolic phenotypes of G6PD-deficient donors recapitulate the basic storage lesion profile that leads to loss of metabolic linkage and rewiring. Donor-related issues affect the storability of RBCs even in the narrow context of this donor subgroup in a way likely relevant to transfusion medicine.

Donor-specific individuality of red blood cell performance during storage is partly a function of serum uric acid levels

Previous investigations in leukoreduced units of red blood cells (RBCs) in mannitol additive solution revealed the close association of uric acid (UA) levels in vivo with the susceptibility of RBCs to storage lesion markers. In this study, we examined whether UA has a similar correlation with the capability of RBCs to cope with the oxidative provocations of storage under different conditions, namely, in CPDA‐1 and in the absence of leukoreduction.

Red blood cell transfusion in surgical cancer patients: Targets, risks, mechanistic understanding and further therapeutic opportunities

Anemia is present in more than half of cancer patients and appears to be an independent prognostic factor of short- and long-term adverse outcomes. It increases in the advanced period of cancer and perioperatively, in patients with solid tumors who undergo surgery. As a result, allogeneic red blood cell (RBC) transfusion is an indispensable treatment in cancer. However, its safety remains controversial, based on several laboratory and clinical data reporting a linkage with increased risk for cancer recurrence, infection and cancer-related mortality. Immunological, inflammatory and thrombotic reactions mediated by the residual leukocytes and platelets, the stored RBCs per se, the biological response modifiers and the plasticizer of the unit may underlie infection and tumor-promoting effects. Although the causality between transfusion and infection has been established, the effects of transfusion on cancer recurrence remain confusing; this is mainly due to the extreme biological heterogeneity that characterizes RBC donations and cancer context. In fact, the functional interplay between donation-associated factors and recipient characteristics, including tumor biology per se, inflammation, infection, coagulation and immune activation state and competence may synergistically and individually define the clinical impact of each transfusion in any given cancer patient. Our understanding of how the potential risk is mediated is important to make RBC transfusion safer and to pave the way for novel, promising and highly personalized strategies for the treatment of anemia in surgical cancer patients.

Red cell transfusion in paediatric patients with thalassaemia and sickle cell disease: Current status, challenges and perspectives

Notwithstanding the high safety level of the currently available blood for transfusion and the decreasing frequency of transfusion-related complications, administration of labile blood products to paediatric patients still poses unique challenges and considerations. The incidence of thalassaemia and sickle cell disease in the paediatric population may be high enough under specific racial and geographical contexts. Red cell transfusion is the cornerstone of β-thalassaemia treatment and one of the most effective ways to prevent or correct specific acute and chronic complications of sickle cell disease. However, this life-saving strategy comes with its own complications, such as additional iron overload, alloimmunization and haemolytic reactions, among others. In paediatrics, the dependency of the transfusion outcome upon disease and other recipient characteristics is more prominent compared with the adults, owing to differences in developmental maturity and physiology that render them more susceptible to common risks, exacerbate the host response to transfused cells, and modify the type or the clinical severity of the transfusion-related morbidity. The adverse branch of red cell transfusion is likely the overall effect of several factors acting synergistically to shape the clinical phenotype of this therapy, including inherent donor/blood unit variables, like antigenicity, red cell deformability and extracellular vesicles, as well as recipient variables, such as history of alloimmunization and inflammation level at time of transfusion. This review focuses on paediatric patients with β-thalassaemia and sickle cell disease as a recipient group with distinct transfusion-related characteristics, and introduces new concepts for consideration, not adequately studied and elucidated so far.