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On the other hand, the geometry16 and membrane cation permeability of bThal+ RBC render them osmoti- cally resistant, a probably advantageous feature for RBC at storage conditions.17 Of note, baseline adult hemoglo- bin A (HbA) and fetal hemoglobin (HbF) levels in the
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general donor population have been found to be positive- ly associated with resistance of stored RBC to stress hemolysis.18 Moreover, bThal+ RBC exhibit low aggrega- bility,19 probably rendering them less susceptible to the storage-induced tendency to cell aggregation. Improved metabolic20 and total cardiovascular risk profiles,21 along with survival following malaria infection22 have been also reported thalassemia traits. The aim of the present study was to clarify whether the homeostasis of these unique RBC acts positively or negatively towards the challenges of blood banking.
Methods
Biological samples and blood unit preparation
Venous blood from n=204 regular blood donors was collected into EDTA, citrate and serum vacutainer tubes (in vivo study). Thirty-eight donors (18 bThal+ and 20 controls) were selected to evaluate RBC storability in citrate-phosphate-dextrose (CPD)/ saline-adenine-glucose-mannitol (SAGM) for 42 days at 4°C. The two donor groups exhibited typical hematological differ- ences between them but minimal baseline variation in sex, age, donation frequency and other demographics (Online Supplementary Table S1). bThal+ trait was confirmed by Hb elec- trophoresis and molecular identification of mutations. The study was approved by the Ethics Committee of the Department of
The favorable storability of bThal+ RBC
of stored RBC,5 such as deficiency in the activity of glu- cose 6-phosphate dehydrogenase (G6PD) resulting both in increased oxidant stress6 and lower recovery in healthy blood donors.7 Like G6PD deficiency, genetic polymor- phisms associated with non-canonical hemoglobin traits are enriched in certain donor populations. Carrier state for beta thalassemia (bThal+) is characterized by mild effects on globin synthesis and RBC survival, and as such, many bThal+ subjects are eligible blood donors.8 Despite their unique hematological profile, that could affect the capacity of enduring storage stress in either a negative or a positive way, little is known about the storability and recovery of bThal+ RBC.
Apart from RBC indices and minor Hb variants,9 bThal+ RBC may exhibit differences in membrane structure,10 deformability and ion exchange,11 among others. Free heme and iron reactions bring about mild but sustained oxidative stress that when combined with decreased plas- ma antioxidant capacity,12 may lead to oxidative defects in skeletal proteins13 and membrane lipids. Augmented protein phosphorylation14 and proteolytic cleavage of band 315 have been also observed as a probable result of caspase-3 activation. Since the N-terminus of band 3 can regulate metabolic fluxes through glycolysis by means of inhibitory binding to glycolytic enzymes, the aforemen- tioned alterations could trigger excessive consumption of glucose through the glycolytic pathway at the expense of NADPH production by the pentose phosphate pathway (PPP),11 with a consequent deficit in the capacity to fuel several antioxidant systems that rely on this cofactor. Many of these bThal+ RBC distortions are typical storage lesion aspects and some of them have already been linked to poor recovery or adverse transfusion effects.
Biology, School of Science, NKUA. Investigations were carried out upon donor consent, in accordance with the principles of the Declaration of Helsinki.
Physiological parameters
Free Hb levels were measured in plasma/supernatant through spectrophotometry,23 followed by the Allen correction. In order to examine the osmotically induced hemolysis, RBC were exposed to solutions of increasing saline (NaCl) concentration and the mean corpuscular fragility (MCF, concentration of NaCl at 50% hemolysis) was calculated. The mechanical fragility index (MFI) was determined by measuring the amount of Hb released in the supernatant of RBC rocked with stainless steel beads for 1 hour (h). Oxidative hemolysis levels were evaluated following treatment of RBC with 17 mM phenylhydrazine (PHZ) for 1 h at 37°C. Reactive oxygen species (ROS) and calci- um accumulation were measured by fluorometry; the extracel- lular antioxidant activity and lipid peroxidation were deter- mined spectrophotometrically; phosphatidylserine (PS) expo- sure and RBC membrane protein carbonylation were estimated by multicolor flow cytometry and western blotting, respectively (see the Online Supplementary Methods for details).
Omics analyses
Preliminary proteomics analyses were performed in stored samples of RBC membranes through a filter-aided sample prepa- ration digestion prior to analysis via nano-ultra performance liq- uid chromatography - tandem mass spectrometer (nano- UHPLC-MS/MS) (Evosep One system coupled to timsTOF Pro mass spectrometer - Bruker Daltonics, Bremen, Germany), as extensively described in prior technical notes.24 Metabolomics analyses were performed as previously reported.25 100 mL of stored RBC were collected on a weekly basis, extracted at 1:6 dilution in methanol:acetonitrile:water (5:3:2) and analyzed by UHPLC-MS (Ultimate 3000 RSLC-Q Exactive, Thermo Fisher) (see the Online Supplementary Methods for details). Metabolite assignment was performed against an in house standard library, as reported,26 through the freely available software Maven (Princeton University, USA). No data pre-processing (neither normalization nor log-transformation) was performed.
Statistical analysis
For statistical analysis (SPSS Version 22.0, IBM Corp, NKUA) Shapiro-Wilk test and detrended normal Q–Q plots (for testing normal distribution and outliers), independent t-test or repeated measures analysis of variance (ANOVA) with Bonferroni-like adjustment (for intergroup differences) and Pearson's or Spearman's tests (for correlation analysis) were used. Variables that exhibited repeatable correlations between each individual storage day (six time points) and fresh blood were used for the construction of in vivo/ex vivo biological networks (Cytoscape 3.7.2). Receiver operating characteristic (ROC) curves were used to find out parameters strongly indicative of bThal+ status in stored RBC. Significance was accepted at P<0.05.
Results
Baseline features and storability of beta thalassemia red blood cells
In a cohort of 204 eligible blood donors the bThal+ sub- group was approximately 9%. As expected, lower Hb concentration and RBC indices but higher RBC count and HbA2 concentration were measured in bThal+ donors (ROC curve: area under the curve [AUC] for mean cor-
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