Dysregulation of bisphosphoglycerate mutase during in vitro maturation of oocytes.

PURPOSE: Oxygen is vital for oocyte maturation; however, oxygen regulation within ovarian follicles is not fully understood. Hemoglobin is abundant within the in vivo matured oocyte, indicating potential function as an oxygen regulator. However, hemoglobin is significantly reduced following in vitro maturation (IVM). The molecule 2,3-bisphosphoglycerate (2,3-BPG) is essential in red blood cells, facilitating release of oxygen from hemoglobin. Towards understanding the role of 2,3-BPG in the oocyte, we characterized gene expression and protein abundance of bisphosphoglycerate mutase (Bpgm), which synthesizes 2,3-BPG, and whether this is altered under low oxygen or hemoglobin addition during IVM. METHODS: Hemoglobin and Bpgm expression within in vivo matured human cumulus cells and mouse cumulus-oocyte complexes (COCs) were evaluated to determine physiological levels of Bpgm. During IVM, Bpgm gene expression and protein abundance were analyzed in the presence or absence of low oxygen (2% and 5% oxygen) or exogenous hemoglobin. RESULTS: The expression of Bpgm was significantly lower than hemoglobin when mouse COCs were matured in vivo. Following IVM at 20% oxygen, Bpgm gene expression and protein abundance were significantly higher compared to in vivo. At 2% oxygen, Bpgm was significantly higher compared to 20% oxygen, while exogenous hemoglobin resulted in significantly lower Bpgm in the COC. CONCLUSION: Hemoglobin and 2,3-BPG may play a role within the maturing COC. This study shows that IVM increases Bpgm within COCs compared to in vivo. Decreasing oxygen concentration and the addition of hemoglobin altered Bpgm, albeit not to levels observed in vivo.

To study the effect of oxygen carrying capacity on expressed changes of erythrocyte membrane protein in different storage times.

Erythrocyte membrane is crucial to maintain the stability of erythrocyte structure. The membrane protein on the surface of erythrocyte membrane enables erythrocyte to have plasticity and pass through the microcirculation without being blocked or destroyed. Decreased deformability of erythrocyte membrane protein will lead to a series of pathological and physiological changes such as tissue and organ ischemia and hypoxia. Therefore, this research collected 30 cases of healthy blood donors, and explored erythrocyte stored at different times relating indicators including effective oxygen uptake (Q), P50, 2,3-DPG, Na+-k+-ATP. Erythrocyte morphology was observed by electron microscopy. Western blot and immunofluorescence assay were used to detect membrane protein EPB41, S1P, GLTP, SPPL2A expression changes of erythrocyte. To explore the effective carry oxygen capacity of erythrocyte at different storage time resulting in the expression change of erythrocyte surface membrane protein.

Effect of phase-change material blood containers on the quality of red blood cells during transportation in environmentally-challenging conditions.

BACKGROUND: The effect of phase-change material blood containers on the quality of stored red blood cells (RBCs) transported in the Qinghai-Tibet Plateau remains to be studied. STUDY DESIGN AND METHODS: RBCs stored in a phase-change material blood container were transported from Chengdu to Tibet and then back to Chengdu. The detection time points were the 1st day of fresh-collected RBCs (group 1), the 14th day of resting refrigerated storage (group 2), and the 14th day of plateau transportation under refrigerated storage in the container (group 3). RBC counts, hemoglobin (HGB) content, free hemoglobin (FHb) content, blood biochemical indexes, hemorheologic indexes and 2,3-DPG content were detected. RESULTS: Compared with group 2, RBC counts and HGB were decreased, and the mean corpuscular volume (MCV), FHb and K+ content were increased in group 3. The glucose consumption and lactic acid production were significantly increased in groups 2 and 3. Compared with group 2, the 2,3-DPG content and whole blood viscosity were decreased in group 3. After resting refrigerated storage and plateau transportation, the RBC quality still met the national standard (GB18469-2012 whole blood and component blood quality requirements). CONCLUSION: The phase-change material blood container can be maintained at a constant temperature under plateau environmental conditions, ensuring that the quality of the stored RBCs is compliant with GB18469-2012 whole blood and component blood quality requirements.

Discovery of metabolic alterations in the serum of patients infected with Plasmodium spp. by high-resolution metabolomics.

INTRODUCTION: Despite the advances in diagnosis and treatment, malaria has still not been eradicated. Metabolic interactions between the host and Plasmodium may present novel targets for malaria control, but such interactions are yet to be deciphered. An exploration of metabolic interactions between humans and two Plasmodium species by high-resolution metabolomics may provide fundamental insights that can aid the development of a new strategy for the control of malaria. OBJECTIVES: This study aimed at exploring the metabolic changes in the sera of patients infected with Plasmodium falciparum and Plasmodium vivax. METHODS: Uni- and multivariate metabolomic analyses were performed on the sera of four groups of patients, namely normal control (N, n = 100), P. falciparum-infected patients (PF, n = 21), P. vivax-infected patients (PV, n = 74), and non-malarial pyretic patients (Pyr, n = 25). RESULTS: Univariate and multivariate analyses of N, PF, and PV groups showed differential metabolic phenotypes and subsequent comparisons in pairs revealed significant features. Pathway enrichment test with significant features showed the affected pathways, namely glycolysis/gluconeogenesis for PF and retinol metabolism for PV. The metabolites belonging to the affected pathways included significantly low 2,3-diphosphoglycerate and glyceraldehyde-3-phosphate in the sera of PF. The sera of PV had significantly low levels of retinol but high levels of retinoic acid. CONCLUSION: Our study reveals metabolic alterations induced by Plasmodium spp. in human serum and would serve as a milestone in the development of novel anti-malarial strategies.

Alzheimer's Disease: Erythrocyte 2,3-diphosphoglycerate Content and Circulating Erythropoietin.

BACKGROUND: Alzheimer's Disease (AD) features the accumulation of ß-amyloid in erythrocytes. The subsequent red cell damage may well affect their oxygen-carrying capabilities. 2,3- diphosphoglycerate (2,3-DPG) binds to the hemoglobin thereby promoting oxygen release. It is theorized that 2,3-DPG is reduced in AD and that the resulting hypoxia triggers erythropoietin (EPO) release. METHODS & OBJECTIVE: To explore this theory, we analyzed red cell 2,3-DPG content and EPO in AD, mild cognitive impairment, and the control group, subjective cognitive impairment. RESULTS: We studied (i) 2,3-DPG in red cells, and (ii) circulating EPO in AD, and both markers were unaffected by dementia. Disturbances of these oxygen-regulatory pathways do not appear to participate in brain hypoxia in AD.

The expression of CD47 and its association with 2,3-DPG levels in stored leuco-reduced blood units.

BACKGROUND: Red blood cell (RBC) aging in transfusion medicine is characterized by alteration of many biochemical and morphological integrity of the cell referred to as red cell storage lesion (RCSL), CD47 is a protective marker expressed on RBCs that salvage the cell from phagocytosis. 2,3-diphosphoglycerate (2,3-DPG) tends to have a greater affinity towards deoxygenated hemoglobin. Any oxygen unloading at tissue capillaries are facilitated by 2,3-DPG, and any alterations in its levels can significantly interfere with oxygen release. Alteration of both CD47 expression and 2,3-DPG levels during red cell storage may serve as markers in the development of RCSL. The aim of this study was to validate the impact of storage time and leuco-depletion on CD47 expression on the RBCs, which could be a prospective marker for detection of RBCs viability and to clarify if the changes in CD47 expression and 2,3-DPG levels are correlated during storage of Packed RBCs. SUBJECTS AND METHODS: One hundred samples from Packed RBCs units were divided into two groups [Group 1 comprised unfiltered packed red cell units (n=50), whereas Group 2 included filtered "leuco-reduced" red cell units (n=50)]. Collection of samples was executed on days 0, 1 and 21. Each sample was measured for 2,3-DPG and alteration of CD47 expression on RBC using flow cytometry. RESULTS: Decreased CD47 expression along the storage period was statistically significant in both groups (P<0.05). Interestingly, the expression of CD47 was significantly higher in group 2 than group 1 on day zero, 1st and 21st days (P<0.05). Additionally, a statistically significant decrease in 2,3-DPG level was detected at day 21 of storage in group 1 compared to group 2 with a P-value of <0.001. There was a significant positive correlation (r=0.570, P<0.001) between CD47 MFI on RBC during storage and the level of 2,3-DPG at day 21 from packed RBCs storage. CONCLUSION: Older unfiltered RBC possesses lower expression of CD47 and low levels of 2,3-DPG, however filtration (leucoreduction) of RBCs units may help to retain considerable levels of 2,3-DPG and CD47 and hence sustains preservation of RBCs through reduction of phagocytosis.

In vitro assessment of quality of citrate-phosphate-dextrose-adenine-1 preserved feline blood collected by a commercial closed system.

BACKGROUND: Optimal procedure for storage of feline blood is needed. Open-collection systems have been employed in feline medicine, thus limiting the possibility for storage. OBJECTIVES: To evaluate indicators of quality of feline blood stored for 35 days at +4°C in a closed-collection system specifically designed for cats. ANIMALS: Eight healthy adult European domestic shorthair cats with a weight of 5-6.8 kg. METHODS: This is a case series study. A bacteriological test, CBC, blood smear, pH, osmotic fragility, 2,3-diphosphoglycerate (2,3-DPG), and adenosine triphosphate (ATP) measurement were performed weekly on whole blood (WB) units from day 1 to day 35 after donation. The hemolysis index, lactate and potassium concentrations, prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen were measured on plasma aliquots. RESULTS: One out of eight blood units (BUs) had bacterial growth (Serratia marcescens) at day 35. No significant differences were found regarding CBC, morphology, pH, and osmotic fragility. Despite high inter-individual variability and low starting levels, significant decreases in the mean concentrations of 2,3-DPG (T0 1.99 mmol/g Hb, SD 0.52, T35 1.25 mmol/g Hb, SD 1.43; P = .003) and ATP (T0 1.45 mmol/g Hb, SD 0.71, T35 0.62 mmol/g Hb, SD 0.51; P < .001) were detected during the study, as opposed to an increase in hemolysis (T0 0.11 mmol/L, SD 0.07, T35 0.84 mmol/L, SD 0.19; P < .001), lactate (T0 3.30 mmol/L, SD 0.86, T35 13.36 mmol/L, SD 2.90; P < .001), and potassium (T0 3.10 mmol/L, SD 0.21, T35 4.12 mmol/L, SD 0.35; P < .001) concentrations. CONCLUSIONS AND CLINICAL IMPORTANCE: The commercial BU kit is appropriate for blood collection and conservation of WB in cats. The maintenance of WB quality indicators during storage is essential for future improvements of feline transfusion medicine.

The relationship between plasma vascular endothelial growth factor and erythrocyte 2,3-bisphosphoglycerate: The putative role of chronic hypoxia.

The non-invasive assessment of chronic tissue hypoxia is difficult. Pulse oximetry only allows the peripheral oxygen saturation to be measured, while the detection of hyperlactataemia needs to take into account the fact that the accumulation of lactic acid may result from several causes other than prolonged tissue hypoxia. Arterial blood oxygen measurement is invasive and often does not give a good indication of the level of tissue hypoxia. Other suggested methods include the use of positron emission tomography, magnetic resonance T2∗ relaxation time measurement, photoacoustics and high-frequency ultrasound. Tissue hypoxia leads to increased levels of hypoxia-inducible factor-1α, which in turn upregulates VEGFA, leading to increased levels of vascular endothelial growth factor (VEGF), which promote angiogenesis. Hypoxia lasting for more than a few hours is associated with increased synthesis in erythrocytes of 2,3-bisphosphoglycerate (BPG), a powerful regulator of the allosteric properties of haemoglobin, via the Rapoport-Luebering phosphoglycerate cycle. We therefore hypothesised that plasma VEGF and erythrocyte BPG levels should be positively correlated. Venous blood samples from 34 patients (18 male, mean age (standard error) 43.4 (3.2) y) were analysed; plasma VEGF was measured using an enzyme-linked immunosorbent assay while the erythrocyte BPG was assessed by quantitative Fourier transform infrared spectrometry following gel electrophoresis. The Pearson product-moment correlation between the two variables was 0.622 (p < 0.0001). Based on our findings, we suggest that it may be useful to measure both erythrocyte BPG and plasma VEGF, together, when assessing chronic hypoxia; elevated levels of both are likely to indicate hypoxia.

A fit-for-purpose LC-MS/MS method for the simultaneous quantitation of ATP and 2,3-DPG in human K2EDTA whole blood.

Many hemolytic anemias results in major metabolic abnormalities: two common metabolite abnormalities include increased levels of 2,3-diphosphoglycerate (2,3-DPG) and decreased levels of adenosine triphosphate (ATP). To better monitor the concentration changes of these metabolites, the development of a reliable LC-MS/MS method to quantitatively profile the concentrations of 2, 3-DPG and ATP in whole blood is essential to understand the effects of investigational therapeutics. Accurate quantification of both compounds imposes great challenges to bioanalytical scientists due to their polar, ionic and endogenous nature. Here we present an LC-MS/MS method for the reliable quantification of 2,3-DPG and ATP from K2EDTA human whole blood (WB) simultaneously. Whole blood samples were spiked with stable isotope labeled internal standards, processed by protein precipitation extraction, and analyzed using zwitterionic ion chromatography-hydrophilic interaction chromatography (ZIC-HILIC) coupled with tandem mass spectrometry. The linear analytical range of the assay was 50-3000µg/mL. The fit-for-purpose method demonstrated excellent accuracy and precision. The overall accuracy was within ±10.5% (%RE) for both analytes and the intra- and inter-assay precision (%CV) were less than 6.7% and 6.2% for both analytes, respectively. ATP and 2,3-DPG were found to be stable in human K2EDTA blood for at least 8h at 4°C, 96days when stored at -70°C and after three freeze/thaw cycles. The assay has been successfully applied to K2EDTA human whole blood samples to support clinical studies.

Hypophosphatemia-induced Cardiomyopathy.

Relatively few studies have been conducted to evaluate the effect of hypophosphatemia on cardiac function. The goal of this review was to determine whether there is an association between hypophosphatemia and cardiac function and to increase awareness of hypophosphatemia-induced cardiomyopathy as a new clinical entity and a reversible cause of heart failure. We searched MEDLINE and PubMed from 1971 until March 2015 for primary studies, which reported the relationship between hypophosphatemia and cardiac function. A total of 837 articles were initially obtained. Of these articles, 826 publications were excluded according to the inclusion and exclusion criteria. In all, 11 articles were included in this review. These articles included 7 case series or case reports, 1 case-control study, 1 pretest versus posttest in a single group and 2 animal studies. In conclusion, the mechanisms of hypophosphatemia in cardiomyopathy have been reported to be a depletion of adenosine triphosphate in myocardial cells and decreased 2,3-diphosphoglycerate in erythrocytes. After correction of hypophosphatemia, left ventricular performance seems to improve in patients with severe hypophosphatemia, but not in those with mild-to-moderate hypophosphatemia. However, analyses of the relationship between cardiac function and hypophosphatemia using clinical end points have not been conducted.

Beneficial Role of Erythrocyte Adenosine A2B Receptor-Mediated AMP-Activated Protein Kinase Activation in High-Altitude Hypoxia.

BACKGROUND: High altitude is a challenging condition caused by insufficient oxygen supply. Inability to adjust to hypoxia may lead to pulmonary edema, stroke, cardiovascular dysfunction, and even death. Thus, understanding the molecular basis of adaptation to high altitude may reveal novel therapeutics to counteract the detrimental consequences of hypoxia. METHODS: Using high-throughput, unbiased metabolomic profiling, we report that the metabolic pathway responsible for production of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), a negative allosteric regulator of hemoglobin-O2 binding affinity, was significantly induced in 21 healthy humans within 2 hours of arrival at 5260 m and further increased after 16 days at 5260 m. RESULTS: This finding led us to discover that plasma adenosine concentrations and soluble CD73 activity rapidly increased at high altitude and were associated with elevated erythrocyte 2,3-BPG levels and O2 releasing capacity. Mouse genetic studies demonstrated that elevated CD73 contributed to hypoxia-induced adenosine accumulation and that elevated adenosine-mediated erythrocyte A2B adenosine receptor activation was beneficial by inducing 2,3-BPG production and triggering O2 release to prevent multiple tissue hypoxia, inflammation, and pulmonary vascular leakage. Mechanistically, we demonstrated that erythrocyte AMP-activated protein kinase was activated in humans at high altitude and that AMP-activated protein kinase is a key protein functioning downstream of the A2B adenosine receptor, phosphorylating and activating BPG mutase and thus inducing 2,3-BPG production and O2 release from erythrocytes. Significantly, preclinical studies demonstrated that activation of AMP-activated protein kinase enhanced BPG mutase activation, 2,3-BPG production, and O2 release capacity in CD73-deficient mice, in erythrocyte-specific A2B adenosine receptor knockouts, and in wild-type mice and in turn reduced tissue hypoxia and inflammation. CONCLUSIONS: Together, human and mouse studies reveal novel mechanisms of hypoxia adaptation and potential therapeutic approaches for counteracting hypoxia-induced tissue damage.

2,3-Diphosphoglycerate Concentrations in Autologous Salvaged Versus Stored Red Blood Cells and in Surgical Patients After Transfusion.

BACKGROUND: Stored red blood cells (RBCs) are deficient in 2,3-diphosphoglycerate (2,3-DPG), but it is unclear how autologous salvaged blood (ASB) compares with stored blood and how rapidly 2,3-DPG levels return to normal after transfusion. Therefore, we compared levels of 2,3-DPG in stored versus ASB RBCs and in patients' blood after transfusion. METHODS: Twenty-four patients undergoing multilevel spine fusion surgery were enrolled. We measured 2,3-DPG and the oxyhemoglobin dissociation curve (P50) in samples taken from the ASB and stored blood bags before transfusion and in blood samples drawn from patients before and after transfusion. RESULTS: The mean storage duration for stored RBCs was 24 ± 8 days. Compared with fresh RBCs, stored RBCs had decreased 2,3-DPG levels (by approximately 90%; P < 0.0001) and a decreased P50 (by approximately 30%; P < 0.0001). However, ASB RBCs did not exhibit these changes. The mean 2,3-DPG concentration decreased by approximately 20% (P < 0.05) in postoperative blood sampled from patients who received 1 to 3 stored RBC units and by approximately 30% (P < 0.01) in those who received ≥4 stored RBC units. 2,3-DPG was unchanged in patients who received no stored blood or ASB alone. After surgery, 2,3-DPG levels recovered gradually over 3 postoperative days in patients who received stored RBCs. CONCLUSIONS: Stored RBCs, but not ASB RBCs, have decreased levels of 2,3-DPG and a left-shift in the oxyhemoglobin dissociation curve. Postoperatively, 2,3-DPG levels remain below preoperative baseline levels for up to 3 postoperative days in patients who receive stored RBCs but are unchanged in those who receive only ASB RBCs.

Relationship between chronic disturbance of 2,3-diphosphoglycerate metabolism in erythrocytes and Alzheimer disease.

Alzheimer disease (AD) is one of the most common neurodegenerative disorders widely occurring among the elderly. The pathogenic mechanisms involved in the development of this disease are still unknown. In AD, in addition to brain, a number of peripheral tissues and cells are affected, including erythrocytes. In this study, we analyzed glycolytic energy metabolism, antioxidant status, glutathione, adenylate and proteolytic systems in erythrocytes from patients with AD and compared with those from age-matched controls and young adult controls. Glycolytic enzymes hexokinase, phosphofructokinase, bisphosphoglycerate mutase and bisphosphoglycerate phosphatase displayed lower activities in agematched controls, and higher activities in AD patients, as compared to those in young adult control subjects. In both aging and AD, oxidative stress is increased in erythrocytes whereas elevated concentrations of hydrogen peroxide and organic hydroperoxides as well as decreased glutathione/glutathione disulfide ratio and glutathione transferase activity can be detected. These oxidative disturbances are also accompanied by reductions in ATP levels, adenine nucleotide pool size and adenylate energy charge. Caspase-3 and calpain activities in age-matched controls and AD patients were about three times those of young adult controls. 2,3-diphosphoglycerate levels were significantly decreased in AD patients. Taken together these data suggest that AD patients are associated with chronic disturbance of 2,3-diphosphoglycerate metabolism in erythrocytes. These defects may play a central role in pathophysiological processes predisposing elderly subjects to dementia.

Oxygen carrying capacity of salvaged blood in patients undergoing off-pump coronary artery bypass grafting surgery: a prospective observational study.

BACKGROUND: Intraoperative cell salvage (ICS), hereby referred to 'mechanical red cell salvage', has been widely used and proven to be an effective way to reduce or avoid the need for allogeneic red blood cells (RBCs)transfusion and its associated complications in surgeries involving major blood loss. However, little is known about the influence of this technique on the functional state of salvaged RBCs. Furthermore, there are no articles that describe the change of free hemoglobin (fHb) in salvage blood during storage, which is a key index of the quality control of salvaged blood. Therefore, in this study, the influence of ICS on the function of salvaged RBCs and the changes of salvaged RBCs during storage were studied with respect to the presence of oxyhemoglobin affinity (recorded as a P50 value) and the level of 2, 3-diphosphoglycerate (2, 3-DPG) and fHb by comparing salvaged RBCs with self-venous RBCs and 2-week-old packed RBCs. METHODS: Fifteen patients undergoing off-pump coronary artery bypass grafting (OPCAB) surgery were enrolled. Blood was collected and processed using a Dideco Electa device. The level of P50, 2, 3-DPG and fHB from salvaged RBCs, venous RBCs and 2-week-old packed RBCs was measured. We also measured the changes of these indicators among salvaged RBCs at 4 h (storage at 21-24 °C) and at 24 h (storage at 1-6 °C). RESULTS: The P50 value of salvaged RBCs at 0 h (28.77 ± 0.27 mmHg) was significantly higher than the value of venous RBCs (27.07 ± 0.23 mmHg, p=0.000) and the value of the 2-week-old packed RBCs (16.26 ± 0.62 mmHg, p=0.000). P50 value did not change obviously at 4 h (p=0.121) and 24 h (p=0.384) compared with the value at 0 h. The 2, 3-DPG value of salvaged RBCs at 0 h (17.94 ± 6.91 µmol/g Hb) was significantly higher than the value of venous RBCs (12.73 ± 6.52 mmHg, p = 0.007) and the value of the 2-week-old packed RBCs (2.62 ± 3.13 mmHg, p=0.000). The level of 2, 3-DPG slightly decreased at 4 h (p=0.380) and 24 h (p=0.425) compared with the value at 0 h. Percentage of hemolysis of the salvaged blood at 0 h(0.51 ± 0.27 %) was significantly higher than the level of venous blood (0.07 ± 0.05 %, p=0.000) and the value of 2-week-old packed RBCs (0.07 ± 0.05 %, p=0.000), and reached 1.11 ± 0.42 % at 4 h (p=0.002) and 1.83 ± 0.77 % at 24 h (p=0.000). CONCLUSIONS: The oxygen transport function of salvaged RBCs at 0 h was not influenced by the cell salvage process and was better than that of the venous RBCs and 2-week-old packed RBCs. At the end of storage, the oxygen transport function of salvaged RBCs did not change obviously, but percentage of hemolysis significantly increased.

Effect of propolis on erythrocyte rheology in experimental mercury intoxication in rats.

In the present study, changes in erythrocyte rheology in association with mercury toxicity and the role of propolis were analyzed in rats. Forty male Wistar Albino rats that were 4-5 months old were used in the study. The control group was administered normal saline intraperitoneal (ip) injections; the mercury chloride group was administered HgCl2 (4 mg/kg, ip); the propolis group was administered propolis (200 mg/kg, by gavage); and the HgCl2+ propolis group was administered HgCl2 (4 mg/kg, ip) + propolis (200 mg/kg, by gavage) for 3 days. The following parameters were analyzed: hematological parameters, plasma potassium (K) levels, methemoglobin, 2,3-DPG, erythrocyte deformability, and hemolysis as a percentage. The results revealed that leukocyte count significantly increased, and a significant decline occurred in the platelet count (p < 0.01). Serum K(+), MetHb, 2, 3-DPG, and hemolysis percentage significantly increased in the rats exposed to mercury (p < 0.01). However, the values of rats administered only with propolis were close to the values of the control group and the changes were avoided by the administration of propolis as protection in the rats exposed to mercury chloride.

The effects of an overnight holding of whole blood at room temperature on haemoglobin modification and in vitro markers of red blood cell aging.

BACKGROUND: Some effects of the red blood cell (RBC) storage lesion are well documented whereas others are not. Whether a period of room temperature hold (RTH) during RBC production enhances the RBC storage lesion has remained controversial. In this study, we compared whole blood (WB)-derived RBCs produced after 24-h RTH with rapidly cooled (RC) RBCs and tested them for classical metabolic markers and signs of oxidative damage. STUDY DESIGN AND METHODS: SAGM-RBCs were prepared from mixed and split pairs (n = 12) of WB units. RBCs prepared after a 24-h period of RTH on day+1 after collection (RTH-RBCs) were compared with RC-RBCs. All RBCs were stored at 4°C for 42 days with assay of in vitro variables on days+1, +15, +22, +29 and +42. The study examined standard quality parameters, glutathione, catalase and superoxide dismutase (SOD) activities, and indicative markers of oxidative cell damage including post-translational haemoglobin modification, malondialdehyde (MDA), and phosphatidylserine expression. RESULTS: RTH-RBCs exhibited decreased levels of potassium (1·98 ± 0·26 vs. 5·23 ± 0·65 mmol/l) and of 2,3-diphosphoglycerate (2,3-DPG) on day+1 compared with RC-RBCs. Haemolysis rate on day+42 was higher in RTH-RBCs than in RC-RBCs (0·52 ± 0·13 vs. 0·37 ± 0·12%). The phosphatidylserine expression amounted to 0·25 ± 0·20% in RTH-RBCs and 0·07 ± 0·12% in RC-RBCs. Haemoglobin modification was not different between both RBC groups. RTH-RBCs showed slightly higher MDA concentration on days +29 and +42. CONCLUSIONS: RC-RBCs and RTH-RBCs show only small differences of classical in vitro parameters and no relevant differences in antioxidative metabolism and oxidative haemoglobin modification. These findings do not explain the loss observed in in vivo survival studies with RBCs.

AS-7 improved in vitro quality of red blood cells prepared from whole blood held overnight at room temperature.

BACKGROUND: Extended room temperature (RT) hold of whole blood (WB) may affect the quality of red blood cell (RBC) components produced from these donations. The availability of better RBC additive solutions (ASs) may help reduce the effects. A new AS, AS-7 (SOLX, Haemonetics Corporation), was investigated for improved in vitro quality of RBCs prepared from WB held overnight at RT. STUDY DESIGN AND METHODS: Sixteen WB units were held for 21.4 hours ± 40 minutes at 22°C on cooling plates before processing. Each pair of ABO-matched WB units were pooled, divided into a WB filter pack containing saline-adenine-glucose-mannitol (control) and a LEUKOSEP WB-filter pack containing SOLX, and processed according to manufacturer's instructions. RBCs were stored at 2 to 6°C and sampled weekly until expiry. Glycophorin A (GPA+) and annexin V-binding microparticles (MPs) were quantitated using flow cytometry. Osmotic fragility, intracellular pH (pHi), adenosine triphosphate (ATP), 2,3-diphosphoglycerate (2,3-DPG), and routine quality variables were measured. Adhesion of RBCs to human endothelial cells (ECs) was evaluated by flow perfusion under low shear stress (0.5 dyne/cm(2) ), similar to low blood flow in microvessels. RESULTS: ATP and 2,3-DPG levels were improved for SOLX-RBCs. SOLX-RBCs maintained higher pHi, increased resistance to hypotonic stress, and reduced numbers of GPA+ MPs. No significant difference was observed between annexin V binding to MPs or adhesion of RBCs to ECs under shear stress. CONCLUSION: SOLX-stored RBCs showed increased osmotic resistance, pHi, and reduced GPA+ MPs and together with higher ATP and 2,3-DPG levels demonstrated improved in vitro RBC quality measures during 42 days of storage.

Effects of storage-aged red blood cell transfusions on endothelial function in hospitalized patients.

BACKGROUND: Clinical and animal studies indicate that transfusions of older stored red blood cells (RBCs) impair clinical outcomes as compared to fresh RBC transfusions. It has been suggested that this effect is due to inhibition of nitric oxide (NO)-mediated vasodilation after transfusion of older RBC units. However, to date this effect has not been identified in human transfusion recipients. STUDY DESIGN AND METHODS: Forty-three hospitalized patients with transfusion orders were randomly assigned to receive either fresh (<14 days) or older stored (>21 days) RBC units. Before transfusion, and at selected time points after the start of transfusion, endothelial function was assessed using noninvasive flow-mediated dilation assays. RESULTS: After transfusion of older RBC units, there was a significant reduction in NO-mediated vasodilation at 24 hours after transfusion (p = 0.045), while fresh RBC transfusions had no effect (p = 0.231). CONCLUSIONS: This study suggests for the first time a significant inhibitory effect of transfused RBC units stored more than 21 days on NO-mediated vasodilation in anemic hospitalized patients. This finding lends further support to the hypothesis that deranged NO signaling mediates adverse clinical effects of older RBC transfusions. Future investigations will be necessary to address possible confounding factors and confirm these results.