Modeling hemoglobin levels of patients chronically transfused with red blood cells.

BACKGROUND: Good models of chronic red blood cell (RBC) transfusion can improve care by accurately estimating time-dependent hemoglobin (Hb) levels and clinically relevant transfusion parameters. STUDY DESIGN AND METHODS: A previously derived mathematical model based on overall Hb mass balance (HMB model) with input parameters of transfused units, transfusion efficiency, RBC lifespan, endogenous Hb, and transfusion interval was applied to three separate clinical studies spanning six transfusion conditions across patient populations with myelodysplastic syndrome (MDS) or thalassemia. RESULTS: The HMB model accurately predicted mean pretransfusion Hb levels for each ensemble cohort of patients with thalassemia or MDS. Dynamic changes in Hb levels were modeled as a function of changes in key input parameters. Improving the 24-h post-transfusion RBC survival from 72% to 86% can be used to either (1) reduce RBC usage by 15%-20% through longer transfusion intervals or (2) increase pretransfusion [Hb] by 8%-11% while maintaining a constant transfusion interval. DISCUSSION: The endogenous Hb level is introduced in the HMB model which represents the patient's self-contribution to overall Hb levels via the autologous RBC lifecycle and was estimated to be 5.0 g/dL for patients with MDS or thalassemia. Transfusion therapy approaches and complementary therapeutics can target multiple, unique model inputs while monitoring net, overall impact on transfusion efficacy. Applying the HMB model to fit individual patient Hb fluctuations will be explored in the future.

Safety of hypoxic red blood cell administration in patients with transfusion-dependent hematological malignancies: An interim analysis.

Anemia is a common symptom of hematological malignancies and red blood cell (RBC) transfusion is the primary supportive treatment, with many patients becoming transfusion dependent. Hemanext Inc. (Lexington, MA, United States) has developed a CE mark certified device to process and store RBCs hypoxically - citrate-phosphatedextrose (CPD)/phosphate-adenine-glucose-guanosine-saline-mannitol (PAGGSM) RBCs, leukocytes-reduced (LR), O2/CO2 reduced - with the aim of improving RBC quality for transfusion. This interim analysis describes the first patients to receive hypoxic RBCs, administered as part of a pilot post-marketing study in Norway. The primary outcome was adverse events (AEs) within 24 h of transfusion initiation and overall up to 7 days ( ± 1 day) post-transfusion. Secondary outcomes included changes in hemoglobin levels post-transfusion. Five patients with hematological malignancies were included (80 % male, mean age 69.8 [SD ± 19.3] years). Prior to the study, patients had been receiving conventional RBC transfusions every two weeks. Patients received 2 units of hypoxic RBCs over 2 h without complication. One mild AE (rhinovirus) was reported two days post-treatment and was deemed unrelated to treatment. The mean ± SD pre-transfusion hemoglobin level was 7.7 ± 0.5 g/dL, evolving to 9.0 ± 0.9 g/dL following administration of hypoxic RBCs; an increase of 17 %. This interim analysis showed that transfusion with hypoxic RBCs processed with the CPD/PAGGSM LR, O2/CO2 reduced system was effective and well tolerated in patients with hematologic malignancies. The overall clinical program will assess whether the use of hypoxic RBCs can reduce transfusion interval versus conventional RBCs in patients requiring acute and chronic transfusions.

The oxygen saturation of red blood cell concentrates: The basis for a novel index of red cell oxidative stress.

BACKGROUND: Oxidative stress is a major driving force in the development of storage lesions in red cell concentrates (RCCs). Unlike manufactured pharmaceuticals, differences in component preparation methods and genetic/physiological status of donors result in nonuniform biochemical characteristics of RCCs. Various characteristics of donated blood on oxygen saturation (SO2 ) distribution were investigated, and a model to estimate potential oxidative stress burden of stored RCC at transfusion is proposed. STUDY DESIGN AND METHODS: The oxygen content of freshly prepared RCCs (770) was quantified noninvasively as fractional hemoglobin saturation (SO2 ) with visible reflectance spectrometry. Using separate RCCs and mimicking typical handling of RCCs during routine storage, evolution of SO2 was followed for construction of an empirical model. Based on this model, the oxygen exposure index (OEI) was formulated to estimate the accumulated oxygen exposure burden of RCC at the time of transfusion. RESULTS: The SO2 of RCCs varied widely at donation (mean 43% ± 1.3%; range 20%-93%). Multivariate regression model showed that sex and processing method had small effects on SO2 (R2  = 0.12), indicating that variability was mainly attributed to other individual donor characteristics. Storage simulation model indicated that median SO2 increased gradually over 6 weeks (approx. 1.3 fold), while OEI increased at a faster rate (approx. eight-fold). CONCLUSION: In addition to storage age, the OEI provides a potential new metric to assess the quality of RCCs at the time of transfusion in terms of their oxidative stress. In future studies, a single noninvasive measurement during storage could link OEI to clinical outcomes in transfusion recipients.

Modelling haemoglobin incremental loss on chronic red blood cell transfusions.

BACKGROUND AND OBJECTIVES: Understanding the impact of red blood cell (RBC) lifespan, initial RBC removal, and transfusion intervals on patient haemoglobin (Hb) levels and total iron exposure is not accessible for chronic transfusion scenarios. This article introduces the first model to help clinicians optimize chronic transfusion intervals to minimize transfusion frequency. MATERIALS AND METHODS: Hb levels and iron exposure from multiple transfusions were calculated from Weibull residual lifespan distributions, the fraction effete RBC removed within 24-h (Xe ) and the nominal Hb increment. Two-unit transfusions of RBCs initiated at patient [Hb] = 7 g/dl were modelled for different RBC lifespans and transfusion intervals from 18 to 90 days, and Xe from 0.1 to 0.5. RESULTS: Increased Xe requires shorter transfusion intervals to achieve steady-state [Hb] of 9 g/dl as follows: 30 days between transfusions at Xe  = 0.5, 36 days at Xe  = 0.4, 42 days at Xe  = 0.3, 48 days at Xe  = 0.2 and 54 days at Xe  = 0.1. The same transfusion interval/Xe pairs result in a steady-state [Hb] = 8 g/dl when the RBC lifespan was halved. By reducing transfused RBC increment loss from 30% to 10%, annual transfusions were decreased by 22% with iron addition decreased by 24%. Acute dosing of iron occurs at the higher values of Xe on the day after a transfusion event. CONCLUSION: Systematic trends in fractional Hb incremental loss Xe have been modelled and have a significant and calculatable impact on transfusion intervals and associated introduction of iron.

Hypoxic storage of red blood cells improves metabolism and post-transfusion recovery.

BACKGROUND: Blood transfusion is a lifesaving intervention for millions of recipients worldwide every year. Storing blood makes this possible but also promotes a series of alterations to the metabolism of the stored erythrocyte. It is unclear whether the metabolic storage lesion is correlated with clinically relevant outcomes and whether strategies aimed at improving the metabolic quality of stored units, such as hypoxic storage, ultimately improve performance in the transfused recipient. STUDY DESIGN AND METHODS: Twelve healthy donor volunteers were recruited in a two-arm cross-sectional study, in which each subject donated 2 units to be stored under standard (normoxic) or hypoxic conditions (Hemanext technology). End-of-storage measurements of hemolysis and autologous posttransfusion recovery (PTR) were correlated to metabolomics measurements at Days 0, 21, and 42. RESULTS: Hypoxic red blood cells (RBCs) showed superior PTR and comparable hemolysis to donor-paired standard units. Hypoxic storage improved energy and redox metabolism (glycolysis and 2,3-diphosphoglycerate), improved glutathione and methionine homeostasis, decreased purine oxidation and membrane lipid remodeling (free fatty acid levels, unsaturation and hydroxylation, acyl-carnitines). Intra- and extracellular metabolites in these pathways (including some dietary purines) showed significant correlations with PTR and hemolysis, though the degree of correlation was influenced by sulfur dioxide (SO2 ) levels. CONCLUSION: Hypoxic storage improves energy and redox metabolism of stored RBCs, which results in improved posttransfusion recoveries in healthy autologous recipients-a Food and Drug Administration gold standard of stored blood quality. In addition, we identified candidate metabolic predictors of PTR for RBCs stored under standard and hypoxic conditions.

Oxidative modifications of glyceraldehyde 3-phosphate dehydrogenase regulate metabolic reprogramming of stored red blood cells.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) plays a key regulatory function in glucose oxidation by mediating fluxes through glycolysis or the pentose phosphate pathway (PPP) in an oxidative stress-dependent fashion. Previous studies documented metabolic reprogramming in stored red blood cells (RBCs) and oxidation of GAPDH at functional residues upon exposure to pro-oxidants diamide and H2O2 Here we hypothesize that routine storage of erythrocyte concentrates promotes metabolic modulation of stored RBCs by targeting functional thiol residues of GAPDH. Progressive increases in PPP/glycolysis ratios were determined via metabolic flux analysis after spiking (13)C1,2,3-glucose in erythrocyte concentrates stored in Additive Solution-3 under blood bank conditions for up to 42 days. Proteomics analyses revealed a storage-dependent oxidation of GAPDH at functional Cys152, 156, 247, and His179. Activity loss by oxidation occurred with increasing storage duration and was progressively irreversible. Irreversibly oxidized GAPDH accumulated in stored erythrocyte membranes and supernatants through storage day 42. By combining state-of-the-art ultra-high-pressure liquid chromatography-mass spectrometry metabolic flux analysis with redox and switch-tag proteomics, we identify for the first time ex vivo functionally relevant reversible and irreversible (sulfinic acid; Cys to dehydroalanine) oxidations of GAPDH without exogenous supplementation of excess pro-oxidant compounds in clinically relevant blood products. Oxidative and metabolic lesions, exacerbated by storage under hyperoxic conditions, were ameliorated by hypoxic storage. Storage-dependent reversible oxidation of GAPDH represents a mechanistic adaptation in stored erythrocytes to promote PPP activation and generate reducing equivalents. Removal of irreversibly oxidized, functionally compromised GAPDH identifies enhanced vesiculation as a self-protective mechanism in ex vivo aging erythrocytes.

Hypoxia modulates the purine salvage pathway and decreases red blood cell and supernatant levels of hypoxanthine during refrigerated storage.

Hypoxanthine catabolism in vivo is potentially dangerous as it fuels production of urate and, most importantly, hydrogen peroxide. However, it is unclear whether accumulation of intracellular and supernatant hypoxanthine in stored red blood cell units is clinically relevant for transfused recipients. Leukoreduced red blood cells from glucose-6-phosphate dehydrogenase-normal or -deficient human volunteers were stored in AS-3 under normoxic, hyperoxic, or hypoxic conditions (with oxygen saturation ranging from <3% to >95%). Red blood cells from healthy human volunteers were also collected at sea level or after 1-7 days at high altitude (>5000 m). Finally, C57BL/6J mouse red blood cells were incubated in vitro with 13C1-aspartate or 13C5-adenosine under normoxic or hypoxic conditions, with or without deoxycoformycin, a purine deaminase inhibitor. Metabolomics analyses were performed on human and mouse red blood cells stored for up to 42 or 14 days, respectively, and correlated with 24 h post-transfusion red blood cell recovery. Hypoxanthine increased in stored red blood cell units as a function of oxygen levels. Stored red blood cells from human glucose-6-phosphate dehydrogenase-deficient donors had higher levels of deaminated purines. Hypoxia in vitro and in vivo decreased purine oxidation and enhanced purine salvage reactions in human and mouse red blood cells, which was partly explained by decreased adenosine monophosphate deaminase activity. In addition, hypoxanthine levels negatively correlated with post-transfusion red blood cell recovery in mice and - preliminarily albeit significantly - in humans. In conclusion, hypoxanthine is an in vitro metabolic marker of the red blood cell storage lesion that negatively correlates with post-transfusion recovery in vivo Storage-dependent hypoxanthine accumulation is ameliorated by hypoxia-induced decreases in purine deamination reaction rates.

Methylation of protein aspartates and deamidated asparagines as a function of blood bank storage and oxidative stress in human red blood cells.

BACKGROUND: Being devoid of de novo protein synthesis capacity, red blood cells (RBCs) have evolved to recycle oxidatively damaged proteins via mechanisms that involve methylation of dehydrated and deamidated aspartate and asparagine residues. Here we hypothesize that such mechanisms are relevant to routine storage in the blood bank. STUDY DESIGN AND METHODS: Within the framework of the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study, packed RBC units (n = 599) were stored under blood bank conditions for 10, 23, and 42 days and profiled for oxidative hemolysis and time-dependent metabolic dysregulation of the trans-sulfuration pathway. RESULTS: In these units, methionine consumption positively correlated with storage age and oxidative hemolysis. Mechanistic studies show that this phenomenon is favored by oxidative stress or hyperoxic storage (sulfur dioxide >95%), and prevented by hypoxia or methyltransferase inhibition. Through a combination of proteomics approaches and 13 C-methionine tracing, we observed oxidation-induced increases in both Asn deamidation to Asp and formation of methyl-Asp on key structural proteins and enzymes, including Band 3, hemoglobin, ankyrin, 4.1, spectrin beta, aldolase, glyceraldehyde 3-phosphate dehydrogenase, biphosphoglycerate mutase, lactate dehydrogenase and catalase. Methylated regions tended to map proximal to the active site (e.g., N316 of glyceraldehyde 3-phosphate dehydrogenase) and/or residues interacting with the N-terminal cytosolic domain of Band 3. CONCLUSION: While methylation of basic amino acid residues serves as an epigenetic modification in nucleated cells, protein methylation at carboxylate side chains and deamidated asparagines is a nonepigenetic posttranslational sensor of oxidative stress and refrigerated storage in anucleated human RBCs.

Clinical and pathological impact of VHL, PBRM1, BAP1, SETD2, KDM6A, and JARID1c in clear cell renal cell carcinoma.

VHL is mutated in the majority of patients with clear cell renal cell carcinoma (ccRCC), with conflicting clinical relevance. Recent studies have identified recurrent mutations in histone modifying and chromatin remodeling genes, including BAP1, PBRM1, SETD2, KDM6A, and JARID1c. Current evidence suggests that BAP1 mutations are associated with aggressive disease. The clinical significance of the remaining genes is unknown. In this study, targeted sequencing of VHL and JARID1c (entire genes) and coding regions of BAP1, PBRM1, SETD2, and KDM6A was performed on 132 ccRCCs and matched normal tissues. Associations between mutations and clinical and pathological outcomes were interrogated. Inactivation of VHL (coding mutation or promoter methylation) was seen in 75% of ccRCCs. Somatic noncoding VHL alterations were identified in 29% of ccRCCs and may be associated with improved overall survival. BAP1 (11%), PBRM1 (33%), SETD2 (16%), JARID1c (4%), and KDM6A (3%) mutations were identified. BAP1-mutated tumors were associated with metastatic disease at presentation (P = 0.023), advanced clinical stage (P = 0.042) and a trend towards shorter recurrence free survival (P = 0.059) when compared with tumors exclusively mutated for PBRM1. Our results support those of recent publications pointing towards a role for BAP1 and PBRM1 mutations in risk stratifying ccRCCs. Further investigation of noncoding alterations in VHL is warranted.

Improved Ion Mobility Separation and Structural Characterization of Steroids using Derivatization Methods.

Steroids are an important class of biomolecules studied for their role in metabolism, development, nutrition, and disease. Although highly sensitive GC- and LC-MS/MS-based methods have been developed for targeted quantitation of known steroid metabolites, emerging techniques including ion mobility (IM) have shown promise in improved analysis and capacity to better identify unknowns in complex biological samples. Herein, we couple LC-IM-MS/MS with structurally selective reactions targeting hydroxyl and carbonyl functional groups to improve IM resolution and structural elucidation. We demonstrate that 1,1-carbonyldiimidazole derivatization of hydroxyl stereoisomer pairs such as testosterone/epitestosterone and androsterone/epiandrosterone results in increased IM resolution with ΔCCS > 15%. Additionally, performing this in parallel with derivatization of the carbonyl group by Girard's Reagent P resulted in unique products based on relative differences in number of each functional group and C17 alkylation. These changes could be easily deciphered using the combination of retention time, collision cross section, accurate mass, and MS/MS fragmentation pattern. Derivatization by Girard's Reagent P, which contains a fixed charge quaternary amine, also increased the ionization efficiency and could be explored for its potential benefit to sensitivity. Overall, the combination of these simple and easy derivatization reactions with LC-IM-MS/MS analysis provides a method for improved analysis of known target analytes while also yielding critical structural information that can be used for identification of potential unknowns.

Hypoxia and hypocapnia storage of γ-irradiated red cell concentrates.

BACKGROUND: γ-irradiation is used to treat red blood cell (RBC) concentrates (RCCs) transfused to immunosuppressed patients. This treatment damages RBCs and increases storage lesions. Several studies have shown the beneficial effect of reducing O2 content during RBC storage. The present research work investigated the effect of γ-irradiation on RCCs stored under normal and hypoxia/hypocapnia conditions. MATERIALS AND METHODS: O2 concentration (measured as oxyhaemoglobin fraction, sO2) and ABO-matched RCCs from whole blood donations, leukoreduced and prepared in phosphate, adenine, glucose, guanosine, saline and mannitol (PAGGSM) were pooled and split in two identical RCCs within 24 h post donation. One bag (Hx) was submitted to O2 and CO2 adsorption for 3 h on an orbital shaker at 22±2 °C and then transferred to a storage bag impermeable to gas. The other bag (Ctrl) was left as it was. The two bags were then stored at 4 °C. γ-irradiation (25 Gy) was applied at day 2 or 14, and the RCCs were stored until day 43. Different parameters (metabolites, haemolysis, morphology) were measured. RESULTS: Starting sO2 values were 63.7±18.4% (n=12) in Ctrl and 20.8±9.8% (n=12) in Hx bags, and reached 90.8±9.1% and 6.6±5.9% at day 43, respectively. As expected, an increase in glycolysis rate was observed after deoxygenation. Extracellular potassium concentrations were identical and reached around 70 mM at expiry with an irradiation-dependent kinetic release. No difference in haemolysis was observed after irradiation on day 2 in either group (<0.40%, p>0.9999). When irradiated at day 14, haemolysis was lower (p=0.033) in RCCs under hypoxia at the end of storage (day 28, 0.67±0.16%) compared to control (1.06±0.33%). Percentages of spherocytes were lower under hypoxia. DISCUSSION: The storage under hypoxia provided equivalent storage when RCCs were irradiated at day 2 and was advantageous when irradiated at day 14. In summary, O2-depletion of RCCs enable a better storage of RBCs, particularly when late irradiation is applied.

Oxygen in Red Blood Cell Concentrates: Influence of Donors' Characteristics and Blood Processing.

Objective: Unexpectedly wide distribution (<10 to >90%) of hemoglobin oxygen saturation (sO2) within red cell concentrates (RCCs) has recently been observed. Causes of such variability are not yet completely explained whereas the roles of oxygen and oxidative lesions during the storage of RCCs are known. The objectives of the present study are to characterize sO2 distribution in RCCs produced in a Swiss blood center and to investigate the influence of processing and donors' characteristics. Methods: The level of sO2 was measured in 1701 leukocyte-depleted RCCs derived from whole blood donations in both top-bottom (TB; component filtered, SAGM) and top-top (TT; whole blood filtration, PAGGSM) RCCs. The sO2 value was measured non-invasively through the PVC bag prior to storage by resonance Raman spectroscopy. Gender, age, blood type, hemoglobin level, and living altitude of donors, as well as process method and time-to-process were recorded. Results: Overall, the sO2 exhibited a wide non-Gaussian distribution with a mean of 51.2 ± 18.5%. Use of top-top kits resulted in a 16% higher sO2 (P < 0.0001) than with top-bottom ones. Waiting time before processing only had a modest impact, but the blood processing itself reduced the sO2 by almost 12% (P < 0.0001). sO2 was also significantly affected by some donors' characteristics. RCCs from men exhibited 25% higher sO2 (P < 0.0001) than those donated by women. Multivariate analysis revealed that the apparent correlation observed with hemoglobin level and age was actually due to multicollinearity with the sex variable. Finally, we noticed no significant differences across blood type but found that altitude of residence was associated with the sO2 (i.e., higher in higher living place). Conclusion: These data confirm wide sO2 distribution in RCCs reported recently. The sO2 was impacted by the processing and also by donors' characteristics such as the gender and the living altitude, but not by the hemoglobin level, blood group and donor age. This study provides new hints on the factors influencing red blood cells storage lesions, since they are known to be related to O2 content within the bags, giving clues to better process and to better store RCCs and therefore potentially improve the efficacy of transfusion.

Transfusion of Anaerobically or Conventionally Stored Blood After Hemorrhagic Shock.

BACKGROUND: Resuscitation from hemorrhagic shock (HS) by blood transfusion restores oxygen (O2) delivery and provides hemodynamic stability. Current regulations allow red blood cells (RBCs) to be stored and used for up to 42 days. During storage, RBCs undergo many structural and functional changes. These storage lesions have been associated with adverse events and increased mortality after transfusion, increasing the need for improved RBC storage protocols. This study evaluates the efficacy of anaerobically stored RBCs to resuscitate rats from severe HS compared with conventionally stored RBCs. METHODS AND RESULTS: Rat RBCs were stored under anaerobic, anaerobic/hypercapnic, or conventional conditions for a period of 3 weeks. Hemorrhage was induced by controlled bleeding, shock was maintained for 30 min, and RBCs were transfused to restore and maintain blood pressure near the prhemorrhage level. All storage conditions met current regulatory 24-h posttransfusion recovery requirements. Transfusion of anaerobically stored RBCs required significantly less RBC volume to restore and maintain hemodynamics. Anaerobic or anaerobic/hypercapnic RBCs restored hemodynamics better than conventionally stored RBCs. Resuscitation with conventionally stored RBCs impaired indices of left ventricular cardiac function, increased hypoxic tissue staining and inflammatory markers, and affected organ function compared with anaerobically stored RBCs. CONCLUSIONS: Resuscitation from HS via transfusion of anaerobically stored RBCs recovered cardiac function, restored hemodynamic stability, and improved outcomes.

Physicochemical stability of lipid injectable emulsions: correlating changes in large globule distributions with phase separation behavior.

Single particle optical sensing (SPOS) and visual inspection were used to characterize a series of lipid injectable emulsions (n=21) featuring three lipid types, two electrolyte conditions, and three pH levels (7.0, 4.75, and 2.5). Seven of the twenty-one sample conditions exhibited phase separation instability by visual inspection within 98 h of emulsion preparation. The phase instability was driven by electrolyte type and pH, and "cracking" phenomena were independent of lipid type despite the base lipids ranging almost two orders of magnitude in PFAT5 levels. Logistic regression analysis showed that the PFAT5 level determined 1h after admixture preparation was not correlated with phase separation behavior. However, PFAT5 measured at later times showed much improved correlations with emulsion instability. PFAT5 was highly correlated with neighboring cumulative distributions termed PFATX where X=2-10 microm. Although the admixtures studied were not clinically relevant, the data demonstrate some limitations of developing empirical correlations between single-point SPOS measurements and emulsion instability. An alternative limit test for emulsion stability based on the rate of change in the large globule counts is proposed to mitigate inherent deficiencies in the current USP Chapter 729 limit test based on single-point determination of PFAT5 values.

Enhancing uniformity and overall quality of red cell concentrate with anaerobic storage.

BACKGROUND: Recent research focused on understanding stored red blood cell (RBC) quality has demonstrated high variability in measures of RBC function and health across units. Studies have historically linked this high variability to variations in processing, storage method, and age. More recently, a large number of studies have focused on differences in donor demographics, donor iron sufficiency, and genetic predisposition of the donor to poor storage, particularly through mechanisms of accelerated oxidative damage. A study was undertaken to evaluate a potential additional source of unit to unit variation in stored RBC: the role of variable percent oxygen saturation (%SO2) levels on blood quality parameters during storage. MATERIALS AND METHODS: %SO2 data from 492 LR-RBC/AS-3 units used for internal and external collaborative research was included in the analysis. Whole blood units were processed into red blood cells, AS-3 added, leucocyte reduced, in compliance with American Association of Blood Banks guidelines. LR-RBC/AS-3 products were subsequently analysed for %SO2 levels within 3-24 hours of phlebotomy using a co-oximeter. Separately, to evaluate the impact of pre-storage as well as increasing levels of %SO2 during storage, a pool-and-split study was performed. Four units of LR-RBC/AS-3 were split 6 ways; "as is" (control), hyperoxygenated to more than 90%, and four levels of pre-storage %SO2. The units were periodically sampled up to 42 days and analysed for %SO2, pCO2, methaemoglobin, ATP, 2,3-BPG as well as with the metabolomics workflow. RESULTS: The measured mean %SO2 in LR-RBC/AS-3 within 24 hours of collection was 45.9±17.5% with (32.7-61.0 IQR). %SO2 in all products increased to approximately 95-100% in three weeks. Measured blood quality parameters including ATP, % haemolysis, methaemoglobin, oxidised lipids, and GSH/GSSG indicated suppressed cellular metabolism and increased red cell degradation in response to higher %SO2 levels. DISCUSSION: The surprisingly high variability in starting %SO2 levels, coupled with negative impacts of high oxygen saturation on red blood cell quality indicates that oxygen levels may be an important and under-appreciated source of unit-to-unit variability in RBC quality.

Metabolism of Citrate and Other Carboxylic Acids in Erythrocytes As a Function of Oxygen Saturation and Refrigerated Storage.

State-of-the-art proteomics technologies have recently helped to elucidate the unanticipated complexity of red blood cell metabolism. One recent example is citrate metabolism, which is catalyzed by cytosolic isoforms of Krebs cycle enzymes that are present and active in mature erythrocytes and was determined using quantitative metabolic flux analysis. In previous studies, we reported significant increases in glycolytic fluxes in red blood cells exposed to hypoxia in vitro or in vivo, an observation relevant to transfusion medicine owing to the potential benefits associated with hypoxic storage of packed red blood cells. Here, using a combination of steady state and quantitative tracing metabolomics experiments with 13C1,2,3-glucose, 13C6-citrate, 13C515N2-glutamine, and 13C1-aspartate via ultra-high performance liquid chromatography coupled on line with mass spectrometry, we observed that hypoxia in vivo and in vitro promotes consumption of citrate and other carboxylates. These metabolic reactions are theoretically explained by the activity of cytosolic malate dehydrogenase 1 and isocitrate dehydrogenase 1 (abundantly represented in the red blood cell proteome), though moonlighting functions of additional enzymes cannot be ruled out. These observations enhance understanding of red blood cell metabolic responses to hypoxia, which could be relevant to understand systemic physiological and pathological responses to high altitude, ischemia, hemorrhage, sepsis, pulmonary hypertension, or hemoglobinopathies. Results from this study will also inform the design and testing of novel additive solutions that optimize red blood cell storage under oxygen-controlled conditions.

Tongue base suspension in children with cerebral palsy and obstructive sleep apnea.

OBJECTIVE: Children with cerebral palsy (CP) are commonly affected by obstructive sleep apnea (OSA). This study examines the efficacy of combined surgical techniques for OSA including tongue base suspension (TBS), using perioperative polysomnograms (PSG) in pediatric patients with CP. STUDY DESIGN: Case series with outcome analysis. SETTING: University based tertiary care children's hospital. METHODS: A 7-year retrospective chart review of children with CP who underwent surgical management for OSA. Surgical procedures, postoperative complications, and perioperative PSG data were examined. Only patients with both preoperative and postoperative PSG results were included in the study. Based upon procedures performed patients fell into 2 equal groups for analysis. RESULTS: Fourteen children were identified. Seven patients (mean age = 6.0 years) underwent combined adenotonsillectomy (T&A), uvulopalatopharyngoplasty (UPPP), and tongue base suspension (TBS). Another 7 patients (mean age = 6.3 years) underwent T&A and UPPP alone. Those who received TBS had a mean preoperative AHI of 27.2 compared to 6.8 in the group that did not have TBS. The AHI decreased by a mean of 16.5 in the TBS group and 5.0 in the non-TBS group. The mean oxygen saturation nadir improved in both the TBS (74.0-84.0) and non-TBS (64.8-84.6) groups. The arousal index also improved in the TBS (33.1-20.7) and non-TBS (11.0-5.8) groups. No surgical complications occurred. CONCLUSION: This study suggests that concomitant surgical approaches for OSA in children with CP are effective. Moderate to severe OSA in this population may safely benefit from the added technique of tongue base suspension.

Genome-wide and fine-resolution association analysis of malaria in West Africa.

We report a genome-wide association (GWA) study of severe malaria in The Gambia. The initial GWA scan included 2,500 children genotyped on the Affymetrix 500K GeneChip, and a replication study included 3,400 children. We used this to examine the performance of GWA methods in Africa. We found considerable population stratification, and also that signals of association at known malaria resistance loci were greatly attenuated owing to weak linkage disequilibrium (LD). To investigate possible solutions to the problem of low LD, we focused on the HbS locus, sequencing this region of the genome in 62 Gambian individuals and then using these data to conduct multipoint imputation in the GWA samples. This increased the signal of association, from P = 4 × 10(-7) to P = 4 × 10(-14), with the peak of the signal located precisely at the HbS causal variant. Our findings provide proof of principle that fine-resolution multipoint imputation, based on population-specific sequencing data, can substantially boost authentic GWA signals and enable fine mapping of causal variants in African populations.