Saline-adenine-glucose-mannitol Research Articles

Multi-component cord blood banking: a proof-of-concept international exercise.

Most public cord blood (CB) banks currently discard more than 80% of umbilical CB units not suitable for hemopoietic stem cell transplant due to low stem cell count. Although CB platelets, plasma, and red blood cells have been used for experimental allogeneic applications in wound healing, corneal ulcer treatment, and neonatal transfusion, no standard procedures for their preparation have been defined internationally. A network of 12 public CB banks in Spain, Italy, Greece, the UK, and Singapore developed a protocol to validate a procedure for the routine production of CB platelet concentrate (CB-PC), CB platelet-poor plasma (CB-PPP), and CB leukoreduced red blood cells (CB-LR-RBC) using locally available equipment and the commercial BioNest ABC and EF medical devices. CB units with >50 mL volume (excluding anticoagulant) and ≥150×109/L platelets were double centrifuged to obtain CB-PC, CB-PPP, and CB-RBC. The CB-RBC were diluted with saline-adenine-glucose-mannitol (SAGM), leukoreduced by filtration, stored at 2-6°C, and tested for hemolysis and potassium (K+) release over 15 days, with gamma irradiation performed on day 14. A set of acceptance criteria was pre-defined. This was for CB-PC: volume ≥5 mL and platelet count 800-1,200×109/L; for CB-PPP: platelet count <50×109/L; and for CB-LR-RBC: volume ≥20 mL, hematocrit 55-65%, residual leukocytes <0.2×106/unit, and hemolysis ≤0.8%. Eight CB banks completed the validation exercise. Compliance with acceptance criteria was 99% for minimum volume and 86.1% for platelet count in CB-PC, and 90% for platelet count in CB-PPP. Compliance in CB-LR-RBC was 85.7% for minimum volume, 98.9% for residual leukocytes, and 90% for hematocrit. Compliance for hemolysis ≤0.8% decreased from 89.0 to 63.2% from day 0 to 15. K+ release increased from 3.0±1.8 to 25.0±7.0 mmol/L from day 0 to 15, respectively. The MultiCord12 protocol was a useful tool to develop preliminary standardization of CB-PC, CB-PPP, and CB-LR-RBC.

The compound effect of irradiation and familial pseudohyperkalemia on potassium leak from red blood cells.

Familial pseudohyperkalemia (FP) is a rare asymptomatic condition characterized by an increased rate of potassium leak from red blood cells (RBC) on refrigeration. Gamma irradiation compromises RBC membrane integrity and accelerates potassium leakage. Here, we compared the effect of irradiation, applied early or late in storage, on FP versus non-FP RBC. Five FP and 10 non-FP individuals from the National Institute for Health Research Cambridge BioResource, UK, and three FP and six non-FP individuals identified by Australian Red Cross Lifeblood consented to the study. Blood was collected according to standard practice in each center, held overnight at 18-24°C, leucocyte-depleted, and processed into red cell concentrates (RCC) in Saline Adenine Glucose Mannitol. On Day 1, RCC were split equally into six Red Cell Splits (RCS). Two RCS remained non-irradiated, two were irradiated on Day 1 and two were irradiated on Day 14. RBCs were tested over cold storage for quality parameters. As expected, non-irradiated FP RCS had significantly higher supernatant potassium levels than controls throughout 28 days of storage (p < .001). When irradiated early, FP RCS released potassium at similar rates to control. When irradiated late, FP RCS supernatants had higher initial post-irradiation potassium concentration than controls but were similar to controls by the end of storage (14 days post-irradiation). No other parameters studied showed a significant difference between FP and control. FP does not increase the rate of potassium leak from irradiated RBCs. Irradiation may cause a membrane defect similar to that in FP RBCs.

Red cells manufactured from lipaemic whole blood donations: Do they have higher haemolysis?

Lipaemia in blood donations is thought to influence haemolysis in stored red blood cell (RBC) components. Higher lipid concentrations are believed to increase red cell fragility, exacerbating haemolysis during collection and subsequent red cell storage. This study aimed to investigate associations between lipoproteins in plasma and haemolysis of red cells stored in saline-adenine-glucose-mannitol (SAGM). Fifty-four plasma and matched RBCs were obtained from lipaemic whole blood donations. Plasma was tested for coagulation factors, triglycerides and cholesterol. Haemolysis, glucose, lactate, extracellular potassium, lactate dehydrogenase and adenosine triphosphate (ATP) were measured in RBC on Days 7, 21 and 42 of storage. Additionally, 20 plasma and matched RBCs from non-lipaemic donations were tested as controls. Lipaemic plasma had significantly higher triglyceride concentrations compared with non-lipaemic plasma. However, there was no significant difference in plasma cholesterol between the two groups. There were no significant differences in glucose, extracellular potassium or ATP concentrations in RBC from either group. There was no significant difference in haemolysis at expiry in lipaemic-derived and control RBC, with a weak correlation between haemolysis and either triglycerides or cholesterol. There was no significant difference in haemolysis in RBC manufactured from lipaemic and non-lipaemic whole blood donations when stored in SAGM; however, the proportion of RBC from lipaemic donations with higher haemolysis was greater than in the controls. There was a weak correlation between red cell haemolysis and plasma triglycerides. Therefore, RBCs derived from lipaemic donations are suitable for blood bank inventories.

Clinical and in vitro evaluation of red blood cells collected and stored in a non-DEHP plasticized bag system.

Di-ethyl-hexyl-phthalate (DEHP) is currently the main plasticizer used for whole blood collection systems. However, in Europe, after May 2025, DEHP may no longer be used above 0.1% (w/w) in medical devices. DEHP stabilizes red cell membranes, thereby suppressing haemolysis during storage. Here we compared in vitro quality parameters of red cell concentrates (RCCs) collected and stored in DEHP-, DINCH- or DINCH/BTHC-PVC hybrid blood bags with saline-adenine-glucose-mannitol (SAGM) or phosphate-adenine-glucose-guanosine-saline-mannitol (PAGGSM) storage solution. Last, we performed haemovigilance surveillance for RCC collected in DINCH-PVC and stored in PAGGSM/BTHC-PVC. In vitro quality parameters of RCC were determined during 42 days of storage. Haemovigilance surveillance was conducted to compare the frequency and type of transfusion reaction. Haemolysis levels were increased in SAGM/BTHC-PVC as compared to SAGM/DEHP-PVC (0.66% ± 0.18% vs. 0.36% ± 0.17%). PAGGSM storage solution was able to adequately suppress haemolysis to levels observed during storage in SAGM/DEHP-PVC, both in BTHC-PVC (0.38% ± 0.12%), and to a slightly lesser extent in DINCH-PVC (0.48% ± 0.17%). A total of 1650 PAGGSM/BTHC-PVC and 5662 SAGM/DEHP-PVC RCC were transfused yielding a transfusion reaction frequency of 0.24% (95% CI 0.0000-0.0048) and 0.44% (95% CI 0.0027-0.0061) respectively. The in vitro quality of RCC stored in PAGGSM/BTHC-PVC and SAGM/DEHP-PVC is comparable. There is no indication that transfusion of erythrocytes stored in PAGGSM/BTHC-PVC results in increased transfusion reaction frequency. These initial results provide a basis for further clinical evaluation to narrow down the confidence interval of transfusion reaction frequency.

Storage of red blood cells in alkaline PAGGGM improves metabolism but has no effect on recovery after transfusion

AbstractAdditive solutions are used to limit changes that red blood cells (RBCs) undergo during storage. Several studies have shown better preservation of glucose and redox metabolism using the alkaline additive solution PAGGGM (phosphate-adenine-glucose-guanosine-gluconate-mannitol). In this randomized open-label intervention trial in 20 healthy volunteers, the effect of storage, PAGGGM vs SAGM (saline-adenine-glucose-mannitol), on posttransfusion recovery (PTR) and metabolic restoration after transfusion was assessed. Subjects received an autologous biotinylated RBC concentrate stored for 35 days in SAGM or PAGGGM. As a reference for the PTR, a 2-day stored autologous biotinylated RBC concentrate stored in SAGM was simultaneously transfused. RBC phenotype and PTR were assessed after transfusion. Biotinylated RBCs were isolated from the circulation for metabolomics analysis up to 24 hours after transfusion. The PTR was significantly higher in the 2-day stored RBCs than in 35-day stored RBCs 2 and 7 days after transfusion: 96% (90 to 99) vs 72% (66 to 89) and 96% (90 to 99) vs 72% (66 to 89), respectively. PTR of SAGM- and PAGGGM-stored RBCs did not differ significantly. Glucose and redox metabolism were better preserved in PAGGGM-stored RBCs. The differences measured in the blood bag remained present only until 1 day after transfusion. No differences in RBC phenotype were found besides an increased complement C3 deposition on 35-day RBCs stored in PAGGGM. Our data indicate that despite better metabolic preservation, PAGGGM is not a suitable alternative for SAGM because storage in PAGGGM did not result in an increased PTR. Finally, RBCs recovered from circulation after transfusion showed reversal of the metabolic storage lesion in vivo within a day. This study is registered in the Dutch trial register (NTR6492).

Storage lesions after irradiation: Comparison between blood stored in citrate phosphate dextrose adenine and saline adenine glucose mannitol

Background and Objectives: Saline adenine glucose mannitol (SAGM) with citrate phosphate dextrose (CPD) or CPD adenine (CPDA) are two common red cell preservatives used in our country. This study analyzed and compared serial biochemical changes on different days of storage in irradiated packed red blood cells stored in CPDA and SAGM solutions. The aim of this study was to see if these changes are influenced by or different, if any, in these two storage solutions. Patients and Methods: Ninety units of RBCs were randomly chosen, with 45 units each collected in CPDA and SAGM, respectively. Nine units each were irradiated by gamma irradiator using cobalt 60 (BI 2000) on days 1, 7, 14, and 21 of their storage, respectively. Nine units were not irradiated and used as controls. All the units were weekly assessed for their plasma levels of potassium, sodium, glucose, lactate dehydrogenase (LDH), and lactate by Clinical Chemistry Auto-analyzer, Beckman Coulter AU680. The values were documented and analyzed by SPSS. Results: Baseline values on day 1 for potassium, lactate, and LDH were similar between SAGM- and CPDA-stored blood bags. Glucose and sodium levels were slightly higher in SAGM bags compared to CPDA. Postirradiation, the changes (increase in K+, Lactate, LDH and decrease in Na+, glucose) were higher in CPDA bags than SAGM bags, and the difference in this trend was not significantly different from that seen in nonirradiated blood bags stored in these two preservative solutions. Conclusion: Storage lesions (biochemical parameters) after irradiation were severe, but paralleled that observed in nonirradiated bags. The storage solution, either SAGM or CPDA, made no difference to these changes.

Effect of storage on biochemical parameters of packed red blood cells of goats in............ 257-261 citrate phosphate dextrose adenine/ saline adenine glucose mannitol

A study was conducted to assess the suitability of citrate phosphate dextrose adenine / saline adenine glucose mannitol as a storage media for packed RBCs of goats. Samples collected from ten apparently healthy goats were utilized for the study. Biochemical studies were carried out on day 0, 14, 28 and 42 days of storage using the parameters, viz. pH, glucose, potassium, malondialdehyde and reduced glutathione. The pH was stable throughout the study, whereas glucose showed significant reduction. Rest of the parameters increased significantly from 0th day to 42nd day. Based on the results, the storage media can be considered to be suitable for storing caprine packed RBCs.

DEHT is a suitable plasticizer option for phthalate-free storage of irradiated red blood cells.

Due to increasing concerns about possible endocrine-disrupting properties, the use of the plasticizer di(2-ethylhexyl) phthalate (DEHP) will be banned in future blood storage. Di(2-ethylhexyl) terephthalate (DEHT) provides sufficient red blood cell (RBC) quality during conventional blood bank storage. It is important that a new plasticizer also maintains acceptable quality during exposure to high cell stress, such as irradiation, which is commonly used to prevent graft-versus-host disease. A total of 59 RBC units were collected and processed in polyvinyl chloride (PVC)-DEHT or PVC-DEHP blood bags combined with either saline-adenine-glucose-mannitol (SAGM) or phosphate-adenine-glucose-guanosine-saline-mannitol (PAGGSM) additive solution. All units were X-ray irradiated on day 2 post-collection. Sampling for assessment of parameters of storage lesion was performed on day 2 pre-irradiation and day 14 and 28 post-irradiation. Though irradiation increased cell stress, DEHT/PAGGSM and current common European preference DEHP/SAGM were equally affected up to 14 days post-irradiation for all measured parameters. At day 28, haemolysis and microvesicle count were slightly increased in DEHT, whereas extracellular potassium ions, glucose, lactate, pH, mean corpuscular volume and microvesicle phosphatidylserine remained unaffected by plasticizer choice throughout storage. No individual unit exceeded 0.8% haemolysis, not even in DEHT/SAGM, the combination overall most affected by irradiation. Of the four combinations, membrane stability was least impacted in DEHP/PAGGSM. We demonstrate that DEHT is a suitable plasticizer for storage of RBCs after X-ray irradiation cell stress. This strengthens the option of DEHT as a viable non-phthalate substitute for DEHP.

Effect of parachute delivery on red blood cell (RBC) and plasma quality measures of blood for transfusion.

BackgroundParachute airdrop offers a rapid transfusion supply option for humanitarian aid and military support. However, its impact on longer‐term RBC survival is undocumented. This study aimed to determine post‐drop quality of RBCs in concentrates (RCC), and both RBCs and plasma in whole blood (WB) during subsequent storage.Study design and methodsTwenty‐two units of leucodepleted RCC in saline, adenine, glucose, mannitol (SAGM) and 22 units of nonclinical issue WB were randomly allocated for air transportation, parachute drop, and subsequent storage (parachute), or simply storage under identical conventional conditions (4 ± 2°C) (control). All blood products were 6–8 days post‐donation. Parachute units were packed into Credo Cubes, (Series 4, 16 L) inside a PeliCase (Peli 0350) and rigged as parachute delivery packs. Packs underwent a 4‐h tactical flight (C130 aircraft), then parachuted from 250 to 400 ft before ground recovery. The units were sampled aseptically before and after airdrop at weekly intervals. A range of assays quantified the RBC storage lesion and coagulation parameters.ResultsBlood units were maintained at 2–6°C and recovered intact after recorded ground impacts of 341–1038 m s−2. All units showed a classical RBC storage lesion and increased RBC microparticles during 42 days of storage. Fibrinogen and clotting factors decreased in WB during storage. Nevertheless, no significant difference was observed between Control and Parachute groups. Air transportation and parachute delivery onto land did not adversely affect, or shorten, the shelf life of fresh RBCs or WB.DiscussionAppropriately packaged aerial delivery by parachute can be successfully used for blood supply.

Modelling of Red Blood Cell Morphological and Deformability Changes during In-Vitro Storage

Storage lesion is a critical issue facing transfusion treatments, and it adversely affects the quality and viability of stored red blood cells (RBCs). RBC deformability is a key indicator of cell health. Deformability measurements of each RBC unit are a key challenge in transfusion medicine research and clinical haematology. In this paper, a numerical study, inspired from the previous research for RBC deformability and morphology predictions, is conducted for the first time, to investigate the deformability and morphology characteristics of RBCs undergoing storage lesion. This study investigates the evolution of the cell shape factor, elongation index and membrane spicule details, where applicable, of discocyte, echinocyte I, echinocyte II, echinocyte III and sphero-echinocyte morphologies during 42 days of in-vitro storage at 4 °C in saline-adenine-glucose-mannitol (SAGM). Computer simulations were performed to investigate the influence of storage lesion-induced membrane structural defects on cell deformability and its recoverability during optical tweezers stretching deformations. The predicted morphology and deformability indicate decreasing quality and viability of stored RBCs undergoing storage lesion. The loss of membrane structural integrity due to the storage lesion further degrades the cell deformability and recoverability during mechanical deformations. This numerical approach provides a potential framework to study the RBC deformation characteristics under varying pathophysiological conditions for better diagnostics and treatments.

Rejuvenation of RBCs: validation of a manufacturing method suitable for clinical use.

Rejuvenation of stored red blood cells (RBCs) increases levels of adenosine 5'-triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) to those of fresh cells. This study aimed to optimize and validate the US-approved process to a UK setting for manufacture and issue of rejuvenated RBCs for a multicenter randomized controlled clinical trial in cardiac surgery. Rejuvenation of leukoreduced RBC units involved adding a solution containing pyruvate, inosine, phosphate, and adenine (Rejuvesol, Zimmer Biomet), warming at 37°C for 60 minutes, then "manual" washing with saline adenine glucose mannitol solution. A laboratory study was conducted on six pools of ABO/D-matched units made the day after donation. On Days 7, 21, and 28 of 4 ± 2°C storage, one unit per pool was rejuvenated and measured over 96 hours for volume, hematocrit, hemolysis, ATP, 2,3-DPG, supernatant potassium, lactate, and purines added (inosine) or produced (hypoxanthine) by rejuvenation. Subsequently, an operational validation (two phases of 32 units each) was undertaken, with results from the first informing a trial component specification applied to the second. Rejuvenation effects were also tested on crossmatch reactivity and RBC antigen profiles. Rejuvenation raised 2,3-DPG to, and ATP above, levels of fresh cells. The final component had potassium and hemolysis values below those of standard storage Days 7 and 21, respectively, containing 1.2% exogenous inosine and 500 to 1900 μmoles/unit of hypoxanthine. The second operational validation met compliance to the trial component specification. Rejuvenation did not adversely affect crossmatch reactivity or RBC antigen profiles. The validated rejuvenation process operates within defined quality limits, preserving RBC immunophenotypes, enabling manufacture for clinical trials.

Hemoglobin Utilization in Asplenic and Non-Splenectomized Transfusion Dependent Thalassemia Patients Supported with Pathogen Reduced Red Blood Cell Concentrates in a Phase 3 Study (SPARC)

Introduction: Transfusion dependent thalassemia (TDT) requires regular transfusion of red cell concentrates (RBCC) to prevent the complications of anemia and excessive erythroid expansion. Despite donor testing, long-term transfusion has a substantial cumulative risk of transfusion-transmitted infection (TTI) due to undetected viruses, bacteria, and protozoa. Splenectomized (-S) TDT patients may have greater TTI morbidity than patients with spleens (+S); but may benefit from reduced use of red blood cell concentrates (RBCC) and reduced transfusion iron (Fe) loading. Pathogen reduction (PR) of RBCC with amustaline-glutathione (A-GSH) offers potential to reduce the risk of TTI. Objectives: To determine, the impact of PR-RBCC on hemoglobin (Hb) use, transfused Fe burden, incidence of RBC antibodies, and safety in -S and +S TDT patients. Methods: TDT patients at 3 sites, not stratified by spleen status, were prospectively enrolled in a two- period cross-over study randomized by sequence for RBCC preparation. Leukocyte reduced PR-RBCC (Test) were treated with 0.2mM amustaline and 20 mM GSH, re-suspended in saline-adenine-glucose mannitol (SAGM), and stored up to 35 days at 4°C. Leukocyte reduced conventional RBCC (Control) were suspended in SAGM and stored for up to 35 days at 4°C. Patients received 6 transfusions in each treatment sequence of Test or Control RBCC over ~ 5 months. Clinicians, blinded to RBCC Hb content and treatment sequence, ordered RBCC to maintain targeted pre-transfusion Hb thresholds of ~ 9-10 g/dL. Transfusion intervals or number of RBCC transfused were adjusted for clinical management. The primary efficacy outcome was assessed by non-inferiority (NI) analysis for Hb use (expressed as g/kg body weight/ day) using a pre-specified NI margin (≤ 15% of the observed Control mean). Results: Overall, mean (SD) Hb content (g) of 1024 Test RBCC = 54.6±5.9 (range: 39-73) and of 1008 Control RBCC = 55.6 ± 5.9 (range: 35-74) and varied widely. By intent-to-treat (ITT), 80 patients (40 +S and 40 -S) were transfused. For ITT patients (Table), the baseline Hb level (BL-Hb, g/dL) at first transfusion of Control periods was significantly lower than at Test periods; but the mean number of RBCC transfused, RBCC storage days, total Hb dose (g), and transfusion intervals were not significantly different for Test and Control. ITT analysis for all transfusion episodes showed Hb use for Test RBCC (0.110 g/kg/d) was not different from Control RBCC (0.112 g/kg/day). Non-inferiority was demonstrated (T-C = - 0.002 g/kg/d: 95% CI: -0.005, 0.001). ITT Test patients received a slightly lower mean total Hb dose (- 14g), and mean pre-transfusion Hb levels declined after 6 transfusions (9.4 to 8.8 g/dL). -S patients had lower BL-Hb levels (g/dL) than S+ patients in Test (9.2 vs 9.7) and Control (8.8 vs 9.2) periods (Table). -S patients received a lower mean total Hb dose of Test than Control RBCC (p=0.019); and had a decline in mean pre-transfusion Hb levels during Test periods (from 9.2 to 8.7 g/dL). Transfusion intervals were significantly longer for -S patients than +S patients with both Test and Control RBCC (p Conclusions: Amustaline-GSH PR treatment of RBCCs offers the potential to reduce TTI risk without impacting Hb use or Fe burden in TDT. However, Hb content of Test and Control RBCC varies widely and may contribute to unexpected changes in pre-transfusion Hb levels. Spleen status affected Hb use comparably for PR-RBCC and Control RBCC, and remains an important factor in assessing transfusion requirements and Fe loading. Disclosures Aydinok:TERUMO: Research Funding; Cerus: Honoraria, Research Funding; CRISPR Tech: Other: DMC; Protagonist: Other: SSC; La Jolla Pharmaceuticals: Research Funding; Celgene: Research Funding; Novartis: Research Funding, Speakers Bureau. Piga:Apopharma: Honoraria, Research Funding; Celgene Corp: Membership on an entity9s Board of Directors or advisory committees, Research Funding; La Jolla: Membership on an entity9s Board of Directors or advisory committees, Research Funding; Bluebird Bio: Honoraria; Acceleron: Research Funding; Novartis: Research Funding. Origa:Novartis: Honoraria; Bluebird Bio: Consultancy; Cerus Corporation: Research Funding; Apopharma: Honoraria. Mufti:Cerus Corporation: Employment, Equity Ownership. Erikson:Cerus Corporation: Employment, Equity Ownership. North:Cerus Corporation: Employment, Equity Ownership. Waldhaus:Cerus Corporation: Employment, Equity Ownership. Ernst:Cerus Corporation: Employment, Equity Ownership. Lin:Cerus Corporation: Employment, Equity Ownership. Huang:Cerus Corporation: Employment, Equity Ownership. Benjamin:Cerus Corporation: Employment, Equity Ownership. Corash:Cerus Corporation: Employment, Equity Ownership.

Complement Deposition and IgG Binding on Stored Red Blood Cells Are Independent of Storage Time

Background: In the Netherlands, red blood cells (RBCs) are allowed to be stored up to 35 days at 2-6 °C in saline-adenine-glucose-mannitol (SAGM). During storage, RBCs undergo several changes that are collectively known as storage lesion. We investigated to what extent complement deposition and antibody binding occurred during RBC storage and investigated phagocytic uptake in vitro. Methods: RBCs were stored for different lengths of time at 2-6 °C in SAGM. Complement deposition and antibody binding were assessed upon storage and after incubation with serum. M1- and M2-type macrophages were generated from blood monocytes to investigate RBC phagocytosis. Results: No complement deposition was directly observed on stored RBCs, while incubation of RBCs with serum resulted in variable donor-dependent C3 deposition and IgG binding, both independent of storage time. Only 1-4% phagocytosis of stored RBCs by macrophages was observed. Conclusion: RBCs are susceptible to complement deposition and antibody binding independent of storage time. Limited phagocytic uptake by macrophages was observed in vitro.

Vitamin E Analogue Protects Red Blood Cells against Storage-Induced Oxidative Damage

Background: To investigate i) the effects of Trolox® or mannitol, which represent two different classes of antioxidants, on oxidative changes generated in manually isolated red blood cells (RBCs) from citrate-phosphate-dextrose (CPD) preserved whole blood, followed by up to 20 days refrigerated storage, and ii) whether Trolox supplemented to the blood bank-manufactured saline-adenine-glucose-mannitol (SAGM) preserved RBC units would offer better storage conditions compared with SAGM alone. Methods: The percentage of hemolysis and extracellular activity of lactate dehydrogenase (LDH) was measured to assess RBC membrane integrity. Lipid peroxidation, reduced glutathione (GSH) levels and total antioxidant capacity (TAC) were quantified by thiobarbituric acid-reactive substances (TBARS), Ellman's reagent and 2, 2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS^.+) based assay, respectively. Results: Trolox was little more effective than mannitol in protecting against progressive RBC hemolysis. Trolox (0.125-3.125 mmol/l) inhibited storage-induced leakage of LDH, lipid peroxidation, and to a lesser extent GSH depletion. Mannitol at these concentrations neither inhibited TBARS formation nor prevented GSH depletion. RBC units stored in SAGM-Trolox had significantly lower hemolysis, LDH leakage, and lipid peroxidation level compared to RBCs stored in SAGM. Conclusion: There is evidence of the beneficial effects of supplementing RBC-additive solutions with membrane-interacting antioxidants such as vitamin E analogues.

The antioxidant capacity of erythrocyte concentrates is increased during the first week of storage and correlated with the uric acid level.

Red blood cells (RBCs) suffer from lesions during cold storage, depending in part on their ability to counterbalance oxidative stress by activating their antioxidant defence. The aim of this study was to monitor the antioxidant power (AOP) in erythrocyte concentrates (ECs) during cold storage. Six ECs were prepared in saline-adenine-glucose-mannitol (SAGM) additive solution and followed during 43days. The AOP was quantified electrochemically using disposable electrode strips and compared with results obtained from a colorimetric assay. Haematological data, data on haemolysis and the extracellular concentration of uric acid were also recorded. Additionally, a kinetic model was developed to extract quantitative kinetic data on the AOP behaviour. The AOP of total ECs and their extracellular samples attained a maximum after 1week of storage prior to decaying and reaching a plateau, as shown by the electrochemical measurements. The observed trend was confirmed with a colorimetric assay. Uric acid had a major contribution to the extracellular AOP. Interestingly, the AOP and uric acid levels were linked to the sex of the donors. The marked increase in AOP during the first week of storage suggests that RBCs are impacted early by the modification of their environment. The AOP behaviour reflects the changes in metabolism activity following the adjustment of the extracellular uric acid level. Knowing the origin, interdonor variability and the effects of the AOP on the RBCs could be beneficial for the storage quality, which will have to be further studied.

Attenuation of Red Blood Cell Storage Lesions with Vitamin C.

Stored red blood cells (RBCs) undergo oxidative stress that induces deleterious metabolic, structural, biochemical, and molecular changes collectively referred to as “storage lesions”. We hypothesized that vitamin C (VitC, reduced or oxidized) would reduce red cell storage lesions, thus prolonging their storage duration. Whole-blood-derived, leuko-reduced, SAGM (saline-adenine-glucose-mannitol)-preserved RBC concentrates were equally divided into four pediatric storage bags and the following additions made: (1) saline (saline); (2) 0.3 mmol/L reduced VitC (Lo VitC); (3) 3 mmol/L reduced VitC (Hi VitC); or (4) 0.3 mmol/L oxidized VitC (dehydroascorbic acid, DHA) as final concentrations. Biochemical and rheological parameters were serially assessed at baseline (prior to supplementation) and Days 7, 21, 42, and 56 for RBC VitC concentration, pH, osmotic fragility by mechanical fragility index, and percent hemolysis, LDH release, glutathione depletion, RBC membrane integrity by scanning electron microscopy, and Western blot for β-spectrin. VitC exposure (reduced and oxidized) significantly increased RBC antioxidant status with varying dynamics and produced trends in reduction in osmotic fragility and increases in membrane integrity. Conclusion: VitC partially protects RBC from oxidative changes during storage. Combining VitC with other antioxidants has the potential to improve long-term storage of RBC.

Dielectric Response of Cytoplasmic Water and Its Connection to the Vitality of Human Red Blood Cells. II. The Influence of Storage.

Maintaining an appropriate inventory of packaged blood products is a critical part of modern medicine. Consequently, the assessment of red blood cell (RBC) functionality is instrumental for the monitoring of the quality of stored RBC (sRBC) in the blood bank. We present a comprehensive study of sRBC lesion kinetics in SAGM (saline, adenine, glucose, mannitol) solution, using microwave dielectric spectroscopy (0.5-50 GHz) and cell deformability. As part of the research, we have isolated the microwave dielectric response of cytoplasmic water in sRBC. The extracted dielectric parameters are sensitive to the age of the cells and, in particular, to the critical moment of transition from discocyte to echinocyte. From the analysis of the dielectric relaxation as a function of storage-duration, we postulate that the behavior is rooted in the delicate interplay between bound and bulk water in the cellular interior. In particular, the microwave dielectric response reflects the moment when the continuous diffusion of oxygen to the cell and the oxygenation of hemoglobin affects the role played by water in the maintenance of cell integrity. These results open a possible new avenue for the noninvasive inspection of stored red blood cells, permitting a true inventory system for the modern blood bank.

Prevention of red cell storage lesion: a comparison of five different additive solutions.

In Europe, red cell concentrates (RCC) are usually stored in SAGM (saline-adenine-glucose-mannitol). During storage, in vitro red cell quality declines, including lowered energy status and increased cell lysis. Recently, several additive solutions (ASs), designed to diminish the decline in in vitro quality during storage, have been developed. These new solutions have mainly been developed to better maintain red blood cell (RBC) 2,3-biphosphoglycerate (2,3 BPG) levels and energy status during storage. High levels of 2,3 BPG allow for better oxygen release while high energy status is necessary for function and survival of RBC in vivo. In a paired study design, RBC ASs were compared for their ability to provide improved in vitro quality during hypothermic storage. For each experiment, 5 whole blood units held overnight were pooled and split. The whole blood units were processed according to the buffy coat method. RBCs were resuspended in either SAGM, PAGGSM, PAG3M, E-Sol 5 or AS-7 and leucoreduced by filtration. RCCs were stored for eight weeks at 2-6 °C and sampled weekly for analysis of in vitro quality parameters. Red cell concentrates stored in PAG3M, E-Sol 5 and AS-7 showed significantly higher lactate production and higher levels of intracellular adenosine triphosphate (ATP) and total adenylate. 2,3 BPG levels rapidly declined during storage in SAGM and PAGGSM. The decline in 2,3 BPG was inhibited during storage in E-Sol 5 and AS-7, while in PAG3M, 2,3 BPG level increased above the initial level till day 35 and remained detectable till day 56. Haemolysis was comparable for all ASs until day 35, upon prolonged storage, haemolysis in SAGM was higher than with the other ASs. As compared to SAGM, storage in PAGGSM, PAG3M, E-Sol 5 and AS-7 better maintained morphological properties. Storage of RBCs in the new generation ASs yield RBCs with more stable metabolite levels and improved overall quality during storage as compared with RBCs stored in SAGM.

The effect of prefreeze rejuvenation on postthaw storage of red blood cells in AS-3 and SAGM.

We investigated whether improving the metabolic status of red blood cell concentrates before freezing could extend the postthaw shelf life beyond 14 days while still meeting the requirements for hemolysis (0.8%) and total adenylate (>82% of original values). At Day 8 after collection, four leukoreduced red blood cell concentrates in saline-adenine-glucose-mannitol (SAGM) were pooled, mixed, and split (n = 4). Of these concentrates, two were rejuvenated in Rejuvesol. In addition, two leukoreduced red blood cell concentrates in phosphate-adenine-glucose-guanosine-gluconate-mannitol (PAGGGM) were pooled, mixed, and split at Day 8 after collection (n = 4). All concentrates were glycerolized, frozen, and stored for at least 2 weeks at -80°C. After thawing and deglycerolization, from each pair, one red blood cell concentrate was resuspended in SAGM, and one was suspended in AS-3. During postthaw storage at 2 to 6°C for 35 days, all concentrates were sampled weekly and analyzed for hematologic, metabolic, and morphologic parameters. Both Rejuvesol and PAGGGM treatment produced increased adenosine triphosphate and total adenylate and 2,3-diphosphoglycerate levels compared with untreated red blood cell concentrates. Regardless of prefreeze Rejuvesol or PAGGGM treatment, postthaw hemolysis remained below 0.8% during 7 days in SAGM and during 35 days in AS-3. At Day 35 of postthaw storage in AS-3, total adenylate in nonrejuvenated red blood cell concentrates had decreased to 72% of the original values; whereas, in prefreeze Rejuvesol-treated and PAGGGM-treated concentrates, adenylate values were still were at 101% and 98%, respectively. Based on maximum allowable hemolysis of 0.8% and total adenylate content greater than 82% of the original value, thawed, prefreeze Rejuvesol-treated or PAGGGM-treated red blood cell concentrates can be stored for 35 days at 2 to 6ºC in AS-3.

Study of Hemolysis During Storage of Blood in the Blood Bank of a Tertiary Health Care Centre.

The aim of RBC storage system in a blood bank is to counteract damage to the metabolic machinery and the membrane, to improve post-transfusion viability. In recent years, the need for strict control over the quality of blood has been emphasised. Such quality indicator includes extend of hemolysis and morphological changes of RBC during storage. This study was design to see extend of hemolysis and level of plasma lactate dehydrogenase (LDH) and plasma potassium, during processing and storage at different intervals under blood bank condition. Forty-six donors were selected and blood units were collected and stored under blood bank conditions. Mean plasma haemoglobin of stored blood was estimated by tetra methyl benzidine method (TMB) and percentage hemolysis was calculated on day 0, 1, 7, 21, 28, 35 and 42days. Similarly plasma LDH and plasma potassium level was also assessed during storage. It was noted that free haemoglobin level and percentage hemolysis progressively increased with storage along with the level of LDH and potassium. However, extend of hemolysis did not exceed the permissible limit of 0.8% up to 42days of storage. 15 blood bags which showed visual hemolysis on day 28 did not exceeded the threshold of 0.8% hemolysis, when interpreted by TMB method. It was concluded that TMB method is better than visual method for determination of hemolysis. The reduced hemolysis at this centre may be accounted for the use of additive solution SAGM (Saline, Adenine, Glucose, Mannitol) and DEHP (di-2-ethyl hexyl phthalate) as plasticizer in blood bags for storage.