Extended storage of red blood cells under anaerobic conditions

BACKGROUND: Red blood cells (RBC) are subject to oxidative stress by reactive oxygen species during refrigerated storage. Near-complete removal of oxygen from red cells during storage should eliminate this contributor to the red cell 'storage lesion'. The in vitro effects of storing red cells under oxygen-depleted conditions for extended periods were investigated, and these were correlated with the observed recoveries after reinfusion. STUDY DESIGN AND METHODS: Units of red cells, obtained after 'soft spin', were placed in a double volume of AS-3 additive solution and subdivided. Oxygen in the test units was depleted by repeated exposure to Ar gas (to O(2) saturation < 4%), and units were stored in anaerobic canisters for up to 15 weeks. Samples were taken weekly to monitor adenosine triphosphate (ATP), 2,3-diphosphoglycerate (2,3-DPG), cell-free haemoglobin, and vesicle production. In a parallel experiment, six units of red cells was depleted of oxygen in a similar manner, stored for 8, 9 and 10 weeks, and reinfused autologously to determine the 24 h post-transfusion recovery via (51)Cr/(99m)Tc radiolabelling. A similar study was also carried out using EAS61 additive solution, which by itself, had shown the ability to support 9-week storage, comparing biochemical profiles and in vivo recovery after aerobic vs. anaerobic storage. RESULTS: Oxygen-depleted AS-3 units had significantly elevated ATP levels compared to controls. They also had significantly lower cell free haemoglobin and vesicle production when RBCs were stored for more than 9 weeks. An average of over 75% post-transfusion survival was observed after 9 weeks of anaerobic storage with less than 0.43% haemolysis. However, no further extension of storage was achieved with EAS61 additive. CONCLUSION: Anaerobic conditions permit acceptable 9-week storage of RBCs using double-volume AS-3 additive solution. It did not synergize with the alkaline, 9-week additive, EAS61, to further lengthen the acceptable storage time. These studies indicate that anaerobic storage may allow reduction in the effect of the storage lesion, but suggest that other factors contribute to limitations of RBC storage as well.     

Up to 21-day banked red blood cells collected by apheresis and stored for 14 days after automated wash at different times of storage

BACKGROUND AND OBJECTIVES: A closed-system technology (ACP-215, Haemonetics, Braintree, MA) enables automated washing and extended storage of frozen red blood cells (RBC). This technology was applied to wash banked RBC for removal of undesirable protein and metabolites before transfusion. We studied protein and metabolite depletion as well as RBC metabolism and viability up to 14 days postwash with regard to various pre-storage times. MATERIALS AND METHODS: Thirty RBC units were collected by means of apheresis and subdivided into three arms based on prewash storage time period (6 days/group 1, 14 days/group 2, 21 days/group 3). Wash efficacy (protein depletion, IgA), RBC metabolism (pH, lactate, potassium, haemolysis) and cell viability (ATP) were analysed immediately and 14 days after washing. RESULTS: Total protein and IgA postwash were lowered by automated wash in all groups and uniformly met EC guidelines. Potassium (mmol/l) was below 1.2 mmol/l postwash and significantly below prewash values in all groups, even after 14 days of storage (prewash vs. postwash; P < 0.05). RBCs washed after 14 and 21 days, respectively, showed significantly lower pH values and lower ATP content than RBCs washed after only 6 days of storage. Haemolysis rate remained significantly below 0.8%, the maximum level recommended by the EC guidelines, immediately and 14 days after washing in all units. CONCLUSION: Our data confirm that RBC units banked up to 21 days can be effectively protein- and potassium-depleted with the ACP-215 independent from prewash storage time. With respect to high ATP levels and pH, postwash storage of 2 weeks should be limited to units not older than 7 days before wash. This new washing technology ensures better standardization in washed RBC and provides blood centres with a logistical alternative to 24-h washed RBC products.     

Commercially available blood storage containers

Plastic blood bags improve the safety and effectiveness of blood component separation and storage. Progress towards optimal storage systems is driven by medical, scientific, business and environmental concerns and is limited by available materials, consumer acceptance and manufacturing and regulatory concerns. Blood bag manufacturers were invited to submit lists of the bags they manufacture. The lists were combined and sorted by planned use. The lists were analysed by experts to assess the degree to which the products attend to scientific problems. Specific issues addressed included the use of di‐ethylhexyl phthalate (DEHP) as plasticizer for polyvinyl chloride (PVC) blood bags, the size, material and thickness of platelet bags, and the fracture resistance of plasma bags. Alternatives to DEHP for red blood cell (RBC) storage exist, but are mostly in a developmental stage. Plastic bags (DEHP‐free, PVC‐free) for platelet storage with better gas diffusion capabilities are widely available. Alternatives for plasma storage with better fracture resistance at low temperatures exist. Most RBC products are stored in DEHP‐plasticized PVC as no fully satisfactory alternative exists that ensures adequate storage with low haemolysis. A variety of alternative platelet storage systems are available, but their significance – other than improved oxygen transport – is poorly understood. The necessity to remove DEHP from blood bags still needs to be determined.     

Elastic properties of stored red blood cells from sickle trait donor units

BACKGROUND AND OBJECTIVES: Red blood cells (RBCs) from patients with sickle cell disease present reduced deformability. The aim of this study was to analyse the elasticity of stored RBCs from patients with the sickle cell trait (AS). MATERIALS AND METHODS: The cell elasticity was studied, using laser optical tweezers, on storage days 1, 14, 21, 28 and 35. RESULTS: The elasticity of RBC from AS units stored for 1, 14 and 21 days was significantly greater compared with that of control RBC cells stored for the same time-period. More than 30% of the cells from AS units stored for 28 or 35 days were very rigid and escaped from the optical trap. CONCLUSIONS: RBCs became rigid during storage, suggesting that haemoglobin S might compromise the cell elasticity.     

Sex-related aspects of the red blood cell storage lesion

BackgroundSeveral factors contribute to the manifestation of red blood cell (RBC) storage lesions, with one of the most interesting being the “donor variation effect”. Since many haematological characteristics of blood donors are sex-dependent, sex hormones and their age-dependent variation may affect the storage profile of RBCs.Materials and methodsFresh blood from 200 healthy male and female donors underwent haematological, biochemical and physiological analysis. Three selected groups of donors (men, n=8; pre-menopausal women, n=8; and post-menopausal women, n=4) exhibiting as similar as possible baseline values were recruited for blood donation in leukoreduced CPD/SAGM units. RBC indices, haemolysis and propensity for haemolysis, reactive oxygen species (ROS) and plasma antioxidant capacity were measured bi-weekly.ResultsFemale blood was characterised by lower plasma antioxidant capacity and free haemoglobin (Hb) levels in vivo, in spite of the higher RBC osmotic fragility, compared to male blood. Comparatively low Hb concentration was also measured in stored RBCs from female donors, as in vivo. Mean corpuscular Hb (MCH), mean corpuscular Hb concentration (MCHC), and plasma antioxidant capacity were also lower in female donors throughout storage, even though baseline levels were equal to those of the male group. There was no difference in propensity of stored RBCs for haemolysis between male and female units but intracellular ROS levels were significantly lower in female RBCs. Increased end-of-storage extracellular potassium and recruitment of protein stress markers (clusterin, Hb) to the RBC membrane were observed in the units of post- vs pre-menopausal female donors at mid-storage onwards.DiscussionDonor’s sex has an impact on Hb concentration and redox parameters of stored RBCs. In addition, menopause seems to promote RBC membrane remodelling, at least during prolonged storage. Our pilot study provides new insights on the different effects on RBC storage lesion according to sex.     

Transitory interruption of recommended storage conditions does not cause significant changes in in vitro parameters of leucocyte‐depleted red blood cells

Background and Objectives Conventional quality control studies of the shelf life of RBC units do not consider cold chain interruptions that occur during cross‐matching or between the release of RBCs from the blood bank and their return from the ward. These interruptions may, however, lead to a considerable loss of quality. On the other hand, differences in the quality of RBCs may derive from the different manufacturing processes employed in various blood centres. Materials and Methods One day after the expiry date of the RBC unit, we analysed complete blood count, blood gas, potassium, LDH, hydroxybutyrate dehydrogenase, glucose, lactate, total and free haemoglobin (Hb) and ATP and compared the results with regard to the frequency of storage interruptions and to two manufacturers of these RBCs. Results We could not find any correlations between the frequency of interruptions (0–11) and these parameters in any of the data sets. However, we found significant differences when comparing the two suppliers. RBCs of manufacturer A (‘A’, inline filtration of whole blood) contained 25% more Hb than those of manufacturer B (‘B’, inline filtration after buffy coat reduction). Sixteen percentage of ‘A’‐RBC, but none of ‘B’‐RBC, exceeded a haemolysis of 0·8%. Conclusions Transitory interruptions of cold chain do not measurably impair the quality of RBCs. The effect on storage of RBCs in the blood bank is not as significant a factor as the differences that exist between RBC manufacturing procedures.     

Early γ‐irradiation and subsequent storage of red cells in SAG‐M additive solution potentiate energy imbalance,microvesiculation and susceptibility to stress‐induced apoptotic cell death

γ‐Irradiation of red blood cell (RBC) concentrates prevents transfusion‐associated graft‐versus‐host disease but may diminish RBC quality. Herein, we show that early γ‐irradiation (25 Gy) of RBC units and their subsequent storage in SAG‐M additive solution altered membrane microvesiculation, supernatant haemoglobin and cytosolic ATP. γ‐Irradiation did not influence phosphatidylserine externalization, a marker of erythrocyte apoptotic cell death (eryptosis), in RBC stored for 42 days. However, shorter periods (4–21 days) of storage accentuated eryptosis in γ‐irradiated RBC versus untreated RBCs following energy depletion, suggesting that γ‐irradiated RBC is primed for stress‐induced eryptosis during storage.     

The use of the mechanical fragility test in evaluating sublethal RBC injury during storage

Background The mechanical fragility index (MFI) is an in vitro measurement of the extent of RBC sublethal injury. Sublethal injury might constitute a component of the RBC storage lesion, thus the MFI was determined serially during routine RBC storage. Methods Leucoreduced AS‐5‐ and SAGM‐preserved RBCs were stored under routine blood bank conditions. The mechanical fragility (MF) of each unit was serially measured during storage. Results For both AS‐5 and SAGM units, male and female RBCs demonstrated statistically significant increases in the MFI during storage. The MFI was significantly lower in AS‐5 units compared to SAGM units throughout storage. Female RBCs had significantly lower MFI vs. male RBCs in both AS‐5 and SAGM units at all times. No significant differences in MFI were observed between ABO groups for both genders for AS‐5 RBCs. Conclusions The MF of RBCs increases during storage. Both gender and preservation solution influenced the MFI; however, the male:female MFI ratios were similar at all time‐points and remained stable, suggesting that gender‐based biological differences exist independent of storage solution. The MF could be a useful test for evaluating the effect of novel interventions intended to mitigate the susceptibility of RBCs to sublethal injury during storage.     

Studies in Red Blood Cell Preservation

Studies were conducted to examine whether an experimental additive solution (EAS-2) containing, in mM: 20 NH₄Cl, 30 Na₂HPO₄, 2 adenine, 110 dextrose, 55 mannitol, pH 7.15, would be useful to extend the storage shelf life of human RBCs. With 6 pairs of split units, ATP concentrations were better maintained for 12 weeks with EAS-2 than with Adsol® (1.8 vs. 1.1 μmol/gHb, respectively, p = 0.002). Autologous 24-hours ⁵¹Cr viability values for split units in the same donors were: on 6 paired units at 8 weeks, EAS-2 87.0±4.5%, Adsol 72.6±2.3%, p = 0.004; on 11 paired units at 9 weeks, EAS-2 79.5±7.1%, Adsol 68.2±10.1%, p = 0.0003. The data suggest that packed RBCs stored for 9 weeks with EAS-2 will be suitable for transfusion following the removal of supernatant with a single washing step.     

Influence of irradiation on in vitro red-blood-cell (RBC) storage variables of leucoreduced RBCs in additive solution PAGGS-M

Background The effect of gamma irradiation on leucoreduced red‐blood‐cells (RBCs) stored in an additive solution (AS) containing phosphate, adenine, glucose, guanosine, saline and mannitol (PAGGS‐M) has not yet been studied, and there are different recommendations about storage time of leucoreduced RBCs after irradiation. Study Design and Methods We studied 63 leucoreduced RBC units. All RBCs were stored in AS PAGGS‐M and leucoreduced on the collection day. Twenty‐one components were irradiated on Day +14 with 30 Gy and 22 served as non‐irradiated controls. Samples were drawn and analysed from these 43 units on Day +7, +14, +21, +28, +35, +42 and +49 from the collection day. From 20 units, no samples were taken earlier than on Day +49. Of these, 10 components had been irradiated on Day +14 with 30 Gy and 10 served as non‐irradiated controls. Results Gamma irradiation induced an enhanced in vitro haemolysis rate in the irradiated components. One of the irradiated units showed a haemolysis rate over the recommended limit of 0·8% on Day +42 and four on Day +49. The leakage of potassium ions from irradiated RBCs started to increase faster than that of unirradiated RBCs from the day of irradiation. Lactate dehydrogenase levels increased faster in irradiated units 3 weeks after irradiation. We showed that taking samples weekly does not affect the final result. Conclusions Our findings show that the European recommendations should not be changed in regard to the limitation of the storageability after irradiation of leucoreduced RBCs. The damage after irradiation and storage cannot be prevented by using the high‐quality AS PAGGS‐M.     

Variable adhesion of different red blood cell products to activated vascular endothelium under flow conditions

Red blood cells (RBCs) that have been stored prior to transfusion show increased adherence to vascular endothelium in vitro, which suggests a potential for stored blood transfusion to impede blood flow in some patients. Transfusion is often required in patients with sepsis or inflammation; however, whether activation of endothelium affects stored RBC-endothelial cell (EC) interactions is unknown. We investigated whether storage time and leukocyte content of RBC products influences the adhesion of RBCs to activated ECs. RBCs from nonleukocyte-reduced (S-RBCs), buffy-coat-poor (BCP-RBCs), and leukocyte-filtered (LF-RBCs) products and cultured EC layers were pretreated with endotoxin, tumor necrosis factor-alpha (TNF-alpha), or medium alone prior to perfusion of the RBCs across the EC layer in a continuous flow microchamber. After a single day of RBC storage, the number of adherent RBCs was increased in the endotoxin and TNF-alpha pretreated groups compared to the unactivated-control group. These differences were statistically significant for S-RBCs and LF-RBC products (P < 0.05). In contrast, there was no significant difference in RBC adherence to activated and unactivated endothelium at other time-points of RBC product storage. The strength of adhesion of stored RBCs from S-RBC products to activated ECs was not altered following treatment; however, endotoxin significantly increased the adhesive strength of LF-RBCs to endothelium. These results demonstrate that while fresh RBCs show increased adhesion to activated endothelium, storage of RBCs did not promote increased adhesion to activated endothelium. However, inflammatory conditions promote stronger adhesion of stored RBCs to ECs, which may contribute to impaired tissue perfusion in some transfusion recipients.     

Deterioration of red blood cell mechanical properties is reduced in anaerobic storage

BackgroundHypothermic storage of red blood cells (RBCs) results in progressive deterioration of the rheological properties of the cells, which may reduce the efficacy of RBC transfusions. Recent studies have suggested that storing RBC units under anaerobic conditions may reduce this storage-induced deterioration.ResultsThe bulk perfusion rates for anaerobically stored RBC were significantly higher than for conventionally stored RBCs over the entire duration of storage for all devices (up to 10% on day 42; up to 14% on day 63). Capillary perfusion rates suggested that anaerobically stored RBC units contained significantly fewer non-deformable RBC capable of transiently plugging microfluidic device capillaries. The number of plugging events caused by these non-deformable RBC increased over the 63 days of hypothermic storage by nearly 16- to 21-fold for conventionally stored units, and by only about 3- to 6-fold for anaerobically stored units.DiscussionThe perfusion measurements suggest that anaerobically stored RBC retain a greater ability to perfuse networks of artificial capillaries compared to conventionally (aerobically) stored RBC. It is likely that anaerobic storage confers this positive effect on the bulk mechanical properties of stored RBC by significantly reducing the number of non-deformable cells present in the overall population of relatively well-preserved RBC.     

Storage of whole blood overnight in different blood bags preceding preparation of blood components: in vitro effects on red blood cells

Background

Routines for the storage of whole blood (WB) overnight for the preparation of blood components on the following day are of increasing interest primarily for logistic reasons. The present study focuses on in vitro effects during storage for 6 weeks on red blood cells (RBC) prepared in different blood containers after being held overnight.

Study design and methods

Five different blood collection systems were used with either inline leucocyte reduction red cell filters for the preparation of RBC, buffy coat (BC) and plasma or WB filters for the preparation of RBC and plasma. A new container with an integrated WB filter removing leucocytes but not platelets was also included for the preparation of leucocyte-reduced RBC, BC and plasma units. Standard CPD solution (63 or 70 mL) and SAG-M solution (100 or 110 mL) were used for the collection of either 450 or 500 mL blood. All WB units were stored at room temperature, either overnight for 18–24 hours (test groups, n=104) or for up to 8 hours (reference groups, n=20). In addition, five test units were stored overnight under refrigeration.

Results

In test groups (overnight storage at room temperature) we found significantly lower levels of extracellular potassium, 2,3-DPG and pH (up to day 28). During storage, higher levels of ATP (Terumo, CaridianBCT until day 35, Fresenius until day 14, Fenwal throughout storage) were seen in test groups than in reference groups. When WB was stored overnight at 2–6°C before WB filtration, the levels of ATP and haemolysis were higher than in the corresponding reference.

Conclusion

Significant differences in in vitro parameters were observed between RBC prepared within 8 hours and 18–24 hours after blood collection. The results were consistent irrespective of the blood container used. New alkaline solutions may decrease the differences.     

Cellular properties of human erythrocytes preserved in saline-adenine-glucose-mannitol in the presence of L-carnitine

L-Carnitine (LC) in the preservation medium during storage of red blood cells (RBC) can improve the mean 24-hr percent recovery in vivo and increase RBC life-span after reinfusion. The purpose of the study was to investigate the differences in the biochemical properties of RBCs stored in the presence or absence of LC, and the cell-age related responses to storage conditions and to LC. RBC concentrates in saline-adenine-glucose-mannitol (SAG-M) were stored in the presence or absence of 5 mM LC at 4 degrees C for up to 8 weeks. RBC subpopulations of different densities were prepared by centrifugation on Stractan density gradient. Cells were sampled at 0, 3, 6, and 8 weeks, and hematological and cellular properties analyzed (MCV, MCHC, 4.1a/4.1b ratio as a cell age parameter, intracellular Na(+) and K(+)). After 6 weeks, MCV of RBC stored in the presence of LC was lower than that of controls (6 weeks MCV: controls 95.4 +/- 1.8 fl; LC 91.5 +/- 2.0 fl; n = 6; P < 0.005). This was due to swelling of control cells, and affected mainly older RBCs. LC appeared to reduce or retard cell swelling. Among the osmotically active substances whose changes during storage could contribute to cell swelling, only intracellular Na(+) and K(+) differed between stored control RBCs and LC-treated cells. LC reduces the swelling of older cells during storage at 4 degrees C in SAG-M, possibly by acting on the permeability of cell membrane to monovalent cations.     

Interlaboratory comparison of red-cell ATP, 2,3-diphosphoglycerate and haemolysis measurements

BACKGROUND AND OBJECTIVES: Red blood cell (RBC) storage systems are licensed based on their ability to prevent haemolysis and maintain RBC 24-h in vivo recovery. Preclinical testing includes measurement of RBC ATP as a surrogate for recovery, 2,3-diphosphoglycerate (DPG) as a surrogate for oxygen affinity, and free haemoglobin, which is indicative of red cell lysis. The reproducibility of RBC ATP, DPG and haemolysis measurements between centres was investigated. MATERIALS AND METHODS: Five, 4-day-old leucoreduced AS-1 RBC units were pooled, aliquotted and shipped on ice to 14 laboratories in the USA and European Union (EU). Each laboratory was to sample the bag twice on day 7 and measure RBC ATP, DPG, haemoglobin and haemolysis levels in triplicate on each sample. The variability of results was assessed by using coefficients of variation (CV) and analysis of variance. RESULTS: Measurements were highly reproducible at the individual sites. Between sites, the CV was 16% for ATP, 35% for DPG, 2% for total haemoglobin and 54% for haemolysis. For ATP and total haemoglobin, 94 and 80% of the variance in measurements was contributed by differences between sites, and more than 80% of the variance for DPG and haemolysis measurements came from markedly discordant results from three sites and one site, respectively. In descending order, mathematical errors, unvalidated analytical methods, a lack of shared standards and fluid handling errors contributed to the variability in measurements from different sites. CONCLUSIONS: While the methods used by laboratories engaged in RBC storage system clinical trials demonstrated good precision, differences in results between laboratories may hinder comparative analysis. Efforts to improve performance should focus on developing robust methods, especially for measuring RBC ATP.     

Prolonged storage of red blood cells affects aminophospholipid translocase activity

BACKGROUND AND OBJECTIVES: Loss of phospholipid asymmetry in the membrane of red blood cells (RBC) results in exposure of phosphatidylserine (PS) and to subsequent removal from the circulation. In this study, we investigated the effect of long-term storage of RBCs on two activities affecting phospholipid asymmetry: the ATP-dependent aminophospholipid translocase (or flippase, transporting PS from the outer to the inner leaflet) and phospholipid scrambling (which will move PS from the inner to the outer leaflet). MATERIALS AND METHODS: Standard leukodepleted RBC concentrates were stored in saline-adenine-glucose-mannitol (SAGM) at 4 degrees C for up to 7 weeks. PS exposure was determined by measurement of AnnexinV-FITC binding to the cells, flippase activity by measurement of the inward translocation of NBD-labelled PS. Scrambling activity was determined by following the inward translocation of fluorescent NBD-phosphatidylcholine. In parallel, intracellular ATP levels were determined. RESULTS: PS exposure amounted to only 1.5 +/- 0.3% positive cells (n = 8) after 5 weeks of storage, which slightly increased to 3.5 +/- 0.7% (n = 8) after 7 weeks of storage. Flippase activity started to decrease after 21 days of storage and reached 81 +/- 5% of the control value after 5 weeks of storage (n = 6) and 59 +/- 6% (n = 6) after 7 weeks. Also in RBC obtained by apheresis, flippase activity decreased upon storage. Scrambling activity remained virtually absent during storage, explaining the low PS exposure despite the decrease in flippase activity. Rejuvenation of RBC after 7 weeks to increase ATP levels only partially restored flippase activity, but in combination with a correction of the intracellular pH to that of fresh cells, almost complete restoration was achieved. The decrease in flippase activity after prolonged storage did make the RBCs more prone to PS exposure after activation of phospholipid scrambling. CONCLUSION: This study shows that, although PS exposure remains low, prolonged storage does compromise the RBC membrane by affecting flippase activity. When the metabolic changes induced by storage are corrected, flippase activity can be restored.     

Improved leucoreduction of red blood cell units prepared after a 24-h hold with the platelet-rich plasma method using newly developed filters

Background and Objectives  A previous study indicated that the extension of whole blood (WB) storage from 8 to 24 h at 20–24 °C before the processing of platelet-rich plasma (PRP)-depleted red blood cell (RBC) units had a negative effect on the efficacy of leucoreduction filters. In this study, we further characterized the phenomenon and tested the leucoreduction capacity of two newly developed filters.
Materials and Methods  Whole blood was stored at 20–24 °C and processed at 4-h intervals between 8 and 24 h postcollection. Components were leucoreduced before storage. Efficacy of novel filters to leucoreduce 24-h-hold PRP-depleted RBC units was also evaluated.
Results  Using a conventional filter, the mean residual white blood cell (WBC) counts in leucoreduced PRP-depleted RBCs were comparable in units prepared within 12 h from collection but gradually increased upon extended preprocessing storage from 0·36 ± 0·03 at 12 h to 0·46 ± 0·21, 0·76 ± 0·54 and 1·72 ± 1·76 × 10⁶ per unit at 16, 20 and 24 h, respectively. However, the mean residual WBC content in 24-h-hold RBCs was reduced to 0·60 ± 0·39 × 10⁶ and 0·46 ± 0·13 × 10⁶ per units using RC2D and the prototypes B-1582 rev B filters, respectively.
Conclusion  For PRP-depleted RBC units, the extension of the WB room temperature storage from 8 to 24 h before processing is likely to require the introduction of newly developed filters having an increased leucoreduction capacity in order to meet the maximal residual WBC guideline in the RBCs.     

Stability of lymphocytes and Epstein-Barr virus during red blood cell storage

BACKGROUND: The Epstein-Barr virus (EBV) establishes and maintains latent infection in B lymphocytes of the healthy adults. Lymphocytes remain viable during red blood cell (RBC) storage. The effect of RBC storage on the stability of EBV-infected B lymphocytes and EBV genome is not known. STUDY DESIGN AND METHODS: Eight randomly selected non-leukoreduced AS-5 RBC units were stored for 42 days under standard blood bank refrigerated at 1-6 degrees C. Cell count and EBV genomes in CD19+ B lymphocytes were measured in fresh products and weekly for 6 weeks. Total white blood cells (CD45+), T lymphocyte (CD3+), and B lymphocyte (CD19+) were quantified by a single platform flow cytometric assay. EBV genomes were quantified by real-time polymerase chain reaction using DNA purified from CD19+ B cells. RESULTS: Viable white blood cell, T and B lymphocytes followed a biphasic decline curve during RBC storage consisting of a steep steady decline during the first 3 weeks followed by a plateau for the remainder of the storage. At the end of the RBC shelf-life, 19% of the original T and B cells remained viable. EBV genomes per 10(5) CD19+ B lymphocytes remained constant during RBC storage. However, the total EBV genomes in the RBC units decline by more than 80% of their original value at the end of RBC storage due to loss of viable B lymphocytes. CONCLUSIONS: The results indicate that lymphocytes and EBV latently infected B cells can survive the normal storage conditions for RBC.     

Hypoxia and hypocapnia storage of γ-irradiated red cell concentrates

Backgroundγ-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 methodsO₂ 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.ResultsStarting 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.DiscussionThe 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.     

Platelets Stored for 6 Days in a Polyolefin Container in a Synthetic Medium with Minimal Plasma Carry-Over

Platelet concentrates (PC) were stored for 6 days in either polyolefin (PO) or polyvinylchloride/di-(2-ethylhexyl)phtalate (PVC/DEHP) bags in 100% plasma or in a synthetic medium with 35 or 10% plasma. For all conditions studied the usual in vitro parameters were well maintained, with a pH above 6.8. In both bag types platelets can be satisfactorily stored for 6 days in a synthetic medium with minimal amounts of residual plasma. For this medium, the PO bag offers a slight advantage with respect to the preservation of platelet ATP content (>80 versus >70% in the PVC bags) and aggregation and adhesion capacity. The adhesion capacity increased in the PO bags, while it decreased in the PVC bags.