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.     

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.     

Influence of late irradiation on the in vitro RBC storage variables of leucoreduced RBCs in SAGM additive solution

Background There exists only few data on in vitro and in vivo effects of gamma irradiation of leucoreduced red blood cells (RBCs). Reported studies reflect the effects of early irradiation and subsequent storage. The effects of irradiation on RBCs shortly before the end of their shelf‐life have not been examined. Study Design and Methods We studied 160 RBC units that were stored in the additive solution saline–adenine–glucose–mannitol and leucoreduced on the collection day. Forty components were irradiated on day +14 with 30 Gy, 40 on day +28, 40 on day +35, and 40 served as nonirradiated controls. In vitro evaluation of all units was performed on days +3, +7, +14, +21, +28, +35, and +42 from the collection day. Results Gamma irradiation induced leakage of potassium ions and lactate dehydrogenase and enhanced in vitro haemolysis rate in the irradiated components, which started to increase faster than that of nonirradiated RBCs from the day of irradiation, i.e. from day +14 in units that were irradiated on day +14, from day +28 in units that were irradiated on day +28, and from day +35 in units that were irradiated on day +35. Conclusions This study presents data on the in vitro quality of leucoreduced RBCs that have been irradiated on days +14, +28, or +35 after collection. Our findings support the proposal that the current limitation of the age of RBCs on the day of gamma irradiation may be replaced by staged limitations depending on the time of irradiation.     

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.     

The effects of polyvinyl chloride and polyolefin blood bags on red blood cells stored in a new additive solution.

BACKGROUND AND OBJECTIVES: Red blood cells (RBCs) must be stored in polyvinyl chloride (PVC) bags plasticized with di-2-ethylhexyl phthalate or a similar plasticizer to achieve their full storage life with conventional storage solutions. Improved storage solutions might remove this requirement and allow blood storage in other plastics. Experimental Additive Solution-61 (EAS-61), which maintains RBCs for 9 weeks with reduced haemolysis and satisfactory 51Cr 24-h recovery, is an appropriate candidate improved RBC storage solution. MATERIALS AND METHODS: Twenty-four units of packed RBCs were pooled in groups of four units, each pool was realiquoted into four units and stored, six pooled units per arm, in one of the following: 100 ml of EAS-61 in PVC; 200 ml of EAS-61 in PVC; 100 ml of EAS-61 in polyolefin (PO); and 200 ml of EAS-61 in PO. Haemolysis, RBC morphology indices, RBC ATP concentrations, and other measures of RBC metabolism and function were measured weekly. RESULTS: RBC haemolysis exceeded 1% by 7 weeks in PO bags containing 100 ml or 200 ml of EAS-61. In PVC bags, haemolysis was less than 1% at 11 weeks. RBC ATP concentrations were 1 mol/g of haemoglobin (Hb) higher at 2 weeks in the PVC-stored units. CONCLUSIONS: RBCs stored in PVC had markedly less haemolysis and higher RBC ATP concentrations than those stored in PO. Haemolysis would limit RBC storage in PO bags to a duration of 6 weeks, even with EAS-61.     

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.     

Stored red‐blood‐cells inhibit platelet function under physiologic flow

Background and Objectives The DiaMed Impact R tests platelet function under close to physiological flow conditions. The machine is designed to use whole blood but by adding back compatible red cells, it can be used to study stored platelet concentrates. To date, red cells ≤14 days old have been used. In this study, the effect on the assay of using red cells stored for up to 60 days was examined. Material and Methods This study looked at buffy coat‐derived platelet concentrates on day 2 of storage along with various stored red‐blood‐cells (RBC). To determine whether the age of the RBC is a factor in supporting adhesion and aggregation, platelets were assayed with either RBC stored between 2 and 60 days or with separated ‘young’ and ‘old’ red cell populations obtained using a centrifugation method and confirmed by percoll gradient analysis. Results A statistically significant difference was observed between red‐blood‐cells stored for ≤20 days compared with those which have been stored for 21–60 days in respect of their ability to support platelet adhesion (SC) and aggregation (AS) (P < 0·01). Separating red cells by centrifugation into top (young population) and bottom (old population) showed that the effect of storage was much greater than was any difference between young and old at the individual time‐points e.g. ‘young’ red cells from stored units were poorer at supporting platelet adhesion and aggregation than ‘young’ red cells from fresh units. Conclusion Results suggest that the red cells should be stored for less than 21 days when using this assay. This assay may also allow assessment of red cell functionality.     

Cytokine generation in whole blood, leukocyte-depleted and temporarily warmed red blood cell concentrates

BACKGROUND AND OBJECTIVES: It has been suggested that inflammatory cytokines such as Interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha (TNF-alpha) and IL-8 might be responsible for a large number of non-antibody-mediated adverse reactions to the transfusion of blood components, especially of platelet concentrates (PCs). The aim of this study was to compare the levels of proinflammatory cytokines in different blood components containing red cells such as buffy-coat-free packed red cells (RBCs), filtered RBCs and whole blood (WB) during storage under several conditions. MATERIALS AND METHODS: WB (CPD-A1, n = 16) was stored for 35 days at 2-6 degrees C; samples were taken on days 0, 21 and 35. Buffy-coat-poor RBCs in additive solution PAGGS-M (n = 16) were divided into halves, one half was leukocyte (WBC)-depleted by filtration on day 0, both halves were stored for 49 days at 2-6 degrees C (samples: days 0, 21, 49). Furthermore, buffy-coat-poor, unfiltered SAG-M RBCs (n = 16) were halved immediately after production and stored at 2-6 degrees C until day 42 (samples: days 0, 21, 42). One half remained at room temperature for 24 h on day 3. Cytokine levels were determined with commercial enzyme-linked immunosorbent assays. RESULTS: Levels of IL-1beta and TNF-alpha rose during storage of WB and RBCs. IL-6 could be detected markedly above the detection threshold in WB only. At the end of storage, we detected IL-8 in 1 of 16 units of WB tested, in 10 of 16 standard PAGGS-M RBCs and in 15 of 16 temporarily warmed SAG-M RBCs. Prestorage filtration of RBCs prevented the accumulation of IL-1beta and TNF-alpha. Temporarily warming of RBCs for 24 h did not cause any substantial increase in cytokine levels other than IL-8. RBCs stored in different additive solutions (PAGGS-M versus SAG-M) showed similar cytokine concentrations during storage. The cytokine content of WB was very similar to that of buffy-coat-poor RBCs. CONCLUSION: Cytokine levels measured in WB and buffy-coat-poor RBCs result in levels which are unlikely to cause febrile reactions even in the case of massive transfusion. We conclude that, according to present knowledge, there is no reason for prestorage filtration of buffy-coat-poor RBCs or WB to avoid febrile transfusion reactions due to cytokine accumulation during storage.     

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.     

Gamma-ray-irradiated red blood cells stored in mannitol-adenine-phosphate medium: rheological evaluation and susceptibility to oxidative stress

BACKGROUND AND OBJECTIVES: To evaluate the rheological properties and the oxidative susceptibility of gamma-ray-irradiated red blood cells (RBCs). MATERIALS AND METHODS: RBCs in mannitol-adenine-phosphate (MAP) medium were irradiated with 35 Gy and stored at 4 degrees C for 4 weeks. The deformability of the RBCs was examined under shear flow in relation to the morphological and biochemical changes. The RBCs were further exposed to 1 mM FeSO(4) and 5 mM ascorbate to examine the oxidative susceptibility. RESULTS: The RBC deformability was decreased during storage, and the impairment was further enhanced by the irradiation, which promoted cell shrinkage and intracellular hemoglobin condensation accompanying potassium loss. Lipid peroxidation and protein aggregation of the RBC membrane as well as echinocytosis were not enhanced by the irradiation. The exposure to free iron did not stimulate the oxidation of the irradiated RBC membrane. CONCLUSION: The decreased deformability of gamma-ray-irradiated RBCs in MAP medium was mainly induced by dehydration due to potassium loss, and the membrane lipids and proteins were stably preserved against oxidative stress.     

Concurrent collection of in-line filtered platelets and red blood cells by apheresis

Multicomponent apheresis procedures offer the possibility to collect standardized blood components as compared to whole blood donations. A new program for the concurrent collection of platelets (PLTs) and red blood cells (RBCs) was evaluated in a prospective study. Apheresis donors ( n=18) underwent concurrent collection of PLTs and RBCs using the Haemonetics MCS+ blood cell separator. Aliquots of PLTs and RBCs were collected during five to six passes of the discontinuous flow procedure. The platelet product was in-line filtered during the last pass of the separation procedure. After collection, saline-adenine-glucose-mannitol (SAGM) preservative solution was automatically added to the RBCs. Thereafter, the RBCs were in-line leukodepleted by gravity filtration at room temperature. The PLTs and RBCs were subsequently stored at 22+/-2 degrees C for 5 days and 4+/-2 degrees C for 35 days, respectively. The following in vitro parameters were evaluated over the storage periods: blood cell counts, glucose, lactate, lactate dehydrogenase, pH, plasma hemoglobin, and potassium. Two ready-to-use blood components from one donor were collected in an average procedure time of 86+/-10 min; 2.47+/-0.74 x 10(11) PLTs were collected in a product volume of 232+/-43 ml. The RBC volume averaged 280+/-20 ml and the hemoglobin content was 56.8+/-2.4 g per unit. The leukocyte contamination of the platelet product was 0.44+/-0.56 x 10(5) and the residual leukocyte content of the RBC product was 0.28+/-0.02 x 10(5). Storage data showed no relevant drop in pH. Day 35 results of the RBC products showed that all of the units had less than 0.8% hemolysis. Standardized PLT and RBC products of good quality can be concurrently collected with the MCS+ blood cell separator. In vitro testing of the products collected and stored for 5 and 35 days, respectively, met the Council of Europe criteria for leukodepleted blood products.     

Alloimmunization is associated with older age of transfused red blood cells in sickle cell disease

Red blood cell (RBC) alloimmunization is a significant clinical complication of sickle cell disease (SCD). It can lead to difficulty with cross‐matching for future transfusions and may sometimes trigger life‐threatening delayed hemolytic transfusion reactions. We conducted a retrospective study to explore the association of clinical complications and age of RBC with alloimmunization in patients with SCD followed at a single institution from 2005 to 2012. One hundred and sixty six patients with a total of 488 RBC transfusions were evaluated. Nineteen patients (11%) developed new alloantibodies following blood transfusions during the period of review. The median age of RBC units was 20 days (interquartile range: 14–27 days). RBC antibody formation was significantly associated with the age of RBC units (P = 0.002), with a hazard ratio of 3.5 (95% CI: 1.71–7.11) for a RBC unit that was 7 days old and 9.8 (95% CI: 2.66–35.97) for a unit that was 35 days old, 28 days after the blood transfusion. No association was observed between RBC alloimmunization and acute vaso‐occlusive complications. Although increased echocardiography‐derived tricuspid regurgitant jet velocity (TRV) was associated with the presence of RBC alloantibodies (P = 0.02), TRV was not significantly associated with alloimmunization when adjusted for patient age and number of transfused RBC units. Our study suggests that RBC antibody formation is significantly associated with older age of RBCs at the time of transfusion. Prospective studies in patients with SCD are required to confirm this finding. Am. J. Hematol. 90:691–695, 2015. © 2015 Wiley Periodicals, Inc.     

Processing cord blood from premature infants into autologous red-blood-cell products for transfusion

Background and Objectives The use of umbilical cord blood (UCB) for transfusion purposes has gained interest the past years. UCB transfusion could serve premature infants, who often need transfusions early in life. Material and Methods We investigated the suitability of different storage media. UCB was collected after 25 ⁰/₇–35 ⁶/₇ gestational weeks and centrifuged to concentrate red cells subsequently stored in saline–adenine–glucose–mannitol (SAGM), or in additive solution‐3 (AS‐3), or stored as whole blood in citrate–phosphate–dextrose–adenine‐1. Quality parameters were measured at 7 day intervals during 35 days and compared to the standard RBC product. Results White‐blood‐cell‐ and platelet counts were higher in the UCB products. In the fractionated units, haemolysis remained below 1·0% in 64% after 14 days, and in 30% after 21 days. Storage in SAGM or AS‐3 showed similar quality. Whole blood UCB showed better pH and haemolysis rates after 21 days. Conclusion UCB can be processed into autologous products for premature infants. Shelf‐life is limited to 14–21 days and compares unfavourably to stored whole blood. Considering the early transfusion needs in these infants, a short shelf‐life would not be a practical objection.     

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.     

Modified formulation of CPDA for storage of whole blood, and of SAGM for storage of red blood cells, to maintain the concentration of 2,3-diphosphoglycerate

BACKGROUND AND OBJECTIVES: A dramatic decrease in the level of 2,3-diphosphoglycerate (2,3-DPG) takes place during the storage of whole blood (WB) in CPDA (citrate-phosphate-dextrose-adenine) and a similar decrease occurs during the storage of red blood cells (RBCs) in SAGM (saline-adenine-glucose-mannitol). The aim of the present study was to prevent this decrease by modifying CPDA and SAGM. MATERIALS AND METHODS: The pH of WB anticoagulant or RBC preservative solution was maintained at 7.6 by autoclaving the dextrose solution separately, by incorporating ascorbic acid and nicotinic acid into both CPDA and SAGM (to produce modified CPDA and SAGM solutions), and by reducing the concentration of adenine and adding citrate to the modified SAGM solution. The concentration of 2,3-DPG in WB after 28 days of storage in modified CPDA, and in RBCs stored in modified SAGM, was compared with that in WB or RBCs stored in unmodified solutions. RESULTS: The initial 2,3-DPG levels were maintained after 28 days in the modified formulations [10.63 +/- 2.58 microM/g of haemoglobin (Hb) in the case of modified CPDA and 12.07 +/- 1.47 microM/g of Hb in the case of modified SAGM], whereas in standard CPDA and SAGM solutions, the concentration of 2,3-DPG decreased to very low levels (0.86 +/- 0.97 microM/g Hb for CPDA and 0.12 +/- 0.008 for SAGM). CONCLUSIONS: Our modification in the formulation of CPDA or SAGM is effective in arresting the dramatic decrease in the level of 2,3-DPG that occurs during storage of WB and RBCs in unmodified solutions.     

Red blood cells metabolome changes upon treatment with different X-ray irradiation doses

The upholding of red blood cells (RBC) quality and the removal of leukocytes are two essential issues in transfusion therapy. Leukodepletion provides optimum results, nonetheless there are cases where irradiation is recommended for some groups of hematological patients such as the ones with chronic graft-vs-host disease, congenital cellular immunodeficiency, and hematopoietic stem cell transplant recipients. The European guidelines suggest irradiation doses from 25 to 50 Gray (Gγ). We evaluated the effect of different prescribed doses (15 to 50 Gγ) of X-ray irradiation on fresh leukodepleted RBCs bags using a novel protocol that provides a controlled irradiation. Biochemical assays integrated with RBCs metabolome profile, assessed by nuclear magnetic resonance spectroscopy, were performed on RBC units supernatant, during 14 days storage. Metabolome analysis evidenced a direct correlation between concentration increase of three metabolites, glycine, glutamine and creatine, and irradiation dose. Higher doses (35 and 50 Gγ) effect on RBC mean corpuscular volume, hemolysis, and ammonia concentration are considerable after 7 and 14 days of storage. Our data show that irradiation with 50 Gγ should be avoided and we suggest that 35 Gγ should be the upper limit. Moreover, we suggest for leukodepleted RBCs units the irradiation with the prescribed dose of 15 Gγ, value at center of bag, and ranging between 13.35–15 Gγ, measured over the entire bag volume, may guarantee the same benefits of a 25 Gγ dose assuring, in addition, a better quality of RBCs.     

Effects of storage time and leucocyte burden of packed and buffy-coat depleted red blood cell units on red cell storage lesion

Background

The red cell storage lesion (RCSL) comprises the biochemical and biomechanical changes that take place during red blood cell (RBC) storage, reducing the survival and function of these cells. Contaminating white blood cells have been major contributors to the RCSL. Markers of RCSL, such as CD47 and phosphatidylserine (PS), on RBC are attracting more attention. The aim of this study was to elucidate the effects of storage time and buffy-coat removal on CD47 and PS expression on RBC. Potassium and free haemoglobin levels in the supernatant plasma were also assessed.

Materials and methods

Forty-three red cell concentrates were divided into two groups [Group 1: packed red cells (n=22); Group 2: red cell units from which the buffy-coat had been removed (n=21)] and samples were collected on days 1, 14 and 28. Flow cytometry was used to monitor changes of CD47 and PS expression on RBC over times. Supernatant potassium was measured and percent of haemolysis calculated.

Results

A significant, progressive decrease in RBC CD47 expression during storage was observed in both groups. The decrease in RBC CD47 expression was significantly less in the buffy-coat-removed group of units than in the other group. The percentage of annexin V-positive cells increased significantly in both groups. Buffy-coat depleted components showed less expression of PS only in the early samples. There were significant, progressive increases in percentage of haemolysis and supernatant potassium during storage in both groups.

Conclusion

RBC stored for more than 14 days exhibited reduced CD47 and increased PS. Buffy coat removal reduced the loss of CD47, but had no impact on plasma haemoglobin, potassium or RBC PS exposure.     

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.     

Evaluation of in vivo and in vitro Quality of Apheresis-Collected RBC Stored for 42 Days

Background and Objectives : New technological developments make it possible to collect red blood cells (RBCs) by apheresis, which allows for better product consistency and has the potential for improved RBC quality. The purpose of these studies was to evaluate the quality and consistency of units of RBCs collected by apheresis using the MCS+® machine (Haemonetics Corp., Braintree, Mass., USA). Materials and Methods : Two studies were performed. In study 1 (n = 10), using containers and CP2D/AS-3 solutions from Medsep Corp. (Covina, Calif. USA), one-unit apheresis RBCs were compared to manually collected RBCs in a random crossover design. In study 2 (n = 12), 6 subjects had one unit collected, while the remaining 6 subjects had two units of RBCs collected with comparison to previously manually collected RBCs from the same donors. Haemonetics containers and solutions were used in study 2. Results : Low RBC volume variability was found for the apheresis collections with a standard deviation of only 6 ml difference between actual and target volumes. Combining the data from the two studies (n = 21 pairs), at 42 days of storage, the apheresis units showed slightly lower hemolysis (0.44±0.26 vs. 0.61±0.50%), lower supernatant potassium levels (50±3 vs. 53±3 mEq/l), and improved tolerance to osmotic shock (47±3 vs. 49±3%) as compared to manual units (p < 0.05). There was no statistically significant difference in RBC ATP (3.0±0.6 vs. 2.9±0.5 μmol/g Hb) or in 24-hour percent recoveries (81±6 for apheresis vs. 81±4% for apheresis red cells). Apheresis RBC quality was not affected by the manufacturer (Haemonetics vs. Medsep) of solutions and containers. Conclusions : RBC units collected by apheresis demonstrated low variability in volume of RBC mass collected, and showed similar RBC properties as compared to manually collected RBCs after processing and after 42 days of storage.     

Optical tweezers for measuring red blood cell elasticity: application to the study of drug response in sickle cell disease

The deformability of erythrocytes is a critical determinant of blood flow in microcirculation. By capturing red blood cells (RBC) with optical tweezers and dragging them through a viscous fluid we were able to measure their overall elasticity. We measured, and compared, the RBC deformability of 15 homozygous patients (HbSS) including five patients taking hydroxyurea (HU) for at least 6 months (HbSS/HU), 10 subjects with sickle cell trait (HbAS) and 35 normal controls. Our results showed that the RBC deformability was significantly lower in haemoglobin S (HbS) subjects (HbSS and HbAS), except for HbSS/HU cells, whose deformability was similar to the normal controls. Our data showed that the laser optical tweezers technique is able to detect differences in HbS RBC from subjects taking HU, and to differentiate RBC from normal controls and HbAS, indicating that this is a very sensitive method and can be applied for detection of drug-response in sickle cell disease.