Composition of the additive solution affects red blood cell integrity after photodynamic treatment

BACKGROUND AND OBJECTIVES: Photodynamic treatment is a promising technique for pathogen inactivation of red blood cell concentrates. For protocol optimization, the influence of the composition of the storage solution on the integrity of phototreated red cells was studied. MATERIALS AND METHODS: Red blood cells were resuspended in the storage solutions SAG-M or AS-3 to a haematocrit (Hct) of 30%. After addition of the photosensitizer, 1,9-dimethylmethylene blue (DMMB) (25 microm), the suspensions were illuminated with red light, and potassium leakage and delayed haemolysis were determined. In some experiments, the cells were washed after illumination and resuspended in modified storage solutions. RESULTS: Illumination of red cells in the presence of DMMB resulted in an immediate, light-dose-dependent increase in potassium leakage. The illumination conditions used induced no detectable haemolysis immediately after photodynamic treatment. Potassium leakage was higher when the illumination was performed in AS-3. In contrast, delayed haemolysis, measured after overnight storage, was considerably lower when cells were stored in AS-3. This protection was mainly a result of the presence of citrate in AS-3. In addition, other impermeant solutes protected against haemolysis. CONCLUSIONS: The additive solution strongly influences the integrity of red cells after photodynamic treatment. Whereas the solution in which the cells are illuminated has a small effect on red cell integrity, the main influence of the additive solution is during post-treatment storage. Red cell integrity is best maintained when illumination is performed in SAG-M followed by storage in AS-3. The presence of non-permeant solutes, such as citrate, in the solution used for storage, prevents haemolysis of the phototreated, cation-permeable cells by counterbalancing the osmotic activity of haemoglobin.     

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.     

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.     

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.     

Development of a combined storage medium for 7-day storage of platelet concentrates and 42-day storage of red cell concentrates

Studies were carried out to examine whether a single additive solution could support both platelet and red cell storage. An ionically balanced electrolyte solution fortified with citrate, glucose and bicarbonate was used. This solution has previously been shown to provide good platelet viability with storage for up to 7 d. Optimization studies demonstrated that with adenine added to this solution (CSM), it also allowed for satisfactory preservation of in vitro red cell parameters for up to 49 d of storage. Confirmatory paired in vivo post-transfusion studies were carried out in which platelets and red cells obtained from the same donor were processed and stored in CSM on one occasion, and in CPD-plasma (platelets) and AS-1 (red cells) on another occasion. Five paired studies were conducted with platelets stored for 5 d and red cells for 42 d; another five paired studies with platelets stored for 7 d and red cells for 49 d. Except for a slight decrease in platelet survival (P less than 0.05) with storage in CSM, there were no statistically significant differences in post-transfusion recoveries and survivals between test and control media, demonstrating that both platelets and red cells may be satisfactorily stored in a combined single additive solution.     

Erythrocyte Endogenous Proteinase Activity during Blood Bank Storage

We studied proteolytic alterations of membrane proteins in ghosts derived from human red blood cells, preserved up to 35 days in the liquid state either as whole blood or with additive solution. The study was carried out by performing sodium dodecyl sulfate polyacrylamide gel electrophoresis of stromal proteins from erythrocytes, either previously treated with proteinase inhibitors or previously incubated in conditions promoting proteolysis. To differentiate the effect of erythrocyte from granulocyte proteinases, the investigation was also carried out in leukocyte-free red cell preparations. The results show: (1) the effects of endogenous proteinases on membrane proteins derived from red cells stored under blood bank conditions; (2) a decrease of proteolytic effects in ghosts derived from red cells which have been submitted to a longer storage; (3) a relevant influence of the red cell resuspending medium before lysis on the time-dependent onset and exhaustion of proteolysis in ghosts. The presence of increased proteolysis in ghosts could be regarded as a marker of molecular lesions induced in red cells by storage under blood bank conditions.     

Studies in Red Blood Cell Preservation 2. Comparison of Vesicle Formation, Morphology, and Membrane Lipids during Storage in AS-1 and CPDA-1

Abstract. The changes in morphology, the quantitative changes in membrane lipids and the shedding of exocytic vesicles by red blood cells (RBC) stored for 42 and 56 days in AS-1 and CPDA-1 were compared. RBC stored in AS-1 shed significantly less vesicle membrane cholesterol, phospholipid and protein and maintained better morphology scores. RBC membrane cholesterol remained higher after 56 days in AS-1 than in CPDA-1. The data suggest that during the first weeks of storage cholesterol is lost from the RBC membrane followed by a larger release of phospholipids accompanied by alterations in the phosphoinositides. The shedding of exocytic vesicles appears to be secondary to the changes in morphology resulting from the perturbation of the membrane lipids.     

Effects of 3-5 log10 pre-storage leucocyte depletion on red cell storage and metabolism

SUMMARY. A new, in-line high-efficiency 3-5 log₁₀ leucodepletion filter system (Leukotrap° RC system) was used to investigate the effect of pre-storage white cell removal on the quality of AS-3 red cell concentrates stored for 42 d at 4°. Median residual white cell content was 4 × 10⁵ when filtration was performed at 22° within 8 h of phlebotomy ( n = 20) and 3.2 × 10⁴ when filtration was performed at 4° 12-24 h after phlebotomy ( n = 24). None exceeded 1 × 10⁶ WBC per red cell product. Filtration was rapid (median 28 min), and red cell loss averaged (mean ± 1 SD) 6.4 ± 0.7%. In a paired study design, post-transfusion recoveries of 42 d stored red cells in the filtered units averaged 84 ± 6% v 82 ± 8% for unfiltered units ( P < 0.05) and post-storage haemolysis, ATP, osmotic fragility, K+ and pH were significantly ( P < 0.05) better in the filtered units. Reduced glycolytic activity was also observed in the filtered units, and there was a correlation between osmotic fragility, glucose consumption, and lactate produced in standard units that was not present in leucodepleted units. In conclusion, this study suggests that leucodepletion of AS-3 red cell concentrates prior to storage results in better maintenance of the integrity of the red cell membrane with reduced glycolytic activity. There was a modest improvement in post-infusion viability sufficient to offset the filtration-induced loss and to result in an equivalent red cell product.     

Storage of ADSOL-preserved red cells at 2.5 and 5.5 degrees C: comparable retention of in vitro properties

Previous studies with CPDA-1 and Nutricel preserving solutions indicated that red cell properties were affected by small differences in storage temperature. The influence of a 3 degrees C differential on the preservation of the in vitro properties of ADSOL-preserved (AS-1) red blood cells was investigated in a paired study. AS-1 red blood cells were stored at 2.5 and 5.5 degrees C for 42 days. Extent of hemolysis, glucose consumption and pH levels were comparable at the two storage temperatures. Lactate levels were slightly, but significantly higher at 5.5 degrees C. ATP levels were slightly but significantly higher at 2.5 degrees C, only during the later part of the storage period. 2.3-DPG levels were slightly better retained at 2.5 degrees C after 7 days of storage. Holding units of whole blood for either 1 or 8 h at ambient temperature after phlebotomy prior to processing did not influence the types of temperature-dependent changes. The differences as a function of storage temperature were small and appear to be of no practical importance in connection with the storage of AS-1 red blood cells.     

In vitro function of buffy coat-derived platelet concentrates stored for 9 days in CompoSol, PASII or 100% plasma in three different storage bags

BACKGROUND AND OBJECTIVES: The aim of the study was to compare the in vitro quality of buffy coat-derived platelet concentrates (PC) during extended storage in plasma or additive solution in three different storage bags. MATERIALS AND METHODS: A pooled and split design was chosen so that identical PCs were produced in either 100% plasma, 70% PASII : 30% plasma or 70% CompoSol : 30% plasma (n = 6 each). This was repeated for three different manufacturers' platelet storage bags (Fresenius, Baxter and Pall). PCs were sampled on days 1, 5, 7 and 9 of storage and tested in vitro using a variety of tests of platelet function. For each bag type, storage in PASII or Composol was compared with plasma (data taken across the entire storage period), and differences occurring with time were analysed for all storage media. RESULTS: The pH of all PCs was > 6.8 at day 9 of storage. In vitro platelet function, as assessed by markers of platelet activation and metabolism, of PCs stored in CompoSol appeared to be similar to that of PCs stored in plasma over 9 days of storage. In contrast, PCs stored in PASII tended to have significantly higher levels of platelet activation (almost a twofold increase in % platelets positive for CD62P by day 5) and lower hypotonic shock response (approximately 40%, by day 7) compared to either PCs stored in 100% plasma or 70% CompoSol. The magnitude of the differences observed between platelet storage media appeared to be dependent on the type of platelet storage bag with the highest degree of platelet activation and lowest hypotonic shock response values being observed in Fresenius bags in combination with PASII. CONCLUSIONS: The maintenance of platelet function in vitro during extended storage of PCs in platelet additive solutions is dependent on the combination of type of additive solution and type of platelet storage bag. For all bag types studied, storage in PASII resulted in poorer platelet function in vitro.     

Half-Strength Citrate CPD Combined with a New Additive Solution for Improved Storage of Red Blood Cells Suitable for Clinical Use

Currently used systems for red blood cell (RBC) collection and storage for transfusion have the disadvantage that the RBC 2,3-bisphosphoglycerate (BPG) concentration is depleted within two weeks of storage, resulting in a left-shift of the oxygen dissociation curve and a temporarily impaired capacity to deliver oxygen. We have studied the effects on red cell metabolism, morphology and in vivo recovery of 49-day storage of RBC, with collection in half-strength citrate CPD (0.5CPD) and storage in an additive solution containing citrate, adenine, mannitol, phosphate and glucose (RAS2). Traditional CPD-SAGM was used for comparison. Component preparation was performed after an initial holding period of the whole blood at ambient temperature for 8 h. The BPG concentration in 0.5CPD-RAS2 RBC was 0.633 ± 0.120mol (mol Hb)^-1 as compared to 0.454±0.138mol (mol Hb)^-1 in CPD-SAGM RBC which implied a decrease to 67 and 48% of normal concentration, respectively. The mean RBC BPG concentration was maintained at the initial level for 28 days in the new system but decreased to very low levels within 14 days in the controls. The total adenine nucleotides were well maintained in both systems, adenosine triphosphate slightly better in the new system. Hemolysis after 49 days was 0.35±0.21% in the new system and 0.72 ±0.25% in the controls (p<0.001). The morphology was better maintained in the new system (p<0.001). The 24-hour posttransfusion survival of 49-day stored RBC was 78.9±7.1%. The membrane leakage of sodium and potassium was not significantly different in the two systems. The concentration of inorganic phosphate (Pi) in the extracellular fluid of 0.5CPD-RAS2 RBC was initially 7.1±0.8 mmol^l-1. It decreased to approximately 5.5 mmol l^-1 during the first 2 weeks of storage and then slowly increased to 9.4±0.9 mmol l^-1 on day 49. Pi in the CPD-SAGM controls was low initially but rose to 6.2±0.4 mmol l^-1 at the end of storage. The new system offers clearcut advantages as compared to traditional systems and does not seem to raise any serious clinical objections against its use even in massive transfusions.     

Multicenter clinical evaluation of red cell concentrates stored up to 6 weeks in MAP, a new additive solution]

The clinical safety and efficacy of transfusion of red cell concentrates stored in MAP solution (MAP-CRC) containing mannitol, adenine, glucose, phosphate and citrate, into 39 anemic patients were evaluated. In 23 patients, infusion of MAP-CRC was alternated with infusion of ordinary CRC as a control. The MAP-CRC and CRC used in this study were stored at 4 degrees C for an average of 38.2 +/- 2.6 days (n = 52) and 18.1 +/- 2.2 days (n = 26), respectively. Red cell recovery was 77.5% for MAP-CRC and 82.5% for CRC, based on calculation of the increase in hemoglobin level one day after transfusion. There were no differences between patients transfused with MAP-CRC and those transfused with CRC in clinical findings or biochemical data. No major side-effects other than pyrexia associated with the underlying infections were seen in patients transfused with MAP-CRC. MAP-CRC stored up to 42 days is apparently as safe and effective as stored CRC. This new additive solution may therefore be useful for the future expansion of the indications for autologous blood transfusion by facilitating the collection and storage of more blood in the liquid state for a longer period, and may also be useful in obtaining more plasma from whole blood as source plasma.     

24-hour 51Cr post-transfusion survival, 51Cr life span and haemolysis of red blood cells stored at 4 degrees C for 56 days in AS-3

BACKGROUND AND OBJECTIVES: Red blood cells (RBC) were collected either by a manual method using a 16-gauge needle or by an apheresis procedure using an 18-gauge needle, and were stored at 4 degrees C in a solution of CP2D (anticoagulant)/AS-3 (Nutricel) for 56 days. The purpose was to compare the outcome of the autotransfused red cells collected by both techniques. MATERIALS AND METHODS: Five healthy male volunteers were studied on two occasions. RESULTS: The autotransfusions of the manual and apheresed RBC resulted in a mean 24-h post-transfusion survival of 71%, a normal mean 51Cr RBC life span, a 2,3 DPG level that was less than 10% of normal, and 0.6% haemolysis. CONCLUSIONS: Whether collected manually or by apheresis, the outcomes were similar for RBC stored at 4 degrees C for 56 days in CP2D/AS-3.     

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.     

Influence of prestorage leucocyte depletion and storage time on rheologic properties of erythrocyte concentrates

BACKGROUND AND OBJECTIVES: Rheological blood properties were studied during storage. MATERIALS AND METHODS: Blood viscosity, erythrocyte morphology and ATP levels were determined in filtered samples (Leukotrap WB filter system) and their unfiltered counterparts during storage with saline-adenine-glucose-mannitol (SAG-M) for 42 days. RESULTS: Prestorage leucocyte depletion decreased blood viscosity at a high shear rate and reduced the degree of anisocytosis of erythrocytes. During storage, erythrocytes underwent a time-dependent echinocytic shape transformation, which increased the suspension viscosity at high and low shear rates. On day 42, high shear viscosity in filtered units remained lower than in unfiltered counterparts, the mean cellular volume and red blood cell distribution width (RDW) were lower and erythrocytic ATP levels were higher. CONCLUSIONS: Prestorage leucocyte depletion by Leukotrap WB filters improves biophysical properties of erythrocyte concentrates throughout storage, which is, however, outweighed by a time-dependent echinocytic shape transformation and deterioration of these properties.     

Extended storage of platelets in SSP platelet additive solution

BACKGROUND AND OBJECTIVES: The aim of this study was to investigate the effects of extended storage of pooled random platelets in SSP+ additive solution (MacoPharma). MATERIALS AND METHODS: Eight buffy coat-derived, pooled, leucoreduced platelet concentrates were prepared in 75% SSP+, 25% plasma using Fresenius/NPBI Composelect thrombocyte polishing filter (TPF) systems. Platelet concentrates were stored for 19 days in polyolefin storage bags and samples for in vitro analysis were taken at various time-points during storage. RESULTS: Platelet yields were lower than seen routinely when platelets are prepared in 100% plasma. The in vitro quality of the platelets stored in SSP+ was maintained until day 9. Glucose was depleted by day 12 and this was accompanied by a rapid fall in pCO2, a rise in pO2 and a cessation of lactate production. ATP and bicarbonate concentrations fell, the platelets began to swell and the ability to swirl decreased. Soluble P-selectin, glycocalicin, and regulated on activation, normal, T-cell expressed, and secreted (RANTES) concentrations increased, as did P-selectin expression. Loss of platelets and an increase in lacate hydrogenase concentration indicated that lysis had occurred. However, the pH remained between 6.4 and 7.4. CONCLUSIONS: The results suggest that SSP+ could be used for platelet storage for up to 9 days. However, the preparation of platelets in the additive requires some optimization. In vivo studies are required to confirm these in vitro results.     

Studies in Red Blood Cell Preservation. 8. Liquid Storage of Red Cells in a Glycerol-Containing Additive Solution

The purpose of the present study was to determine whether a hypotonic additive containing a low concentration of glycerol as a membrane permeable solute would improve the liquid storage of red blood cells (RBCs). Packed RBCs were stored either with 200 ml of an experimental additive solution, EAS 25, containing (m M ): glycerol 150, adenine 2, glucose 110, mannitol 55, and NaCl 50, or with 100 ml/unit of a conventional additive solution Adsol®. The results show that the adenosine triphosphate values, hemolysis, potassium leakage, and the morphology scores of RBCs were significantly better with EAS 25 than with Adsol up to 84 days of storage. The ATP values were significantly different only after the first 42 days of storage. The mean corpuscular volumes (MCVs) of the RBCs were significantly higher throughout in the experimental additive accompanied by decreased microvesiculation as compared to Adsol. The total microvesicle membrane protein shed by 100 ml of RBCs was 47.92±12.31 mg in Adsol and 18.96±5.49 mg in EAS 25 (p<0.001). The larger MCVs of the RBCs in EAS 25 may have a favorable effect on maintaining membrane integrity by decreasing the loss of membrane by microvesiculation.     

Two-step process of cytoskeletal structural damage during long-term storage of packed red blood cells

BackgroundStorage of packed red blood cells (PRBC) for 42 days causes morphological, structural, and functional changes in the red cells. To assess the quality of stored PRBC, it is important to evaluate the main components of the product. The aim of this study was to evaluate the kinetics of the structural transformations in the cytoskeleton of red cells during long-term storage (up to 42 days).Materials and methodsBags of PRBC were stored with CPD/SAGM solution at +4 °C. Cytoskeletal parameters were measured on days 3, 12, 19, 21, 24, 28, 35, and 42 of storage to determine their changes. Atomic force microscopy was used to obtain images and analyse the parameters of the cytoskeletal network. As the storage time increased, a general PRBC test was performed. Membrane fixatives were not used at any stage of the preparation of the specimens for cytoskeletal imaging.ResultsWhen PRBC were stored for 42 days, the main changes to the cytoskeletal mesh included rupture of filaments, merger of small pores into larger ones, a decrease of the number of pores, thickening of filaments, and an increase of membrane stiffness. A process of irreversible changes to the cytoskeleton started on days 19–21. A kinetic model of changes in the parameters of the cytoskeletal mesh with time of PRBC storage was created.DiscussionTwo stages of impairment in cytoskeletal elements were found: rupture of filaments and clustering of protein components. The typical time of development and specifics of these stages are discussed. The consequences of the altered configuration of the cytoskeleton are also discussed. Destruction of the red cell cytoskeleton can have a negative effect on the efficacy of blood transfusion and increase the risk of post-transfusion complications. Our findings can be used in clinical medicine to evaluate the quality of PRBC for blood transfusion as well as for studies of the molecular organisation of red cells undergoing various types of physical and chemical treatment.     

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.     

Storage of buffy-coat-derived platelets in additive solutions at 4 degrees C and 22 degrees C: flow cytometry analysis of platelet glycoprotein expression

BACKGROUND: The aim of our in vitro study is to compare the effects on platelet membrane glycoproteins that play an important role in the main functions of platelets, when platelets are stored for a period of 21 days at 4 degrees C or 22 degrees C. STUDY DESIGN AND METHODS: Platelet concentrates (PC) were prepared from pooled buffy-coats (BC) for paired studies (total eight pools from 80 BCs) by using the OrbiSac system. We divided each pool into two PCs and stored them at 4 degrees C or 22 degrees C. RESULTS: The activation marker CD62 remained almost unchanged during storage in all units. The expression of CD63 was higher in PCs stored at 22 degrees C than in those stored at 4 degrees C. No significant difference in CD41 expression was detected over time. The expression of CD42b declined during storage and even more in PCs stored at 4 degrees C until day 21 [day 14: mean flourscence intensity: 32.5 +/- 13.1 vs. 46.5 +/- 19.1], but the percentage of platelets expressing CD42b remained high in platelets stored at 4 degrees C, but gradually decreased at 22 degrees C (day 14: 95.0 +/- 1.5 vs. 59.0 +/- 9.9). Storage at 4 degrees C reduced the rate of glycolysis and maintained the pH better after day 10 than in PCs stored at 22 degrees C (day 14: 7.009 +/- 0.067 vs. 7.233 +/- 0.125). The concentration of regulated upon activation of normal T-cells expressed and secreted was higher in PCs stored at 22 degrees C than at 4 degrees C (day 7: 414.7 +/- 32.3 vs. 49.6 +/- 19.0). No response to extent of shape change and no swirling were detected at 4 degrees C. CONCLUSION: Platelets stored at 4 degrees C retain their in vitro characteristics better than those stored at 22 degrees C, except for parameters that reflect changes in shape. Storage at 4 degrees C is not associated with an increased expression of glycoprotein (GpIb, GpIIb/IIIa) and platelet activation markers (CD62p and CD63) as compared with storage at 22 degrees C.