Formation of phosphatidylethanol in vitro in red blood cells from healthy volunteers and chronic alcoholics

Phosphatidylethanol (PEth) is an abnormal phospholipid, formed only in the presence of ethanol via a transphosphatidylation reaction of phospholipase D (PLD). PEth in blood is a promising new marker of alcohol abuse. Blood PEth is found almost exclusively in red cells. This study was performed to investigate a possible PEth formation in human red cells from alcoholics and healthy individuals, at physiologically relevant ethanol concentrations. Blood was drawn from six healthy volunteers (controls) and six chronic inpatient alcoholics. Hematological analyses were performed, and red blood cells were separated and incubated in plasma with ethanol to study PEth formation. Lipids were extracted and PEth analyzed with high pressure liquid chromatography and evaporative light-scattering detection. Incubation of red cells in 50 mM ethanol yielded detectable PEth after 12 hours. Formation of PEth was concentration dependent at 10 to 50 mM ethanol. In vitro formation of PEth was significantly higher (P <.001) in red cells from alcoholics (5.2 +/- 1.1 micromol/l) compared to controls (2.4 +/- 0.6 micromol/l) (mean +/- SD). A significant correlation (P <.01) was observed between initial mean corpuscular volume and accumulated PEth. This study demonstrates that PEth is formed in human red cells at physiologically relevant ethanol concentrations. Alcoholics accumulate about twice as much PEth than controls. The accumulation rate of PEth is slower in red cells compared to rates reported for other tissues.     

Procoagulant activity of long-term stored red blood cells due to phosphatidylserine exposure

Objective/Background: Phosphatidylserine (PS) exposure on the outer membrane surface is a known storage lesion of red blood cells (RBCs). Procoagulant activity (PCA) of PRBC units has been shown to increase during storage. However, the PCA and the relation to PS of banked RBCs remain unknown. The aim of this study is to investigate the PCA and the correlation with PS exposure on banked RBCs. Materials and Methods: Human normal leucodepleted RBC concentrates were stored in saline–adenine–glucose–mannitol (SAGM) solution at 4 °C for up to 42 days. PS exposure was measured by lactadherin and annexin V. PCA of RBCs was determined by one‐step recalcification assay and further determined by prothrombinase and tenase assays. The correlation between PCA and PS exposure of RBCs was measured by inhibition tests with lactadherin or annexin V. Results: Lactadherin detected 1·5% PS‐positive stored RBCs vs 0·5% for annexin V after 14 days of storage, which significantly reached 18·4 vs 4·5% after 42 days of storage. Potent PCA was observed on day 14 and continuously increased. PCA of RBCs on day 42 was inhibited 94% by lactadherin vs 45% by annexin V. Incubation at 37 °C in fresh heparinised plasma partially reversed PS exposure and PCA of RBCs stored for 14 days but had no effect on these parameters for cells stored for 42 days. Conclusions: PCA and PS exposure of stored RBCs increased during storage and showed significant positive correlation. PCA of long‐term stored RBCs may not be reversed by transfusion.     

Supernates from stored red blood cells inhibit platelet aggregation

BACKGROUND: Previously, we reported that red blood cells (RBCs) stored in AS‐5 accumulated proinflammatory substances during storage. We observed in those studies that supernates from nonleukoreduced (NLR) RBCs reduced mean anti‐CD41a‐fluorescein isothiocyanate (FITC) fluorescence on platelets (PLTs), indicative of decreased expression of glycoprotein (GP)IIb/IIIa on the PLT membrane. The objective of this study was to determine if supernates from stored RBCs impaired PLT aggregation as a consequence of reduction in GPIIb/IIIa expression. STUDY DESIGN AND METHODS: Leukoreduced (LR) and NLR RBC units were prepared in AS‐5 and stored at 1 to 6°C for 6 weeks. Supernates from RBC samples collected every 2 weeks were mixed with freshly collected type‐matched blood and incubated for 30 minutes at 37°C. PLTs in each incubated blood sample were evaluated for GPIIb/IIIa expression by flow cytometry and for aggregation response to collagen by whole blood aggregometry. RESULTS: Supernates from stored NLR RBCs reduced CD41a‐FITC fluorescence on PLTs by 15% to 31%. A reduction in fluorescence was induced by supernates of RBCs stored for 14 days and increased as storage time increased. Supernates from Day 42 NLR RBCs reduced the mean amplitude of PLT aggregation by 31% compared to Day 0 supernates and lengthened the time before onset of aggregation by 21%. In addition, amplitude correlated directly and lag time correlated inversely with CD41a‐FITC fluorescence in all samples. Supernates from prestorage LR RBCs did not affect PLT CD41a‐FITC fluorescence or aggregation response. CONCLUSIONS: Substances that decrease expression of GPIIb/IIIa and inhibit PLT aggregation accumulate in NLR RBCs. Accumulation of this material is prevented by leukoreduction.     

Rejuvenation improves roller pump-induced physical stress resistance of fresh and stored red blood cells

BACKGROUND: Retrospective studies on transfusion recipients suggested that transfusion of older red blood cells (RBCs) was associated with higher morbidity. Similar studies were also done on cardiac surgery patients who were placed on cardiac bypass pumps. It is possible that stored RBCs are more fragile and could be more easily damaged by these pumps, thus leading to additional morbidity. STUDY DESIGN AND METHODS: Fresh and stored (42 days) RBCs, rejuvenated and nonrejuvenated, were compared in resistance to physical stress, induced by a roller pump, and osmotic fragility changes during physical stress to model RBCs going through cardiac bypass instruments. In addition, posttransfusion in vivo recovery was evaluated in an immunodeficient mouse model to minimize species differences between transfusion product and recipient. RESULTS: Fresh RBCs were more resistant to both osmotic and physical stress than stored cells. After 2 hours of physical stress, the osmotic stress resistance of fresh cells declined and was the same as for stored cells. Rejuvenated fresh cells did not demonstrate a decline in osmotic resistance during the stress test and both fresh and stored cells had the same improved resistance to osmotic stress before and after the physical stress. Rejuvenation slightly improved recovery of fresh RBCs but almost doubled the recovery of stored cells in the mouse model. CONCLUSIONS: Our studies suggest that rejuvenation improves roller pump–induced physical and osmotic stress resistance of stored RBCs.     

Transfusion of fresh murine red blood cells reverses adverse effects of older stored red blood cells

BACKGROUND: Although a subset of recent studies have suggested that red blood cell (RBC) storage length is associated with adverse patient outcomes, others have shown no such relationship. Adults may be transfused with RBC units of different storage lengths, and existing studies do not take into consideration that fresh RBCs may alter responses to concurrently transfused stored RBCs. To test this possibility, we utilized a murine model and investigated transfusion outcomes of fresh, stored, or fresh‐plus‐stored RBCs. STUDY DESIGN AND METHODS: Fresh, 14‐day‐stored or fresh plus 14‐day‐stored leukoreduced RBCs from HOD‐transgenic donors (with RBC‐specific expression of hen egg lysozyme, ovalbumin, and human Duffy^b) were transfused into naïve C57BL/6 recipients. Serum cytokines and anti‐HOD alloimmunization were evaluated after transfusion. RESULTS: In six of six experiments (n = 90 mice total), a proinflammatory serum cytokine storm of interleukin‐6, keratinocyte‐derived chemokine/CXCL1, and monocyte chemoattractant protein‐1 was observed in transfusion recipients of stored but not fresh RBCs, along with high degrees of anti‐HOD alloimmunization. However, concurrent transfusion of fresh HOD RBCs along with stored HOD RBCs significantly decreased these adverse outcomes (p < 0.05). CONCLUSIONS: These results are consistent with fresh murine HOD RBCs losing protective properties during storage, and introduce a previously unrecognized variable in RBC storage studies. If translatable to humans, uniform “old blood” groups may be needed in future clinical studies to more accurately investigate the biologic effects of older RBC units.     

Effects of room-temperature exposure on bacterial growth in stored red cells

Experiments were performed to determine the effect of room-temperature (RT) exposure on the growth rates of selected bacteria by deliberate contamination of AS-1 packed red cell (RBC) units contained in PL146 plastic bags. Isolates of Staphylococcus aureus, S. epidermidis, Streptococcus faecalis, Pseudomonas aeruginosa, Citrobacter freundii, Enterobacter agglomerans, and Yersinia enterocolitica were inoculated into 3- to 5-day-old packed RBC units. The units were refrigerated at 4 to 6 degrees C for 14 days. On two occasions during this period, one-half of the units were removed from the refrigerator and held at 25 to 26 degrees C for 2 hours. Timed samples taken during the periods at RT revealed stable or decreasing bacterial counts in the majority of exposed units, and in no instance was a marked increase in bacterial growth observed. Samples taken after subsequent prolonged rerefrigeration showed no significant increase in bacterial counts as compared with those in control units that were refrigerated constantly. These results indicate that RT exposure of contaminated RBC units does not result in accelerated bacterial proliferation over a period of 2 hours and that it may, in fact, have a detrimental effect on bacterial growth.     

Effect of bilirubin on stored red blood cells.

Bilirubin may cause haemolysis of red cells, especially in the presence of light. The possible dependence of this effect on the metabolic state of the cells has been investigated. For this purpose erythrocytes were stored up to five days to deplete the concentration of ATP. Every day a constant amount of bilirubin was added to the cells. On third day of storage bilirubin induced haemolysis, which was aggravated rapidly during further storage. Bilirubin-induced haemolysis could be reduced after increasing the ATP levels by incubation of the red cells with inosine.     

Depressed blood group antigens on red cells from a Mexican donor

Red cells from a Mexican blood donor had depressed H, IF, ID, i, Sda, Sdx, D, LW, Dib, Vel and JMH antigens. A wide range of other blood group antigens had normal activity. The unusual red cell phenotype may be caused by a membrane anomaly that alters orientation of certain cell surface components. The specificities of the depressed red cell antigens in this phenomenon differ from those affected in Melanesians with ovalocytosis or in individuals with the In(Lu) inhibitor gene.     

Manufacture of red cells in additive solution from whole blood refrigerated for 5 days or remanufactured from red cells stored in plasma

Background and Objectives: To investigate methods for the production of red cell concentrates (RCC) in saline, adenine, glucose and mannitol (SAG‐M), from whole blood or red cells stored in plasma for 5 or 6 days and to provide evidence that exchange transfusion RCC in citrate phosphate dextrose (CPD) plasma or citrate, phosphate, dextrose, adenine (CPDA‐1) plasma are of comparable quality. Methods and Materials: Ten RCC in SAG‐M were produced following the remanufacture of red cells in CPD plasma on day 5/6 or after 5 days hold as leucodepleted CPD whole blood. In addition, 10 RCC in CPD plasma and 9 in CPDA‐1 plasma were stored without further processing. Units were assessed for red cell parameters including haemolysis, adenosine triphosphate (ATP), 2,3‐diphosphoglycerate (2,3‐DPG) and extracellular potassium. Results: Units in SAG‐M produced by remanufacture of RCC in plasma or by delayed manufacture of whole blood had comparable levels of haemolysis, ATP and 2,3‐DPG. Furthermore, these units underwent biochemical changes similar to reference SAG‐M units, with the exception of haemolysis which was greater at the end of shelf life and supernatant potassium which was lower following remanufacture. As expected, the decline in ATP was greater in red cells stored in CPD plasma compared with CPDA‐1 plasma. In general, units in CPD plasma were of similar quality at day 28 compared to those in CPDA‐1 plasma at day 35. Conclusions: RCC produced following the remanufacture of RCC in plasma or the delayed manufacture of whole blood are of acceptable in vitro quality and should be assigned the same shelf life as standard RCC in SAG‐M.     

Supernatant from stored red cells activates neutrophils

Bioreactive substances including cytokines and lipids accumulate during storage of red blood cells (RBCs) but their clinical importance is uncertain. The goal of this study was to evaluate the effect of stored RBC supernatant on neutrophil activity in vitro . Packed RBCs (PRBCs) were collected and divided into two aliquots, one leukodepleted and the other nonleukodepleted. Plasma supernatant from PRBCs were collected on days 1, 8, 15, 29 and 35 and its effect on neutrophil expression of CD11b, CD16 and oxidative burst was measured by flow cytometry. Levels of tumour necrosis factor alpha (TNFα) and interleukin-8 (IL8) were also measured. The supernatant from PRBC units stored for greater than 15 days activated and primed neutrophils as evidenced by an increase CD11b and CD16 expression and oxidative burst. The greatest effect was seen in the oldest concentrates (35-day-old) ( P  < 0.008). Leukodepletion abrogated the effects of stored supernatant on CD11b and CD16 expression ( P  < 0.02) but did not reduce priming of the neutrophil oxidative burst ( P  > 0.1). Very low levels of IL8 and TNFα were detected in stored supernatants. Stored PRBC supernatant contains substances which directly enhance neutrophil expression of adhesion protein CD11b, CD16 and prime neutrophil oxidative burst. The exceedingly low level of IL8 and TNFα found in this study suggests that other factors may play a more important role in neutrophil priming and activation.     

Transfusion of stored red blood cells adhere in the rat microvasculature

BACKGROUND: Ex vivo storage of red blood cells (RBCS) for transfusions is associated with a “storage lesion,” which decreases RBC deformability and increases RBC adhesiveness to vascular endothelium. This may impair microcirculatory flow with deleterious effects on oxygen delivery after transfusion. Previous studies have shown that human RBCs adhere to endothelial monolayers in vitro with prolonged storage and is reduced by prestorage leukoreduction (LR). The objective of this study was to determine whether duration of RBC storage and LR influence RBC adhesion in vivo in capillaries. STUDY DESIGN AND METHODS: Rat RBCs were collected and stored in CPDA‐1 under standard blood bank conditions. Three RBC products were compared: 1) fresh RBCs, less than 24 hours of storage (n = 6); 2) nonleukoreduced (NLR) RBCs stored for 7 days (n = 6); and 3) prestorage LR RBCs stored for 7 days (n = 6). RBCs were labeled with fluorescein isothiocyanate (FITC) 24 hours before transfusion and reinjected in an isovolemic manner into healthy rats. The FITC‐labeled RBCs were visualized in the extensor digitorum longus muscle using intravital video microscopy (20× magnification). The number of RBCs adherent in capillaries was counted 1 hour after transfusion in 10 random fields and the median values were compared with one‐way analysis of variance. RESULTS: Stored RBCs showed increased levels of adherence in capillaries compared to their fresh counterparts (p < 0.05). Prestorage LR decreased RBC adherence to levels equivalent to those of fresh RBCs (p < 0.05 for stored LR vs. stored NLR). CONCLUSION: Rat RBCs stored under conditions that closely mimicked clinical transfusion adhere in capillaries. The decreased RBC adherence with LR suggest a direct effect of white blood cells or their byproducts on RBC deformability and/or adhesiveness to microvascular endothelium. Further study will examine the mechanism of adherence and the impact it has on microcirculatory flow and oxygen delivery in the critically ill host.     

In vivo viability of red blood cells stored in CPDA-2

The marginal viability of erythrocytes stored for 35 days as red blood cell concentrates in citrate-phosphate-dextrose-adenine-one (CPDA-1) was attributed to inadequate nutrient support with adenine and glucose. In an effort to improve the viability of red blood cells following extended storage, a new CPD-adenine and 1.4 times more glucose than CPDA-1. The efficacy of CPDA-2 was evaluated in vivo by measurement of 24-hour postinfusion recovery of 51Cr-labeled erythrocytes which had been stored as whole blood or red blood cell concentrates for 5 to 8 weeks. All red blood cell concentrates, and the whole blood units stored for 35 and 42 days, were held at room temperature for 8 hours prior to processing and/or refrigeration. CPDA-2 yielded significantly higher 51Cr survivals than CPDA-1 and exceeded the accepted criterion for anticoagulant preservative efficacy of 70 percent postinfusion survival of red blood cells after storage for a period of 42 days. Preliminary data supports possible usage to 49 days. Plasma glucose and red blood cell ATP concentration were maintained better in CPDA-2 than in CPDA-1. When compared to historical controls for CPD and CPDA-1 the data suggest that red blood cells stored in CPDA-02 will have superior viability throughout the entire storage period. CPDA-2 is a candidate to replace CPDA-1 as the anticoagulant-preservative solution of choice for red blood cell concentrates.     

In vivo viability of stored red blood cells derived from riboflavin plus ultraviolet light-treated whole blood

BACKGROUND: A novel system using ultraviolet (UV) light and riboflavin (Mirasol System, CaridianBCT Biotechnologies) to fragment nucleic acids has been developed to treat whole blood (WB), aiming at the reduction of potential pathogen load and white blood cell inactivation. We evaluated stored red blood cell (RBC) metabolic status and viability, in vitro and in vivo, of riboflavin/UV light–treated WB (IMPROVE study). STUDY DESIGN AND METHODS: The study compared recovery and survival of RBCs obtained from nonleukoreduced WB treated using three different UV light energies (22, 33, or 44 J/mL_RBC). After treatment, WB from 12 subjects was separated into components and tested at the beginning and end of component storage. After 42 days of storage, an aliquot of RBCs was radiolabeled and autologously reinfused into subjects for analysis of 24‐hour recovery and survival of RBCs. RESULTS: Eleven subjects completed the in vivo study. No device‐related adverse events were observed. By Day 42 of storage, a significant change in the concentrations of sodium and potassium was observed. Five subjects had a 24‐hour RBC recovery of 75% or more with no significant differences among the energy groups. RBC t_1/2 was 24 ± 9 days for the combined three groups. Significant correlations between 24‐hour RBC recovery and survival, hemolysis, adenosine triphosphate (ATP), and CO₂ levels were observed. CONCLUSIONS: This study shows that key RBC quality variables, hemolysis, and ATP concentration may be predictive of their 24‐hour recovery and t_1/2 survival. These variables will now be used to assess modifications to the system including storage duration, storage temperature, and appropriate energy dose for treatment.