T.J. Greenwalt

Erythrocyte Membrane Vesiculation and Changes in Membrane Composition during Storage in Citrate-Phosphate-Dextrose-Adenine-1

Abstract. Serial studies were made of the membranes of the erythrocytes and the vesicles shed during storage of blood in polyvinyl chloride containers for 35 days in citrate‐phosphate‐dextrose‐adenine anticoagulant. Special precautions were taken to eliminate artifacts created by contaminating leukocytes, platelets and red blood cell ghosts. A total of 15.6% of the cholesterol and 5.2% of the phospholipids of the membranes was lost with no gross change in the gel electrophoretic patterns. The quantity of vesicles found in the supernatant plasma increased during storage and their membranes were characterized by the absence of spectrin, ankyrin, and periodic acid Schiff bands 2 and 3. The ratio of lipids to protein in the vesicles increased as they accumulated perhaps reflecting a rearrangement of the erythrocyte membrane constituents during prolonged maintenance at 4°C.

Studies in Red Blood Cell Preservation: 4. Plasma Vesicle Hemoglobin Exceeds Free Hemoglobin

Abstract. Studies were designed to find out how much of the plasma hemoglobin (Hb) in whole blood was in microvesicles and how much was free Hb after 21 days of storage in citrate‐phosphate‐dextrose anticoagulant and to determine the effect of the plasticizer, di‐(2‐ethylhexyl)phthalate (DEHP). The total plasma Hb in polyolefin (PO) containers without DEHP was much higher than in polyvinyl chloride (PVC) with the plasticizer (p=0.004). Less than 30% of the Hb was in free solution in either type of container. The addition of 300 μg/ml of DEHP to the plasma in the PO containers resulted in marked reduction in the microvesiculation (p<0.01) but did not affect the level of free Hb. RBC hypotonic fragility and morphology scores were significantly improved. It is concluded that microvesiculation contributes more to plasma Hb concentration than free Hb during storage.

Studies in Red Blood Cell Preservation

The purpose of this study was to examine whether vesiculation of RBC plays a significant role in their rejuvenation. Outdated units of Adsol® blood, were divided into two aliquots and incubated with equal volumes of a solution of 100 mM pyruvate and inosine, 103 mM phosphate and 5 mM adenine (PIPA) or 0.9% saline. Following 1 h incubation, vesicles were isolated from the supernatants and quantitated for hemoglobin content. Restoration of RBC ATP, 2,3‐DPG, morphology, and osmotic fragility after rejuvenation was satisfactory. The postrejuvenation mean corpuscular volumes (88.2+6.9 fl) were significantly lower (p<0.001) than the prerejuvenation (94.6+6.8 fl) and control (104.0+7.3 fl) volumes. The hemoglobin shed in vesicles during rejuvenation was significantly greater than in the saline controls (0.44+0.31 vs. 0.18+0.10 mg/dl RBCs; p = 0.026). These data suggest that the decreased MCV following rejuvenation is in part due to membrane loss in exocytic vesiculation.

Buffering and dilution in red blood cell storage.

BACKGROUND:  Red blood cell (RBC) storage solutions work in a narrow pH range between 7.2 and 6.4. While keeping RBC within that pH range, ATP production can be increased by buffering or dilution.

Successful storage of RBCs for 10 weeks in a new additive solution.

BACKGROUND: This study explored the effect of storing packed RBCs suspended in 200 mL of an alkaline, hypotonic, experimental additive solution (EAS 61).

Studies in Red Blood Cell Preservation 1. Effect of the Other Formed Elements

Abstract. The purpose of this study was to determine if the removal of most of the leukocytes and platelets would affect the in vitro characteristics of stored red blood cells (RBC). Fresh RBC concentrates prepared by removing platelet‐rich plasma and filtration through Imugard IG 500 filters were compared with unmanipulated units after storage for up to 56 days. The filtered units were significantly better after 56 days storage for supernatant K+ (p = 0.001), hemolysis (p = 0.05), total vesicle membrane protein shed (p = 0.03), and RBC morphology score (p = 0.04). These differences occurred even though ATP levels were well maintained in both groups. The measurements that did not differ significantly were pH, hematocrit, ATP, 2,3‐DPG, glucose and supernatant Na+. It is suggested that enzymes, leukotrienes, catecholamines and eicosanoids released by degenerating leukocytes and platelets may be inimical to RBC. Some may act as agonists on a‐adrenergic and cholinergic muscarinic receptors present on RBC membranes.

The effects of phosphate, pH, and AS volume on RBCs stored in saline-adenine-glucose-mannitol solutions

BACKGROUND: RBC ATP concentrations are the most important correlate of RBC viability. Tests were performed to determine whether increased AS volume, pH, and phosphate content increased stored RBC ATP concentrations.

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

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.

Biochemical and structural changes in RBCs stored with different plasticizers: the role of hexanol

BACKGROUND: PVC containers are plasticized with di(2‐ethyl)hexylphthalate (DEHP) or a related phthalate. The toxicity of DEHP has been questioned. It has been proposed to use butyryltrihexylcitrate (BTHC) as the plasticizer. The purpose of this study was to determine if hexanol, a component of BTHC, plays a role in the preservation of RBCs stored in BTHC‐plasticized PVC bags.

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.