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. Some hemolysis of red blood cells (RBC) is expected during blood bank storage. It has been shown that part of the hemoglobin (Hb) in the suspending medium is free and part is encapsulated in microvescicles shed by the RBC [1], The amount of hemolysis and microvesiculation that occurs has been noted to be less when blood is stored in polyvinyl chloride (PVC) containers in which di-(2-ethylhexyl)phthalate (DEHP) is used as the plasticizer [1]. The DEHP that leaches into the plasma has been shown to decrease hemolysis, microvesiculation, and the increase in osmotic fragility which RBC undergo during refrigerated storage [2-9]. The posttransfusion survival of RBC stored in DEHP-plasticized PVC has been found superior to RBC stored in PVC bags plasticized with tri-2-ethylhexyl-trimellitate (TEHTM) which is much less leachable. The protective effect of the DEHP is believed to be due to its insertion into the RBC membrane [6-9], The present studies were done to quantify how much of the ‘hemolysis’ that has been observed during the storage of RBC is dissolved Hb and how much is Hb encapsulated in microvesicles.

The how and why of exocytic vesicles

“…4). 106 We saw no differences in the size of the vesicles in either preparation; it is possible, however, that the nanovesicles (diameter, 60 nm), first reported by Allan and associates 54 were centrifuged down with the microvesicles under the conditions we used.…”

RBC‐derived vesicles during storage: ultrastructure, protein composition, oxidation, and signaling components

“…It has been suggested that microvesiculation contributes more to the increase in the plasma Hb occurring during storage than free Hb. 25 Based on our microscopic data, it seems probable that the plasma Hb found in prolonged liquid storage is not so effectively enclosed into vesicles, but it can be released from them.…”

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

“…The adenine nucleotides are better maintained in RAS2 than in SAGM and the spontaneous storage hemolysis is significantly lower from the second week of storage. It has recently been demonstrated [26] that most of the free he moglobin in stored blood originates from microvesicles. Microvesiculation implies an irreversible loss of mem brane cholesterol and phospholipids, occurring when mi crovesicles are budded off from RBC spicules (mainly in echinocytes-3 and echino-spherocytes).…”