Bregt Roerdinkholder-Stoelwinder

The proteome of red cell membranes and vesicles during storage in blood bank conditions

BACKGROUND During storage of red cells (RBCs) for transfusion, RBCs undergo a number of biochemical and morphologic changes. To be able to identify the mechanisms underlying these storage lesions, a proteomic analysis of the membranes of RBCs and their vesicles was performed during various periods of storage in blood bank conditions. STUDY DESIGN AND METHODS RBCs and vesicles were isolated from RBCs after various storage periods. The proteins of RBC membranes and vesicles were separated by gel electrophoresis and identified by a semiquantitative proteomic analysis. RESULTS Our findings confirm previous data, such as a storage-associated increase in hemoglobin binding to the membrane and aggregation and degradation of the integral membrane protein band 3, suggesting a remodeling of the RBC membrane during storage. Our data also show storage-dependent changes in the membrane association of proteasome and chaperone proteins, metabolic enzymes, small G proteins, and signal transduction proteins. Vesicles display similar changes in their protein composition during storage. CONCLUSION The results of this analysis indicate that the storage-related changes in the RBC membrane are the results of disturbance and/or acceleration of physiologic processes such as cellular aging, including vesicle formation. The latter may serve to remove damaged membrane patches that would otherwise lead to accelerated RBC removal. These data provide a framework for future studies toward the development of better storage conditions and a reduction of the side effects of RBC transfusion.

Erythrocyte vesiculation: a self-protective mechanism?

Previous studies demonstrated that 20% of haemoglobin is lost from circulating erythrocytes during their total lifespan by vesiculation. To study whether removal molecules other than membrane‐bound haemoglobin were present in erythrocyte‐derived vesicles, flow cytometry and immunoblot analysis were employed to examine the presence of phosphatidylserine (PS) and IgG, and senescent cell antigens respectively. It was demonstrated that 67% of glycophorin A‐positive vesicles exposed PS, and that half of these vesicles also contained IgG. Immunoblot analysis revealed the presence of a breakdown product of band 3 that reacted with antibodies directed against senescent erythrocyte antigen‐associated band 3 sequences. In contrast, only the oldest erythrocytes contained senescent cell antigens and IgG, and only 0·1% of erythrocytes, of all ages, exposed PS. It was concluded that vesiculation constitutes a mechanism for the removal of erythrocyte membrane patches containing removal molecules, thereby postponing the untimely elimination of otherwise healthy erythrocytes. Consequently, these same removal molecules mediate the rapid removal of erythrocyte‐derived vesicles from the circulation.

Survival of red blood cells after transfusion: a comparison between red cells concentrates of different storage periods

BACKGROUND: The use of fresh red blood cells (RBCs) is recommended for critically ill patients and patients undergoing surgery, although there is no conclusive evidence that this is beneficial. In this follow‐up study, the short‐term and the long‐term recovery of irradiated, leukoreduced RBCs transfused after either a short storage (SS) or a long storage (LS) period were compared. By consecutive transfusion of RBCs with a SS and LS period, a direct comparison of their survival within the same patient was possible.

DETERMINANTS OF RED-BLOOD-CELL DEFORMABILITY IN RELATION TO CELL AGE

Abstract:  Red blood cell (RBC) deformability was determined with an ektacytometer in fractions separated on the basis of differences in cell volume or density. Deformability was measured with ektacytometry (rpm‐scan and osmo‐scan). We studied three groups of RBC fractions: 1. By counterflow centrifugation we obtained fractions of different cell age which showed a slight decrease in mean corpuscular haemoglobin concentration (MCHC) and an increase in surface‐to‐volume (S/V) ratio in fractions with older cells. 2. By Percoll fractionation fractions were obtained which showed a pronounced increase in (MCHC) but no change in S/V ratio. 3. By a combination of both fractionation techniques, fractions were obtained which showed an increased MCHC and an increase in S/V ratio. Deformability in group 1,2 and 3 showed respectively no change, a moderate decrease and a pronounced decrease in fractions of older cells. A decline in deformability occurs during the aging process of the red blood cell. This decline in deformability in old red cells is greater than originally thought. This decline is the result of an increase in haemoglobin concentration and a second factor, probably a decrease in membrane elasticity.

Quantification of loss of haemoglobin components from the circulating red blood cell in vivo.

Abstract:  Previous studies have shown that a considerable amount of haemoglobin is lost from the intact red cell during its lifespan. The aim of this study was to determine the relative contribution of all the haemoglobin components to this process. Therefore, the relative amount of haemoglobins A0, A2, F and the glycated haemoglobins were determined in 24 fractions of different cell age. These fractions were obtained by the combination of counterflow and density centrifugation. When the absolute amount of all haemoglobin components were calculated using the MCH‐values of each fraction, it appeared that the mean red cell loss of haemoglobins A0, A2, F, an unknown X and “rest” comprised, respectively, 440, 23, 1, 4 and 1 amol per cell, while the mean gain of the glycated haemoglobins was 84 amol per cell. This resulted in a net loss of 385 amol of haemoglobin per cell. One of the glycated haemoglobins (HbA1e2) turned out to be the product of further carbamylation. It was concluded that in the first half of the red cell lifespan HbA0 and HbA2 decreased by glycation and carbamylation and that in the second half some of the HbA0 and HbA2 but also some of the glycated and carbamylated haemoglobin components leave the red cell. The total loss amounted to about 20%.

Red cell concentrates of hemochromatosis patients comply with the storage guidelines for transfusion purposes

BACKGROUND: Therapeutic phlebotomy is the preferred treatment for iron overload associated with hemochromatosis. In the Netherlands, red blood cell concentrates (RCCs) from hemochromatosis patients are not used for transfusion purposes. In this study, their storage performance was compared with that of control donors as a first step in the evaluation of their potential usefulness for transfusion.

The proteome of red cell membranes and vesicles during storage in blood bank conditions: RBC MEMBRANE PROTEOME DURING STORAGE

BACKGROUND: During storage of red cells (RBCs) for transfusion, RBCs undergo a number of biochemical and morphologic changes. To be able to identify the mechanisms underlying these storage lesions, a proteomic analysis of the membranes of RBCs and their vesicles was performed during various periods of storage in blood bank conditions.