Arthur W. Rowe

Cryopreservation of granulocytes for transfusion: Studies on human granulocyte isolation, the effect of glycerol on lysosomes, kinetics of glycerol uptake and cryopreservation with dimethyl sulfoxide and glycerol

Abstract Existing methods for the cryopreservation of granulocytes employ primarily dimethyl sulfoxide (Me 2 SO) rather than glycerol as the cryoprotective additive of choice. Although Me 2 SO has been demonstrated to be an effective cryoprotective additive for granulocyte preservation to yield viable cells (dye exclusion, phagocytosis, etc.), the inherent toxicity and clinical objections of Me 2 SO as a cryoprotective additive for granulocyte preservation preclude its extensive and routine use in patients. Therefore, glycerol, with its important advantage of nontoxicity, has been investigated for its potential usefulness as a cryoprotective additive for preserving human granulocytes for transfusion. Granulocyte preparations were isolated from impure leukocyte concentrates obtained from the buffy coats of human whole blood. Studies on the isolation and purification of the granulocytes involved separation by sedimentation with dextran, removal of red cells by hypotonic shock with water, resuspension with Plasmatein and further purification by centrifugation. Intact viable granulocytes were obtained with a purity in excess of 90%. Lysosomes were studied as indicators of cryoinjury in granulocytes using β-glucuronidase as the key marker enzyme. This enzyme has been characterized as a sensitive indicator of damage to lysosomes and a direct linear relationship has been established between damage to granulocytes by freezing and amount of lysosomal enzyme released. Addition or presence of the cryoprotectant, glycerol, did not appear to have any adverse effect on lysosomes of intact granulocytes. Studies on the permeation kinetics of glycerol in granulocytes indicated that the additive was freely permeable and did not cause any potentially damaging osmotic changes in cell volume. Granulocytes in various concentrations of glycerol were then frozen at slow, moderate, and rapid cooling rates. Based on the small amount of β-glucuronidase released, good preservation of granulocyte lysosomes has been obtained with a slow cooling rate of 5 °C/min and a concentration of 15% glycerol. Further studies now are necessary to define those conditions of cooling rate and glycerol concentration required to develop a simple method for optimal preservation of granulocytes based on additional functional criteria of viability.

Duffy and duffy-related human antigens in primates

Duffy-related Fy3, Fy5, and Fs antigens on red cells of various species of primates were characterized and compared to the findings of others. While most species appear to be monomorphic for the Fy b and Fy 3 antigens, Macaca mulatta, Macaca fascicularis , and Pan troglodytes are polymorphic for Fy b with the gene frequencies ranging for Fy b from 0.72 to 0.80 and for Fy from 0.20 to 0.28. It may be postulated that Fy:3 and Fy(a-b-) could constitute the oldest phenotypes common to several species in primate evolution, while Fy5 and Fy a appear to be more recent antigens particular to the gorilla and human lineages respectively.

Increase in ATPase activity in red cell membranes as a function of freezing regimen

Abstract The effects of freezing on the membrane of red blood cells were examined using membranes prepared from fresh human blood by one-step hemolysis with phosphate buffer of pH 5.8. Membrane-associated adenosinetriphosphatase (ATPase) was used as an indicator of freezing effects. Freezing and thawing resulted in marked increase of ATPase activity, as compared to that of unfrozen membranes. The rate of freezing was a main factor in the increase of ATPase activity, the faster rate causing the greater increase. The increase of ATPase activity by freezing appeared attributable to the increase in accessibility of ATP to ATPase arising from disruption in membranes.

Droplet-Frozen Sensitized Red Cells for Gm Typing

Summary

Biochemistry of Whole Blood in Poly(Ethylene‐Co‐Ethylacrylate) Experimental Blood Containers

Abstract. The biochemical status of whole blood stored in containers fabricated of ethylene ethylacrylate (EEA) film was monitored at several times during 4 weeks of storage at 4°C. Fifteen biochemical indicators were studied to reflect on erythrocyte integrity, cellular metabolism, plasma protein stability, and microaggregate formation. Comparison to storage in polyvinyl chloride (PVC) containers was made by distributing aliquots from each unit of blood among the containers being compared. Whole blood in EEA developed significantly higher levels of plasma hemoglobin, erythrocyte osmotic fragility, and D‐glycerate‐2,3‐diphosphate (2,3‐DPG), and somewhat greater glucose utilization, lactate production, and pH. These biochemical differences were not of great magnitude and the data suggest that EEA containers are compatible with the storage of whole blood.

Cryopreservation of tissue and solid organs for transplantation: Edited by A. B. Glassman and J. Umlas. American Association of Blood Banks (AABB), Arlington, Virginia 1983. 112 pp., (

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An alloantigen was detected in the cytoplasm of blood cells, predominantly in hemolyzed erythrocytes, by radioimmunoassays developed with antibodies from a hemophiliac. The frequency of the antigen varies in different races. Studies in Caucasian families revealed an autosomal‐dominant inheritance of the antigen.