Leslie L. Lenny

Transfusions to group O subjects of 2 units of red cells enzymatically converted from group B to group O

Background: It has previously been shown that full‐unit (200 mL) transfusions of red cells (RBCs) enzymatically converted from group B to group O by treatment with α‐galactosidase (ECO RBCs) are both safe and efficacious for normal group O or A subjects.

Further Evidence for the Presence of A Antigen on Group B Erythrocytes through the Use of Specific Exoglycosidases

Abstract. Certain antibodies have shown the ability to detect small amounts of A antigenic structures on certain group B cells. These rare cells that reverse group as B, are designated here as B(A) cells. Among the anti‐A antibodies capable of detecting these cells are MHO4 (an IgM murine monoclonal antibody) and polyclonal anti‐A derived from blood group O donors. The latter (anti‐A, B) have been adsorbed exhaustively with normal B cells, to deplete the serum of antibodies to group B antigen. The cell specificity detected by these antibodies can be removed only by an α‐Nacetylgalactosaminidase (A‐zyme) but not by an α‐galactosidase (B‐zyme). Inhibition studies show that these reactions can be inhibited by A secretor saliva and cannot be inhibited by B secretor saliva. Moreover, papain treatment of normal group B cells not previously agglutinable with these antibodies, now causes these cells to become reactive, and this specificity, too, is removed only by A‐zyme. These results suggest that low levels of blood group A antigen are being recognized by these antibodies and that these structures can exist not only on B(A) cells but on all group B erythrocytes.

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.

Photochemical decontamination of red cell concentrates with the silicon phthalocyanine Pc 4 and red light

Virus inactivation in red blood cell concentrates (RBCC) is being studied in order to increase the safety of the blood supply. For this purpose we have been studying the silicon phthalocyanine (Pc 4), a photosensitizer activated with red light. Two approaches were used to achieve enhanced selectivity of Pc 4 for virus inactivation. One was formulation of Pc 4 in liposomes that reduce its binding to red cells. The other was the use of a light emitting diode (LED) array emitting at 700 nm. Vesicular stomatitis virus (VSV) infectivity served as an endpoint for virus kill in treated RBCC. Red cell hemolysis and circulatory survival in rabbits served as measures for red cell damage. Treatment of small aliquots of human RBCC with 2 μM Pc 4 in liposomes and 10 J/cm2 of 700 nm LED light in the presence of the quenchers of reactive oxygen species glutathione and trolox resulted in 6 log10 inactivation of VSV. Under these conditions hemolysis of treated red cells stored at 4 °C for 21 days was only slightly above that of control cells. Rabbit RBCC similarly treated circulated with a half life of 7.5 days compared with 10.5 days of control. It is concluded that Pc 4 used as described here may be useful for viral decontamination of RBCC, pending toxicological and clinical studies.