Perhaps the most distinguishing feature of eosinophil leucocytes is the presence in their cytoplasm of numerous acidophilic granules. This investigation was undertaken in the hope that knowledge about the nature of these granules might shed light on possible functions of eosinophils.We report here techniques that allow separation of horse and rat eosinophils free of other cell types, and methods for isolating their cytoplasmic granules. Peroxidase and a variety of hydrolytic enzymes were found to be associated with eosinophil granules. RESULTS Isolation of Eosinophils from Horse Blood and from Rat Peritoneal Washings.--Eosinophils constitute only a minor proportion of the cells in normal horse blood, and are present together with mast cells and macrophages in rat peritoneal fluid. Our first efforts were accordingly directed towards separation of eosinophils from other cell types. Such a separation was accomplished b y centrifugation in media of high specific gravity, thus taking advantage of the unusually high density of the eosinophil as compared to erythrocytes and other leucocytes. Detailed aspects of the procedures employed were as follows : -Horse Blood Eosinophils.--A horse was bled from the jugular vein, 250 ml of blood being collected and mixed with one-tenth volume of 0.1 u sodium citrate to prevent dotting. The blood was then dispensed into test tubes and allowed to stand for 45 minutes, during which time erythrocytes sedimented spontaneously. The supernatant plasma, containing platelets, leucocytes, and a few red cells, was then aspirated and spun at 100 g for 10 minutes, depositing white ceils but not platelets. After decanting the supernate, the leucocyte button was suspended in 5 m136 per cent bovine albumin.Albumin powder (Bovine fraction V), obtained from Armour Laboratories, Chicago, was dissolved in isotonic saline at 60 per cent, and adjusted to pH 7 by addition of 1 ~ NaOH. The albumin solution was then dialyzed twice with mixing for 2 hours against 100 volumes of 0.9 per cent saline. Increase in volume of the albumin solution resulting from neutralization

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I t was shown in the preceding report (1) that eosinophil granules are lysosome-like structures, similar in m a n y regards to cytoplasmic granules of rabbit polymorphonuclear leucocytes. Lysis of granules in intact polymorphonuclear leucocytes during phagocytosis has recently been demonstrated (2). The present study was designed to determine whether or not eosinophil granules also disrupt during phagocytosis. MethodsEosinophils were separated from mixed horse leucocytes by sedimentation through 36 per cent albumin as described in the accompanying paper (1). They were then washed 3 times in physiological saline and finally suspended in saline at approximately 108 per ml, The suspension was held at room temperature in a stoppered lusteroid tube.Serum was obtained from fresh clotted horse blood. Frequently the serum was adsorbed with glass powder prior to use. 10 ml serum was mixed with 1 gm of 325 mesh borosilicate glass powder over the course of 1 hour at room temperature, followed by centrifugation at 100 g for 10 minutes to remove the glass. On some occasions phenol red indicator was added to the serum to permit adjustment of the pH to approximately 7 with CO2.Immune serum was obtained from a horse which had been injected weekly X 5 intravenously with 5 ml volumes of human red cells or of yeast cell walls (zymosan). Human red cells were washed thrice in saline and a 5 per cent suspension was then prepared. Zymosan (Standard Brands, Inc., New York City) was boiled in physiological saline, washed on the centrifuge three times, and finally suspended at 10 nag per ml in saline. Horse serum containing precipitating antibodies against human albumin was obtained from Sylvana Chemical Company, Orange, New Jersey. Heat inactivation of serum was for 30 minutes at 56°C.The particulates to be engulfed included: (a) human red cells from the same person whose red cells were employed to immunize the horse, (b) zymosan, and (c) an antigen-antibody precipitate prepared by mixing 1.5 mg of crystalline human albumin with 1 ml of horse antihuman albumin serum, followed by centrifugafion and three washings in saline.Thin 1~eparatlons on glass slides were made as described previously (2). Slides and coverslips were coated with formvar by dipping them into a 0.2 per cent solution in ethylene dichlo-

Methods are described for the isolation of human eosinophils and basophils from donor blood. Using these cell preparations Charcot-Leyden crystals were found to originate from both eosinophils and basophils when the cells were suspended in hypotonic saline solution. The crystals formed also when saline extracts of eosinophils and basophils were concentrated by ultrafiltration through dialysis tubing. Fractions of eosinophils were prepared and the crystals were obtained from the cytoplasmic fraction but not from the nuclear or granular fractions. Chemical studies showed the crystals to be protein in nature and some evidence is presented which suggests that RNA may decrease the tendency for the protein to crystallize out of solution.

Platelet concentrates were prepared in plastic packs of polyvinyl chloride with tri(2-ethylhexyl) trimellitate as plasticizer. They were stored, with gentle shaking, at room temperature for periods up to 7 days before labelling with isotope and reinfusing. In vivo survival studies, platelet counts, pH and electron microscopy indicated that platelet concentrates prepared in the new plastic were superior to those prepared in the standard pack currently in use. Oxygen was found to diffuse through the new pack more rapidly than through the standard pack. A shelflife of up to 1 week at room temperature seems possible for platelet concentrates prepared in the new plastic.