Fabian J. Lionetti

Preservation of Human Granulocytes: II. Characteristics of Granulocytes Obtained by Counterflow Centrifugation

Human granulocytes were isolated from whole blood and from the buffy coat of blood by counterflow centrifugation using the Beckman JE‐6 rotor. Ninety‐one per cent of granulocytes in the human blood sample were isolated in 59 studies. The differential white blood cell count showed 97 per cent segmented neutrophils, 1 per cent lymphocytes and 2 per cent others. The ratio of polymorphonuclear white blood cells to red blood cells was 9:1 when the granulocytes were isolated from whole blood and 40:1 when the granulocytes were isolated from the buffy coat of CPD‐collected blood. The size distribution showed a single symmetrical peak. Measurements of oxygen consumption, chemotaxis, and phagocytosis in granulocytes isolated by counter‐flow centrifugation were similar to those in granulocytes isolated from blood by dextran sedimentation. The granulocytes had normal adenine and guanine di‐ and trinucleotide levels.

In Vitro Studies of Cryopreserved Baboon Granulocytes

Granulocytes were isolated from the buffy coat of baboon blood by counterflow centrifugation. They were frozen in polypropylene tubes in 2.0 ml volumes containing 1 × 107 granulocytes. The medium consisted of 5% DMSO, 6% HES, 4% human serum albumin, and 6 mM glucose in Normosol‐R, pH 7.1. Granulocytes were cooled to 4 C for 30 minutes, then cooled at 4 C per minute to –80 C and stored for one to three weeks in liquid nitrogen at –197 C. Cooling rates of 1 C and 10 C per minute were less efficacious. Tubes were thawed manually with swirling for 130 seconds at 42 C in a water bath. The yield after thawing was 98 ± 14 per cent.

Cryopreservation of human red cells in liquid nitrogen with hydroxyethyl starch

Summary Erythrocytes were frozen in plastic bags in liquid nitrogen with 14% (w/v) hydroxyethylstarch (HES). Both a small volume (25.0 ml) and a full unit system (405 ml) were employed. Small volume freezings were investigated to optimize container configuration, conditions, composition of media, and postthawed stability of cells. Based on this experience a method for full unit freezing of packed erythrocytes was devised. The results of the 10 most recent experiments with 25.0 ml freezing gave postthawed cell recoveries of 99.2 ± 0.3%. Stability for 1 hr in 0.15 M NaCl at 22.0°C was 85.9 ± 4.3%. The cells gained 17 mequiv of Na + and lost 21 mequiv of K + . The ATP decreased 23% while 2,3 DPG was unchanged. In the large volume method using all the packed red cells in a unit after removal of platelet rich plasma and buffy coat, a single bag was sandwiched between large perforated aluminum plates. The results of eight recent freezings and short-term storage in liquid nitrogen vapor gave cell recoveries of 97.2 ± 1.1%, and stabilities in saline of 75.7 ± 1.8%. Relatively small losses of ATP and 2,3 PDG were observed. The cells gained 33 mequiv of Na + and lost 31 mequiv of K + . Assuming acceptable 24-hr posttransfusion survival can be achieved, the feasibility of freezing full units of red cells in a one-step procedure is now demonstrated.

Preservation of human granulocytes. III. Liquid preservation studied by electronic sizing.

Human granulocytes were isolated from blood either by counterflow centrifugation in a Beckman JE‐6 rotor or by sedimentation of the red blood cells with dextran and centrifugation of the granulocyte‐rich plasma. The stability of the granulocytes was assessed during storage in the liquid state by measurements of granulocyte loss, volume distribution characteristics, and ability to produce fluorescein in their cytoplasm and to exclude ethidium bromide from their nuclei. After storage at 4 C for 2 days in phosphate‐buffered saline with a pH of 7.1 containing 5 g/dl human albumin + 0.46 g/dl dextrose or 1 g/dl Physiogel + 0.46 g/dl dextrose, the granulocytes were adequately preserved from the in vitro measurements.

Enzymes of human saliva, II. Parotid saliva total esterases.

ODIUM beta-naphthylacetate has been employed as a substrate for the demonstration of nonspecific esterases in tissues and for the colorimetric determination of esterases in saliva.2 Although this substrate is most readily attacked by the esterases of liver and blood serum, it can also be hydrolyzed by the lipases found in pancreatic homogenates.3 The ability of acetyland pseudo-cholinesterases to split /3-naphthylacetate has been indicated by studies employing brain homogenate, serum cholinesterase, and red cell membranes.4 Therefore, in the measurement of the enzymatic hydrolysis of ft-naphthylacetate by human saliva the term total esterase activity has been employed to encompass activity originating from nonspecific esterases, cholinesterases, and lipases. The results given here represent a partial kinetic characterization of the total esterase activity of human parotid saliva.

Improved method for the cryopreservation of human red cells in liquid nitrogen with hydroxyethyl starch.

Refinement of a method described previously (Cryobiology 12, 110–118, April, 1975) made possible routine freezing of full units of packed erythrocytes after separation of platelet rich plasma, and buffy coats. The volume frozen was 405 ml which included packed red cells (190–220 ml), plasma (43–73 ml), and cryo-HES (142 ml, final concentration 14% wv). The units could be frozen with or without shaking by direct immersion in liquid nitrogen. Thawing was accomplished by transferring units quickly from liquid nitrogen storage to a shaking water bath at 54 °C. The average yield from units of red cells was 98.4%. The stability to a 50-fold dilution in 0.15 m NaCl was 87.8%. Thawing rate was the critical variable in producing the most stable thawed cells. Plasma expander HES was usable but the thawed units were more viscous and about 7% less stable. Red cells prewashed with 0.15 m NaCl and frozen without plasma showed no significant changes in cellular yield or stability. The optimum resuspension medium was 3% glucose. A morphologic study of cells fixed in 1% glutaraldehyde revealed that before freezing red cells were partially dehydrated in 14% HES. These were smooth, flat discs. Cells fixed on thawing were extensively dehydrated and seen as large, thin, smooth, flat discs with approximately 10% echinocytes. On dilution with 6% glucose (1:1) these swelled and reverted to biconcave discocytes except for approximately 5% echinocytes. Storage in liquid nitrogen measured in groups of three units of 15 units for 0, 3, 6, 9, and 12 weeks revealed normal postthawed oxygen delivery (P-50). The greatest measurable effect of freezing red cells in HES was a loss of cellular K+ compensated by a corresponding increase in Na+.