Platelet Preservation. Ii. Preservation of Canine Platelet Concentrates by Freezing in Solutions of Glycerol Plasma*

Abstract

The successful long-term preservation of bull semen by freezing in glycerol solutions provided the impetus for application of this technique to other cells and tissues (1). Among the hematopoietic elements, the red blood cells (2-7) and the bone marrow (8) have been preserved by this method. In the case of bone marrow, evidence that a high percentage of viable cells emerges from glycerol freezing is lacking, since there is no adequate quantitation of a transfused bone marrow dose-response curve. A few viable primordial elements of the marrow (reticulum cells, hemocytoblasts) may be adequate to provide a "take." By analogy only a small percentage of viable bull sperm may be required to cause conception. Only in the case of red blood cells is there evidence in man and laboratory animals that a high percentage of cells (90 per cent) survives glycerol freezing and is capable of living a normal life span after transfusion into a compatible recipient (7).The red blood cell in several respects lends itself readily to in vitro manipulation. It survives the trauma of contact with foreign surfaces, anticoagulation (9), centrifugation, acidification (10), cooling (40 C), supercooling (-3 C) (11), and warming (40 to 370 C). Despite bizarre morphology and ample evidence of chemical alteration with storage (12), the red blood cell survives the lesion incurred in leaving the body by its remarkable capacity to be reconstituted on renewal of contact with the blood stream (13,14).Platelet preservation by glycerol freezing (15)(16)(17)(18)(19)(20), however, has lagged behind that of the red blood cell for several reasons. Platelets are not handled in vitro with the same facility as red blood cells. The very nature and function of the platelet renders it susceptible to irreversible damage *This investigation was supported in part by a research grant from the Department of the Army, Office of the Surgeon General (DA-49-007-MD-701). from the moment it leaves its intravascular milieu (21-23). This irreversible damage may shorten the platelet life span without altering its morphological appearance or clot-retraction function (18,(24)(25)(26). Any worthwhile preservation procedure, therefore, must retain in the preserved platelets the capacity to survive normally in vivo (16-19, 25, 26) as well as the capacity to perform clot-retraction and thromboplastic functions in vitro (27,28).The canine was selected as the animal model for development of platelet preservation techniques. This animal offers several advantages over smaller animals. Volumes of blood nearly comparable (350 to 400 ml) with those obtained in standard phlebotomies in humans (450 to 500 ml) are obtained with ease, thus permitting experience in handling platelets comparable in volume with that anticipated in man. The dog is relatively longlived compared with smaller laboratory animals, permitting observations in the same animal over many years. This has permitted a linear experience with various methods for platelet preservation using the same animal over and over again...

Glycerol Treated Human Red Cells Frozen with Liquid Nitrogen

“…In their work on platelet preservation Cohen and Gardner [2] used for the removal of glycerol an extracellular osmotic gradient of sorbitol, to prevent damage to the thrombocytes. This destruction is caused by the slower diffusion of glycerol out of the cells in com parison with water.…”

“…After thawing the glycerol can be removed without special equipment by washing with a 16% sorbitol solution for maintaining an osmotic gradient [2].…”

Glycerol Treated Human Red Cells Frozen with Liquid Nitrogen

“…I n a final concentration of 17.5% (w/v), glycerol gives a sufficient protection of the red cells. After thawing the glycerol can be removed without special equipment by washing with a 16% sorbitol solution for maintaining an osmotic gradient [2].…”