THE PRESERVATION OF LIVING RED BLOOD CELLS IN VITRO : I. METHODS OF PRESERVATION.
The erythrocytes of some species are much damaged when handled in salt solutions, as in washing with the centrifuge after the ordinary method. The injury is mechanical in character. It may express itself in hemolysis only after the cells have been kept for some days. It is greatest in the case of dog corpuscles, and well marked with sheep and rabbit cells. The fragility of the red cells, as indicated by washing or shaking them in salt solution is different, not only for different species, but for different individuals. It varies independently of the resistance to hypotonic solutions. The protection of fragile erythrocytes during washing is essential if they are to be preserved in vitro for any considerable time. The addition of a little gelatin ((1/8) per cent) to the wash fluid suffices for this purpose, and by its use the period of survival in salt solutions of washed rabbit, sheep, and dog cells is greatly prolonged. Plasma, like gelatin, has marked protective properties. Though gelatin acts as a protective for red cells it is not preservative of them in the real sense. Cells do not last longer when it is added to the fluids in which they are kept. Locke's solution, though better probably than Ringer's solution, or a sodium chloride solution, as a medium in which to keep red cells, is ultimately harmful. The addition of innocuous colloids does not improve it. But the sugars, especially dextrose and saccharose, have a remarkable power to prevent its injurious action, and they possess, in addition, preservative qualities. Cells washed in gelatin-Locke's and placed in a mixture of Locke's solution with an isotonic, watery solution of a sugar remain intact for a long time,-nearly 2 months in the case of sheep cells. The kept cells go easily into suspension free of clumps, they pass readily through paper filters, take up and give off oxygen, and when used for the Wassermann reaction behave exactly as do fresh cells of the same individual. The best preservative solutions are approximately isotonic with the blood serum. If the cells are to be much handled gelatin should be present, for the sugars do not protect against mechanical injury. Different preservative mixtures are required for the cells of different species. Dog cells last longest in fluids containing dextrin as well as a sugar. The mixture best for red cells is not necessarily best for leukocytes. A simple and practical method of keeping rabbit and human erythrocytes is in citrated whole blood to which sugar solution is added. In citrated blood, as such, human red cells tend to break down rather rapidly, no matter what the proportion of citrate. Hemolysis is well marked after little more than a week. But in a mixture of 3 parts of human blood, 2 parts of isotonic citrate solution (3.8 per cent sodium citrate in water), and 5 parts of isotonic dextrose solution (5.4 per cent dextrose in water), the cells remain intact for about 4 weeks. Rabbit red cells can be kept for more than 3 weeks in citrated blood; and the addition of sugar lengthens the preservation only a little. The results differ strikingly with the amount of citrate employed. Hemolysis occurs relatively early when the smallest quantity is used that will prevent clotting. The optimum mixture has 3 parts of rabbit blood to 2 of isotonic citrate solution. In the second part of this paper experiments are detailed which prove that cells preserved by the methods here recorded function excellently when reintroduced into the body.
Laboratories of The Rockefeller Institute for Medical Research.
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Turner