(1) When human red cells are hemolyzed in very hypotonic media (NaCl of a tonicity of 0.167) and when the tonicity is restored, by adding appropriate amounts of NaCl, to tonicities such as 0.3, 0.5, 1.0, and 1.7, the mean volume of the ghosts appears to be linear with the reciprocal of the tonicity. This might lead one to conclude that the ghosts are osmometers and that their volume is governed by simple osmotic considerations such as those expressed by a modified van’t Hoff-Mariotte law. Examination of the shapes and volumes of individual ghosts by a technic which combines phase optics, electronic flash (exposure time 0.001 second) and photography shows that three distinct populations of ghost coexist in any tonicity. These are spherical ghosts with a mean volume of 150 µ3, discoidal biconcave ghosts with a mean volume of 85 µ3, and crenated ghosts with a smaller volume which can be calculated. The most likely reason for this complexity is that the shape and volume of the ghost depends partly on its structure, and not altogether on the tonicity of the surrounding medium. Simple osmotic laws have no real application to systems of this kind.
(2) The changes in ghost volume and shape, as they depend on the duration of storage of the blood, at 4 C, from which the ghosts are prepared, and as they reflect changes in ghost structure, can be expressed simply. Crenated and discoidal ghosts certainly have some of the elements of red cell structure; the spherical ghost, which soon fragments and gives rise to myelin forms, may also retain some of the original elements of structure, but the fragment and the myelin form have certainly lost them. The latter objects are so small and light that they are not thrown down into the ghost column in the hematocrit tube, and so, as ghost structure disappears with increasing time of storage of the red cells from which they are prepared, a discrepancy appears between the volume of the ghost column as measured by the hematocrit and the volume which one would expect. This discrepancy can be used as a measure of the extent to which ghost structure is lost, and there comes a time, as the duration of red cell storage is increased, when the ghosts prepared from these red cells begin to be replaced by breakdown products such as myelin forms, etc.
(3) The less the efficiency of the conditions of red cell preservation, the shorter is this time. In human blood rendered incoagulable with heparin, structural breakdown in the preceding sense and measured by a simple expression which changes sign when the loss of structure has reached a certain point, occurs after about 26 days. In human blood preserved in ACD, structural breakdown measured in an identical manner occurs after about 55 to 60 days. In human blood preserved in ACD-inosine, structural breakdown does not occur until about 75 to 80 days.
These results are based on a large amount of preliminary work of an exploratory nature and then on three runs with heparin, five runs with ACD and five runs with ACD-Inosine.