Real-time imaging during centrifugation

Gary Boas

People who lose blood or whose bodies aren’t producing enough can receive transfusions. However, they sometimes need only certain blood components, such as red blood cells, plasma and platelets. In these cases, centrifugation is used to separate the components from the whole blood.

But does centrifugation alter the blood? Studies comparing human red blood cells before and after centrifugation have shown that the procedure doesn’t affect the shape or volume of the cells. We still do not know, however, whether and how these change during centrifugation. Researchers at Yale University in New Haven, Conn., and at the Marine Biological Laboratory in Woods Hole, Mass., have described changes in human and Amphiuma salamander red blood cells during centrifugation using a specially designed microscope.

The study was made possible, said Yale researcher Joseph F. Hoffman, by a centrifuge polarizing microscope. Developed by Shinya Inoué of the Marine Biological Laboratory in collaboration with Olympus America Inc. of Melville, N.Y., and Hamamatsu Photonics KK of Hamamatsu City, Japan, the instrument enables video observation at the microscopic level, and thus allowed them to record stratification of the various components in living cells during centrifugation. A timing circuit triggers a 6-ns pulse from a 532-nm Nd:YAG laser as individual cells pass below a 40x, 0.55-numerical-aperture objective. A photodiode acquires sequential images with a resolution of 0.5 μm.

Reversible changes

Hoffman and Inoué observed the red blood cells suspended in solutions and spinning at rates of 200 to 11,700 rpm. They analyzed the data by playing back the video sequences at various speeds and listening to audio commentary that they had recorded in real time.

The experiments demonstrated that centrifugation changes a red cell’s shape and the distribution of its various components, but that this is completely reversible — indicating that the changes cause no damage.

In human cells, the researchers observed two major changes in the cells’ appearance at higher centrifugation speeds. First is a change of shape caused by sedimentation of hemoglobin inside the cell. This leads to a bulging of the lower part of the cell, producing a sort of baglike shape. Here, however, the upper part of the cell maintains its original biconcave (dimpled) form, suggesting that the original shape is not dependent on the presence of hemoglobin. In any event, the cells immediately revert to the original shape when they touch cells in the classical rouleau formation, in which cells are stacked on top of one another like a roll of coins.

Experiments with the specially designed microscope revealed significant shape changes during centrifugation, particularly at high speeds. Sedimentation of hemoglobin molecules led to a bulging in the lower parts of the cells. Because the upper parts remained relatively thin, the cells took on baglike aspects. Reprinted with permission of PNAS.

Changes in the salamander red cells were different, although they also proved reversible.

The most interesting question remains, however: What is the basis of the cells’ normal, biconcave shape? This question has been asked since 1827, Hoffman said, when use of a new microscope, corrected for chromatic aberration, definitively established the biconcave shape of red blood cells. Studying shape transformations of abnormal cells with the centrifuge polarizing microscope might help provide an answer, he added.

PNAS, Feb. 21, 2006, pp. 2971-2976.