The sera of 8 patients with acquired hemolytic anemia associated with elevated levels of cold agglutinins were studied by various procedures of zone electrophoresis. The agglutinating activity was found associated with proteins of variable mobility in the different cases. The majority represented "fast" γ-globulins. The 4 sera with the highest titers of cold agglutinins showed distinguishable abnormal electrophoretic components. The titers correlated with the height of the abnormal components.

Ultracentrifugal analysis of the electrophoretic fractions indicated that the cold agglutinins were associated with proteins having a sedimentation coefficient of approximately 19 S. The abnormal component from the serum with the highest biological activity showed almost no contamination with lower molecular weight proteins. The amount of 19 S material found correlated with the titer of agglutinating activity.

The high molecular weight character of the cold agglutinins was confirmed by procedures of density gradient zone centrifugation. The biological activity sedimented with proteins of the 19 S class in all the sera including those of relatively low titer with which no abnormal electrophoretic components were observed.

Dissociation of the abnormal high molecular weight components was possible by means of certain sulfhydryl compounds. This resulted in disappearance of cold agglutinin activity.

Some of the cases could be classified as macroglobulinemias because of the very large content of high molecular weight components. However, the same disease picture occurred without recognizable elevation of these components.

The sera of 3 patients with severe acquired hemolytic anemia of the warm type associated with warm incomplete antibodies failed to show similar abnormal electrophoretic components and the antibody activity sedimented with proteins of the 7 S class.

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