Studies on the Use of Enzyme-Treated Red Cells in Tests for Rh Sensitization

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Abstract
Summary: The use of enzyme-treated red cells is a sensitive and specific method for testing for Rh-Hr antibodies and antibodies of other specificities. The most satisfactory results have so far been obtained with trypsin, papain, and ficin. For best results red cells should be washed three times with saline solution before treating them with enzyme, in order to remove the enzyme inhibitor present in normal human plasma. The sensitivity of the reactions increases with the period of incubation of the red cells with the enzyme preparation. Most satisfactory results were obtained by incubating the cells with the enzyme for a period of one hour at body temperature. Prolonged treatment with the enzyme may give rise to non-specific clumping. The action of the enzyme can be stopped at any time by washing the cells with saline in order to remove the enzyme. Chilling slows or stops the action of the enzyme, but this can be started again by rewarming the mixture. The use of enzyme-treated cells gives more sensitive results in tests for Rh sensitization than other standard tests hitherto introduced, and equals them in specificity. For example, the titers obtained in general are, on the average, four to five times as high as by the albumin-plasma conglutination technique. In no case of Rh sensitization encountered by us to date has the test with enzyme-treated cells failed to demonstrate the presence of Rh antibodies, although in several of these cases the other tests were negative. The use of trypsinated cells is a sensitive method of demonstrating autosensitization. Autosensitization, as detected in this manner, may be present without producing clinical symptoms. Normal human serum is approximately ⅛ as active as a 1 per cent solution of crystallized soy bean inhibitor in preventing the action of trypsin on red cells. Trypsin inhibitor is present in umbilical cord serum in approximately the same concentration as in adult serum, and is also present in equal concentration in the serum of sensitized Rh-negative individuals. Normal human serum inhibits the action of papain, whereas soy bean inhibitor, which is a more potent trypsin inhibitor, has no effect on papain. Thus the action of the inhibitors is specific. Evidence is produced to show that the enzymes do not damage any of the hemagglutinogen loci, but act instead upon a substance, probably a serum protein, which is intimately bound into the surface of the red cells. The clumping which results when enzyme-treated cells are coated with antibody appears to be related to conglutination rather than agglutination. Diagrammatic representations of these concepts are offered.