Anti–Granulocyte Antibody Screening with Extracted Granulocyte Antigens by a Micro–Mixed Passive Hemagglutination Method

Abstract

Background and Objectives: Serologic tests for granulocyte antibodies, i.e., the granulocyte agglutination test and the granulocyte immunofluorescence test, require panels of typed granulocytes that cannot be preserved for more than a few hours. We have developed a new method in which granulocyte antigens, extrcated into saline containing 3% sucrose, are coated onto U–type Terasaki plates. With this new method, we evaluated the micro–mixed passive hemagglutination test (EG–MPHA) for screening for granulocyte antibodies. Materials and Methods: We tested the ability of the EG–MPHA to detect granulocyte antigens using 5 human antibodies specific for NA1, NA2, NB1, 5b, and Sar^a, and 8 different monoclonal antibodies for NA1, CD11a, CD11b, CD13, CD16, CD18 and HLA class I. Sera from 94 alloimmunized patients were screened by the chloroquine–treated EG–MPHA method. Results: NA1, NA2, NB1, 5b, Sar^a, CD11a, CD11b, CD13, CD16, CD18 and HLA class I antigens were present in the extracted granulocyte antigen preparation. CD11b and HLA class I antigens were removed when the extracted granulocyte antigens were treated with chloroquine. Granulocyte antibody screening of sera from alloimmunized patients showed that approximately 30% of the anti–HLA–positive and 10% of the anti–HLA–negative sera were positive for granulocyte antibody by the chloroquine–treated EG–MPHA. The extracted granulocyte antigen panels could be stored frozen for at least 1 year at –80°C. Conclusion: This new method is preferable for screening for granulocyte antibodies. In addition, it has the advantage of requiring only 5 μl of serum for each test.