BACKGROUND: Granulocyte specific antibodies are associated with several clinical conditions including febrile transfusion reaction and transfusion-related acute lung injury as well as immune neutropenias. The identification of granulocyte specific antibodies is important for the diagnosis of these disorders. However, there have been rarely confirmed clinical reports in Korea since the testing techniques are complicated and difficult to maintain. In this study, development of in-house indicator cells and renewedly establishment of the mixed passive hemagglutination assay (MPHA) as a serologic test to detect and identify granulocyte specific antibodies were conducted. METHODS: The in-house indicator cells for MPHA were made by sensitizing human Rh(D) positive O RBCs with human IgG anti-Rh(D) (DiaMed AG, Switzerland) and then combining with AHG anti-IgG (Immucor Inc., USA). To determine the optimal conditions, various combinations of anti-Rh(D) IgG sensitization strengths of indicator cells, microwell coated antigens (intact granulocyte vs. extracted granulocyte) and reaction conditions were compared. RESULTS: The best test conditions for MPHA were as follows: optimal results were obtained with the anti-Rh(D) sensitization dilutions of 1/64-1/192 and the reaction condition of 4 hours incubation at room temperature in humid chamber. Extracted granulocytes coated at the plate showed better results than intact granulocytes. HLA antigens were completely removed from extracted granulocyte antigens after acidified chloroquine treatment. CONCLUSION: Granulocyte MPHA using in-house anti-Rh(D) sensitized indicator cells was developed for the first time in Korea. The newly established MPHA would be effectively used for the diagnosis and treatment of disorders associated with granulocyte specific antigen-antibody reactions in Korea.