A Novel Method Using Extracted Human Neutrophil Antigens From HNA Gene-Transfected Cell Lines for Detection of Antibodies Against Human Neutrophil Antigens

Abstract 3272

Antibodies against Human Neutrophil Antigen (anti-HNA antibodies) have been implicated in immune neutropenia and transfusion-related acute lung injury. Those have been usually analyzed by Granulocyte indirect immunofluorescence test by flow-cytometry (GIFT-FCM), which requires fresh blood for panel neutrophils that cannot be preserved more than a few hours. Furthermore, specificity for anti-HNA antibodies cannot be easily identified by GIFT-FCM because of several antigens on membrane of panel neutrophil. In this study, we have developed a novel method designated as KY-Luminex Method, which was modified from Monoclonal antibody-specific immobilization of granulocyte antigens.

For KY-Luminex Method, extracted human neutrophil antigens (eHNAs) were established from gene-transfected cell lines (KY-1a, -1b, -1c, -2a, -4a, -4b, -5a, -5b) that express HNA-1a, -1b, -1c, -2a, -4a, -4b, -5a, -5b, respectively. These eHNAs were immobilized by monoclonal antibodies on microspheres (eHNA-microspheres). Sera were tested to the eHNA-microspheres in wells of a 96-well microplate, respectively. The reactivities of the sera were analyzed by Luminex¹⁰⁰.

From May 2010 to March 2012, sera were obtained from 101 patients with autoimmune neutropenia in children. The age distribution was from 2 months to 72 months. Sixty-three patients were females and 38 were males.

As shown in Table, the sera from 74 patients showed positive reaction both in standard GIFT-FCM and KY-Luminex method. The sera from 11 patients showed positive in GIFT-FCM, and negative in KY-Luminex. These sera finally proved to have antibodies against antigens other than neutrophil such as anti-HLA antibody. The sera from 13 patients showed negative in GIFT-FCM, and positive in KY-Luminex. The sera from 3 patients showed negative both in GIFT-FCM and KY-Luminex. Sera with positive reaction against each panel neutrophil in GIFT-FCM have tendency to react against multiple antigens in KY-luminex method. Especially, majoryty of analyzed sera had antibodies against HNA-4a, 4b, 5a and 5b. No antibodies against each antigens were detected in sera from normal controls by KY-luminex method.

These results indicate that KY-luminex method has superior to GIFT-FCM in both sensitivity and specificity of anti-HNA antibodies. Furthermore, eHNA-microspheres are stable at 4 centigrade for more than a half year, and many samples such as 96 samples could be analyzes at once in simple and easy method.