PMID: 15373582Sep 18, 2004Paper

Detection of granulocyte antibodies by flow cytometry without the use of pure granulocyte isolates

Immunohematology
K M KiekhaeferD F Stroncek

Abstract

Established methods used to detect serum antibodies to granulocytes require the isolation of granulocytes. Flow cytometric analysis of granulocytes with monoclonal antibodies eliminates the need for granulocyte isolation. The purpose of this study was to develop a method to evaluate reactions of antibodies to granulocytes without separating granulocytes from other leukocytes. Three screening cell samples for granulocyte antibody detection were prepared from whole-blood samples in which the red blood cells (RBCs) were lysed and remaining leukocytes tested against sera at 4 degrees C. Binding of human alloantibodies to the screening cells was determined by flow cytometric analysis using phycoerythrin-conjugated antibody to human immunoglobulin. Forward and side scatter were used to analyze granulocytes separately from other leukocytes. The assay was validated by testing granulocytes with reference alloantibodies directed to NA1, NA2, 5b, and Mart antigens. Samples from 32 patients were tested, and the results of the assays were compared with the results of testing the samples in a granulocyte immunofluorescence (GIF) assay performed by a reference laboratory. In the whole-blood flow cytometric (WBFC) assay the mean fluorescence i...Continue Reading

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