Rapid screening of granulocyte antibodies with a novel assay: flow cytometric granulocyte immunofluorescence test.
BACKGROUND
White blood cell (WBC)-associated antibodies can lead to severe pulmonary transfusion reactions (transfusion-related acute lung injury [TRALI]). Investigation of a large number of blood donor samples using the standard granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT) proved to be difficult to perform due to the time-consuming process and the large quantity of test cells required. This study describes the novel flow cytometric GIFT (Flow-GIFT) method for a rapid detection of granulocyte antibodies by flow cytometric analysis.


METHODS
A total of 141 sera were analyzed for the presence of granulocyte antibodies that were previously associated with suspected TRALI. As test cells whole blood samples from human neutrophil antigen (HNA)-typed donors were isolated using cell sedimentation in a ficoll density gradient. WBCs were incubated with the respective serum and binding of antibodies to the test cells was detected using fluorescein isothiocyanate-conjugated anti-human antibody. Standard GIFT and GAT were performed as reference methods.


RESULTS
Seven sera containing anti-HNA-3a, CD16, and HLA Class I were negative in the standard GIFT and eight sera containing anti-HNA-2a, anti-CD16, and anti-HLA Class I were not detected in the GAT. The novel Flow-GIFT was able to detect all granulocyte antibodies, which were only detectable in a combination of standard GIFT and GAT. In serial dilution tests, the Flow-GIFT detected the antibodies at higher dilutions than the reference methods GIFT and GAT.


CONCLUSIONS
The Flow-GIFT method permits rapid detection of granulocyte antibodies requiring fewer donor test cells. This method is ideal for automation and will potentially open the way for screening of granulocyte antibodies in a large donor population.
Nguyen
Xuan Duc
XD
Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University, Red-Cross Blood Service of Baden-Württemberg-Hessen, Mannheim, Germany. xuan-duc.nguyen@medma.uni-heidelberg.de
Flesch
Brigitte
B
Sachs
Ulrich J
UJ
Kroll
Hartmut
H
Klüter
Harald
H
Müller-Steinhardt
Michael
M
D000906:Antibodies / Q000032:analysis*; D000918:Antibody Specificity; D000094902:Blood Banking / Q000379:methods*; D001803:Blood Transfusion; D002469:Cell Separation / Q000379:methods; D005362:Ficoll; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique / Q000379:methods*; D058851:GPI-Linked Proteins; D006098:Granulocytes / Q000166:cytology / Q000276:immunology*; D015395:Histocompatibility Antigens Class I / Q000276:immunology; D006801:Humans; D007201:Indicator Dilution Techniques; D007519:Isoantigens / Q000276:immunology; D008214:Lymphocytes / Q000166:cytology; D009000:Monocytes / Q000166:cytology; D017452:Receptors, IgG / Q000276:immunology
D000906:Antibodies; C428200:FCGR3B protein, human; D058851:GPI-Linked Proteins; C518134:HNA-3a antigen, human; D015395:Histocompatibility Antigens Class I; D007519:Isoantigens; D017452:Receptors, IgG; D005362:Ficoll