Methods for the detection of granulocyte antibodies
International workshops revealed a combination of several methods as the best means for the detection of granulocyte antibodies.
With the Granulocyte Agglutination Test (GAT) we use the feature that some granulocyte antibodies directly aggregate/agglutinate granulocytes.
In this test, a panel of granulocytes is incubated with the serum in question on a microtiter plate. Aggregation requires some hours and is subsequently evaluated by microscope.
Especially for HNA-3a antibodies, which are often involved in severe TRALI reactions, the GAT is the best method for detection.
Depending on the antibody specificity, the aggregates show different shapes.
On the other hand, not all granulocyte antibodies show positive agglutination. To detect the non-agglutinating antibodies, we perform the Granulocyte Immunofluorescence Test (GIFT). By using a FITC-conjugated antibody it is possible to detect antibodies bound to the granulocyte membrane. The evaluation is done either by flow cytometry or by fluorescence microscopy. Microscopic evaluation is more time-consuming than flow cytometry and needs a lot of experience, but according to the results of the International Workshop for Granulocyte Serology, microscopic evaluation is preferable. In contrast to flowcytometric evaluation, microscopy offers the chance to identify high fluorescence signals coming from damaged cells as false positive signals. Furthermore, it is possible to distinguish the fluorescence pattern of antigen-bound antibodies from that caused by unspecific adsorption of immune complexes via Fc? receptors. By using adequate secondary antibodies it is possible to differentiate between IgG-, IgM- and IgA-antibodies against granulocytes.
HLA class I antibodies react similar to HNA antibodies in GIFT and GAT. To distinguish them from HNA antibodies, we use the Lymphocyte Immunofluorescence Test (LIFT). This test is performed analogous to the GIFT, but the evaluation is made by flow cytometry. Since lymphocytes are stable and by the majority do not have Fc receptors, there are no problems due to unspecific staining. The LIFT is also useful in identifying anti-HNA-3a because this antigen is also expressed on lymphocytes. GIFT and GAT, optionally combined with LIFT, are adequate tests for granulocyte antibody screening. Due to their short life span, the typed test cells always must be freshly isolated from donor blood. An elegant method for identifying the specificity of a granulocyte reactive antibody or for analysis of antibody mixtures is the glycoprotein-specific ELISA (Monoclonal Antibody Immobilization of Granulocyte Antigens = MAIGA). MAIGA can be performed with all antigens which can be captured by a monoclonal antibody. There is only one exception: a monoclonal antibody against HNA-3 antigens does not exist. Since the MAIGA is a laborious test, it is not suitable for antibody screening. Tab. 2 shows the reaction patterns of different HNA antibody specificities.