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Michael Woetzel, Sabine Schroeder, Ulrich Sack
Differentiation of anti-platelet antibodies using microspheres and flow cytometry Einsatz von Mikrosphären und Durchflusszytometrie für die Differenzierung von Antithrombozyten-Antikörpern
Keywords: anti-platelet antibodies, flow cytometry, microspheres, monoclonal antibody immobilization of platelet antigens (MAIPA), multiplexed testing
Background: Allo- and autoantibodies against membrane structures of human platelets cause different types of immune thrombocytopenic disorders and require a sensitive and specific assay. The well-established monoclonal antibody immobilization of platelet antigens (MAIPA) test is able to distinguish between specifically bound immunoglobulins and coating of membrane-associated immune complexes; but MAIPA is rather time consuming and requires 2×107 platelets or more. For patients with very low platelet count and for little children with small blood volume, this may present a serious limitation.
Methods: The advantages of flow cytometry and of modern microsphere technology were combined with the selectivity of MAIPA to overcome the disadvantages of this assay. In order to compare different types of beads, i) goat anti-mouse Ig-labelled magnetic beads, ii) dyed polymeric beads designed for multiplexed assays, and iii) blanc polymeric beads have been studied. After loading with glycoprotein-specific or anti-?2-microglobulin-specific antibodies, these beads could be stored for months at 4°C without substantial loss of sensitivity.
Glycoproteins from platelet membranes, loaded with platelet-specific auto-/alloantibodies, have been immobilized on the bead surfaces during detergent solubilization of platelets prior to labelling with anti-human IgG-Biotin and Extravidin® R-Phycoerythrin.
Results: Magnetic beads are very easy to handle, show strong signals and low nonspecific binding, but have stronger autofluorescence compared to polymeric beads. Polymeric beads have low autofluorescence but stronger tendency to nonspecific binding. All types of beads tested in this study can be used to detect antibodies against platelet antigens, even against glycoproteins with low frequency such as anti-HPA-5 autoantibodies and with as few as 106 platelets or less. In multiplexed settings, they are suitable for simultaneous detection of multiple antibodies.
Conclusions: Flow cytometry is a fast and sensitive method for a bead-based MAIPA assay and is especially useful for low platelet counts and for pediatric patients. It is further improved by the use of multiplexed beads.
LaboratoriumsMedizin, Walter de Gruyter
Print ISSN: 0025-8466
Volume: 29, 10/2005
Pages: 368 - 376