A Flow Cytometric Approach to Quantitative Estimation of Platelet Surface Immunoglobulin G

Flow cytometry (FC) was used to estimate platelet-surface IgG (PSIgG) by quantifying the fluorescence of platelets incubated with a fluorescein isothio-cyanate (FLTC)-labelled polyclonal goat anti-human IgG antibody or FITC-labelled non-immune goat IgG. Results were expressed as relative fluorescence intensity (RFI) defined as the ratio of specific fluorescence (mean fluorescence of platelets incubated with the FITC anti-IgG) over non-specific fluorescence (mean fluorescence of platelets incubated with FITC non-immune goat IgG). A normal range was formed by analysing platelets from 71 healthy subjects. Platelets from 16 patients with a firm clinical diagnosis of immune-mediated thrombocytopenia had a mean RFI significantly higher (p <0.001) than the controls, whereas platelets from 9 patients thought to have non-immune thrombocytopenia had an RFI not significantly different from the normal controls. From a prospectively studied group of 62 patients with no clinically obvious cause for their thrombocytopenia or impaired platelet function 35.5% had raised PSIgG. In order to express the results as number of IgG molecules per platelet, reference curves were created by using FC to nieasurc PSIgG of platelets coated with known amounts of a chimeric IgG (human IgG with murine hypervariable region) monoclonal antibody to the glycoprotein IIb-IIIa complex. Normal platelets had an average 1,463 (SD = 927) molecules of PSIgG. In patients with immune-mediated thrombocytopenia the levels ranged from 690 to 32,328 (mean 11,535) molecules per platelet. Flow-cytometric PSIgG estimation was sensitive, fast and easy to perform and therefore suitable for both research and clinical service purposes.