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

Abstract

Flow cytometry (FC) was used to estimate platelet-surface IgG (PSIgG) by quantifying the fluorescence of platelets incubated with a fluorescein isothiocyanate (FITC)-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 measure PSIgG of platelets coated with known amounts of a chimeric IgG (human IgG with murine hypervariable region) monoclonal antibody to the glycoprotein Ilb-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.

Prospective evaluation of a new platelet glycoprotein (GP)-specific assay (PakAuto) in the diagnosis of autoimmune thrombocytopenia (AITP)

“…Phase II assays were designed to measure either surface or total PAIgG [7,8]. However, it was later shown that PAIgG was elevated in a wide spectrum of nonimmune TP disorders.…”

Platelet surface IgG in patients receiving infusions of Fab fragments of a chimaeric monoclonal antibody to glycoprotein IIb-IIIa

“…The mean fluorescence channel number (MFC) was used as an arbitrary unit of platelet fluorescence intensity. It has been shown in other studies [15] that, over the range of fluorescence intensities encountered in these measurements, the MFC number is, for practical purposes, a linear function of the amount of antigen (in this case 7E3) present on the platelet surface. 7 had risen to 3.88 ± 0 51, which was significantly higher than the RFI at baseline (P < 0 01) and at 24h (P = 003).…”

“…Fluctuations in PSIgG were in the same direction as fluctuations in PS7E3 up to day 5, but on day 8 the level of PSIgG had remained high despite a fall in PS7E3 to < 50% of the level reached at the end of the infusion. Using a formula derived as described previously [15], it was estimated that on day 8 this patient's platelets were bearing approx. 12 000 IgG molecules on their surface (level of normal controls 1460 ± 927 (mean + s.d.…”

A novel method for simultaneous analysis of specific platelet antibodies: SASPA

“…These antibodies can target specific platelet glycoproteins (GP), human leucocyte antigen (HLA) class I antigens or phospholipids (Kurata et al, 1993;Alcorta et al, 1996;Macchi et al, 1996Macchi et al, , 1997Cuadrado et al, 1997;Panzer et al, 1997;Kiefel et al, 2001;Ranasinghe et al, 2001;Cines & Blanchette, 2002). The detection of these antibodies with high sensitivity and characterization of their specificity is important for diagnosis and specific therapy.The platelet immunofluorescence test (PIFT) has been widely used as a screening method for the detection of platelet antibodies (von dem Borne et al, 1978;Christopoulos et al, 1993;Porcelijn and von dem Borne, 1998;Hagenström et al, 2000). In order to differentiate the detected antibodies, the monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay has been mostly used as the reference assay (Kiefel et al, 1987).…”

“…The platelet immunofluorescence test (PIFT) has been widely used as a screening method for the detection of platelet antibodies (von dem Borne et al, 1978;Christopoulos et al, 1993;Porcelijn and von dem Borne, 1998;Hagenström et al, 2000). In order to differentiate the detected antibodies, the monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay has been mostly used as the reference assay (Kiefel et al, 1987).…”