Establishment and clinical application of flow cytometric bead assay in detecting platelet-specific autoantibodies

Abstract

Objective To establish a novel method to detect autoantibodies against platelatespecific receptors by flow cytometric bead assay and study its clinical application. Methods The beads were coated with monoclonal antibodies SZ2, SZ22, SZ21 and 7E3 against platelet GP Ⅰ b, GP Ⅱ b, GP Ⅲa and GP Ⅱ b/Ⅲ a, respectively. Captured platelet glycoprotein and beads complex was detected by FITC labeled polyclonal goat antihuman immunoglobulin using flow cytometer. The platelet samples that reacted with antibodies (SZ2, SZ22, SZ21 and 7E3) negatively and positively were tested, respectively. Each sample was repeated 20 times to generate intra-day CV for the MFI and once a day for 8 days to generate inter-day CV values. The 85 ITP patients, 17 NITP patients and 50 controls from the First Affiliated Hospital of Soochow University during March 2006 to December 2008 were included in the studies. The sensitivity and specificity of these four platelet antibodies to diagnose ITP were analyzed using ROC curve. The results were compared with MAIPA. Results The CV of the intra-day-assay for samples negative to antibody SZ2, SZ22,SZ21 and 7E3 were 3.26%, 2. 86%, 1.65% and 4. 94%, respectively; While the CV of the intra-day-assay for samples positive to antibody SZ2, SZ22, SZ21 and 7E3 were 6. 16%, 4. 88%, 5.20% and 5. 85%,respectively. The CV of the inter-day-assay for samples negative to antibody SZ2, SZ22, SZ21 and 7E3 were 5. 86%, 4. 74%, 5.69% and 7.56%, respectively; While the CV of the inter-day-assay for samples positive to antibody SZ2, SZ22, SZ21 and 7E3 were 7.53%, 5.49%, 7.11% and 6.25%,respectively. The MFI for SZ2 in ITP group, NITP group and healthy control group were 1.49(0. 88-16. 24),1.12(1.00-1.33), 1.01 (0. 83-1.37), respectively, which showed significant differences (H = 36.89,P＜0.01). The MFI for SZ22 in the three groups were 1.55 (0.84-11.30), 1.13(1.03-1.29), 0.98(0. 85-1.24), respectively (H=28.41, P ＜0.01). The MFI of SZ21 were 1.50 (0.87-11.04), 1.13(0.97-1.32), 1.05 (0.85-1.48), respectively (H=54.42, P＜0. 01). The MFI for7E3 were 1.51(0. 84-9.81), 1.05(0.86-1.13), 1.03 (0.74-1.28), respectively (H =31.97, P ＜0.01). Based on ROC analysis, with cut-off values of 1.37, 1. 24, 1.48 and 1.28 for SZ2, SZ22, SZ21 and 7E3,respectively, the AUC were 0. 86, 0.90, 0. 87 and 0. 84, respectively. The sensitivities of the assays were 58. 82% (50/85), 52. 94% (45/85), 52.94% (45/85) and 51.76% (44/85), respectively. When all four antibodies were used, the sensitivity was increased to 74. 12% (63/85), which was higher than that of MAIPA [ 50. 59% (43/85) ,χ2 = 6. 78, P ＜ 0. 05) ]. Conclusion Flow cytometric bead assay can be used to detect four platelet-specific autoantibodies simultaneously, and may be a useful method to aid in the diagnosis of ITP. Key words: Flow cytometry; Autoantibodies; Glycoprotein; Thrombocytopenia