Analysis of anti-D
Anti-D quantification was performed using flow cytometry. To increase sensitivity erythrocytes were pretreated with the enzyme papain. All dilutions were made in ID-CellStab (Bio-Rad Laboratories Inc) with 0.5% BSA (A7906, Sigma-Aldrich) as additive. The calibration standard was the 2nd BRITISH STANDARD 1992 Anti-D (Rho) Antibodies (code: 73/515, NIBSC, Potters Bar, UK). In short, 50 µL 0.8% erythrocytes (R0r) and 100 µL standard or sample was incubated for 30 min at 37°C. After washing with 3 x 200 µL PBS, 50 µL mouse anti-human IgG-PE (Southern Biotech) diluted 100-fold was added and incubated for 30 min at RT. After an additional wash as above, the erythrocytes were dispersed in 250 µl PBS and analysed on Cytomics FC500 MPL. The mean fluorescense intensity (MFI) on 10,000 gated cells were measured and imported to SoftMaxPro (Molecular Devices) which was used for constructing the calibration curve and calculation of anti-D concentration. The LOD was 1 IU/L (0.2 µg/L).