3-P

Abstract

Aim Luminex based analysis of HLA antibody concentration in serum (represneted as mean fluorescence intensity-MFI) is increasingly used for patient management. However, different factors contribute to the variation in these MFI values including a real biological condition, type of antigen, disease or assay, drug treatment and technical variation. Here, we evaluated MFI variation for HLA class I and II single antigen assay when performed by different technologist for sera known to have multiple HLA abs of varied strength. Methods A total of 6 highly sensitized samples (3 samples each for HLA class I and II) were tested by three technologists using labscreen ® Single antigen assay. To ensure uniformity, all experiments were performed according to the manufacturer’s guidelines on the same day using same reagents, centrifuges & Luminex analyzer and reactions were performed on the same position of the tray. Mean, standard deviation (STD), and coefficient variance (CV) was calculated. Results Significant technical variation was observed, where average CV was negatively correlated with the strength of antibody. Higher CV was observed for abs with MFI of 10,000 or more. Differences were significant for HLA Class I abs and showed a trend of significance for HLA class II abs.[ Table 1 ]A total of 60 HLA class I and 83 HLA class II abs were detected. Significantly lower CV was observed in case of HLA class I than HLA class II abs (avg CV = 8.3% and 16.5% respectively), though this was primarily due to the variation in quantification of HLA-DP abs (avg CV = 29%). Conclusions Technical variation in MFI values for different HLA abs was inversely proportional to the strength of the antibody. Our results suggest that contribution from technical variation should be considered in interpreting single antigen assay results especially when a longitudinal study for patient serum is being performed.