Development of Real-time PCR and Melting Curve Analysis for the Rapid Detection of DEL with RHD (c.1222T>C) or RHD (c.1227G>A).

BACKGROUND: DEL, a variant of RhD, is difficult to detect in routine blood bank testing owing to its extremely low levels of D antigen expression. However, DEL is capable of alloimmunizing a patient when transfused into RhDnegative individuals.
METHODS: In this study, we developed real-time PCR and melting curve analysis for the rapid detection of the DEL phenotype.
RESULTS: Of the 325 serologically RhD-negative individuals involved in the study, 56 (17.2%) had melting temperatures distinguishable from complete RHD absence as follows: 53 RHD (c.1227G>A) DEL specimens had a plateau at 54 - 56°C and a peak at 61.95°C, while 3 RHD (c.1222T>C) DEL had a melting temperature of 62.62°C. All DEL results were identical to those obtained by multiplex single-base primer extension reactions.
CONCLUSIONS: The rapid DEL genotyping method developed in this study will be useful for screening DEL in serologically RhD-negative donors and preventing the alloimmunization of RhD-negative individuals by DEL.