Bioingeniøren (Mar 2014)
RHD genotyping by allele specific “Loop-mediated isothermal DNA Amplification”
Abstract
Background: The “Rh” cluster, with its main antigen D, is the most important blood group system after “ABO”. Because anti-D antibodies can cause hemolytic disease of the fetus and newborn as well as hemolytic transfusion reactions, phenotyping for RhD is routinely used in every immunohematology laboratory. As it is not always possible to determine RhD antigens by serology alone, several RHD genotyping methods have been developed; these, however, necessitate advanced equipment and specially trained operators. The aim of this study is to introduce loop mediated isothermal amplification (LAMP) as a robust, fast and simple method for RHD genotyping. Material and methods: Fifteen persons were feno- and genotyped for RhD by standard serology and our proprietary LAMP protocol, using isolated DNA and cheek cells, respectively. Analytic sensitivity and specificity was determined by serial dilutions of solutions containing RHD DNA and RHD/RHCE DNA. Results and conclusion: LAMP is highly sensitive; it detects as little as 9.6 pg RHD positive DNA per reaction. There is complete agreement between genotypes and phenotypes, while it is possible to specifically detect 0.4 percent RHD DNA in a background of RHCE DNA. This shows that LAMP may be capable of detecting cell free fetal RHD positive DNA in plasma from RhD negative pregnant women. Clinical evaluation of LAMP RHD genotyping, however, is pending to date. The method also works with heat treated epithelia cells as template, which demonstrates that the method does not require purified DNA for successful amplification.
