BACKGROUND: When a pregnant woman has an antibody with the potential to cause hemolytic disease of the fetus and newborn, it is beneficial to determine whether her fetus has the corresponding antigen to assess risk. In many countries this is now done routinely for RhD, by testing cell-free fetal DNA in the maternal plasma. Similar tests for K, C, c, and E are reported. STUDY DESIGN AND METHODS: Real-time quantitative polymerase chain reaction incorporating an allele-specific primer was developed for detecting the K allele of KEL and the C, c, and E alleles of RHCE. These methods were used to test DNA isolated from plasma of pregnant women with antibodies to K, C, c, or E. Accuracy of the tests was determined by comparing results with serologic tests performed on cord red blood cells (RBCs) after delivery or by molecular genotyping on DNA obtained from fetal cells. RESULTS: The K test incorporated an allele-specific primer with two locked nucleic acids and a mismatch. In 70 tests, including 27 K+ fetuses, only one false-negative and no false-positive results were obtained. The C, c, and E tests, performed on 13, 44, and 46 samples, respectively, gave rise to no false results. CONCLUSION: Reliable methods have been developed for predicting fetal K, C, c, and E phenotypes, by testing fetal DNA in the plasma samples of pregnant women whose RBCs lack the corresponding antigens. These methods are now being used routinely in a diagnostic service in the United Kingdom.
BACKGROUND: When a pregnant woman has an antibody with the potential to cause hemolytic disease of the fetus and newborn, it is beneficial to determine whether her fetus has the corresponding antigen to assess risk. In many countries this is now done routinely for RhD, by testing cell-free fetal DNA in the maternal plasma. Similar tests for K, C, c, and E are reported. STUDY DESIGN AND METHODS: Real-time quantitative polymerase chain reaction incorporating an allele-specific primer was developed for detecting the K allele of KEL and the C, c, and E alleles of RHCE. These methods were used to test DNA isolated from plasma of pregnant women with antibodies to K, C, c, or E. Accuracy of the tests was determined by comparing results with serologic tests performed on cord red blood cells (RBCs) after delivery or by molecular genotyping on DNA obtained from fetal cells. RESULTS: The K test incorporated an allele-specific primer with two locked nucleic acids and a mismatch. In 70 tests, including 27 K+ fetuses, only one false-negative and no false-positive results were obtained. The C, c, and E tests, performed on 13, 44, and 46 samples, respectively, gave rise to no false results. CONCLUSION: Reliable methods have been developed for predicting fetal K, C, c, and E phenotypes, by testing fetal DNA in the plasma samples of pregnant women whose RBCs lack the corresponding antigens. These methods are now being used routinely in a diagnostic service in the United Kingdom.