SENSITIVE DETERMINATION OF THE RHD GENOTYPE IN MIXED SAMPLES USING FLUORESCENCE-BASED POLYMERASE CHAIN-REACTION

Objectives: Prenatal determination of the fetal RhD status in pregnanc ies of Rh-negative (Rh-neg) women by polymerase chain reaction (PCR) o n DNA has become of increasing importance. Since determination of the RhD genotype in these cases is usually performed in samples containing both maternal Rh-neg and fetal Rh-neg or Rh-positive (Rh-pos) DNA, th e sensitivity and specificity of PCR-based DNA detection are of crucia l importance in the diagnosis of the fetal RhD status. Methods: We dev eloped a method for RhD typing using the PCR and a fluorescence-based detection method that allows us to determine RhD-pos DNA even if it is mixed with large amounts of Rh-neg DNA. Results: Using this approach, Rh-pos DNA could be detected in dilutions with Rh-neg DNA of as high as 1 in 10,000 (Rh-pos/Rh-neg). Furthermore PCR amplification could al so be carried out on DNA samples from persons with weak (D-weak) or pa rtial (D-cat) RhD antigens. Conclusion: This method will be valuable i n prenatal RhD typing after amniocentesis or after the isolation of fe tal cells from maternal peripheral blood.