Sensitive determination of the RhD genotype in mixed samples using fluorescence-based polymerase chain reaction.

ABSTRACT

OBJECTIVES: Prenatal determination of the fetal RhD status in pregnancies of Rh-negative (Rh-neg) women by polymerase chain reaction (PCR) on 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, the sensitivity and specificity of PCR-based DNA detection are of crucial importance in the diagnosis of the fetal RhD status. METHODS: We developed 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 also be carried out on DNA samples from persons with weak (Dweak) or partial (Dcat) RhD antigens. CONCLUSION: This method will be valuable in prenatal RhD typing after amniocentesis or after the isolation of fetal cells from maternal peripheral blood.