ObjectiveTo determine the fetal Rhcgenotype by using the polymerase chain reaction (PCR) amplification procedure and maternal blood at the different steps of the fetal cell enrichment process.MethodsMaternal peripheral venous blood samples were obtained from 11 pregnant women homozygous for the C antigen before amniocentesis. Three were not alloimmunized and eight were alloimmunized. The fathers were known to be heterozygous or homozygous for the c antigen by serologic testing. The mononuclear cell layer was isolated from maternal blood and flow sorted using monoclonal antibodies to CD36 or CD71 and glycophorin A. This was followed by PCR of the blood, mononuclear cells, and the sorted cells with allele-specific primers to RhCcgenes. Gel electrophoresis was performed to predict fetal Rhcgenotype. The fetal RhCcgenotype was confirmed by serologic and DNA testing.ResultsAll infants were positive for the Rhcgene. The positive fetal Rhcgenotype was determined correctly in three of the 11 maternal blood samples without enrichment, in six of the nine mononuclear cell samples, and in seven of the eight sorted cell samples. The fetal genotype from one sorted sample was predicted to be homozygousC. One infant was determined by serology on cord blood to be negative for the c antigen, but repeated infant DNA amplification was consistent with thecgenotype.ConclusionNoninvasive fetal Rhcgenotyping can be determined by PCR amplification of the rare fetal cells in maternal blood. These data reaffirm that enrichment of maternal blood for fetal cells is necessary to improve the sensitivity of the test.