RHD GENOTYPE DETERMINATION BY SINGLE SPERM CELL ANALYSIS

OBJECTIVE: An Rh-negative woman with preexisting anti-D antibodies may affect some or all subsequent fetuses, depending on the genotype of h er Rh-positive partner. Currently, a reliable technique for an absolut e determination of RhD genotype is not available. This study was initi ated to develop an accurate method for RhD genotyping in men. STUDY DE SIGN: RhD genotype was determined by deoxyribonucleic acid amplificati on of a D-specific sequence in single sperm cell samples. Micromanipul ation techniques were used for sampling of single sperm cells, which w ere further amplified by multiplex nested polymerase chain reaction at the RhD locus. A RhD sequence amplification product was expected in a ll of the successfully amplified samples from Rh-positive homozygotes, in some of the samples from heterozygotes, and in none of the samples form Rh-negative subjects. RESULTS: RhD genotype was accurately deter mined in 10 of 10 donors, A total of 132 single sperm cells were analy zed (8 to 17 samples per donor), of which 96 were successfully amplifi ed as assessed by an internal control. As expected, the specific regio n of the RhD gene was amplified in all, some, and none of the signal-p ositive sperm samples from Rh-positive homozygotes, heterozygotes, and Rh-negative subjects, respectively, allowing accurate determination o f the genotype. CONCLUSION: An accurate diagnosis of the RhD genotype can be attained from single sperm cell analysis by means of polymerase chain reaction and may have major clinical applications in the manage ment of Rh isoimmunization.