title: Rh genotyping: avoiding false-negative and false-positive results among individuals of African ancestry.
abstract: High homology, variant alleles, and silent alleles have made the development of completely reliable genotyping assays for the RHD and RHC alleles difficult. An RHD pseudogene (RHDPsi) possessing a 37-bp insertion within exon 4 is common among serologically RhD-negative individuals of African descent and generates false-positive results in previously reported RhD genotyping assays. Genotyping RhC is problematic due to exon 2 homology between RHD and RHC; however, an RHC-specific 109-bp insertion within intron 2 has been reported useful for genotyping. Primers flanking the exon 4 insertion point were used for detection of RHD and RHDPsi among a total of 231 serotyped individuals: 134 African American, 85 Caucasian, and 12 RhD serotype-negative/genotype-positive, D-sensitized women. Primers flanking the RHC-specific intron 2 insertion were used to genotype 282 serotyped individuals (128 African American, 154 Caucasian) and were compared to RHC genotyping using the exon 1 RhC-specific nt48 cytosine polymorphism. Complete correlation was observed between genotyping with the RHDPsi primer pair and serotyping among 219 individuals and 10/12 previous RHD false-positive genotyping results were resolved. RHDPsi was detected in 19% (n = 4/21) of RhD seronegative African Americans and 4.4% (n = 5/113) of RhD seropositive African Americans. When using the 109-bp intron 2 insertion for genotyping of RHC, a 23.9% (n = 11/46) false-negative rate was observed among African American RhCc serotyped heterozygotes. Utilization of the exon 1 nt48 cytosine for indirect genotyping of RHC yielded a 7.2% (n = 4/55) and 56.3% (n = 45/80) false-positive rate among Rhcc Caucasians and African Americans, respectively. We conclude that these additional reactions, though not sufficient alone, can be useful supplements to existing Rh genotyping assays.
authors.affiliation: Diagnostic Laboratories, The Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin, USA.
authors.forename: G C
authors.initials: GC
authors.lastname: Ekman
authors.forename: R
authors.initials: R
authors.lastname: Billingsly
authors.forename: M J
authors.initials: MJ
authors.lastname: Hessner
mesh_terms: D000483:Alleles; D000906:Antibodies / Q000097:blood; D044383:Black People; D003596:Cytosine; D004247:DNA / Q000097:blood; D005091:Exons; D005188:False Negative Reactions; D005189:False Positive Reactions; D005260:Female; D005787:Gene Frequency; D005838:Genotype; D006801:Humans; D016130:Immunophenotyping; D007438:Introns; D011247:Pregnancy; D012203:Rh Isoimmunization / Q000235:genetics; D012204:Rh-Hr Blood-Group System / Q000235:genetics*; D001741:Black or African American
chemical_list: D000906:Antibodies; D012204:Rh-Hr Blood-Group System; D003596:Cytosine; D004247:DNA