Rapid Rh D genotyping by polymerase chain reaction-based amplification of DNA.
Rh (rhesus) D is the dominant antigen of the Rh blood group system. Recent advances in characterization of the nucleotide sequence of the cDNA(s) encoding the Rh D polypeptide allow the determination of the Rh D genotype at the DNA level. This can be of help in cases in which red blood cells are not available for phenotyping, eg, when in concerns a fetus. We have tested three independent DNA typing methods based on the polymerase chain reaction (PCR) for their suitability to determine the Rh D genotype. DNA derived from peripheral blood mononuclear cells from 234 Rh-phenotyped healthy donors (178 Rh D positive and 56 Rh D negative) was used in the PCR. The Rh D genotypes, as determined with a method based on the allele-specific amplification of the 3' noncoding region of the Rh D gene described by Bennett et al (N Engl J Med 329:607, 1993), were not concordant with the serologically established phenotypes in all cases. We have encountered 5 discrepant results, ie, 3 false-positive and 2 false-negative (a father and child). Rh D genotyping with the second method was performed by PCR amplification of exon 7 of the D gene with allele-specific primers. In all donors phenotyped as D positive tested so far (n = 178), the results of molecular genotyping with this method were concordant with the serologic results, whereas a false-positive result was obtained in one of the D-negative donors (also false-positive in the first method). Complete agreement was found between genotypes determined in the third method, based on a 600-bp deletion in intron 4 of the Rh D gene described by Arce et al (Blood 82:651, 1993), and serologically determined phenotypes. The Rh blood group system is complex, and unknown polymorphisms at the DNA level are expected to exist. Therefore, although genotypes determined by the method of Arce et al were in agreement with serotypes, it cannot yet be regarded as the golden standard. More experience with this or other methods is still needed.
Simsek
S
S
Central Laboratory, The Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Faas
B H
BH
Bleeker
P M
PM
Overbeeke
M A
MA
Cuijpers
H T
HT
van der Schoot
C E
CE
von dem Borne
A E
AE
D000483:Alleles; D001483:Base Sequence; D001788:Blood Grouping and Crossmatching / Q000379:methods*; D015139:Blotting, Southern; D017931:DNA Primers / Q000737:chemistry; D005796:Genes; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012204:Rh-Hr Blood-Group System / Q000235:genetics*; D012703:Serotyping
D017931:DNA Primers; D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen