Determination of Fetal RHD Genotype Including the RHD Pseudogene in Maternal Plasma.
OBJECTIVE
To examine the accuracy of fetal RHD genotype and RHD pseudogene determination in a multiethnical population.


METHODS
Prospective study involving D-negative pregnant women. Cell-free DNA was extracted from 1 ml of maternal plasma by an automated system (MagNA Pure Compact, Roche) and real-time PCR was performed in triplicate targeting the RHD gene exons 5 and 7. Inconclusive samples underwent RHD pseudogene testing by real-time PCR analysis employing novel primers and probe.


RESULTS
A positive result was observed in 128/185 (69.2%) samples and negative in 50 (27.0%). Umbilical cord blood phenotype confirmed all cases with a positive or negative PCR result. Seven (3.8%) cases were found inconclusive (exon 7 amplification only) and RHD pseudogene testing with both conventional and real-time PCR demonstrated a positive result in five of them, while two samples were also RHD pseudogene negative.


CONCLUSIONS
Real-time PCR targeting RHD exons 5 and 7 simultaneously in maternal plasma is an accurate method for the diagnosis of fetal D genotype in our population. The RHD pseudogene real-time PCR assay is feasible and is particularly useful in populations with a high prevalence of this allele.
Fundação Pró-Sangue/Hemocentro de São Paulo, São Paulo, Brazil.
Karen Chinoca
KC
Ziza
Department of Obstetrics and Gynecology, Hospital das Clínicas, São Paulo University Medical School, São Paulo, Brazil.
Adolfo Wenjaw
AW
Liao
Fundação Pró-Sangue/Hemocentro de São Paulo, São Paulo, Brazil.
Marcia
M
Dezan
Fundação Pró-Sangue/Hemocentro de São Paulo, São Paulo, Brazil.
Carla Luana
CL
Dinardo
Fundação Pró-Sangue/Hemocentro de São Paulo, São Paulo, Brazil.
Eduardo
E
Jens
Department of Obstetrics and Gynecology, Hospital das Clínicas, São Paulo University Medical School, São Paulo, Brazil.
Rossana Pulcineli Vieira
RPV
Francisco
Fundação Pró-Sangue/Hemocentro de São Paulo, São Paulo, Brazil.
Alfredo Mendrone
AM
Junior
Department of Obstetrics and Gynecology, Hospital das Clínicas, São Paulo University Medical School, São Paulo, Brazil.
Marcelo
M
Zugaib
Fundação Pró-Sangue/Hemocentro de São Paulo, São Paulo, Brazil.
José Eduardo
JE
Levi
D000328:Adult; D004247:DNA / Q000097:blood / Q000737:chemistry / Q000235:genetics; D005260:Female; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*; D011446:Prospective Studies; D011544:Pseudogenes / Q000235:genetics*; D012204:Rh-Hr Blood-Group System / Q000235:genetics*; D012680:Sensitivity and Specificity; D055815:Young Adult
D012204:Rh-Hr Blood-Group System; D004247:DNA
DNA/analysis; RhD Blood group system; genotyping techniques; prenatal diagnosis/methods; sensitivity and specificity