The use of polymerase chain reaction to determine fetal RhD status Judith Pratt Rossiter, MD," Karin J. Blakemore, MD,b Thomas S. KickIer, MD; Laura M. Kasch, BS," Adib N. Khouzami, MD,b Eva K. Pressman, MD,b Anthony C. Sciscione, DO,b and Haig H. Kazazian, Jr., MD" Baltimore, Maryland OBJECTIVE: Our purpose was (1) to establish the accuracy of a deoxyribonucleic acid amplification method in determination of RhD status in adult blood samples, including weak D variants (previously referred to as DU) and a D mosaic, and (2) to apply the method to determine fetal RhD status in alloimmunized pregnancies. STUDY DESIGN: Twenty-five adult blood samples, including five weak D variants and one D mosaic, were analyzed with a polymerase chain reaction to determine RhD type. The method was then applied to amniotic fluid samples obtained by amniocentesis from three RhD-negative women with known RhD sensitization. RESULTS: RhD type determined by polymerase chain reaction for all adult blood samples agreed with serologic typing results. All weak D variants and the D mosaic gave results consistent with RhD positivity. Fetal RhD status was determined in each of the three alloimmunized pregnancies, and obstetric management decisions were made on the basis of these results. CONCLUSIONS: This polymerase chain reaction method allows rapid and accurate determinations of fetal RhD status by amniocentesis. Fetal blood sampling or serial amniocenteses may be avoided when the fetus is RhD negative, and plans for surveillance and intervention can be confidently made if the fetus is RhD positive. However, before the widespread use of this assay, its sensitivity and specificity must be established. Because weak D variants and a D mosaic demonstrated RhD-positive status by polymerase chain reaction, the method described is applicable to these RhD variants. (AM J OSSTET GYNECOL 1994;171:1047-51.) Key words: Polymerase chain reaction, Rh alloimmunization, amniocentesis, RhD variants Although the incidence of RhD alloimmunization has greatly decreased with the use of Rh-immune globulin for sensitization prophylaxis, this disorder and the com- plications arising from its antepartum management persist today. A recent study estimated that the inci- dence of Rh-hemolytic disease of the newborn is 10.6 per 10,000 births in the United States. 1 Current man- agement strategies for RhD alloimmunization include serial amniocenteses to evaluate the extent of fetal hemolysis by optical densitometry2 or fetal blood sam- pling to directly determine fetal blood type serologi- cally," or both. The first alternative carries the risk associated with multiple procedures and is limited by the indirect nature of the assay. The second alternative From the Center for Medical Genetics, a the Department of Gynecology and Obstetrics, b and the-Department of Pathology,' The Johns Hopkins University School of Medicine. Presented at the Fourteenth Annual Meeting of the Society of Perinatal Obstetricians, Las Vegas, Nevada, January 24-29, 1994. Reprint requests: Judith Pratt Rossiter, MD, The Center for Medical Genetics, The Johns flopkins Hospital, Blalock 10-08, 600 North Wolfe St., Baltimore, MD 21287. Copyright © 1994 by Mosby-Year Book, Inc. 0002-9378/94 $3.00 + 0 6/6/57828 allows definitive fetal blood typing, but it also involves a I % to 3% risk of procedure-related fetal loss. 4 Over the past several years a great deal of progress has been made in the characterization of the Rh antigens. The Rh D, C, and E antigens were found to be carried by distinct polypeptide chains, 5 which are homologous but nonidentical. 6 Subsequently, the Rh genes have been cloned from complementary deoxyribonucleic acid (cDNA) libraries by several groupS.7-9 There are two genes encoding the Rh antigens. The gene for D, the antigen, is absent in RhD-negative individuals. 9 . 10 It has 92% amino acid homology with the RhCc/Ee gene, which encodes the C/c and E/e antigens. B • 9 Recently, Bennett et al. II reported the use of poly- merase chain reaction (PCR) to determine fetal RhD type from amniotic fluid with the use of two sets of oligonucleotide primers. One pair promotes amplifica- tion of a portion of the RhD gene and the other, a portion of the RhCc/Ee gene, which provides an inter- nal control for the PCR, because all individuals should have a PCR product from the RhCc/Ee gene. Arce et al. 9 described regions in the RhD and RhCc/Ee genes that are homologous, allowing amplification from both 1047