RHD Gene Detection, Fetal
RHD Gene Detection, Fetal
This test is used to determine fetal Rh D status in Rh D-negative pregnant individuals and assess risk for Rh D alloimmunization-associated hemolytic disease of the fetus and newborn (HDFN).
Test Summary
RHD Gene Detection, Fetal
Test code: 16592
Clinical use
- Determine fetal Rh D status in Rh D–negative pregnant individuals
- Assess risk for Rh D alloimmunization-associated hemolytic disease of the fetus and newborn (HDFN)
Clinical background
Expression of Rh D antigen on fetal red blood cells can promote a maternal immune response from Rh D–negative pregnant individuals resulting in a high risk of HDFN.1 Routine Rh immune globulin (RhIG) prophylaxis in Rh D–negative patients can prevent alloimmunization-associated HDFN, but approximately 40% of these Rh D–negative individuals carry an Rh D–negative fetus who is not at risk.2 Thus, early determination of fetal Rh D status may provide a more accurate assessment of HDFN risk and could reduce the use of prenatal RhIG prophylaxis.
For Rh D–negative pregnant individuals, fetal Rh D status can be determined from paternal RHD genotype; homozygous Rh D–negative paternal Rh D indicates that the fetus is not at risk for HDFN. When the paternal RHD genotype is heterozygous or the paternal specimen is unavailable, fetal Rh D phenotype or RHD genotype is conventionally assessed with cordocentesis, amniocentesis, or chorionic villus sampling (CVS). However, these invasive procedures may cause maternal or fetal hemorrhage, which may exacerbate alloimmunization, result in increased fetal morbidity, or even cause fetal loss.3–5
Genotyping of cell-free DNA (cfDNA) isolated from maternal plasma is a noninvasive alternative methodology and can be performed as early as 10 weeks’ gestation. Since being introduced in 1993, this assay has been refined and achieved a high overall diagnostic performance (sensitivity >99%; specificity >98%) compared with the phenotyping gold standard by serology.6,7 In response to a national shortage of RhIG, the American College of Obstetricians and Gynecologists (ACOG) recommended, when available, paternal RHD genotyping to assess risk for Rh D alloimmunization. ACOG also supported the use of fetal cfDNA as an alternative to amniocentesis to determine fetal Rh D status under a set of well-defined conditions of RhIG scarcity.8
Quest Diagnostics offers RHD Gene Detection screening test, Fetal (test code 16592) for Rh D–negative pregnant individuals to determine the fetal Rh D status when paternal RHD genotype is heterozygous or unavailable. This test is performed on placental cfDNA isolated from a maternal blood specimen. Unlike other cfDNA screening tests for fetal Rh D that are often included as part of a panel evaluation of genetic abnormalities, this is a standalone test. This single-exon (exon 4) amplification approach has demonstrated similar performance (sensitivity >99% and specificity >99%) as multiple-exon approaches in Sweden9–11 but has not been evaluated in the United States, which has a more racially and ethnically heterogeneous population.
RHD genotype varies among populations and does not always correspond to Rh D phenotype (Table). Rh D–negative phenotype is more common in White (15%) than Black (5% to 8%) populations and is rare in Asian populations.2 Among Rh D–negative White individuals, genotype is predominately deletion of the RHD gene. However, among Rh D–negative Black individuals, 54% have deletion of the RHD genes, 24% have an RHD pseudogene (RHDΨ), and up to 22% have an RHD-CE-Ds hybrid gene.12 The latter genotypes have implications for the results of cfDNA screening (see "Interpretive information").
Table. Common RHD Genotypes and Corresponding Phenotypes
Genotype |
Phenotype12 |
2 copies of RHD gene |
Rh D–positive |
1 copy of RHD gene |
Rh D–positive |
0 copies of RHD gene |
Rh D–negative |
1 copy of RHD pseudogene (RHDΨ) |
Rh D–negative |
1 copy of RHD-CE-Ds hybrid gene |
Rh D–negative |
Individuals suitable for testing
- Rh D–negative pregnant individuals after 10 weeks' gestation
Method
- Qualitative real-time polymerase chain reaction amplification and detection RHD exon 4 from cfDNA
- Detection of GAPDH housekeeping gene as an endogenous control
Interpretive information
A “not detected” result for fetal cfDNA isolated from a maternal specimen is predictive of an Rh D–negative phenotype in the fetus. The fetus is considered at reduced risk for HDFN.
A “detected” result for fetal cfDNA isolated from a maternal specimen is predictive of an Rh D–positive phenotype in the fetus. The fetus is considered at increased risk for HDFN.
A “indeterminate” result is rare and may be caused by the presence of unknown or unreported RHD alleles. In these cases, the risk of HDFN for the fetus is not ruled out.10 Submission of a new sample may be considered.
In rare cases, RHD genotypes associated with absence or partial expression of the Rh D antigen (Table) may cause discrepancies between cfDNA screening results and serological phenotypes.7,11 A fetus with a screen-positive result may have an Rh D–negative phenotype because of variants (eg, RHD pseudogene10) not detectable by this assay.
Additional assistance in interpretation of results is available from Quest genomic science specialists at 1.866.GENE.INFO (1.866.436.3463).
References
- American College of Obstetricians and Gynecologists' Committee on Practice Bulletins—Obstetrics. Practice Bulletin No. 181: prevention of Rh D alloimmunization. Obstet Gynecol. 2017;130(2):e57-e70. doi:10.1097/aog.0000000000002232
- American College of Obstetricians and Gynecologists' Committee on Practice Bulletins—Obstetrics. Practice Bulletin No. 192: management of alloimmunization during pregnancy. Obstet Gynecol. 2018;131(3):e82-e90. doi:10.1097/aog.0000000000002528
- Ghidini A, Sepulveda W, Lockwood CJ, et al. Complications of fetal blood sampling. Am J Obstet Gynecol. 1993;168(5):1339-1344. doi:10.1016/s0002-9378(11)90761-3
- Odibo AO, Gray DL, Dicke JM, et al. Revisiting the fetal loss rate after second-trimester genetic amniocentesis. Obstet Gynecol. 2008;111(3):589-595. doi:10.1097/aog.0b013e318162eb53
- Blakemore KJ, Baumgarten A, Schoenfeld-Dimaio M, et al. Rise in maternal serum α-fetoprotein concentration after chorionic villus sampling and the possibility of isoimmunization. Am J Obstet Gynecol. 1986;155(5):988-993. doi:10.1016/0002-9378(86)90332-7
- Lo YMD, Bowell PJ, Selinger M, et al. Prenatal determination of fetal RhD status by analysis of peripheral blood of rhesus negative mothers. Lancet. 1993;341(8853):1147-1148. doi:10.1016/0140-6736(93)93161-s
- Alshehri AA, Jackson DE. Non-invasive prenatal fetal blood group genotype and its application in the management of hemolytic disease of fetus and newborn: systematic review and meta-analysis. Transfus Med Rev. 2021;35(2):85-94. doi:10.1016/j.tmrv.2021.02.001
- ACOG clinical practice update: paternal and fetal genotyping in the management of alloimmunization in pregnancy. Obstet Gynecol. 2024;144(2):e47-e49. doi:10.1097/aog.0000000000005630
- Wikman AT, Tiblad E, Karlsson A, et al. Noninvasive single-exon fetal RHD determination in a routine screening program in early pregnancy. Obstet Gynecol. 2012;120(2, Part 1):227-234. doi:10.1097/aog.0b013e31825d33d9
- Uzunel M, Tiblad E, Mörtberg A, et al. Single-exon approach to non-invasive fetal RHD screening in early pregnancy: an update after 10 years’ experience. Vox Sang. 2022;117(11):1296-1301. doi:10.1111/vox.13348
- Isakson P, Pardi C. Evaluation of an automated platform for non-invasive single-exon fetal RHD genotyping early in pregnancy. Blood Transfus. 2023;21(6):472-478. doi:10.2450/2023.0267-22
- Singleton BK, Green CA, Avent ND, et al. The presence of an RHD pseudogene containing a 37 base pair duplication and a nonsense mutation in Africans with the Rh D–negative blood group phenotype. Blood. 2000;95(1):12-18. doi:10.1182/blood.v95.1.12
Content reviewed 7/2025
This test is used to determine fetal Rh D status in Rh D-negative pregnant individuals and assess risk for Rh D alloimmunization-associated hemolytic disease of the fetus and newborn (HDFN).
Test Summary
RHD Gene Detection, Fetal
Test code: 16592
Clinical use
- Determine fetal Rh D status in Rh D–negative pregnant individuals
- Assess risk for Rh D alloimmunization-associated hemolytic disease of the fetus and newborn (HDFN)
Clinical background
Expression of Rh D antigen on fetal red blood cells can promote a maternal immune response from Rh D–negative pregnant individuals resulting in a high risk of HDFN.1 Routine Rh immune globulin (RhIG) prophylaxis in Rh D–negative patients can prevent alloimmunization-associated HDFN, but approximately 40% of these Rh D–negative individuals carry an Rh D–negative fetus who is not at risk.2 Thus, early determination of fetal Rh D status may provide a more accurate assessment of HDFN risk and could reduce the use of prenatal RhIG prophylaxis.
For Rh D–negative pregnant individuals, fetal Rh D status can be determined from paternal RHD genotype; homozygous Rh D–negative paternal Rh D indicates that the fetus is not at risk for HDFN. When the paternal RHD genotype is heterozygous or the paternal specimen is unavailable, fetal Rh D phenotype or RHD genotype is conventionally assessed with cordocentesis, amniocentesis, or chorionic villus sampling (CVS). However, these invasive procedures may cause maternal or fetal hemorrhage, which may exacerbate alloimmunization, result in increased fetal morbidity, or even cause fetal loss.3–5
Genotyping of cell-free DNA (cfDNA) isolated from maternal plasma is a noninvasive alternative methodology and can be performed as early as 10 weeks’ gestation. Since being introduced in 1993, this assay has been refined and achieved a high overall diagnostic performance (sensitivity >99%; specificity >98%) compared with the phenotyping gold standard by serology.6,7 In response to a national shortage of RhIG, the American College of Obstetricians and Gynecologists (ACOG) recommended, when available, paternal RHD genotyping to assess risk for Rh D alloimmunization. ACOG also supported the use of fetal cfDNA as an alternative to amniocentesis to determine fetal Rh D status under a set of well-defined conditions of RhIG scarcity.8
Quest Diagnostics offers RHD Gene Detection screening test, Fetal (test code 16592) for Rh D–negative pregnant individuals to determine the fetal Rh D status when paternal RHD genotype is heterozygous or unavailable. This test is performed on placental cfDNA isolated from a maternal blood specimen. Unlike other cfDNA screening tests for fetal Rh D that are often included as part of a panel evaluation of genetic abnormalities, this is a standalone test. This single-exon (exon 4) amplification approach has demonstrated similar performance (sensitivity >99% and specificity >99%) as multiple-exon approaches in Sweden9–11 but has not been evaluated in the United States, which has a more racially and ethnically heterogeneous population.
RHD genotype varies among populations and does not always correspond to Rh D phenotype (Table). Rh D–negative phenotype is more common in White (15%) than Black (5% to 8%) populations and is rare in Asian populations.2 Among Rh D–negative White individuals, genotype is predominately deletion of the RHD gene. However, among Rh D–negative Black individuals, 54% have deletion of the RHD genes, 24% have an RHD pseudogene (RHDΨ), and up to 22% have an RHD-CE-Ds hybrid gene.12 The latter genotypes have implications for the results of cfDNA screening (see "Interpretive information").
Table. Common RHD Genotypes and Corresponding Phenotypes
Genotype |
Phenotype12 |
2 copies of RHD gene |
Rh D–positive |
1 copy of RHD gene |
Rh D–positive |
0 copies of RHD gene |
Rh D–negative |
1 copy of RHD pseudogene (RHDΨ) |
Rh D–negative |
1 copy of RHD-CE-Ds hybrid gene |
Rh D–negative |
Individuals suitable for testing
- Rh D–negative pregnant individuals after 10 weeks' gestation
Method
- Qualitative real-time polymerase chain reaction amplification and detection RHD exon 4 from cfDNA
- Detection of GAPDH housekeeping gene as an endogenous control
Interpretive information
A “not detected” result for fetal cfDNA isolated from a maternal specimen is predictive of an Rh D–negative phenotype in the fetus. The fetus is considered at reduced risk for HDFN.
A “detected” result for fetal cfDNA isolated from a maternal specimen is predictive of an Rh D–positive phenotype in the fetus. The fetus is considered at increased risk for HDFN.
A “indeterminate” result is rare and may be caused by the presence of unknown or unreported RHD alleles. In these cases, the risk of HDFN for the fetus is not ruled out.10 Submission of a new sample may be considered.
In rare cases, RHD genotypes associated with absence or partial expression of the Rh D antigen (Table) may cause discrepancies between cfDNA screening results and serological phenotypes.7,11 A fetus with a screen-positive result may have an Rh D–negative phenotype because of variants (eg, RHD pseudogene10) not detectable by this assay.
Additional assistance in interpretation of results is available from Quest genomic science specialists at 1.866.GENE.INFO (1.866.436.3463).
References
- American College of Obstetricians and Gynecologists' Committee on Practice Bulletins—Obstetrics. Practice Bulletin No. 181: prevention of Rh D alloimmunization. Obstet Gynecol. 2017;130(2):e57-e70. doi:10.1097/aog.0000000000002232
- American College of Obstetricians and Gynecologists' Committee on Practice Bulletins—Obstetrics. Practice Bulletin No. 192: management of alloimmunization during pregnancy. Obstet Gynecol. 2018;131(3):e82-e90. doi:10.1097/aog.0000000000002528
- Ghidini A, Sepulveda W, Lockwood CJ, et al. Complications of fetal blood sampling. Am J Obstet Gynecol. 1993;168(5):1339-1344. doi:10.1016/s0002-9378(11)90761-3
- Odibo AO, Gray DL, Dicke JM, et al. Revisiting the fetal loss rate after second-trimester genetic amniocentesis. Obstet Gynecol. 2008;111(3):589-595. doi:10.1097/aog.0b013e318162eb53
- Blakemore KJ, Baumgarten A, Schoenfeld-Dimaio M, et al. Rise in maternal serum α-fetoprotein concentration after chorionic villus sampling and the possibility of isoimmunization. Am J Obstet Gynecol. 1986;155(5):988-993. doi:10.1016/0002-9378(86)90332-7
- Lo YMD, Bowell PJ, Selinger M, et al. Prenatal determination of fetal RhD status by analysis of peripheral blood of rhesus negative mothers. Lancet. 1993;341(8853):1147-1148. doi:10.1016/0140-6736(93)93161-s
- Alshehri AA, Jackson DE. Non-invasive prenatal fetal blood group genotype and its application in the management of hemolytic disease of fetus and newborn: systematic review and meta-analysis. Transfus Med Rev. 2021;35(2):85-94. doi:10.1016/j.tmrv.2021.02.001
- ACOG clinical practice update: paternal and fetal genotyping in the management of alloimmunization in pregnancy. Obstet Gynecol. 2024;144(2):e47-e49. doi:10.1097/aog.0000000000005630
- Wikman AT, Tiblad E, Karlsson A, et al. Noninvasive single-exon fetal RHD determination in a routine screening program in early pregnancy. Obstet Gynecol. 2012;120(2, Part 1):227-234. doi:10.1097/aog.0b013e31825d33d9
- Uzunel M, Tiblad E, Mörtberg A, et al. Single-exon approach to non-invasive fetal RHD screening in early pregnancy: an update after 10 years’ experience. Vox Sang. 2022;117(11):1296-1301. doi:10.1111/vox.13348
- Isakson P, Pardi C. Evaluation of an automated platform for non-invasive single-exon fetal RHD genotyping early in pregnancy. Blood Transfus. 2023;21(6):472-478. doi:10.2450/2023.0267-22
- Singleton BK, Green CA, Avent ND, et al. The presence of an RHD pseudogene containing a 37 base pair duplication and a nonsense mutation in Africans with the Rh D–negative blood group phenotype. Blood. 2000;95(1):12-18. doi:10.1182/blood.v95.1.12
Content reviewed 7/2025