Medical Policy

Policy Num:       11.003.014
Policy Name:     Noninvasive Fetal RHD Genotyping Using Cell-Free Fetal DNA

Policy ID:           [11.003.014] [Ac / B / M- / P-] [2.04.108]

Last Review:       September 12, 2024
Next Review:       September 20, 2025
 

Related Policies: None

Noninvasive Fetal RHD Genotyping Using Cell-Free Fetal DNA

Summary

Rhesus D (RhD)-negative women who are exposed to RhD-positive red blood cells can develop anti-RhD antibodies, which can cross the placenta and cause fetal anemia. If undiagnosed and untreated, alloimmunization can cause significant perinatal morbidity and mortality. Determining the RhD status of the fetus may guide subsequent management of the pregnancy. Hence, the use of cell-free fetal DNA in maternal blood has been proposed as a noninvasive method to determine fetal RHD genotype.

Summary of Evidence

For individuals who are pregnant and have RhD-negative blood type who receive noninvasive RHD genotyping of the fetus using cell-free DNA from maternal plasma, the evidence includes a meta-analysis and additional prospective studies (for clinical validity) and no direct evidence for clinical utility. Relevant outcomes are test validity, morbid events, medication use, and treatment-related morbidity. Clinical validity studies have demonstrated that the sensitivity and specificity of the test are high; however, the false-negative test rate, which is low, is not zero, potentially leading to alloimmunization of the RhD-negative mothers in these cases. It is uncertain whether RHD genotyping using cell-free fetal DNA will lead to improved health outcomes. The evidence is insufficient to determine the effects of the technology on health outcomes.

Additional Information

Not applicable.

Objective

The objective of this evidence review is to evaluate whether noninvasive fetal RHD genotyping using cell-free fetal DNA improves the net health outcome in individuals who are pregnant and have Rhesus D-negative blood type.

policy Statements

Noninvasive fetal RHD genotyping using cell-free fetal DNA is considered investigational.

 Policy Guidelines

Genetics Nomenclature Update

The Human Genome Variation Society nomenclature is used to report information on variants found in DNA and serves as an international standard in DNA diagnostics. It is being implemented for genetic testing medical evidence review updates starting in 2017 (see Table PG1). The Society's nomenclature is recommended by the Human Variome Project, the HUman Genome Organization, and by the Human Genome Variation Society itself.

The American College of Medical Genetics and Genomics and the Association for Molecular Pathology standards and guidelines for interpretation of sequence variants represent expert opinion from both organizations, in addition to the College of American Pathologists. These recommendations primarily apply to genetic tests used in clinical laboratories, including genotyping, single genes, panels, exomes, and genomes. Table PG2 shows the recommended standard terminology-"pathogenic," "likely pathogenic," "uncertain significance," "likely benign," and "benign"-to describe variants identified that cause Mendelian disorders.

Table PG1. Nomenclature to Report on Variants Found in DNA

Table PG2. ACMG-AMP Standards and Guidelines for Variant Classification

American College of Medical Genetics and Genomics; AMP: Association for Molecular Pathology.

Coding

Please see the Codes table for details.

Benefit Application

BlueCard/National Account Issues

Some plans may have contract or benefit exclusions for genetic testing.

Benefits are determined by the group contract, member benefit booklet, and/or individual subscriber certificate in effect at the time services were rendered. Benefit products or negotiated coverages may have all or some of the services discussed in this medical policy excluded from their coverage.

Background

Alloimmunization

Alloimmunization refers to the development of antibodies in a patient whose blood type is Rhesus D (RhD)-negative and who is exposed to RhD-positive red blood cells (RBCs). This most commonly occurs from fetal-placental hemorrhage and entry of fetal blood cells into the maternal circulation. The management of an RhD-negative pregnant patient who is not alloimmunized and is carrying a known RhD-positive fetus or a fetus whose RhD status is unknown involves administration of RhD immunoglobulin during pregnancy to prevent the formation of anti-RhD antibodies. If the patient is already alloimmunized, monitoring the levels of anti-RhD antibody titers for the development of fetal anemia is performed. Noninvasive and invasive tests to determine fetal RhD status exist.

Rhesus Blood Groups

The Rhesus (Rh) system includes more than 100 antigen varieties found on RBCs. Rhesus D is the most common and the most immunogenic. When people have the RhD antigen on their RBCs, they are considered to be RhD-positive; if their RBCs lack the antigen, they are considered to be RhD-negative. The RhD antigen is inherited in an autosomally dominant fashion, and a person may be heterozygous (Dd; approximately 60% of RhD-positive people) or homozygous (DD; approximately 40% of RhD-positive people). Homozygotes always pass the RhD antigen to their offspring, whereas heterozygotes have a 50% chance of passing the antigen to their offspring. A person who is RhD-negative does not have the Rh antigen. Although nomenclature refers to RhD-negative as dd, there is no small d antigen (ie, they lack the RHD gene and the corresponding RhD antigen).

Rhesus D-negative status varies across ethnic groups and is 15% in White populations, 5% to 8% in Black populations, and 1% to 2% in Asians and Native Americans.

In the White population, almost all RhD-negative individuals are homozygous for a deletion of the RHD gene. However, in Black populations, only 18% of RhD-negative individuals are homozygous for an RHD deletion, and 66% of RhD-negative Black individuals have an inactive RHD pseudogene (RHDy).^1, There are also numerous rare variants of the D antigen, which are recognized by weakness of expression of D and/or by the absence of some of the epitopes of D. Some individuals with variant D antigens can make antibodies to 1 or more epitopes of the D antigen if exposed to RhD-positive RBCs.^1,

Rhesus D-negative women can have a fetus that is RhD-positive if the fetus inherits the RhD-positive antigen from the paternal father.

Causes of Alloimmunization

By 30 days of gestation, the RhD antigen is expressed on the RBC membrane, and alloimmunization can occur when fetal RhD-positive RBCs enter maternal circulation and the RhD-negative mother develops anti-D antibodies.^2, Once anti-D antibodies are present in a pregnant woman's circulation, they can cross the placenta and destroy fetal RBCs.

The production of anti-D antibodies in RhD-negative women is highly variable and significantly affected by several factors, including the volume of fetomaternal hemorrhage, the degree of the maternal immune response, concurrent ABO incompatibility, and fetal homozygosity versus heterozygosity for the D antigen. Therefore, although about 10% of pregnancies are RhD-incompatible, less than 20% of RhD-incompatible pregnancies actually lead to maternal alloimmunization.

Small fetomaternal hemorrhages of RhD-positive fetal RBCs into the circulation of an RhD-negative woman occur in nearly all pregnancies, and incidence of fetomaternal hemorrhage increases as the pregnancy progresses: 7% in the first trimester, 16% in the second trimester, and 29% in the third trimester, with the greatest risk of RhD alloimmunization occurring at birth (15 to 50%). Transplacental hemorrhage accounts for almost all cases of maternal RhD alloimmunization.

Fetomaternal hemorrhage can also be associated with miscarriage, pregnancy termination, ectopic pregnancy, invasive in utero procedures (eg, amniocentesis), in utero fetal death, maternal abdominal trauma, antepartum maternal hemorrhage, and external cephalic version. Other causes of alloimmunization include inadvertent transfusion of RhD-positive blood and RhD-mismatched allogeneic hematopoietic cell transplantation.

Consequences of Alloimmunization

Immunoglobulin G antibody-mediated hemolysis of fetal RBCs, known as hemolytic disease of the fetus and newborn, varies in severity and manifestations. The anemia can range from mild to severe, with associated hyperbilirubinemia and jaundice. In severe cases, hemolysis may lead to extramedullary hematopoiesis and reticuloendothelial clearance of fetal RBCs, which may result in hepatosplenomegaly, decreased liver function, hypoproteinemia, ascites, and anasarca. When accompanied by high-output cardiac failure and pericardial effusion, this condition is known as hydrops fetalis, which without intervention, is often fatal. Intensive neonatal care, including emergent exchange transfusion, is required.

Cases of hemolysis in the newborn that do not result in fetal hydrops can still lead to kernicterus, a neurologic condition observed in infants with severe hyperbilirubinemia due to the deposition of unconjugated bilirubin in the brain. Symptoms that manifest several days after delivery can include poor feeding, inactivity, loss of the Moro reflex, bulging fontanelle, and seizures. The 10% of infants who survive may develop spastic choreoathetosis, deafness, and/or mental retardation.

Hemolytic disease in the fetus or newborn was once a major contributor to perinatal morbidity and mortality. However, the widespread adoption of antenatal and postpartum use of RhD immunoglobulin in developed countries resulted in a major decrease in the frequency of this disease. In developing countries without prophylaxis programs, stillbirth occurs in 14% of affected pregnancies, and 50% of pregnancy survivors either die in the neonatal period or develop a cerebral injury.^3,

Prevention of Alloimmunization

There are 4 RhD immunoglobulin products available in the U.S., all of which undergo micropore filtration to eliminate viral transmission.^3, To date, no reported cases of viral infection related to RhD immunoglobulin administration have been reported in the U.S.^3, Theoretically, the Creutzfeldt-Jakob disease agent could be transmitted by the use of RhD immunoglobulin. Local adverse reactions may occur, including redness, swelling, and mild pain at the site of injection, and hypersensitivity reactions.

The American College of Obstetricians and Gynecologists and the American Association of Blood Banks have recommended the first dose of Rh_o(D) immunoglobulin (eg, RhoGAM) be given at 28 weeks of gestation (or earlier if there's been an invasive event), followed by a postpartum dose given within 72 hours of delivery.

Diagnosis of Alloimmunization

The diagnosis of alloimmunization is based on detection of anti-RhD antibodies in the maternal serum. The most common test for determining antibodies in serum is the indirect Coombs test.^3, The maternal serum is incubated with known RhD-positive RBCs. Any anti-RhD antibody present in the maternal serum will adhere to the RBCs. The RBCs are then washed and suspended in Coombs serum, which is antihuman globulin. Red blood cells coated with maternal anti-RhD will agglutinate, which is referred to as a positive indirect Coombs test. The indirect Coombs titer is the value used to direct management of pregnant alloimmunized women.

Management of Alloimmunization During Pregnancy

A patient's first alloimmunized pregnancy involves minimal fetal or neonatal disease. Subsequent pregnancies are associated with more severe degrees of fetal anemia. Treatment of an alloimmunized pregnancy requires monitoring maternal anti-D antibody titers and serial ultrasound assessment of middle cerebral artery peak systolic velocity of the fetus.

If severe fetal anemia is present near term, delivery is performed. If severe anemia is detected remote from term, intrauterine fetal blood transfusions may be performed.

Determining Fetal Rhesus D Status

The American College of Obstetrician and Gynecologists has recommended that all pregnant women be tested during their first prenatal visit for ABO blood group typing and RhD type, and be screened for the presence of anti-RBC antibodies. These laboratory tests should be repeated for each subsequent pregnancy. The American Association of Blood Banks has also recommended that antibody screening be repeated before administration of anti-D immunoglobulin at 28 weeks of gestation, postpartum, and at the time of any event during pregnancy.

If the mother is determined to be RhD-negative, the paternal RhD status should also be determined at the initial management of a pregnancy. If paternity is certain and the father is RhD-negative, the fetus will be RhD-negative, and further assessment and intervention are unnecessary. If the father is RhD-positive, he can be either homozygous or heterozygous for the D allele. If homozygous for the D allele (ie, D/D), then the fetus is RhD-positive. If the paternal genotype is heterozygous for Rh status or is unknown, determination of the RhD status of the fetus is the next step to assess the RhD compatibility of the pregnancy (first or any subsequent pregnancy).

Invasive and noninvasive testing methods to determine the RhD status of a fetus are available. These procedures use polymerase chain reaction assays to assess the fetal cellular elements in amniotic fluid by amniocentesis or chorionic villus sampling (CVS). Although CVS can be performed earlier in a pregnancy, amniocentesis is preferred because CVS is associated with disruption of the villi and the potential for larger fetomaternal hemorrhage and worsening alloimmunization if the fetus is RhD-positive. The sensitivity and specificity of fetal RHD genotyping by polymerase chain reaction are reported as 98.7% and 100%, respectively, with positive and negative predictive values of 100% and 96.9%, respectively.^4,

Noninvasive testing involves molecular analysis of cell-free fetal DNA (cffDNA) in the maternal plasma or serum. Lo et al (1998) showed that about 3% of cffDNA in the plasma of first-trimester pregnant women is of fetal origin, with this percentage rising to 6% in the third trimester.^5, Fetal DNA cannot be separated from maternal DNA, but if the pregnant woman is RhD-negative, the presence of specific exons of the RHD gene, which are not normally present in the circulation of an RhD-negative patient, predicts an RhD-positive fetus. The use of cffDNA has been proposed as a noninvasive alternative to obtaining fetal tissue by invasive methods, which are associated with a risk of miscarriage.^1,

The large quantity of maternal DNA compared with fetal DNA in the maternal circulation complicates the inclusion of satisfactory internal controls to test for successful amplification of fetal DNA. Therefore, reactions to detect Y chromosome-linked gene(s) can be included in the test, which will be positive when the fetus is a male.^1, When Y chromosome-linked genes are not detected, tests for variants may be performed to determine whether the result is derived from fetal not maternal DNA.

Use of cffDNA testing to determine the fetal RHD genotype is the standard of care in many European countries.^3,

Regulatory Status

Clinical laboratories may develop and validate tests in-house and market them as a laboratory service; laboratory-developed tests must meet the general regulatory standards of the Clinical Laboratory Improvement Amendments. Laboratories that offer laboratory-developed tests must be licensed by the Clinical Laboratory Improvement Amendments for high-complexity testing. To date, the U.S. Food and Drug Administration has chosen not to require any regulatory review of this test.

Sequenom offers the SensiGene™ Fetal RHD Genotyping test, performed by proprietary SEQureDx™ technology. The assay targets exons 4, 5, and 7 of the RHD gene located on chromosome 1, psi (ψ) pseudogene in exon 4, and assay controls, which are 3 targets on the Y chromosome (SRY, TTTY, DBY) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based nucleic acid analysis. The company claims that uses of its test include:

•         Clarifying fetal RhD status without testing the father, thereby avoiding the cost of paternity testing and paternal genotyping

•         Clarifying fetalRhDstatus when maternal anti-D titers are unclear

•         Identifying the RhD-negative fetus in mothers who are opposed to immunization(s) and vaccines

•         Identifying RhD-negative sensitized patients

•         Avoiding invasive testing by CVS or genetic amniocentesis.

Another noninvasive RhD test is the Unity Screen™ test from BillionToOne. In addition to testing for RhD, the test evaluates the C, c, D, E, Fy^a, and K antigens, aneuploidy, and recessive conditions including cystic fibrosis, spinal muscular atrophy, sickle cell disease, alpha and beta thalassemia, and fragile X syndrome.

Rationale

This evidence review was created in November 2013 and has been updated regularly with searches of the PubMed database. The most recent literature update was performed through July 1, 2024.

Evidence reviews assess whether a medical test is clinically useful. A useful test provides information to make a clinical management decision that improves the net health outcome. That is, the balance of benefits and harms is better when the test is used to manage the condition than when another test or no test is used to manage the condition.

The first step in assessing a medical test is to formulate the clinical context and purpose of the test. The test must be technically reliable, clinically valid, and clinically useful for that purpose. Evidence reviews assess the evidence on whether a test is clinically valid and clinically useful. Technical reliability is outside the scope of these reviews, and credible information on technical reliability is available from other sources.

Promotion of greater diversity and inclusion in clinical research of historically marginalized groups (e.g., People of Color [African-American, Asian, Black, Latino and Native American]; LGBTQIA (Lesbian, Gay, Bisexual, Transgender, Queer, Intersex, Asexual); Women; and People with Disabilities [Physical and Invisible]) allows policy populations to be more reflective of and findings more applicable to our diverse members. While we also strive to use inclusive language related to these groups in our policies, use of gender-specific nouns (e.g., women, men, sisters, etc.) will continue when reflective of language used in publications describing study populations.

Population Reference No. 1

Testing Pregnant Women with Rhesus D-negative Blood Type

Clinical Context and Test Purpose

The purpose of genetic testing of individuals who are pregnant and have Rhesus D (RhD)-negative blood type is to determine the RhD status of the fetus to guide pregnancy management, including avoidance of invasive testing (chorionic villus sampling or amniocentesis) and administration of anti-D immunoglobulin.

The questions addressed in this evidence review include:

• Does RHD genotyping reduce the need for invasive testing by chorionic villus sampling or amniocentesis?

• Does RHD genotyping guide the administration of anti-D immunoglobulin during pregnancy?

• Does RHD genotyping lead to improved pregnancy outcomes?

The following PICO was used to select literature to inform this review.

Populations

The relevant population of interest includes individuals who are pregnant and have an RhD-negative blood type.

Interventions

The test being considered is noninvasive RHD genotyping of the fetus using cell-free DNA from maternal plasma.

Comparators

The following practices are currently being used: invasive methods to determine fetal Rhesus (Rh) status and management based on maternal RhD status.

Outcomes

The general outcomes of interest are test validity, morbid events, medication use, and treatment-related morbidity. The potential beneficial outcomes of primary interest are the avoidance of invasive testing (chorionic villus sampling or amniocentesis) and avoidance of unnecessary administration of RhD immunoglobulin.

Potentially harmful outcomes are those resulting from false-positive or false-negative test results. False-positive test results can lead to unnecessary administration of RhD immunoglobulins during pregnancy. False-negative test results can lead to lack of RhD immunoglobulin administration, development of maternal alloimmunization to RhD, and current and future pregnancy complications due to maternal alloantibodies to RhD.

Outcomes may be measured at various times. During a first pregnancy, testing may be conducted to detect the development of maternal alloimmunization to RhD and minimal-to-mild fetal or neonatal disease. In subsequent pregnancies, testing may be conducted to detect pregnancy complications due to maternal alloimmunization to RhD and potentially severe fetal or neonatal hemolytic anemia.

Study Selection Criteria

For the evaluation of clinical validity, studies that meet the following eligibility criteria were considered:

• Reported on the accuracy of the marketed version of the technology

• Included a suitable reference standard

• Patient/sample clinical characteristics were described

• Patient/sample selection criteria were described.

Clinically Valid

A test must detect the presence or absence of a condition, the risk of developing a condition in the future, or treatment response (beneficial or adverse).

Review of Evidence

Systematic Reviews

Zhu et al (2014) published a meta-analysis of studies on the diagnostic accuracy of noninvasive fetal RHD genotyping using cell-free fetal DNA (cffDNA).^6, Reviewers identified 37 studies conducted in RhD-negative pregnant women that had been published by the end of 2013. The studies included 11129 samples, and 352 inconclusive samples were excluded. When all data were pooled, the sensitivity of fetal RHD genotyping was 99% and the specificity was 98%. Diagnostic accuracy was higher in samples collected in the first trimester (99.0%) than in those collected in the second (98.3%) or third (96.4%) trimesters.

Observational Studies

Chitty et al (2014)^7, published a prospective study from the U.K. that was not included in the Zhu et al (2014) meta-analysis. Samples from 2288 RhD-negative women who initiated prenatal care before 24 weeks of gestation were analyzed using RHD genotyping. Overall, the sensitivity of the test was 99.34% and the specificity was 94.91%. The likelihood of correctly detecting RhD status in the fetus increased with gestational age, with high levels of accuracy after 11 weeks. In samples taken before 11 completed weeks of gestation, the sensitivity was 96.85% and the specificity was 94.40%; at 14 to 17 weeks of gestation, the sensitivity was 99.67% and specificity was 95.34%. These findings of increased diagnostic accuracy as pregnancies advanced differ from those of the Zhu et al (2014) meta-analysis, which found the highest diagnostic accuracy in the first trimester.

Two key studies reporting on the clinical validity of fetal RHD genotyping with the Sequenom assay, which is commercially available in the U.S., are detailed next, and findings are summarized in Table 1.

Moise et al (2012) analyzed samples from 120 patients enrolled prospectively from multiple centers.^3, All were RhD-negative pregnant patients with no evidence of alloimmunization. The samples were analyzed using the SensiGene Fetal RHD test using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to detect control and fetal-specific DNA signals. The determination of fetal sex was defined as follows: 3 Y chromosome markers is a male fetus, 2 markers are inconclusive, and 1 or no marker is a female fetus. The algorithm for RHD determination was defined as follows: pseudogene present is inconclusive, 3 RHD markers present is an RHD-positive fetus, 2 markers present is inconclusive, 1 or no marker is an RHD-negative fetus. If the results were RHD-positive and male, the fetus was determined to be RHD-positive and male, and if RHD-negative and male results were noted, the fetus was determined to be RHD-negative and male. If the results were RHD-positive and female, the fetus was determined to be RHD-positive and female. If an RHD-negative and female result was noted, reflex testing was performed with a panel of 92 single nucleotide variants. If a minimum of 6 informative paternal alleles (uniquely and unambiguously fetal in nature) were detected, the result was an RHD-negative, female fetus. If fewer than 6 alleles were detected, the sample was reported as inconclusive. Cord blood was obtained at delivery and RhD typing was determined using standard serologic methods. Phenotype assessment of the newborns was used to assign sex. The pregnant patients underwent planned venipunctures during 3 time periods in gestation: 11 to 13, 16 to 19, and 28 to 29 weeks. At the second blood draw, 2 patients were not evaluated because they did not return during the prescribed gestational age window; and at the time of the third-trimester blood draw, 7 patients did not have a sample obtained.

Median gestational ages of the first-, second-, and third-trimester samplings were 12.4 weeks (range, 10.6 to 13.9 weeks), 17.6 weeks (range, 16 to 20.9 weeks), and 28.7 weeks (range, 27.9 to 33.9 weeks), respectively. Three samples in the first trimester and 2 in the second trimester were insufficient in quantity to perform the DNA assay (1.4% of the total samples). Twenty-two samples (6.3% of the total samples; 2.5% of the patients) were deemed inconclusive. In 23% of these inclusive cases, there was an RHD-negative, female result, but an insufficient number of paternal single nucleotide variants detected to confirm the presence of fetal DNA. In the remaining 77% of the inconclusive results (4.8% of the total samples), the RHD pseudogene (RHDy) was detected, and the sample was deemed inconclusive. Erroneous results were observed for 6 (1.7%) of the samples, and included discrepancies in 4 (1.1%) RHD genotyping tests and 2 (0.6%) fetal sex determinations following data unblinding. Three cases of RhD typing were false-positives (cffDNA was RHD-positive but neonatal serology RhD-negative) and one case was a false-negative (cffDNA was RHD-negative but neonatal serology RhD-positive). Accuracy for determination of the RHD status of the fetus was 99.1%, 99.1%, and 98.1%, respectively for each of the 3 consecutive trimesters of pregnancy, and accuracy of fetal sex determination was 99.1%, 99.1%, and 100%, respectively.

Bombard et al (2011) analyzed the performance of the SensiGene Fetal RHD Genotyping test in 2 cohorts.^8, Cohort 1 used as a reference point the clinical RhD serotype obtained from cord blood at delivery. Samples from cohort 2 were originally genotyped at a single Sequenom location and results were used for clinical validation of genotyping performed at another Sequenom facility.

In cohort 1, RHD genotyping was performed on 236 maternal plasma samples from singleton, nonsensitized pregnancies with documented fetal RhD serology ^8, . The samples were obtained at 11 to 13 weeks of gestation. The ethnic origin of the pregnant women was White (77.1%), African (19.1%), mixed-race (3.4%), and South Asian (0.4%). Neonatal RhD phenotype, determined by serology at the time of birth, was positive in 69.1% of samples and negative in 30.9% of samples. In 2 (0.9%) of the 236 samples, the results were classified as invalid. In the 234 (99.1%) samples with sufficient DNA, the result was conclusive in 207 (88.5%) samples, inconclusive in 16 (6.8%) samples; and y-positive/RHD variant in 11 (4.7%) samples. In the 207 samples with a conclusive result, the neonatal RhD phenotype was positive in 142 (68.6%) samples and negative in 65 (31.4%) samples. The Fetal RHD Genotyping test correctly predicted the neonatal RhD phenotype in 201 (97.1%) of 207 samples (95% confidence interval [CI], 93.5% to 98.8%). In the 142 samples with RhD-positive fetuses, the test predicted that the fetus was positive in 138 and was negative in 4, for an RhD-positive sensitivity of 97.2% (95% CI, 93.0% to 98.9%). In 63 of the 65 samples with RhD-negative fetuses, the Fetal RHD Genotyping test predicted that the fetus was negative and, in the remaining 2, that it was positive, for an RhD-positive specificity of 96.9% (95% CI, 89.5% to 99.1%). The test predicted that the fetus was RhD-positive in 140 samples, of which 138 were predicted correctly, for a positive predictive value of 98.6% (95% CI, 94.9% to 99.6%). The test predicted that the fetus was RhD-negative in 67 samples, of which 63 were predicted correctly, for a negative predictive value for RhD-positive fetuses of 94.0% (95% CI, 85.6% to 97.6%).

Cohort 2 consisted of 205 samples from 6 to 30 weeks of gestation. Testing sought to detect the presence of RHD exon sequences 4, 5, and 7, the RHDy, and 3 Y chromosome sequences (SRY, DBY, TTTY2), using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based nucleic acid analysis (the Fetal RHD Genotyping laboratory-developed test). The laboratory performing the assays for both cohorts was blinded to the sex and fetal RHD genotype. In cohort 2, the test correctly classified 198 of 199 patients, for a test accuracy of 99.5%, with a sensitivity and specificity for prediction of RHD genotype of 100.0% and 98.3%, respectively.

Table 1. Sequenom SensiGene Clinical Validation Studies

RhD: Rhesus D.

Moise et al (2016) analyzed blood samples collected in each trimester of pregnancy for 520 nonalloimmunized RhD-negative patients in a prospective, observational study using the Fetal RHD Genotyping test.^9, Inconclusive results secondary to the presence of the RHDy or an RHD variant were noted in 5.6%, 5.7%, and 6.1% of the first-, second-, and third-trimester samples, respectively. The false-positive rates for RhD (an RhD-negative fetus with an RHD-positive result) was 1.54% (95% CI, 0.42% to 5.44%), 1.53% (95% CI, 0.42% to 5.40%), and 0.82% (95% CI, 0.04% to 4.50%), respectively, across the 3 trimesters. There was only 1 (0.32%) false-negative diagnosis (an RhD-positive fetus with an RHD-negative result), which occurred in the first trimester (95% CI, 0.08% to 1.78%). Genotyping for mismatches across repeated samples revealed that this error was related to mislabeling of samples from 2 patients collected on the same day at a collection site. Overall test results were in agreement across all 3 trimesters (p>.99).

The Unity screen, which assesses RhD, K1, Fy^a, C, c, and E antigens, demonstrated 100% sensitivity and specificity in a validation study in 1683 clinical samples.^10, No prospective clinical studies of this assay have been published.

Section Summary: Clinically Valid

The clinical sensitivity of RHD genotyping is high. However, there is variability in the sensitivity based on the trimester when the test is performed. Clinical validation studies have found the false-negative rates ranging from 0.5% to 2.0%. False-negative results in this clinical context would lead to lack of RhD immunoglobulin administration, development of maternal alloimmunization to RhD, and current and future pregnancy complications due to maternal alloantibodies to RhD compared with standard management of RhD-negative pregnant women.

Clinically Useful

A test is clinically useful if the use of the results informs management decisions that improve the net health outcome of care. The net health outcome can be improved if patients receive correct therapy, or more effective therapy, or avoid unnecessary therapy, or avoid unnecessary testing.

Review of Evidence

Direct Evidence

Direct evidence of clinical utility is provided by studies that have compared health outcomes for patients managed with and without the test. Because these are intervention studies, the preferred evidence would be from randomized controlled trials.

No published data were identified showing that fetal RHD genotyping leads to improved health outcomes.

Chain of Evidence

Indirect evidence on clinical utility rests on clinical validity. If the evidence is insufficient to demonstrate test performance, no inferences can be made about clinical utility.

The possible clinical utility of RHD genotyping using cffDNA includes the following scenarios. In the RhD-negative, nonalloimmunized pregnant patient:

• Avoidance of unnecessary anti-D immunoglobulin if the fetus is RhD-negative;

• Avoidance of invasive procedures to obtain fetal tissue when the paternity is unknown or the father is heterozygous for the D antigen.

In the RhD-negative, alloimmunized pregnant patient:

• Avoidance of invasive procedures to obtain fetal tissue if the RhD-negative pregnant woman is alloimmunized to determine fetal RhD status;

• Avoidance of serial antibody testing in the mother and middle cerebral artery surveillance of the fetus if the fetus is determined to be RhD-negative.

This type of testing could lead to the avoidance of the use of anti-D immunoglobulin (eg, RhoGAM) in RhD-negative mothers with RhD-negative fetuses. However, the false-negative test rate, while low, is not zero, and a certain percentage of RhD-negative women will develop alloimmunization to RhD-positive fetuses. Other issues that need to be defined include the optimal timing of testing during the pregnancy.

Section Summary: Clinically Useful

Direct evidence of the clinical utility of RHD genotyping using cffDNA is lacking. There is potential clinical utility in avoidance of unnecessary anti-D immunoglobulin administration, avoidance of invasive procedures to determine fetal RhD status, avoidance of serial antibody testing in alloimmunized pregnant patients, and avoidance of middle cerebral artery surveillance in an RhD-negative fetus. However, a certain percentage of RhD-negative women will develop alloimmunization to RhD-positive fetuses due to false-negative test results.

For individuals who are pregnant and have Rhesus D (RhD)-negative blood type who receive noninvasive RHD genotyping of the fetus using cell-free DNA from maternal plasma, the evidence includes a meta-analysis and additional prospective studies (for clinical validity) and no direct evidence for clinical utility. Relevant outcomes are test validity, morbid events, medication use, and treatment-related morbidity. Clinical validity studies have demonstrated that the sensitivity and specificity of the test are high; however, the false-negative test rate, while low, is not zero, potentially leading to alloimmunization of the RhD-negative mothers in these cases. It is uncertain whether RHD genotyping using cffDNA will lead to improved health outcomes. The evidence is insufficient to determine that the technology results in an improvement in the net health outcome.

Supplemental Information

The purpose of the following information is to provide reference material. Inclusion does not imply endorsement or alignment with the evidence review conclusions.

Practice Guidelines and Position Statements

Guidelines or position statements will be considered for inclusion in ‘Supplemental Information' if they were issued by, or jointly by, a US professional society, an international society with US representation, or National Institute for Health and Care Excellence (NICE). Priority will be given to guidelines that are informed by a systematic review, include strength of evidence ratings, and include a description of management of conflict of interest.

American College of Obstetricians and Gynecologists

In 2018, the American College of Obstetricians and Gynecologists reaffirmed its 2006 position that detection of fetal Rhesus D (RhD) using molecular analysis of maternal plasma or serum can be assessed in the second trimester with an accuracy greater than 99% but that this test is not a widely used clinical tool.^11,12,This statement was last reaffirmed in 2024.

In its 2017 Practice Bulletin Number 181 on the prevention of RhD alloimmunization, the College stated that "Despite the improved accuracies noted with noninvasive fetal RHD genotyping, cost comparisons with current routine prophylaxis of anti-D immunoglobulin at 28 weeks of gestation have not shown a consistent benefit and, thus, this test is not routinely recommended."^13,This statement was last reaffirmed in 2024.

Sperling et al (2018) compared the guidelines from the American College of Obstetricians and Gynecologists as well as 3 international guidelines on the prevention of RhD alloimmunization.^14, All 4 guidelines recommended that all women have an antibody screen with an indirect Coombs test at prenatal intake and at 24 to 28 weeks. None currently recommend screening with cell-free fetal DNA.

A 2024 Practice Advisory statement on RhD immune globulin shortages endorses using noninvasive prenatal testing with cell-free fetal DNA in the setting of a shortage to help with supply conservation efforts.^15, Postpartum administration should be prioritized first, followed by 28 weeks of antepartum prophylaxis if there is sufficient supply.

U.S. Preventive Services Task Force Recommendations

No U.S. Preventive Services Task Force recommendations addressing fetal RHD genotyping were identified.

Medicare National Coverage

There is no national coverage determination. In the absence of a national coverage determination, coverage decisions are left to the discretion of local Medicare carriers.

Ongoing and Unpublished Clinical Trials

A search of ClinicalTrials.gov in July 2024 did not identify any ongoing or unpublished phase 3 trials that would likely influence this review.

References

1. Daniels G, Finning K, Martin P, et al. Fetal RhD genotyping: a more efficient use of anti-D immunoglobulin. Transfus Clin Biol. Dec 2007; 14(6): 568-71. PMID 18436463
2. Moise K. Overview of Rhesus (Rh) alloimmunization in pregnancy. In: Lockwood CJ, ed. UpToDate. Waltham, MA: UpToDate; 2013.
3. Moise KJ, Argoti PS. Management and prevention of red cell alloimmunization in pregnancy: a systematic review. Obstet Gynecol. Nov 2012; 120(5): 1132-9. PMID 23090532
4. Van den Veyver IB, Moise KJ. Fetal RhD typing by polymerase chain reaction in pregnancies complicated by rhesus alloimmunization. Obstet Gynecol. Dec 1996; 88(6): 1061-7. PMID 8942854
5. Lo YM, Tein MS, Lau TK, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet. Apr 1998; 62(4): 768-75. PMID 9529358
6. Zhu YJ, Zheng YR, Li L, et al. Diagnostic accuracy of non-invasive fetal RhD genotyping using cell-free fetal DNA: a meta analysis. J Matern Fetal Neonatal Med. Dec 2014; 27(18): 1839-44. PMID 24422551
7. Chitty LS, Finning K, Wade A, et al. Diagnostic accuracy of routine antenatal determination of fetal RHD status across gestation: population based cohort study. BMJ. Sep 04 2014; 349: g5243. PMID 25190055
8. Bombard AT, Akolekar R, Farkas DH, et al. Fetal RHD genotype detection from circulating cell-free fetal DNA in maternal plasma in non-sensitized RhD negative women. Prenat Diagn. Aug 2011; 31(8): 802-8. PMID 21626507
9. Moise KJ, Gandhi M, Boring NH, et al. Circulating Cell-Free DNA to Determine the Fetal RHD Status in All Three Trimesters of Pregnancy. Obstet Gynecol. Dec 2016; 128(6): 1340-1346. PMID 27824757
10. Alford B, Landry BP, Hou S, et al. Validation of a non-invasive prenatal test for fetal RhD, C, c, E, K and Fy a antigens. Sci Rep. Aug 07 2023; 13(1): 12786. PMID 37550335
11. American College of Obstetricians and Gynecologists. ACOG Practice Bulletin No. 75: Management of alloimmunization during pregnancy. Obstet Gynecol. Aug 2006; 108(2): 457-64. PMID 16880320
12. ACOG Practice Bulletin No. 192 Summary: Management of Alloimmunization During Pregnancy. Obstet Gynecol. Mar 2018; 131(3): 611-612. PMID 29470338
13. Practice Bulletin No. 181: Prevention of Rh D Alloimmunization. Obstet Gynecol. Aug 2017; 130(2): e57-e70. PMID 28742673
14. Sperling JD, Dahlke JD, Sutton D, et al. Prevention of RhD Alloimmunization: A Comparison of Four National Guidelines. Am J Perinatol. Jan 2018; 35(2): 110-119. PMID 28910850
15. American College of Obstetricians and Gynecologists. Practice Advisory: Rho(D) Immune Globulin Shortages. July 2024. https://www.acog.org/clinical/clinical-guidance/practice-advisory/articles/2024/03/rhod-immune-globulin-shortages. Accessed July 24, 2024.

Codes

Applicable Modifiers

As per Correct Coding Guidelines

Policy History