Noninvasive Fetal RHD Genotyping Using Cell-Free Fetal DNA

POLICY NUMBER

A.2.04.108

DESCRIPTION

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. The use of cell-free fetal DNA in maternal blood has been proposed as a noninvasive method to determine fetal RHD genotype.

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 individual 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 individual 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 (i.e., 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). There are also numerous rare variants of the D antigen, which are recognized by weakness of expression of D and/or by absence of some of the epitopes of D. Some individuals with variant D antigens can make antibodies to one or more epitopes of the D antigen if exposed to RhD-positive RBCs.

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 red blood cell (RBC) membrane, and alloimmunization can occur when fetal RhD-positive RBCs enter maternal circulation and the RhD-negative mother develops anti-D antibodies. 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 individuals 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 individual 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 (e.g., 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 cerebral injury.

Prevention of Alloimmunization

There are four RhD immunoglobulin products available in the U.S., all of which undergo micropore filtration to eliminate viral transmission. To date, no reported cases of viral infection related to RhD immunoglobulin administration have been reported in the U.S. 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 (ACOG) and the American Association of Blood Banks (AABB) have recommended that the first dose of Rh_o(D) immunoglobulin (e.g., 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. 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

An individual'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 Obstetricians 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 he is homozygous for the D allele (i.e., 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 (PCR) 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.

Noninvasive testing involves molecular analysis of cell-free fetal DNA (cffDNA) in the maternal plasma or serum. Lo and colleagues 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. 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.

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. When Y chromosome-linked genes are not detected, tests for variants may be performed to determine whether the result is derived from fetal rather than maternal DNA.

Use of cell-free fetal DNA testing to determine the fetal RHD genotype is the standard of care in many European countries.

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

The proprietary SensiGene™ Fetal RHD Genotyping test SEQureDx™ technology was marketed by Sequenom. 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 fetal RHD status when maternal anti-D titers are unclear.

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

• IdentifyingRhD-negative sensitized patients.

• Avoiding invasive testing by chorionic villus sampling or genetic amniocentesis.

At the time of the 2025 policy update, the availability of the SensiGene™ Fetal RHD Genotyping test could not be confirmed. The policy no longer considers this test.

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, Fyª, and K antigens, aneuploidy, and recessive conditions including cystic fibrosis, spinal muscular atrophy, sickle cell disease, alpha and beta thalassemia, and fragile X syndrome. The Unity Screen test uses a proprietary technology (Quantitative Counting Templates) to quantify fetal DNA with as little as a single base pair alteration. The quantitative counting templates are traceable synthetic DNA fragments that are added to the patient's sample. After amplification, the number of fragments is added to a calculation that determines the number of DNA fragments of interest in the patient sample.

Natera offers an add-on fetal RhD test to its noninvasive prenatal Panorama™ test, which uses next-generation sequencing technology. The manufacturer states that Panorama is the only single nucleotide polymorphism-based noninvasive prenatal test. More than 13,000 single nucleotide polymorphisms are included in the screening test. The assay involves DNA primers to regions that specifically identify the RHD psi (ψ) pseudogene.

POLICY

Measurement of cell-free DNA for fetal genotyping for RhD antigen may be medically necessary when all of the following criteria are met:

1. Pregnancy may be at risk for alloimmunization due to maternal RhD negative status or the presence of maternal red cell antigen antibodies; and

2. Paternal antigen typing is unavailable or heterozygous; and

3. Amniocentesis is declined or contraindicated.

POLICY EXCEPTIONS

None

POLICY GUIDELINES

The coverage guidelines outlined in the Medical Policy Manual should not be used in lieu of the Member's specific benefit plan language.

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 1). 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 (ACMG) and the Association for Molecular Pathology (AMP) 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 2 shows the recommended standard terminology—“pathogenic,” “likely pathogenic,” “uncertain significance,” “likely benign,” and “benign”—to describe variants identified that cause Mendelian disorders.

Table 1. Nomenclature to Report on Variants Found in DNA

Table 2. ACMG-AMP Standards and Guidelines for Variant Classification

Investigative is defined as the use of any treatment procedure, facility, equipment, drug, device, or supply not yet recognized as a generally accepted standard of good medical practice for the treatment of the condition being treated and; therefore, is not considered medically necessary. For the definition of Investigative, “generally accepted standards of medical practice” means standards that are based on credible scientific evidence published in peer-reviewed medical literature generally recognized by the relevant medical community, and physician specialty society recommendations, and the views of medical practitioners practicing in relevant clinical areas and any other relevant factors. In order for equipment, devices, drugs or supplies [i.e, technologies], to be considered not investigative, the technology must have final approval from the appropriate governmental bodies, and scientific evidence must permit conclusions concerning the effect of the technology on health outcomes, and the technology must improve the net health outcome, and the technology must be as beneficial as any established alternative and the improvement must be attainable outside the testing/investigational setting.

POLICY HISTORY

04/01/2014: Approved by Medical Policy Advisory Committee.

01/12/2015: Policy reviewed; no changes.

07/20/2015: Code Reference section updated for ICD-10.

02/17/2016: Policy title changed from "Fetal RHD Genotyping Using Maternal Plasma" to "Noninvasive Fetal RHD Genotyping Using Cell-Free Fetal DNA." Policy description updated regarding laboratory-developed tests. Investigational statement updated to state that noninvasive fetal RHD genotyping using cell-free fetal DNA is considered investigational. It previously stated: Fetal RHD genotyping using maternal plasma is considered investigational. Investigative definition updated in policy guidelines section. Code Reference section updated to add CPT code 81403.

06/07/2016: Policy number A.2.04.108 added.

06/16/2017: Policy description updated regarding tests. Policy statement unchanged.

06/27/2017: Code Reference section updated to revise code description for CPT code 81403, effective 07/01/2017.

12/22/2017: Code Reference section updated to revise description for CPT code 81403 effective 01/01/2018.

06/13/2018: Policy reviewed. Policy statement unchanged. Policy Guidelines updated to add genetics nomenclature and genetic counseling information.

09/09/2019: Policy reviewed; no changes.

09/23/2020: Policy reviewed. Policy statement unchanged. Policy Guidelines updated to remove genetic counseling information. Code Reference section updated to add new CPT code 0222U, effective 10/01/2020.

12/07/2021: Policy description updated. Policy statement unchanged.

04/26/2023: Policy description updated. Policy statement unchanged. Code Reference section updated to add CPT code 0198U.

09/11/2023: Policy description updated. Policy statement unchanged.

09/10/2024: Policy description updated regarding tests. Policy statement unchanged.

10/15/2025: Policy description updated regarding tests. Added policy statement that the measurement of cell-free DNA for fetal genotyping for RhD antigen may be medically necessary when all of the listed criteria are met. It previously stated: Noninvasive fetal RHD genotyping using cell-free fetal DNA is considered investigational. Code Reference section updated to change CPT codes 81403 and 81479 from Investigational to Medically Necessary. Added ICD-10 diagnosis codes O36.0110 - O36.0199 and Z31.82.

SOURCE(S)

Blue Cross and Blue Shield Association Policy # 2.04.108

CODE REFERENCE

This may not be a comprehensive list of procedure codes applicable to this policy.

The code(s) listed below are ONLY medically necessary if the procedure is performed according to the "Policy" section of this document.

Medically Necessary Codes

Investigational Codes

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