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 if fetal RhD status is unknown, involves administration of RhD immunoglobulin at standardized 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.

Rh Blood Groups

The Rh (Rhesus) system includes more than 100 antigen varieties found on RBCs. RhD 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; »60% of RhD-positive people) or homozygous (DD; »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).

RhD-negative status varies across ethnic groups and is 15% in whites, 5% to 8% in blacks, 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 blacks 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 if exposed to RhD-positive RBCs, can make antibodies to one or more epitopes of the D antigen.^1,

RhD-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 be caused 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 vs 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 occurs in nearly all pregnancies, and percentages of fetomaternal hemorrhage increase 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%-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 a cerebral injury.^3,

Prevention of Alloimmunization

There are four 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 (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.^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. RBCs 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 RhD 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 (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 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 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.

The 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 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
• Identifying RhD-negative sensitized patients
• Avoiding invasive testing by CVS or genetic amniocentesis.

• None

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


Policy Guidelines: (Information to guide medical necessity determination based on the criteria contained within the policy statements above.)

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 policy 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

Previous	Updated	Definition
Mutation	Disease-associated variant	Disease-associated change in the DNA sequence
	Variant	Change in the DNA sequence
	Familial variant	Disease-associated variant identified in a proband for use in subsequent targeted genetic testing in first-degree relatives

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

Variant Classification	Definition
Pathogenic	Disease-causing change in the DNA sequence
Likely pathogenic	Likely disease-causing change in the DNA sequence
Variant of uncertain significance	Change in DNA sequence with uncertain effects on disease
Likely benign	Likely benign change in the DNA sequence
Benign	Benign change in the DNA sequence

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

Genetic Counseling
Experts recommend formal genetic counseling for patients who are at risk for inherited disorders and who wish to undergo genetic testing. Interpreting the results of genetic tests and understanding risk factors can be difficult for some patients; genetic counseling helps individuals understand the impact of genetic testing, including the possible effects the test results could have on the individual or their family members. It should be noted that genetic counseling may alter the utilization of genetic testing substantially and may reduce inappropriate testing; further, genetic counseling should be performed by an individual with experience and expertise in genetic medicine and genetic testing methods.

Medicare Advantage Coverage:
There is no National Coverage Determination (NCD) or Local Coverage Determination (LCD) for jurisdiction JL for this service. Therefore, Medicare Advantage Products will follow the Horizon BCBSNJ Medical Policy.

Medicaid Coverage:
For members enrolled in Medicaid and NJ FamilyCare plans, Horizon BCBSNJ applies the above medical policy.

FIDE-SNP Coverage:
For members enrolled in a Fully Integrated Dual Eligible Special Needs Plan (FIDE-SNP): (1) to the extent the service is covered under the Medicare portion of the member’s benefit package, the above Medicare Coverage statement applies; and (2) to the extent the service is not covered under the Medicare portion of the member’s benefit package, the above Medicaid Coverage statement applies.

[RATIONALE: This policy was created in 2013 and has been updated regularly with searches of the PubMed database. The most recent literature update was performed through June 10, 2019.

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.

Testing Pregnant Women with RhesusD-negative Blood Type

Clinical Context and Test Purpose

The purpose of genetic testing of individuals who are pregnant and have 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 policy include:

1. Does Rhesus D (RHD) genotyping reduce the need for invasive testing by chorionic villus sampling or amniocentesis?
2. Does RHD genotyping guide the administration of anti-D immunoglobulin during pregnancy?
3. Does RHD genotyping lead to improved pregnancy outcomes?

Patients

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.

The primary setting would be in the obstetrics population where maternal blood type and RhD status are determined during the prenatal period and RhD-negative patients are monitored and/or treated to prevent alloimmunization to RhD.

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 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.

Technically Reliable

Assessment of technical reliability focuses on specific tests and operators and requires a review of unpublished and often proprietary information. Review of specific tests, operators, and unpublished data are outside the scope of this policy and alternative sources exist. This policy focuses on the clinical validity and clinical utility.

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).

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.

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.

Table 1. Sequenom SensiGene Clinical Validation Studies

Author	Accuracy for RhD Status Determination, %	False-Negative Rate RhD Determination, %
Moise et al (2012)3,	98.1%-99.1%, depending on trimester when test performed	0.45
Bombard et al (2011)8,
Cohort 1	97.1	1.9
Cohort 2	99.5	0

RhD: Rhesus D.

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-flightmass spectrometry to detect control and fetal-specific DNA signals. The determination of fetal sex was defined as follows: three Y chromosome markers is a male fetus, two markers are inconclusive, and one or no marker is a female fetus. The algorithm for RHD determination was defined as follows: pseudogene present is inconclusive, three RHD markers present is an RHD-positive fetus, two markers present is inconclusive, one 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 six informative paternal alleles (uniquely and unambiguously fetal in nature) were detected, the result was an RHD-negative, female fetus. If fewer thansix alleles were detected, the sample was reported as inconclusive. Cord blood was obtained at delivery and RhD typing was determined using standard serologic methods, and 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,^6/7 16 to 19,^6/7 and 28 to 29^6/7weeks. At the second blood draw, two 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, seven patients did not have a sample obtained.

Median gestational ages of the first-, second-, and third-trimester samplings were 12.4 weeks (range, 10.6-13.9 weeks), 17.6 weeks (range, 16-20.9 weeks), and 28.7 weeks (range, 27.9-33.9 weeks), respectively. Three samples in the first trimester and two 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: 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 one 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. 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, 7, the RHDy, and three, 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.

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 two patients collected on the same day at a collection site. Overall test results were in agreement across all 3 trimesters (p>0.99).

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.

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.

• Avoidance of invasive procedures to obtain fetal tissue if 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.

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 patient, 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.

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. The 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 cffDNA will lead to improved health outcomes. The evidence is insufficient to determine the effects of the technology on health outcomes.

SUPPLEMENTAL INFORMATION

Practice Guidelines and Position Statements

American Association of Blood Banks

The American Association of Blood Banks has not issued specific practice guidelines or recommendations on the use of fetal Rhesus D (RHD) genotyping.

American College of Obstetricians and Gynecologists

The American College of Obstetricians and Gynecologists (2018) reaffirmed its 2006 position that detection of fetal 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.^10,11,

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."^12,

Sperling et al (2018) compared the guidelines from the American College of Obstetricians and Gynecologists as well as 3 international on the prevention of RhD alloimmunization.^13, 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.

U.S. Preventive Services Task Force Recommendations

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

Ongoing and Unpublished Clinical Trials

A search of ClinicalTrials.gov in June 2019 did not identify any ongoing or unpublished phase 3 trials that would likely influence this review..]
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Horizon BCBSNJ Medical Policy Development Process:

This Horizon BCBSNJ Medical Policy (the “Medical Policy”) has been developed by Horizon BCBSNJ’s Medical Policy Committee (the “Committee”) consistent with generally accepted standards of medical practice, and reflects Horizon BCBSNJ’s view of the subject health care services, supplies or procedures, and in what circumstances they are deemed to be medically necessary or experimental/ investigational in nature. This Medical Policy also considers whether and to what degree the subject health care services, supplies or procedures are clinically appropriate, in terms of type, frequency, extent, site and duration and if they are considered effective for the illnesses, injuries or diseases discussed. Where relevant, this Medical Policy considers whether the subject health care services, supplies or procedures are being requested primarily for the convenience of the covered person or the health care provider. It may also consider whether the services, supplies or procedures are more costly than an alternative service or sequence of services, supplies or procedures that are at least as likely to produce equivalent therapeutic or diagnostic results as to the diagnosis or treatment of the relevant illness, injury or disease. In reaching its conclusion regarding what it considers to be the generally accepted standards of medical practice, the Committee reviews and considers the following: all credible scientific evidence published in peer-reviewed medical literature generally recognized by the relevant medical community, physician and health care provider specialty society recommendations, the views of physicians and health care providers practicing in relevant clinical areas (including, but not limited to, the prevailing opinion within the appropriate specialty) and any other relevant factor as determined by applicable State and Federal laws and regulations.

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Index:
Noninvasive Fetal RHD Genotyping Using Cell-Free Fetal DNA
Fetal RHD Genotyping Using Maternal Plasma
RHD Genotyping Using Maternal Plasma
SensiGene Fetal RHD Genotyping test
SensiGene

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):568-571. PMID 18436463.

2. Moise K. Overview of Rhesus (Rh) alloimmunization in pregnancy. In: Lockwood CJ, ed. UpToDate. Waltham, MA: UpToDate; 2013.

3. Moise KJ, Jr., Argoti PS. Management and prevention of red cell alloimmunization in pregnancy: a systematic review. Obstet Gynecol. Nov 2012;120(5):1132-1139. PMID 23090532.

4. Van den Veyver IB, Moise KJ, Jr. Fetal RhD typing by polymerase chain reaction in pregnancies complicated by rhesus alloimmunization. Obstet Gynecol. Dec 1996;88(6):1061-1067. 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):768-775. 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):1839-1844. 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):802-808. PMID 21626507.

9. Moise KJ, Jr., 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. American College of Obstetricians and Gynecologists. ACOG Practice Bulletin No. 75: Management of alloimmunization during pregnancy. Obstet Gynecol. Aug 2006;108(2):457-464. PMID 16880320.

11. ACOG Practice Bulletin No. 192 Summary: Management of alloimmunization during pregnancy. Obstet Gynecol. Mar 2018;131(3):611-612. PMID 29470338.

12. Committee on Practice Bulletins-Obstetrics. Practice Bulletin No. 181: Prevention of Rh D Alloimmunization. Obstet Gynecol. Aug 2017;130(2):e57-e70. PMID 28742673.

13. 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.

Codes:
(The list of codes is not intended to be all-inclusive and is included below for informational purposes only. Inclusion or exclusion of a procedure, diagnosis, drug or device code(s) does not constitute or imply authorization, certification, approval, offer of coverage or guarantee of payment.)

CPT*

81403