Please note: In 2003, the CTF updated its Grades of Recommendations to include an "I Recommendation" for situations where insufficient evidence exists to allow a recommendation to be made.  (Formerly, these situations were captured under a "C Recommendation".)  This change is not retroactive, and all "C Recommendations" made prior to 2003 have not been reevaluated in light of the new "I" recommendation grade.  For a discussion of these recommendation grades, please link to the 2003 article in the Canadian Medical Association Journal here.

Screening for D (Rh) Sensitization in Pregnancy

Overview
In 1979 the Canadian Task Force found good evidence for screening for blood group incompatibility in pregnancy. This review updates the previous recommendation and is based on the recent review of the evidence by the U.S. Preventive Services Task Force for Screening for D (Rh) Incompatibility. We now find that there is good to fair evidence to recommend that all pregnant women should receive D (formerly Rh) blood typing and antibody screening at their first prenatal visit and, if D negative, repeat antibody screening at 24-28 weeks’ gestation. Unless the father is known to be D negative, unsensitized D negative women should receive D immunoglobulin (D Ig) at 28-29 weeks’ gestation, within 72 hours after delivery of a D positive infant, and after induced abortion or amniocentesis. It may also be prudent to administer D immunoglobulin to unsensitized D negative women after spontaneous abortion, ectopic pregnancy or other obstetrical procedures or complications. The effectiveness of prevention in these situations has never been proven by controlled trials but is inferred considering their similarity with other clinical conditions where prevention has been proven to be effective. There is some controversy on the effectiveness of the administration of D immunoglobulin after chorionic villus sampling (CVS). One study reported results suggesting that it can create adverse effects to the fetus. However these results were preliminary ones and did not control for important confounding variables.

Burden of Suffering
Some degree of fetal-maternal transplacental hemorrhage occurs in 75% of all pregnancies. This phenomenon is not dangerous to the fetus unless there is incompatibility between the mother and her fetus with respect to the D antigen of the red blood cells. D incompatibility exists when a D negative woman is pregnant with a D positive fetus. This occurs in up to 9-10% of pregnancies, depending on race. The incidence in Canadians of caucasian extraction is about 15%, 7% in American blacks, and as low as 1% in North American Indians and Inuit. The majority of fetuses (70%) of D negative women are D positive. About 35,000 D-negative women become pregnant annually. They carry 24,500 D positive fetuses who are at risk of developing D isoimmunization. If no preventive measures are taken, 0.7-1.8% of these women will become isoimmunized antenatally, developing D antibody through exposure to fetal blood; 8-17% will become isoimmunized at delivery, 3-6% after spontaneous or therapeutic abortion, and 2-5% after amniocentesis. Once a woman is isoimmunized, the risk to the fetus increases in subsequent D positive pregnancies because maternal D Ig antibodies are produced earlier in the pregnancy and in greater amount. In isoimmunized D negative women the risk of fetal isoimmunization disease increases with the number of pregnancies.

Once isoimmunization has occurred, the severity of fetal hemolysis varies. In 50% of the cases the fetus is very mildly affected and does not require postpartum treatment. However, without treatment, 25-30% of these offspring will have some degree of hemolytic anemia and hyperbilirubinemia, another 20-25% will be hydropic and many will die either in utero or during the neonatal period.

Since the introduction of routine postpartum prophylaxis in the 1960s, the crude incidence of D isoimmunization in Canada and the United States has fallen from 9.1-10.3 cases to 1.3 cases per 1,000 total births. Hemolytic disease of the fetus or newborn due to D isoimmunization (also called erythroblastosis fetalis) now accounts for only 4-5 deaths per 100,000 births, although this may be an underestimate as early intrauterine deaths are not always reported. However, this decline in fetal and neonatal mortality from D hemolytic disease cannot be attributed exclusively to the effectiveness of primary prevention and early treatment. It has been estimated that 30-40% of the recent decline in disease incidence is attributable to smaller family size, since the incidence of D hemolytic disease increases with increasing birth order. Smaller family size also contributes to the decreasing case fatality rate, since the first affected infant in a family generally has less severe disease. Since the 1940s, the case fatality rate has fallen from about 50% to 2-6%. This decline has also been associated with the introduction of interventions such as amniotic fluid spectrophotometry, exchange transfusion, amniocentesis, intrauterine fetal transfusion, and improved care of both the mother and the premature erythroblastotic infant.

Maneuver
Hemagglutination is the established reference standard for the determination of D blood type. The indirect antiglobulin (Coomb’s) test (IAGT) is the reference standard for detecting anti-D antibody in women who have been sensitized to D positive blood. The IAGT will also detect other maternal antibodies that may cause hemolytic disease.

Effectiveness of Screening and Treatment
No therapeutic intervention, with the exception of intravenous serum globulin and plasma exchange, can suppress the isoimmunization process once it is initiated. Primary prevention of D immunization process is therefore the objective. Early detection of D negative blood type in the pregnant woman is of substantial benefit if the patient is not yet isoimmunized and the father is not known to be D negative. Administration of D Immunoglobulin [D Ig, or Rho(D) Immune Globulin (Human)] to an unsensitized D negative woman after delivery of a D positive fetus will prevent maternal isoimmunization and consequent hemolytic disease in subsequent D positive offspring. The efficacy of D Ig prophylaxis was demonstrated convincingly in a series of controlled clinical trials in the early 1960s.<1,2> Despite various minor flaws in study design, these trials showed that isoimmunization did not occur in any of the women who received a full dose of D Ig postpartum and who were not sensitized at the time it was administered. These findings led to the introduction in 1968 of routine postpartum prophylaxis following licensure of D Ig.

The most frequent cause of apparent failure of postpartum prophylaxis is antenatal isoimmunization, which happens in 0.7-1.8% of pregnant women at risk. Although sample selection and other design features were not optimal, non-randomized controlled trials have shown that the administration of D Ig at 28 weeks’ gestation, when combined with postpartum administration, reduces the incidence of isoimmunization to 0.2% of women at risk.<3-5>

Since D isoimmunization during pregnancy is caused by transplacental hemorrhage, the risk of isoimmunization increases whenever such hemorrhage is likely to occur, including after abortion, amniocentesis, chorionic villus sampling (CVS), cordocentesis, ectopic pregnancy, fetal manipulation (e.g. external version procedures) or surgery, antepartum hemorrhage, antepartum fetal death and stillbirth.<6,7> The Task Force makes a number of recommendations which include these procedures in the protocol (see Chapter 7 on neural tube defects, Chapter 8 on Down Syndrome, Chapter 19 on cystic fibrosis and Chapter 46 on unintended teen pregnancy). Studies documenting the effectiveness of D Ig prophylaxis are available for only a few of these indications, however. In a non-randomized trial of D Ig administration after amniocentesis, control Rh-negative women delivering Rh-positive infants were more likely to become isoimmunized than were those receiving D Ig (5.2% vs. 0%), although because of small numbers this difference was not statistically significant. Case series describing D Ig administration after amniocentesis have demonstrated isoimmunization rates as low as 0-0.5%. In a case series of D Ig after termination of pregnancy, the isoimmunization rate was 0.4%,<8> compared to 2.6% among a series of patients who did not receive D Ig described by the same authors.<9>

Some controversy exists concerning the value of D Ig administration after CVS. In the preliminary analysis of a controlled trial planned to evaluate the safety of transcervical CVS compared to transabdominal CVS and amniocentesis,<10> Smidt-Jensen and Philip reported some data on a randomized experiment of D Ig administration in the group allocated to either modality of CVS. All women allocated to amniocentesis received D Ig. Among Rh-negative women delivering Rh-positive infants, similar rates of isoimmunization were seen in both intervention (D Ig after CVS) (2.3%) and control (no D Ig after CVS) (1.1%) groups. Although the women who received D Ig experienced twice as many unintended fetal losses as did controls (6.9% vs. 3.8%), the difference was not statistically significant, possibly due to small sample size. Also, the authors did not control for the effect of the chorionic villus sampling technique. Transabdominal CVS was associated with a lower rate of fetal loss than transcervical CVS. Finally, data were not available for all participants. A full report of this study has yet to be published. Meanwhile, it cannot be considered as evidence of an adverse effect of D Ig administration after CVS and the controversy will persist.

The standard postpartum dose of D Ig (300 mg) contains sufficient anti-D immunoglobulin to prevent sensitization to at least 15 mL of D positive fetal red blood cells (RBCs), or approximately 30 mL of fetal blood; a "minidose" (50 mg) prevents sensitization to 2.5 mL of D positive fetal RBCs. For women with transplacental hemorrhages >30 mL of fetal blood, the risk of D isoimmunization developing after the full post-partum D Ig dose is 30-35%. The incidence of fetal-maternal hemorrhage >30 mL is 0.1-0.7% for all D negative pregnancies, but is 1.7-2.5% after complicated vaginal and cesarian deliveries, and 4.5% after stillbirth. Several methods for detecting excess fetal-maternal hemorrhage are available. Acid elution (Kleihauer-Betke) is both sensitive and specific when done correctly but is subject to substantial laboratory and technological error. Flow cytometry is also highly sensitive and specific, but is technically difficult to perform. The erythrocyte rosette test is simple to perform and highly sensitive (99-100%) for the presence of >15 mL of D positive fetal RBCs, but specificity is low. Positive results must be confirmed by more specific tests such as acid elution and flow cytometry.

In clinical practice, combined antenatal and postnatal prophylaxis will prevent isoimmunization in 96% of women at risk.<5> The remaining cases are due to failure to give D Ig when indicated, administration of an insufficient dose, or treatment failure (i.e., isoimmunization occurring before 28 weeks or transplacental hemorrhage that was too large or occurred too late in pregnancy to be prevented by the standard antepartum dose). Thus, human error causes 22-50% of these cases.<5> Indeed, while clinicians almost always administer D Ig postpartum and after induced abortion, administration rates have been documented to be lower for other obstetric procedures and complications: 81-88% after spontaneous abortion, 36-60% after ectopic pregnancy, 31% after antepartum hemorrhage, and 14% after amniocentesis.

D immunoglobulin has few adverse effects. Some fetuses will become weakly direct-antiglobulin-positive following antenatal administration, but resulting anemia and hyperbilirubinemia in the newborn are very rare.<3> All plasma for D Ig production is screened for infectious diseases as required by the Food and Drug Administration; no cases of HIV infection from D Ig have been reported. The evidence is therefore compelling that early detection and prophylaxis of the unsensitized D negative woman is both safe and effective in preventing isoimmunization and thus in preventing D hemolytic disease.

Early detection is also beneficial for D negative women who are already isoimmunized and are carrying D positive offspring, because early intervention may improve clinical outcome. Decisions to intervene depend on the validity of screening tests in predicting the degree of fetal anemia. Obstetric history, maternal antibody titers and ultrasound are currently used to determine the need for more invasive tests during isoimmunized pregnancies, but in the absence of hydrops none of these reliably distinguishes mild from severe hemolytic disease.<6> Immunologic tests on maternal serum show promise in predicting disease severity. In the third trimester, serial amniotic fluid spectrophotometry has been found to correctly predict disease severity (i.e., cord hemoglobin and need for neonatal therapy) in 94-99% of cases. In the second trimester, however, this test has insufficient sensitivity or specificity for predicting the need for intervention. Determination of fetal hemoglobin and D blood type by ultrasound-guided cordocentesis, which can be performed in the second trimester, quantifies the degree of anemia, can be followed by fetal transfusion if indicated, and allows referral of those with D negative babies to routine care. However, case series have demonstrated complication rates of 2-7% and procedure-related fetal mortality rates of 0.5-1%.<7>

In the presence of severe fetal anemia, early intervention appears to offer substantial improvement in clinical outcome. Current perinatal survival after ultrasound-guided intravascular transfusion at experienced centers is 62-86% for hydropic fetuses and >90% for those without hydrops. Once pulmonary maturity is established, the fetus can be delivered early and exchange transfusion performed with only 1% mortality risk.

Recommendations of Others
In 1989, the U.S. Preventive Services Task Force recommended that all pregnant women should receive ABO/Rh blood typing testing for anti-Rh(D) antibody at their first prenatal visit. Unsensitized Rh-negative women should receive Rh(D) immune globulin (D Ig) at 28-29 weeks’ gestation and within 72 hours after delivery, as well as after spontaneous or therapeutic abortion, ectopic pregnancy, amniocentesis, antepartum placental hemorrhage, or a transfusion of Rh-positive blood products.<11>

The Society of Obstetricians and Gynecologists of Canada and the American College of Obstetricians and Gynecologists (ACOG) recommend D blood typing and antibody screening at the first prenatal visit and repeat D antibody screening at 24-28 weeks of pregnancy for D-negative women.<6,12> Both groups recommend offering D Ig to all unsensitized D-negative women at 28 weeks gestation, and to those at increased risk of sensitization because of delivery of a D positive infant, antepartum hemorrhage, spontaneous or induced abortion, amniocentesis, external version procedures or ectopic pregnancy, within 72 hours of the event. They also recommend D Ig administration to unsensitized D-negative women who have CVS, cordocentesis, antepartum fetal death, fetal surgery, or transfusion of D positive blood products, and measuring fetal blood cell levels in the mother when antepartum placental hemorrhage occurs.

Conclusions and Recommendations
There is excellent evidence for the efficacy and effectiveness of blood typing, anti-D antibody screening and postpartum D Ig prophylaxis. Antepartum prophylaxis offers some additional benefit, although some critics have argued that the total impact of antepartum prophylaxis on the incidence of D disease is relatively small. Other studies support the cost effectiveness of antepartum prophylaxis.<5> The cost effectiveness of D Ig after obstetric procedures and complications is unknown.

All pregnant women should receive D blood typing and antibody testing at their first prenatal visit (A Recommendation). D blood typing and antibody screening should also be performed before elective procedures such as amniocentesis and therapeutic abortion (B Recommendation). For purposes of blood typing and prophylaxis, D^u nd D positive blood types should be considered equivalent.<6> Unless the father is known to be D negative, a repeat D antibody test should be performed at 24-28 weeks’ gestation on all unsensitized D negative women, followed by the administration of a full (300 mg) dose of D Ig if antibody-negative. If a D (or D^u) positive infant is delivered, the dose should be repeated postpartum, preferably within 72 hours after delivery. Unless the father is known to be D negative, all unsensitized D negative women should receive a full dose of D Ig after elective abortion (50 mg before 13 weeks) or amniocentesis (B Recommendation). There is currently insufficient evidence to recommend for or against the routine administration of D Ig after other obstetric procedures or complications such as chorionic villus sampling, ectopic pregnancy termination, cordocentesis, fetal surgery or manipulation (including external version), antepartum placental hemorrhage, antepartum fetal death, and stillbirth. It may also be prudent to administer D immunoglobulin to unsensitized D negative women after spontaneous abortion, ectopic pregnancy or other obstetrical procedures or complications (C Recommendation). The effectiveness of prevention in these situations has never been proven by controlled trials but is inferred considering their similarity to other clinical conditions where prevention has been proven to be effective. There is some controversy about the effectiveness of the administration of D immunoglobulin after CVS. One study reported results suggesting that D Ig can create adverse effects to the fetus. However these were preliminary results and lacked control for important confounding variables.

Routine screening for excess maternal hemorrhage is not recommended, because the risk of D isoimmunization from excess hemorrhage is 0.3% for all D negative pregnancies (see above). For women at increased risk for transplacental hemorrhages >30 mL because of antepartum placental hemorrhage, stillbirth, or cesarian or complicated vaginal delivery,<6> the yield from screening is likely to be higher. In these patients, therefore, physicians may wish to test for fetal RBCs in the maternal circulation, using either quantitative methods (acid elution or flow cytometry), or the erythrocyte rosette test with follow-up of positive results by quantitative methods. If transplacental hemorrhage exceeds 30 mL, 20 mg D Ig per 1 mL of fetal red blood cells should be given.<6>

Unanswered Questions (Research Agenda)
The safety and effectiveness of D Ig administration following CVS requires further study.

Evidence
The literature was identified with a MEDLINE search in English for the years 1989 to December 1993, using the following key words: RH-HR blood group system, RH Isoimmunization, immunoglobulins, amniocentesis, ultrasound, blood transfusion intrauterine and erythroblastosis fetalis. This review was initiated in November 1992 and the recommendations finalized by the Task Force in June 1993.

Acknowledgements
The Task Force would like to thank T.F. Baskett, MB, BCh, FRCS, Head, Department of Gynecology, Halifax Infirmary, Halifax, Nova Scotia, Professor, Dalhousie University, Halifax, Nova Scotia for reviewing the draft chapter.

Full Citation
Beaulieu M.D. Screening for D (Rh) sensitization in pregnancy. In: Canadian Task Force on the Periodic Health Examination. Canadian Guide to Clinical Preventive Health Care. Ottawa: Health Canada, 1994; 116-24.