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Hemolytic Disease of the Fetus and Newborn

Last Literature Review: February 2026 Last Update:

Medical Experts

Contributor
Contributor

Mao

Rong Mao, MD, FACMG
Rong Mao, MD, FACMG
Professor and Co-Director of Laboratory Genetics and Genomics Fellowship, University of Utah
Medical Director, Molecular Genetics and Genomics, ARUP Laboratories
Contributor

Tang

Mei San Tang, MD
Mei San Tang, MD
Assistant Professor (Clinical), University of Utah
Medical Director, Immunohematology Reference Lab and Blood Services, ARUP Laboratories

Hemolytic disease of the fetus and newborn (HDFN), also known as hemolytic disease of the newborn (HDN) or alloimmune HDFN, is a potentially severe alloimmune condition that may, on rare occasions, result in stillbirth or neonatal death. ,  HDFN occurs when fetal red blood cells (RBCs) are destroyed by maternal alloantibodies that have crossed into fetal circulation, leading to hemolysis and anemia.  Maternal alloantibodies may develop against fetal RBC antigen(s) not possessed by the mother and typically result from previous transfusion or pregnancy.  Previously, most cases of severe HDFN were due to the D antigen in the Rh system; however, the introduction of anti-D immune globulin prophylaxis has dramatically decreased the incidence of HDFN in the United States. ABO blood group incompatibility is now the most common cause of HDFN. ,  Laboratory testing for HDFN may be useful for prenatal risk assessment, pre- and postnatal diagnosis, and prenatal monitoring of disease progression.

Quick Answers for Clinicians

Is noninvasive fetal genotyping for RHD available to guide selective anti-D immune globulin prophylaxis?

Noninvasive RHD genotyping for determination of fetal D status is only available in a limited manner in the United States and therefore is not used in the standard of care. Up to 40% of D-negative individuals will carry a D-negative fetus, which could render routine administration of anti-D immune globulin prophylaxis unnecessary.  The use of noninvasive fetal genotyping has been suggested to determine if a fetus is D-negative and assess the need for anti-D immune globulin prophylaxis.  However, cost-benefit analyses do not show a consistent benefit from the use of fetal genotyping at this time.  Noninvasive fetal genotyping is not currently routine in the U.S. and may only be considered in special situations (such as during a shortage of available anti-D immune globulin and increased patient triage). , 

What types of events may increase the risk of alloimmunization, and how does alloimmunization affect testing for hemolytic disease of the fetus and newborn?

Any ante- or perinatal event that introduces a sufficient number of nonself red blood cells (RBCs) into maternal circulation may lead to alloimmunization. ,  Common reasons for maternal alloimmunization include previous pregnancy, blood transfusion, fetomaternal hemorrhage, and transplantation; possible association with intravenous drug abuse has also been described. Fetomaternal alloimmunization may occur with ectopic pregnancies, miscarriages, antenatal bleeding, multifetal gestation, placenta previa or abruption, and delivery. ,  A number of obstetric procedures may also result in fetomaternal alloimmunization, including amniocentesis, chorionic villus sampling, uterine curettage, external cephalic version, and other surgeries. ,  The use of chorionic villus biopsy is discouraged in testing for hemolytic disease of the fetus and newborn (HDFN) because it is more likely to cause fetomaternal hemorrhage and worsen alloimmunization than other techniques (e.g., amniocentesis). 

What are the clinical implications of hemolytic disease of the fetus and newborn?

Hemolytic disease of the fetus and newborn (HDFN) may present with fetal anemia, hyperbilirubinemia, and jaundice due to hemolysis.  If left untreated, hydrops fetalis, premature birth, stillbirth, or neonatal death may occur.  The severity of HDFN varies greatly and depends on antigen specificity and the amount and type of antibodies present. 

Which red blood cell antigens are associated with hemolytic disease of the fetus and newborn?

Antibodies against red blood cell (RBC) antigens in the Rh system are often associated with hemolytic disease of the fetus and newborn (HDFN), and the majority of these HDFN-associated Rh antibodies are directed to the D, C, c, and E antigens.  Anti-K is another important HDFN-associated alloantibody, as the K antigen from the Kell system is expressed on early red cell precursors; anti-K consequently causes less hemolysis but more severe HDFN. Less commonly, alloantibodies to the Kp(a), Kp(b), Ku, Js(a), Js(b), Fy(a), Fy(b), S, s, and U antigens have also been implicated in moderate to severe HDFN.  Of note, immunoglobulin G (IgG) antibodies (but not IgM antibodies) cause HDFN because only IgG antibodies can cross the placenta. ,  Hence, even though anti-A and anti-B alloantibodies are often present as IgM, anti-A and anti-B alloantibodies are also present as IgG antibodies in individuals with type O blood and are capable of causing mild to moderate HDFN in non-O infants born to mothers with type O blood. , , 

Indications for Testing

Laboratory testing for HDFN is used to:

  • Assess risk for developing disease
  • Diagnose HDFN, both pre- and postnatally
  • Monitor disease progression

Laboratory Testing

Risk Assessment and Diagnosis

Maternal Testing

ABO/D typing and an antibody screen are recommended for all pregnant individuals to assess the risk of HDFN development, identify alloantibodies, and determine the need for anti-D immune globulin prophylaxis. 

Maternal Blood Typing

Testing for maternal ABO blood group and D type is recommended for all pregnant individuals during their first prenatal visit. ,  A small subset of mothers may have a serologic weak D type. A serologic weak D type is defined as ≤2+ reactivity with immediate spin (i.e., without addition of antihuman globulin) on D forward typing or a negative initial result that becomes positive after the addition of antihuman globulin (i.e., a serologic weak D test). RHD genotyping is required to determine whether mothers with serologic weak D results are at risk of alloimmunization against the D antigen. Weak D genotypes 1, 2, and 3 are not associated with alloimmunization to the D antigen, whereas other genotyping results may suggest risk, in which case anti-D immune globulin prophylaxis would be required. 

Maternal Testing for Unexpected Red Blood Cell Antibodies

Testing for unexpected RBC antibodies with an antibody screen is recommended at the first prenatal visit for each pregnancy. , ,  If detected, anti-D antibodies should be investigated to determine whether they are the result of sensitization or previous injection of anti-D immune globulin; if they are due to the latter cause, continued anti-D immune globulin treatment is recommended, as indicated.  Antibody screening is also recommended before anti-D immune globulin treatment at 28 weeks of gestation, during the postpartum period, and in association with any pregnancy event that may result in fetomaternal hemorrhage to confirm that D sensitization has not occurred. ,  Anti-D immune globulin treatment is only recommended for mothers who were not previously sensitized to the D antigen.

Antibody testing is also useful for ensuring compatibility of blood products, should they be needed. 

Paternal and Fetal Testing

If an unexpected alloantibody associated with HDFN is identified in a pregnant individual, determination of the paternal and/or fetal status for the implicated antigen is recommended to inform monitoring. ,  If testing reveals that the fetus is negative for the antigen of interest in a sensitized mother, no further action is required; however, if the fetus is positive for the antigen of interest in a sensitized mother, monitoring is required to ensure appropriate intervention.

Determination of Paternal Genotype

If the father of the fetus is negative for the antigen to which the mother has been alloimmunized (and paternity is assured), no further testing is necessary.  If the father is D positive and the mother is D negative and sensitized (e.g., due to a blood transfusion or a previous pregnancy without anti-D immune globulin prophylaxis), homozygosity or heterozygosity for RHD can be determined by means of RHD genotyping.  If the father is homozygous for the conventional RHD allele, then all of his offspring will be D positive; if heterozygous, there is a 50% chance that the fetus will be D positive.  For the other non-D minor RBC antigens, except when rare variant alleles are suspected, antigen typing using serology is often sufficient for determination of zygosity. 

Determination of Fetal Genotype

If the father of the fetus is heterozygous for the antigen to which the mother has been alloimmunized, or if the father is unknown or unavailable for testing, fetal genotyping can be used to determine the risk of HDFN. ,  In some cases, it may be reasonable to postpone fetal genotyping until maternal antibody titers would merit fetal monitoring because invasive testing does carry risks of miscarriage and alloimmunization. 

Polymerase chain reaction (PCR) testing of amniotic fluid obtained via amniocentesis is the most common invasive technique used to determine the fetal genotype.  The use of chorionic villus samples is not recommended due to the risk of fetomaternal hemorrhage.  Noninvasive fetal RHD genotyping with maternal cell-free, fetal-derived DNA can be used to avoid these procedure-related risks; however, this testing is not routinely performed in the U.S. at this time. Noninvasive genotyping for other HDFN-associated antigens may also be employed in the future but is not common practice in the U.S. currently. ,  If the results of noninvasive genotyping are inconclusive, or if the antigen in question cannot be noninvasively genotyped, invasive testing may be warranted.  If the fetus is negative for the antigen in question, further testing may not be necessary, although noninvasive monitoring can be considered. 

Fetal Status Monitoring

Maternal Antibody Titers

Antibody titers can be used to monitor the risk of HDFN in pregnancies affected by alloimmunization.  In mothers who are alloimmunized and have not had a previous HDFN-affected pregnancy, the titer of maternal antibodies against the antigen in question can be used to determine whether intervention may be necessary.  A critical antibody titer indicates significant risk for HDFN and hydrops fetalis and requires fetal monitoring with ultrasound. , 

Definitions of critical titer thresholds vary, ranging between 1:8 and 1:32 at most centers. The recommended frequency of antibody titers also varies; the American College of Obstetricians and Gynecologists (ACOG) notes that monitoring may occur at monthly intervals following an initial titer of 1:8 or less. Serial antibody titers should not be used to monitor fetal status once the titer has reached a critical threshold, and they should not be used in a mother who has had a previous pregnancy affected by HDFN due to the same antibody.  Ultrasound monitoring of the fetus should then be performed. ,  Importantly, Kell antibodies do not show correlation with fetal status and should not be used to guide care. 

The titers of anti-K alloantibodies or antibodies to the other antigens in the Kell system may not correlate with fetal status. ,  An institution’s definition for the critical titer for anti-Kell may be lower than for other antibodies given its association with severe HDFN. Referral to a specialist for monitoring with fetal ultrasound is recommended once any anti-Kell antibodies are detected. 

Fetal Monitoring

Imaging, specifically noninvasive fetal middle cerebral artery (MCA) peak systolic velocity (PSV) Doppler ultrasound, is preferred for monitoring the progression of HDFN to determine the need for fetal transfusion or early delivery.  MCA PSV Doppler ultrasound requires a trained practitioner, and the fetus must be of an appropriate age.  When MCA PSV reaches the critical threshold, intrauterine transfusion may be performed. The accuracy of MCA Doppler assessment is reduced after 35 weeks of gestation. 

Postnatal Testing

Fetomaternal Hemorrhage Testing

Testing for fetomaternal hemorrhage is recommended for all D-negative individuals who have given birth to D-positive infants to guide anti-D immune globulin treatment.  The rosette fetal RBC assay is generally used as a first-line test for hemorrhage.  A quantitative test to determine the percentage of fetal RBCs in the maternal circulation, such as the Kleihauer-Betke test or flow cytometry, is the recommended follow-up approach after a positive rosette fetal RBC assay result.  These results are then used to calculate the appropriate dose of anti-D immune globulin prophylaxis.

Cord Blood Testing

Cord blood testing is recommended whenever a mother has known clinically significant RBC antibodies.  Cord blood testing may also be performed if a mother has type O blood and/or is D negative. ABO/D typing and/or a direct antiglobulin test (DAT) may be performed. In the case of a D-negative mother, if the neonatal D type is initially negative, a serologic weak D test is performed to determine whether or not anti-D immune globulin prophylaxis is needed.

Hemoglobin and bilirubin levels can be assessed to determine the degree of anemia and hemolysis in the neonate.  Regular hemoglobin and bilirubin testing may be warranted to manage anemia and hemolysis. 

ARUP Laboratory Tests

Risk Assessment and Diagnosis

Maternal Testing
Paternal Testing
Fetal Testing

Postnatal Testing

Fetomaternal Hemorrhage Testing
Cord Blood Testing

References

  1. AABB - Perinatal issues in transfusion medicine

    Andrews J, Clarke G. Perinatal issues in transfusion medicine. In: Cohn CS, Delaney M, Johnson ST, et al, eds. Technical Manual. 21st ed. Association for the Advancement of Blood and Biotherapies; 2023:709-728.