Hemolytic Disease of the Fetus and Newborn

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 the 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.  At one time, most cases of severe HDFN were due to the RhD antigen; 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.   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

What types of events may increase the risk of alloimmunization, and how does this 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 the maternal circulation may lead to alloimmunization.   Common reasons for maternal alloimmunization include blood transfusion, fetomaternal hemorrhage, and intravenous drug abuse. 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.   Therefore, 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 (eg, 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).  Alloantibodies to RhD, along with Rhc, RhC, RhE, and Rhe antigens, may lead to HDFN of varying severity.  Antibodies to Kell antigens may result in severe HDFN.  Anti-A and anti-B antibodies of the ABO blood group in blood type O mothers commonly cause mild to moderate HDFN.  Other antigens associated with HDFN include Duffy, Kidd, MNSs, MSSs, Lutheran, Diego, Xg, P, and numerous public and private antigens.  Of note, immunoglobulin G (IgG) antibodies (but not IgM antibodies) cause HDFN because only IgG antibodies can cross the placenta.  

Should noninvasive fetal genotyping be used to guide selective anti-D immune globulin prophylaxis?

Currently, the prophylactic administration of anti-D immune globulin is routine in RhD-negative women without existing anti-D antibodies.  Up to 40% of RhD-negative women will carry an RhD-negative fetus, which could render routine administration of anti-D immune globulin prophylaxis unnecessary.  The use of noninvasive fetal genotyping to determine if a fetus is RhD-negative and thus determine the need for anti-D immune globulin prophylaxis has been suggested as a potential measure to prevent unnecessary exposure to the drug, but cost-benefit analyses do not show a consistent benefit from the use of fetal genotyping at this time.  Noninvasive fetal genotyping is therefore not currently routine in the United States, although this strategy may become more useful as the cost of genotyping decreases. 

Which testing algorithms are related to this topic?

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/Rh typing and an antibody screen are recommended for all pregnant women 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 RhD type is recommended for all pregnant women during the first prenatal visit.   A small subset of mothers may be a serologic weak D type. A serologic weak D is defined as ≤2+ reactivity on RhD forward typing, or a negative initial result that becomes positive after the addition of antihuman globulin (ie, a serologic weak D test). 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.  Weak D genotyping is not currently performed at ARUP Laboratories.

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-RhD antibodies should be investigated to determine whether they are the result of sensitization or previous injection of anti-D immune globulin; if the latter, 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, postpartum, and in association with any pregnancy event that may result in fetomaternal hemorrhage to confirm that RhD sensitization has not occurred.   Anti-D immune globulin treatment is only recommended for mothers who were not previously sensitized to RhD. 

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 mother, 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. If testing indicates that 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 RhD positive, and the RhD-negative mother is sensitized (eg, 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 D antigen, then all of his offspring will be RhD positive; if heterozygous, there is a 50% chance that the fetus will be RhD positive.  Genotyping can also be used to determine the paternal genotype for any other HDFN-associated antigen (eg, RhC/c, RhE/e, and Kell), but antigen typing using serology is often sufficient for specificities other than RhD. 

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 or alloimmunization. 

Polymerase chain reaction (PCR) on 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, it 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 currently in the U.S.   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 alloimmunized mothers who 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 from 8-32 at most centers. The recommended frequency of antibody titers also varies; the American College of Obstetricians and Gynecologists (ACOG) recommends monitoring for anti-D and anti-C every 4 weeks up to 28 weeks of gestation, and then every 2 weeks until delivery.  For all other antibodies except anti-Kell, ACOG recommends testing at 28 weeks.  Serial antibody titers should not be used to monitor fetal status in a mother who has had a previous pregnancy affected by HDFN due to the same antibody. 

Anti-Kell antibody titers may not correlate with fetal status.   An institution’s definition for the critical titer for anti-Kell may be lower (eg, 8) 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.  The Royal College of Obstetricians and Gynaecologists recommends testing for anti-Kell antibodies every 4 weeks through 28 weeks of gestation, and every 2 weeks thereafter. 

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.  Weekly monitoring is appropriate for pregnancies complicated by anti-D, anti-c, and anti-Kell antibodies; for all other antibodies, monitoring every 1-2 weeks is reasonable.  When MCA PSV reaches the critical threshold, intrauterine transfusion may be performed.

Postnatal Testing

Fetomaternal Hemorrhage Testing

Testing for fetomaternal hemorrhage is recommended for all RhD-negative women who have given birth to RhD-positive infants to guide anti-D immune globulin treatment.  The rosette fetal RBC assay is generally used as a first 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 fetal RBC rosette assay result.  These results are then used to calculate the appropriate dose of anti-D immune globulin.

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 is blood type O and/or RhD negative. ABO/RhD typing and/or a direct antiglobulin test (DAT) may be performed. In the case of an RhD-negative mother, if the neonatal RhD type is initially negative, a serologic weak D test is performed to determine whether or not anti-D immune globulin 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

Initial prenatal screen; determine presence of RBC antibodies 

Reflex patterns: If rapid plasma reagin (RPR) is weakly reactive or reactive, then a titer will be added; if antibody screen is positive, then antibody identification will be added; if results for hepatitis B virus surface antigen (HBsAg), prenatal are reactive, then HBsAg confirmation, prenatal will be added

Method

Automated Cell Count/Differential/Semi-Quantitative Charcoal Agglutination/Qualitative Chemiluminescent Immunoassay/Semi-Quantitative Chemiluminescent Immunoassay/Hemagglutination/Solid Phase

Includes RPR with reflex to titer; rubella antibody, IgG; ABO-Rh prenatal; antibody screen RBC with reflex to identification; CBC; HBsAg with reflex to confirmation, prenatal

Assess risk for HDFN

Includes K, Fya, Fyb, Jka, Jkb, S, s (k, cellano, testing performed if indicated)

Determine presence of RBC antibodies

Reflex patterns: for positive results for females 15-45 years of age, antibody identification, RBC (prenatal only) is added; for all other positive results, antibody ID package is added

Paternal and Fetal Testing

Determine the number of copies of the RHD gene

Assess risk for alloimmune HDFN or hemolytic transfusion reaction

Assess risk for alloimmune HDFN in the fetus due to RHCE gene-related alloimmunization

Initial screen for K antigen status (not for fetal use)

Fetal Status Monitoring

Maternal Antibody Titers

Determine presence of RBC antibodies

Reflex patterns: for positive results for females 15-45 years of age, antibody identification, RBC (prenatal only) is added; for all other positive results, antibody ID package is added

Postnatal Testing

Fetomaternal Hemorrhage Testing

Detect and quantify the extent of fetomaternal hemorrhage in pregnant or postpartum women

Cord Blood Testing

Determine neonatal blood type

Identify maternal antibodies bound to RBC antigens in the fetal circulation

Assess for hyperbilirubinemia

Medical Experts

Contributor
Contributor

Leonard

 

Nicole Leonard, MD

Pathology Resident, University of Utah

Contributor

Metcalf

Ryan A. Metcalf, MD, CQA(ASQ)
Assistant Professor of Clnical Pathology, University of Utah
Medical Director, Blood Services and Immunohematology Reference Laboratory, ARUP Laboratories

References

Additional Resources
Resources from the ARUP Institute for Clinical and Experimental Pathology®