Hemolytic Disease of the Newborn

  • Diagnosis
  • Screening
  • Monitoring
  • Background
  • Lab Tests
  • References
  • Related Content

Indications for Testing

  • Assess risk for alloimmune hemolytic disease of the newborn in fetus or father of pregnancy

Laboratory Testing

  • Prenatal testing
    • Amniotic bilirubin scan (also known as ΔOD450)
      • Amniotic fluid testing is invasive, and bilirubin scan is not typically performed unless fetal transfusion is recommended (amniotic fluid can be collected during transfusion treatment)
        • Preferred testing is middle cerebral artery (MCA) ultrasound (see Imaging)
      • Scanning spectrophotometry performed on amniotic fluid (serial measurement recommended)
        • Change in optical density at a wavelength of 450 nm is proportional to bilirubin concentration
      • Results interpreted using a Liley or Queenan chart (Queenan chart is reported to have higher diagnostic accuracy for identifying severe anemia)
  • Pre- and postnatal testing
    • Fetal hemoglobin determination – aids in detecting fetomaternal hemorrhage
    • Maternal serum antibody Rh titer – >15 IU/mL indicates high risk of severe fetal anemia
    • Noninvasive Prenatal Testing (NIPT) Rh screening – determines fetal RhD
    • Initial postnatal lab test – bilirubin
  • Antigen genotyping to assess risk for Rh disease in an infant
    • RhD – most important antibody test (RhD causes ~50% of maternal alloimmunization cases)
      • Fetal testing for RhD
      • Paternal testing for RHD heterozygosity or homozygosity
        • Autosomal recessive inheritance
        • If father is homozygous for the RHD allele, offspring can be assumed to be RhD positive, negating need for fetal RhD testing
    • RhCc/ RhEe – fetal tests for fetomaternal antigen incompatibility (usually ordered together)
    • Kell K/k – test for fetomaternal or transplant-related K hemolytic risk
      • Consider after RhD and ABO incompatibility have been ruled out
      • Autosomal dominant inheritance
      • If father is homozygous for Kell allele, all offspring can be assumed to be Kell positive, negating need for fetal Kell testing

Imaging

  • MCA Doppler ultrasound (85% accurate)
    • MCA imaging is superior to ΔOD450 amniotic fluid testing and is preferred in most cases of suspected or identified Rh incompatibility

Differential Diagnosis

  • Genotyping for RHD – only necessary if the mother is Rh negative and has alloantibodies
    • Paternal testing – if the father is homozygous D, all offspring are positive and are at risk for hemolytic disease of the newborn
    • Fetal testing – if the father is heterozygous D or unknown
  • MCA Doppler ultrasound (noninvasive) – useful in monitoring pregnancy if Rh incompatibility is suspected or identified

Hemolytic disease of the newborn (HDN) is a potentially fatal alloimmune condition where fetal red blood cells are destroyed by transplacentally acquired maternal antibodies. RhD is the most common offending paternal antigen.

Epidemiology

  • Incidence – 6-7/1,000 live RhD births in the U.S. (CDC 2001)
    • Dramatic decrease since introduction of anti-D immunoglobulin
  • Ethnicity
    • Incidence of RhD negativity
      • Caucasian – 15%
      • African American – 5%
      • Asian – <1%
    • Incidence of Kell antigen positivity
      • Up to 25% in Arabs
      • 9% in Caucasians
      • 2% in African Americans
      • K homozygosity is rare
      • ~4% of K negative (k/k) mothers will deliver a K positive fetus with potential hemolytic disease of the newborn

Inheritance

  • Autosomal recessive – RhD
  • Autosomal dominant – Kell

Risk Factors

  • Rh negative mother in combination with one or more of the following
    • Rh positive paternal partner
    • Previous blood transfusion
    • Failure to receive anti-D immunoglobulin (RhoGAM)
      • During and following a previous pregnancy
      • Transplacental hemorrhage following an unrecognized miscarriage
  • Kell negative mother in combination with
    • Kell positive paternal partner
    • Previous blood transfusion
    • Previous pregnancy with K positive baby

Pathophysiology

  • 13% of hydrops fetalis (severe HDN) is caused by antigen-antibody mediated red-cell hemolysis from previously transplacentally transferred maternal antibodies
  • >40 different implicated alloantibodies that vary across ethnic groups
    • Antibodies associated with HDN
      • Anti-Rh (D, C, c, E, and e)
      • Anti-A and Anti-B in mothers of blood type O
      • Anti-Kell (25 identified antigens)
      • Anti-Duffy (Fya and Fyb)
      • Anti-Kidd (Jka and Jkb)
      • Anti-K (K1)
      • Anti-MNSU (M, N, S, s, and U)
      • Anti-A and Anti-B in mothers of blood type O
    • Anti-D is the most common cause of HDN, followed by anti-c, anti-K, and anti-E
  • HDN mediated disease
    • Rh or Kell negative mother may be sensitized to antigens from a positive fetus during previous pregnancy
      • Antibodies cross the placenta and cause immune destruction of red blood cells in fetus
  • Anemia due to hemolysis leads to reduced oxygen delivery, resulting in the following complications
    • Endothelial damage
    • Increase in capillary permeability with fetal hypoproteinemia and ascites
    • Extramedullary hematopoiesis with decreased liver function
    • Reduced protein synthesis, culminating in the hydrops process

Clinical Presentation (varies with disease severity)

  • Symptoms may be noted as early as 20 weeks in utero
  • Fetal hemolytic anemia – may be severe
  • Jaundice
  • Hepatosplenomegaly
  • Erythroblastosis
  • Hydrops fetalis
  • Stillbirth

Prevention

  • Fetal hemoglobin determination for fetomaternal hemorrhage is used to assess need for RhoGAM in Rh negative mothers during and following pregnancy

Indications for Laboratory Testing

Tests generally appear in the order most useful for common clinical situations.
Click on number for test-specific information in the ARUP Laboratory Test Directory

Antigen Testing, Rh Phenotype 0013019
Method: Hemagglutination

Limitations

Assess maternal, paternal, or fetal Rh status after delivery

Antigen testing for D, C, E, c, e

Amniotic Bilirubin Scan 0080276
Method: Quantitative Spectrophotometry

Limitations

Bloody amniotic fluid compromises accuracy

Follow Up

Ultrasound measurement of the middle cerebral artery blood velocity can estimate fetal anemia 

Fetal Hemoglobin Determination for Fetomaternal Hemorrhage 2001743
Method: Quantitative Flow Cytometry

RhD Antigen (RhD) Genotyping 0051368
Method: Polymerase Chain Reaction/Fluorescence Monitoring

Limitations

This assay cannot differentiate between typical RhD and weak RhD alleles

Most rare mutations in the RHD gene (ie, missense, nonsense, insertions, gene fusion, or small deletions) will not be detected by this test

  • In these cases, the sample may be misinterpreted as being RhD-positive (false-positive)

Rare diagnostic errors may result from primer-site mutations

Bloody amniotic fluid specimens may give false-negative results due to maternal-cell contamination

Follow Up

Fetuses predicted to be unaffected should continue to be monitored by noninvasive means

RhCc Antigen (RHCE) Genotyping 0050421
Method: Polymerase Chain Reaction/Fluorescence Monitoring

Limitations

Bloody amniotic fluid specimens may give false-negative results because of maternal cell contamination

Specificity may be compromised by mutations in primer sites or those outside the RHCE exons examined

Weak or no expression of the Cc/Ee antigens may result from RHCE gene alterations such as RHCE-D-CE hybrids; other hybrids allow for expression of the C, c, or e antigens on the RHD allele

Genotyping may result in false-negative RhC, Rhc, or Rhe predictions due to RHCE-D-CE fusion genes

Clinical sensitivity: unknown

Follow Up

Fetuses predicted to be unaffected should continue to be monitored by noninvasive means

RhEe Antigen (RHCE) Genotyping 0050423
Method: Polymerase Chain Reaction/Fluorescence Monitoring

Limitations

Bloody amniotic fluid specimens may give false-negative results from maternal cell contamination

Specificity may be compromised by mutations in primer sites or those outside the RHCE exons examined

Weak or no expression of Cc/Ee antigens may result from RHCE gene alterations such as RHCE-D-CE hybrids; other hybrids allow for expression of the C, c, or e antigens on the RHD allele

Genotyping may result in false-negative RhC, Rhc, or Rhe predictions due to RHCE-D-CE fusion genes

Clinical sensitivity unknown

Follow Up

Fetuses predicted to be unaffected should continue to be monitored by noninvasive means

Kell K/k Antigen (KEL) Genotyping 0051644
Method: Polymerase Chain Reaction/Fluorescence Monitoring

Limitations

Only evaluates Kell antigen K/k

Bloody amniotic fluid specimens may give false-negative results due to maternal cell contamination

Diagnostic errors can occur due to rare sequence variation

Kell Antigen Typing - Patient 2007731
Method: Hemagglutination

Non-Invasive Prenatal Testing for RhD Genotyping, Fetal 2009077
Method: Mass Spectrometry

Limitations

Mother must be at least 10 weeks pregnant and have Rh negative blood type

Test is specific for RHD gene – other causes of alloimmune hemolytic disease will not be detected

Rare mutations  (eg, missense, nonsense, insertions, gene fusion, or small deletions) will not be detected

  • In these cases, the sample may test as RHD-positive and be misinterpreted as RhD-positive (false-positive)

Additional Tests Available

Hemoglobin F 0081348
Method: High Performance Liquid Chromatography

Kleihauer-Betke Stain for Fetal Hemoglobin 0040105
Method: Semi-Quantitative Acid Elution Eosin Stain/Microscopy

Comments

Do not use to detect fetomaternal hemorrhage

ABO-Rh Type 0010025
Method: Hemagglutination

Bilirubin, Direct, Serum or Plasma 0020033
Method: Quantitative Spectrophotometry

Guidelines

U.S. Preventive Services Task Force. Screening of infants for hyperbilirubinemia to prevent chronic bilirubin encephalopathy: recommendation statement. Am Fam Physician. 2010; 82(4): 408-10. PubMed

General References

Denomme G, Fernandes B. Fetal blood group genotyping. Transfusion. 2007; 47(1 Suppl): 64S-8S. PubMed

Grenache D. Hemolytic Disease of the Newborn. In Gronowski AM. Handbook of Clinical Laboratory Testing during Pregnancy, Totowa, NJ: Humana Press, 2004.

Illanes S, Soothill P. Management of red cell alloimmunisation in pregnancy: the non-invasive monitoring of the disease. Prenat Diagn. 2010; 30(7): 668-73. PubMed

LILEY A. Errors in the assessment of hemolytic disease from amniotic fluid. Am J Obstet Gynecol. 1963; 86: 485-94. PubMed

Oepkes D, Seaward G, Vandenbussche F, Windrim R, Kingdom J, Beyene J, Kanhai H, Ohlsson A, Ryan G, DIAMOND Study Group. Doppler ultrasonography versus amniocentesis to predict fetal anemia. N Engl J Med. 2006; 355(2): 156-64. PubMed

Pirelli K, Pietz B, Johnson S, Pinder H, Bellissimo D. Molecular determination of RHD zygosity: predicting risk of hemolytic disease of the fetus and newborn related to anti-D. Prenat Diagn. 2010; 30(12-13): 1207-12. PubMed

Queenan J, Tomai T, Ural S, King J. Deviation in amniotic fluid optical density at a wavelength of 450 nm in Rh-immunized pregnancies from 14 to 40 weeks' gestation: a proposal for clinical management. Am J Obstet Gynecol. 1993; 168(5): 1370-6. PubMed

Schwartz H, Haberman B, Ruddy R. Hyperbilirubinemia: current guidelines and emerging therapies. Pediatr Emerg Care. 2011; 27(9): 884-9. PubMed

Zimring J, Welniak L, Semple J, Ness P, Slichter S, Spitalnik S, NHLBI Alloimmunization Working Group. Current problems and future directions of transfusion-induced alloimmunization: summary of an NHLBI working group. Transfusion. 2011; 51(2): 435-41. PubMed

References from the ARUP Institute for Clinical and Experimental Pathology®

Bornhorst J, Cousin R, Pili A, Erickson A, Ashwood E. Evaluation of the efficacy of chloroform extraction of amniotic fluid bilirubin. Clin Chem. 2006; 52(11): 2120-1. PubMed

Christensen R, Nussenzveig R, Yaish H, Henry E, Eggert L, Agarwal A. Causes of hemolysis in neonates with extreme hyperbilirubinemia. J Perinatol. 2014; 34(8): 616-9. PubMed

Yaish H, Christensen R, Agarwal A. A neonate with Coombs-negative hemolytic jaundice with spherocytes but normal erythrocyte indices: a rare case of autosomal-recessive hereditary spherocytosis due to alpha-spectrin deficiency. J Perinatol. 2013; 33(5): 404-6. PubMed

Medical Reviewers

Last Update: January 2016