Hemolytic Disease of the Newborn

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

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 peak systolic velocity (MCA-PSV) 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-PSV Doppler ultrasound (85% accurate)
    • MCA-PSV 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-PSV Doppler ultrasound (noninvasive) – useful in monitoring pregnancy if Rh incompatibility is suspected or identified

Hemolytic disease of the newborn (HDN), also known as alloimmune HDN or erythroblastosis fetalis, 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.
    • 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

  • RhD – autosomal recessive
  • Kell – autosomal dominant

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
    • Examples of 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
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 variants 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 variants

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 due to maternal cell contamination

Specificity may be compromised by variants 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 due to maternal cell contamination

Specificity may be compromised by variants 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 variants (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)

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 GA, Fernandes BJ. 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 AW. Errors in the assessment of hemolytic disease from amniotic fluid. Am J Obstet Gynecol. 1963; 86: 485-94. PubMed

Oepkes D, Seaward G, Vandenbussche FP, Windrim R, Kingdom J, Beyene J, Kanhai HH, 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 KJ, Pietz BC, Johnson ST, Pinder HL, Bellissimo DB. 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 JT, Tomai TP, Ural SH, King JC. 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 HP, Haberman BE, Ruddy RM. Hyperbilirubinemia: current guidelines and emerging therapies. Pediatr Emerg Care. 2011; 27(9): 884-9. PubMed

Zimring JC, Welniak L, Semple JW, Ness PM, Slichter SJ, Spitalnik SL, 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 JA, Cousin R, Pili AT, Erickson A, Ashwood ER. Evaluation of the efficacy of chloroform extraction of amniotic fluid bilirubin. Clin Chem. 2006; 52(11): 2120-1. PubMed

Christensen RD, Nussenzveig RH, Yaish HM, Henry E, Eggert LD, Agarwal AM. Causes of hemolysis in neonates with extreme hyperbilirubinemia. J Perinatol. 2014; 34(8): 616-9. PubMed

Yaish HM, Christensen RD, 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

Bayrak-Toydemir, Pinar, MD, PhD, Medical Director, Molecular Genetics and Genomics at ARUP Laboratories; Associate Professor of Clinical Pathology, University of Utah

Blaylock, Robert C., MD, Medical Director, Blood Services, and Phlebotomy and Support Services, Immunohematology Reference, Clinical Laboratory and Transfusion Services at the University of Utah Hospitals and Clinics at ARUP Laboratories ; Associate Professor of Clinical Pathology, University of Utah

Grenache, David G., PhD, Medical Director, Special Chemistry; Co-Director, Electrophoresis and Manual Endocrinology; Chief Medical Director, Clinical Chemistry at ARUP Laboratories; Associate Professor of Clinical Pathology, University of Utah

Heikal, Nahla, MD, MS, Assistant Medical Director, Immunology and Hemostasis/Thrombosis at ARUP Laboratories; Assistant Professor of Clinical Pathology, University of Utah

LaGrave, Danielle, MS, LCGC, Licensed Genetic Counselor, Maternal Serum Screening and Cytogenetics at ARUP Laboratories; Faculty, Genetic Counseling Graduate Program, Human Genetics, University of Utah

Lyon, Elaine, PhD, Medical Director, Genetics, and Co-Medical Director, Pharmacogenomics at ARUP Laboratories; Associate Professor of Clinical Pathology, University of Utah

Mao, Rong, MD, Medical Director, Molecular Genetics and Genomics at ARUP Laboratories; Associate Professor of Clinical Pathology and Co-director, Clinical Medical Genetics Fellowship Program, University of Utah

Velden, Sara, MS, LCGC, Genetic Counselor, Molecular Genetics and Cytogenetics at ARUP Laboratories

Wittwer, Carl T., MD, PhD, Medical Director and Technical Vice President, General Flow Cytometry at ARUP Laboratories; Professor of Clinical Pathology, University of Utah

Last Update: August 2016