Hemolytic Disease of the Newborn

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

Indications for Testing

  • Assess risk for alloimmune hemolytic disease of the newborn (HDN)

Laboratory Testing

  • Initial screening
    • Prenatal screening panel at first prenatal visit – should include RBC antibody detection test
      • Positive result – follow up with diagnostic testing to determine risk for alloimmune HDN
  • Follow-up testing to determine risk for HDN
    • Antigen genotyping
      • RhD antigen genotyping
        • Paternal testing – peripheral blood specimen
          • Determine RHD genotype (heterozygous or homozygous) in RhD phenotypically positive individual when reproductive partner has clinically significant alloantibody
          • Assess paternal contribution for risk of alloimmune HDN
        • Fetal testing
          • Appropriate only when mother is Rh-negative and father is heterozygous for RHD genotyping or unavailable for testing
          • Assess risk of alloimmune HDN in fetus
          • Two options for testing
            • Circulating cell-free DNA (cfDNA) for [DGG1] RHD genotyping (not available at ARUP Laboratories)
              • cfDNA is isolated from maternal plasma in maternal whole blood specimen to determine fetal RHD genotype and assess risk for HDN
            • RHD genotyping
              • Performed on amniotic fluid or chorionic villus sampling (CVS)
        • Follow-up
          • Fetuses predicted to be unaffected should continue to be monitored by noninvasive means – middle cerebral arterial (MCA) peak systolic velocity (PSV) Doppler ultrasound preferred; refer to Monitoring section for more information
          • Maternal serum antibody Rh titer
      • RHCE and KEL antigen genotyping
    • Amniotic bilirubin scan (also known as delta OD450 [ΔOD450])
      • May be used to assess alloimmune HDN and follow progression of disease to determine need for fetal transfusion or early delivery; however, noninvasive MCA PSV Doppler ultrasound is preferred testing
      • ΔOD450 is invasive and is not typically performed unless fetal transfusion is recommended (amniotic fluid can be collected during transfusion treatment)
      • May be evaluated in conjunction with fetal Rh genotyping
      • 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)

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

  • Initial screening
    • Prenatal screening panel at first prenatal visit – should include RBC antibody detection test
      • Positive result – follow up with diagnostic testing to determine risk for alloimmune hemolytic disease of the newborn (refer to Diagnosis section)

 

  • 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 births with maternal RhD alloimmunization in the U.S.
    • Dramatic decrease since introduction of anti-D immunoglobulin
  • Ethnicity
    • Incidence of RhD negativity
      • Caucasians – 15%
      • African Americans – 5%
      • Asians – <1%
    • Incidence of Kell antigen positivity (KEL1)
      • Up to 25% in Arabs
      • 9% in Caucasians
      • 2% in African Americans
      • K homozygosity is rare

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
  • >50 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 RBCs 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.

Prenatal Reflexive Panel 0095044
Method: Automated Cell Count/Differential/Semi-Quantitative Charcoal Agglutination/Qualitative Chemiluminescent Immunoassay/Semi-Quantitative Chemiluminescent Immunoassay/Hemagglutination/Solid Phase

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

Limitations 

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)

Diagnostic errors can occur due to rare sequence variations

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

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 

Evaluates only the KEL1 (K) and KEL2 (k) alleles of the KEL gene  

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

Diagnostic errors can occur due to rare sequence variations

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

Content Reviewed: 
December 2016

Last Update: August 2017