Rh Disease - Hemolytic Disease of the Newborn

Diagnosis

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)
      • Liley Curve

        Reproduced with permission from Humana Press.  Liley Chart. (Used with permission from Grenache, 2004, 231)

      • Queenan chart


        Reproduced with permission from Humana Press.  Queenan Chart. (Used with permission from Grenache, 2004, 235)

  • Postnatal testing
    • Initial lab test postnatal – bilirubin
  • 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
  • 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
        • 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 antigen incompatibility
      • If father is homozygous for KEL1, all of his offspring can be assumed to be KEL1-positive, negating need for fetal KEL1 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

Screening

  • Genotyping for RHD – only necessary if the mother is Rh- 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

Monitoring

  • MCA Doppler ultrasound (noninvasive) – useful in monitoring pregnancy if Rh incompatibility is suspected or identified

Clinical Background

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 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%

Inheritance

  • Autosomal recessive

Risk Factors

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

Pathophysiology

  • 13% of hydrops fetalis (severe HDN) is caused by antigen-antibody mediated red-cell hemolysis
  • Predominant mechanism – Rh-mediated disease
    • Rh blood group is composed of 2 genes – RHD and RHCE
    • Rh- mother may be sensitized to Rh antigens by an Rh+ fetus during previous pregnancy
      • Antibodies cross the placenta and cause immune destruction of Rh+ red blood cells in fetus
  • >40 different implicated alloantibodies that vary across ethnic groups
    • Non-Rh antigens can also cause HDN
    • Antibodies associated with HDN
      • Anti-Rh (D, C, c, E, and e)
      • Anti-Kell (24 members)
      • 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
  • 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)

  • Fetal hemolytic anemia
  • Jaundice
  • Hepatosplenomegaly
  • Erythroblastosis
  • Hydrops fetalis
  • Stillbirth

Treatment

  • >90% survival if anemia is diagnosed and treated early
  • In utero transfusion if fetal anemia is severe
  • Early delivery if clinically indicated

Prevention

  • Fetal hemoglobin determination for fetomaternal hemorrhage is used to assess need for RhoGAM in Rh- 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
Test Name and Number Recommended Use Limitations Follow Up
Antigen Testing, Rh Phenotype 0013019
Method: Hemagglutination

Assess maternal, paternal, or fetal Rh status after delivery

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

   
Amniotic Bilirubin Scan 0080276
Method: Quantitative Spectrophotometry

Assess alloimmune hemolytic disease of the fetus

Follow progression of disease to determine need for fetal transfusion or early delivery

Evaluate in conjunction with fetal Rh genotyping

Bloody amniotic fluid compromises accuracy

Ultrasound measurement of the middle cerebral artery blood velocity can estimate fetal anemia
Fetal Hemoglobin Determination for Fetomaternal Hemorrhage 2001743
Method: Quantitative Flow Cytometry

Detect and quantify the extent of fetomaternal hemorrhage in pregnant or postpartum women and to assess the need for Rh immune globulin (eg, RhoGAM) for fetomaternal hemorrhage

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

Determine paternal RHD genotype (heterozygous or homozygous) in phenotypically positive individual when reproductive partner has clinically significant alloantibody

  • Include father’s ethnicity and RhD phenotyping result with order
  • If father of pregnancy is homozygous for RHD allele, all of his offspring can be assumed to be RhD-positive, negating the need for fetal RHD testing

Determine fetal genotype when mother has clinically significant alloantibody AND father is heterozygous for RHD or not available for testing

  • Include parent’s ethnicity and RhD phenotyping result with order

Clinical sensitivity ≥98%

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

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

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

Fetal testing when the mother has a clinically significant alloantibody level, and the father is phenotypically positive for the RHCE gene encoding the corresponding antigen

Polymorphisms tested – c.48C>G (p.W16C); c.201A>G (p.S67S), c.203A>G (p.N68S) and an intron 2 insertion

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

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

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

Fetal testing when mother has clinically significant alloantibody level and father is phenotypically positive for the RHCE gene encoding the corresponding antigen

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

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

Fetal testing when mother has clinically significant alloantibody and father is either heterozygous for KEL1 or not available for testing

Paternal testing in a KEL1 RBC antigen-positive individual to determine KEL1 heterozygosity or homozygosity when the mother is KEL1-negative by RBC antigen typing

Clinical sensitivity 99%

KEL antigens other than KEL1/KEL2 are not evaluated by this assay

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

 
Kell Antigen Typing - Patient 2007731
Method: Hemagglutination

Identify Kell antibody type

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

Use to determine fetal RHD and fetal gender

RHD genotyping is appropriate only when the mother is Rh-negative and the father is heterozygous for RHD or not available for testing

Useful after 10 weeks gestation

Clinical sensitivity – 98%

Analytic sensitivity/specificity – 99%/98.3%

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
 
Click the plus sign to expand the table of additional tests.
Test Name and NumberComments
Hemoglobin F 0081348
Method: High Performance Liquid Chromatography
Kleihauer-Betke Stain for Fetal Hemoglobin 0040105
Method: Semi-Quantitative Acid Elution Eosin Stain/Microscopy

Do not use to detect fetomaternal hemorrhage

ABO-Rh Type 0010025
Method: Hemagglutination
Bilirubin, Direct, Serum or Plasma 0020033
Method: Quantitative Spectrophotometry