Rh Disease - Hemolytic Disease of the Newborn

Dx
Screen
Monitor
Background
Lab Tests

Diagnosis

Indications for Testing

High level of suspicion for disease (previous affected pregnancy or other known risk factors)

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
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

Infectious diseases
- Parvovirus
- TORCH syndrome
Chromosomal abnormalities
Hereditary blood disorders
- Hereditary hemolytic disease – eg, hereditary spherocytosis, elliptocytosis, pyruvate kinase deficiency
- Hemoglobinopathies (thalassemias)

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 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
>50 different implicated alloantibodies that vary across ethnic groups
- Non-Rh antigens can also cause HDN
- Antibodies
  - Anti-Rh (D, C, c, E, and e)
  - Anti-Kell (K and k)
  - Anti-Duffy (Fya)
  - Anti-Kidd (Jka and Jkb)
- 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)

Anemia
Erythroblastosis
Fetal hemolytic anemia
Hepatosplenomegaly
Hydrops fetalis
Jaundice
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 Rh status Assess fetal Rh status after pregnancy 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	Use for fetal testing for RhD when the mother has clinically significant alloantibody and the father is either heterozygous for RHD or not available for testing Use for paternal testing for RHD heterozygosity or homozygosity when the mother has clinically significant alloantibody Clinical sensitivity ≥98%	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 Most rare mutations in the RHD gene (ie, missense, nonsense, insertions, gene fusion, or small deletions) will not be detected by this assay, and the sample may be misinterpreted as being Rh-positive	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	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

Additional Tests Available

Click the plus sign to expand the table of additional tests.

Test Name and Number	Comments
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