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Disease Categories > Perinatal Disease

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Hemolytic Disease of the Newborn - Rh Disease

Hemolytic disease of the newborn (HDN) is a potentially fatal disease caused by immune destruction of fetal red blood cells via transplacentally acquired maternal antibodies.

Epidemiology

Incidence – 6-7/1,000 live births in U.S. (CDC 2002)
- Dramatic decrease in cases since introduction of anti-D immunoglobulin
Population incidence of RhD negativity
- Caucasian – 15%
- Afro American – 5%
- Asian – <1%

Risk Factors

Rh negative mother
- Partnered with Rh positive father
- Sensitized by previous blood transfusion
- Unrecognized miscarriage with transplacental hemorrhage
- Failure to receive anti-D immunoglobulin during and following previous pregnancy

Pathophysiology

Predominant mechanism
- Rh blood group is composed of 2 genes – RhD and RhCE
- Rh negative mother can be sensitized to Rh antigens by an Rh positive fetus in previous pregnancy
- Antibodies cross the placenta and cause immune destruction of Rh positive fetal red blood cells
Non-Rh antigens can also cause HDN (see below)

Types of Antibodies

More than 50 different implicated alloantibodies which vary across ethnic groups
Anti-D is the most common cause of HDN followed by anti-c, anti-K, and anti-E
Antibodies
- Anti-Rh (D, C, c, E, and e)
- Anti-Kell (K and k)
- Anti-Duffy (Fy^a)
- Anti-Kidd (Jk^a and Jk^b)

Clinical Presentation (varies with disease severity)

Anemia
Jaundice
Hepatosplenomegaly
Fetal hemolytic anemia
Hydrops fetalis (severe HDN)
Stillbirth

Diagnosis

High level of suspicion – previous affected pregnancy, known risk factors
Laboratory testing
- Amniotic bilirubin scan (also known as delta OD450)
  - Scanning spectrophotometry performed on amniotic fluid. The change in optical density at a wavelength of 450 nm is proportional to the concentration of bilirubin.
  - Serial measurements are recommended.
  - Results interpreted using a Liley or Queenan chart. The Queenan chart has been reported to have higher diagnostic accuracy for identifying severe anemia.
Genotyping for RhD – only necessary if mother is Rh negative and has alloantibody
- Paternal testing – if father is homozygous D, all offspring are positive and are at risk for HDN
- Fetal testing – if father is heterozygous D or unknown
Hemoglobin F – aids in detecting maternofetal hemorrhage

Differential Diagnosis

Parvovirus
TORCH syndrome

Disease Monitoring

Doppler ultrasonography (noninvasive) – measures velocity of blood flow through the middle cerebral artery.

Treatment

In utero transfusion if fetal anemia is severe
Early delivery if clinically indicated

Indications for Ordering

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	Maternal testing to assess Rh status Fetal testing after pregnancy Includes D, C, E, c, e
Kell Antigen Genotyping, (KEL1/KEL2) 0051644 Method: Polymerase Chain Reaction/Fluorescence Monitoring	Fetal testing when the mother has clinically significant alloantibody and the father of the pregnancy 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 his reproductive partner is KEL1-negative by RBC antigen typing	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 If the father of the pregnancy is determined to be homozygous for the KEL1 allele, all of his offspring can be assumed to be KEL1-positive, negating the need for fetal KEL1 testing
Amniotic Bilirubin Scan 0080276 Method: Spectrophotometry (Delta OD 450 nm)	Assess alloimmune hemolytic disease of the fetus Follow progression of disease to determine need for fetal transfusion or early delivery	Bloody amniotic fluid compromises the accuracy of results	Evaluate in conjunction with fetal Rh genotyping Ultrasound measurement of the middle cerebral artery blood velocity can estimate fetal anemia
Rh Genotyping D Antigen 0051368 Method: Polymerase Chain Reaction/Fluorescence Monitoring	Paternal testing to determine RhD heterozygosity or homozygosity in a phenotypically positive individual when his reproductive partner has clinically significant alloantibody If the father of the pregnancy is determined to be homozygous for the RhD allele, all of his offspring can be assumed to be RhD positive, negating the need for fetal RhD testing Fetal testing when the mother has clinically significant alloantibody and the father of the pregnancy is either heterozygous for RhD or not available for testing	Most rare mutations in the RhD gene (ie. missense, nonsense, insertions, gene fusion, or small deletions) will not be detected by this assay. In these cases, the sample may be misinterpreted as being Rh-positive
Rh Genotyping - C/c Antigen, Fetal 0050421 Method: Polymerase Chain Reaction/Fluorescent Monitoring	Fetal testing when a pregnant woman has a clinically significant alloantibody level and the father of the pregnancy is phenotypically positive for the Rh gene encoding the corresponding antigen	Bloody amniotic fluid samples may give false-negative results due to maternal cell contamination Individuals with weak or no expression of the Cc/Ee antigens may result from RhCE gene alterations such as RhCE-D-CE gene 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-DCE fusion genes
Rh Genotyping - E/e Antigen, Fetal 0050423 Method: Polymerase Chain Reaction/Fluorescent Monitoring	Fetal testing when a pregnant woman has a clinically significant alloantibody level and the father of the pregnancy is phenotypically positive for the Rh gene encoding the corresponding antigen	Bloody amniotic fluid samples may give false-negative results due to maternal cell contamination Individuals with weak or no expression of the Cc/Ee antigens may result from RhCE gene alterations such as RhCE-D-CE gene 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-DCE fusion genes

Additional Tests Available

Click on number for test-specific information in the ARUP Laboratory Test Directory

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	Should not be used to detect feto-maternal hemorrhage

Additional Information

Liley Chart
(Used with permission from Grenache, 2004, 231)

Queenan Chart
(Used with permission from Grenache, 2004, 235)

Guidelines

Management of hyperbilirubinemia in the newborn infant 35 or more weeks of gestation. American Academy of Pediatrics - Medical Specialty Society. 1994 Oct (revised 2004 Jul). 20 pages. NGC:003716 (Link to NGC)

Screening for Rh(D) incompatibility: recommendation statement. United States Preventive Services Task Force - Independent Expert Panel. 2004 Feb 24. 4 pages. NGC:003455 (Link to NGC)

Cited References

Grenache DG. Hemolytic Disease of the Newborn. In Gronowski AM, ed. Handbook of Clinical Laboratory Testing during Pregnancy. Totowa, NJ: Humana Press, 2004. pp. 219-244.

General References

Eder AF. Update on HDFN: new information on long-standing controversies. Immunohematol. 2006;22(4):188-195. (Link to PubMed)

LILEY AW. Errors in the assessment of hemolytic disease from amniotic fluid. Am J Obstet Gynecol. 1963;86:485-494. (Link to PubMed)

Moise KJ Jr. Management of rhesus alloimmunization in pregnancy. Obstet Gynecol. 2002;100(3):600-611. (Link to PubMed)

Murray NA, Roberts IA. Haemolytic disease of the newborn. Arch Dis Child Fetal Neonatal Ed. 2007;92(2):F83-F88. (Link to PubMed)

Oepkes D, Seaward PG, Vandenbussche FP, Windrim R, Kingdom J, Beyene J, Kanhai HH, Ohlsson A, Ryan G. Doppler ultrasonography versus amniocentesis to predict fetal anemia. N Engl J Med. 2006;355(2):156-164. (Link to 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):1370-1376. (Link to PubMed)

References from the ARUP Institute for Clinical and Experimental Pathology®

Bornhorst JA, Cousin R, Pili AT, Erickson JA, Ashwood ER. Evaluation of the efficacy of chloroform extraction of amniotic fluid bilirubin. Clin Chem. 2006;52(11):2120-2121. (Link to PubMed)

Reviewed by

Ashwood, Edward R., M.D. Senior Vice President, Director of Laboratories and Chief Medical Officer at ARUP Laboratories; Professor of Pathology, University of Utah

Bayrak-Toydemir, Pinar , M.D., Ph.D. Assistant Medical Director, Molecular Genetics at ARUP Laboratories; Assistant Professor (Clinical), Pathology, University of Utah

Grenache, David G., Ph.D. Medical Director, Special Chemistry at ARUP Laboratories; Assistant Professor, Clinical Pathology, University of Utah

Comprehensive Review: July 2008
Last Update: July 2008