Hemolytic Disease of the Newborn

Clinical Background

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*)
- Anti-Kidd (Jk* and Jk**)

Clinical Presentation (varies with disease severity)

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

Treatment

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

See Also

Hemoglobinopathies

Hemolytic Anemias

Tests

Tests generally appear in the order most useful for common clinical situations

Test name: Antigen Testing, Rh Phenotype

ARUP #: 0013019

Methodology: Hemagglutination

Use:

Maternal testing to assess Rh status

Fetal testing after pregnancy

Includes D, C, E, c, e

Test name: Kell Antigen Genotyping, (KEL1/KEL2)

ARUP #: 0051644

Methodology: Polymerase Chain Reaction/Fluorescence Monitoring

Use:

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

Limitations:

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

Test name: Amniotic Bilirubin Scan

ARUP #: 0080276

Methodology: Spectrophotometry (Delta OD 450 nm)

Use:

Assess alloimmune hemolytic disease of the fetus

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

Limitations:

Bloody amniotic fluid compromises the accuracy of results

Follow-up:

Evaluate in conjunction with fetal Rh genotyping

Ultrasound measurement of the middle cerebral artery blood velocity can estimate fetal anemia

Test name: Rh Genotyping D Antigen

ARUP #: 0051368

Methodology: Polymerase Chain Reaction/Fluorescence Monitoring

Use:

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

Limitations:

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

Test name: RhCc Antigen (RHCE) Genotyping

ARUP #: 0050421

Methodology: Polymerase Chain Reaction/Fluorescent Monitoring

Use:

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

Limitations:

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

Test name: RhEe Antigen (RHCE) Genotyping

ARUP #: 0050423

Methodology: Polymerase Chain Reaction/Fluorescent Monitoring

Use:

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

Limitations:

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

Test name: Hemoglobin F

ARUP #: 0081348

Methodology: High Performance Liquid Chromatography

Comments:

Test name: Kleihauer-Betke Stain for Fetal Hemoglobin

ARUP #: 0040105

Methodology: Semi-Quantitative Acid Elution Eosin Stain/Microscopy

Comments: Should not be used to detect feto-maternal hemorrhage

Test name: ABO-Rh Type

ARUP #: 0010025

Methodology: Hemagglutination

Comments:

Additional Information

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

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

References

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

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

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.

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

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

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

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.

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.

References from the ARUP Institute for Clinical and Experimental Pathology Research®

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.

Medical Reviewers

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