Alloimmune Hemolytic Disease of the Fetus and Newborn (RhCc, RhEe, RhD, or Kell Antigen Genotyping)

RhC/c (RHCE) Antigen Genotyping 3002002
Method: Polymerase Chain Reaction/Fluorescence Monitoring

Genotyping to assess Rh blood group RHCE*2 (C) and RHCE*4 (c)

Use to determine paternal RhCc genotype when reproductive partner has clinically significant alloantibody

Determine fetal RhCc genotype when mother has clinically significant alloantibody AND father is either heterozygous or unavailable for testing

Immucor PreciseType HEA Molecular BeadChip which is FDA-approved for clinical testing

RhD Gene (RHD) Copy Number 0051368
Method: Polymerase Chain Reaction/Fluorescence Monitoring

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

Determine fetal RHD gene copy number when mother has clinically significant alloantibody AND father is either heterozygous for RHD or unavailable for testing

This test does not assess for weak or partial RHD genotypes

RhE/e (RHCE) Antigen Genotyping 3002003
Method: Polymerase Chain Reaction/Fluorescence Monitoring

Genotyping to assess Rh blood group RHCE*3 (E) and RHCE*5 (e)

Use to determine paternal RhEe genotype when reproductive partner has clinically significant alloantibody

Determine fetal RhEe genotype when mother has clinically significant alloantibody AND father is either heterozygous or unavailable for testing

Immucor PreciseType HEA Molecular BeadChip which is FDA-approved for clinical testing

Kell K/k (KEL) Antigen Genotyping 3002001
Method: Polymerase Chain Reaction/Fluorescence Monitoring

Genotyping to assess Kell blood group KEL*01 (K), KEL*02 (k)

Use to determine paternal K/k genotype when reproductive partner has clinically significant alloantibody

Determine fetal K/k genotype when mother has clinically significant alloantibody AND father is either heterozygous or unavailable for testing

Immucor PreciseType HEA Molecular BeadChip which is FDA-approved for clinical testing

Related Test
Antigen Testing, Rh Phenotype 0013019
Method: Hemagglutination

Antigen testing for D, C, E, c, e to assess maternal, paternal, or newborn Rh phenotype status

The Rh blood group is a complex human blood group. Rh antigens are encoded by two genes, RHD and RHCE, and are highly immunogenic. Antibodies against Rh antigens are the major cause of alloimmune hemolytic disease of the newborn (HDFN). 

The Kell blood group is complex with many antigens encoded by the KEL gene. The two major codominant alleles are KEL*01 and KEL*02 with corresponding K and k antigens. The K antigen is one of the most clinically significant antigens as anti-K causes severe fetal anemia by suppressing fetal red blood cell (RBC) synthesis.  Kell isoimmunization is the third most common cause of HDFN. 

In obstetrics, RBC antigen genotyping is useful when the mother has a clinically significant alloantibody level and the father is phenotypically positive for the corresponding antigen. Paternal genotyping can be used to assess risk for HDFN in offspring by determining copy number for the antigen of interest. Fetal genotyping is useful when the father of the fetus is either heterozygous for the corresponding antigen or unavailable for testing.

DISEASE OVERVIEW

Incidence of HDFN

  • 13% of hydrops fetalis is caused by antigen/antibody-mediated RBC hemolysis
  • 6-7/1,000 live births with maternal RhD alloimmunization in the U.S. 
  • RhD antigen causes ~50% of clinically significant maternal alloimmunization cases 
    • Anti-c one of the most common causes of severe HDFN, after anti-D
    • Anti-C, anti-E, and anti-e are less common causes of HDFN
      • When symptoms occur, they are usually mild to moderate 
  • ~4% of K negative (k/k) mothers will deliver a K positive baby with potential for HDFN 

Symptoms of HDFN

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

Physiology

  • Transplacentally transferred maternal IgG antibodies attack fetal RBCs in response to foreign, paternally inherited antigens in fetus
  • >50 different RBC antigens are known to be associated with maternal alloimmunization and HDFN
  • Despite routine screening and treatment, anti-D alloimmunization may still occur in some RhD-negative women due to:
    • Blood transfusion
    • Unrecognized miscarriages
    • Failure to receive prophylactic anti-D immunoglobulin during and following pregnancy

GENETICS

Genes and Variants
Blood Group Gene Allele Antigen (ISBT #)  ISBT Genotype Variants Tested Variants Used to Predict Allele Phenotype Frequency  
Rh

RHCE (NM_020485.5)

C

RH2

RHCE*2

c.307C>T; p.Pro103Ser

109bp insertion

c.307T; p.Ser103

109bp insertion

C: 68%

AA: 27%

A: 93%

c

RH4

RHCE*4

c.307C; p.Pro103

C: 80%

AA: 98%

A: 47%

E

RH3

RHCE*3

c.676G>C; p.Ala226Pro

c.676C; p.Pro226

C: 29%

AA: 22%

A: 39%

e

RH5

RHCE*5

c.676G; p.Ala226

C: 98%

AA: 98%

A: 96%

RHD (NM_016124.4)

D

RH1

RHD*01

Presence of the RHD exons 5, 7, and a 37 base pair insertion in the intron 3/exon 4 boundary. Allelic height ratios are used to determine the number of copies of RHD as compared to RHCE.

C: 15% RhD negative

AA: 8% RhD negative

A: <1% RhD negative

Kell KEL (NM_000420.2)

K

KEL1

KEL*01

c.578C>T; p.Thr193Met

c.578T; p.Met193

C: 9%

AA: 2%

A: Rare

Iranian Jews: 12%

Arabs: up to 25%

k

KEL2

KEL*02

c.578C; p.Thr193

C: 99.8%

AA: 100%

ISBT, International Society of Blood Transfusion

Variants Resulting in RhD Negative Phenotypes

  • RhD-negative Caucasians
    • Most have complete deletions of both copies of RHD gene
      • Rarely, a nonfunctional RHD gene is due to sequence variant, insertions (eg, pseudogene at border of intron 3/exon 4), or a nonfunctional RHD-CE-D fusion gene
  • RhD-negative African Americans
    • 25% have a 37-base-pair insertion inactivating the gene
    • Most others have a nonfunctional fusion gene or complete gene deletion 
  • RhD-negative Asians
    • 72% have partial or complete gene deletion 
    • Remainder have sequence variant(s) or a nonfunctional fusion gene

Inheritance

Codominant

TEST INTERPRETATION

Clinical Sensitivity

  • RhD: 98%   
  • RhCc, RhEe, and Kell: 99%

Analytical Sensitivity/Specificity

RhD: 99%

RhCc, RhEe, and Kell: 99%

Results

  • RhCc Genotyping
    • Homozygosity for C allele is predictive of RhC+c- phenotype
    • Cc compound heterozygosity is predictive of RhC+c+ phenotype
    • Homozygosity for c allele is predictive of RhC-c+ phenotype
  • RhD Copy Number
    • Presence of one or two copies of RHD gene predicts RhD positive phenotype
    • No copies of RHD gene predicts RhD negative phenotype
    • Inconclusive results may occur due to:
      • Presence of RHD exon 5 but absence of exon 7, or vice versa
      • Presence of the 37-base-pair insertion seen in African Americans
  • RhEe Genotyping
    • Homozygosity for E allele is predictive of RhE+e- phenotype
    • Ee compound heterozygosity is predictive of RhE+e+ phenotype
    • Homozygosity for e allele is predictive of RhE-e+ phenotype
  • Kell Genotyping
    • Homozygosity for K allele is predictive of K+k- phenotype
    • Kk compound heterozygosity is predictive of K+k+ phenotype
    • Homozygosity for k allele is predictive of K-k+ phenotype
  • Fetuses predicted to be unaffected following prenatal genotyping should continue to be monitored by noninvasive means for the development of erythroblastosis or hydrops

Limitations

  • Bloody amniotic fluid specimens may give false-negative results due to maternal cell contamination
  • Rare nucleotide changes leading to altered or partial antigen expression may not be detected
  • Genotypes resulting in Rh null phenotypes will not be assessed
  • Weak or partial RHD genotypes are not assessed
  • This assay is occasionally limited in predicting genotype due to extreme variation in the Rh locus. False-negative RhC or Rhc predictions may result due to RHCE-D-CE fusion genes.
  • Diagnostic errors can occur due to rare sequence variations
  • Abnormal signal intensities may result in indeterminate genotyping results
  • Patients who have had hematopoietic stem cell transplants may have inconclusive results on this test
References 
  1. Dean L. Blood Groups and Red Cell Antigens [Internet]. Chapter 7, The Rh blood group. National Center for Biotechnology Information (US). Bethesda, MD [Accessed: Sep 2019]
  2. Dean L. Blood Groups and Red Cell Antigens [Internet]. Chapter 8, The Kell blood group. National Center for Biotechnology Information (US). Bethesda, MD [Accessed: Jun 2019]
  3. Martin JA, Hamilton BE, Ventura SJ, Menacker F, Park MM, Sutton PD. Births: final data for 2001. Natl Vital Stat Rep. 2002; 51(2): 1-102. PubMed
  4. Avent ND, Reid ME. The Rh blood group system: a review. Blood. 2000; 95(2): 375-87. PubMed
  5. Klein HG, Anstee DJ. Mollison's Blood Transfusion in Clinical Medicine. 12th ed. Chichester, UK: John Wiley & Sons, 2014.
  6. Moise KJ. Fetal anemia due to non-Rhesus-D red-cell alloimmunization. Semin Fetal Neonatal Med. 2008; 13(4): 207-14. PubMed
  7. Reid ME, Lomas-Francis C, Olsson ML. The Blood Group Antigen FactsBook, 3rd Edition. Academic Press, 2012.
  8. Singleton BK, Green CA, Avent ND, Martin PG, Smart E, Daka A, Narter-Olaga EG, Hawthorne LM, Daniels G. The presence of an RHD pseudogene containing a 37 base pair duplication and a nonsense mutation in africans with the Rh D-negative blood group phenotype. Blood. 2000; 95(1): 12-8. PubMed
  9. Okuda H, Kawano M, Iwamoto S, Tanaka M, Seno T, Okubo Y, Kajii E. The RHD gene is highly detectable in RhD-negative Japanese donors. J Clin Invest. 1997; 100(2): 373-9. PubMed
  10. Daniels G. The molecular genetics of blood group polymorphism. Transpl Immunol. 2005; 14(3-4): 143-53. PubMed

Last Update: November 2019