Red Blood Cell Antigen Genotyping

Typical Testing Strategy

Phenotype Testing
  • Evaluates specific RBC antigen presence by serology
  • Results can aid in selecting antigen negative RBC units

Serological testing includes K, Fya, Fyb, Jka, Jkb, S, s (k, cellano, testing performed if indicated) to assess maternal or paternal RBC phenotype status.

Antigen Testing, Rh Phenotype 0013019
Method: Hemagglutination

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

Genotype Testing
  • May help resolve serological discrepancies or challenges
  • Results can aid in selecting antigen negative RBC units if recently transfused
  • Assess risk for HDFN for couples with a previously affected pregnancy
  • Test for rare variants not detected by phenotype testing
Red Blood Cell Antigen Genotyping 3001053
Method: Polymerase chain reaction followed by fluorescent probe ligation and detection
  • Assess risk for hemolytic transfusion reactions or alloimmune HDFN by genotyping for common and rare RBC antigens.
  • Detects 24 variants associated with 35 RBC antigens and phenotypic variants, in addition to the hemoglobin S variant.
  • Only clinical platform for RBC genotype testing currently approved by the U.S. Food and Drug Administration (Wilkinson, 2016).

See Related Tests below for antigen-specific genotyping and phenotyping tests.

Testing is useful in determining allelic variants predicting red blood cell (RBC) antigen phenotypes for patients with recent history of transfusion or with conflicting serological antibody results due to partial, variant, or weak expression antigens. Also useful as an aid in management of hemolytic disease of the fetus and newborn (HDFN).

Disease Overview

Prevalence and/or Incidence

Erythrocyte alloimmunization occurs in up to 58% of sickle cell patients, up to 35% in other transfusion-dependent patients, and in approximately 0.8% of all pregnant women.

Symptoms

Transfusion reactions or HDFN can occur due to alloimmunization:

  • Intravascular hemolysis: hemoglobinuria, jaundice, shock
  • Extravascular hemolysis: fever and chills
  • HDFN: fetal hemolytic anemia, hepatosplenomegaly, jaundice, erythroblastosis, neurological damage, hydrops fetalis

Clinical presentation is variable and dependent upon the specific antibody and recipient factors

Pathophysiology

Extravascular Hemolysis

Removal of antibody-bound RBCs via splenic macrophages

Intravascular Hemolysis

  • Rare for non-ABO antibodies
  • Donor RBCs are destroyed by the recipient's antibodies while they are still inside blood vessels
  • Hemoglobin released into plasma and excreted in urine; bilirubin accumulates in blood

HDN-Mediated Disease

  • Caused by antigen-antibody mediated red-cell hemolysis from previously transferred maternal antibodies
  • Examples of antibodies associated with HDFN:
    • Rh (Ant-D, -C, -c, -E, and -e)
    • Kell (Anti-K, -k, -Kpa, -Kpb, -Jsa and -Jsb)
    • Duffy (Anti-Fya and -Fyb)
    • MNS (Anti-M, -N, -S, -s, and -U)
    • Kidd (Anti-Jka and -Jkb)
  • Anti-D is the most common cause of HDFN, followed by anti-c, anti-K, and anti-E; anti-Jka and -Jkb is rare cause
  • Antibodies cross the placenta and cause immune destruction of RBCs in fetus, leads to hydrops fetalis

Genetics

See Genes and Variants Tested table below

Inheritance

Typically codominant for RBC antigens; autosomal recessive for HbS  

Test Interpretation

Sensitivity/Specificity

  • Clinical sensitivity and specificity
    •  >99% for C, c, E, e, K, k, Jka, Jkb, Fya, Fyb, M, N, S, s
    • Unknown for Kpa, Kpb, Jsa, Jsb, Lua, Lub, Dia, Dib, Coa, Cob, Doa, Dob, Joa, Hy, LWa, LWb, Sc1, Sc2, U, V, VS, HbS
  • Analytical sensitivity and specificity
    • >99% for C, c, E, e, K, k, Jka, Jkb, Fya, Fyb, M, N, S, s
    • Unknown for Kpa, Kpb, Jsa, Jsb, Lua, Lub, Dia, Dib, Coa, Cob, Doa, Dob, Joa, Hy, LWa, LWb, Sc1, Sc2, U, V, VS, HbS

Results

  • Predicted phenotype for each RBC antigen tested will be provided
    • If identified, weak alleles will be reported
  • Possible variant
    •  Reported for allele combinations that have not widely been reported in the literature
  • Indeterminate
    • Abnormal signal intensities may result in inability to predict genotype resulting in indeterminate results for all tested antigens and HbS

Limitations

  • Only the variants listed will be interrogated
  • Does not test for Rh D, a major cause of HDFN 
  • Patients who have had allogeneic hematopoietic stem cell transplants may have inconclusive results on this test
  • Diagnostic/phenotype prediction errors can occur due to
    • Rare variants affecting primer/probe binding
    • Molecular events that affect the blood-group antigen expression and phenotypes that are not detected by this assay (ie, certain null phenotypes or other phenotypes with altered expression)
  • This assay is not designed to diagnose sickle cell disease
Genes and Variants Tested
Blood Group Allele Antigen (ISBT #) ISBT Genotype Variants Tested Variants Used to Predict Allele Phenotype Frequency1,2

Rh

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%

V

RH10

RHCE*01.20.01

RHCE*01.20.02

RHCE*01.20.04

RHCE*01.20.05

c.733C>G; p.Leu245Val

c.1006G>T; p.Gly336Cys

c.733G; p.Val245

C: 1%

AA: 30%

VS

RH20

c.[733C>G; 1006G>T]; p.[Leu245Val; Gly336Cys]

AA: 26-40%

All other populations: <0.01%

Kell

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%

Kpa

KEL3

KEL*03

c.841C>T; p.Arg281Trp

c.841T; p.Trp281

C: 2%

AA: <0.01%

Kpb

KEL4

KEL*04

c.841C; p.Arg281

All populations: 100%

Jsa

KEL6

KEL*06

c.1790T>C; p.Leu597Pro

c.1790C; p.Pro597

C: <0.01%

AA: 20%

Jsb

KEL7

KEL*07

c.1790T; p.Leu597

C: 100%

AA: 99%

Duffy

Fya

FY1

FY*01

c.125G>A; p.Gly42Asp

c.125G; p.Gly42

C: 66%

AA: 10%

A: 99%

Thai: 97%

Fyb

FY2

FY*02

c.125A; p.Asp42

C: 83%

AA: 23%

A: 18.5%

Thai: 31%

Chinese: 9.2%

Fyb-67C (Fyb ES)

GATA FY-2

FY*02N.01

c.-67T>C

c.-67T>C

C: rare

AA: 68%

A: rare

Fyx

FY2W

FY*02M

c.265C>T; p.Arg89Cys

c.265C>T; p.Arg89Cys

rare

Kidd

Jka

JK1

JK*01

c.838G>A; p.Asp280Asn

c.838G; p.Asp280

C: 77%

AA: 92%

A: 72%

Jkb

JK2

JK*02

c.838A; p.Asn280

C: 74%

AA: 49%

A: 76%

MNS

M

MNS1

GYPA*01

c.59T>C; p.Leu20Ser

c.59C; p.Ser20

C: 78%

A: 74%

N

MNS2

GYPA*02

c.59T; p.Leu20

C: 72%

AA: 75%

S

MNS3

GYPB*03

c.143C>T; p.Thr48Met

c.143T; p.Met48

C: 55%

AA: 31%

s

MNS4

GYPB*04

c.143C; p.Thr48

C: 89%

AA: 93%

U

MNS5

 

 

 

C: 99.9%

AA: 99%

Silencing S (Uvar)

MNS-3,w5

GYPB*03N.01

GYPB*03N.02

GYPB*03N.03

GYPB*03N.04

c.230C>T; p.Thr77Met

or

c.270+5G>T

c.230C>T; p.Thr77Met

or

c.270+5G>T

rare

Silencing S (Uneg)

MNS-3,-4,-5

Deletion of GYPB exons 2-6

 

rare

Lutheran

Lua

LU1

LU*01

c.230G>A; p.Arg77His

c.230A; p.His77

C: 8%

AA: 5%

Lub

LU2

LU*02

c.230G; p.Arg77

All populations: 99.8%

Dombrock

Doa

DO1

DO*01

c. 793G>A; p.Asp265Asn

c.793A; p.Asn265

C: 67%

AA: 55%

Japanese: 24%

Thai: 14%

Dob

DO2

DO*02

c.793G; p.Asp265

C: 82%

AA: 89%

Hy

DO4

DO*04

c.323G>T; p.Gly108Val

c.323G; p.Gly108

AA: >99%

Most populations: 100%

 

DO-4

 

c.323T; p.Val108

rare

Joa

DO5

DO*05

c.350C>T; p.Thr117Ile

c.350C; p.Thr117

AA: >99%

Most populations: 100%

 

DO-5

 

c.350T; p.Ile117

rare

Landsteiner-Wiener (LW)

LWa

LW5

LW*05

c.299A>G; p.Gln100Arg

c.299G; p.Arg100

All populations: 100%

LWb

LW7

LW*07

c.299A; p.Gln100

Most populations: Rare

Estonians: 8%

Finns: 6%

Latvians and Lithuanians: 5%

Poles and Russians: 2%

Other Europeans:<1%

Diego

Dia

DI1

DI*01

c.2561C>T; p.Pro854Leu

c.2561T; p.Leu854

Most populations: 0.01%

South American Indians: 2% -54%

Japanese: 12%

Chinese: 5%

Hispanics: 1%

Poles: 0.47%

Dib

DI2

DI*02

c.2561C; p.Pro854

Most populations: 100%

Native Americans: 99%

Colton

Coa

CO1

CO*01

c.134C>T; p.Ala45Val

c.134C; p.Ala45

All populations: 99.5%

Cob

CO2

CO*02

c.134T, p.Val45

All populations: 10%

Scianna

Sc1

SC1

SC*01

c.169G>A; p.Gly57Arg

c.169G; p.Gly57

All populations: >99%

Sc2

SC2

SC*02

c.169A; p.Arg57

Europeans: 1%; more common in Mennonites

Hemoglobin S

HbS

 

 

HBB c.20A>T; Glu6Val

c.20A>T; Glu6Val

AA: 10%

1Reid, Marion E., Lomas-Francis, Christine, and Olsson, Martin L. Blood Group Antigen Facts Book, Third Edition. Oxford: Academic Press, 2012. Print.

2Dean L. Blood Groups and Red Cell Antigens [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2005. Chapter 9, The Duffy blood group. Available from: https://www.ncbi.nlm.nih.gov/books/NBK2271/

A, Asian; AA, African American; C, Caucasian; ISBT, International Society for Blood Transfusion (ISBT) Committee on Terminology for Red Cell Surface Antigens

Reference transcripts: RHCE (NM_020485.5), KEL (NM_000420.2), ACKR1 (NM_002036.3; alt reference NM_001122951.2), SLC14A1 (NM_015865.6), GYPA (NM_002099.7), GYPB (NM_002100.5), BCAM (NM_005581.4), ART4 (NM_021071.2), ICAM4 (NM_001544.4), SLC4A1 (NM_000342.3), AQP1 (NM_198098.3), ERMAP (NM_018538.3)

References 

Avent ND. Large-scale blood group genotyping: clinical implications. Br J Haematol. 2009; 144(1): 3-13. PubMed

Chou ST, Jackson T, Vege S, Smith-Whitley K, Friedman DF, Westhoff CM. High prevalence of red blood cell alloimmunization in sickle cell disease despite transfusion from Rh-matched minority donors. Blood. 2013; 122(6): 1062-71. PubMed

Cruz Rd, Mota MA, Conti FM, Pereira RA, Kutner JM, Aravechia MG, Castilho L. Prevalence of erythrocyte alloimmunization in polytransfused patients. Einstein (Sao Paulo). 2011; 9(2): 173-8. PubMed

Gassner C, Hyland C, Red Cell Immunogenetics and Blood Group Terminology. Names for FY (ISBT 008) Blood Group Alleles. 4.1. International Society of Blood Transfusion. Amsterdam, the Netherlands [Accessed: Jan 2019]

Mitra R, Mishra N, Rath GP. Blood groups systems. Indian J Anaesth. 2014; 58(5): 524-8. PubMed

Wilkinson DS. Clinical Utility of Genotyping Human Erythrocyte Antigens. Lab Med. 2016; 47(3): e28-31. PubMed

Last Update: February 2019