O - Swisstransfusion

ABO-Blutgruppensystem
Swisstransfusion 2012
Bern – 7. September 2012
E. A. Scharberg
Institut für Transfusionsmedizin und Immunhämatologie Baden-Baden
DRK-Blutspendedienst Baden-Württemberg-Hessen

Grundlage der ABO-Blutgruppen
Fuc
O
Gal
GlcNAc
Fuc
A
GalNAc
Gal
GlcNAc
Fuc
B
Gal
Gal
GlcNAc
Glykosyltransferase
(ABO-Gen)
Erythrozyt

Genetik der ABO-Blutgruppen
Chromosom 9
ABO-Gen
~15 kb
genom. DNA
Exon 1 2 3 4 5 6 7
Exon 6 Exon 7
Allele 261 467 657 703 796 802 803 930 1059
A 1 (wt) G C C G C G G G C
A 2 - T - - - - - - del
B - - T A A - C A -
O 1 del - - - - - - - -
O 2 - - - - - A - - -
Phänotyp
A 1
A 2
B
O
O
‣ 4 Positionen in der DNA-Sequenz sind für die Spezifität und
Aktivität der kodierten Glykosyltransferase entscheidend und
ermöglichen die Unterscheidung der Blutgruppen A 1 , A 2 , B und O

Serumeigenschaften:
Anti-A1: ++
Anti-A2: negativ
Anti-B: negativ
Anti-O (H): negativ
Genotyp: O0301/B101

Serumeigenschaften :
Anti-A1: ++
Anti-A2: negativ
Anti-B: negativ
Anti-O (H): negativ
Genotyp:
A3(neu)/B101
(107T>C exon 7 A201)

Serumeigenschaften:
Anti-A1: ++
Anti-A2: negativ
Anti-B: negativ
Anti-O (H): negativ
Genotyp:
A3(neu)/B101
107T>C exon 7 A201

Anti-A1: +
Anti-A2: neg
Anti-B: ++++
Anti-O/H: neg
Genotyp:
Ax01/O01

Bx03/O02

Ax03/O03
Ax01/O01

• Routine ABO, (RhD, RhCcEe, K) phenotyping of first and second time
donors on Olympus PK 7200 with monoclonal anti-A, anti-B reagents
and reverse typing (A1, A2, B, O red cells)
• Unclear results in ABO proved in tube test (incubation 30 minutes
at room temperature) with monoclonal anti-A, anti-B reagents and
reverse typing (A1, A2, B, O red cells)
Since May 2004
• Samples with weak or negative A and B antigens and missing or
weak (+) corresponding isoagglutinins
Low resolution ABO genotyping for A, A2, B, O01, O03 alleles (PCR-SSP)
• If no correlation between phenotype and genotype
ABO subtyping for 36 rare alleles (Ael, Ax, Aw, Bx, Bw, etc.) (PCR-SSP)
• If still no correlation between phenotype and genotype
Exon amplification and sequencing of entire ABO gene from genomic DNA

Results
Donors with aberrant ABO phenotypes
• May 2004 thru December 2007: 206,262 first and second time donors
• 412 samples with aberrant ABO phenotypes (0.20%)
45 samples with weak antigens
Ax01 (646T>A): n=15
Aw06 (502C>G): n=5
Aw13 (2T>C): n=3
Ael01 (804insG): n=2
Ax03, Aw04, Aw09,
B(A)03: n=4 (one each)
novel variant (Bw21): n=1
347 samples with negative antigens
and weak or absent isoagglutinins
non-deletional O alleles
(O03, Aw08, O51): n=255
other alleles (Ax03, Ax01): n=6
novel variants (Bw20,
Aw15): n=3
molecular basis unknown: 123
molecular basis unknown: n=15

Geno-/Phänotypen im ABO-System
serologische
Nachweisgrenze
Phänotyp 0 A el A x /A weak A 3 A 2 A 1
Allele O01 O03 Ael01 Aw01 A301 A201 A101
O02 Aw08 Ael02 Ax01 A302 A202 A102
... ... Ax03 ... ... ... ... ...
‣ Die Genotyp-Phänotyp-Korrelation bei den seltenen Varianten ist ein
quantitativer Effekt.
‣ D.h. es ändert sich nicht das Antigen sondern die Antigendichte.
‣ Dieser quantitative Effekt ist genetisch nicht verlässich bestimmbar.

Clinical significance of non-deletional O alleles
in blood donors
Transfusion practice:
No deferral of donors with missing or weak isoagglutinins
Red cell units with clear serological ABO antigen typing
used for transfusions
No transfusion reaction reported
Do some of the non-deletional O alleles have
low antigen expression that might
boost the isoagglutinins of recipients

• Plasma or serum samples of all 6,387 patients tested in our
laboratory from January 1 thru December 31, 2007 stored at -30°C.
• Look back procedure (January 2008) identifying patients who
randomly received ABO variants with non-deletional O alleles
• In patients with follow up samples Titration of anti-A and anti-B
in samples at the time of the cross-match of the ABO variant
and in follow up samples in gel technique using A1 and B red cells:
• Neutral gel cards: incubation 15 minutes at room temperature
• Antiglobulin gel cards: incubation 15 minutes at 37°C

• 20,566 red cell units cross-matched for 6,387 patients tested in our
laboratory from January 1 thru December 31, 2007 stored at -30°C.
• 35 patients received red cell units with non-deletional O alleles
21 of them had follow up samples (6 to 286 days after transfusion)
17 patients (all group O) received units with O03 allele
2 patients (both group A) received units with Bw20 allele
1 patient (group O) received a unit with Aw08 allele
1 patient (group O) received a unit with Ax03 allele

Variant ABO alleles in this study
*O01
261delG
*O03
*Aw08
53G>T 220C>T 297A>G 526C>G 802G>A
*Ax03
220C>T 297A>G 526C>G 802G>A
488C>T
*Bw20
646T>A 771C>T
681G>A 829G>A
817insG
297A>G 526C>G 703G>A 803G>C
657C>T 796C>A

Anti-A Titer before and after Transfusion of Red Cell Units
with O03 Allels
1000
100
Titer
10
1
1
2
3
4
5
6
7
8
9
Sample
10
11
12
13
14
15
16
17
IgG after Transfusion
IgG before Transfusion
IgM after Transfusion
IgM before Transfusion

Anti-B Titer before and after Transfusion of Red Cell Units
with O03 Allels
100
10
Titer
1
1
2
3
4
5
6
7
8
9
Sample
10
11
12
13
14
15
16
17
IgG after Transfusion
IgG before Transfusion
IgM after Transfusion
IgM before Transfusion

Anti-A Titer before and after Transfusion of
Red Cell Units with Aw08 and Ax03 Alleles
1000
100
10
Titer
1
IgG after
Transfusion
Aw08
Ax03
IgM after
Transfusion
IgG before
Transfusion
IgM before
Transfusion

Anti-B Titer before and after Transfusion of
Red Cell Units with Aw08, Ax03 and Bw20 Alleles
100
10
Titer
Aw08
1
Ax03
Bw20-1
Bw20-2
IgM before
Transfusion
IgM after
Transfusion
IgG before
Transfusion
IgG after
Transfusion

Conclusions:
• The increasing number of known ABO variants with non-deletional
alleles raises the question of their clinical significance – especially in
blood donors.
• Transfusion of red cell units with the variant ABO alleles O03, Aw08,
and Ax03 typed and transfused as group O,
and Bw20 typed and transfused as group A
did not boost anti-A and anti-B titers in blood group O/A recipients .
• For these variants no donor referral because of missing or weak
isoagglutinins seems to be necessary .

Exon 2
wild type:
mutant:
98G>T
T T G T T T G G g t a a g a c
T T G T T T G K g t a a g a c
intron 2
Provisional Allele Name: Aw32, GenBank JX398332

Intron 1 (reverse) IVS1+2insT
wild type:
mutant:
t g c a c t c - a c C G G C C
t g c a c t c a m c S G S C M
intron 1
exon 1
Provisional Allele Name: O76, GenBank JX519571

Exon 3
wild type:
mutant:
107T>C
T A C G G G G T C C T A A G C
T A C G G G G Y C C T A A G C
Provisional Allele Name: A309, GenBank JX398333

Exon 7
wild type:
mutant:
815G>A
T T C G G G G G G T C G G T G
T T C G G G G R G T C G G T G
Provisional Allele Name: O75, GenBank JX519569

Exon 7
wild type:
mutant:
926A>G
A A C A A G T A C C T G C T G
A A C A A G T R C C T G C T G
Provisional Allele Name: Aw30, GenBank JX398330

Genetische Vielfalt im ABO System
‣ 238 Genvarianten (Stand Juni 2012)
‣ A-Allele (96): Phänotyp
*A1 9 Allele A 1
*A2 21 Allele A 2
*A3 8 Allele A 3
*Ael 9 Allele A el
*Aw 29 Allele A weak
*Ax 20 Allele A weak
‣ B-Allele (68): Phänotyp
*B1 18 Allele B
*B3 8 Allele B 3
*Bel 6 Allele B el
*Bw 27 Allele B weak
*Bx 9 Allele B weak
‣ O-Allele (74):
Phänotyp
*O1 (261delG) 57 Allele 0
*O2 (non-261delG) 17 Allele 0; schwache oder fehlende
Isoagglutinine
The Blood Group Antigen Gene Mutation Database
www.ncbi.nlm.nih.gov/gv/mhc/xslcgi.cgicmd=bgmut/home

6 days before!!!

Acknowledgments
Peter Bugert
Gaby Rink
Institut für Transfusionsmedizin und Immunologie
DRK-BSD Baden-Württemberg-Hessen
Mannheim
Nureyla Kanbur
Raphaela Baudendistel
Sabine Roth
Susanne Seyboth
Ekkehard Richter
Institut für Transfusionsmedizin und Immunhämatologie
Baden-Württemberg-Hessen
Baden-Baden

Thank you!

Schwaches A-Antigen

Mischfeldagglutination nach
ABO-ungleicher Transfusion

A-Antigen-Verlust (MDS-Patient)

Thank you!