Monitoring humoral immune
responses in SOT - ABO blood
group system & isoagglutinin test
Dong Il Won
Kyungpook National University School of Medicine
Department of Clinical Pathology
ABO Antibodies
MFI ratio
(Ab level)
Levels of ABO antibodies
in 120 healthy blood donors
Stussi G, et al., 2005
ABO IgG subclass
 74 patients with 141 HLA-DSA
 IgG1 (111/141; 78%)
 IgG2 (69/141; 49%)
 IgG3 (51/141; 36%)
 IgG4 (28/141; 20%)
ABO IgG
MFI ratio
total IgG
cord
blood
•
Serum IgG subclass levels in
healthy infants and children.
•
ABO IgG subclass analysis in sera
of 15 blood group O donors
Various methods
to measure ABO antibody levels
• Tube hemagglutination technique (TT)
– Saline:
IgM
– Antiglobulin: IgG
– traditionally used
• Column agglutination technique (CAT)
– Simple
– Low intercenter variation
– Most popular
Various methods
to measure ABO antibody levels (others)
• ELISA
– Rieben R, et al. Transfusion 1991;31:607.
• Surface plasmon resonance
– Kimura S, et al. Transfusion 2005;45:56.
• RBCs modified with KODE technology
– Frame, et al. Transfusion 2007;47:876.
• Flow cytometry (FC)
– Using RBC
• Tanabe K. Transplantation 2007;84:S13.
• Seebach’s protocol
– Stussi G, Br J Haematol 2005;130:954.
– Yung GP, Transplantation 2007;84:S20.
• Won & Kim.
Optimized Flow Cytometry to Measure
Anti-ABO Immunoglobulin G. Lab Med 2012;43:281.
– Using latex beads
• Sundbäck M, Transplantation 2007;84:S24.
Interpretation of TT reactions
Macrosopically Observed Findings
Grading
Score
One solid agglutinate
4+
12
Sevevral large agglutinates
3+
10
Medium-size agglutinates, clear bacground
2+
8
Small agglutinates, turbid background
1+
5
Very small agglutinates, turbid background
1+w
4
w+ or +/-
2
0
0
Barely visible agglutination, turbid background
No agglutination
Mixtures of agglutinated and unagglutinated RBCs
(mixed field)
mf
Complete hemolysis
H
Partial hemolysis , some RBCs remain
PH
Examples of TT
End Point
=
=
=
•
•
The titer is determined from the highest dilution of serum that gives a reaction ≥ 1+ (score 5).
This may differ significantly from the titration endpoint (shown in parentheses), as with the reactions of
non-ABO antibody with high-titer, low-avidity characteristics, manifested by Sample 3.
Interpretation of CAT
gel or beads
End Point
X
ABO titer predicts AMR?
Antibody-Mediated Rejection
• Tobian, 2010:
• 46 cases of ABO-incompatible KT
– Using TT
N = 46
post-KT titer≥ 64
AMR
No AMR
x 31
• Post-KT titer
– With a titer ≥ 64
• Sensitivity 57.1%
• Specificity 79.5%
■
TPE
@ AMR
weeks (post-KT)
Low intercenter variation of CAT?
•
Evaluation summary report
for the 2nd proficiency test,
2013, Korea.
•
Severe variation between
enrolled laboratories is seen,
even for CAT AHG.
Other Disadvantages of CAT
• Polyspecific Coombs’ serum in Korea
– Detect IgG, IgM, IgA, & C’
• FC: IgG (γ-chain)-specific
• Not fully objective reading
– 1+ or w+
• FC: definite cutoff
• High cost
– ≥ 10 gel microcolumns / sample
• FC: only 1 tube
?
Intracenter variation for ABO Ab titer
Flow
cytometry
•
•
Tube
technique
Flow showed excellent reproducibility and no measurement deviation.
Both CAT and TT assays showed a fourfold difference.
ABO antibody by Flow Cytometry
No Consensus Protocol
Reagents for Flow ABO IgG
• RBC
– Fixed with Karnovsky buffer
• 5% glutaraldehyde and 4% paraformaldehyde in 0.08 M
sodium phosphate buffer
• Antiglobulin
– Fab, not F(ab’)2
– PE-conjugated Antiglobulin rather than FITC-
• Patient serum
– Treated with DTT to destroy IgM
Protocol for Flow ABO IgG
DTT
Sensitization
℃
MFI ratio
Staining
Won, 2012
MFI ratio
• Sensitization at 4°C
Stussi, 2005
IgM
MFI ratio
IgG
ABO IgG levels: Flow vs. CAT
Log (MFI ratio) = 0.863 x log (CAT titer) + 0.342
r=0.895, p=0.01
• 17 cases of ABOi KT (Maryknoll & KNUH)
· 14 patients (KNUH)
Comparison of baseline data
between the high and low MFI ratio groups.
Group
Gende
Primary
r/age renal disease
Baseline
HLA
MM
ABO
(D  R)
MFI raio
CAT titer
Rituximab
(mg)
#PP
High MFI ratio
(N=7)
1
M/58
DM
3
BO
810
1024
700
13
2
F/57
CGN
3
A O
800
1024
500
12
3
M/17
CGN
3
BO
544
128
500
6
4
M/57
CGN
5
A O
526
512
500
7
5
F/50
DM
5
A O
308
64
500
5
6
M/33
CTIN
2
A O
260
32
200
6
7
M/48
CGN
3
BA
201
8
500
3
8
M/38
CGN
1
AB  B
172
16
500
6
9
M/51
CGN
0
BO
134
128
700
6
10
M/18
CGN
2
90
16
200
4
11
M/45
CGN
5
AB  A
A B
18
8
200
3
12
M/68
CGN
5
BA
13.6
16
500
2
13
14
F/34
CTIN
3
AB  B
2.2
4
200
3
M/31
CGN
1
AB  A
1.7
8
200
4
Low MFI ratio
(N=7)
Required Number
of Plasma Pheresis
The number of plasma pheresis
according to baseline MFI ratio by Flow
r=0.908, p=0.01
Baseline MFI ratio
Comparison of Outcome
between the high MFI ratio and low MFI ratio groups.
Baseline
Group
at KT
Serum Cr, post-KT
F/U
Duration (M)
MFI ratio
CAT Titer
MFI ratio
CAT Titer
at 1 M
Final
1
810
1024
9.8
32
1.4
1.6
9
2
800
1024
17.5
32
0.8
0.7
11
3
544
128
6.9
8
1.0
1.4
3
4
526
512
3.8
8
1.3
1.4
18
5
308
64
32.6
8
0.9
0.9
2
6
260
32
7.5
4
1.3
1.4
7
7
201
8
10.4
4
1.1
1.4
7
8
172
16
8.8
2
1.0
1.1
7
9
134
128
3.2
8
1.4
1.6
5
10
90
16
1.1
4
1.4
1.4
6
11
18
8
4.1
2
1.2
1.3
16
12
13.6
16
5.1
4
1.2
1.5
2
13
2.2
4
4.2
4
0.8
1.0
2
14
1.7
8
1.4
2
1.3
1.5
21
High MFI ratio
(N=7)
Low MFI ratio
(N=7)
Serum Cr (mg/dL)
Comparison of the serum creatinine level
between the high and the low MFI ratio groups
(baseline)
Post-transplant
Comparison of complications
between the high and low MFI ratio groups
Group
High MFI ratio
(N=7)
Low MFI ratio
(N=7)
Postoperative bleeding
2
1
Herpes simplex/zoster
1/2
0/0
0
0
0/0
0/0
Acute rejection
Graft loss/patient death
ABO IgG: Flow vs. CAT
: Concordant case (B to A)
128
: PP/IVIG
176.7
16
CAT
128
14
Flow
64
12
Cr
32
10
16
8
8
6
4
4
2
2
1
0
2/10
2/15
2/20
Rituximab
FK506 0.05 mg/kg po bid
2/25
3/2
ABOi KT
MMF 500~750 mg po bid
PDS 0.25 mg/kg po bid
PDS IV
3/7
3/12
Cr
ABO Titer
or
MFI ratio
256
ABO IgG: Flow vs. CAT
: Discordant case (A to O)
: PP/IVIG
2,048
CAT
Flow
512
8
256
128
6
32
4
16
8
2
4
ABOi KT
1/21
1/1
12/12
11/22
11/2
10/13
9/23
9/3
8/14
7/25
7/5
6/15
5/26
0
5/6
2
Cr
64
4/16
or
MFI ratio
1,024
ABO Titer
10
ABO IgG subclass
Activating classical complement pathway
: IgG3 >IgG1>IgG2>>IgG4
ABO IgG
MFI ratio
total IgG
 74 patients with 141 HLA-DSA
 IgG1 (111/141; 78%)
 IgG2 (69/141; 49%)
 IgG3 (51/141; 36%)
 IgG4 (28/141; 20%)
cord
blood
•
Serum IgG subclass levels in
healthy infants and children.
•
ABO IgG subclass analysis in sera
of 15 blood group O donors
Complement-activating IgG
• IgG subtype
– 2° Ab for IgG1-4 subclasses
• Zymed Laboratories (biotin-)
• Southern Biotech (PE-)
• Complement detection
– anti-C4d FITC
• Biomedica
– C3b is more sensitive marker
than C1q or C4d.
• Watanabe J. 2006.
HLA Ab
C1q- and C4d-Luminex assay
C1q assay
C4d assay
HLA Ab
1° Ab
2° Ab
•
Complement source
•
Reporter Ab PE
Take Home Messages
• There are various methods for ABO IgG
– Common discordant results
• Flow cytometry is another approach.
• Further studies
– Complement-activating IgG is important for
graft rejection?
Thank You!

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