the efficient use of rhig - centro nazionale sangue...fc glycopeptides: mass spectrometric analysis...
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![Page 1: The efficient use of RhIg - Centro Nazionale Sangue...Fc glycopeptides: Mass spectrometric analysis 23 April, 2018 | 5 Glycan species Glycan structure IgG1 E 293 EQYNSTYR 301 a P01857b](https://reader030.vdocuments.mx/reader030/viewer/2022040406/5ea6bb1cb8499e332c7ba33b/html5/thumbnails/1.jpg)
| 1 23 April, 2018
C. Ellen van der Schoot
Department Experimental Immunohematology
Sanquin, Amsterdam, the Netherlands
The efficient use of RhIg
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| 2 23 April, 2018
Increasing the efficiency of RhIg
1. Increasing the biological activity of RhIg
• Effect of Fc-core glycosylation of anti-D immunoglobulin
2. Decreasing the unnecessary use of RhIg
• Guiding of immunoprophylaxis by fetal RHD typing
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Fc-glycans
April 23, 2018 | 3
Asn297
Sialic acid
Galactose
Fucose
Bisected
GlcNac
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Glycans affect binding to FcgR
Low Fc-fucosylation
results in 50x
enhanced binding
To FcγRIIIa
Dekkers et al. Front Immun 2017
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Fc glycopeptides: Mass spectrometric analysis
| 5 23 April, 2018
Glycan species Glycan structure
IgG1
E293EQYNSTYR301a
P01857b
[M+H]+
No glycan - 1190.5198
G0
2487.9880
G0F
2634.0459
G1
2650.0408
G1F
2796.0987
G0N
2691.0674
G0FN
2837.1253
G2
2812.0936
G2F
2958.1515
G1N
2853.1202
G1FN
2999.1781
G2N
3015.1730
G2FN
3161.2309
Total IgG1
pep pep
pep
pep
pep
pep
pep
G0F G1F
G2F
* *
pep
G1 pep
G2
* *
A
G0
Majority of IgG1 in plasma is fucosylated
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Anti-D IgG is low - fucosylated
| 6 23 April, 2018
Total IgG1
Anti-D IgG1
2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 m/z
pep pep
pep
pep
pep
pep
pep
G0F G1F
G2F
pep
pep
pep
G0
G1
G2
pep
G2F pep
G1F
pep
*
*
*
* *
pep
G1 pep
G2
* *
A
B
G0
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Fc-fucosylation of anti-D IgG is variable and
related to pathogenicity
| 7 23 April, 2018
F u c o s y la tio n (% )
* * * , p :< 0 .0 0 0 1
0 2 0 4 0 6 0 8 0 1 0 0
0
2 0
4 0
6 0
8 0
1 0 0
T o ta l Ig G
An
ti-D
an
tib
od
y
8 10 12 14 160
20
40
60
80
100
R2=0.467p= 0.010
NK-cell ADCC (% lysis)
An
ti-D
Ig
G1
-fu
co
sy
lati
on
(%
)
0 5 10 15 200
20
40
60
80
100R2=0.289,P: *, 0.037
Haemoglobin (g/dl)
An
ti-D
Ig
G1
-fu
co
sy
lati
on
(%
)
NK-cell ADCC R2=0.467,p=0.010
Neonatal Hb R2=0.289, p=0.038
Fucosylation(%) p<0.0001
Kapur ,Della Valle , Br J Haemat 2016
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Fc-fucosylation of anti-D IgG from
hyperimmune donors is variably low
| 8 23 April, 2018
Pre
gnant w
omen
HID
fem
ale
HID
mal
e
0
20
40
60
80
100
An
ti-D
fu
co
syla
tio
n (
%)
NS
**
*
A
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Anti-D preparations display variable
decreases in Fc-fucosylation
| 9 23 April, 2018
Rh
oG
am
(a
)
Rh
oG
am
(b
)
Rh
oG
am
(c
)
Pa
rto
bu
lin
SD
F
Rh
op
hy
lac
CG
Rh
op
hy
lac
QC
G
LF
B C
RT
S
D-G
am
Rh
es
on
ati
v
Rh
eD
Qu
in 2
00
7
Rh
eD
Qu
in 2
01
4
0
5
10
15
20
IgG
-bis
ec
tio
n (
%)
50
60
70
80
90
100
Total IgG
Anti-D IgG
IgG
-fu
co
syla
tio
n (
%)
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Efficacy of anti-D appears to be related to Fc-
fucosylation
• Human polyclonal anti-D immunoglobulin:
• Low level of fucosylation:
• high ADCC, very rapid RBC clearance, prevented D-immunization.
• Moab anti-D produced in human B cell lines (BRAD3, BRAD5)
• Medium level of fucosylation
• medium ADCC, fast clearance, prevented D-immunization in 93% subjects
•
• Moab anti-D produced in human-mouse hetero hybridoma: (Fog1, AD1, G7,
G12)
• High level of fucosylation
• low ADCC, variable and slow clearance, stimulated D-immunization.
| 10 23 April, 2018 Kumpel et al. submitted
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Conclusion (1)
Production of RhIg from hyperimmune donors with low fucosylated anti-D Ig
might result in more potent RhIg
| 11 23 April, 2018
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Guiding Rh immunoprophylaxis by fetal RHD
typing
• 40% of D-neg women carry a D-neg child and do not need Rh-Ig
• Fetal DNA is present in low concentration in maternal plasma from week 5
| 12 23 April, 2018 Illustration: Lo, 2007
placenta plasma
of pregnant woman
Mixture of DNA
from mother and
DNA from fetus
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Dutch screening program
Pregnancy
12th week
ABO typing
D typing
IEAscreen
27th week
ABO typing
D typing
IEAscreen
Fetal RHD
typing
30th week
Antenatal anti-D Ig
prophylaxis if RHD-
positive fetus
After birth
Postnatal anti-D Ig
prophylaxis if fetal RHD
typing was positive
During first 15 months:
Cord blood analysis
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Technical approach
• Centralized at one laboratory (Sanquin, Amsterdam)
• DNA isolation from 1 ml of EDTA plasma in 27th week of
pregnancy
• First 15 months comparison with cord blood
DNA
Purification
DNA
PCR
Setup
Amplification
Detection
Report
Electronic
Result
Plasma
Pipetting
DNA
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23 april 2018 | 15
Design fetal RHD typing
RHDexon 1 exon 10 exon 10 exon 1
RHCERHDexon 1 exon 10 exon 10 exon 1
RHCE
RHD-PCR Multiplex
exon 5: not amplified in majority of RHD variants
in Caucasians: RHD*DVI
in Blacks: RHD*Ψ; RHD*01N.06 and RHD*03N.01 (r’s)
exon 7: present in most RHD variants
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First 25.500 samples:
Diagnostic accuracy of at least 99.1%
CB-serology
Plasma-PCR
Pos Neg
Pos 15,826 225
(0.87%)
Neg 9
(0.03%) 9,740
| 16 de Haas et al. BMJ 2016
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Systemic analysis of false results
• False negative fetal RHD typing:
• No antenatal prophylaxis : 0.61% risk of alloimmunization
• No postnatal prophylaxis: 17% risk of alloimmunization
• False positive fetal RHD typing:
• Unneccessary RhIg is given, no clinical consequences
| 17 Thurik et al. Prenat Diagn 2016
Stegman et al. Brit J Haematol 2016
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Potential effect of PCR guided ante- and
postnatal immunoprophylaxis
Unnecessary
antenatal anti-
D
No antenatal
anti-D, while
at risk
No postnatal
anti-D, while
at risk
Old program
(no PCR, only cord
blood)
38,3% 0% 0,09%
New program
(only PCR, no cord
blood)
0,43% 0,03% 0,03%
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Accuracy of implemented fetal RHD typing
(Denmark, the Netherlands, Finland)
Samples TRUE Pos
False Pos
TRUE Neg
FALSE Neg
sensitivity %
specificity %
Clausen et al., 2014
12688 7636 41 4706 11 99.86 99.14
de Haas et al., 2016
25789 15816 225 9739 9 99.94 97.74
Haimila et al., 2017
10814 7080 7 3640 1 99.99 99.81
Total 49291 21 99.93
| 19 23 April, 2018
van der Schoot et al. Curr Opin Hem 2017
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Conclusion (2)
Implementation of fetal RHD typing has resulted in decreased
unnecessary use of antenatal RhIg, without loss of efficiency of the
program
| 20 23 April, 2018
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Acknowledgements
• Fc-glycosylation
• Gestur Vidarsson
• Rick Kapur
• Gillian Dekkers
• Lussy Della Valle
• Myrthe Sonneveld
• Manfred Wuhrer (LUMC, Leiden)
• Belinda Kumpel (Bristol)
• Fetal RHD genotyping
• Masja de Haas
• Barbera Veldhuisen
• Florentine Thurik
• Peter Scheffer
• Aicha Ait Soussan
• Tamara Stegman
• Lieve Christiaens (UMCU, Utrecht)
| 21 23 April, 2018