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50 Years Later: The Current Status of
the Epidemiology, Pathophysiology,
and Treatment of Rh Disease
10/13/2018
Faculty Disclosures
The following faculty have no relevant financial relationships to disclose:
– Jeanne Hendrickson MD
– Ellen Van Der Schoot MD, PhD
– Vinod Bhutani MBBS
The following faculty have a relevant financial relationship:
– Steven Spitalnik MD
Kedrion Biopharma: Consultant
Theranos, Inc.: Consultant
Tioma, Inc.: Consultant
Hemanext: Consultant
New York Genome Center: Consultant
BloodWorks Research Institute: Consultant
www.aabb.org 2
Learning Objectives
• Describe current hypotheses explaining the potential mechanism(s) of action of Rh immune globulin
• Describe how to use modern approaches of Rh typing, including the use of molecular genetic tests, to identify pregnant women who should, or should not, receive Rh immune globulin prophylaxis
• Describe the epidemiology of Rh disease in less well-developed countries and identify ways of establishing this therapeutic intervention in these settings
www.aabb.org 3
Jeanne Hendrickson, MDOctober 13, 2018
Immunoprophylaxis Against Red Blood Cell Antigens: Successes and Failures
The speaker has no disclosures
Objectives
To review the clinical significance of red blood cell (RBC) alloantibodies
To provide an overview of the success of RhIg
To discuss a murine model involving KELIg
Case
A 30 yo P2G1 presented to OB care at 10 weeks of pregnancy
Initial maternal antibody screen showed an anti-K alloantibody Dad tested positive for the K antigen on his RBCs
Mom’s anti-K titer increased during pregnancy
The baby was noted to have anemia and evidence of early hydrops fetalis by middle cerebral artery doppler testing
Intrauterine Transfusions (IUT) with K Negative RBCs were Begun
Case
After the 2nd intrauterine transfusion, the mother developed an anti-c alloantibody in addition to her anti-K alloantibody
The baby was delivered by C/S at 35 weeks of gestation, after a total of 7 intrauterine transfusions
He presented at 4 weeks of age with a Hgb of 6.6 g/dL and retic <0.5%
Antibody screen (IAT) positive for anti-K and anti-c
Required transfusion therapy until 4 months of life
RBC Alloantibodies
May develop following transfusion of products containing foreign RBC alloantigens
3-5% of transfused patients
May occur during pregnancy/following delivery of a fetus/infant whose RBCs contain paternally derived alloantigens foreign to the mother
~6/1000 pregnancies
RBC Alloimmunization
Occurs in approximately 3-5% of “general” transfused adults
Karafin et al, BJH 2018
REDS-III Recipient Database, with 6,597 alloimmunized patients
RBC Alloimmunization
The responder/non-responder ratio spikes in females during childbearing years
Grey bars = femaleBlack bars = male
6,597 responders and 30,569 non-responders
Karafin et al, BJH 2018
REDS-III Donor Data: RBC Antibody Screen Positivity is higher in females than in males through all age ranges
Karafin et al, in press, Transfusion
>600,000donors
Never
tran
sfu
sed
, n
ever
pre
gn
an
t
Never
tran
sfu
sed
, ever
pre
gn
an
t
Ever
tran
sfu
sed
, n
ever
pre
gn
an
t
Ever
tran
sfu
sed
, ever
pre
gn
an
t
0
2
4
6
% P
os
itiv
e
n /a n /a
Black bars = malesGrey bars = females
REDS-III Data: Past Pregnancy Accounts for Most Positive RBC Antibody Screens in Female Blood Donors
Karafin et al, in press, Transfusion
Never
tran
sfu
sed
, n
ever
pre
gn
an
t
Never
tran
sfu
sed
, ever
pre
gn
an
t
Ever
tran
sfu
sed
, n
ever
pre
gn
an
t
Ever
tran
sfu
sed
, ever
pre
gn
an
t
0
2
4
6
% P
os
itiv
e
n /a n /a
Black bars = malesGrey bars = females
REDS-III Data: Past Pregnancy Accounts for Most Positive RBC Antibody Screens in Female Blood Donors
Karafin et al, in press, Transfusion
>131,000 females were never transfused and ever pregnant;
>2000 females were ever transfused and never pregnant
>8000 females were ever transfused and ever pregnant
1800+
57
448
Rh
D
Rh
CE K J
k
Du
ffy
MN
S
Oth
er
0
5 0
1 0 0
1 5 0
F e m a le : t ra n s fu s io n v s p r e g n a n c y
Nu
mb
er o
f fe
ma
le d
on
ors
T ra n s fu s io n
P re g n a n c y
REDS-III Data: Most Anti-D Antibodies Detected Were Induced Through Pregnancy
Karafin et al, in press, Transfusion
Did these women not receive RhIg, or did RhIg fail?
Prevention of HDFN: Rh(D)
Polyclonal anti-D is one of the most successful immunotherapies to date for Rh(D) prophylaxis
Mechanism of action is unclear
Studies support and refute multiple potential mechanisms of action
Antigen blocking/steric hindrance
RBC clearance
FcRIIb mediated inhibition
Other
Despite decades of research, no monoclonal anti-D has been deemed successful enough at preventing Rh(D) immunization to be licensed for use during pregnancy
J OB GYN CA 2018
RhIg is Strongly Recommended in Many Situations, with High Quality Evidence
Prevention of HDFN: Rh(D)
Does RhIg solely prevent anti-D antibodies, or might it also mitigate the formation of other RBC alloantibodies?
O/A and O/B mother/baby pairs have lower rates of RBC alloimmunization during pregnancy, presumably due to premature clearance of fetal RBCs
Does RhIg clear enough fetal RBCs to have the same benefit?
Prevention of HDFN: Non-Rh(D)
No “antigen specific” therapy current exists to prevent or to mitigate the dangers of an existing non-Rh(D) alloantibody in pregnancy
TPE, IVIG, IUT IUT is associated with a relatively high risk of new
antibody formation
Transfusion perspective:
Phenotypic matching
K negative RBCs for females of childbearing age
Given the logistic difficulties of completing mechanistic studies in humans, we decided that studies using a transgenic mouse model with RBC specific expression of a clinically significant human blood group system (KEL) may increase our understanding of the induction and prevention of RBC alloimmunization.
Development of Transgenic Mice with RBC Specific Expression of Human KEL Glycoprotein Antigen
Smith et al, Transfusion 2012
Anti-Jsb Anti-Kpb Anti-Cellano
Transfusion Induced KEL RBC Alloimmunization
Wild type B6 recipients
Evaluation for anti-KEL responses by flow crossmatch with KEL or B6 RBCs
RBCs from Transgenic KEL donors
RBC transfusion
Hypothesis: Passive Infusion of Polyclonal Anti-KEL Will Prevent Active Anti-KEL Formation in our Murine Model
Characterization of Effect of Polyclonal Anti-KEL on Active Alloantibody Response
Wild Type Recipients
KELIg Infusion or Saline Control
Analysis of Antibody Response
Analysis of RBC Clearance Patterns
Analysis of Antigen/Antibody Interactions
Transfusion of (Labeled) KEL and B6 RBCs
Polyclonal Anti-KEL (“KELIg”) Prevents KEL Alloimmunization
Passive KELIg or IgG enriched KELIg
KEL RBC Transfusion
Recipient Anti-KEL Alloimmune Responses
KE
L R
BC
s a
lon
e
Passiv
e K
EL
Ig, K
EL
RB
Cs
0
5 0
1 0 0
1 5 0
R e c ip ie n t A n t i-K E L G ly c o p ro te in Ig G R e s p o n s e s
Ad
jus
ted
M
FI
Passive IgG enriched KELIg, then KEL RBCsKEL RBCs alone
Anti-KEL Glycoprotein
Stowell et al, Haematologica 2015
At First Glance, KEL RBCs Transfused into Recipients Treated with KELIg Appear to be Rapidly Cleared
0 .0 0 .5 1 .0 1 .5
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
D a y s P o s t-T ra n s fu s io n
MF
I
P a s s iv e K E L Ig
N o K E L Ig
K E L R B C R e c o v e ry a s M e a s u re d
b y K E L A n tig e n D e te c tio n
Recipient without
KELIg
Recipient with
KELIg
Stain recovered RBCs with polyclonal anti-KEL
Secondary is anti-mouse Igs
Stowell et al, Haematologica 2015
At Second Glance, About 50% of Incompatible KEL RBCs Remain in Circulation
Stowell et al, Haematologica 2015
Passive KELIg or IgG enriched KELIg
KEL RBC Transfusion
RBC Clearance Patterns
The KEL RBCs Remaining in Circulation have no KEL Antigen Detectable by Flow Cytometry
100 101 102 103 104
FSC-H
100
101
102
103
104
SS
C-H
100 101 102 103 104100
101
102
103
104S
SC
-H
FL1 (KEL x uGFP RBCs)
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
K E L A n tig e n D e te c tio n
o n u G F P K E L R B C s
MF
I
Co
ntr
ol
2 h
ou
rs
Co
ntr
ol
10
min
ute
sC
on
tro
l 2
4 h
ou
rs
Inc
om
pa
t 2
ho
urs
Inc
om
pa
t 1
0 m
inu
tes
Inc
om
pa
t 2
4 h
ou
rs
Evaluate KEL antigen on uGFP RBCs using polyclonal anti-KEL
Stowell et al, Haematologica 2015
The KEL RBCs Remaining in Circulation also have no KEL Antigen Detectable by Western Blot
Liu et al, Blood 2016
KEL Antigen Modulation is Not Unique to This Murine
Model
Zimring et al, TMR 2009
Zimring et al, TMR 2009
Antigen Modulation Has Been Described in Multiple Blood Group Systems in Humans
Antigen Modulation Has Also Been Described to Rh(D), in a Child with ITP Treated with RhIg
Stowell et al, Transfusion 2018
Pre-RhIg Post-RhIg
Clearance of Incompatible KEL RBCs Involves Complement and FcγR Pathways
Girard-Pierce et al, Blood 2013
+/- Passive KELIg
administration
KEL RBC
transfusion
Recipient anti-KEL alloimmune responses
Wild type, FcγR KO, C3KO, or FcγR KO x C3 KO (Double KO) recipients
Wild
typ
e
FcR
KO
C3K
O
Do
ub
le K
O
Mu
MT
0
5 0 0
1 0 0 0
A n ti-K E L Ig G in M ic e T ra n s fu s e d
W ith Im m u n o p ro p h y la x is
Ad
jus
ted
M
FI
KELIg Prevents Alloimmunization in FcγR KO or C3 KO Mice, but Fails to Prevent Alloimmunizationin Double KO Mice Lacking FcγR and C3
Liu et al, Blood 2016
B6
FcR
KO
C3 K
O
FcR
x C
3 d
ou
ble
KO
B6, w
ith
an
t i-K
EL
FcR
KO
, w
ith
an
t i-K
EL
C3 K
O, w
ith
an
t i-K
EL
FcR
x C
3 d
ou
ble
KO
, w
ith
an
t i-K
EL
0
5 0
1 0 0
1 5 0
2 0 0
K E L A n t ig e n D e te c te d
o n T ra n s fu s e d R B C s
MF
I
Bars shown for each group are:
10 minutes, 1 hour, and 24
hours post-transfusion
KEL Antigen Modulation Does Not Occur in FcγR x C3 Double KO Mice
Liu et al, Blood 2016
KELIg Working Model, Circa 2016:
Westhoff, Blood 2016
Is antigen modulation critical to the mechanism of action of KELIg?
Will KELIg fail in the presence of an adjuvant?
Poly (I:C) and KELIg
KEL RBC Transfusion
Recipient Anti-KEL Alloimmune Responses
Poly (I:C) is a double stranded RNA that mimics viral-like inflammation
Inflammation with Poly (I:C) Increases Alloimmunization in Every Murine Model We Have Studied to Date
Anti-HEL IgG by ELISA
LR H
OD
PIC
LR H
OD
0
1
2
3O
D 4
15 Anti-hGPA by flow crossmatch
hGPA
PIC
hGPA
0
200
400
600
800
1000
Ad
juste
d
MF
I
Hendrickson et al, Transfusion 2006 and 2010; Smith et al, Blood 2012; Stowell et al, Transfusion 2013.
Pretreatment with Poly (I:C) Around the Time of KELIg Infusion Leads to KELIg Immunoprophylaxis Failure
Poly (I:C) and KELIg
KEL RBC Transfusion
Recipient Anti-KEL Alloimmune Responses
No
KE
LIg
KE
LIg
Po
ly (
I:C
) + K
EL
Ig
0
2 0 0 0
4 0 0 0
6 0 0 0
R e c ip ie n t A n t i-K E L G ly c o p ro te in Ig G R e s p o n s e s
Ad
jus
ted
M
FI
Modulation of the KEL Antigen Occurs Equally Well in Mice Treated with Poly (I:C) or Not
No
KE
LIg
KE
LIg
Po
ly (
I:C
) + K
EL
Ig
0
1 0 0
2 0 0
3 0 0
4 0 0
K E L A n t ig e n M o d u la t io n
MF
I
10 min, 1 hr, 24 hrs, 7d post-transfusion
Clearance of KEL RBCs Occurs at Least as Rapidly if Not More So in Mice Treated with Poly (I:C)
10 min, 1 hr, 24 hrs, 7d post-transfusion
No
KE
LIg
KE
LIg
Po
ly (
I:C
) + K
EL
Ig
0
5 0
1 0 0
1 5 0
K E L R B C C le a ra n c e
Pe
rc
en
tag
e
What Pathways Are Most Important for Poly (I:C) Induced RBC Alloimmunization?
Gibb et al, JI 2017
A B
*
* *
Gibb et al, JCI 2017 and Transfusion 2017
Type 1 IFN Signaling is Important for Anti-KEL Responses in General and for Responses to Poly (I:C)
General KEL Responses:
Responses to Poly (I:C):
- + P o ly( I:C )
0
1 0 0
2 0 0
3 0 0
4 0 0
A n ti-K 1 Ig G
Ad
jus
ted
MF
I
W T IF N A R 1-/ -
0
2 0
4 0
6 0
A n ti-K 1 Ig G
Ad
jus
ted
M
FI
+ Poly (I:C)
Days 0, 5, 10, 21, 28 post-transfusion
Treatment of Animals With Exogenous Type I IFN Also Leads to KELIg Immunoprophylaxis Failure
KE
L R
BC
s o
nly
KE
LIg
Po
ly (
I:C
) + K
EL
Ig
Exo
gen
ou
s T
yp
e 1
IF
N
0
2 0 0 0
4 0 0 0
6 0 0 0
8 0 0 0
1 0 0 0 0
A n ti-K E L G ly c o p ro te in Ig G
MF
IExogenous type 1 IFN and KELIg
KEL RBC Transfusion
Recipient Anti-KEL Alloimmune Responses
KELIg Working Model, Circa 2018
KELIg efficacy does not appear to be fully dependent on its ability to modulate the KEL antigen or to clear RBCs
The KEL antigen is rapidly modulated in the presence of poly (I:C) or exogenous type 1 IFN, yet immunoprophylaxis failure still occurs
Each “failure” leads to additional questions
Summary
RBC alloimmunization remains a relatively “common” complication of pregnancy (and transfusion)
RhIg is one of the most successful immunomodulatory therapies to date The mechanism of action remains poorly understood
Animal studies of KELIg have increased our understanding of immunoprophylaxis (a bit) But many questions remain
Back to the Beginning. . .
A 30 yo P2G1 with anti-K alloantibody: Could primary pregnancy associated anti-K
alloimmunization have been prevented with “KELIg” type therapy?
Would this woman have had a lower risk of forming an anti-K antibody if she had been RhD negative and received RhIg?
Thank YouNHLBI
NIDDK
Yale Cooperative Center of Excellence in Hematology
Lab members (Jingchun Liu, David Gibb, Manju Santhanakrishnan, Dong Liu, and others)
Collaborators at Yale (Steph Eisenbarth’s Lab)
Collaborators at Emory (Sean Stowell’s Lab)
Collaborators in Seattle (Jim Ziming’s Lab, Krystal Hudson’s Lab)
22 oktober 2018 | 1
Guiding Rh-immunopropylaxis by
non-invasive fetal RHD typing
Ellen van der Schoot
Department Experimental Immunohematology, Sanquin Research; Amsterdam, the Netherlands
Guiding immunoprophylaxis
Immunoprophylaxis is indicated in RhD-negative women pregnant of an RhD
positive child
Based on RHD allele frequencies in a Caucasian population
an RhD-negative mother has
60% chance to carry an RhD-positive child
40% chance to carry an RhD-negative child
Fetal RHD status can be determined on cell free fetal DNA
22 oktober 2018 | 2
Source of fetal DNA: syncitiotrophoblast
| 3
22 October, 2018
Maternal blood
Chorionic villi
Syncitiotrobhoblast
Cell-free fetal DNA in maternal plasma
Illustration: Lo, 2007
placenta plasma
of pregnant woman
Mixture of DNA
from mother and
DNA from fetus
Excess of maternal cell-free DNA:
• First trimester: 3% fetal DNA (range: 0,5% - 12%)
• Last trimester: 6% fetal DNA (range: 2% - 15%)
Cell free DNA is derived from apoptotic cells
• Present in plasma as nucleosomes
• Majority of cell free fetal DNA < 143 bp
• Cell free maternal DNA: majority >143bp:
• Mainly derived from maternal leukocytes
• Increased in various conditions: e.g. sepsis, autoimmune diseases, pregnancy
| 5
22 October, 2018
Illustration Lo 2010
Concentration of cell free fetal DNA
• Earliest presence: 5 weeks of gestation
• Very low concentration of fetal DNA in maternal plasma
• 16th week: 25 genome equivalents/mL of plasma (range 3-70 geq/mL)
• 30th week: 290 genome equivalents/mL of plasma (range 50-1000
geq/mL)
• Half-life: 45 minutes
| 6
22 October, 2018
October 22, 2018 | 7
Fetal typing: three challenges!!!
1 . Very low concentration
25 -500 genome equivalents/mL
2.. Still no universal control for the presence of fetal DNA
=> Sensitivity
3. Excess of maternal cell-free DNA:
3 – 6 % fetal DNA
=> Specificity
Dutch fetal RHD screening program
since July 2011 (organized by RIVM)
Pregnancy of D-neg women
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 evaluation year:
Cord blood sent to Sanquin for
serology and DNA analysis
Fully automated approach
• Centralized at one laboratory (Sanquin, Amsterdam)
• 7-8 cc EDTA anti-coagulated blood
• DNA isolation from 1 ml of plasma
• Robotic workstation for PCR setup
• RQ-PCR in triplicate (15 µL input/well), 50 cycli
• NO fetal identifier, NO total DNA control
• Real-time PCR
• 25 µL PCR
Plasma
separation
Purification
RNA/DNA
PCR
Setup
Amplification
Detection
Report
Electronic Result
Xiril MagnaPure 96 Xiril StepOnePlus
22 oktober 2018 | 10
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: 80% RHD*Ψ;
RHD*03N.01 (r’s or Ccdes-1)
RHD*01N.06 (Ccdes-2)
exon 7: present in most RHD variants
Results of the first 15 months
22 oktober 2018 | 11
33,673
blood samples
32,237
RhD- pregnants
1,436
RhD+ pregnants
19,877(61.7%)
RHD+ results
12,360(38.3%)
RHD- results
16,051(80.8%)Cord blood known
9,748 (78.9%)Cord blood known
15,823 (61.33%)
Serology RhD+
228 (0.88%)
Serology RhD-
9,740 (37.75%)
Serology RhD-
*+1 false-negative result determined in hospital
8 (0.03%)*
Serology RhD+
Systemic analysis of false results
• False negative fetal RHD typing:
• No antenatal prophylaxis : 0.30 % extra risk of alloimmunization
(Koelewijn et al. Transfusion 2008)
• No postnatal prophylaxis: 17% extra risk of alloimmunization
• False positive fetal RHD typing:
• Unneccessary RhIg is given, no clinical consequences
22 oktober 2018 | 12
22 oktober 2018 | 1322 oktober 2018 | 13
Repeat testing (manual DNA isolation)
- DNA fingerprinting of cord blood and maternal blood
- RHD-PCR on cord blood
- Repeat RHD PCR; mRASSF1a, DYS14 and alb-PCR
Results
- Sample mix up: n=0
- Fetal DNA concentration low: n=7
- Technical failure or putative technical failure: n=2
Nine false negative results
NO fetal RH variants were found, unlikely since all known
RH-variants with D-expression will be positive in our assay
How to decrease false negative results
• Increase the input of plasma?
• For 99.96% of the women (15824/15831) 1 ml is sufficiënt
• Fetal DNA concentration is highly variable
=> to cover all women 10 ml of plasma would be needed, not feasible22 oktober 2018 | 14
How to decrease false negative results
• Increase the input of plasma?
• For 99.96% of the women (15824/15831) 1 ml is sufficient :
• Implement a positive control for the presence of fetal DNA?
• No simple universal fetal DNA marker is available for RQ-PCR
• Inclusion of synthetic DNA sequence, added to the plasma and tested
in triplex PCR together with RHD PCR?
• Control for DNA isolation (pipetting errors of robotic DNA isolation)
• Control for PCR inhibitors in plasma
• 1000 copies of plasmid is added to 1 ml plasma (unpublished results)
22 oktober 2018 | 15
NO
YES
NO
Most false negative results are caused by (biological?) variation
in fetal DNA concentration
| 16
Evaluation of false positive results (0.87%)
non-specific amplification
0.45%
D-pos fetal variants0.09%
D-neg fetal variants0.09%
D-neg maternal variants0.21%
Vanishing twin0.03%
PCR algorithm is aimed to
prevent false negative results
Placental chimerism (Thurik et al. Prenatal Diagnosis 2016)
False negative cord blood
D-neg RHD variants(Stegmann et al. BJH
2016 )
Maternal RHD-variants
22 oktober 2018 | 17
Amplification of
maternal RHD DNA
hides fetal DNA
=> Prophylaxis advised
How to deal with these mothers:
- analyze maternal RHD gene?
- issue result on a single exon?
- guide postnatal prophylaxis by cord blood?
Maternal RHD
Fetal RHD
~1% of Dutch serologically RhD-’negative’
women carry an RhD-negative RHD variant
gene• 362 women with variant RhD-negative RHD allele:
0.96% (95% CI 0.86 % - 1.06%)
55% D-neg (47% RHD*Ψ) :: 11 novel alleles in 14 women
3 normal RHD alleles in 3 women
16% Del (2.6% RHD*01EL01) 2 novel alleles in 9 women
3% weak D (2.2% RHD*W01 or *W02)
26% Partial D (16% RHD*06) 2 novel alleles in 2 women
0.05% novel alleles
0.43% detected by genotyping and missed by serology
October 22, 2018 | 18Stegmann et al. BJH 2016
Fetal RHD typing in mothers with RHD*Ψ and
RHD*DVI on only RHD exon 5 is not reliable
Variant
mother
Number Cord
available
"Child positive“
exon 5
"Child negative"
Dpseudo 122 87 61 (4 false) 26 (1 false)
DVI 46 40 21 (0 false) 19 (2 false, 9 DVI)
<32UD
Exon7Exon5
<3234-40
Exon7Exon5
“Child negative”“Child positive”
RHD*pseudogene and RHD*DVI: 63% of maternal variants
Maternal amplification of RHD exon 7, mother negative for RHD exon 5
Amplification of maternal exon7
inhibits fetal exon5 PCR
22 oktober 2018 | 20
Exon 7 Exon 5
RHD-negative mother with RHD positive child
RHD-pseudogene positive mother with RHD positive child
Conclusions
• Fully automated and robotized fetal RHD typing is efficient and reliable
(99.1% concurrence, 9 false negative results)
• The design of the exon5-exon7 PCR does not allow reliable prediction of
fetal RhD-negativity in the majority of women carrying variant RHD gene
• Antenatal and postnatal prophylaxis can be safely guided by fetal RHD
typing
| 21
Unnecessary
antenatal anti-D
No antenatal anti-
D, while at risk
No postnatal anti-
D, while at risk
Old program
(no PCR, only CB)38,3% 0% 0,09%
New program
(only PCR, no CB)0,43% 0,03% 0,03%
Accuracy of implemented fetal RHD typing
SamplesTRUEPos
FALSE Pos
TRUENeg
FALSENeg
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
| 22
22 October, 2018
van der Schoot et al. Curr Opin Hem 2017
Future perspectives
• Multiplex PCR with extra amplifications negative on RHD pseudogene
• Fetal RHD typing earlier in pregnancy
• To guide antenatal prophylaxis after obstetric procedures or immature deliveries
before 30th week
• Sensitivity of fetal RHD typing in week 11-13 slightly lower (99.12%)
• Reducing costs for women opting for NIPT
• by including RHD typing in targeted NIPT tests
• by Including HPA-1a typing in targeted NIPT tests
22 oktober 2018 | 23
Acknowledgements
| 24
Florentine
Thurik
Aicha Ait
Soussan
Lieve Page-
Christiaens
Masja
de Haas
Barbera
Veldhuisen
Heleen
Woortmeijer
50 Years Later:
The Current Status of the Epidemiology,
Pathophysiology, and Treatment
of Rh Disease
50 Years Later:
The Current Status of the Epidemiology,
Pathophysiology, and Treatment
of Rh Disease
Steven SpitalnikIntroduction
Jeanne HendricksonPathophysiological mechanisms
Ellen van der SchootState-of-the-art practice
Vinod BhutaniIssues remaining in less well developed countries
50 Years Later:
The Current Status of the Epidemiology,
Pathophysiology, and Treatment
of Rh Disease
Steven SpitalnikIntroduction
Jeanne HendricksonPathophysiological mechanisms
Ellen van der SchootState-of-the-art practice
Vinod BhutaniIssues remaining in less well developed countries
50 Years Later:
The Current Status of the Epidemiology,
Pathophysiology, and Treatment
of Rh Disease
Steven SpitalnikIntroduction & Global Perspective
Jeanne HendricksonPathophysiological mechanisms
Ellen van der SchootState-of-the-art practice
50 years of preventing Rh disease
and the need to “complete the job”
Steven L. Spitalnik, M.D.
Laboratory of Transfusion Biology
Hemanext: Advisory Board
Tioma, Inc: Consultant
BloodWorks Research Inst.: Advisory Board
Kedrion Biopharma: Consultant
Potential Conflicts of Interest
David Zimmerman
Karl Landsteiner
ABO: Vienna, 1900
M, N, P: New York, early 20th century
Karl Landsteiner
ABO: Vienna, 1900
M, N, P: New York, early 20th century
Philip Levine Alexander Wiener
Landsteiner’s students
Co-discoverers of Rh
Philip Levine Alexander Wiener
Landsteiner’s students
Co-discoverers of Rh
Hemolytic Disease
of the Fetus & Newborn
Erythroblastosis fetalis
Fetal hydrops
Neonatal anemia and jaundice
Kernicterus
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Maternal IgG to an RBC alloantigen crosses
placenta, binds to fetal RBCs, and destroys them.
Diamond, Chown, et al.
Rh immune globulin to protect
against maternal alloimmunization to D
Columbia University/Presbyterian Hospital
Vincent Freda, MD: Obstetrics & Gynecology
John Gorman, MD: Pathology (Blood Bank)
John Gorman in 2016
131 Years
This upside-down idea was entirely “doable” in 1871.
It just didn’t occur to anyone for 131 years.
First clear glass Heinz ketchup bottle introduced in 1871,
the squeezable in 1983, the upside down in 2002. It took
131 years before the upside-down idea was thought of
and implemented.
Theobald Smith
Journal of Experimental Medicine 11:241-256, 1909
Theobald Smith
Ideas that were circulating at the time
Alloimmunization and Rh disease rarely seen in
first pregnancy (different than ABO HDFN)
Fetal outcomes get progressively worse with
subsequent Rh-incompatible pregnancies
Maternal-fetal ABO incompatibility is protective
(e.g. Group A fetus & Group O mother)
When do fetal RBCs enter the maternal
circulation?
The plan (NYC & Liverpool)
Inject all Rh(D)-negative primigravidas at
delivery with a source of IgG anti-D
(hyperimmune plasma, purified gamma
globulin, etc.)
Do not treat with IgG anti-D before delivery
Monitor the health of the first neonate, the
development of anti-D, and the outcome of
the next pregnancy
The plan (NYC & Liverpool)
Science 151:828-830, 1966
The plan (NYC & Liverpool)
New Engl J Med 277:1022-1023, 1967
How does it really work?
Antibody-mediated immunosuppression (AMIS)
Clearing RBCs before immune system can
“see” them
Cloaking the Rh antigen
Fc receptor-mediated mechanism
Other?
Remains unknown
How does it really work?
Antibody-mediated immunosuppression (AMIS)
Clearing RBCs before immune system can
“see” them
Cloaking the Rh antigen
Fc receptor-mediated mechanism
Other?
Remains unknown
When do fetal RBCs enter the
maternal circulation?
Alvin Zipursky
Kleihauer-Betke test
Post GR et al. Laboratory Hematology 18:11-13, 2012
Fetal RBC
Maternal RBC
Lancet February 29, 1959, pages 451-452
Fetal RBCs in maternal circulation
during pregnancy
Bruce Chown John Bowman
Canadian Medical Association Journal 118:623-627, 1978
Antenatal RhIg:
“surprising” results
IgG anti-D (RhIg) does cross the placenta
Anti-D is detected in fetal plasma
Anti-D does bind to fetal RBCs (i.e., DAT+)
No hyperbilirubinemia no “disease”
Enhances effectiveness in preventing
alloimmunization to Rh(D)
More fruits of Rh disease research
Amniocentesis
Liley curve
Fetal blood sampling
Intrauterine transfusion
Exchange transfusion
“Bili-lights”
Prenatal diagnosis with cell-free DNA
“Cell-free” DNA
New activities at Columbia
&
New York-Presbyterian Hospital
/Users//Documents/Fundraising/anniversary/3698 John G. Gorman Lectureship 2016/3698 Gorman1609230036.jpg
Lead
The panel (with Gorman looming)
The first patient
Dr. David Landers/Marianne Cummins/Alvin Zipursky
Malcolm Pollack/Charlie Clark/Pam Freda
Next generation
Jeanne Hendrickson (Yale): Keynote Speaker
http://newsroom.cumc.columbia.edu/blog/2018/02/22/rhogam-
at-50-a-columbia-drug-still-saving-lives-of-newborns/
https://vimeo.com/254747528
Not yet…
Rh disease: Are we done?
Not yet…
Rh disease: Are we done?
“Gap analysis”
Gioacchino De Giorgi (Kedrion), et al.
Unpublished data
Rh disease: Are we done?
“True” gap =
(doses needed) -
(actual doses given)
Rh disease: Are we done?
“True” gap =
(ante-partum doses + post-partum doses)-
(actual doses given)
Rh disease: Are we done?
Rh disease: Are we done?
The number of anti-D doses
administered was not available for 27countries
33 (17%) countries for which PP IP isfully satisfied
11 (6%) countries with a gap between
PP IP demand and supply not higherthan 30%
2 (1%) countries with a gap between
PP IP demand and supply between30% and 50%
25 (13%) countries with a gap between
PP IP demand and supply between50% and 80%
101 (51%) countries with a gap
between PP IP demand and supplyhigher than 80%
Rh disease: Are we done?
Rh disease: Are we done?
Actual doses
Rh disease: Are we done?
Dosed needed for
post-partum prophylaxis
Actual doses
Rh disease: Are we done?
Doses needed for routine
ante-partum prophylaxis
Dosed needed for
post-partum prophylaxis
Actual doses
Rh disease: Are we done?
Doses needed for routine
ante-partum prophylaxis
Dosed needed for
post-partum prophylaxis
Actual doses
Rh disease: Are we done?
Doses needed for routine
ante-partum prophylaxis
Dosed needed for
post-partum prophylaxis
Actual doses
Rh disease: Are we done?
Existing gap (if only provide post-partum):
>2,600,000 doses
At ~$50/dose = ~$230,000,000
Rh disease: Are we done?
Existing gap (if only provide post-partum):
>2,600,000 doses
At ~$50/dose = ~$230,000,000
Rh disease: Are we done?
Existing gap (if only provide post-partum):
>2,600,000 doses
At ~$50/dose = ~$230,000,000
Rh disease: Are we done?
Rh disease: Are we done?
November 1, 2018
Columbia University
Thank you