50 years later: the current status of the epidemiology ...€¦ · the speaker has no disclosures....

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


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


>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


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


At Second Glance, About 50% of Incompatible KEL RBCs Remain in Circulation


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


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


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


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


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


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


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

http://safenoe.org/cocoon/safeorg/info/public/index.xhtml

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:



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:



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

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


Science 151:828-830, 1966


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

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

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

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?

“Gap analysis”

Gioacchino De Giorgi (Kedrion), et al.

Unpublished data


“True” gap =

(doses needed) -

(actual doses given)


“True” gap =

(ante-partum doses + post-partum doses)-

(actual doses given)



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%


PP IP demand and supply between30% and 50%


PP IP demand and supply between50% and 80%

101 (51%) countries with a gap

between PP IP demand and supplyhigher than 80%


Actual doses


Dosed needed for

post-partum prophylaxis

Actual doses


Doses needed for routine

ante-partum prophylaxis

Dosed needed for

post-partum prophylaxis

Actual doses


Existing gap (if only provide post-partum):

>2,600,000 doses

At ~$50/dose = ~$230,000,000


November 1, 2018

Columbia University

Thank you