1. science
2. physics
3. electromagnetism

Phasing in RHD Genotyping
What is the Impact?
Greg Denomme, PhD, FCSMLS(D)
Director of Immunohematology and Transfusion
Services
Objectives
¾ Understand the differences among
category/partial, weak, and Del variants
¾ Present the problem of Rh phenotyping in
transfusion and pregnancy
¾ Provide information on a recommendation to
resolve weak D phenotypes
Introduction to RH (004)
¾ RH is one of the most complex blood
group system; 45+ antigens
¾ RhD “inferred” in 1940 by testing human
RBCs with a rabbit anti-Rhesus monkey
RBC antisera
¾S
Subsequently,
b
tl Rh status
t t was discovered
di
d
to be unrelated to the Rhesus monkey
protein (LW)
¾ The Rh proteins are part of a complex
that includes RhD Rhce, RhAG, LW,
Duffy, and Band 3
1
What is D?
¾ Multi-pass membrane protein encoded by RHD at
1p36.13-p34
¾ Differs from Rhce by 34 to 37 amino acids (C or c)
¾ Its’ expression on RBCs defines the Rh-positive
phenotype
h
t
– gene is
i absent
b
t iin ((most)
t) Rh
Rh-negatives!!
ti
!!
¾ Highly immunogenic:
“When a relatively large amount of D-positive red cells (200 ml or
more) is transfused to D-negative subjects, within 2-5 months antiD can be detected in the plasma of some 85% of the recipients.”
Mollison’s Blood Transfusion in Clinical Medicine, 11th ed, pg
183
Distribution of D
Population
Study
Rh+
Rh-
Caucasian
Kopec 1970 Wagner 1995
Garratty 2004
82%
18%
Tunisia, Nigeria
Ranque 1961
Enosolease 2008
92-94%
6-8%
India
Makroo 2013
94%
6%
Basques/Morocco
Goti 1958
Messerlin 1951
71%
29%
China
Shao 2002
>99%
0.3%
Weinstock ,C., Blood Transfusion 2014;12:3-6
Frequency of weak D expression
Hopkins
Scotland
1967
0.56%
Garretta
France
1974
0.66%
Beck
USA
1990
0.2%
Jenkins
USA
2004
0.4%
Flegel
Germany
2006
0.4%
2
Classification of RHD
¾ r’ haplotype: in trans with D (Ceppellini effect)
¾ Partial and Category D: hybrid RHD alleles
ƒ DVI, types I, II, III
yp
single
g amino acid changes
g
¾ Weak D “types”:
ƒ Weak D Type 2
¾ Del: detection by adsorption/elution
ƒ K409K, plus several others
¾ RHD-deletion and non-functional RHD alleles
Ceppelini Effect
Partial and Category D
¾ Arise from hybrid RHD/RHCE genes and missense
mutations to regions of RHD encoding parts of D
external to RBC membrane
¾ React weakly with some monoclonal anti-D reagents
‘altered or missing epitope’
¾ May make anti-D to ‘missing’ epitope of D
¾ if transfused - should receive Rh-negative RBCs
¾ if pregnant - are candidates for RhIG
3
Weak D Types
¾ Arise from missense mutations to regions of RHD
encoding transmembrane/cytoplasmic portion of D
¾ Weak D types may or may not make anti-D
ƒ The absence of the ability to make alloanti-D is not
(never was) included in the definition of weak D types
¾ Can type as Rh-positive or Rh-negative by direct
agglutination with monoclonal anti-D reagents
¾ Weak D types 1 – 3 have not been reported to make
alloanti-D
Rh D polypeptide
Grey: partial D
Black: weak D
Flegel WA. Transfus Clin Bio 2006;13:4-12
Toronto Study (reagent discrepancy)
Two anti-D direct tests were performed routinely
¾ 33,864 D phenotypings performed over an 18 month
interval
¾ 55 of 5672 potential Rh
Rh-negative
negative patients were tube test
positive for one anti-D (0.98%)
¾ 54 were tube test negative using one FDA-approved reagent but
positive (2+ or less) using another government-approved
antisera
4
Michigan Study (reagent change)
Change from manual testing to instrument platform
¾ 10 ‘historical’ discrepancies
evaluated
Anti-D Score
ProVue
Gamma
Clone
Bioclone
8
8
5
8
8
0
8
10
5
¾ repeat instrument testing:
Include an evaluation by tube
t t methods
test
th d carefully
f ll ffollowing
ll i
manufacturer’s instructions
¾ Perform molecular analysis
8
7
0
8
10
5
8
10
6
8
10
5
8
8
5
8
7
0
8
7
0
Alberta Study (policy change – no IAT)
Beginning January 1, 2007 the IAT for the detect of weak
D was discontinued for prenatal patients
¾ For 209 of 88,972 phenotypings (0.23%) the current RhD
type differed from the historical RhD result
prevalence of “Du”))
ƒ Consistent with studies on the p
¾ All RhD results that were discrepant with historical
results were sent for molecular analysis
ƒ Patient returned to the Rh clinic the following week
¾ Recommendation for RhIG prophylaxis was made on the
basis of the RHD allele present
ƒ Weak D types 1 – 4 were deemed Rh-positive
Toronto Study (Reagent Discrepancy)
20 functional RHD alleles detected; 1 wildtype (HDN)
51% were Weak D types 1, 2, and 3
¾ 34 Weak D Types (PCR-RFLP):
¾ 16 weak D Type 1
¾ 1 weak D Type 3
yp 5
¾ 1 weak D Type
8 weak D Type 2
6 weak D Type 4
2 weak D Type
yp 42
¾ 7 DAR
¾ 6 DVa or DVa-like alleles:
¾ 3 DVa(Kou.) 1 DVaHK(E233K) 1 DVa-like 1 DTO (Novel)
¾ DFR, DAU-4, DAU-5 (Novel), DAU-6 (Novel)
¾ Compound heterozygotes: DAR/DAU-2, DAU-0/Cdes
¾ Not identified: a possible DIIIa, DVa, DAR, or DOL
5
Resolution of D discrepancies
¾ Conserve Rh-negative blood for D-negative recipients
(high risk of making anti-D)
¾ Avoid the administration of RhIG to women who do not
need it
¾ Resolve early in pregnancy to eliminate false-positive
rosette tests
¾ Resolved discrepancies (Rh+) avoids delay at
discharge after delivery of an Rh+ infant
What’s Next?
¾ A group of representatives and experts convened to provide a
recommendation for the resolution of Weak D phenotypes
Sandler SG. et al. TRANSFUSION 2015;55:680-9
¾ The weak D phenotype is defined as ≤2+ hemagglutination for
initial testing (any technique), but moderate to strong
hemagglutination with AHG (when performed)
¾ RHD genotyping
t i provides
id clinical
li i l d
decision-making
i i
ki iinformation
f
ti
¾ Recommend that RHD genotyping be performed to resolve
discordant results and when a serological weak D phenotype is
detected.
¾ Weak D types 1, 2, 3 can be deemed Rh-positive for the
purposes of RhIG prophylaxis and transfusion
¾ Impact…
Rh Discrepancy Algorithm
Sandler SG, et al. Transfusion 2015;55:680-9
6
Impact for Pregnancy
3,953,000 Live births
3,812,000 Pregnancies
556,500 RhD-negative
16,700 Serological
Weak D
RHD Genotyping
13,360 weak D
types 1, 2
or 3
24,700 unnecessary
ante- and postpartum
RhIg injections
25,000 unnecessary injections of RhIG
Sandler SG, et al. Transfusion 2015;55:680-9
Impact for Transfusion
5,000,000 Individuals Transfused
Annually in US
730,000 RhD Negative
21,900 Serological
Weak D
RHD Genotyping
17,520 weak D
types 1, 2
or 3
Could receive RhD+ Units
(47,700 units)
50,000 unnecessary Rh-negative transfusions prevented
Sandler SG, et al. Transfusion 2015;55:680-9
Impact if deemed Rh-negative!
Inappropriate use of blood products
RHD Allele
OB
TR
NB
Weak D types
12
8
5
12 OB patients received RhIG
4 transfusion recipients received 12 Rh-neg pRBCs
Impact
DAR
3
1
3
3 OB patients received RhIG
Potential transfusion recipient was not transfused.
DVa and DVa-like
1
1
5
1 OB patient an delivered an Rh-neg infant
Potential transfusion recipient not transfused
DAU, DFR, DTO
3
2
2
2 OB patient delivered an Rh+ infant
Neither potential transfusion recipient transfused
Total:
Total:
20 RhIG
7 Rhig
12 Rh-negative pRBCs
0 Rh-negative pRBCs
7
Michigan Study (Reagent Change)
Anti-D Score
PV GA
BI
Genotype
MoAb
8
8
5
DAR
DV
8
8
0
DAR
DV
8
10
5
Weak D type 1
8
7
0
Weak D type 1
8
10
5
Unknown
8
10
6
Exon 4-5 CE hybrid
8
10
5
Weak D type 2
8
8
5
Weak D type 2
8
7
0
Weak D type 2
8
7
0
Weak D type 2
Alberta Study (Policy Change)
Analysis ’07 to ’08 = 88,972 Total discrepancies = 209 (0.23% of total)
DNA Typing Results
# of Patients
Rh Status
Assigned
RHIG
Recommended
% of DNA Results
Received
Weak D Type 1
60
Pos
No
29.0
Weak D Type 2
19
Pos
No
9.2
Weak D Type 3
38
Pos
No
18.4
Weak D Type 4
15
Pos
No
7.2
DAR
2
Neg
Yes
1.0
Partial DVI Type I
3
Neg
Yes
Partial DVI Type II
1
Neg
Yes
0.5
DVI Type II
2
Neg
Yes
1.0
DVa partial
1
Neg
Yes
0.5
Partial DVA-like
1
Neg
Yes
0.5
Unclassified
31.4
65
Neg
Yes
Pending
2
TBD
TBD
TOTAL
209
57%
1.3
43%
Blood Inventory
America’s Blood Centers, Newsletter April 3, 2015
8
Conclusions
¾ Rh D discrepancies are resolved using RHD genotyping:
) MoAbs cannot identify weak D types 1, 2, 3
¾ Laboratories who change methods, reagents, or drop the
IAT as a routine test on all patients can implement a
recommended clinical practice to resolve discrepancies.
Pregnant
g
women and transfusion recipients
p
expressing
p
g a weak D type
yp 1,2,3
, , have
not been reported to make anti-D. Therefore, for clinical purposes this patient is
deemed Rh positive and is not a candidate for antenatal/postpartum Rh immune
globulin prophylaxis.*
This patient can receive Rh positive blood if red cell transfusions are necessary.
* This patient may be reported as Rh negative by other institutions depending on
the anti-D and the methods used to resolve Rh D discrepancies.
In light of the equivocal D typing with regulatory reagents and the presence of a
partial D, this patient is deemed Rh negative for the purposes of Rh prophylaxis
and red cell transfusion.
Future of RHD ‘genotyping’
¾ 3-5% Rh-negative RBCs go to Weak D type recipients
¾ 3-5% RhIG doses go to women with Weak D types
How often do you need to address Rh status? [~0.5%]
¾ Molecular testing is a permanent solution:
ƒ Weak D types 1,2, 3 are Rh+ pregnancy/Tx recipient (and donor)
• Avoid a blood product where it is not needed (in pregnancy!)
• Informed consent not required for the administration of RhIG
• Mitigate RhIG shortages, potential spread of infectious agent
• Avoid the use of Rh- blood products when an Rh+ will do!
Questions?
9