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The myth and mystery of RhD
Joyce Poole
International Blood Group Reference Laboratory Bristol UK
Quotient Biodiagnostics Industry Workshop AABB San Diego 2011
• Reasons why myth and mystery surround RhD!
• Case studies with learning points: pregnancy, transfusion, donor-related
• IBGRL approaches and use of ALBAclone anti-D panel
• UK typing protocols for D typing and treating D variant patients
Objectives of my talk
• Secret or hidden
• Puzzling
Mystery?
Why is D difficult
•Not a simple antigen
•Nomenclature is confusing
• D typing is not straightforward
• Controversial
The mystery of RhD
Complex
Clinically significant!
•Huge molecular diversity
• >150 variant D antigens
• >50 ways of being D negative/Del
The mystery of RhD
Complexity
• Not derived from an amino acid polymorphism but from presence of RhD protein
• D expression dependent on different epitopes along the RhD protein
• Epitopes are conformation-dependent
• Antigen expression varies quantitatively and qualitatively
The mystery of RhD
Confusing Nomenclature
•D+
•D-
D category D mosaic
Partial D Weak D
Weak partial D D variant
The mystery of RhD
Controversial
D+ or D- ?
The mystery of RhD
Do we treat the patient as D+ or D-?
The myth of RhD
Widely held but false notion
D typing is routine test and should therefore be straightforward!
D typing
How do we do it ?
Column
Like this balancing act, D typing can be difficult
for the majority of us
GUIDELINES
Compatibility procedures in blood transfusion laboratories
BCSH British Committee for Standards in Haematology/
Blood Transfusion
(Transfusion Medicine 2004:14:59-73)
Patients
• Test in duplicate with IgM monoclonal anti-D
• [Two anti-D or same one twice]
• Exception for full automation - single anti-D
• Anti-D should not detect DVI
D typing patients (1)
UK GUIDELINES
• The IAT should not be used
• Anti-CDE is of no value and is not recommended
[Misinterpretation of r’ and r’’ as D+]
UK GUIDELINES
D typing patients (2)
Non-compliance with guidelines!
• 24% not performing duplicate D typing are using
manual systems
• 5% using IAT anti-D for D neg pre-transfusion samples (3% in 2002) [recommended against]
• 6% include an anti-CDE reagent (10% 2002)
• 9 labs using one anti-D that detects DVI
• <1% diluting anti-D! (5% in 2002)
Data from UK NEQAS exercise late 2005
Patient D typing in UK
Donors
D typing donors
• Adopt procedures to maximise detection of weak D and partial D as D positive
• Determined on each donation
• D group in doubt?
Safer to classify as D+
Pregnant Woman (SR)
• Typed as normal D+ (Ro) but with allo anti-D in plasma
• All anti-D’s positive with her cells • No Ig anti-D given • Referred for RHD sequencing
Case Study
609 654 667 674 807
186 602 667 819
Novel DIII
RHD Psuedogene
609 654 667 674 807
RHD Psuedogene
Normal RHD
Twin son and daughter of SR - mutations in RHD
Patient SR - mutations in RHD
10 exons of RHD
10 exons of RHD
Transfusion Recipient
• Elderly male patient - normal D+ with allo anti-D
• Transfused in 1975 with 4 units of D+ • RHD sequence – exon 4 mutation G520A (V174M)
• Characteristic of weak D type 33 • Transfused D negative from now on
Case Study
Blood Donor • Female donor typed as D- (r’’r) • Transfused to a D- recipient who made anti-D!
• Referred to IBGRL for elucidation • Very weak expression of D • Rh genotype D+
Case Study
RHD sequence: No mutations in RHD (or CE or RHAG)
Learning points
• Partial D and weak D can both present as normal D
• Some variants will only be detected if they have made anti-D
• Weak D’s can make allo anti-D • Important to detect very weak expression of D on donor cells to prevent immunisation
Case Studies
IBGRL referrals
UK hospitals Overseas reference
labs
Blood Centre
IBGRL
We do not do routine patient or donor typing
Reasons for referral • Pregnant female - do we give antenatal immunoglobulin anti-D?
• Patient is D+ with anti-D - is it allo or auto?
• Is this a weak D or partial D?
• Do we treat as D+ or D-?
IBGRL referrals
• 4 routine anti-D reagents that detect weak D + most partial D between them (+ C, Cw, c, E, e)
• ALBAclone IgG anti-D panel (12)
Clear-cut pattern Not clear
IBGRL referrals
Report
Refer for molecular analysis (RHD sequence)
1
2
411 RhD referrals in 5 years • Variants that gave clear patterns of reactivity vs the ALBAclone panel – DHK/DAU-4 : 17 – DVII : 12 – DVI : 9 – DFR : 7 – DMH : 6 – DOL : 6 – DAU-5 : 12 – DAR-E : 6 – Plus many others of even lower incidence
• Variants that can give ambiguous patterns of reactivity vs the ALBAclone panel – Weak D type 1 : 74 – Weak D type 2 : 79 – Weak D type 4.2.2 (DAR) : 50
• Why the variation? – Different individuals express different amounts of the RhD protein
– C in trans (R1*r’ and R2*r’) weakens expression of RhD
High referral rate
The variants DAU-5 and DV type 1 gave an identical serological pattern
+ + + + +/- + - + + + + + - - - (+)/
-
+ + + - + + + + + + + + + + - -
(+)/- + + - - + - + - - + - - - - -
+ + + + + + - + + + + + + + - -
+ + + - - + - + + + + - - - - -
+ + + - + + + + + + + + + + - -
+ + + - + + + + + + + + + + - -
+ - + - + + + + + + + + + +/- - -
(+)/-
+ + - - - - + - - - - - - - -
+ + + + + + - - - - (+) - (+) + + -
+ + + + + + - + + + + + + - - -
+ + + + + - - + + - + + - - + -
Kit
ID
Wk
D
Ty
pe
1 &
2
DII
&
DN
U
D I
II
D I
V
D V
DC
S
D V
I
D V
II
DO
L
DF
R
DM
H
DA
R
DA
R-E
DH
K /
DA
U-4
DB
T
RO
Ha
r
A
B
C
D
E
F
G
H
I
J
K
L
A novel finding
Diagrammatic representation of RhD
Extracellular
Trans-
membrane
Intracellular
Phe223Val Glu233Gln
Thr379Met
DAU-5: Phe223Val Glu233Gln Thr379Met
DV type 1: Phe223Val Glu233Gln
RhD model
90° rotation
Thr379
Phe223 Glu233
Thr379
Phe223
Glu233
Extracellular
Intracellular
DAU-5: Phe223Val, Glu233Gln, Thr379Met
DV type 1: Phe223Val, Glu233Gln
D CE RhAG
Does the similarity in reaction
profile matter?
• The clinical care of a patient with a DAR-E or DV type 1 is same
• Clear cut positive and negative reactions indicate loss of epitopes (partial D)
• Anti-D production possible
• Treat as D-
• Identifying weak D 1, 2 and 3 is important……………
NO
Patients • Identified weak D types 1,2 and 3 treat as D+
• Weak D type 4 onwards treat as D-
• Partial D treat as D-
UK Blood Service Policy
Transfusion to D variants
D- D+
Ig anti-D
prophylaxis
No Ig anti-D
prophylaxis
Inadequate rr
blood supply
May be
immunised
to make anti-D Inappropriate
use
of Ig anti-D
RISK
Unlikely in weak D types 1,2 and 3
D- blood D+ blood
Recommended