The summary provides an overview of the key points and mysteries surrounding RhD typing. It begins with the complex nature and confusing nomenclature of RhD, explaining there are over 150 variants and 50 ways to be RhD negative. Case studies are presented that show weak and partial D can present as normal D, and variants only detected if they make anti-D. Guidelines are discussed for RhD typing of patients and donors. The importance of detecting very weak D expression in donors to prevent immunization is highlighted.

1. The myth and
mystery of RhD
Quotient Biodiagnostics Industry Workshop
AABB San Diego 2011
Joyce Poole
International Blood Group Reference Laboratory
Bristol UK

2. Objectives of my talk
• 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

3. Mystery?
• Secret or hidden
• Puzzling

4. The mystery of RhD
Why is D difficult
• Not a simple antigen Complex
• Nomenclature is confusing
• D typing is not straightforward
• Controversial
Clinically significant!

5. The mystery of RhD
• Huge molecular diversity
• >150 variant D antigens
• >50 ways of being D
negative/Del

6. 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

7. The mystery of RhD
Confusing Nomenclature
• D+ D category D mosaic
Partial D Weak D
Weak partial D D variant
• D-

8. The mystery of RhD
Controversial
D+ or D- ?
Do we treat the patient
as D+ or D-?

9. The myth of RhD
Widely held but
false notion
D typing is routine test and
should therefore be straightforward!

10. D typing
How do we do it ?
Column

11. Like this balancing act, D typing can be difficult
for the majority of us

12. GUIDELINES
Compatibility procedures in blood
transfusion laboratories
BCSH British Committee for
Standards in Haematology/
Blood Transfusion
(Transfusion Medicine 2004:14:59-73)
Patients

13. D typing patients (1)
UK GUIDELINES
• 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

14. D typing patients (2)
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+]

15. Non-compliance with guidelines!
Patient D typing in UK
• 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

16. Donors

17. 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+

18. Case Study
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

19. 609 654 667 674 807
RHD Psuedogene
10 exons of RHD
Novel DIII
186 602 667 819
Patient SR - mutations in RHD
609 654 667 674 807
RHD Psuedogene
10 exons of RHD
Normal RHD
Twin son and daughter of SR - mutations in RHD

20. Case Study
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

21. 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+
RHD sequence: No mutations in RHD (or CE or RHAG)

22. Case Studies
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

23. IBGRL referrals
Overseas reference
UK hospitals
labs
Blood Centre
IBGRL
We do not do routine patient or donor typing

24. IBGRL referrals
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-?

25. IBGRL referrals
• 4 routine anti-D reagents that
1
detect weak D + most partial D
between them (+ C, Cw, c, E, e)
• ALBAclone IgG anti-D panel (12)
2
Clear-cut pattern Not clear
Refer for
molecular analysis
Report (RHD sequence)

26. 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

27. • Variants that can give
ambiguous patterns of
reactivity vs the ALBAclone
panel
High
– Weak D type 1 : 74 referral
– Weak D type 2 : 79 rate
– 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

28. A novel finding
The variants DAU-5 and DV type 1
gave an identical serological pattern
DII & DNU
Type 1 & 2
(+)/
+ + + + +/- + - + + + + + - - -
DAR-E
DAU-4
DHK /
Kit ID
D VII
DMH
-
ROHar
WkD
DAR
DOL
D III
D IV
D VI
DBT
DFR
DCS
+ + + - + DV + + + + + + + + + - -
A (+)/- + + - - + - + - - + - - - - -
B + + + + + + - + + + + + + + - -
C + + + - - + - + + + + - - - - -
D + + + - + + + + + + + + + + - -
E + + + - + + + + + + + + + + - -
F + - + - + + + + + + + + + +/- - -
G (+)/
+ + - - - - + - - - - - - - -
H -
I + + + + + + - - - - (+) - (+) + + -
J + + + + + + - + + + + + + - - -
K + + + + + - - + + - + + - - + -
L

29. Diagrammatic representation of RhD
DAU-5: Phe223Val Glu233Gln Thr379Met
DV type 1: Phe223Val Glu233Gln
Phe223Val Glu233Gln
Extracellular
Trans-
membrane
Intracellular
Thr379Met

30. RhD model
DAU-5: Phe223Val, Glu233Gln, Thr379Met
DV type 1: Phe223Val, Glu233Gln
Extracellular
Glu233 Phe223 Glu233
Phe223
90° rotation
Thr379
D Thr379
CE
Intracellular
RhAG

31. Does the similarity in reaction NO
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……………

32. UK Blood Service Policy
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-

33. Transfusion to D variants
RISK
D- D+
D- blood D+ blood
Ig anti-D No Ig anti-D
prophylaxis prophylaxis
May be
Inadequate rr
immunised
blood supply
to make anti-D
Inappropriate
use Unlikely in weak D types 1,2 and 3
of Ig anti-D