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