Session 3 - Reasons behind RhD Myth & Mystery

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

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

Mystery?• Secret or hidden• Puzzling

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

The mystery of RhD• 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 category D mosaic Partial D Weak D Weak partial D D variant• D-

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

The myth of RhD Widely held but false notion D typing is routine test andshould therefore be straightforward!

D typingHow do we do it ? Column

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

GUIDELINESCompatibility procedures in blood transfusion laboratoriesBCSH British Committee for Standards in Haematology/ Blood Transfusion (Transfusion Medicine 2004:14:59-73) Patients

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

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+]

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

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+

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

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 Psuedogene10 exons of RHD Normal RHD Twin son and daughter of SR - mutations in RHD

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

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)

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

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

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-?

IBGRL referrals• 4 routine anti-D reagents that1 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)

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

• 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

A novel findingThe 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

Diagrammatic representation of RhD DAU-5: Phe223Val Glu233Gln Thr379Met DV type 1: Phe223Val Glu233Gln Phe223Val Glu233GlnExtracellularTrans-membraneIntracellular Thr379Met

RhD modelDAU-5: Phe223Val, Glu233Gln, Thr379MetDV type 1: Phe223Val, Glu233GlnExtracellular Glu233 Phe223 Glu233 Phe223 90° rotation Thr379 D Thr379 CE Intracellular RhAG

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……………

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-

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

Session 3 - Reasons behind RhD Myth & Mystery

by QBD18940 on Nov 28, 2011

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Session 3 - Reasons behind RhD Myth & Mystery — Presentation Transcript