Rhesus D: The clinical importance of molecular typing

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1 Rhesus D: The clinical importance of molecular typing Vicky Van Sandt Apr. Sarah Mahieu

2 Rhesus: role and structure Rh proteins: important components of the RBC membrane Rh complex in RBC membrane: RhCcEe + RhD protein = Rh core complex, associated with 2 RhAG (Rhesus Associated Glycoproteine) = Rh50 Function: transport of ammonium and CO 2 (protection of the RBC in the kidneys and liver)

3 Rhesus: frequency in caucasians Haplotype FR Haplotype W Frequency Antigens CDe R 1 0,40 C,D,e cde r 0,38 c,e cde R 2 0,14 c,d,e cde R 0 0,025 c,d,e cde r 0,013 c,e Cde r 0,009 C,e CDE R z rare C,D,E CdE r y very rare C,E

4 Rhesus: structure Rh proteins are encoded by the Rh Locus = 2 highly homologous (98,6 %) genes, RhD and RHCE RhD - Recombinations - Gene conversions - Mutations - Deletions / Insertions - Gene duplications RhD complex genetic background

5 Rhesus: structure Rh CE Rh D Gen sequenties 90% homologous Same protein structure!!!

6 Rhesus: immunisation D antigen is defined by multiple conformational epitopes Immunisation risk with non compatible transfusion:!! RhD: HIGH immunisation risk > 50% production of allo antibodies Rh EeCc CORRECT TYPING OF RhD and Rh subgroups VERY IMPORTANT!

7 Rhesus D phenotypes Phenotypes D+ positive Partial D Weak D Very weak D (Del) D- negative Rh null phenotype

8 Partial D Mutation = EXTRACELLULAR Effect antigen presenting surface Susceptible to immune respons

9 Partial D: Tranfusion advise White Black Asian Immune respons Tranfusion Advise D positive 85% 92% 99% No D + Weak D TYPE 1 TYPE 2 TYPE 3 Weak D TYPE 4.0/4.1. Partial D type <1% Yes D- D negative 15% 5% <1% Yes D-

10 Weak D Mutantion = INTRA MEMBRANE OR INTRA CELLULAR POSSIBLE: effect on antigen presenting surface AND/OR reduced expression on RBC membrane POSSIBLE susceptible to immune respons

11 Weak D: Tranfusion advise White Black Asian Immune respons Tranfusion Advise D positive 85% 92% 99% No D + Weak D TYPE 1 TYPE 2 TYPE 3 <1% No D+ Weak D TYPE 4.0/4.1. Partial D type <1% Yes D- D negative 15% 5% <1% Yes D-

12 Weak D TYPE 1,2,3 Mutations do NOT effect Antigen presenting surface Frequency: Type 1: 70% of all weak D s in whites Type 2: 18% of all weak D s in whites Type 3: 4% of all weak D s whites 90% of all weak D

13 Weak D type 1,2,3: Tranfusion advise White Black Asian Immune respons Tranfusion Advise D positive 85% 92% 99% No D + Weak D TYPE 1 TYPE 2 TYPE 3 <1% No D+ Weak D TYPE 4.0/4.1. <0,1% Yes D- Partial D type <1% Yes D- D negative 15% 5% <1% Yes D-

14 Weak D other than type 1,2,3 Rhesus alert: Weak D Type Allo Antibody titer : 1:128 Weak D Type 11 Allo Antibody titer : 1:256 Weak D Type 15 Allo Antibody titer: low titer.

15 Clinical importance of correct typing DONORS: PATIENTS:!! weak or partial Ds must be typed D POSITIVE WEAK D TYPE 1,2 and 3 patients should recieve D POS transfusion no immunisation risk no need for RhIg administration no waste of D NEG Other WEAK D and PARTIAL D SHOULD be typed D NEG immunisation risk

16 Serological D typing Agglutination D+ D- D variants +/- = No problem?

17 Serological D typing Different serological results with different reagens Serology CANNOT discriminate clinical relevant D types from weak D type 1,2,3 Pirenne et al., 2007 Transfusion 47: Some D variants remain UNDETECTED!

18 Antigen density ANTIGEN DENSITY = Very variable: ( ) AND NOT always correlated to the immunogenicity!! -

19 Molecular D typing SSP-PCR : primer with specific nucleotide on 3 Amplification patern is determined by presence/absence of delations/mutations

20 Molecular D typing Strategy: STEP 1: WEAK D TYPE BAGene Detection of the most frequent anti-d 90 % identification 10% is further analyzed in STEP 2

21 Molecular D typing Strategy: STAP 2: PARTIAL D TYPE BAGene Detection of the most frequent partial D D POS en D NEG

22 Molecular D typing: 10 month evaluation weak D type 1 Weak D type 2 Weak D type 3 Weak D type 4.0/4.1 Weak D type 4.2 Weak D type 5 Weak D type 11 Weak D type 15 Partial D D NEG D POS TOTAL D NEG D POS 15% D POS 67,5% WEAK D TYPE 1,2,3 Partial D Weak D type 15 Weak D type 11 Weak D type 5 Weak D type 4.2 Weak D type 4.0/4.1 Weak D type 3 weak D type 1 81,9% TA: D POS 6,8 % D NEG 4% Other WEAK D TYPE 6 % PARTIAL D Weak D type 2 18,1% TA: D NEG

23 When molecular typing Name DTM DM SG TUBE GEL SEROLOGY TA D+?? Molecular Typing DIIIb variant Weak D TYPE 4.0/4.1 Weak D TYPE 11 MOLECULAR TA D- D- D- DIII very high AG density, can serologically not be distinguished from D+; VGH MMP ?? Weak D TYPE 2 Weak D TYPE 1 D+ D+ Freq: very Low!! KVDV - - D- Weak D TYPE 1 D+ SEROLOGICAL: 3+ result in combination with 2+, 1+ OR THIS ALLOWS DETECTION OF 99% OF D VARIANTS

24 Typing of donors: Screening of D+ donors for partial/weak D? NO, partial/weak D considered as D+ donors Screening of D- donors for partial/weak D? NO, study Westhoff et al. (Transfusion, Volume 47, Aug 2007) 2427 D- donors molecular typed: 2424/2427 (99,88%) real D- 1 donor Del: low immunogenicity 1 donor weak D type 1 1 donor weak D type 2 Almost all weak D s have E and/or C In RKV: ddccee/ddccee are considerd as positive donors

25 When molecular typing of RhD NO clear serological typing result AND the patients need to recieve blood in the future Send EDTA sample + order for molecular typing RhD to BTC and mention reason: - weak or contradicting serological result - anti-d present: auto/allo? -

26 Limitations of molecular typing Discovery of new alleles/mutations: 2/77 were discoverd as new mutations by SBT Kits are made for a certain etnic population: problems with multi-etnic patients

27 To Summarize Rh genotyping offers a powerful adjunct to serologic testing, especially in samples with serological discrepancies High throughput platforms necessary to advance patient care and to eliminate or reduce Rhesus allo-immunisation

Investigating the prospect of. develop optimal transfusion

Investigating the prospect of. develop optimal transfusion Investigating the prospect of applying RHD genotyping to develop optimal transfusion strategies for weak D patients in Ireland Paula Holton 1, Diarmaid O Donghaile 2, Sorcha Ni Loingsigh 2, Mark Lambert