Rh Molecular Testing

Transcription

1 Rh Molecular Testing

2 Future Webinars October 31 December 6 HLA: Transfusion and Transplant Running a Remote Transfusion Service Link to register: All Content 2015 Immucor, Inc.

3 Continuing Education PACE, Florida and California DHS 1.0 Contact Hours Each attendee must register to receive CE at: Registration deadline is October 13, 2017 Certificates will be sent via only to those who have registered by October 27, 2017 All Content 2015 Immucor, Inc.

4 Course content is for information and illustration purposes only. Immucor makes no representation or warranties about the accuracy or reliability of the information presented, and this information is not to be used for clinical or maintenance evaluations. The opinions contained in this presentation are those of the presenter and do not necessarily reflect those of Immucor. All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc.

5 RH Molecular Testing An Immucor Webinar JOANN MOULDS PHD, MT(ASCP)SBB IMMUNOHEMATOLOGY CONSULTANT

6 History of RH - Act I 25 y.o. group O woman delivered a stillborn fetus* Transfused with 500 ml of blood from her husband Hemolytic reaction: chills, pain in legs and back Antibody reacted with 83/104 (80%) group O donors Different from the ABO, MN, P blood groups *Levine P, Stetson RE. An Unusual Case of Intra-Group Agglutination. JAMA 1939;113(2):

7 History of RH- Act II Macaca mulatta (Rhesus monkey) red blood cells injected to rabbit Rabbit produced an antibody named anti-rhesus Anti-Rhesus agglutinates RBCs of 85% random individuals Antibody referred to as anti-rh (Rh for Rhesus)* Apparently identical to Levine & Stetson s case of HDN 1941: Levine published a full account of the etiology of their case *Landsteiner K, Wiener AS. An agglutinable factor in human blood recognized by immune sera for rhesus blood Proc. Soc. Exp. Biol. and Med. 1940;43:223.

8 History of Rh- Act III Newborn RBC typing: Human anti-rh: can differentiate Rh+ and Rh- Rabbit anti-rh: both Rh+ and Rh- typed positive Human & rabbit anti-rh react against 2 different antigens Human antibody named anti-rh, defining Rh blood group Rabbit antibody named anti-lw, defining LW (or Rhesus ) Antibody described by Levine & Stetson later identified as anti-rh(d)

9 Nomenclature: Who is Right, Who is Wrong? Wiener Fisher Rosenfield Wiener: Rh-Hr terminology One locus, multiple allelic forms Fisher-Race: CDE terminology Three closely linked loci producing antithetical antigens: D or d; C or c; E or e Rosenfield: Numerical terminology Represent serological data free of bias and divorced from speculative implication R 1 DCe RH 1,2,-3,-4,5 r dce RH -1,-2,-3,4,5 R 2 DcE RH 1,-2,3,4,-5 R 0 Dce RH 1,-2,-3,4,5 r dce RH -1,-2,3,4,-5 r dce RH -1,2,-3,-4,5 R z DCE RH 1,2,3,-4,-5 R y dce RH -1,2,3,-4,-5

10 They Were All Wrong. Rh genes at 1p36.11 RHD and RHCE originated by ancestral duplication Two DNA segments Rh box border RHD RHD Rh box RHD Rh box RHCE RHCE 5 Closely linked genes: Each gene has10 exons ~94% homology ,500 >5,000 >5,000 13,500 ATG STOP STOP ATG

11 Rh null: No D, Cc or Ee antigens Due to mutations in RHAG gene (regulator type) or deleted RHD paired with silent RHCE allele (amorph) D : Lacks Cc, Ee; has enhanced D Due to mutation in RHCE gene Rh negative: Lacks D antigen Deletion of RHD gene most common in Caucasians In African descent: RHD pseudogene & hybrids In Asians: Del

12 D Negative Polymorphism Caucasian: Frequency ~15% Due to misalignment of the two chromosomes in trans during meiosis Molecular assays for RHD zygosity: Hybrid Rhesus box PCR Pst I RFLP

13 RhD Negative Genotypes in Blacks Percent South Africa Tunisia Brazil (S.P.) Brazil (Camp.) USA (Shv) Deletion Pseudogene 66 < Hybrid Hybrid/Ψ # Studied

14 Weak D Polymorphism Negative on direct agglutination testing (IS); positive only at IAT phase: initially named D U Mutations result in aa substitution in intramembranous portion (>90 SNPs) Folding & integration of the Rh protein into membrane is affected Reduction of D antigen >10 fold Expresses all D epitopes Weak D types 1, 2, 3 don t form ALLO anti-d NH 2 Type 52 (92T>C) Type 6 (29G>A) Type 37 (399G>T) Type 71 (29G>C) Type 51 (594A>T) (602C>G) Type 23 (634G>T) Type 1 (809T>G) Type 43 (605T>C) Type 53 (740T>G) Type 34 (809T>A) Type 12 (830G>A) Type 22 (1224G>C) SNPs causing Weak D and position of aa. change in Rh protein Type 72 (1212C>A) COOH Type 73 (1241C>T)

15 Partial D Polymorphism D IVb Type 2 Evans+ D antigen is composed of >30 epitopes on the extracellular portion of the RBC membrane D V Type 1 D V Type 5 667T>G 697G>C 697G>A KOU, FK D w + DHK, DYO D w - Normal D expression dependent on linear & 3-D conformational arrangements Due to single aa changes or hybrids D VI Type 2 DBT Type 2 Weak Partial D G>A BARC+ Rh32+ Partial D carriers CAN form allo anti-d RHD mutations causing Partial D

16 Clinical Considerations for Partial D Donors: have stimulated allo anti-d but rare Patients: Partial D has less risk of forming anti-d than Rh negative individuals who have no RhD protein Those missing a single Rh epitope can make antibody to the missing piece Partial Ds are not necessarily compatible with each other

17 RhD Genotypes In SCD Patients* Normal D D neg Homozygous variant or heter. & silenced D 2 anti-d 8 anti-d 8 anti-d (5 deletion) (includes r s ) *Moulds, JM. LifeShare Blood Centers unpublished data

18 Clinical Relevance of Rh ~20% of D- develop anti-d after exposure to small volume of D+ blood Rh antibodies cause severe hemolytic transfusion reactions (HTR) and hemolytic disease of the fetus and newborn (HDFN) HTR: o o Donor: Typed negative when in fact weak or partial antigen present induce antibody Recipient: Typed positive when in fact weak or partial antigen present produce antibody HDFN (Mother) o o Typed positive when in fact weak or partial antigen present produce antibody Typed negative when in fact weak (do not produce antibody) unnecessary interventions Accurate Rh phenotype identification remains problematic despite advances in serological methods and reagents

19 Comprehensive Transfusion Medicine Survey by CAP Sandler SG, et al. Arch Pathol Lab Med 2014:138; QUESTION Routinely perform weak D-AHG on patients Weak D reported as RhD positive Transfusion of RhD negative to weak D recipient Recommend RhIg to weak D transfused with RhD+ Rh type performed on all pregnancies at delivery 58.2% 19.8% 50.7% 46.9% 43.5% 49.2% 19.9% 15.7% 39.8% (32.3%) 48% (21.6%)

20 Rh Working Group Representatives from: AABB CAP ABC ACOG ARC US Armed Services Scientific consultants

21 Patient Neg/Donor Pos is confusing to everyone including physicians 95% of women with weak D don t make anti-d Waste of RhIg Unnecessary exposure to blood product 95% of weak D transfusion recipients do not need Rh negative blood

22 Rh Neg Rh Discrepant/Inconclusive Rh typing RT Rh Pos RhIG candidate RhD neg. for transfusion RHD Genotype for weak D Not RhIG candidate RhD+ for transfusion No weak D type 1,2,3 RhIG candidate RhD negative for transfusion Weak D type 1,2,3 Not RhIG candidate RhD+ for transfusion

23 Problems With The 2+ Rule Differences in typing reagents polyclonal vs monoclonal vs blends Method used automated, eg. PK 7200 manual: gel, tube, etc. Presence of C or E in trans

24 46% minorities 485,836 5,830 4,858 Sandler SG, et. al. Transfusion 2015;55:680-9

25 Financial Implications Kacker et al., Transfusion 2015;55:2095 Modeled a dynamic US population, tracking pregnancyrelated costs, and contemplating the diversity of races It compared the expected financial impact of two strategies: Weak D treated as D, at risk of HDFN and received RhIG Weak D genotyped for weak D type 1, 2, or 3 managed as D+ Average costs of tests + RhIg administrations per pregnancy over 10 yrs. Conservative strategy: $34.75 Alternative strategy: $34.73 Over 10 years RHD genotyping is cost-saving if <$256/test

26 Availability of labs doing RH genotyping Cost of testing - depends on test; $ FDA approved tests - none TAT - 2 to 5 days Convincing OBs - is giving RhIg safer?

27 Full RHD gene sequencing (10 exons) Sequencing exons encoding weak D 1,2,3 Microarray RHD assays Allele specific PCR or RFLP

28 Full RHD gene sequencing (10 exons) will detect all mutations in coding regions Sequencing exons encoding weak D 1,2,3 will detect relevant weak D mutations will detect other mutations in exons 6, 9, 1 RHD Gene

29 Microarray RHD assays vary by manufacturer all detect weak D 1, 2, 3 some detect partial RHD

30 RhD BeadChip Assay RhD negative: RhD deletion, DΨ, 5 hybrids, 16X, 269X Del: 1227A, IVS3+1G>A Weak D: 1, 1.1, 2, 3, 5, 14, 17, 29, 34, 40, 41, 47, 51 Partial D: DBS0,1,2; DAR, DAR-E, DAU 1,2,3,4,5; DOL 1,2,3; DBT 1,2; DIII a,b,c; DIII 4,6,7; DIVa,b; DIVa-2, DIV 3,4,5; DV 1,2,4,5,6,7,8,9; DVI, DCS 1,2; DFR1,2,3,4; DHMi, DNB, DUC2, cehar, DFV, weak D 4.0,4.1,4.3,11,15

31 Policies regarding RH will continue to evolve but will most certainly involve RH genotyping

32 Questions? You are all muted Q&A following session Type in questions All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc.

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34 Continuing Education PACE, Florida and California DHS 1.0 Contact Hours Each attendee must register to receive CE at: Registration deadline is October 13, 2017 Certificates will be sent via only to those who have registered by October 27, 2017 All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc.

35 Thank you! All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc.