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Phasing in RHD Genotyping What is the Impact? Greg Denomme, PhD, FCSMLS(D) Director of Immunohematology and Transfusion Services Objectives ¾ Understand the differences among category/partial, weak, and Del variants ¾ Present the problem of Rh phenotyping in transfusion and pregnancy ¾ Provide information on a recommendation to resolve weak D phenotypes Introduction to RH (004) ¾ RH is one of the most complex blood group system; 45+ antigens ¾ RhD “inferred” in 1940 by testing human RBCs with a rabbit anti-Rhesus monkey RBC antisera ¾S Subsequently, b tl Rh status t t was discovered di d to be unrelated to the Rhesus monkey protein (LW) ¾ The Rh proteins are part of a complex that includes RhD Rhce, RhAG, LW, Duffy, and Band 3 1 What is D? ¾ Multi-pass membrane protein encoded by RHD at 1p36.13-p34 ¾ Differs from Rhce by 34 to 37 amino acids (C or c) ¾ Its’ expression on RBCs defines the Rh-positive phenotype h t – gene is i absent b t iin ((most) t) Rh Rh-negatives!! ti !! ¾ Highly immunogenic: “When a relatively large amount of D-positive red cells (200 ml or more) is transfused to D-negative subjects, within 2-5 months antiD can be detected in the plasma of some 85% of the recipients.” Mollison’s Blood Transfusion in Clinical Medicine, 11th ed, pg 183 Distribution of D Population Study Rh+ Rh- Caucasian Kopec 1970 Wagner 1995 Garratty 2004 82% 18% Tunisia, Nigeria Ranque 1961 Enosolease 2008 92-94% 6-8% India Makroo 2013 94% 6% Basques/Morocco Goti 1958 Messerlin 1951 71% 29% China Shao 2002 >99% 0.3% Weinstock ,C., Blood Transfusion 2014;12:3-6 Frequency of weak D expression Hopkins Scotland 1967 0.56% Garretta France 1974 0.66% Beck USA 1990 0.2% Jenkins USA 2004 0.4% Flegel Germany 2006 0.4% 2 Classification of RHD ¾ r’ haplotype: in trans with D (Ceppellini effect) ¾ Partial and Category D: hybrid RHD alleles DVI, types I, II, III yp single g amino acid changes g ¾ Weak D “types”: Weak D Type 2 ¾ Del: detection by adsorption/elution K409K, plus several others ¾ RHD-deletion and non-functional RHD alleles Ceppelini Effect Partial and Category D ¾ Arise from hybrid RHD/RHCE genes and missense mutations to regions of RHD encoding parts of D external to RBC membrane ¾ React weakly with some monoclonal anti-D reagents ‘altered or missing epitope’ ¾ May make anti-D to ‘missing’ epitope of D ¾ if transfused - should receive Rh-negative RBCs ¾ if pregnant - are candidates for RhIG 3 Weak D Types ¾ Arise from missense mutations to regions of RHD encoding transmembrane/cytoplasmic portion of D ¾ Weak D types may or may not make anti-D The absence of the ability to make alloanti-D is not (never was) included in the definition of weak D types ¾ Can type as Rh-positive or Rh-negative by direct agglutination with monoclonal anti-D reagents ¾ Weak D types 1 – 3 have not been reported to make alloanti-D Rh D polypeptide Grey: partial D Black: weak D Flegel WA. Transfus Clin Bio 2006;13:4-12 Toronto Study (reagent discrepancy) Two anti-D direct tests were performed routinely ¾ 33,864 D phenotypings performed over an 18 month interval ¾ 55 of 5672 potential Rh Rh-negative negative patients were tube test positive for one anti-D (0.98%) ¾ 54 were tube test negative using one FDA-approved reagent but positive (2+ or less) using another government-approved antisera 4 Michigan Study (reagent change) Change from manual testing to instrument platform ¾ 10 ‘historical’ discrepancies evaluated Anti-D Score ProVue Gamma Clone Bioclone 8 8 5 8 8 0 8 10 5 ¾ repeat instrument testing: Include an evaluation by tube t t methods test th d carefully f ll ffollowing ll i manufacturer’s instructions ¾ Perform molecular analysis 8 7 0 8 10 5 8 10 6 8 10 5 8 8 5 8 7 0 8 7 0 Alberta Study (policy change – no IAT) Beginning January 1, 2007 the IAT for the detect of weak D was discontinued for prenatal patients ¾ For 209 of 88,972 phenotypings (0.23%) the current RhD type differed from the historical RhD result prevalence of “Du”)) Consistent with studies on the p ¾ All RhD results that were discrepant with historical results were sent for molecular analysis Patient returned to the Rh clinic the following week ¾ Recommendation for RhIG prophylaxis was made on the basis of the RHD allele present Weak D types 1 – 4 were deemed Rh-positive Toronto Study (Reagent Discrepancy) 20 functional RHD alleles detected; 1 wildtype (HDN) 51% were Weak D types 1, 2, and 3 ¾ 34 Weak D Types (PCR-RFLP): ¾ 16 weak D Type 1 ¾ 1 weak D Type 3 yp 5 ¾ 1 weak D Type 8 weak D Type 2 6 weak D Type 4 2 weak D Type yp 42 ¾ 7 DAR ¾ 6 DVa or DVa-like alleles: ¾ 3 DVa(Kou.) 1 DVaHK(E233K) 1 DVa-like 1 DTO (Novel) ¾ DFR, DAU-4, DAU-5 (Novel), DAU-6 (Novel) ¾ Compound heterozygotes: DAR/DAU-2, DAU-0/Cdes ¾ Not identified: a possible DIIIa, DVa, DAR, or DOL 5 Resolution of D discrepancies ¾ Conserve Rh-negative blood for D-negative recipients (high risk of making anti-D) ¾ Avoid the administration of RhIG to women who do not need it ¾ Resolve early in pregnancy to eliminate false-positive rosette tests ¾ Resolved discrepancies (Rh+) avoids delay at discharge after delivery of an Rh+ infant What’s Next? ¾ A group of representatives and experts convened to provide a recommendation for the resolution of Weak D phenotypes Sandler SG. et al. TRANSFUSION 2015;55:680-9 ¾ The weak D phenotype is defined as ≤2+ hemagglutination for initial testing (any technique), but moderate to strong hemagglutination with AHG (when performed) ¾ RHD genotyping t i provides id clinical li i l d decision-making i i ki iinformation f ti ¾ Recommend that RHD genotyping be performed to resolve discordant results and when a serological weak D phenotype is detected. ¾ Weak D types 1, 2, 3 can be deemed Rh-positive for the purposes of RhIG prophylaxis and transfusion ¾ Impact… Rh Discrepancy Algorithm Sandler SG, et al. Transfusion 2015;55:680-9 6 Impact for Pregnancy 3,953,000 Live births 3,812,000 Pregnancies 556,500 RhD-negative 16,700 Serological Weak D RHD Genotyping 13,360 weak D types 1, 2 or 3 24,700 unnecessary ante- and postpartum RhIg injections 25,000 unnecessary injections of RhIG Sandler SG, et al. Transfusion 2015;55:680-9 Impact for Transfusion 5,000,000 Individuals Transfused Annually in US 730,000 RhD Negative 21,900 Serological Weak D RHD Genotyping 17,520 weak D types 1, 2 or 3 Could receive RhD+ Units (47,700 units) 50,000 unnecessary Rh-negative transfusions prevented Sandler SG, et al. Transfusion 2015;55:680-9 Impact if deemed Rh-negative! Inappropriate use of blood products RHD Allele OB TR NB Weak D types 12 8 5 12 OB patients received RhIG 4 transfusion recipients received 12 Rh-neg pRBCs Impact DAR 3 1 3 3 OB patients received RhIG Potential transfusion recipient was not transfused. DVa and DVa-like 1 1 5 1 OB patient an delivered an Rh-neg infant Potential transfusion recipient not transfused DAU, DFR, DTO 3 2 2 2 OB patient delivered an Rh+ infant Neither potential transfusion recipient transfused Total: Total: 20 RhIG 7 Rhig 12 Rh-negative pRBCs 0 Rh-negative pRBCs 7 Michigan Study (Reagent Change) Anti-D Score PV GA BI Genotype MoAb 8 8 5 DAR DV 8 8 0 DAR DV 8 10 5 Weak D type 1 8 7 0 Weak D type 1 8 10 5 Unknown 8 10 6 Exon 4-5 CE hybrid 8 10 5 Weak D type 2 8 8 5 Weak D type 2 8 7 0 Weak D type 2 8 7 0 Weak D type 2 Alberta Study (Policy Change) Analysis ’07 to ’08 = 88,972 Total discrepancies = 209 (0.23% of total) DNA Typing Results # of Patients Rh Status Assigned RHIG Recommended % of DNA Results Received Weak D Type 1 60 Pos No 29.0 Weak D Type 2 19 Pos No 9.2 Weak D Type 3 38 Pos No 18.4 Weak D Type 4 15 Pos No 7.2 DAR 2 Neg Yes 1.0 Partial DVI Type I 3 Neg Yes Partial DVI Type II 1 Neg Yes 0.5 DVI Type II 2 Neg Yes 1.0 DVa partial 1 Neg Yes 0.5 Partial DVA-like 1 Neg Yes 0.5 Unclassified 31.4 65 Neg Yes Pending 2 TBD TBD TOTAL 209 57% 1.3 43% Blood Inventory America’s Blood Centers, Newsletter April 3, 2015 8 Conclusions ¾ Rh D discrepancies are resolved using RHD genotyping: ) MoAbs cannot identify weak D types 1, 2, 3 ¾ Laboratories who change methods, reagents, or drop the IAT as a routine test on all patients can implement a recommended clinical practice to resolve discrepancies. Pregnant g women and transfusion recipients p expressing p g a weak D type yp 1,2,3 , , have not been reported to make anti-D. Therefore, for clinical purposes this patient is deemed Rh positive and is not a candidate for antenatal/postpartum Rh immune globulin prophylaxis.* This patient can receive Rh positive blood if red cell transfusions are necessary. * This patient may be reported as Rh negative by other institutions depending on the anti-D and the methods used to resolve Rh D discrepancies. In light of the equivocal D typing with regulatory reagents and the presence of a partial D, this patient is deemed Rh negative for the purposes of Rh prophylaxis and red cell transfusion. Future of RHD ‘genotyping’ ¾ 3-5% Rh-negative RBCs go to Weak D type recipients ¾ 3-5% RhIG doses go to women with Weak D types How often do you need to address Rh status? [~0.5%] ¾ Molecular testing is a permanent solution: Weak D types 1,2, 3 are Rh+ pregnancy/Tx recipient (and donor) • Avoid a blood product where it is not needed (in pregnancy!) • Informed consent not required for the administration of RhIG • Mitigate RhIG shortages, potential spread of infectious agent • Avoid the use of Rh- blood products when an Rh+ will do! Questions? 9