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2 Handouts Program-Handouts.aspx 2 All Content Immucor, Inc.

3 2019 Advanced Track Webinars Link to register: 3 All Content Immucor, Inc.

4 2019 Advanced Track Webinars Link to register: 4 All Content Immucor, Inc.

5 Link to register: 5 All Content Immucor, Inc.

6 learn.immucor.com 6 All Content Immucor, Inc.

7 Continuing Education PACE, Florida and California DHS 1.0 Contact Hours Each attendee must register to receive CE at: Registration deadline is March 8, 2019 Certificates will be sent via only to those who have registered March 22, All Content Immucor, Inc.

8 Questions? You are all muted Q&A following session - Type in questions 8 All Content Immucor, Inc.

9 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. 9 All Content Immucor, Inc.

10 From C to e and the Rhs in Between Margaret A. Keller, PhD Sr. Director, National Molecular Laboratory February, 2019

11 Objectives Review the Rh proteins and the RH genes Review Rh terminology Illustrate the challenges of serologic Rh antigen typings and how molecular methods can help Discuss the AABB recommendations regarding use of RHD genotyping to resolve a serologic weak D phenotype Use cases to illustrate how RHD genotyping can aid in determination of risk of alloimmunization Demonstrate how RH allele matching can be used to find compatible blood for a patient with an antibody to a high prevalence Rh antigen 11

12 The RH Blood Group System The 5 principle antigens (D, C, c, E, e) were identified using serologic methods There are 62 antigens to date There are many complex phenotypes and partial antigen phenotypes There are two genes (RHD and RHCE) that encode the two proteins (RhD and RhCE) that encode these antigens There is enormous genetic variation, especially in individuals of African ancestry There are ~400 RHD alleles There are >100 RHCE alleles

13 The genes and proteins RHD RHCE C/c E/e RhD RhCE Modified from Westhoff, 2010

14 Rh Terminology Fisher-Race (three gene theory) Dce R 0 DCe R 1 DcE R 2 DCE dce dce r R Z dce r dce X r r Y Wiener (one gene theory) X D C E

15 There are 3 mechanisms to be RhD negative RHD Gene Deletion by Unequal Crossover RHD Pseudogene 37 bp insertion Causes frameshift mutation 3 missense mutations M218I, F223V, S225F Premature stop codon at residue 269 RHD SMP1 RHCE b RHD SMP1 RHCE SMP1 + RHCE RHD-CE-D Hybrid Gene b RHD RHD SMP1 RHCE RHD exon RHCE exon expresses no D antigen expresses altered C antigen Schematic modified from Wagner and Flegel, Blood 2000,95:3662

16 Genetic Variation in RHD Weak Ds 145 different weak Ds caused by SNPs -alter quantity of protein, but not the surface D- epitope -individuals do not usually make anti-d Del 46 different DEL alleles -Very weak expression of D, type D- -can stimulate anti-d in D- patients Partial Ds ~130 alleles, including those with amino acid variants that alter D epitopes and hybrid genes -they have altered epitopes -can type strongly D+ -can type serologically weak D -may not be recognized until they make anti-d Diagrams courtesy of Lilian Castilho

17 Challenges of Serologic Typing 17 Simple epitope (eg. K) vs complex epitopes (eg. RhD) Antigen variants can be missed (eg. partial D) Antigens can be missed due to weak expression (eg. Weak D, D el ) Reagents can cross-react with other antigens (eg. cehar, cecf) D 17

18 Challenges of Serologic Typing Serologic methods vary Tube Gel Solid phase Serologic reagents vary or are unavailable Monoclonal Polyclonal Blends Patient source 18

19 19 Challenges of Serologic Typing Expression level can hamper detection (eg. weak D) RBC RBC RBC RBC 19

20 Challenges of Serologic Typing mab1 mab2 mab1 mab2 X RhD Partial RhD 20

21 Harnessing the differences Organizations AABB America s Blood Centers American College of OBGYN American Red Cross Armed Service Blood Program College of American Pathologists 21

22 It s Time to Phase In RHD Genotyping 22 *Sandler SG, Flegel WA, Westhoff CM et al. It s time to phase in RHD genotyping for patients with a serologic weak D phenotype. Transfusion (3):680-9.

23 Weak D Alleles and Alloimmunization Weak D types 1, 2 and 3 are not associated with allo anti-d The most common weak D types in Caucasians RHD*01W01 c.809g (270G) RHD*01W02 c.1154c (70Q) RHD*01W03 c.8g (3C) 23 Flegel WA. Curr Opin Hematol Nov;13(6): Pham BN et al Transfusion :

24 Weak D Alleles and Alloimmunization Weak D types associated with allo-anti-d Type 4.2, 11, 15, 21, 57 Weak D type 4.0 cases of anti-d in Germany, France, Tunisia and the US Weak D type 4.1 No cases of anti-d in Germany and France Wagner et al. Blood : Flegel WA. Curr Opin Hematol : Le Marechal et al. Transfusion : McGann et al. Immunohematology 2010, 26:

25 Harnessing the differences Organizations AABB America s Blood Centers American College of OBGYN American Red Cross Armed Service Blood Program College of American Pathologists 25

26 Harnessing the differences One Hospital Blood Bank s Practice: Two typing for first time patients are performed with two methods Luo et al Blood Transfusion 16(3):

27 Selection Criteria Evaluated Three criteria were used to determine if the sample should be sent for genotyping: Discrepancy between 2 methods and Gel reaction strength at least 2+ stronger than tube Serologic weak reaction strength <2+ regardless of testing method if both tube test and Gel performed OR <2+ if only Gel test performed OR <1+ if only tube test performed Presence of anti-d in D positive patient with no history of RhIg in last 3 months Luo et al Blood Transfusion 16(3):

28 Serologic Results, Gel and Tube Testing Patients

29 Serologic Results, Gel and Tube Testing Patients Luo et al Blood Transfusion 16(3):

30 Differences in anti-d reactivity can be used Outcomes to identify patients expressing D variants A total of 50 patients ranging from newborn to 93 years in age were identified to be genotyped Genomic testing confirmed D variants in 49/50 cases Positive predictive value (PPV) of 98% 1/50 (2%) was D+ with anti-d with 1 conventional RHD allele Auto-adsorption was not performed Identified more partial D+ cases than other approaches Identified 39/50 (78%) partial D+ cases May be due to race/ethnicity of cohort (54% AA, 32% Hispanic) May be due to two test method approach (51% of partial D alleles found based on this criteria) Luo et al Blood Transfusion 16(3):

31 Putting Rh Alloimmunization into Perspective: Over-treatment 85% of Caucasians who are serologic weak D are weak D types 1, 2 or 3 They do NOT need RhIg, leaving only 15% who do. 31

32 Putting Rh Alloimmunization into Perspective: Hiding in Plain Sight 10% of African Americans are Partial D BUT many of these will test strongly D+ by automated testing methods 32

33 Case - Is it really D? 28 year old Caucasian female Pregnant Types D+ at immediate spin (w+ to 3+) with 3 of 4 sources of anti-d Typed D- at IS and at AHG with the fourth anti-d 33

34 Case - Is it really D? Allele 1 RHD RHCE RHD*01N.01 RHCE*ce Allele 2 733C>G 712A>G 697C>G 667G>T RHD*01N.01 L245V, M238V, Q233E, V223F RHCE*ceHAR Predicted Phenotype: D-, C-, E- c+ e+ DHAR+ FPPT+ The patient is not predicted to express D. They carry the RHCE*ceHAR allele Individuals with a RHCE*ceHAR allele express D epitope(s) that type strongly D+ with some monoclonal anti-d reagents Such individuals should be considered D negative 34

35 Case - New Mom, New Discrepancy 32 year old Caucasian female, post-partum RhD typing discrepancy Patient typed repeatedly RhD negative with negative Ab screen Post-partum, both patient and infant type RhD negative at IS and positive (3+) at AHG. RHD genotyping performed 35

36 Case New Mom, New Discrepancy Allele 1 RHD*01 Allele 2 RHD*01N.01 Probable RHD Genotype: RHD*01 / RHD*01N.01 (hemizygous) Predicted RhD phenotype: D+ COMMENTS: Targeted genomic testing did not detect variants associated with common partial or weak RhD antigens including weak D types 1, 2 and 3. The patient may carry a rare or novel RHD allele not interrogated by this medium resolution testing. 36

37 Resolution of Molecular Testing Methods Low Resolution Medium Resolution High Resolution Gel-based methods SSP-PCR for known SNPs PCR-RFLP for known SNPs High melt resolution analysis Arrays Immucor BeadChip and Grifols IDCore Real-time PCR-based genotyping Single base extension (SBE) using MALDI-TOF DNA sequence analysis Exon scanning cdna analysis NextGen Sequencing 37

38 Ruling out what the array doesn t Genomic sequence analysis/exon scanning TESTING PERFORMED RESULT RHD Variants Method Analyte: Nucleotide (Amino Acid) Nucleotide(s) which differ from consensus Exon 1 None Exon 2 329C Exon 3 None Exon 4 None RHD SEQUENCING gdna seq Exon 5 Exon 6 None None Exon 7 None Exon 8 None Exon 9 None Exon 10 None 38

39 Case New Mom, New Discrepancy 329T>C (Leu110Pro) Allele 1 RHD*DVII.1 Allele 2 RHD*01N.01 Predicted Phenotype: Partial D+ Comments: The patient may be at risk for production of allo-anti-d. 39

40 Case - Tip of the Iceberg 26 year old African American female with vaginal bleed RBCs type D+ and C+ Ordered RH genotyping 40

41 Case Tip of the Iceberg 1048G>C Allele 1 RHD*1048C (D350H) Allele 2 186C>T (L62F) 410C>T (A137V)) 455A>C(N152T) Expresses partial RhD RHD*DIIIa-CE(4-7)-D Does not express RhD Predicted Phenotype: Partial D+ altered C+ 41

42 Case Tip of the Iceberg 186G>T 410C>T 455A>C Allele 1 Allele 2 RHD*DIIIa-CE(4-7)-D 1048G>C RHD*1048C(D350H) G336C L245V W16C RHCE*ce48C,733G,1006T T342I RHCE*ceTI W16C Predicted Phenotype: Partial D+, Altered C+, E- partial c+ partial e+ Go(a+) The patient is at risk for production of allo-anti-d, -C, -c, -e, -f 42

43 Case: Does the patient need Rh null? International Request to the American Rare Donor Program (ARDP) for Rh null RBCs for 18-year old patient with sickle cell disease History of anti-c, -M, -Do a, -Wr a, -hr B, -Hr B Patient s RH genes were genotyped RHD*01 / RHD-CE(4-7)-D RHCE*ce48C,733G,1006T/RHCE*ce733G

44 Case: Does the patient need Rh null? Anti-hr B detected at Red Cross National IRL in Philadelphia Sample was non-reactive with 2 of 2 examples of RHD*01 RHCE*ce733G homozygous cells at PEG IgG-AGT phase

45 Premise of RH Allele Matching Individuals who express partial Rh antigens (and have made alloantibodies to the Rh antigen) would be expected to be compatible with donor cells with the same (or similar) partial antigens

46 Premise of RH Allele Matching PATIENT Considering patients with two distinct alleles A donor with the same 2 distinct alleles = Tier 1 A donor homozygous for one of the alleles = Tier 2 A donor with a different allele with a similar phenotype = Tier 3 (exception hr S - alleles) TIER 1 TIER 2 TIER 2 TIER 3

47 Premise of RH Allele Matching The patient s HYPOTHETICAL RHCE alleles: Allele 1 Allele 2 RHCE X RHCE Y RHCE Z RHCE X Tier 2 Tier 1 Tier 3 RHCE Y Tier 2 Tier 3 RHCE Z Tier 3 Tier 1 match has exactly the alleles of the patient Tier 2 has no allele that the patient does not Tier 3 has similar but not identical allele(s) rg X / Y Then repeat the process with RHD alleles

48 Case: Does the patient need Rh null? RH Allele Punnett Square FOR THIS CASE Patient: RHCE*ce733G/RHCE*ce48C,733G,1006T RHD-CE(4-7)-D / RHD*01 RHD* Allele 1 Allele 2 RHD*01 RHD-CE(4-7)-D RHD*DAU0 Pseudo RHD*01N.01 RHD* RHD-CE(4-7)-D RHD*DAU Pseudo 1 1 RHD*01N.01 1 RHCE*ce Allele 1 Allele 2 ce733g ce48c,733g ce48c,7333g,1006t ce733g ce48c,733g 3 3 ce48c,733g,1006t 2

49 Case: Does the patient need Rh null? DONORS IDENTIFIED FOR THIS PATIENT Patient: RHCE*ce733G / RHCE*ce48C,733G,1006T RHD-CE(4-7)-D / RHD*01 RH Haplotype N Tier RHD*01/RHD-CE(4-7)-D, RHCE*ce733G/RHCE*ce48C,733G,1006T 41 1 RHD*01 (homoz/hemiz) RHCE*ce733G/RHCE*ce48C,733G,1006T 4 2 RHD-CE(4-7)-D (homoz/hemiz), RHCE*ce48C,733G,1006T homoz 36 2 RHD*01/RHD-CE(4-7)-D, RHCE*ce48C,733G,1006T homoz 7 2 RHD*01 (homoz/hemiz);rhce*ce48c,733g,1006t/rhce*ce733g 6 2 RHD-CE(4-7)-D (homoz/hemiz), RHCE*ce48C,733G,1006T / RHCE*ce733G 5 2 RHD*01 (hemiz/homoz), RHCE*ce733G homoz 88 2 RHD*01/RHD-CE(4-7)-D, RHCE*ce733G homoz 1 2 RHD*01N.01 homozygous RHCE*ce733G homoz 1 2 RHD not tested, homozygous RHCE*ce733G homoz RHD*01N.01 homozygous, RHCE*ce48C,733G / RHCE*ce733G 2 3 RHD*01 (homoz/hemiz), RHCE*ce48C,733G homoz 7 3 RHD*01/RHD-CE(4-7)-D, RHCE*ce48C,733G/RHCE*ce48C,733G,1006T 3 3

50 Summary 1. The RH blood group system is very complex, with many antigens and much genetic variation 2. Variant antigens can case cross-reactivity, typing discrepancies and can put patients at risk for alloimmunization 3. Molecular methods can be used to resolve discrepancies, identify antigen variants and assess risk of alloimmunization 4. Blood donors expressing variant Rh antigens can be matched to Rh alloimmunized patients with the same or similar antigens 50

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52 Questions? You are all muted Q&A following session - Type in questions 52 All Content Immucor, Inc.

53 2019 Advanced Track Webinars Link to register: 53 All Content Immucor, Inc.

54 Questions? You are all muted Q&A following session - Type in questions 54 All Content Immucor, Inc.

55 Continuing Education PACE, Florida and California DHS 1.0 Contact Hours Each attendee must register to receive CE at: Registration deadline is March 8, 2019 Certificates will be sent via only to those who have registered March 22, All Content Immucor, Inc.

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