How Do I Implement RHD Genotyping in the Transfusion Service Chelsea Hayes, MD Assistant Professor, Division of Transfusion Medicine Department of Pathology and Laboratory Medicine Cedars-Sinai, Los Angeles, CA

Disclosures I have no relevant financial relationships to disclose.

Learning Objectives Explain the guidelines used to implement RHD genotyping in the transfusion service. Review transfusion recommendations based on RHD genotyping results. Review challenges of RHD genotyping.

CAP Survey (2014) >3000 labs questioned regarding approach to testing and interpreting weaker than expected serologic D typing results (weak D phenotypes). Most labs perform the “weak D test” (antihuman globulin test) on all apparent RhD-negative blood donors and newborns, per AABB standards. Discrepancy regarding patient testing: Perform “weak D test” following a weaker than expected D typing result Interpret a weaker than expected initial result as RhD-negative Interpret a weaker than expected initial result as RhD-positive No standardized approach to testing and interpreting weak D phenotypes. Sandler SG et al. Arch Pathol Lab Med. 2014 May; 138(5):620-5.

Why do we care? Serologic weak D phenotypes may indicate the presence of an altered D antigen: Weak D Partial D Weak D: All major D epitopes present on RBC surface Intracellular changes to RhD protein Usually NOT at risk of forming anti-D antibodies RhIG (RhoGAM) and RhD-negative RBCs unnecessary

Why do we care? Partial D: Different/missing D epitopes on RBC surface Extracellular changes to RhD protein At RISK of forming anti-D antibodies RhIG and RhD-negative RBCs necessary Weak vs. Partial D cannot be distinguished using serologic testing.

AABB/CAP Work Group on RHD Genotyping Responsible for developing recommendations to clarify clinical issues related to D typing in patients with a serologic weak D phenotype. Definition of a serologic weak D phenotype: ≤2+ reactivity in initial testing, but moderate to strong agglutination with antihuman globulin (weak D test). Work Group Recommendation: Perform RHD genotyping with discordant or serologic weak D typing results: 1. Females of childbearing potential 2. Transfusion recipients Sandler SG, et al. Transfusion 2015;55:680-689.

AABB/CAP Work Group on RHD Genotyping Goal: Identify weak D patients (weak D types 1, 2 or 3) who can safely be treated as RhD-positive. 1. Females of childbearing potential Avoid 24,700 doses of RhIG annually 2. Transfusion recipients Avoid transfusion of 47,700 RhD-negative RBCs annually Transfusion Recommendations: Weak D type 1, 2 or 3 DETECTED = RhD-positive Weak D type 1, 2 or 3 NOT DETECTED = RhD-negative

Cedars-Sinai 886 licensed beds Annually: 55,000 transfusions 7,000 babies delivered 32,000 operating room procedures 85,000 emergency department visits

Cedars-Sinai Transfusion Service Policy Perform RHD genotyping: Serologic weak D phenotype Weak to 2+ on initial testing (primarily gel) Discrepant results 2 samples with a difference of at least 2+ reactivity Apparent RhD-positive patient with an anti-D antibody

Cedars-Sinai Transfusion Service Policy Order RHD genotyping to detect presence of altered D antigen Initially sent-out Now completed in-house Already performing molecular testing to predict extended RBC phenotype (detects 38 RBC antigens) Turnaround time is typically 2-3 weeks While RHD genotyping results are pending: Restrict patient to RhD-negative RBCs Patient is considered eligible for RhIG

Review patient history to rule out: Mislabeled sample – repeat testing Massive transfusion Stem cell transplant Large fetal-maternal bleed

Immucor RHD BeadChip 80+ variant RHD alleles Does not detect all variants 8 wells/slide – batch 3-4 slides Research Use Only http://www.immucor.com/en-us/Products/Documents/BioArray_RH_SalesSheet.pdf

Transfusion Recommendations Weak D type 1, 2 or 3 → Manage as RhD-positive Not at risk of forming anti-D antibodies Transfuse RhD-positive RBCs Not a candidate for RhIG → Increase availability of RhD-negative RBCs and avoid unnecessary RhIG NOT weak D type 1, 2 or 3 → Manage as RhD-negative Considered at risk of forming anti-D antibodies Restrict to RhD-negative RBCs Eligible for RhIG → Prevent D-alloimmunization

Cedars-Sinai RHD Genotyping Results Tested 22 serologic weak D patients in 15 months → Weak D types 1 or 2 = 13 (59%) Weak D Types 1, 2 or 3 Number of Patients Reason for Testing Weak D type 1 9 SWD Weak D type 2 4 Other Genotyping Results Weak D type 4.0 or 4.3 1 Weak D type 5 DVI 3 RHD psi or DV type 1 or DBS2 DIVa type 2 or DIVa type2/DIIIaCE(4-7)-D SWD and anti-D Dllla/DIIIa-CE(4-7)-D Possible D (sequencing not performed) *SWD = serologic weak D phenotype

Cedars-Sinai RHD Genotyping Results Weak D types 1 or 2 = 13 (59%) 4/4 obstetric patients received RhIG while results were pending None have presented with a subsequent pregnancy No transfusion of RhD-positive RBCs since genotyping Genotyping has not yet resulted in a reduction of unnecessary RhIG or conservation of any RhD-negative RBCs.

RHD Genotyping Challenges 1. Long turn-around-time Patients are being treated as RhD-negative while genotyping results are pending No immediate change in practice Benefits only exist with future encounters, assuming patient returns 2. Provider and patient education Some providers are uncomfortable withholding RhIG or RhD-negative RBCs from weak D type 1, 2 or 3 patients. Some patients may be uncomfortable suddenly receiving RhD-positive RBCs. 3. No centralized reporting system Results would not be available if patient were treated at another institution.

RHD Genotyping Challenges 4. Current approach does not identify: Weak D types 1, 2 or 3 that are negative on initial testing Solution: Perform “Weak D” test or RHD genotype all RhD-negative patients Partial D that reacts strongly (≥3+) on initial testing Solution: RHD genotype all RhD-positive patients 5. Cost to Transfusion Service Send-out: Approximately $400 In-house testing: Approximately $150 RHD genotyping pregnant women with serologic weak D phenotypes would be cost-saving when the cost is below $256* *Kacker S, et al. Transfusion. 2015 Sep;55(9):2095-103.

Example Case 57-year-old male with metastatic bladder cancer undergoing chemotherapy and radiation, admitted with anemia. RhD typing = 1+ reactivity on initial gel testing Interpreted as a “serologic weak D” RHD genotyping ordered Received RhD-negative RBCs while waiting for results Weak D type 1 → Not at risk of alloimmunization Patient later returned for transfusion and RhD-positive RBCs were provided Patient refused RhD-positive RBCs! Received 16 units of RhD-negative RBCs since genotyping 

Summary Perform RHD genotyping on weaker than expected or discrepant D typing results. Weak D types 1, 2 or 3 → treat as RhD-positive Reduce unnecessary use of RhIG and RhD-negative RBCs Limitations of RHD genotyping include cost, long turn-around-time, and current lack of understanding among providers and patients. We are hopeful that RHD genotyping will lead to more consistent and personalized patient care and improved utilization of RhIG and RhD-negative RBCs.

References Sandler SG, Flegel WA, Westhoff CM, Denomme GA, Delaney M, Keller MA, Johnson, ST, Katz L, Queenan JT, Vassallo RR, Simon, CD. It’s time to phase in RHD genotyping for patients with a serologic weak D phenotype. Transfusion 2015;55:680-689. Haspel RL, Westhoff CM. How do I manage Rh typing in obstetric patients? Transfusion 2015,55:470-4. Kacker S, Vassallo R, Keller MA, Westhoff CM, Frick KD, Sandler SG, Tobian AA. Financial implications of RHD genotyping of pregnant women with a serologic weak D phenotype. Transfusion. 2015 Sep;55(9):2095-103. Anne Paxton. Groups urge phase in of RHD genotyping. CAP Today. October 2015. Sue Johnson, MSTM, MT(ASCP)SBB. Serologic Weak D Phenotype to RHD Genotyping: How will I know? Immucor User’s Group Meeting, San Ramon, CA. March 8, 2016. Willy A. Flegel, MD; Susan D. Roseff, MD; Ashok Tholpady, MD. Phasing-In RHD Genotyping. Arch Pathol Lab Med 2014 May;138 Sandler SG, Roseff SD, Domen RE, Shaz B, Gottschall JL. Policies and procedures related to testing for weak D phenotypes and administration of Rh immune globulin: results and recommendations related to supplemental questions in the Comprehensive Transfusion Medicine survey of the College of American Pathologists. Arch Pathol Lab Med. 2014 May; 138(5):620-5.