1. 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

2. Disclosures
I have no relevant financial relationships to disclose.

3. 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.

4. 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 May; 138(5):620-5.

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

6. 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.

7. 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:

8. 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

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

10. 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

11. 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

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

13. Immucor RHD BeadChip 80+ variant RHD alleles
Does not detect all variants
8 wells/slide – batch 3-4 slides
Research Use Only

14. 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

16. 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

17. 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.

18. 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.

19. 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 Sep;55(9):

20. 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 

21. 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.

22. 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:
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 Sep;55(9):
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 May; 138(5):620-5.