1. Clinical Applications of Molecular Panel with Focus on RHD genotyping.
S. Gerald Sandler, MD, FACP, FCAP
Professor of Medicine and Pathology
Georgetown University School of Medicine
and
Medical Director, Transfusion Medicine
MedStar Georgetown University Hospital
Washington, DC

2. Objectives.
● Describe AABB-CAP Work Group on RHD Genotyping‘s recommendations on using RHD genotype to resolve serologic weak D phenotypes
● Review the implementation of RHD genotyping in the transfusion service by explaining the guidelines used to perform genotyping and transfusion recommendations based on genotype results

3. Disclosures
No disclosures or conflicts of interest

4. Historical ● 1946 Du, the 1st D antigen variant
● Anti-D in serum of a Du patient
● st edition of AABB Standards
Donor’s RBC’s: testing for Du required
Transfusion recipients’ RBCs : anti-D serum was “sufficient”
● st fatal case of HDFN associated with Du mother
● 1981: Standards 10th edition
Test mother’s RBCs same as a blood donor’s
Addressed RhIG for 1st time
● Agre et al: Du → weak D
● Serologic weak D determined to be molecularly-defined (weak D type 1, type 2, etc.)

5. Historical 2014 – CAP Survey on Policies and Procedures for Testing Weak D
● 3100 laboratories
● 19.8% performed a weak D test on samples testing negative by anti-D
● 49.2% transfuse RhD-negative RBCs for serologic weak D
● No standard practice in US for managing a serologic weak D
● “ Opportunity exists for a multi-organizational collaboration among obstetricians, transfusion medicine specialists, serologists and molecular scientists to establish a nationwide uniform practice.”
Sandler, SG et al for the CAP Transfusion Medicine Resource Committee. Policies and procedures related to testing for weak D phenotypes and administration of Rh immune globulin. Arch Pathol Lab Med 2014;138:620-5.

6. AABB-CAP Interorganizational Work Group on RHD Genotyping
 Organizational Representatives
Susan T. Johnson, MT(ASCP)SBB (AABB)
Louis Katz, MD (ABC)
John T. Queenan, MD (ACOG)
Ralph Vassallo, MD (ARC)
Col. Clayton D. Simon, MD (ASBP)
S. Gerald Sandler, MD (CAP, Chair)
Scientific Consultants 
Megan Delaney, DO, MPH
Gregory A. Denomme, PhD, FCMLS
Willy (Bill) Flegel, MD
Margaret Keller, PhD
Connie M. Westhoff, SBB, PhD

7. Serological Weak D Phenotypes Work Group’s Findings
0.2 – 1.0 % of Caucasians inherit RHD genes that code for serological weak D phenotypes
Prevalence varies by race and ethnicity
Reports of the prevalence vary depending on the method
Manual tube versus automated analyzer
Anti-D reagent (polyspecific serum versus monoclonal blend)
Potentiating reagents (bromelin)

8. Serological Weak D Phenotypes Work Group’s Findings Molecularly-Defined Weak D Types
One or more amino acid substitutions in the RhD protein within or below the RBC membrane causing decreased antigen expression on the RBC surface
-- Sandler SG, Chen L, Flegel WA. Serological weak D phenotypes: a review and guidance for interpreting the RhD blood type using the RHD genotype. Brit J Haematol 2017, in press.

9. -- Flegel WA, Denomme GA, Yazer MH. Transfus Apher Sci. ;44(1):81-91.
Serological Weak D Phenotypes Work Group’s Findings Partial D Phenotypes
Partial Ds are associated with amino acid substitutions in the RhD protein on the RBC surface and lack D epitopes
-- Flegel WA, Denomme GA, Yazer MH. Transfus Apher Sci. ;44(1):81-91.

10. Serological Weak D Phenotypes Work Group’s Findings
Transfusion recipients with weak D types 1, 2 or 3 are not at risk for forming alloanti-D when exposed to conventional RhD-positive RBCs
Approximately 95% of Caucasians in central European with a serological weak D phenotype have weak D type 1, 2, or 3 and can be managed safely as RhD-positive
Alloimmunization has been observed with certain other weak D types, including weak D type 4.2 , type 11, type 15, type 21, and type 57

11. Work Group’s Recommendations
RHD genotyping should be performed whenever a discordant RhD typing result and/or a serologic weak D phenotype is detected in patients, including pregnant women, newborns, and potential transfusion recipients.
For women with a serological weak D phenotype associated with an RHD genotype other than weak D type 1, 2 or 3, the Work Group recommends conventional prophylaxis with RhIG.

12. Potential Benefits of RHD Genotyping Pregnant Women
3,953,000 Live births
3,812,000 Pregnancies
556,500 RhD-negative
16,700 Serologic Weak D
13,360 weak D types 1, 2
or 3
RHD Genotyping
24,700 unnecessary
ante- and postpartum
RhIg injections

13. Potential Benefit of RHD Genotyping Transfusion Recipients
5,000,000 Individuals Transfused Annually in US
730,000 RhD Negative
21,900 Serologic
Weak D
17,520 weak D types 1, 2
or 3
RHD Genotyping
Could receive RhD-positive RBCs
(47,700 units)

14. Recommended Management of Routine RhD Typing Results in Patients
RhD-Negative Serological Weak D RhD-Positive
∙ Candidate for RhIG Send for RHD Genotyping ∙ Not a Candidate for RhIG
∙ RhD-negative for ∙ RhD positive for Transfusion transfusion
Weak D type 1, 2 or Weak D type 1, 2 or 3
not detected detected
∙ Risk for forming anti-D ∙ No risk of forming anti-D
∙ Candidate for RhIG ∙ Not a candidate for RhIG
∙ Rh-negative for transfusion ∙ Rh-positive for transfusion
--Interorganizational Work Group on RHD Genotyping

15. Joint Statement on Phasing-In RHD Genotyping for Pregnant Women and Other Females of Childbearing Potential With a Serologic Weak D Phenotype
“. . . RHD  genotyping is recommended whenever a weak D phenotype is detected by routine Rh blood typing of pregnant women and other females of childbearing potential . . .”
AABB America’s Blood Centers
American College of Obstetricians and Gynecologists
American Red Cross Armed Services Blood Program
College of American Pathologists
July 2015