Phasing-In RHD Genotyping

In this issue, Sandler and colleagues¹  report the results of the College of American Pathologists (CAP) J-B Transfusion Medicine (Comprehensive) and Educational Survey, in which more than 3100 institutions describe how they perform Rh typing for blood donors, pregnant women, and hospital patients. In accordance with American Association of Blood Banks (AABB) Standards for Blood Banks and Transfusion Services,²  most hospital laboratories reported that they do not routinely perform a serologic weak-D test on pregnant women or transfusion recipients. This practice results in most pregnant women and hospital patients with a weak-D phenotype being categorized and managed as Rh⁻ (Table 1).^2,3  In contrast, a weak-D test is performed routinely on blood donors whose red blood cells test D⁻ by direct agglutination, resulting in most blood donors with a weak D being categorized and managed as Rh⁺.²  This 50-year practice appears to be relatively safe,⁴  and there are only a few published reports of persons with a weak-D phenotype forming anti-D antibodies.^5–8  However, this practice confuses patients, blood donors, and caregivers and uses Rh immune globulin (RhIG) and Rh⁻ red blood cells for many persons with a weak D, who could be safely managed as Rh⁺, if their genotypes were known.^3,9,10  The CAP Transfusion Medicine Resource Committee (TMRC) reviewed this practice in the context of the current state of science for RHD genotyping.¹  The TMRC concluded that selective integration of RHD genotyping of weak D phenotypes could improve the accuracy of Rh typing results, thereby reducing unnecessary administration of RhIG in women with a weak D phenotype, and decrease transfusions of Rh⁻ red blood cells in recipients with a weak D phenotype.¹ 

The process of phasing-in RHD genotyping in clinical practice has begun in many hospitals, but as the CAP survey indicates, most pregnant women and hospital patients in the United States continue to have their Rh type determined by dated serologic methods.¹  Those laboratories that do not routinely perform weak-D tests for patients typing Rh⁻ by direct agglutination with anti-D should now begin to introduce Rh typing reagents and procedures selected to detect, not to avoid detection of, weak-D phenotypes.

We recently encountered a 27-year-old North African woman who was designated as Rh⁻ for a cesarean section. Her medical history and laboratory test results are representative of a common subset of patients¹¹  and illustrate how RHD genotyping can improve the management of patients with a weak-D phenotype. We have summarized recommended guidance for diagnostic testing and clinical decision making in women with a weak-D phenotype after delivery of a D⁺ newborn (Table 2).

The woman's routine postpartum blood sample result was strongly positive by a rosette fetal bleed screening test, suggesting the presence of D⁺ fetal red blood cells in her circulation (fetomaternal hemorrhage). However, a quantitative, acid-elution (Kleihauer-Betke) assay result was negative, indicating that the D⁺ red blood cells in her circulation did not contain a significant amount of hemoglobin F; that is, the red blood cells were not of fetal origin. A weak-D test was positive, confirming the clinical impression that her red blood cells expressed an inherited weak-D phenotype. Red blood cells from approximately 0.2% to 1.0% of white people express a weak-D phenotype.¹²  A weak-D phenotype has been reported in 0.1% to 10% of all pregnancies that were initially typed as D⁻.^13–15  We estimate that approximately 90% of patients in the United States with a weak-D phenotype will have one of the prevalent RHD genotypes (types 1, 2, 3, or 4.1).^5,7,11,16  Women with one of these RHD genotypes may be managed as Rh⁺ and do not require RhIG for prenatal or postpartum Rh immunoprophylaxis.^7,17  However, that decision can only be made by RHD genotyping. Even monoclonal anti-D reagents, which were initially believed to be capable of identifying RHD genotypes, cannot distinguish among the most prevalent weak-D genotypes (Table 3).^6,18,19  We performed molecular testing on our patient²⁰  and established that she had inherited the uncommon weak-D type 25,²¹  which required management as Rh⁻ for purposes of Rh immunoprophylaxis and transfusion of red blood cells.

The second step in phasing-in RHD genotyping will be establishing standardized, cost-effective, RHD genotyping protocols for laboratories. Most hospitals will not have a sufficient volume of patients with a weak-D phenotype to justify establishing in-hospital RHD genotyping services. Hospitals are likely to refer blood samples to regional reference laboratories where high test volumes will support both basic and complex genotyping services. A molecular test in D⁻ pregnancies may pay for itself by avoiding the costs associated with often unnecessary multiple administrations of RhIG.^4,17,22  Presently, there are no US Food and Drug Administration–approved molecular test kits for determining the Rh type, but several unlicensed products are marketed commercially in the United States. These products use polymerase chain reaction with detection by gels, bioarray chips, or bead chips. Any of these test kits and test platforms can be used for patient care as tests of either “high” or “moderate” under the Clinical Laboratory Improvement Amendments of 1988.

Based on the results of its 2012 survey and review of the science of RHD genotyping, the CAP TMRC has recommended a multiorganizational collaboration among obstetricians, transfusion medicine specialists, serologists, and molecular scientists to update current practice guidelines and establish a nationwide, uniform practice.¹  The CAP and AABB have formed the Work Group on Phasing-In RHD Genotyping. We believe that the time has come to transition from serologic to molecular methods for managing weak-D phenotypes. Our case illustrates how easily this transition can be accomplished. In conclusion, we support the CAP TMRC's initiative.

We thank S. Gerald Sandler, MD, and Harvey G. Klein, MD, for reviewing the manuscript; A. Hallie Lee-Stroka, MT(ASCP)SBB; Neil Bangs, MS MT(ASCP)SBB; Sherry L. Sheldon, MT(ASCP)SBB; and Debrean Ann Loy, MT(ASCP)ASQ, for performing serology; David Allan Stiles, MS, and Supatta Mary Lucas, MLT(ASCP), for performing RHD sequencing; and Kshitij Srivastava, PhD, for nucleotide sequence data entry.