RH genetic variation and the impact for typing and personalized transfusion strategies: a narrative review

Introduction

Rationale

The purpose of this narrative review is to provide the reader with updated guidance on the clinical utility identifying patients at risk for allo-anti-D formation as well as strategies that can be used to determine candidacy for Rh immune globulin in pregnant women. This narrative also reviews the genetic complexity behind phenotypes lacking high prevalence antigens encoded by the RHCE gene and describes an effective strategy that has been used for more than a decade in the US to personalize donor unit selection in Rh alloimmunized patients based on RH allele information. This strategy can be used in alloimmunized patients in whom transfusion has become seemingly impossible. The objective is to provide the reader with information that can be used to reduce Rh alloimmunization and increase personalized patient care related to red cell alloimmunization. I present the following article in accordance with the Narrative Review reporting checklist (available at https://aob.amegroups.com/article/view/10.21037/aob-22-6/rc).

Methods

Peer-reviewed literatures published in English and listed in PubMed from 1960 to January 2022 were reviewed while compiling this work. In addition, AABB guidance documents and AABB standards as well as guidelines published by the American Society of Hematology and the British Society of Haematology were reviewed and cited where appropriate. The allele tables on the website of the ISBT Working Party for Red Cell Immunogenetics and Blood Group Terminology were also reviewed and referenced. Relevant peer-reviewed abstracts by the author were reviewed and compiled data from such work is included where appropriate (see Table 1).

The RH blood group system

The RH blood group system includes two genes (RHD and RHCE), both with 10 exons, that encode a total of 56 antigens. While the RHD gene encodes one common antigen, RhD or RH1, the RHCE gene encodes two sets of antithetical common antigens, big C (RH2) and little c (RH4) and big E (RH3) and little e (RH5). Expression of RhD and RhCE antigens requires co-expression with the RhAG protein in a multimeric protein complex.

RH locus

The RH locus, located on Chromosome 1, includes the highly homologous and highly polymorphic RHD and RHCE genes. The RhD and Rhce polypeptides are multipass transmembrane proteins that are part of a multiprotein complex on the red cell surface. The RhD polypeptide carries the RhD antigen with multiple epitopes and can express neoepitopes such as Go^a (RH30) or DAK (RH54). The RHCE gene has four normal allele types (RHCE*ce, RHCE*Ce, RHCE*cE and RHCE*CE) that encode Rhce polypeptides expressing antithetical antigens c or C and e or E.

The RHD gene

The RHD gene (NG_007494) includes 10 exons and encompasses ~58,000 base pairs of DNA on chromosome 1. It is polymorphic with variants including deletions, gene conversions, and non-synonymous single nucleotide variants (1).

There are several mechanisms and more than 80 RHD alleles that are associated with a null or RhD negative phenotype (see Table 2). RHD*01N.01, deletion of the gene’s 10 exons is the most common RhD negative allele in Caucasians while RHD*08N.01, the silenced RHD allele is the most common RhD negative allele in individuals of African descent. There are several RHD alleles (RHD*01N.02 – RHD*01N.05) that do not express the D antigen due to gene conversion involving replacement of multiple exons with those from the RHCE gene. In addition, several RHD alleles involve gene conversion that abolishes RhD expression and gains expression of a variant of the C antigen, with RHD*DIIIa-CE(4-7)-D or RHD*03N.01 the most common in African Americans. Finally, there are RHD alleles in which splicing defects abolish RhD expression (e.g., RHD*01N.24), those with insertion/deletion polymorphisms that lead to frameshifts and premature termination (ex. RHD*01N.11) and those with nonsense mutations (ex. RHD*01N.18) as well as those with nonsynonymous mutations where the mechanism is less clear (ex. RHD*01N.80).

RhD antigen

The RhD polypeptide expresses the RhD or RH1 antigen. RHD alleles that encode RhD antigens have historically been categorized on one of three groups (weak, partial or DEL). Partial RhD antigens are defined as antigens in which one or more epitopes recognized by monoclonal antibodies is lacking.

As is the case with some other blood group antigens, including other antigens in the RH blood group system, expression is not binary, meaning that classifying the phenotype as positive or negative is incomplete and inaccurate. Though the red cells may test positive with an anti-D reagent, if the RHD gene sequence differs from the consensus sequence the antigen may be different. These differences may impact copy number, gain or loss of antigen epitopes or both as well as reactivity with human or reagent anti-D. Panels of monoclonal antibodies have been used to investigate the epitope loss in RhD variants (2) and flow cytometry has been used to assess D antigen sites per red blood cell (RBC) in subjects expressing RhD variants (3).

Changing terminology

In 1946, the weak D phenotype was initially described and defined as red cells that agglutinate with some but not all anti-D reagents and was given the term D^U (4). When anti-D sensitizes the RBCs but they do not directly agglutinate, visualization can be enhanced by the use of antiglobulin sera. Subsequently, the US put in place policies requiring blood donor RBCs that type D- to be retested using antiglobulin (“weak D test”) and if agglutination was seen, to be classified as D+ (5). This additional testing is not required of pregnant women who type D-; instead, they are given Rh immunoglobulin (RhIg). The term “D mosaic” was coined in 1962 to describe red cells that express RhD antigen that is missing one or more epitopes defined by reactivity with monoclonal anti-D antisera (6). While initially the D^U term was used to define a red cell with a quantitative but not qualitative difference in the D antigen, this terminology was fraught with challenges. First because there is no D^U antigen or anti-D^U antibody. Second because some D^U+ individuals, initially thought to carry little risk of allo-anti-D formation, were found to make allo-anti-D. The term D^U was replaced with “weak D” in 1992 (7).

Changing guidance

Since the 1990s, our understanding of the complexity of RHD genetic variation and its impact on the RhD antigen has grown significantly. In 2013, Geoffrey Daniels proposed using the term “D variant” to avoid confusion with antigens that can type weakly D+, yet are associated with epitope loss and allo-anti-D production (8). In 2014, Sandler et al. presented results of a College of American Pathology (CAP) survey of 3,100 US hospitals about policies and procedures around testing individuals with weak D phenotypes and administration of RhIg which highlighted the lack of standard practice in the US (9). In 2015, an inter-organizational work group published a commentary (10) with two key take aways, both illustrated in Figure 1.

• A serologic weak D phenotype was redefined as reactivity of RBCs with an anti-D reagent giving no or weak (≤2+) reactivity in initial testing, but agglutinating moderately or strongly with antihuman globulin.
• The RhD phenotype of individuals with a serologic weak D phenotype should be resolved by using RHD genotyping to identify the RHD alleles carried by the patient. In this way, individuals found to carry RHD variant alleles weak D type 1, 2 or 3 (RHD*01W.1, RHD*01W.2, RHD*01W.3) are not considered at risk for allo-anti-D (10), and would avoid RhIg administration.

Figure 1 Algorithm for resolving serologic weak D phenotype test results by RHD genotyping to determine candidacy for RhIG and RhD type for transfusions. RhIG, Rh immunoglobulin.

Based on the number of live births and transfusion recipients and ethnic background of the US population, this strategy was estimated to identify more than 13,000 women of child-bearing age in whom more than 24,000 doses of RhIg would not be needed. The recommendation to “phase-in” RHD genotyping, specifically of women of child-bearing age with a serologic phenotype was endorsed by the AABB and CAP (11). Financial modeling suggested that this approach could add clinical benefit without additional costs (12).

Strategies for identifying those at risk

Since the release of the joint statement (11), there has been a growing appreciation for the ability of molecular methods to assess a patient’s risk of alloimmunization to RhD more precisely. However, besides the flow chart from Sandler et al. (10) reproduced in Figure 1 and a review (13), there is no specific guidance for how a hospital blood bank would identify patients that may benefit from RHD genotyping. Further, many hospital blood banks have automated antigen typing platforms, whether solid phase or gel technology and these platforms may or may not give the user the ability to easily review results of specimens demonstrating weaker reactivities. Several healthcare systems have evaluated algorithms to select samples for RHD genotyping. A repeating theme is the use of differential anti-D reactivity between two methods or reagents to identify those who likely carry RHD variant alleles and express RhD variant antigens. It is important to emphasize that serologic testing cannot accurately distinguish between RhD variant antigens that put the patient at risk of alloimmunization or not. RHD genotyping of these individuals can identify the specific alleles and that information can be used to assess risk of alloimmunization. This strategy can focus genotyping efforts on patients who would benefit the most, since it is not currently cost-effective to perform RHD genotyping of all patients and blood donors.

There have been several publications that describe the use of anti-D reactivity differences to identify patients who could benefit from RHD genotyping (14-19) (see Table 3). These studies share a two reagent and/or two-method strategy and are highly effective at identifying samples for which RHD genotyping predicts expression of RhD variants. Importantly, monoclonal anti-D panels are now recognized as being ineffective at differentiating patients at risk of allo-anti-D while RHD genotyping can differentiate samples carrying weak D types 1, 2 and 3 from samples carrying partial D alleles. The two-test approach does not require additional testing in laboratories accredited by AABB, since these organizations already require two determinations of ABO/Rh for pre-transfusion testing for allogeneic transfusion (31).

Benefit of RHD genotyping: avoiding overtreatment

Among Caucasians, in whom a serologic weak D phenotype is found at a prevalence of 0.2–1% (15,32), 73–80% of these will be found to carry RHD alleles weak D type 1, 2 or 3 (RHD*01W.1, RHD*01W.2, RHD*01W.3) (10,15). Thus, in a primarily Caucasian population, the main benefit of implementing a process to identify patients with serologic weak D phenotype for RHD genotyping will be identification of those women of childbearing age who do not require RhIg prophylaxis. Importantly, the benefits and the outcome of such an approach may be different in other ethnic groups.

Benefit of RHD genotyping: identifying those at risk who are hiding in plain sight

Among non-Caucasians, in whom a serologic weak D phenotype is more often associated with inheritance of a partial D allele, implementing an RHD genotyping strategy can identify patients who may have typed D+ on an automated testing instrument and who may not have been identified as at risk of allo-anti-D otherwise. Though some monoclonal anti-D reagents will be non-reactive with red cells expressing a partial D phenotype, it is common for such cells to react strongly with some anti-D reagents typically used on automated platforms in the US yet weakly or not at all by manual tube agglutination method. Implementing a process to test women of child-bearing age by two different methods and/or anti-D reagents will help to identify women who may express a partial D phenotype and who could benefit from RHD genotyping to accurately assess candidacy for RhIg. A prerequisite for this approach is prenatal care with access to laboratory testing in a timeframe that allows for appropriate RhIg administration during and shortly after delivery.

Findings of a large national US reference laboratory

Keller et al. (20) described RHD genotyping of 879 patientsamples referred to a large national molecular laboratory from across the US in calendar year 2020. These specimens were identified by hospitals based on their own criteria, which were not provided. The race/ethnicity of the cohort was 41% African American (AA), 41% Caucasian (CAUC), 11.1% “other”, 3.2% Hispanic, 1.1% race not provided, 0.7% mixed race, 0.6% Asian and 0.3% Native American. RHD genotyping predicted 63.5% to express a D variant with 31.5% hemizygous or homozygous for Weak D Types 1, 2 and 3. Among Caucasian patients, the majority (61.7%) carried Weak D Types 1, 2 and 3. Among African American patients, 58% were predicted to express a partial D phenotype with 16 different alleles identified, of which the most common RHD variant allele was weak D type 4.0 (Table 2). The RHD alleles identified in this large US cohort are listed in Table 4. The breakdown of patients by race/ethnicity that were found to carry Weak D Types 1, 2 or 3 or other RHD alleles encoding partial D are listed in Table 5 and Table 6. This study is the largest of several published studies (Table 3). Approximately two-thirds (63%) of subjects were found to carry a variant RhD antigen. This percentage is lower than the other studies where subjects were identified using a specific algorithm, such as that used in Luo et al. or Horn et al. (17,18). Interestingly, when the large study (20) is compared to Hudgins et al. (19), in which one-tenth the number of subjects were tested, a similar percent of RHD variants were identified (65% vs. 63%) and though the sample size of Keller et al. was more than ten-fold larger than that of Hudgins et al., it identified 19 distinct RHD variant alleles while Hudgins identified 15 alleles. This may suggest that the number of alleles involved in typing discrepancies in the US population is small and mostly known.

Phasing out serologic weak D result

In 2020, Flegel et al. published a commentary (33) that suggested that blood banks and Immunohematology reference laboratories should stop releasing reports with “weak D+” or “serologic weak D+” interpretations as a final result. Instead, the authors suggest that such cases should be referred to molecular reference laboratories where genotyping can be used to identify the RHD alleles. With knowledge of the RHD alleles that are responsible for the weak D phenotype, the risk of alloimmunization can be more accurately assessed. In pregnant women and women of child-bearing age, this information is critical to determine candidacy for RhIG; in all patients, the information can be used to determine which require RhD negative blood products. This process offers a genome-informed and personalized approach to RhD alloimmunization risk assessment. Furthermore, since RHD alleles associated with partial D phenotypes are often co-inherited with RHCE alleles encoding partial antigens or lacking high prevalence antigens, this information may uncover additional Rh alloimmunization risk. The American Red Cross National Molecular Laboratory saw a 6.3% increase in service requests for RHD genotyping of patients from FY19 (July 2018 to June 2019) to FY2020 (July 2019 to June 2020) followed by a 23.8% increase from FY2020 to FY2021 (July 2020 to June 2021). The later increase may have been influenced by the Flegel et al. publication (33) which was released electronically in March 2020.

Personalized transfusion support for patients lacking high prevalence antigens in the RH system

In patient populations where chronic transfusion is a therapeutic modality (ex. sickle cell anemia and thalassemia), RBC phenotype matching has been used for more than twenty-five years (34-37). More recently, prophylactic red cell antigen matching for common RBC antigens has become commonplace (38,39). Matching for C, E and K is recommended by the British Society of Haematology (40). The American Society of Hematology (ASH) recently recommended matching for C, E and K as the standard of care for all patients with sickle cell disease, optimally before the first transfusion (41). The ASH guidelines also recommend that when RH genotyping identifies a partial C antigen (either by RHD*03N.01 or RHCE*Ce.10) in a patient without a conventional RHCE allele expressing the C antigen, the patient should receive C- red cell products to avoid allo-anti-C. In addition, genotyping for red cell antigens is preferred over serologic phenotyping due to increased accuracy and the ability to predict phenotypes for antigens for which commercial reagents are not available or not reliable (41-43).

Personalized transfusion support for patients with sickle cell disease

In patients with SCD, it has been appreciated for some time that since this population is predominantly of African descent, and since in the USA the blood donor base is primarily Caucasian, there is a high likelihood of mismatches in what had been termed “minor” blood group antigens (i.e., Fy^a, Fy^b, Jk^a, Jk^b, S, s). In the US, the desire to “extend” the match from ABO/Rh and C and E antigens to these additional antigens resulted in a variety of donor recruitment programs aimed at collecting RBC units from African American donors (44). However, individuals of African descent have extensive genetic variation in the RH locus, with not only partial RhD phenotypes but loss of high prevalence antigens such as hr^B, hr^S and Hr (45). While RBC units from Caucasians with normal Rh antigens may put patients of African descent with partial Rh antigens at risk of alloantibodies in the RH system, RBC units from African descent donors expressing neoepitopes such as Go^a in individuals with RHD*DIVa can alloimmunize patients of African descent who do not express the same RhD variant antigen(s) (46). A study of Brazilian patients with SCD found that those who typed D+ with unexpected anti-D carried RHD alleles encoding partial D antigens (47). Additionally, patients of African descent who type C+ with anti-C and/or e+ with anti-e often carry variant RHCE alleles encoding partial antigens. In the case of the C+ patient with anti-C, they may not express the C antigen but instead a hybrid RHD-RHCE allele that encodes a partial C antigen (48).

There has been increasing interest in molecular matching for patients with SCD, including matching at the allele level for RH variants (49). In 2017, Fasano et al. (50) noted that more studies would be needed to determine if use of RH-allele matched RBC units would be a feasible approach to prevent Rh alloimmunization. The feasibility of such an approach was later examined using virtual simulations (51). The study involved use of identical matches, haplotype matches and less restrictive matches that classified RHD*DAU0 as equivalent to RHD*01 and RHCE*ce48C equivalent to RHCE*ce. A recent modeling study provides evidence that some RhD variants may be “benign” based on impact of amino acid substitutions on tertiary structure of the RhD molecule (52). Chou et al. (51) noted that the success of prophylactic RH allele matching protocol would be dependent on improved recruitment of African American blood donors, an issue also highlighted by Karafin et al. (53).

Identifying patients lacking hr^B (RH31) and/or hr^S (RH19)

There are multiple RHCE alleles that predict loss of high-prevalence antigens hr^B (RH31) and/or hr^S (RH19) (42) (Table 7). This is an important distinction from most other blood group antigens and should be taken into account when designing a process for selection of RBC units based on RH alleles and their predicted phenotypes. In addition, there are no commercial reagents to screen for donors lacking either of these antigens such that molecular screening is the primary means of donor identification. While many of the RHCE*ce alleles that encode Rhce molecules lacking hr^B include the single nucleotide variant (SNV) C>G at coding position 733 in the RHCE gene (c.733C>G), not all alleles carrying c.733G are associated with an hr^B- phenotype and for those that do, most but not all will express both V and VS, since the V is lost when the c.733G is coinherited with c.1006T. And there are RHCE*ce alleles such as RHCE*ceAG that do not carry c.733G yet encode an hr^B- phenotype. The RHCE*ce alleles that encode hr^S- phenotype are varied in the SNVs they carry; several carry c.712 and some gain expression of the low prevalence antigen RH49 (STEM). This heterogeneity requires, at minimum, medium-resolution RHCE genotype information for selection of donor units based on RH alleles of the donor and patient. While the commonly used commercial red cell genotyping kits are low-resolution, containing a small number of markers that are insufficient to assign alleles with confidence, additional testing and/or medium-resolution assays include additional SNVs that allow allele assignments with increased accuracy (54). High-resolution methodologies such as Sanger sequencing of genomic DNA or cDNA or Next Generation sequencing provide the greatest accuracy, but analysis and interpretation are labor-intensive and complex (55).

Personalized transfusion support for alloimmunized patients using RH allele selection

The American Rare Donor Program (ARDP) aids in identifying rare blood products for alloimmunized patients (56). These products include units lacking high prevalence antigens hr^B and hr^S (defined by genotype) in the RH blood group system. In a review of ARDP activities from 2005 and 2010 (56), there were 10 requests for hr^B- or hr^S-units in 2005, and since 2010 these requests are filled with RH genotype-selected units, when possible (56,57). In 2013, the process or RH allele selection used by the ARDP was formalized (58) and additional reports of its use have been presented (59). In 2020, the ARDP received 126 requests for RH allele selected red cell units for 53 patients. An analysis of these requests (60) showed that 49% of these patients carried at least one RHCE*ce733G allele. Though the ISBT Working Party on Red Cell Immunogenetics and Blood Group Terminology currently classifies this allele as having an hr^B+^vw/- phenotype, allo-anti-hr^B has been reported (61,62). Therefore, ARDP considers this allele to be associated with risk of allo-anti-hr^B and provides RH allele selected units upon request.

As Chou et al. noted (51), large numbers of well characterized AA donors would be needed to put into practice routine selection of donor units based on RH alleles. To that end, the American Red Cross National Molecular Laboratory developed high-throughput assays to test E-, K-, hemoglobin (Hgb) S negative donors of African descent for both RHCE (63) and RHD (64) variants. In an analysis of more than 6,000 predominantly African American donors tested by HemoID DQS red cell genotyping panel (Agena Bioscience), 564 donors predicted to be E-, K-, HgbS- and predicted to be hr^B+^vw/- or hr^B- or hr^S negative or E-e+^w were selected for further characterization. Genotyping was performed in 96-well format with custom RHCE (10 SNVs) and RHD (18 SNVs) oligonucleotide extension assays designed for MALDI-TOF. Table 8 shows that 82 (14.5%) of those characterized were predicted to be homozygous for RHCE*ce733G and predicted to be hr^B+^vw/-. All of these carried a normal RHD*01 allele. While 5% were predicted to be hr^B- and only 0.5% predicted to be hr^S-. It has not been well appreciated that the E- hr^S- phenotype is at least an order of magnitude rarer than the E- hr^B- phenotype. Also of note, nearly half of the hr^B- and hr^s- donors were predicted to express a partial D phenotype. There is a strong association between loss of the high prevalence antigens hr^B and/or hr^S and a partial D phenotype. In addition, individuals who are found to carry the RHCE*ce48C,733G,1006T allele may carry the RHD*DIIIa-CE(4-7)-D allele and together are associated with the r’s phenotype. This phenotype is associated with expression of a partial C antigen and in patients who do not express a normal C antigen, with risk of allo-anti-C. Another important factor derived from RHD and RHCE co-inheritance data is the finding that most RHCE*ce733G alleles are linked to a normal RHD*01 allele encoding a D+ phenotype.

The RH allele selection process

Importantly, precise RH allele selection requires genotype information for RHCE SNVs not interrogated by commonly used low-resolution, commercially-available red cell genotyping panels. The ARDP requires RHCE c.48G>C, c.340C>T, c.697C>G, c.712A>G, c.733C>G, c.1006G>T and c.1025C>T genotype results for submission of a rare donor based on hr^B- phenotype and RHCE c.48G>C, c.667G>T, c.712A>G, 818C>T and 916A>G for submission of a rare donor based on hr^S- phenotype. Since most laboratories are not performing high-resolution testing, the alleles assigned to the patient are considered the “probable alleles” to convey that there is the potential that the patient carries other variants not interrogated that may change the allele assignment and, in some cases, the predicted phenotype. It is also noteworthy that with more laboratories performing high-resolution methods like full gene or exon sequencing of RHD and/or RHCE alleles, additional RH alleles are likely to be identified and the allele assignments made based on medium resolution genotyping methods may need to be updated. A Punnett square is generated for each patient for whom RH allele selected units are requested, based on the RH alleles they carry. Though a Punnett square could be generated with all known RHD and RHCE alleles, those that are Tier 1, 2 and 3 and for which there are donors in the American Rare Donor Program are typically included. This approach could be modified based on the population and the RH characterization of rare donors.

The use of a Punnett square allows donors with the same or similar alleles to be assigned to Tiers.

Table 9 defines the tiers and how they are assigned. Notably, with RHD, zygosity plays a role as well as an appreciation that some RHD variant alleles such as RHD*DIIIa-CE(4-7)-D do not express RhD but instead express an partial C antigen. RHCE alleles “drive” allele selection since matching is typically performed due to anti-e, -e-like, -hr^B, -hr^S or -Hr antibodies. Importantly patients who carry two different RHCE alleles generally have more potentially compatible donors since the list of donors would include donors carrying the same combination of alleles as the patient (Tier 1) as well as those homozygous for either allele (Tier 2) or those carrying different alleles predicted to encode a similar phenotype.

A clinical case example of RH allele selection

The ARDP received a request for blood from a blood center outside of the US for an 18-year old female with sickle cell disease. The patient was A positive with anti-C, -e, -Wr^a and an antibody to a high-prevalence antigen in the Rh system. This antibody was found to be anti-hr^B. RHCE genotyping predicted that the patient to carry two different RHCE alleles: RHCE*ceVS.03 [also known as RHCE*ce48C,733G,1006T] and RHCE*ceVS.03 [also known as RHCE*ce733G]. RHD genotyping predicted the patient to carry two different RHD alleles: RHD*03N.01 [also known as RHD*DIIIa-CE(4-7)-D] and RHD*01. Based on this information, the patient has a predicted phenotype of D+ partial C+ E- partial e+ partial c+ V+ VS+ hr^S+ and hr^B+^vw/-. The patient’s plasma was non-reactive at PEG IgG-AGT phase with RBCs from two Tier 2 donors carrying RHD*01 (homozygous or hemizygous) and homozygous for RHCE*ce733G. Tables 10-15 illustrates the use of a Punnett Square of RHCE or RHD alleles in RH allele selection of donors based on a patient’s RH alleles, with Table 10 showing the use of a Punnett Square using RHCE common alleles for illustrative purposes only. The RHCE Punnett square in Table 13 and RHD Punnett square in Table 14 are applicable to the clinical case example. Table 16 lists the allele combinations and number of ABO/Rh compatible donors carrying RH alleles predicted to have the same or similar phenotype as the patient. This case was initially presented at ISBT in 2019 (62). A frozen RBC unit from a Tier 2 donor was shipped. The unit was stored and was reported to have been thawed at a later date and transfused to the patient without incident.

A Review of ARDP activities involving RH allele selection

A review of ARDP cases from the year 2020 involving RH allele selected donor units showed that of the 1,045 requests for rare blood, 126 (12%) requests involving 53 patients were for RH allele selected units. Nearly half (26 of 53) of these patients carried the RHCE*ce733G allele, with 11 (42%) reported to have anti-hr^B and 5 (19%) allo-anti-e or allo-anti-e-like antibody with 10 (38%) having warm reactive autoantibodies, 3 (12%) having cold reactive autoantibodies and 11 (42%) have multiple alloantibodies. At that time, the ARDP database included 1,979 donors that carried the RHCE*ce733G allele with 982 (50%) being homozygous. Of the requests for allele-selected units for patients with an RHCE*ce733G allele, 6 (8%) were unable to be filled; these 6 requests required the units to also be negative for C, E, K, S, Fy^a with three requiring Fy(b-), two requiring N- and two requiring Jk(b-) units. This analysis illustrates that the ability to fill RH allele selected unit requests is impacted not only by the number of donors with RH allele information but also by the presence of multiple alloantibodies in the patient. ABO type compatibility also plays a role in the number of donors whose units could fill such requests (65).

Challenges to RH allele selection: ambiguous phenotypes

A factor that complicates using RH allele selected donor units for Rh alloimmunized patients with partial Rh antigens is ambiguous phenotypes. These include the hr^B+^vw/-phenotype assigned to red cells carrying the RHCE*ce733G allele. This phenotype is confusing to clinicians who need to be able to interpret alloimmunization risk, to manufacturers of genotyping kits that include algorithms to assign predicted phenotypes as well as to reference laboratories asked to provide transfusion recommendations based on molecular reports. Ambiguity also lies in the alloimmunization risk for anti-D in patients carrying the RHD*DAU0 and RHD*weak D type 4.0 alleles (52). This is also true of the RHCE*ce48C allele found in more than 10% of African American blood donors (66) for which ISBT Working Party on Red Cell Immunogenetics and Blood Group Terminology (1) assigns a phenotype of e+^weak yet allo-anti-e has been reported in individuals homozygous for this allele (67). Finally, transfusion medicine professionals are trained to provide antigen negative blood products for alloimmunized patients. However, an RH allele selected donor unit may type positive for one or more of the Rh antigens for which a patient may have antibodies, yet the unit would be predicted to be compatible since both the donor and the patient express the same or similar partial antigen(s). Finally, neither hospital computer systems nor blood establishment computer systems are likely equipped to store RH allele information such that it can be used by transfusion medicine professionals in the selection of blood products. Due to the many RHCE alleles expressing partial e antigens, some with incomplete information regarding clinically relevant Rh antigen phenotype classifications, it would be beneficial for the transfusion medicine community to classify this group of antigens e^VAR; this approach would be similar to that taken with U^VAR (68) and would allow databases to use this “new” antigen to overcome limitations of the current binary status (e+ or e−) (69).

Discussion/summary

Though there are limitations to our current understanding the genetics of the RH blood group system and the implications of the many variant alleles and resulting antigens on alloimmunization risk, the benefits of using RH allele information for clinical care are clear. Multiple studies, many of which were reviewed here, have demonstrated clear benefit to the use of RHD genotyping to provide allele information that can be used to assess alloimmunization risk. The value of this methodology is most obvious in women of child-bearing age to determine who would benefit from Rh immune prophylaxis and who does not require it. Also, in patients with variant RHCE alleles, selection of RBC units from donors with the same or similar alleles holds great promise to deliver personalized transfusion medicine, especially in patient populations where chronic transfusion is associated with high rates of alloimmunization. Whereas the use of RH allele selected donor units is currently limited to patients with Rh alloantibodies, if donor centers can attract and retain donors of African descent and can identify those with variant RH alleles lacking high prevalence antigens, there would be the potential to use this approach prophylactically.

Both in reducing risk of allo-anti-D formation and in identification and selection of compatible blood products for patients who have made antibodies to high prevalence antigens, molecular methods can be instrumental in developing a personalized approach to patient care.

Acknowledgments

I thank Sandra T Nance, MS, MT(ASCP) SBB, retired, for critical review of the manuscript.

Funding: None.

Footnote

Provenance and Peer Review: This article was commissioned by the Guest Editor (Lilian Castilho) for the series “Serology and Molecular Biology of the Rh System” published in Annals of Blood. The article has undergone external peer review.

Reporting Checklist: The author has completed the Narrative Review reporting checklist. Available at https://aob.amegroups.com/article/view/10.21037/aob-22-6/rc

Conflicts of Interest: The author has completed the ICMJE uniform disclosure form (available at https://aob.amegroups.com/article/view/10.21037/aob-22-6/coif). The series “Serology and Molecular Biology of the Rh System” was commissioned by the editorial office without any funding or sponsorship. MAK reports that she is an employee of American Red Cross. She also reports support for travel to Annual Meetings (Boston and Chicago) from American Society Clinical Pathology and she is the secretary of ISBT Working Party and South Central Association of Blood Banks. The author has no other conflicts of interest to declare.

Ethical Statement: The author is accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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