Red cell genotyping: real world use

In this issue of Transfusion Medicine, Hui et al describe the experiences at two London trusts (ICHNT, LNWH) of obtaining an extended red cell antigen profile by genotype for patients with sickle cell disease (SCD), with a focus on RH variants and associated alloimmunization.¹ Overall, approximately half of the patients had extended red cell genotypes performed and 20% had RH variants. A small proportion of these patients (10%) had antibodies associated with the RH variant identified. Their work highlights several questions for hematologists and transfusion medicine specialists who care for patients with SCD. First, should all patients with SCD have a red cell antigen genotype, and how comprehensive does it need to be? Second, how should the red cell antigen genotype be used to better inform transfusion support and donor choice for alloimmunized and non-alloimmunized patients with RH variants?

In the United Kingdom (UK), a centralized transfusion service and universal health care allow for more uniform testing and a single laboratory database. Despite this, Hui et al found that only 71% of patients at ICHNT (372 of 482) and 61% of patients at LNWH (211 of 346) had either genotypic or serologic red cell antigen profiles in accordance with British Society of Haematology (BSH) guidelines.² Red cell antigen genotyping was the predominant method used: 64% (311 of 481) and 52% (181 of 346) of patients in the two trusts, respectively. Most but not all patients had additional genotyping assays to identify RH variants. Pediatric patients were less likely than adults to have had a red cell antigen genotype or serologic phenotype (85% vs. 65% in ICHNT and 64% vs. 55% in LNWH), even though children have the potential to receive more blood transfusions over their lifetimes.

Both the BSH² and the American Society of Hematology (ASH)³ guidelines for transfusion of individuals with SCD recommend that all patients have red cell antigen typing performed at initial presentation. Having this information at baseline is needed to provide Rh (C, E or C, c, E, e) and K-matched transfusions. ASH guidelines suggest than an extended red cell antigen profile be obtained for all patients with SCD at the earliest opportunity, and that genotyping is preferred over serologic phenotyping as it provides additional antigen information and increased accuracy for C and Fy^b antigens. The extended antigen profile also guides antibody evaluation and donor selection when patients are at high risk of a hemolytic transfusion reaction (HTR). Genotyping platforms may test over 30 red cell antigens, including many for which no serologic testing is available (ie. Do^a, Do^b, U),⁴ but immunization against which can be associated with an HTR. Knowledge of the antigens that a transfused individual lacks can facilitate antibody identification and guide donor red cell selection for subsequent transfusion.

In the United States (US), patient insurance coverage varies considerably, provider practices are non-uniform, and hospitals typically have contracts with one or more donor blood centers and immunogenomic reference laboratories. One practical barrier in the US is the frequent need for insurance pre-authorization for red cell antigen genotyping. While the Human Erythrocyte Antigen (HEA) assay is a Food and Drug Association (FDA) approved test in the US for an extended red cell antigen profile, the variants assayed by higher resolution RH genotyping is highly dependent on reference laboratory. RHD and RHCE arrays (Immucor) test for the majority of common variants found in individuals of African background, but laboratory-developed tests supplement the RH arrays to identify several frequent single nucleotide polymorphisms (SNPs) that result in partial antigen expression and thus have clinical significance.⁵ This includes the RHD c.1136T change that identifies DAU variants in RHD and the RHCE c.254G change associated with a partial e antigen, which have an allele frequency of 20% and 5%, respectively, among individuals of African descent.⁶

Clinical guidelines for transfusion support for SCD from both the BSH² and ASH³ recommend prophylactic Rh (C, E or C/c, E/e) and K matching, which reduces but does not eliminate Rh alloimmunization. The persistence of Rh alloantibody formation despite serologic Rh matching results from a high prevalence of RH variants in both patients and donors that result in partial antigen expression (missing an epitope(s) of RhD or RhCE proteins), loss of high-prevalence Rh antigens (hr^B, hr^S), or expression of Rh antigens with novel epitopes (ie. V/VS, Go^a).^5-8 A high index of suspicion should be maintained for the presence of RH variants in patients who have antibodies to Rh antigens despite exclusively receiving Rh- and K-matched red cell transfusions. These patients should have RH genotyping performed to inform future transfusions. In our experience, Rh antibodies also form in patients who have conventional alleles corresponding to the Rh antibody formed, and we suspect exposure to donor cells expressing variant Rh and subsequent anti-Rh immunization.^5,6

There are a few scenarios for which prophylactic antigen matching based on the RH genotype can prevent alloimmunization and associated HTRs. Patients identified by genotype with the hybrid RHD*DIIIa-CE(4-7)-D or RHCE*CeRN alleles, which encode partial C antigen and do not have a conventional RHCE*Ce or *CE allele, should be transfused with red cells lacking C antigen to prevent allo-anti-C development.^3,9 Increasing experience also suggests providing prophylactic RhD-negative red cells to RhD+ patients who have RHD variants and exclusive expression of partial D antigen. Examples of “at risk” RHD alleles are DAU3, DAU4, DAU5, and DOL.

Not all patients with RH variants will form alloantibodies and not all alloantibodies associated with RH variants will cause clinically significant HTRs.⁵ The number of red cell exposures prior to antibody formation varies considerably and may reach hundreds of antigen-positive red cell units before a patient with the corresponding antigen variant becomes immunized. The authors note that 4 of 8 patients with RH variants and the corresponding Rh antibody received many transfusions with antigen-positive red cells but had no clinical or laboratory evidence of hemolysis, suggesting that these Rh antibodies are not uniformly associated with poor transfusion outcome.¹ However, this was a small study with a limited number of patients, and each of the 4 patients had unique RH genotypes and transfusion strategies. Three of the patients had partial e, two of whom had an anti-e antibody identified and one who had a pan-reactive antibody but no antigen specificity. One of the patients who formed anti-e was subsequently transfused with multiple e+ red cells without any clinical event. The other patient with anti-e was also re-exposed to e+ red cells but treated concomitantly with intravenous immunoglobulin (IVIG) and steroids to prevent HTRs. The fourth patient reported had formed anti-D and anti-C but her genotype showed partial C only. She was subsequently transfused only D- and C- red cells after the antibodies were detected. We caution that the risks associated with transfusion of antigen-positive red cells to immunized patients with the corresponding partial Rh protein require further study with a significantly larger cohort. Individual outcomes are likely highly dependent on the patient’s RH genotype and which Rh epitopes their red cells lack.

For patients with RH variants associated with partial Rh antigen expression and who form the corresponding antibody, we recommend providing antigen-negative units if possible to prevent recrudescence of the antibody and potential HTR. Finding compatible red cells lacking D, C, or E for those immunized against D, C or E antigens is straightforward. For patients with anti-e who are E+, transfusion with E+ e− red cells is appropriate. However, for patients with anti-e who are E−, exposing them to E+ e− red cells carries significant risk for anti-E development and associated hemolysis. E− e+ red cells that are RH genotype-matched would be the ideal choice, but this is available only in very limited circumstances. If the anti-e is still demonstrable in the patient’s plasma and transfusion cannot be avoided, one should consider concomitant treatment with immunosuppressive therapy (steroids, IVIG), particularly if the patient had clinical or laboratory evidence of red cell hemolysis at the time of anti-e formation. At our institution, we have several E− patients with partial e who formed anti-e, and were subsequently re-exposed with e+ red cells once the antibody was no longer demonstrating and did not show evidence of poor transfusion outcome.

As the number of patients and donors for whom RH genotyping has been performed grows, additional insight will be gained for the specific RH variants that pose the greatest risk for alloimmunization and poor transfusion outcomes. This will better inform how to incorporate patient and donor RH genotypes in red cell matching protocols to prevent alloimmunization due to RH genetic diversity. As new technologies emerge to RH genotype in a high-throughput and cost-effective manner, matching patients and donors by RH alleles will have the potential to further mitigate or eliminate Rh alloimmunization.