In this article, we examine the latest in red blood cell genotyping techniques and applicable settings which are becoming common in transfusion medicine
Vol. 24 • Issue 9 • Page 32
Red blood cell (RBC) genotyping is becoming more commonplace in transfusion medicine and obstetrics. RBC genotyping predicts blood group antigen expression by molecular testing, mostly of peripheral blood mononuclear cell-derived DNA. This testing is based on the knowledge of what genes encode the antigens and what genetic variation; in many cases, single nucleotide polymorphisms (SNPs) are present that alter the protein product resulting in the gain, loss or modification of an antigen.1
Predicting antigen expression through DNA-based methods can be superior to or more efficient than serologic detection of the antigens. First, blood group antigens that are expressed weakly (e.g., RhD, Fyb) can be missed by serologic antigen typing depending on the reagent or method used. Second, there are antigens for which there is no commercially available antisera and poorly characterized patient-source antisera (eg., hrB, hrS, Joa, Hy, U). Third, a recent RBC transfusion or positive direct antiglobulin test (DAT) can hamper the ability to obtain an RBC phenotype even with treatments to remove the transfused cells or antibodies.2 Fourth, many blood group antigens exist in variant forms that cannot be distinguished by antisera, such that the person can be antigen positive but make an allo-antibody (e.g., e+ with anti-e).3 Lastly, RBC genotyping using array-based methods offer multi-parallel testing in which multiple samples can be interrogated for multiple analytes (often SNPs) simultaneously, resulting in efficiencies of scale. Thus, RBC genotyping has found its way into reference laboratories, donor centers and larger blood banks where it is used in both patient and donor testing.
In the setting of patient care, there are several scenarios where RBC genotyping should be considered. It has been estimated that 4 percent of hospitalized patients in the U.S. have one or more RBC antibodies. However, in patients with specific diagnoses, especially those whose treatment includes chronic transfusion, the frequency of alloantibodies is much higher.
In patients with sickle cell disease and thalassemia, for example, the estimates can reach as high as 45 percent4 and 22.6 percent,5 respectively. Patients who present with RBC antibodies have been referred to as “responders” and are considered at increased risk of additional antibodies. In these specific patient populations, or in patients in whom multiple transfusions or a chronic transfusion regimen is likely, some institutions obtain an extended RBC phenotype, either serologically or now, more commonly, via RBC genotyping using a human erythrocyte antigen (HEA) SNP panel. There are approximately 100,000 patients with sickle cell disease in the U.S.,6 and this patient population includes both sporadically and chronically transfused patients – the latter group receiving multiple units every 3-4 weeks either by simple or exchange transfusion. Surprisingly, there is no standard of care for how RBCs are matched to patients with sickle cell disease,6,7 with some institutions matching for C, c, E, e and K antigens prophylactically and others only providing antigen negative blood after alloantibodies are formed.7
There is a large amount of genetic variation in the RHD and RHCE genes in individuals of African descent. These variant alleles can encode altered or weakened antigens and can result in loss of a high prevalence antigens (eg., hrB, hrS) or gain of low prevalence antigens.8 Some show cross-reactivity.9 The complexity of variation in RH is high and continues to grow, including weak, altered and partial antigens. RH genotyping is best suited for reference laboratories where expertise and use of both commercially available genotyping kits and lab-developed tests are critical to most accurately assigning alleles and predicting phenotypes.10,11
RBC Genotyping Settings
Most recently, there has been increased awareness of the value of RBC genotyping in the setting of pregnancy.12 Rate of maternal alloimmunization to fetal RhD occurs 6.3 per 1000 live births13 and can result in hemolytic disease of the fetus and newborn (HDFN). Currently, in the pregnant woman who types RhD-negative at immediate spin (IS), alloimmunization risk is managed by the use of Rh immune globulin (RhIg). However, when a serologic weak D phenotype (RBCs type <=2 with anti-D at IS), RHD genotyping can determine if the patient has one of three variant RHD alleles, weak D types 1, 2 and 3, for which RhIg is not needed. These weak D types are most common in Caucasians, where their frequency is estimated to be 80 percent or higher in the 0.2-1 percent of individuals who show the serologic weak D phenotype.14
In the setting of the donor center, RBC genotyping can be beneficial to efficiently identify donors lacking multiple common or high prevalence antigens. The use of commercially available HEA genotyping panels that test for multiple SNPs in multiple samples simultaneously has become standard in large donor centers. In late 2014, the Immucor HEA panel PreciseType™ HEA Molecular BeadChip became the first RBC genotyping product to obtain FDA licensure as an in vitro diagnostic. It is marketed in a 96-test format suitable for donor screening and in an 8-test format suitable for patient testing.
Resolution of antigen typing discrepancies, in both patients and donors, can be aided by RBC genotyping. Weak or partial antigens in the RH blood group system, especially involving the D antigen, are well appreciated.15 Typing discrepancies involving the D antigen are frequent because:
- the serologic weak D phenotype is common in Caucasians,
- partial D antigens, where one or more epitopes are lost, are common in individuals of African descent and
- there are requirements for the use of different anti-D reagents for donor and patient testing.
However, there are weak forms of other blood group antigens, including Fyb, Jka and U and these are known to cause typing discrepancies.16 Such discrepancies can often be resolved through RBC genotyping.
Various techniques are used for RBC genotyping. These include SNP assays, Sanger sequencing of genomic DNA and cDNA. The SNP assays utilize a variety of approaches, including differentiation based on restriction digestion (e.g., PCR-RFLP) or based on sequence specific PCR (SSP-PCR), both followed by gel-based detection; on-bead extension, followed by fluorescence detection (BeadChip by Immucor, ID Core XT by Progenika); and extension fragment mass determination (HemoID by Agena). These approaches have differences in licensure (PreciseType is the only FDA-licensed product), throughput (BeadChip, IDCore XT and HemoID are multi-parallel approaches) and resolution (Sanger sequencing can identify a novel or rare mutation not interrogated by a SNP array). NextGen sequencing is showing promise in HLA typing;17 its feasibiility and utility in blood group antigens has yet to be fully demonstrated.18 Although a majority of RBC genotyping may take place in large donor centers and reference labs, it is important for hospital labs and laboratorians to familiarize themselves with the available testing and when it is appropriate.19
- Blumenfeld OO, Patnaik SK. 2004. Allelic genes of blood group antigens: a source of human mutations and cSNPs documented in the Blood Group Antigen Gene Mutation Database. Hum Mutat 23:8-16.
- Horn T, Hamilton J, Kosanke J, Hare V, Kluver W, Keller MA. Ability of Commonly Used RBC Phenotyping Pretreatments to Yield an Accurate Antigen Profile. 2014. Transfusion 54(2):165A.
- Pham BN, Peyrard T, Juszczak G, Beolet M et al. Analysis of RhCE variants among 806 individuals in France: considerations for transfusion safety, with emphasis on patients with sickle cell disease. 2011.Transfusion;51:1249-1260.
- Chou ST, Jackson T, Vege S, Smith-Whitley K, Friedman DF and CM Westhoff. 2013. Blood. 122(6):1062-1071.
- supos, T, Karageorga M, Ladis V et al. Red cell alloantibodies in patients with thalassemia. Vox Sang 1990, 58:50-55.
- Management of sickle cell disease: summary of the 2014 evidence-based report by expert panel members.JAMA 2014. 312(10):1033-1048.
- Meny, GM. Transfusion protocols for patients with sickle cell disease: working toward consensus? 2012.Immunohematol 28(1):1-2.
- Halter Hipsky, C, Lomas-Francis, Fuchisawa A, Reid ME et al. RHCE*ceCF encodes partial c and partial e but not CELO, an antigen antithetical to Crawford. 2011. Transfusion 51 25-31.
- Westhoff CM, Vege S, Halter Hipsky C, Hue-Roye K. RHCE*ceTI encodes partial c and partial e and is often in cis to RHD*DIVa. 2012. Transfusion. 2013 Apr;53(4):741-6.
- Crowley, JA, T. Horn and MA Keller. RHCE Characterization using both commercial genotyping arrays and lab developed tests. 2013 Transfusion 53(2S):172A.
- Horn, T, J. Crowley, MA Keller. Feasibility of the RHD BeadChipTM kit to identify and characterize RHD variant alleles. 2013 Transfusion 53(2S):172A.
- Sandler SG, Flegel WA, Westhoff CM, Denomme GA, Delaney M, Keller MA, Katz L, Queenan JT, Johnson ST, Vassallo R, CD Simon. It’s time to phase in RHD genotyping for patients with a serological weak D phenotype. Transfusion 55(3):680-689.
- Martin JA, Hamilton BE, Sutton PD, Ventura SJ, Menacker F, Munson ML. Births: final data for 2002. Natl Vital Stat Rep. Dec 17 2003;52(10):1-113.
- Garratty G. Do we need to be more concerned about weak D antigens? Transfusion 2005;45:1547-51.
- Horn T, Crowley J, MA Keller. RhD Typing Discrepancies resolved through RHD Genotyping: A Patient Perspective. 2014. Transfusion 54(2):29A.
- Keller, MA, Crowley, J, Horn T. Kidd Antigen Discrepancies: Genotype-predicted Phenotype versus Serologic Phenotype. International Society for Blood Transfusion Annual Congress. Seoul, Korea 2014.
- Mayor NP, Robinson J, McWhinnie AJM, Ranade S et al. HLA Typing for the Next Generation. PLoS One2015 DOI:10.1371/journal.pone.0127153
- McBean RS, Hyland CA and RL Flower. Approaches to determination of a full profile of blood group genotypes: single nucleotide variant mapping and massively parallel sequencing. Comput Struct Biotechnol J2014 11(19):147-51.
- Sandler, Keller J, Horn T, Landenberg A, MA Keller. A model for integrating molecular-based testing in transfusion services. Blood Transfusion (in press).