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Traditional serological methods, which have been used for decades to evaluate the human erythrocyte antigen (HEA) composition of recipient and donor specimens, have some serious limitations. Specific reagent antisera are not available for all clinically relevant antigens (eg, V antigen). Reagent antisera are expensive, and serological testing is labor intensive. The results of serological testing are subjective and semiquantitative (eg, microscopic, weak, 1+, 2+, 3+, 4+), and may vary from one technologist to another. Further, in many clinical situations, serological testing may be difficult or impossible.
Recent developments in nucleic acid–based testing (molecular diagnostics) have made it possible to genotype HEA in the clinical laboratory. Most allelic variations occur due to single nucleotide polymorphisms (SNPs), which can be detected and from which the phenotypes can be predicted. HEA genotyping offers several technical and clinical advantages compared with serological testing. The Immucor PreciseType HEA Test is the first and currently the only platform for clinical testing approved by the United States Food and Drug Administration (FDA), to our knowledge.
Testing for human erythrocyte antigens (HEAs) has been an important part of pretransfusion testing for decades. Traditionally, this testing has been performed on patient or donor unit red blood cells (RBCs), using reagent antisera with defined antigenic specificity and an agglutination end point. However, in many situations, this type of serological testing has severe limitations. Recent developments in molecular diagnostics testing, which have overcome the limitations of serological testing, enable laboratories to detect the coding sequence at specific sites, from which the antigen status can be deduced. At a molecular level, antigenic variations are based on DNA sequence variations in the genes that code for the HEA, such as single nucleotide polymorphisms (SNPs), insertions, or deletions. For example, a single nucleotide change in codon 226 of the RHCE gene (676G>C) is the basis of the E/e alleles (proline vs alanine in amino acid position 226).
There are many limitations of serological testing for HEAs.1 For instance, antisera with antigen specificity are required. Also, for many clinically relevant HEAs, there are no commercially available antisera (eg, V antigen). If one must test for multiple antigens, the cost of the specific antisera can be substantial. Serological testing is labor intensive and requires an experienced technologist to obtain consistent results with the subjective end point of agglutination testing. Figure 1 summarizes some of the limitations of serological testing for HEAs.
As a result of the limitations discussed earlier herein, there are many clinical situations in which HEA genotyping can provide essential information that is difficult or impossible to obtain with serological methods for HEA testing.2,3 Patients who have recently or frequently received a transfusion (eg, patients with sickle cell disease or thalassemia) have a mixture of RBC phenotypes, but HEA genotyping uses the white blood cells (WBCs) of the recipient as a DNA source, so the “foreign” RBCs do not interfere with the genotyping. Genotyping makes this kind of testing practical for a wider range of variants, including rarer variants that are usually not detectible with routine serological reagents. Patients with a positive direct antiglobulin test (DAT) require extensive processing in order to perform serological HEA testing. A positive DAT result does not interfere with the HEA genotyping test result, which can be helpful in working up patients with warm autoantibody (eg, those who have autoimmune hemolytic anemia). HEA genotyping may also facilitate working up patients with warm antibodies of unknown specificity, antibodies to high frequency antigens, and patients with multiple antibodies. HEA genotyping provides information to allow detailed genetic matching of donor and recipient, which will minimize alloimmunization and allow blood collection centers to identify and bank rare types of donor blood. It is even possible to genotype certain fetal antigens, such as RHD, using cell free DNA obtained from circulating maternal blood. The clinical indications for HEA genotyping are summarized in Figure 2.
Although initial startup costs are relatively high, the detailed and expanded phenotypes obtained by HEA genotyping are relatively inexpensive on a cost-per-antigen basis. The advantages of HEA genotyping are summarized in Figure 3.
The HEA genotyping platform in use on our laboratory is the Immucor PreciseType (Immunocor, Inc) molecular bead chip test.4,5 Other platforms have been described in the literature,2,6 but, to our knowledge, the Immucor PreciseType HEA Test is the first and currently the only platform for clinical testing approved by the United States Food and Drug Administration (FDA). The procedure starts with extraction of DNA from ethylenediaminetetraacetic acid (EDTA)–collected whole blood, using the QIAcube (Qiagen, Inc). The DNA segments of interest (which contain the sequence variations that are the basis for the phenotypic variations) are amplified by a multiplexed polymerase chain reaction (PCR). The resulting PCR product is treated to remove residual primers and deoxynucleotide triphosphates and to generate single-stranded DNA. The amplified, single-stranded DNA then anneals with oligonucleotide allele-specific probes that are attached to microscopic beads (each approximately 3.2 microns in diameter). Those beads have been dispersed onto a chip containing approximately 4000 wells; each well is large enough to accommodate only 1 bead. The bead chips come in 8-chip and 96-chip formats. The beads have a characteristic fluorescent signature specific for each allele-specific probe. The location of the beads (with their attached allele-specific probes) on the chip is documented at the manufacturing site. When the amplified DNA is perfectly matched to the probe, it undergoes elongation and incorporates a fluorescently labeled nucleotide (a process called elongation mediated multiplexed analysis of polymorphisms). Only perfectly matched DNA segments will elongate and incorporate the fluorescent label. The bead fluorescence profile is captured by a fluorescence microscope and analyzed by the BioArray Solutions Information System (BASIS; Immunocor, Inc.), which translates the fluorescence profile into genotype and phenotype.
As with all nucleic amplification procedures, it is important to separate preamplification steps from postamplification steps to avoid amplicon contamination in the preamplification steps. This separation of steps is usually accomplished by performing preamplification and postamplification in separate rooms and maintaining strict workflow controls (ie, once workers have entered the postamplification areas, they do not return to the preamplification areas without putting on new, clean personal protective equipment).
The antigen coverage for the Immucor PreciseType is shown in Figure 4. The coverage includes 11 blood groups and 24 RBC polymorphisms associated with 38 antigens and phenotypic variants in a single test. The test also detects the mutation leading to the production of sickle hemoglobin (Hb S).
There are certain potential pitfalls with HEA genotyping. For instance, HEA genotyping requires technologists to use molecular laboratory techniques and to have the ability to handle microliter volumes of reagents: these are skills that traditionally trained blood-bank staff members may not possess. However, these skills are relatively easy to learn.
Also, there may be unknown polymorphisms in the coding region that alter the protein structure and antigenicity. Only known mutations for which the appropriate primer pairs are included in the amplification mix will be detected. In addition, there may be polymorphisms in the promotor or enhancer regions that prevent gene expression, despite the presence of the correct coding gene sequence. Epigenetic or posttranslational alterations in the epitope are not detected by HEA genotyping. Despite these limitations, HEA offers an effective way to overcome many of the shortcomings of traditional serological methods of antigen typing.
A specimen from a patient with sickle-cell disease who required transfusion yielded positive results via antibody screening. Further serological testing revealed anti-e in the plasma of the patient. However, the RBCs of the patient tested e-antigen positive. HEA genotyping confirmed the e-antigen genotype but also detected the presence of DNA sequence variants for the presence of V and Vs expression. Expression of V and Vs is known to alter the expression of a normal e-antigen gene in such a way that the individual may recognize a normally expressed e-antigen (that he/she may receive in donor blood) as foreign; subsequently, his/her body may make an anti-e-like antibody, despite having a serologically normal e-antigen.7 Coincidentally, this patient had serological antigen typing results of Fya(+) and Fyb(-). HEA genotyping revealed a genotype of Fya(+) and Fyb(+). However the genotyping also revealed a GATA box mutation in the Fyb gene that is associated with absent expression of Fyb on red blood cells but not other tissues, despite a normal coding gene for Fyb. Hence, people with this genotype do not require Fyb(-) blood.8
In another case, a woman presented with a delayed hemolytic transfusion reaction. A panreactive antibody was present in her plasma. HEA genotyping revealed low signal strength, indicative of absent DNA template, in 3 different sequences related to the glycophorin B (GYPB) gene. This genotype corresponds to the S(-), s(-), U(-) phenotype.9 This information is helpful to the blood supplier in finding appropriate units for this patient.
As HEA genotyping technology becomes more widely deployed, it is likely that genotyping of all donor units and recipients will become routine, allowing extensive antigen matching of donors and recipients. The feasibility of this approach has been proven at several medical centers.10,11 The genotyping panels will offer expanded antigen coverage and include other rare variants. Some centers already include variants of the D antigen (eg, partial D). As the genotyping expands, transfusion services will require more extensive data-management systems to cope with increasingly large amounts of data.12
SNP analysis of genomic DNA provides extended RBC phenotyping. HEA genotyping overcomes several shortcomings of traditional serological testing and may lead to routine, extensive antigen matching of donor and recipient, thereby reducing the incidence of alloimmunization.
I thank Neil Bangs, MS, MT(ASCP)SBB, for many helpful discussions and for performing a critical reading of this manuscript.