Flegel WA EDITORIAL

ABO genotyping: the quest for clinical applications

Willy A. Flegel
Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MD, United States of America

ABO was the first blood group system to be described and applied to clinical practice. While ABO is also the first blood group system defined at the gene level1, the earliest clinical genotyping for any blood group system was published on RHD2. Today, typical clinical applications for blood group genotyping are performed for RH, FY and other protein based blood group systems, yet rarely for ABO3. A seminal publication heralded ABO genotyping for clinical application in 19954, after a similar, but technically more demanding, method had been published the year before5. The methods for ABO4 and RH 6 based on modular PCR with sequence specific priming (PCR-SSP) brought about the first commercial blood typing kits7,8, which are still available today. ABO is covered by only one9 of the two10,11 commercial blood group genotyping platforms, compared recently in a case report12, Table S4. ABO is not addressed by a laboratory developed molecular test13 nor by the largest published study14,15 on donor screening, both of which are based on the same technique for blood group genotyping13. A second laboratory developed molecular test16 with arguably the highest throughput available today17 also does not cover ABO. Why isn't ABO genotyping more widely used in the clinical setting? ABO is one of the most widely used clinical tests for any phenotype and among the least expensive. In most hospitals worldwide, laboratory personnel can perform the ABO test within ten minutes, on a 24/7 basis, when covering traumas or other surgical emergencies. The principle of antibody based testing for ABO remains unchanged from the original test method of agglutination in tubes developed over 110 years ago. It is one of the most reliable tests for clinically relevant phenotypes with error rates in blood donors as low as 1 in 500,000 or less. Has ABO genotyping become the victim of its own ABO serology success? Even among advocates of molecular immunohaematology, some researchers anticipate

that it will take 10 years or more to bring ABO genotyping into routine blood center use. And they may well be right, perhaps as a consequence of a self fulfilling prophecy. In this issue, Liu and colleagues18 from Wuhan, Hubei Province in China describe a quantitative real time PCR method (qRT-PCR) for ABO allele detection. This approach may become a specialised clinical application for ABO genotyping to monitor haematopoietic progenitor cell (HPC) engraftment after ABO mismatched transplantation. ABO gene expression may be an early marker for engraftment of the erythroid cell lineage, including red blood cells (RBCs). Five nucleotide positions in the ABO gene, which are known to distinguish common ABO alleles, were used by Liu et al.18 This technical approach, similar to published test kits4,8 but novel in applying a qRT-PCR platform, would allow the sensitive detection of ABO alleles occurring in donor rather than recipient cells after HPC transplantation. The ABO qRT-PCR adds to our toolset and was shown to be specific and sensitive for the target nucleotide polymorphisms, when in vitro mixes of various ABO alleles were assayed. As expected for qRT-PCR, the molecular assay should easily exceed any sensitivity that could be achieved by serology alone. While qRT-PCR for ABO alleles is a promising technique, its clinical efficacy in monitoring RBC engraftment after HPC transplantation has to be demonstrated in subsequent studies involving clinical samples. While a very sensitive method to detect RBC engraftment may not be needed, the ABO qRT-PCR would match the other molecular methods currently used for chimerism detection following HPC transplantation. Detection of ABO mRNA indicates the presence of cells with transcription of the ABO gene18. The detection of mRNA transcribed from a given ABO allele, carried by the donor but lacking in the recipient, proves unequivocally the engraftment of cells from the donor. However, the ABO gene
Blood Transfus 2013; 11: 6-9 DOI 10.2450/2012.0250-12

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Molecular typing for the ABO gene

expression may not be strictly limited to RBC or the erythroid cell lineage. It is unclear which haematopoietic cells express the ABO genes and, if they do, in which temporal order following HPC engraftment. Most evidence has been gathered from studying cell lines in vitro and applies to blood group proteins, but not to ABO19,20. It is encouraging that non-erythroid cell lineages, in particular during their early differentiation steps, showed no ABO gene expression. For example, mesenchymal stem cells express neither the ABO gene nor the genes of other major blood group systems21. Genes with high polymorphism and an expression pattern that is strictly limited to the erythroid lineage are good candidates for monitoring erythroid engraftment. Most blood group system do not fit this pattern22, but ABO may. Hence, the method devised by Liu et al.18 is a welcome tool for studying differentiation and expression patterns in vivo during the engraftment process following HPC transplantation. Their method may allow establishing the temporal pattern of gene expression for the donor's ABO allele(s) as compared to other donor specific alleles indicative of the various haematopoietic cells of erythroid and non-erythroid cell lineages. The specificity of ABO genotyping devised by Liu et al.18 is likely to exceed 99% in any population tested so far, which is sufficient for the proposed clinical application. Recipient and donor pairs in whom the assay is not specific will be recognised, when a sample of the recipient before transplantation and of the donor HPC unit are tested as controls. For general routine, however, large scale ABO genotyping, which approaches a specificity exceeding only 99%, is unacceptable. While the specificity of RBC genotyping kits and platforms exceeds that of serology for most blood groups14-16, this is not true for ABO today9,23. New ABO alleles identified during the BloodGen project9 and ongoing since24 indicate that a comprehensive cataloguing of ABO alleles, a prerequisite for clinically reliable ABO genotyping, is still incomplete23. What defines a sufficiently complete catalogue of ABO alleles? The answer lies in the specificity that one is aiming for. An error rate, similar to the rate of serology, such as 1 in 1,000,000, represents a specificity of 99.9999%, which approximates

the Six Sigma strategy. To establish a catalogue of ABO alleles sufficient for a six sigma approach to ABO genotyping requires testing of 3,285,000 individuals in the relevant population (assuming a 95% confidence interval for rare events calculated by the Poisson distribution). These figures are within the reach of large blood donor services. How can we establish a sufficiently complete tally of ABO alleles? The premier and most conceivable way is ABO genotyping in large blood donor cohorts and comparison of the molecular results to the known donor ABO phenotype. Discrepancies will be immediately recognised and subsequently resolved by molecular studies. Ethnically mixed countries may represent the best donor populations to perform these studies. Multinational biobanks are suitable sources and should be utilised; they are unlikely to provide for millions of distinct samples. The presence of a gene or its mRNA is not directly indicative of the expression of its associated structure, such as a sugar or protein, a well known fact in the field of genotyping and phenotype prediction. This fact will need to be considered when attempting to replace ABO serology by molecular methods, not at all an academic exercise. Once large scale screens for ABO alleles are attempted, which molecular mechanism should we expect to find? Three groups of molecular mechanisms are influencing the gene expression: (i) epigenetics: (ii) epistasis; and (iii) molecular genetics. Blood group antigens in general and the ABO antigens in particular are prime examples for researching such mechanisms in the era of genomics25 and for utilising the knowledge in clinical applications. DNA methylation, as one of the epigenetic mechanisms, has been shown to occur in the ABO gene: hypomethylation of CpG islands in the ABO gene promoter was associated with the expression of the ABO antigens in cell lines26. Second, epistasis, the modification of a phenotype of one gene by another gene, has been known in ABO since 195227, when Bhende et al.28 showed in 2 patients and 1 donor that the ABO antigens were not expressed, because another gene product was lacking. This other gene became known as H transferase29 along with its extensive allele polymorphism29-32. Third, molecular genetics mechanisms explain the vast majority of blood group antigens, caused by genetic

Blood Transfus 2013; 11: 6-9 DOI 10.2450/2012.0250-12 7

Flegel WA

variation in exons or in other parts of the gene. In fact, the single nucleotide deletion in exon 6 of the ABO gene resulting in the lack of A and B antigen expression and the phenotype blood group O, was the first example of a genetic variant shown in any blood group gene1. Today, several hundred examples of genetic variations in exons are known to affect gene expression among the 33 blood group systems. Genetic variations in gene segments other than the exons, such as the promoter, 5' and 3' untranslated regions and the introns, are observed less commonly, yet are equally important. Current examples are a tissue (erythroid cell)-specific factor binding to the ABO promoter and an enhancer protein binding ABO intron 1, which control the expression of the ABO gene33. A better understanding of donors and their blood components by blood group genotyping will facilitate "precision medicine"34. We cannot know, what we don't know, until the molecular evidence is fully explored. The enormous practical clinical relevance of ABO serology is proven, and we should not continue to forgo large scale in-depth exploration of the ABO blood group system at the molecular level. Molecular immunohaematology, including ABO genotyping, is not just another technology looking for an application: matching patients and blood units by genotype, which we dubbed "dry matching"34, is known to benefit some patients. Dry matching may not be affordable for all patients, but cannot hurt any patient with the current safeguards in place. Many clinical applications that could be implemented using serology, but are not, will fall into place once blood group genotyping becomes standard. For example, platelets from donors of the A2 subgroup, easy to identify by serologic and molecular methods alike, do not carry the A antigen35, making these components fully blood group O compatible36. They also lack anti-A, a useful feature in ABO incompatible stem cell transplantation36. This arguably minor, yet definitive improvement is unlikely to be featured, or even considered, in cost efficiency analyses. There are likely many similar small incremental improvements that in their sum add up to a substantial benefit for patient care34. Transfusion medicine specialists should explore ABO genotyping for research 25 and particularly

routine applications9. There may be more gain in benefits to our patients than currently appreciated. Applying molecular immunohaematology widely in clinical settings, including ABO genotyping, has been shown to improve patient care to a level that cannot be achieved by any available serology alone.

Acknowledgement
The author thanks Harvey G. Klein for discussing and reviewing the manuscript, Gregory A. Denomme for sharing unpublished data, and Kshitij Srivastava for researching references and discussing the molecular gene expression mechanism; and acknowledges Sherry L. Sheldon and Elizabeth J. Furlong for English editing. This work was supported by the Intramural Research Program of the NIH Clinical Center.

Statement of disclaimer
The views expressed do not necessarily represent the view of the National Institutes of Health, the Department of Health and Human Services, or the U.S. Federal Government. The Author declares no competing interests relevant to this article.

References
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Arrived: 31 October 2012 - Revision accepted: 14 November 2012 Correspondence: Willy A. Flegel National Institutes of Health, Clinical Center Department of Transfusion Medicine Building 10, Room 1C711 (MSC-1184) 10 Center Drive Bethesda, MD 20892-1184, USA e-mail: bill.egel@nih.gov

Blood Transfus 2013; 11: 6-9 DOI 10.2450/2012.0250-12 9