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Transfus Med Hemother. 2009 June; 36(3): 230–231.
Published online 2009 May 11. doi:  10.1159/000215117
PMCID: PMC2980534

Will Genotyping Replace Serology in Future Routine Blood Grouping? – Opinion 3

This is the opinion of a classical blood group serologist working in this field for 40 years. Using the tools of serology, nearly all problems in the daily transfusion routine could be solved. The upcoming PCR tests for RBC typing were a welcome help for clarification of dubious reactions some of them being mentioned in this place. In rare situations lack of difficultly available or even not existent test reagents prevented testing of particular antigens and hindered the confirmation of suspected specificities of antibodies against RBC antigens. Some of the commercially available test antibodies produced only partial agglutination patterns of the RBCs under investigation, sometimes leading to doubts in the interpretation of the results. This unwanted phenomenon was observed especially when tests for M, N, Lutheran, and Duffy b antigens were performed. With the appearance of the monoclonal antibodies some of these problems could be solved. Furthermore, over the years the number of ABO discrepancies revealing a misfit between forward and reverse grouping piled up and awaits an explanation. With the availability of DNA typing for RBC antigens in the 1990s an explanation of the phenomenon could be offered: presence of the respective ABO gene on the nucleotide level and lack of antigen expression on the RBC membrane turned out to be the cause for the discrepancies in many individuals, or the presence of variant ABO genes resulted in serologically nondetectable ABO antigens [1]. Also the acquired B phenomenon, a potential cause for ABO-incompatible transfusions [2] could easily be resolved using DNA analysis, demonstrating the lack of the B gene. The Bombay phenotype which could be diagnosed by serological means using an anti-H lectin gained final proof by genotyping. Blood samples giving mixed-field reactions in tube and slide tests or two-cell population patterns in micro-column tests could be traced back to their real genetic background: Reactions of this type were found in polytransfused patients, in recipients of bone marrow or stem cell transplants and in leukemic patients loosing partially their ABO antigens. In some individuals showing two-cell populations in tests for Rh antigens, DNA sequencing revealed deletions at the RH gene locus developing on the basis of hematological disorders [3]. In the rare chimeras where the minor cell population can reflect the true genotype DNA analysis is a perfect tool for safe diagnosis. Genotyping of different blood group antigens poses a great step forward in patients with positive DAT, in polytransfused patients, and in blood samples showing polyagglutination where serological procedures often fail to give correct results. As far as problems in ABO typing are concerned, clinical transfusion problems regarding the selection of compatible blood are managed by circumventing the present iso-antibodies. But as long as guidelines require reverse grouping (what I strongly recommend), DNA typing for ABO grouping needs serologic antibody test for confirmation. With the exception of rare patients presenting antibodies against high-frequency antigens, or patients with antibody mixtures, compatible cross-match-negative RBCs could be provided for the vast majority of the recipients. Serologic methods can be handled easily, they are quick and simple, and they cause mostly tolerable costs. Costs explode when donors have to be tested serologically on a large scale for certain antigens to find suitable blood units for patients with problematic antibodies.

Particularly for smaller transfusion laboratories, which cannot afford DNA technology, the antigen-antibody reaction-based serological methods will be useful tools also in future, especially in urgent pretransfusion testing for unit selection and compatibility testing.

High-throughput genotyping will become quicker and cheaper offering the chance to test the donor population for the whole antigen profile of the most important blood group systems and to facilitate the provision of suitable blood units for patients with multiple antibodies. Further large-scale donor genotyping for the absence of high-frequency antigens and the collection and cryopreservation of the respective units will provide a broader basis for the collaboration of national and international blood centers in covering the demands of recipients with antibodies against these high-frequency antigens. The migration and mixture of populations will change the antigen composition of future donors and recipients and the profile of the appearing antibodies. The annual statistics of the antibodies found in recipients and pregnant women help determine what blood group markers should be tested primarily. The extension to all major blood group systems will be rational.

Genotyping of the whole RBC antigen profile of potential blood recipients makes sense only for individuals requiring chronic transfusion support, to minimize alloimmunization by selection of antigen-negative RBCs. For patients sporadically being transfused the risk of immunization seems to be bearable even in view of the ongoing efforts to screen the donor population for the antigens of the most important RBC systems.

Screening of phenotypically RhD-positive and weak D-positive pregnant women on the DNA level would indicate the women prone to produce allo-anti-D after exposure by an RhD-positive fetus. As a consequence the serologically RhD-positive individuals belonging to category DVII should receive anti-D prophylaxis, whereas the prophylaxis can be omitted in all weak D types except the types 4,2, 15, and 38. This is an economically interesting issue because independent of the number of pregnancies the test has to be done only once in lifetime.

The idea of matching the antigen profiles of recipients and donors to prevent exposure to foreign antigens has practical limitations. Regarding the clinically most important blood group systems ABO, Rh, Kell, Kidd and Duffy, the chance to have the individually required appropriate number of units on the shelf is minimal. The ulterior motive for this strategy was to skip the serological cross-match and to replace it by electronic matching. Taking into account all the possible incompatibilities with blood group markers not tested for, even when DNA technology will be implemented on a broader basis (private antigens), this approach remains a form of the Russian roulette. The author would refuse to receive a RBC unit without a serologic cross-match!

One should never forget that the biological playground for blood group incompatibilities is the protein and sugar level, and not the nucleotide level. The question may be raised whether genotyping is the only reliable method to test for antigens or whether there could occur pitfalls in the technology. Whatever technique is used, we have to be aware of possible methodology-dependent faults in the procedure. In serology antibodies are used for antigen typing. These antibodies may not always be of optimal quality, or an appropriate antibody may not bind to the antigen because of antigenic variants. In genotyping the primer may not bind to the target nucleotide sequence due to mutations that do not necessarily alter the functionality of the resulting antigen. Amplification does not occur, leading to the conclusion that the respective gene is missing! In contrast the primer binds to the sequence searched for indicating the presence of the gene, but the respective antigen is not expressed due to a mutation in the promoter of the gene, rendering the gene inactive. These are two examples that under certain circumstances serology and genotyping do not always give identical typing results. When testing a specific individual, these complexities should be kept in mind. The problem has no major influence on large-scale screening of blood donors because to the opinion of the author the antigens tested by genotyping should be confirmed by serology before the selected blood unit is released for the prospective recipient. A positive side effect of full antigen genotyping is the possibility to find suitably matched donors for the production of cell panels for antibody identification.

Surveillance and clinical guidance of pregnancies threatened by maternal antibodies will in near future surely be done by fetal genotyping from maternal plasma. So far typing of the nominated father was an unsafe method for prediction of antigen positivity of the fetus – be it heterozygosity or the ‘pater semper incertus’ consideration. The latter situation is a typical example for the future coexistence of genotyping and serology as antibodies can only be documented by serology.

Blood grouping in the context of transfusion medicine means testing for antigens and antibodies! Blood group-specific antigen determinants are proteins or sugars, and their reaction partners, the immunoglobulins, are proteins containing sugarSso, why not testing for antibodies on the nucleotide level? It has been estimated that an IgG molecule can be produced in at least more than 2.6 × 106 different ways [4], which of course does not imply that we would have to perform the same number of tests. In transfusion medicine we look for antibody specificities, and the specificity is predominantly defined by the sequence of amino acids forming the tips of the six polypeptide loops of the antigen binding site of the antibody molecule. The specific arrangement of the amino acids is the result of the exposure to an antigen. The RhD antigen for example is composed of at least 30 different epitopes. Consequently anti-D antibodies, despite showing the same specificity, differ from each other. Therefore sequence-specific typing with primers or sequencing of all antibody specificities directed to blood group markers would be a never-ending task.

Genotyping of blood group antigens in the above mentioned situations is an enormous progress and will have a great impact on optimization of patient care in transfusion medicine and mother-child blood group incompatibilities.


1. Wagner FF, Blasczyk R, Seltsam A. Nondeletional ABO*O alleles frequently cause blood donor typing problems. Transfusion. 2005;45:1331–1334. [PubMed]
2. Garratty G, Arndt P, Co A, Rodberg K, Furmanski M. Fatal hemolytic transfusion reaction resulting from ABO mistyping of a patient with acquired B antigen detectable only by some monoclonal anti-B reagents. Transfusion. 1996;36:351–357. [PubMed]
3. Körmöczi GF, Dauber EM, Haas OA, Legler TJ, Clausen FB, Fritsch G, Raderer M, Buchta C, Petzer AL, Schönitzer D, Mayr WR, Gassner C. Mosaicism due to myeloid lineage restricted loss of heterozygosity as cause of spontaneous Rh phenotype splitting. Blood. 2007;110:2148–2157. [PubMed]
4. Lefranc MP, Lefranc G. The Immunoglobulin Facts Book. London: Academic Press; 2001.

Articles from Transfusion Medicine and Hemotherapy are provided here courtesy of Karger Publishers