Goodbye to agglutination and all that?

In the first edition of their seminal text, Race and Sanger1 noted, “The great bulk of blood group knowledge has been gathered from the results of simple agglutination tests. A serum containing a known antibody is added to a saline suspension of red cells. If the cells carry the equivalent antigen they are agglutinated; if no agglutination occurs it is concluded that the cells lack the antigen.” This simple method, in various guises, underpinned the provision of blood safe for transfusion throughout the 20th century. Landsteiner discovered the blood groups with this method. Coombs, Mourant, and Race modified the method so that IgG blood group antibodies could be detected easily and opened a door to the discovery of many new blood groups. Kohler and Milstein showed the way to generate monoclonal antibodies for blood typing and now potent standardized ABO and Rh reagents are widely available for use by the agglutination method. Agglutination has been used as the endpoint for various automated methods of blood typing for mass donor testing2 and for more user-friendly blood typing procedures like the gel test.3 In the past decade, the pivotal role of agglutination in blood grouping has been somewhat eroded by the emergence of molecular methods. Once the genes encoding the major blood group–active red cell (RBC) antigens had been cloned, it was a relatively simple matter to correlate differences in DNA sequence with surface antigen expression.4 A widely used application of this knowledge has been the determination of fetal blood group in pregnancies where the fetus is at risk from Rh hemolytic disease of the newborn. Initially, this was achieved by analysis of fetal amniocyte DNA.5 More recently, this method has been superseded by analysis of fetal DNA present in the maternal circulation.6 Setting aside the ABO and Rh blood group systems, it is generally found that blood group antigens expressed on RBCs and platelets (PLTs) result from single-nucleotide substitutions in the coding sequence of the relevant blood group gene. Single-nucleotide polymorphisms are generally termed SNPs (phonetically, SNIPS). There is huge interest in developing rapid screening methods for the detection of the large numbers of SNPs in the human genome (Sachidanandam and colleagues7 report the mapping of 1.42 million SNPs in the human genome). This interest is driven by the potential for identifying patterns of SNPs that correlate with predisposition to common diseases like cancer and cardiac disease. As a result, numerous high-throughput SNP screening technologies are available and can be applied to the determination of blood group polymorphisms. In this issue of TRANSFUSION, four groups describe their experience with different SNPs detection technologies. Bugert and coworkers8 and Beiboer and coworkers9 focus on the detection of SNPs encoding antigens expressed on PLTs, whereas Hashmi and colleagues10 consider SNPs encoding RBC antigens and Denomme and van Oene11 examine SNPs relevant to both cell types. Taken together, these articles demonstrate clearly the effectiveness of this approach and establish large-scale screening of blood donations by detection of SNPs as a realistic possibility. How then will this technology impact on the world of blood banking? Provision of antigen-matched blood for individuals who are refractory to PLTs or RBCs because of the presence of an antibody or mixture of antibodies is a problem for blood banks worldwide. This is particularly true for patients with diseases like sickle cell disease or thalassemia, which necessitate regular transfusions over long periods of time. Screening blood bank donations for compatible blood when the patient has a mixture of relatively uncommon antibodies can be difficult, sometimes impossible, with agglutination because rare antisera are difficult to obtain. Molecular typing provides an alternative approach, which has been used on a small scale for several years.12 The articles in this issue provide the proof of principle for large-scale automated screening of donations making it feasible for blood centers to type all donors for a large number of SNPs and create a database that could be interrogated to find least mismatched blood for problem cases. One can envisage kits suitable for molecular typing of individual patients being used at the blood bank and electronic interrogation of the blood center database for selection of the most suitable donations available. The articles in this issue of TRANSFUSION address an aspect of blood bank practice that concerns provision of blood for the problem patient with irregular antibodies. They do not (with the exception of Denomme and van Oene who report on Rh typing) address the provision of blood for the majority of recipients of transfusion where ABO and D compatibility is the main concern. Here, the method of typing remains the simple and reliable agglutination test with potent and well-standardized monoclonal reagents. If, as seems likely within the next decade, large-scale automated screening for SNPs in all patients becomes a routine feature of the pathology laboratory (as an aid to diagnosis and to identify a patient's susceptibility to disease, thereby informing the selection of treatment regimes and consideration of the value of lifestyle changes), it might become desirable and cost-effective to add automated blood group typing by SNPs profiling to the same process. An electronic link between the pathology laboratory and blood center would allow automatic identification of the most compatible blood available. This, should it come to pass, would certainly change working practices and revolutionize the way transfusion is managed. Molecular typing for ABO and Rh antigens, however, is not a simple matter of identifying one or two SNPs.13,14 The genetic basis of both antigen systems is complex and will require the careful design of multiple reactions before a “bulletproof” molecular typing system suitable for all racial groups is achieved. In the case of ABO typing, detection of 10 to 15 SNPs is a likely requirement (M.L. Olsson, personal communication, 2005). Do the articles in this issue portend the passing of agglutination as the basis for blood typing in the wealthy countries of the world? Not in the short term, because there is more work to do regarding molecular methods for ABO and D typing before routine application can be contemplated, but in the longer term blood grouping may be swept along with the molecular revolution. Does this mean antibody screening will no longer be necessary? If the patient's genotype is matched to the donors, the possibility of antibody production in response to transfusion will be minimized, although not entirely eliminated. Molecular methods are not error-free, natural selection will not cease (mutations will continue to occur), not all blood group antigens have been cloned, and not all will be typed. There will still be problems for the reference laboratory but perhaps not so many. Hopefully, the multitransfused patient with so many blood group antibodies that compatible blood cannot be found will no longer be seen. The technology already exists to avert this latter situation as the articles from Bugert coworkers, Beiboer colleagues, Hashmi coworkers, and Denomme and van Oene in this issue of TRANSFUSION clearly demonstrate.