1.0 Introduction
Serological blood grouping has been used successfully for decades to ensure safety in blood transfusion. However blood incompatibility continues to be a significant challenge in transfusion medicine reflecting the inherent limitations of the hemagglutination based serological blood typing. Some of the limitations of serological blood grouping include weak reactivity of some antibodies that are clinically significant; weak expression of some red cell antigens; the subjective nature of the available tests and lack of universal methods of detecting and identifying antibodies. In addition there are several issues related to the reagents used which include the wide variability of the available reagents, the different reactivity of monoclonal antibodies compared polyclonal antibodies and the lack of reagent grade antibodies. These technological limitations of the traditional blood grouping have in some cases proved dangerous especially where a foetus is Rhesus positive and the mother is negative or in patients that need multiple blood transfusion (Epstein, et al., 2006).
Serological blood typing simply identifies the major blood group antigens on the surface of red blood cells (RBCs) to determine if the blood group is A, B, AB or O and if it is Rhesus (RHD) positive or negative. However there are other blood types, bringing the total discovered blood types to 29, which are not clinically significant for a single transfusion. However in patients requiring multiple transfusions, for example those with haemophilia or leukaemia or sickle cell anaemia, alloimmunisation (dangerous immune reaction against the donated blood cells) may develop over time because of a mismatch of the less significant blood cells antigens/markers. In the fetal blood typing the complication often arises because of the heterogenicity of the Rhesus factor, which will be covered in another section of the paper, which may not be detected by the traditional blood typing (Foundation for Genomics and Population Health, 2006).
The limitation and the resulting dangers of the serological blood typing have resulted in the long search for more accurate typing techniques. The advancement in medical genetics including the human genome project has provided an excellent opportunity to resolve some of these limitations. With the determination of the molecular basis of the clinically significant blood group polymorphisms and the cloning of almost all genes associated to blood groups it has become possible to determine a person’s blood group from their DNA with a high level of accuracy (Daniels & Bromilow, 2010). DNA based techniques have attracted a lot of attention due to their potential to advance the blood typing technology. These technologies are in use the USA and more in Europe to confirm RHD gene, to manage availability and use of rare blood groups, to resolve RHD and AVO discrepancies, to verify the true genotype when an antigen is weakly expressed, to identify RHD variants that could be at risk for anti-D alloimmmunization, to genotype multiple transfused patients, to screen for antigen negative and rare blood types and many more applications (Epstein, et al., 2006). The overwhelming attention to the DNA based typing has contributed to the growing number of test kits and thus there have been attempts to standardize and validate molecular blood typing techniques for large scale use and to prove their superiority over the serological typing. One such initiative is the BloodGen, an international consortium of researchers which was established in 2000 (Foundation for Genomics and Population Health, 2006).
1.1 Research objectives
The objective of this research paper is to explore molecular blood typing focusing on the application, the underlying principle, the advantages and the limitation.
2.0 Overview of the principles of molecular blood typing
Given the wide variety of the molecular typing techniques available, which include and not limited to, multiplex PCR, allele specific PCR, PCR-RFLP (restriction fragment length polymorphisms) real-time PCR polysequenceing, it would be impossible to describe all the procedures in this research paper. However they all have one principle in common; blood groups are dependent on the combination of antigens which are products of specific genes thus demonstration/detection of these genes is used in determining the blood groups that people belong to (Drake, 2006). It is also apparent that most of the molecular typing techniques are PCR (polymerase chain reaction) based.
The PCR based tests usually involve three basic steps. The first step is DNA extraction using well documented (standard) procedures and restricting (cutting into specific Polymorphisms/ gene variations) the DNA. The DNA sources, as will be seen in the application section, can be varied depending on the application however the emphasis is on obtaining high quality (in terms of purity DNA). The next step is preparing the PCR mix and running a PCR reaction. The PCR mix contains the extracted DNA, specific primers, Taq Polymerase and buffers while the PCR reaction replicates the DNA fragments. The third step is the detection/visualization of the specific DNA fragments thus determining the blood type. Detection can be by various techniques including hybridization followed by visualization of radioactivity or fluorescence and gel electrophoresis (Epstein, et al., 2006).
The most recent DNA based typing involves micro arrays the most popular one being the bloodchip which was developed by a group led by Professor Neil Avent at the University of the West England (Drake, 2006). The bloodchip uses a gene chip, a microscopic glass slide with the capacity of performing up to 300 tests, to test the extracted DNA. Chemicals are added to the DNA on the slide resulting in a fluorescent reaction and on placing the chip on a scanner up to nine clinically important blood types can be identified. This technology allows rapid and accurate identification of a larger number of minor blood types hence reducing the cases of alloimmunisation. According to Professor Marion Scott the Bloodchip test will be a lifesaver for people suffering from illnesses that demand multiple transfusions such as sickle cell anaemia, haemophilia and thelassaemia (BBC news, 2006).
3.0 Applications
Molecular blood typing has found wide application particularly in transfusion medicine. It is however worth noting that in each of the applications there are certain limitations which will be discussed in this section. The two major applications of molecular of blood typing and their particular limitations are:
3.1 Foetal blood grouping
Blood groups are as a result of inherited antigens and one of the most complicated blood group systems is the rhesus system which has several variants. Where the father of a child is Rhesus positive (D+) and the mother is D- the foetus may inherit the Rhesus factor in which case the maternal immune system develops antibodies against the rhesus factor (Bethesda, 2005). The sensitization of the maternal immune system usually occurs when the fetal cells leak into the maternal circulation during labour as a result of fetal-maternal haemorrhage or earlier in the pregnancy due to a miscarriage, prenatal bleeding, chorionic villus sampling or abortion. As a result of the sensitization of the maternal system the risk of haemolytic disease of the newborn (HDN) also called erythroblastosis fetalis increases in subsequent pregnancies (Bethesda, 2005).
It is therefore a routine prenatal and antenatal practice to predict the RhD group of the foetus to assist in evaluating the risk of erythroblastosis fetalis. Accurate prediction helps in monitoring the pregnancy where the foetus is D+ or avoiding unnecessary intervention where it is D-. In most countries it is common practice to administer anti-D immunoglobulin at around 28-34 weeks’ gestation to prevent D immunization. This therapy is occasioned by the complexity of the Rhesus system and the evidence that other blood groups such as the kidd, kell, Duffy, s and MNS blood groups may contribute to HDN. Unfortunately some of the Rhesus variants as well as the rare blood groups are often not detected by the traditional serological typing (Bethesda, 2005).
Basically two RH genes have been identify, RHD and RHCE, both located on the short arm of chromosome number 1. RHD gene encodes the antigens D while the RHCE gene encodes the antigen C, c, E and e. As such the RH blood group system has been said to be the most complex blood grouping system. RH genes introduce a major challenge in both serological and molecular blood typing. For instance the RHD ψ, common in Africans, has all the RHD exons but doesn’t express the D antigen on the RBCs thus will be detected as D+ by molecular typing but D- by serological typing. In other variants such as such as RHDVI, parts of RHD may be replaced by the equivalent region of RHCE, yet a variant D antigen is present on the red cells (Daniels & Bromilow, 2010). As such currently there are three ways to be Rhesus negative; by complete deletion of the RHD (in Caucasians), by a pseudo gene (the gene is intact but has some mutations-in Africans) and by a gene hybrid. Of particular clinical significant is the presence of partial/weak D phenotypes as a result of weakly expressed single nucleotide polymorphism (SNPs) and a series of hybrid RHD genes. These partial “Ds” are not detected by serological tests thus are clumped together as D negative. The molecular blood typing offers a great opportunity to detect the week “Ds” but must be designed in a way that takes into account the mentioned RHD variants in order to reduce false predictions resulting from these variants. Most labs use real time quantitative PCR to determine the presence of the RHD gene which can be misleading because the presence of the RHD gene doesn’t necessarily mean that the person has D antigens. BloodChips are designed to counter this limitation thus accurately evaluate the mother thus take the necessary intervention or avoid unnecessary intervention (Epstein, et al., 2006).
The major limitation of molecular blood typing in this application has to do with obtaining high quality DNA. Before 2001 foetal DNA was obtained by amniocentesis or chorionic villus sampling but the procedures are invasive and are associated with high risks of spontaneous abortion and transplacental haemorrhage which could result in maternal immune sensitization. Cell-free fetal DNA from the placenta can be obtained from the maternal blood and increases throughout the pregnancy. Unfortunately this DNA can not be separated from the maternal DNA but is still used to reliably predict the fetal D type from the beginning of the second trimester thus avoiding invasive procedures. Cell-free fetal DNA from maternal plasma can therefore be used to determine fetal K type and RH C, c and E (Daniels & Bromilow, 2010).
3.2 Blood typing of patients and donors
With compatibility of the donor and the recipient being determined serologically blood transfusion is safe but patients requiring multiple transfusions have been found to develop antibodies against the rare antigens in imperfectly matched blood (An, 2006). This process, referred to as alloimmunisation may lead to serious and often fatal complication. Since most of these “minor” but clinically significantly ntigens arise from SNPs they can easily and rapidly detected by molecular technologies. The conventional molecular methods, involving the use of restriction enzymes or allele-specific primers, are being replaced by technologies with a potential for higher throughput, such as those involving microarrays or coloured microbeads, in which numerous SNPs can be analyzed in a single test. These high throughput techniques have made it possible to type numerous blood donors for the most clinically important blood groups hence establish a large database of fully typed donors. Such a database would be useful in providing safe blood for transfusion dependant patients such as sickle cell anaemia. There is a lot of research into developing tests that can detect the so called null phenotypes, resulting from inactivating gene mutations within the genes. Though these phenotypes are rare they can cause complication and their frequencies may be significant in certain populations (Daniels & Bromilow, 2010). There are various situations in which molecular blood typing is useful.
Conventional serological tests often fail to determine blood group phenotypes of patients that have recently received multiple transfusions due to the presence of the donor’s antibodies and antigens. Molecular based tests on the other hand reveal the recipient’s blood group genotype and not the donors (probably due to the low quantity of nucleated cells in transfused blood) hence predict the recipient’s phenotype. This is particularly useful in transfusion-dependant patients as it helps in providing perfectly matched blood hence prevents transfusion related complications. Molecular testing is also useful when red cells give a positive direct antiglobulin test, making serological testing difficult. This usually applies to patients with autoimmune haemolytic anaemia as a fully predicted phenotype provides clues to which clinically significant alloantibodies might be masked by the presence of an autoantibody. Molecular tests can also be used in testing patients and donors when serological reagents are of poor quality or in short supply. For example, anti-Doa and -Dob can be haemolytic, yet satisfactory serological reagents for antigen testing are not available. Some Rh variants are relatively common in people of African origin, but are difficult to define serologically. Molecular tests can be employed to assist in finding suitable blood for sickle cell disease patients (Daniels & Bromilow, 2010). Also the several variants of the D blood group phenotypes that cannot be distinguished by serological tests require molecular methods for their identification (Epstein, et al., 2006). The DEL is one of the rare D phenotype that is undetectable by serological method but since it is associated with a mutation of the RHD gene it can be detected by molecular tests. Transfusion DEL RBCs can immunize a D- recipient to produce anti-D thus in some parts of Europe it is common practice to screen all apparent D- for RHD which was is only possible with molecular testing.
4.0 Advantages and limitations of molecular blood typing
Molecular blood testing techniques have enormous advantages and even greater potential. One of the greatest advantages is the ability to detect rare blood groups that become clinically significant particularly in cases where multiple blood transfusions are required. Another advantage is the accuracy of the tests in determining blood groups. This is particularly important in determining the Rhesus blood group system which has many variants that are impossible to distinguish by serological tests. Finally the molecular tests can be used to resolve cases of unusual serological findings. User friendly test kits based on PCR-sequence-specific priming (PCR-ssp) have been designed are already in use in Europe to facilitate examination of weak, unclear or unexpected serological results (Department of Production Molecular Genetics BAG, 2007). However molecular tests may not replace routine serological typing in the near future due to some inherent limitations and disadvantages (Epstein, et al., 2006).
Molecular blood typing is cost prohibitive thus not routinely used in countries and hospitals with strained budgets. In addition to the cost factor molecular tests are complex thus require highly trained personnel. The other major limitation is that there are many variations of molecular tests hence there is need for standardization of the test. They also require high quality of DNA which is often not easy to obtain particularly in fetal blood typing. There is also the fact that the genotype is not always the phenotype for example in the case of the RHD variants and there are many genetic variants that are yet to be discovered (Epstein, et al., 2006). Due to these inherent limitations of the molecular blood typing most of the techniques available are only used as supplementary tests. In case of discrepancies or unclear genotype findings transfusion is usually guided by the serological tests particularly where gene sequence analysis, which is recommended for final clarification, is not available (Prager, 2007).
5.0 Conclusion
Most of the genes responsible for the human blood groups have been discovered and cloned making it possible to determine a person’s blood group from their DNA with high degree of accuracy. This has significantly improved the safety of blood transfusion particularly in patients requiring multiple blood transfusions. It has also provided a more accurate means of determining the RhD group of the foetus hence asses the risk of HDN. This determination of clinically relevant, rare or weak variants of blood group systems on a genetic basis provides greater safety in the assessment of blood donors, recipients and pregnant women (BAG Health Care, 2010). However due to the complexity of the blood groups, the cost of the molecular tests and the complexity of the test itself, molecular blood typing is not the method of choice for routine ABO and D type determination. The complexity of the RH blood grouping systems has particularly proved a major challenge and there is need to develop a kit that will provide comprehensive and accurate determination of the RH factor. A lot of research is required to address the limitations of the molecular typing as well as to standardize and evaluate the several molecular tests that are available.
References
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