Blood group molecular genotyping

Transcription

1 STATE OF THE ART 4B-PL5 ISBT Science Series (2011) 6, ª 2011 The Author(s). ISBT Science Series ª 2011 International Society of Blood Transfusion Blood group molecular genotyping C. Jungbauer Austrian Red Cross, Blood Service, Vienna Blood Centre, Wiedner Hauptstrasse, Wien, Austria DNA-based typing methods of red cell antigens are applied in several fields. Genotyping is used to clarify problems in patient serology. It is also increasingly applied for routine mass-scale typing of blood donors for minor red cell antigens and screening for donors with rare blood types. An advance in this field is the non-invasive fetal RHD diagnostics in pregnancies of D-negative women to determine the fetal RHD. Reference centres for immunohaematology commonly use molecular methods to clarify problems, discrepancies or unusual results in patient serology. It is often applied in individuals with variant RhD expression to detect the contributing weak D or partial D types. Genotyping is also used to identify the individual ABO and RhD blood group in patients after mismatched transfusion. In some patients with positive direct antiglobulin test (DAT) it is necessary to type for antigens by DNA techniques. Genotyping is also helpful when reagents are not available or only weakly reactive, or to confirm weakly expressed antigens. Blood establishments are constantly challenged with the blood supply for patients carrying irregular red cell antibodies. A high number of donors have to be typed to find compatible blood units. Increasingly, DNA-based methods are used alongside standard serological typing. The advantages of molecular methods are the wider range of different antigens available for typing and that some DNA methods are less expensive than phenotyping. Extensive donor antigen typing leads to short response times, from request to issuing blood units. Genotyping is also used for identifying donors with extremely weak RhD expression (DEL) to prevent mistyping as RhD-negative. Non-invasive fetal typing of RHD from maternal blood in pregnancies of D-negative women is implemented in many Caucasian blood establishments. It allows that D-negative fetuses can be identified at early stage. In these cases, no anti-d immunoglobulin is required, which provides a more efficient use of this human blood derivate. Furthermore, in anti-d alloimmunized women, the assay allows to accurately identify fetuses at risk for haemolytic disease. Key words: genotyping, immunohematology, molecular typing, red cell antigens. Introduction The International Society of Blood Transfusion (ISBT) has by now authenticated 307 RBC antigens. Most of them are assigned to one of 30 blood group systems (International Blood Group Reference Laboratory, co.uk). For the majority of the antigens and a vast number Correspondence: Christof Jungbauer, Austrian Red Cross, Blood Service, Vienna Blood Centre, Wiedner Hauptstrasse 32, 1040 Wien, Austria jungbauer@redcross.at of rare variants the molecular polymorphism is known at DNA level, which makes them accessible for genotyping (Blood Group Antigen Mutation Database, [1,2]. As antigens are serologically defined all are theoretically available for phenotyping. However, practically a constant supply with regulated commercial antigens is limited to about 30 specificities. For some further specificities the centres are depending on the use and exchange of rare patient materials. 399

2 400 C. Jungbauer If applicable, genotyping allows independence from the availability of serological reagents. It makes a wider spectrum of antigens accessible for routine typing. It complements antigen typing in cases where serology faces difficulties (e.g. positive DAT) or in situations that are complex (e.g. clarification of Rh-variants). Most routine RBC genotyping methods rely on testing of one or a few nucleotides for the prediction of a certain phenotype. This assumes that the relevant alleles in a population are known and the corresponding nucleotide polymorphisms are covered by the assay. If the assay is adequately chosen for the defined problem the genotyping result also will provide reasonable accuracy [1 7.] Patient typing Reference centres for immunohaematology use molecular methods to clarify problems, discrepancies or unusual results in patient serology. Genotyping often applied in individuals with variant RhD expression to detect the contributing weak D or partial D types [8,9]. It is also used to identify the individual ABO and RhD blood group in recently transfused patients who did not receive their individual blood group. Subsequently the original blood group can be issued for further transfusions. In some patients with positive direct antiglobulin test, it is necessary to type for antigens by DNA techniques. Genotyping is also helpful where reagents are not available or only weakly reactive, or to confirm weakly expressed antigens. Numerous publications refer to applications listed below that are routinely applied or that are thought to have potential in future [2 5]: (1) Genotyping recently (multiply) transfused recipients to determine the genuine blood type. (2) Resolving ABO, RhD and other antigen discrepancies. (3) Identifying RhD variants that are at risk for anti-d alloimmunization [10]. (4) Help to distinguish alloantibody from autoantibody. (5) Confirming the true genotype when the antigen is weakly expressed (e.g. DEL). (6) If the antibody is weakly reactive or not available (e.g. Do a,do b,js a,js b ). (7) Antigen typing if phenotyping is prevented by a positive DAT. (8) Chronically transfusion-dependent patients to reduce (further) alloimmunization risk (e.g. sickle cell anaemia, Thalassaemia) [11]. (9) Fetal blood group typing. ABO and RhD discrepancies are frequently forwarded to reference laboratories for genetic testing. Mostly RhD discrepancies are referred. Many RhD variants can routinely be distinguished with commercial and in-house assays. The RHD and RHCE locus is a recombination hotspot and highly polymorphic. Variants are caused by single and multiple nucleotide exchanges, hybrid genes, insertions or deletions, splice site mutations and premature stop codons (RhesusBase, ~fwagner/rh/rb/). The number of known alleles is constantly increasing [8,12]. As the prevalent alleles differ between populations, assays for RhD variants have to be adapted to the specific population. Essentially, the pragmatic view is to identify individuals with variant D who are at risk to become alloimmunized if transfused with D- positive blood. In return, patients with variant D, who are very unlikely to get alloimmunized, could be transfused with D-positive blood units. Some centres or transfusion services implemented regulations in this direction. For example, Swiss recommendations suggest to transfuse patients with weak D type 1 3 with D-positive blood (Swiss Association of Transfusion Medicine, Recently transfused patients might present with mixed field agglutination in ABO) and RhD at admission to a hospital. The detection of the individual blood group may prevent unnecessary wastage of group O or RhD-negative blood units [13]. Patients who chronically receive transfusions due to haemoglobinopathies or other chronic haematological disorders are at risk to gradually become alloimmunized to one or several antigens. Therefore, decreasing phenotype frequencies of compatible blood complicate the supply of appropriate blood units. It has been suggested that this patient group should receive extendedly matched units to minimize alloimmunization risk. This implies that both, recipient and numerous donors will need comprehensive typing [14]. In the field of haemolytic disease of the fetus and newborn (HDFN), related testing is progressing at the moment. Non-invasive fetal genotyping from maternal plasma (predominantly for RHD) replaces invasive percutaneous sampling procedures for fetal blood grouping purposes. It is referred to in an own section below. Donor typing To supply patients with red cell alloantibodies with appropriate blood units which are lacking the corresponding antigen, blood establishments have to type a subset of their donors blood units for several minor RBC antigens. While it is easy to find and select units compatible for most of the common alloantibody specificities, it is very demanding if the phenotype frequency of the compatible blood type is rare. This can occur due to a combination of antibody specificities or a specificity to a high frequency antigen (HFA). High numbers of antigen typing results in the donor databases of the blood centres usually enable short response

3 Blood group molecular genotyping 401 times from the moment of request by the hospital to distribution of the blood unit. To avoid on-demand screening for appropriate units, many blood centres operate donor typing programmes to type a subset of their repeat donors for many minor red cell antigens. Regulated commercial serological reagents are available for a limited range of approximately 30 antigens. Especially larger centres frequently have to supply patients with antibody specificities where no regulated reagents are accessible. Using molecular methods provide independence from the availability of serological reagents and make a wide spectrum of antigens available for routine typing. Many different approaches for mass-scale DNA-based donor typing methods have been used. Each of the methods provides advantages and disadvantages. The assays differ in methodology, technical capabilities, costs, number and selection of the red cell antigens included. The different characteristics have been summarised in some reviews [6,15,16]. Different views on the selection of antigens to be included in donor typing programmes are discussed in the sections below. Typing of minor red cell antigens The ABO and RhD antigens are routinely typed serologically. Kell and RhCcEe are also frequently phenotyped in blood establishments and therefore need not be included in their large-scale molecular typing programmes. ABO phenotyping is extremely reliable due to the combination of forward and reverse typing. Routine ABO-genotyping of donors provides no added value. For RhD the situation is different; there is a great variety in D expression. While the focus in patients is on identification of patients who are at risk of becoming alloimmunized when transfused with D-positive blood, the focus in donors is screening for serologically RhD-negative mistyped donors, whose blood may express minute amounts of D, which might cause anti-d alloimmunization in the D-negative recipient. This aspect of quality assurance of apparently RhD-negative donors is discussed below. In contrast, most minor antigens are not routinely phenotyped. Therefore, typing the right antigens is a significant advantage for a fast and reliable blood supply for patients with red cell antibodies. Considerations, which antigens should be included in a donor typing, may regard the prevalence and the clinical significance of the antibody. The prevalence of an alloantibody-specificity may vary between different ethnical groups, but most of the frequent common antibody specificities will be found in any population. They are generally taken into account in case of a positive antibody screening as they are referred to in guidelines (e.g. British guidelines for compatibility procedures in blood transfusion laboratories: D, C, c, E, e, K, k, Fy a,fy b, Jk a,jk b,s,s,m,n,le a ) and included in commercial antibody-differentiation panels. If the assay or technical platform allows typing for a higher number of antigens, there could also be considerations to type for other antigens, where no serological reagents are available or where the reagents are usually not sufficiently reactive (e.g. anti-do a, anti-do b, anti-hy, anti- Jo b ) [5]. Screening for rare blood types The blood supply for patients with antibodies directed to HFA is critical and many of the patients may not be transfused with appropriate blood units in time [17]. While serological reagents for common specificities are available on a regular base, blood centres are often short of reagents for screening hundreds to thousands of their donors for a rare blood type (e.g., Kp b ),Lu b ),Yt a )). Therefore, DNA-based screening programmes can provide a beneficial alternative. Most commercial and in-house assays published, test for less than 10 HFAs [18]. The occurrence of phenotypes varies between populations. For example, while the RhD or Fy a negative phenotype is rare in Asia, it is 15% respectively 34% in the Caucasian population. The prevalence (and the clinical significance) of the HFA-antibodies in a population should influence the decision whether the HFA-antigen should be included into an assay or not. Incidences of HFA-antibodies have been reported for different populations [18,19]. The selection of rare blood types included in the Austrian Red Cross screening program was mainly based on two incidence reports of HFA-antibodies in the Western European population. Firstly, Seltsam showed that two-third of the HFA-specificities were directed to only four antigens (Kp b, Vel, Lu b,yt a ) [17]. Secondly, the Swiss Red Cross had 344 antibody incidences to high-prevalence antigens listed (Hein Hustinx, SRK Blutspendedienst Bern AG). The multiplex-ssp-pcr assay included 12 HFA-genotypes, which covered 65% of the HFA incidences according to these sources. Not all HFA-antigens are accessible for molecular blood group genotyping yet (e.g. ISBT 901 series of high incidence antigens: Vel, Lan, At a, etc.). Other rare types require more efforts due to a complex genetic background (e.g. O h ) and some would be revealed in the serological routine work (e.g. O h,d--,tj a ) [20,21]. Quality assurance in RhD-negative donors Due to a variety of very weekly expressed RHD-alleles, false-negative immunological RhD typing results do occur [22]. Alloimmunizations by blood units of these individuals have been reported [23,24].

4 402 C. Jungbauer The prevalence of D and variant-d phenotypes and the causative alleles widely differ from population to population. Flegel et al. showed that 0.21% of serologically apparently RhD-negative German donors carried a RHD-gene and 0.1% had alleles expressing DEL phenotypes that could be detected by special serological adsorption and elution techniques [25]. The expression of a weak D-, partial D- or DEL-alleles can be further suppressed by the trans position effect described by Ceppellini [26], therefore the detection of a RHD-gene is more frequent in apparently D-negative individuals who are C-positive or E-positive [27]. In Southeast Asia, about 0.3% of the population appears to be RhD negative in routine testing. Several RHD-alleles code for negative phenotypes. Nevertheless, 17 28% of apparently RhD-negative individuals were reported to express DEL phenotypes [28,29]. Genotyping apparently RhD-negative donors for RHD can help to identify some individuals with an extremely weak RhD expression and prevent the distribution of red cell units that might bear some risk for RhD-alloimmunization in RhD-negative recipients. This might be of special importance in regions with a high frequency of DEL-types like in Asia. Current application of DNA-based donor typing programmes and costs Different groups published methods feasible for high throughput donor typing. Methods included conventional PCR, Real-time PCR, pyrosequencing, microarray and mass spectrometry assays. Some are in-house tests, others commercial tests, which are partly certified for in vitro diagnosis. Due to the significant methodological differences, the assays vary in technical capabilities, antigen selection, throughput and costs [6,16,25,30]. Beside initial publications on methodology that mostly were evaluated in a small collective, unfortunately only few publications report on ongoing genotyping activities and give benchmark data. A Canadian group reported on the effectiveness of genotyping donors for 22 red cell and platelet antigens to achieve a 95% hit rate in the supply of patients with RBC alloantibodies [30,31]. Three European Red Cross blood centres (Springe, Germany; Bern, Switzerland; Vienna, Austria) tested , 1500 and donors for 16, 30 and 35 RBC antigens using inhouse SSP-PCR assays ([32] and personal communication; Christoph Niederhauser, SRK Blutspendedienst Bern AG). The costs for PCR-reagents and DNA extraction were below USD 0.2 per antigen, which is less than for phenotyping. Naturally, the cost benefit of low-price methods may be balanced by disadvantages, such as deficiencies in automated reporting, manual interfering (as a frequent source of error) and lack of certification for in vitro diagnostic (screening character of the result). Commercial assays might not meet these low costs. Depending on method, acquisition or test costs will be comparable to serology or even be a multiple (e.g. micro arrays, mass spectrometry). Fetal typing Beside donor typing, blood establishments are to a greater extent routinely engaged with non-invasive prenatal RHD typing in RhD-negative pregnant women. Less frequent it is also used for the prediction of other fetal antigens (e.g. KEL, RHc) [30,33 35]. While prior to the development of non-invasive fetal typing, methods where relying on invasive procedures, such as chorionic villus sampling, percutaneous amniotic fluid or umbilical cord blood sampling, the non-invasive method is based on detection of RHD sequences from cell-free fetal DNA in the maternal plasma [36]. From the user perspective, two contexts may be distinguished: firstly, where the pregnant woman is already alloimmunized to D. In this case, fetal RHD typing can provide the information if a fetus is D-positive (62% in the Caucasian population) or D-negative (38% in the Caucasian population) at an early stage of pregnancy. D-positive fetuses are at risk of HDFN and need periodic check-ups during pregnancy. D-negative fetuses do not have RhD-related HDFN risk. Secondly, this method can be used in non-alloimmunized pregnant women as basis for the indication and the more efficient use of anti-d immunoglobulin, as all efforts to replace this limited human plasma derivative by monoclonal antibodies have not yet been successful [37]. Conclusions Genotyping will play a more important role in the routine work of blood establishments in future. It will not replace serological methods, but it allows that a wider spectrum of minor antigens is accessible in donor typing. It will contribute to increased quality of red cell reference laboratory work. By giving better insight in serologically complex situations, genotyping supports decision making and will provide high accuracy of results. It allows donors and patients to be typed for a broad spectrum of antigens, including rare blood types, and will therefore be useful for extended antigen profiles. Non-invasive fetal typing can prevent inadequate application of anti-d immunoglobulin or determine the risk of HDFN at a very early stage of pregnancy. Blood group molecular genotyping will contribute making transfusion medicine more personalized and patient orientated.

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