Molecular DNA-based testing for blood group antigens: recipient donor focus

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1 STATE OF THE ART 1A-H01-01 ISBT Science Series (2013) 8, 1 5 ISBT Science Series 2013 International Society of Blood Transfusion Molecular DNA-based testing for blood group antigens: recipient donor focus C. M. Westhoff Immunohematology and Genomics, New York Blood Center, New York, NY, USA DNA-based testing for blood group antigens has become commonplace in a number of clinical situations to improve transfusion therapy for blood transfusion recipients. These include typing for antigens in patients who are multiply transfused to determine which blood group antibodies may be present, typing when the red blood cells (RBCs) are coated with immunoglobulin, and typing for uncommon antigens, especially when no serologic reagent is available. Other clinical applications include resolving ABO typing discrepancies for patients awaiting transplant, or determination of the original type of a bone marrow transplant recipient, or confirmation of an ABO subgroup in a kidney donor. Testing for the specific mutation associated with depressed Kell system antigens in McLeod syndrome may also have value because different mutations appear to be associated with prognosis. Applications in prenatal medicine include discrimination between weak D and partial D to determine if a pregnant woman is a candidate for Rh immune prophylaxis, and assessing the risk for hemolytic disease of the fetus or newborn and neonatal thrombocytopenia. Assessing risk includes testing the paternal sample for the target antigen, and if positive testing for gene copy number (zygosity) and testing the fetus from amniocentesis or non-invasive maternal plasma sample. Donor centers are now able to screen large numbers of donors to label antigen-negative RBC products. DNA typing will change transfusion practice by allowing patients facing chronic transfusion therapy to have a more comprehensive blood group antigen profile performed, and to receive donor units antigenmatched for more than ABO and RhD to reduce alloimmunization. A patient who makes an alloantibody to a RBC antigen is forever at risk for his or her lifetime; for a serious transfusion reaction if ever requiring transfusion in an emergency, and also for a high risk pregnancy if a woman of child bearing age. Prevention of alloimmunization is an important advance in transfusion safety and is now possible with electronic patient medical records and testing and labeling of blood products for additional blood group antigens. Key words: blood groups, blood group antigens, DNA based testing, molecular matching, molecular testing Introduction Determination of blood group antigens with DNA methods (genotyping) is an indirect method for predicting an individual s blood group phenotype, in contrast to direct testing by serological methods using a specific antibody (phenotyping). The results of typing by DNA are often reported as a predicted type to distinguish the results from testing done by serological methods. Correspondence: Connie M. Westhoff, Director, Immunohematology and Genomics, New York Blood Center, 310 East 67th Street, New York, NY 10065, USA cwesthoff@nybloodcenter.org 1

2 2 C. M. Westhoff Most blood group antigens result from single nucleotide gene polymorphisms (SNPs) inherited in a straightforward Mendelian manner, making assay design and interpretation fairly straightforward [1]. However, ABO and Rh are more complex. There are over 100 different alleles encoding the glycosyltransferases responsible for ABO type, and a single point mutation in an A or B allele can result in an inactive transferase, that is a group O phenotype [2]. Next generation sequencing technology would potentially allow routine ABO typing by DNA methods. For the Rh system, testing for the common antigens D, C/c and E/e is fairly straightforward in most individuals, but antigen expression is more complex in diverse ethnic groups. There are over 200 RHD alleles encoding weak D or partial D phenotypes ( base.atspace.com/), and more than 100 RHCE alleles encoding weak, altered or novel hybrid Rh proteins [3,4]. RH genotyping, particularly in minorities, requires sampling of multiple regions of the gene(s) and algorithms for interpretation. The most commonly used methods for determination of red cell, human platelet antigen (HPA) and neutrophil antigens use sequence-specific primer polymerase chain reaction (SSP-PCR) or allele-specific PCR. For manual methods, gel electrophoresis is then used to separate the PCR products for fragment size determination, or alternatively, the assay may include digestion of the PCR products with a restriction enzyme, restriction fragment length polymorphism, followed by electrophoresis and visualization of the fragments. For resolution of discrepancies and to identify new alleles, specialty referral laboratories use methods similar to those used for high resolution human leucocyte antigen typing, that is genespecific amplification of coding exons followed by sequencing, or alternatively, gene-specific cdna amplification and sequencing. These methods are used to investigate new alleles and resolve serological and molecular discrepancies. Semi-automated approaches include realtime PCR using florescent probes with quantitative and qualitative automated read-out. Lastly, high-throughput automated methods increase the number of target alleles in the PCR reaction allowing determination of numerous antigens in a single assay. Most platforms are based on florescent bead technology. Applications of DNA-based molecular testing Clinical transfusion medicine Type patients who have been recently transfused In patients receiving chronic or massive transfusion, the presence of donor red blood cells (RBCs) often makes RBC typing by serological agglutination inaccurate. DNA typing overcomes this limitation and avoids time consuming and cumbersome cell separation methods to isolate and type the patient s reticulocytes. DNA assays for blood groups avoid interference from donor-derived DNA by targeting and amplifying a region of the gene common to all alleles. This allows reliable blood group determination with DNA prepared from a blood sample collected after transfusion. In transfusion-dependent patients who produce alloantibodies, an extended antigen profile is important to determine additional blood group antigens to which the patient can become sensitized. Type RBCs coated with immunoglobulin [positive direct antiglobulin test (DAT)] In patients with RBCs coated with immunoglobulin, with or without autoimmune haemolytic anaemia, the presence of bound immunoglobulin often makes RBC typing by serological methods invalid. Immunoglobulin G (IgG) removal techniques are not always effective and can destroy or weaken the antigen of interest. For patients with serum autoantibody, DNA testing allows determination of an extended antigen profile to select antigen-negative RBCs for transfusion. This avoids the use of least incompatible blood for transfusion, and allows transfusion of units antigen-matched for clinically significant blood group antigens to prevent delayed transfusion reactions and circumvent additional alloimmunization. Importantly, this approach can improve patient care and testing turn-around time by eliminating the need for repeat absorptions to remove the autoantibody to rule out new underlying RBC alloantibodies. Determination of D status Altered expression of D antigen occurs in 2% of Caucasians, <1% of Asians and approximately 4% of Black and Hispanic groups. Routine serological D-typing reagents cannot distinguish RBCs with weak D or partial D, and distinction between these is of clinical importance because the latter are at risk for anti-d. RHD genotyping strategies that sample multiple regions of RHD can discriminate weak D and partial D phenotypes. Females of child-bearing age with partial D would potentially benefit from receiving D-negative RBCs for transfusion to avoid future pregnancy complications and be considered for Rh immune globulin (RhIG) prophylaxis. Many transfusion services error on the side of caution and transfuse patients with D-negative RBCs if the serological D-typing reaction strength is weaker than expected (<3+ to 4+). This results in unnecessary use of the limited D-negative blood supply. DNA testing to discriminate weak D from partial D is important for patient care and for responsible management of D-negative blood resources.

3 Clinical application of blood group genomics 3 Alloantibody versus autoantibody For patients presenting with RBCs that type positive for an antigen with an apparent antibody of the same specificity in the serum or plasma, a DNA-based investigation is helpful for transfusion management. If the sample is predicted to be antigen positive by SNP testing, it should be further investigated by high resolution gene sequencing. The samples may have a novel amino acid change in the protein carrying the blood group antigen. These result in new epitopes and altered (or partial) expression of the conventional antigen. Sickle cell disease (SCD) and chronic transfusion therapy Alloimmunization is a serious complication of chronic transfusion, particularly in patients with sickle cell disease and b-thalassaemia requiring long-term transfusion support. Antibody production can result in delayed haemolytic transfusion reactions (DHTR). Patients with SCD undergoing a DHTR are at risk for life-threatening anaemia, pain crisis, acute chest syndrome and/or acute renal failure, and patients may experience hyper-haemolysis, in which haemoglobin levels drop below pre-transfusion levels due to bystander haemolysis of the patients own antigen-negative red cells. Diagnosis and management of DHTR are difficult in this patient population as symptoms mimic a painful episode and laboratory tests used to diagnose DHTR, elevated lactate dehydrogenase and unconjugated bilirubin levels, are already abnormal. Alloimmunization also results in significant delay in transfusion, increases costs, is associated with risk for production of additional antibodies and often results in a chronic positive DAT with apparent warm autoantibody, that is panagglutinins, which complicate further workups and transfusion therapy. Many programmes attempt to prevent or reduce the risk and incidence of alloantibody production by transfusing RBCs that are antigen matched for D, C, E and K (and a few include Fya/b, Jka/b and S). The most frequent antibodies encountered in patients with SCD receiving units antigen matched for D, C, E and K, have Rh specificities. These antibodies include anti-d, -C and e in patients whose RBC type serologically as D+, C+ and e+. RH genotyping has revealed that these patients have RHD and/or RHCE alleles with amino acid changes that encode altered or partial antigens. For example, approximately 23% of the SCD patients with C+ RBCs do not have a conventional RhCe protein. In these patients, the C antigen is expressed from a hybrid RHD gene that has lost expression of D, but encodes a C epitope. More than one-third of patients with this hybrid gene encoding a C+ phenotype make anti-c or Ce. RH genotyping can identify these patients who are better served on C rather than C+ transfusion protocols. Anti-D in D+ patients with SCD is associated with RHD alleles encoding partial D antigens detected by RHD genotyping. Anti-e in e+ patients with altered Rhce proteins are more problematic to manage. Transfusion with e blood will expose them to the E antigen, and most are E and at risk for anti-e. In summary, RH genotyping expands and extends matching for Rh in this patient population and is important for transfusion management. Prenatal practice Testing for Rh in pregnant women Serological typing for Rh(D) cannot distinguish women whose RBCs lack some epitopes of D (partial D) and who are at risk for anti-d. RBCs with partial D type as D+, some in direct tests and others by indirect tests, but these women would potentially benefit from receiving RhIG prophylaxis if they deliver a D+ fetus. Serological testing cannot distinguish weak reactivity due to missing epitopes (partial D) from weak reactivity due to quantitative reduced antigen (weak D). RHD genotyping can distinguish weak D from partial D to guide RhIG prophylaxis and blood transfusion recommendations. Haemolytic disease of the fetus and newborn (HDFN) The identification of a clinically significant alloantibody in a pregnant woman relies on demonstration of an IgG antibody by serological testing, but management of the pregnancy is aided by testing of a paternal sample, and if indicated, the fetus to assess risk for HDFN. Paternal testing Antigen Typing: The RBCs of father should be tested for the corresponding antigen. If the RBCs are negative, the fetus is not at risk. If the father is positive, zygosity testing can determine if the father is homozygous or heterozygous for the gene expressing the antigen, particularly when there is no allelic antigen, or no antisera to detect the allelic product. Zygosity testing: DNA testing of paternal samples is most often done when testing for possible HDFN due to anti-d or anti-k. For maternal anti-k, testing the paternal RBCs for expression of the allelic k antigen should be performed, but many centres do not have a licenced reagent available and genetic counsellors often request DNA testing. For maternal anti-d, RHD zygosity testing by DNA methods is the only method to determine the paternal gene copy number. A number of different genetic events cause a D-negative phenotype and multiple

4 4 C. M. Westhoff assays must be done to accurately determine RHD zygosity, especially in minority ethnic groups. If the father is RHD homozygous, all children will be D+ and monitoring of the pregnancy will be required. If the father is heterozygous, the fetus has a 50% chance of being at risk. The D type of the fetus should be determined to prevent invasive and unnecessary testing so the mother need not be aggressively monitored or receive immune modulating agents. Fetal testing Amniocentesis: To determine the fetal antigen status, fetal DNA can be isolated from cells obtained by amniocentesis. Non-invasive fetal testing from the maternal plasma: The discovery that cell-free, fetal-derived DNA is present in maternal plasma by approximately 5 weeks of gestation allows maternal plasma to be used as a source of fetal DNA to determine the antigen status of the fetus. This is particularly successful for D typing because the D- negative phenotype in the majority of samples is due to the absence of the RHD gene. Testing for the presence or absence of a gene is less demanding than testing for a single gene polymorphism or SNP. This approach is being used in some European countries to test the maternal plasma for the presence of a fetal RHD gene to eliminate the unnecessary administration of antepartum Rh immune globulin to the approximately 40% of D-negative women who are carrying a D-negative fetus. Neonatal alloimmune thrombocytopenia (NAIT) A diagnosis of NAIT is based on demonstrating HPA-specific antibody in maternal serum and identifying an incompatibility between the parents by HPA platelet genotyping. Twenty-eight HPAs have been characterized, but incompatibility in HPA-1 accounts for approximately 80% of all cases. Platelet genotyping is used to confirm the HPA status of the mother and to type a paternal sample. If the father is homozygous for the target HPA, all children will be positive and monitoring of the pregnancy will be required. If the father is heterozygous, the fetus has a 50% chance of being at risk. The HPA type of the fetus should be determined to prevent invasive and unnecessary testing. To determine the fetal HPA antigen status, fetal DNA can be isolated from cells obtained by amniocentesis. Non-invasive fetal testing from the maternal plasma has been reported and may become more readily available in the future. Other clinical applications McLeod syndrome The McLeod phenotype, characterized by weak expression of RBC Kell system antigens and absence of Kx antigen, is encoded by the X-linked gene, XK. This X-linked syndrome is manifested only in males and is associated with late onset of clinical or subclinical myopathy, neurodegeneration and central nervous system manifestations. The syndrome may be under-diagnosed and the physical characteristics, which often develop only after the fourth decade of life, include muscular and neurological problems. Over 30 different XK gene mutations associated with a McLeod phenotype have been found. Different XK mutations appear to have different clinical effects and may account for the variability in prognosis [5]. Sequencing of XK to determine the specific type of mutation in individuals with McLeod phenotypes has clinical prognostic value. ABO typing discrepancies in patients DNA testing is useful to resolve patient typing discrepancies, confirm subgroup status and determine the original blood type of patients massively transfused, or the original blood type of transplant recipients by testing a buccal sample. The ability to accurately determine an individual s antigen status eliminates the use of group O RBCs and AB plasma for transfusion in the situation of ABO typing discrepancy. ABO genotyping can aid in the differentiation of subgroup alleles, particularly to confirm A2 subgroup in kidney donors who may have been transfused, or whose RBCs give discordant reactivity in serological testing with anti-a1 reagents. Accurate determination of ABO often requires gene sequencing. Donor centre applications Typing for antigens for which there are no commercial reagents DNA-based typing has become the standard to identify antigen-negative units for which there are no serological reagents. One of the most often used is to type for Dombrock (Doa/b). Antibodies to these antigens are clinically significant, but patient serum antibodies are often weak, have poor avidity, disappear over time and are almost always present with other blood group specificities that interfere with screening donor units. As Dombrock antibodies are difficult to detect, matching of patients with donors for Dombrock antigens should be considered in patients when survival of transfused red cells is compromised and complex mixtures of antibodies are present, even when no specific serum antibody to Dombrock antigens is demonstrable by serological testing. Confirming D type of donors Donor centres must perform a test for weak D to attempt to avoid labelling a product as D-negative that might result in anti-d in response to transfused RBCs. It is well-

5 Clinical application of blood group genomics 5 known that some donor RBCs with very weak D expression are not typed as D+ with current serological reagents and are labelled as D for transfusion. The prevalence of weak D RBCs not detected by serological reagents is approximately 01% and although the clinical significance has not been established, donor RBCs with weak D expression have been associated with alloimmunization [6]. RHD genotyping can improve donor testing by confirming D phenotypes [7]. High-throughput screening for donor inventory and rare or uncommon antigens The ability to screen for multiple minor antigens in a single assay format has been a significant aid to donor centres to provide antigen-negative products and to provide antigen-matched products for patients. Although DNA methods are not yet Food & Drug Administration (USA) licensed to label donor units, some are CE marked. Resolving donor ABO discrepancies to retain donors Donor samples with ABO typing results that are discordant with results from a previous donation must be investigated. Donors with depressed antigen or antibody expression and RBC reactions that do not match the plasma reactions cannot be labelled for transfusion. Determination of ABO by DNA-based methods can resolve these, DNA methods offer the potential to be a confirmatory test. Confirmatory testing by DNA would avoid the loss of donor units and retain group O donors with depressed antibody titres in the donor pool. Discrepancies between serology (phenotype) and DNA (genotype) When differences between serological and DNA testing occur, it is important to investigate. This can indicate the presence of a novel allele or genetic variant, particularly when testing individuals from diverse ethnic groups. The primary cause of discrepancies between the serological phenotype and DNA genotype when testing donors by large-scale DNA typing has been traced to human recording errors. Other common causes of discrepancies include the presence in the sample of an FYX allele. This allele encodes an amino acid change causing a Fy(b+w) weak phenotype. The prevalence of FYX encoding Fy(b+w) in Caucasians is as high as 2% [8]. DNA testing interrogates a single or few SNPs associated with antigen expression and cannot sample every nucleotide in the gene. Consequently, discrepancies will occur when typing patients who have rare or novel silencing mutations that cause loss of antigen expression. These can be specific to a particular ethnic group. For example, silencing mutations responsible for S-s-U- phenotypes are common in African ethnic groups, and markers for these should be included when typing these populations. The Fy(a b ) phenotype found in African Blacks is caused by a mutation in the promoter region of FYB, which disrupts a binding site for the erythroid transcription factor GATA-1 and results in the loss of Duffy expression on RBCs [9]. For accuracy, the GATA mutation must be included when typing for Duffy in African groups. Importantly, expression of the protein on endothelium is not altered and Fy(a b ) individuals with GATA-1 mutations are not at risk for anti-fyb. Silencing mutations associated with loss of Kidd antigen expression occur more often in Asians, whereas nucleotide changes encoding amino acid changes that weaken Kidd expression are see in Blacks. Disclosure The author has no conflicts of interest to declare. References 1 Westhoff C: Molecular testing for transfusion medicine. Curr Opin Hematol 2006; 13: Storry JR, Olsson M: The ABO blood group system revisited: a review and update. Immunohematology 2009; 25: Chou ST, Westhoff CM: Molecular biology of the Rh system: clinical considerations for transfusion in sickle cell disease.hematology Am Soc Hematol Educ Program 2009: Chou ST, Westhoff CM: The role of molecular immunohematology in sickle cell disease. Transfus Apheres Sci 2011; 44: Jung HH, Hergersberg M, Vogt M, et al. : McLeod phenotype associated with a XK missense mutation without hematologic, neuromuscular, or cerebral involvement. Transfusion 2003; 43: Wagner T, Kormoczi GF, Buchta C, et al. : Anti-D immunization by DEL red blood cells. Transfusion 2005; 45: Flegel WA, von Zabern I, Wagner FF: Six years experience performing RHD genotyping to confirm D- red blood cell units in Germany for preventing anti-d immunizations. Transfusion 2009; 49: Reid ME, Lomas-Frances C, Olsson ML, editors: The Blood Group Antigen FACTS book, 3rd edn. Elsevier, Tournamille C, Colin Y, Cartron JP, et al. : Disruption of a GATA motif in the Duffy gene promoter abolishes erythroid gene expression in Duffy-negative individuals. Nat Genet 1995; 10: