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Background and Objectives: Next generation sequencing (NGS) has promising applications in transfusion medicine. Exome sequencing (ES) is increasingly used in the clinical setting, and blood group interpretation is an additional value that could be extracted from existing data sets. We provide the first release of an open-source software tailored for this purpose and describe its validation with three blood group systems., Materials and Methods: The DTM-Tools algorithm was designed and used to analyse 1018 ES NGS files from the ClinSeq ® cohort. Predictions were correlated with serology for 5 antigens in a subset of 108 blood samples. Discrepancies were investigated with alternative phenotyping and genotyping methods, including a long-read NGS platform., Results: Of 116 genomic variants queried, those corresponding to 18 known KEL, FY and JK alleles were identified in this cohort. 596 additional exonic variants were identified KEL, ACKR1 and SLC14A1, including 58 predicted frameshifts. Software predictions were validated by serology in 108 participants; one case in the FY blood group and three cases in the JK blood group were discrepant. Investigation revealed that these discrepancies resulted from (1) clerical error, (2) serologic failure to detect weak antigenic expression and (3) a frameshift variant absent in blood group databases., Conclusion: DTM-Tools can be employed for rapid Kell, Duffy and Kidd blood group antigen prediction from existing ES data sets; for discrepancies detected in the validation data set, software predictions proved accurate. DTM-Tools is open-source and in continuous development., (© 2020 International Society of Blood Transfusion.)

Background and Objectives: Paper biodiagnostics for blood typing are novel, cheap, fast and easy to use. Agglutinated red blood cells cannot travel through the porous structure of paper, indicating a positive antibody-antigen interaction has occurred. Conversely, non-agglutinated blood can disperse and wick through the paper structure with the ease to indicate a negative result. This principle has been demonstrated to detect blood group phenotypes: ABO and RhD. However, typing for red blood cell antigens such as Rh, Kell, Duffy and Kidd has not yet been explored on paper., Materials and Methods: Two paper testing methods - an elution and a direct flow-through method - were investigated to detect red blood cell antigens excluding the ABO system and RhD. Antigens explored include the following: C, c, E, e, K, k, Fy(a), Fy(b), Jk(a), Jk(b), M, N, S and s, P1, Le(a) and Le(b). The variables tested include the following: reaction time and reagent concentration. The importance of antibody type/structure for successful agglutination on paper was confirmed., Results: Some blood group phenotypes showed less agglutination due to weaker antibody-antigen interactions. Most blood groups with antibodies available as IgM, such as C, c, E, e, K and k, and Jk(a) and Jk(b), and P1, were successful using both methods. However, other blood groups, especially those with antibodies only available as polyclonal antibodies, were unsuccessful and require further scrutiny., Conclusion: Paper can be used as an alternative blood grouping diagnostic tool for selected blood group phenotypes., (© 2014 International Society of Blood Transfusion.)

Background and Objectives: The aim of the study was to evaluate, in an international collaboration, four lyophilised genomic DNA preparations, selected from genotyped and phenotyped donors by the study organisers, for their suitability to standardise and control blood group genotyping procedures for common ancestral Caucasian and Black African alleles., Materials and Methods: Twenty-nine laboratories performed 'blind' testing of replicated ampoules of the candidate reference reagents, RBC1 (10/232), RBC4 (10/236), RBC5 (10/238) and RBC12 (10/234), using a range of genotyping procedures, most commonly classical PCR using allele or sequence specific primers., Results: The majority of laboratories reported blood group genotypes in accordance with those determined by the study organisers and the serological phenotypes. Despite an overall high level of accuracy in genotyping, the identified errors and inconsistencies, and the limited genotyping capabilities of many laboratories, confirmed the need for validated reference materials to control test procedures., Conclusions: The establishment of RBC1, RBC4, RBC5 and RBC12 as World Health Organization Reference Reagents will facilitate international standardisation of blood group genotyping and ensure that such tests are sufficiently sensitive and specific., (© 2012 The Authors(s). Vox Sanguinis © 2012 International Society of Blood Transfusion.)

Blood group antigens, present on the cell membrane of red blood cells and platelets, can be defined either serologically or predicted based on the genotypes of genes encoding for blood group antigens. At present, the molecular basis of many antigens of the 30 blood group systems and 17 human platelet antigens is known. In many laboratories, blood group genotyping assays are routinely used for diagnostics in cases where patient red cells cannot be used for serological typing due to the presence of auto-antibodies or after recent transfusions. In addition, DNA genotyping is used to support (un)-expected serological findings. Fetal genotyping is routinely performed when there is a risk of alloimmune-mediated red cell or platelet destruction. In case of patient blood group antigen typing, it is important that a genotyping result is quickly available to support the selection of donor blood, and high-throughput of the genotyping method is not a prerequisite. In addition, genotyping of blood donors will be extremely useful to obtain donor blood with rare phenotypes, for example lacking a high-frequency antigen, and to obtain a fully typed donor database to be used for a better matching between recipient and donor to prevent adverse transfusion reactions. Serological typing of large cohorts of donors is a labour-intensive and expensive exercise and hampered by the lack of sufficient amounts of approved typing reagents for all blood group systems of interest. Currently, high-throughput genotyping based on DNA micro-arrays is a very feasible method to obtain a large pool of well-typed blood donors. Several systems for high-throughput blood group genotyping are developed and will be discussed in this review.

Background: Blood samples from patients with sickle cell disease (SCD) present to transfusion service with numerous antibodies, making the searching for compatible red blood cells (RBC) a challenge. To overcome this problem we developed an effective strategy to meet needs of supplying RBC-compatible units to SCD patients using DNA arrays., Methods: We selected DNA samples from 144 SCD patients with multiple (receiving > 5 units) transfusions previously phenotyped for ABO, Rh(D, C, c, E, e), K1, Fy(a) and Jk(a). We also selected DNA samples from 948 Brazilian blood donors whose ABO/RhD phenotype matched that of the patients. All samples were analysed by DNA array analysis (HEA Beadchip(TM), Bioarray Solutions) to determine polymorphisms associated with antigen expression for 11 blood group systems (Rh, Kell, Kidd, Duffy, MNS, Dombrock, Lutheran, Landsteiner-Wiener, Diego, Colton, Scianna); and one mutation associated with haemoglobinopathies., Results: Based on genotype results we were able to predict phenotype-compatible donors needed in order to provide compatible units to this group of patients. Based on their ABO/Rh phenotype we were able to find in this pool of donors compatible units for 134 SCD patients., Conclusion: Blood group genotyping by DNA array contributes to the management of transfusions in SCD patients by facilitating the transfusion support with antigen-matched blood. It has the potential to improve the life of thousands of SCD-transfused patients by reducing mortality due to transfusion reactions and immunization.

In order to screen for antigen-negative blood donors, it is necessary to have appropriate, potent antisera in sufficient volume. Anti-Do(a) and anti-Do(b) are notoriously weakly reactive antibodies, available only in small volumes, usually in sera containing other alloantibodies, and often deteriorate on storage. Thus, it has not been possible to test large numbers of blood samples to find Do (a-) or Do (b-) blood donors. At the NYBC, we now type selected donors for DOA and DOB by DNA analysis. Initially, we tested DNA prepared from donors who had been typed by hemagglutination for one or both antigens. We found that four donors, whose RBCs previously typed as Do (a+b-), had both DOA and DOB alleles, and when retested, the RBCs were Do (a+b+w). We have now tested over 300 donors for DO by PCR-RFLP using either Eam1105 I or BseRI restriction enzymes. Blood from DOA/DOA donors has survived better than "crossmatch compatible" blood for patients with anti-Do(b) and such results suggest that anti-Do(b) is a more frequent cause of transfusion reactions than reported. Furthermore, we have demonstrated that PCR-RFLP can be used to screen for antigen-negative donors in other blood group systems when appropriate antisera are not available. When interpreting the results, it is important to remember that the genotype may not reflect the phenotype. Our strategy has been to perform DNA analysis for the DO alleles on those donors who have been shown by hemagglutination to lack antigens corresponding to multiple alloantibodies in patients' plasma. In this way, we have been able to supply rare blood to numerous patients, whose serum contained at least 5 additional alloantibodies of clinical significance.

Background and Objectives: During the past 10 years several DNA-typing methods have been developed to complement routine serological typing for determination of polymorphisms in the ABO, RH, KEL, JK and FY blood group genes. However, the molecular basis of blood groups can differ widely between ethnic groups. The purpose of this study was to evaluate selected DNA-based methods for phenotype prediction in a population not previously investigated., Materials and Methods: Blood samples from a random sample of Jordanian blood donors were collected and red cells isolated from these blood samples were phenotyped for common ABO (n = 150) and KEL/FY/JK (n = 90) antigens. RHD-negative and -positive donors were selected for RH typing (n = 120 and 30, respectively). DNA was prepared and blood group genotyping performed according to selected methods in current use. Discordant samples required further investigation by extended serology and DNA sequencing., Results: The degree of concordance between phenotype and genotype was high, but some exceptions were noted. Two of 14 A2/A2B samples lacked all mutations associated with known A2 alleles of the ABO system. RH typing revealed four samples with the c(cyt48) marker, causing false-positive RHC typing. A single D-negative sample was positive for D-specific exon 10 markers. The RHD pseudogene was not found in the 150 donors tested. Nine samples revealed discrepancies that were associated with unknown silent or weakly expressing Fyb-like alleles., Conclusions: With the exception of the FY system, we conclude that the molecular background of the clinically important blood group antigens studied here is similar to that reported for Caucasoids.

The increasing pace of development in molecular biological techniques during the last 10-15 years has had a direct effect on mass testing and diagnostic applications, including blood screening. Nucleic acid amplification techniques (NAT), usually based on the polymerase chain reaction (PCR), have been successfully applied to blood grouping and implemented recently in screening of blood donations for hepatitis C virus (HCV). The majority of microarray technologies involve an amplification step, yet the main benefits of this technology come from simultaneous analysis of thousands of analytes. Microarrays were developed to utilize the huge amount of information provided by genome projects, but they have clear potential in mass screening and diagnostics. The application of microarray technology may revolutionize blood testing, providing for the first time the prospect of an integrated platform for comprehensive donor and donation testing, replacing multiple individual assays. Design features of a blood-testing chip and various technologies with potential application in this field are discussed in this review.

Background and Objectives: The prenatal determination of fetal blood group status by molecular techniques has been used in the clinical management of alloimmunised pregnancies for seven years, in particular for the definition of fetal Rh D, c and E, K, Fya and Jka status. This has arisen in response to the definition of the molecular bases of human blood group polymorphism., Materials and Methods: PCR-based amplification assays have been designed to define fetal blood group status, where the source of template DNA is normally derived from amniotic fluid or chorionic villus. Recently, non-invasive methods have been explored to obtain fetal DNA from maternal peripheral blood., Results: PCR-based tests are now available to screen for all fetal medicine significant blood group antigens. The Rh system is the most complex, and assays to define Rh genotype have been modified in response to our increased understanding of the molecular biology of this blood group system., Conclusion: Prenatal diagnosis of fetal blood group status is now in widespread use in the clinical management of HDN. Non-invasive testing, if applied in the clinical setting may invoke a dramatic increase in the numbers of pregnancies that may be analysed prenatally.

With the integration of laboratory information systems into transfusion services, it is now possible to develop standard operating procedures (SOPs) for an electronic crossmatch (EXM) to replace the immediate-spin crossmatch for detecting ABO incompatibility between the blood sample submitted for pre-transfusion testing and the donor unit selected for transfusion. Essential to the safety of an EXM are requirements that: 1) the computer contains logic to prevent assignment and release of ABO incompatible blood; 2) no clinically significant antibodies are detected in the recipient's serum/plasma and there is no record of previous detection of such antibodies; 3) there are concordant results of at least two determinations of the recipient's ABO type on record, one of which is from a current sample; 4) critical elements of the system have been validated on-site; and, 5) there are mechanisms to verify the correct entry of data prior to release of blood. EXM procedures are in use in at least 10 North American facilities, Scandinavia, Hong Kong and Australia. Some provide blood at remote sites lacking laboratory services. Experience has shown that a combination of properly programmed computer software and carefully developed SOPs can provide a safe and efficient means of detecting ABO incompatibility without performing a serological crossmatch.

Background and Objectives: A patient experienced a severe haemolytic transfusion reaction. Neither the haemolytic property nor the specificity of the causative antibody had been sufficiently recognised when performing a microcolumn gel test., Materials and Methods: Subsequent to the transfusion reaction, the serological property and specificity of the causative antibody were analysed. Tube and gel test methods were compared, as were various reagent red cell specimens and their constituents., Results: A haemolytic anti-Vel was detected in the tube test. In contrast, the particular commercial gel test kit used did not reveal the haemolytic property or specificity of the antibody. Our experiments suggest that this was apparently due to the presence of EDTA in the low ionic strength saline solution of the test kit., Conclusions: In rare cases life-treatening haemolytic activity of an irregular blood group antibody may be undetected by a commericial microcolum gel test kit in which EDTA is a constituent.

Phospholipid depletion of human erythrocyte membranes by phospholipase A2 modification markedly decreased binding of two monoclonal antibodies to the Wrb (Wright) blood group determinant on glycophorin A. Binding of seven monoclonal antibodies to other glycophorin A determinants, including anti-M and anti-N, was unaffected by phospholipid depletion. These results indicate that Wrb antigen expression is phospholipid-dependent in human erythrocyte membranes.

50 Cad (+) blood samples were tested using various anti-Cad reagents. Results demonstrated a wide Cad antigenic expression, even among the members of one family. In particular experimental procedures, almost all Cad (+) cells were polyagglutinable.