A New Strategy To Identify Rare Blood Donors: Single Polymerase Chain Reaction Multiplex Snapshot Reaction For Detection Of 16 Blood Group Alleles

Background. As an alternative to phenotyping, large-scale DNA-based assays, which are feasible for high-throughput donor red blood cell typing, were developed for determination of blood group polymorphisms. However, high-throughput genotyping platforms based on these technologies are still expensive and the inclusion of single nucleotide polymorphisms and analysis of the alleles depend on the manufacturer's determination. To overcome this limitation and in order to develop an assay to enable the screening of rare donors, we developed a SNaPshot assay for analysis of nine single nucleotide polymorphisms related to antigens that are difficult to assess using conventional serology. Materials and methods. The single polymerase chain reaction multiplex SNaPshot reaction was optimized to identify nine single nucleotide polymorphisms determining 16 alleles: KEL*3/KEL*4, KEL*6/KEL*7, DI*1/DI*2, DI*3/DI*4, YT*1/YT*2, CO*1/CO*2, DO*1/DO*2, DO*4, DO*5. We designed a single multiplex PCR with primers encompassing the blood group single nucleotide polymorphisms and performed an internal reaction with probe primers able to discriminate the alleles after fragment analysis. The SNaPshot assay was validated with 140 known alleles previously determined by PCR restriction fragment length polymorphism. Results. We were able to simultaneous detect nine single nucleotide polymorphisms defining 16 blood group alleles on an assay based on a multiplex PCR combined with a single base extension using genomic DNA. Discussion. This study demonstrates a robust genotyping strategy for conducting rare donor screening which can be applied in blood centers and could be an important tool for identifying antigen-negative donors and, therefore, for providing rare blood. © SIMTI Servizi Srl.12SUPPL.1s256s263Jungbauer, C., Routine use of DNA testing for red cell antigens in blood centres (2011) Transfus Apher Sci, 45, pp. 61-68Nance, S.T., How to find, recruit and maintain rare blood donors (2009) Curr Opin Hematol, 16, pp. 503-508Veldhuisen, B., Van Der Schoot, C.E., De Haas, M., Blood group genotyping: From patient to high-throughput donor screening (2009) Vox Sang, 97, pp. 198-206Moulds, J.M., Future of molecular testing for red blood cell antigens (2010) Clin Lab Med, 30, pp. 419-429Patnaik, S.K., Helmberg, W., Blumenfeld, O.O., BGMUT: NCBI dbRBC database of allelic variations of genes encoding antigens of blood group systems (2012) Nucleic Acids Res, 40, pp. D1023-D1029Vallone, P.M., Butler, J.M., AutoDimer: A screening tool for primer-dimer and hairpin structures (2004) Biotechniques, 37, pp. 226-231Baleotti Jr., W., Rios, M., Reid, M.E., Dombrock gene analysis in Brazilian people reveals novel alleles (2006) Vox Sang, 91, pp. 81-87Rios, M., Hue-Roye, K., Oyen, R., Insights into the Holleyand Joseph- phenotypes (2002) Transfusion, 42, pp. 52-58Baleotti Jr., W., Rios, M., Reid, M.E., A novel DI*A allele without the Band 3-Memphis mutation in Amazonian Indians (2003) Vox Sang, 84, pp. 326-330Arnoni, C., Latini, F.R.M., Person, R.M., Padronização das técnicas de PCR-RFLP para genotipagem dos alelos KEL*3/ KEL*4 e KEL*5/KEL*6 (2011) Rev Bras Hematol Hemoter, 33 (SUPPL.2), pp. 332-488Baleotti Jr., W., Suzuki, R.B., Ruiz, M., A PCR-RFLP strategy for Wright typing (2011) Rev Bras Hematol Hemoter, 33 (SUPPL. 2), pp. 332-488Brazilian Real - United States Dollar Exchange Rate from Central Bank of Brazil, , http://www4.bcb.gov.br/pec/taxas, April 1st to April 30th, 27/03/2013Daniels, G., The molecular genetics of blood group polymorphism (2009) Hum Genet, 126, pp. 729-742Logdberg, L., Reid, M.E., Zelinski, T., Human blood group genes 2010: Chromosomal locations and cloning strategies revisited (2011) Transfus Med Rev, 25, pp. 36-46Di Cristofaro, J., Silvy, M., Chiaroni, J., Bailly, P., Single PCR multiplex SNaPshot reaction for detection of eleven blood group nucleotide polymorphisms: Optimization, validation, and one year of routine clinical use (2010) J Mol Diagn, 12, pp. 453-460Ferri, G., Pelotti, S., Multiplex ABO genotyping by minisequencing (2009) Methods Mol Biol, 496, pp. 51-58Palacajornsuk, P., Halter, C., Isakova, V., Detection of blood group genes using multiplex SNaPshot method (2009) Transfusion, 49, pp. 740-749Silvy, M., Simon, S., Gouvitsos, J., Weak D and DEL alleles detected by routine SNaPshot genotyping: Identification of four novel RHD alleles (2011) Transfusion, 51, pp. 401-411Silvy, M., Di Cristofaro, J., Beley, S., Identification of RHCE and KEL alleles in large cohorts of Afro-Caribbean and Comorian donors by multiplex SNaPshot and fragment assays: A transfusion support for sickle cell disease patients (2011) Br J Haematol, 154, pp. 260-270Pastinen, T., Kurg, A., Metspalu, A., Minisequencing: A specific tool for DNA analysis and diagnostics on oligonucleotide arrays (1997) Genome Res, 7, pp. 606-614Syvanen, A.C., From gels to chips: "Minisequencing" primer extension for analysis of point mutations and single nucleotide polymorphisms (1999) Hum Mutat, 13, pp. 1-10Information notebook (2011) Blood and Hemoderivates Brasília, , Ministério da Saúde. Secretaria de Atenção à Saúde. Coordenação-Geral de Sangue e Hemoderivados. Hemotherapy production. Unified Health System - SUS Brazil - (Public and private contractors). Private non-contracted services by Unified Health System (SUS Brazil). 4th edSantos, N.P., Ribeiro-Rodrigues, E.M., Ribeiro-Dos-Santos, A.K., Assessing individual interethnic admixture and population substructure using a 48-insertion-deletion (INSEL) ancestry-informative marker (AIM) panel (2010) Hum Mutat, 31, pp. 184-190Storry, J.R., Human blood groups: Inheritance and importance in transfusion medicine (2003) J Infus Nurs, 26, pp. 367-37

Evaluation Of Anti-d Reagents In The Detection Of Weak D And Partial D Antigens [avaliação De Reagentes Anti-d Na Detecção Dos Antígenos D Fraco E D Parcial]

Monoclonal antibodies (MoAb) anti-D IgG and IgM are being developed in order to replace polyclonal antibodies. However, there are few studies about the selection of these MoAb to routinely detect weak D and partial D antigens. A total of 56 weak D blood samples were analyzed with anti-D IgG and IgM MoAb in order to evaluate their reactivity. Molecular analyses were also performed to characterize the weak D types and the presence of partial D. Antigen densities were determined by flow cytometry. Weak D type 1, 3 and 4 samples with high antigen density were reactive with the MoAb anti-D IgM at room temperature while weak D type 2 and the partial D samples with low antigen density were detected with MoAb anti-D IgG using the indirect antiglobulin test. Our results show that anti-D IgM does not detect partial D samples reactive as weak D and thus can be used for routine testing of patients. As the anti-D IgG detected all the weak D and partial D with low antigen density, it should be used in the routine testing of donors.284269274Lomas-Francis, C., Tippett, P., Thompson, K.M., Demonstration of seven epitopes on the D antigen using human monoclonal anti-D antibodies and red cells from D categories (1984) Vox Sang, 57, pp. 261-264Tippett, P., Lomas-Francis, C., Wallace, M., The Rh antigen D: Partial D antigen and associated low incidence antigen (1996) Vox Sang, 70, pp. 123-131Huang, C.H., Chen, Y., Reid, M.E., Ghosh, S., Genetic recombination at the human RH locus: A family study of the red cell Evans phenotype reveals a transfer of exons 2-6 from the to the RHCE gene (1996) American Journal Genetic, 59, pp. 825-833Rouillac C, Colin Y, Hughes-Jones NC, Boelet MD, Ambrosio AM, Cartron JP, et al. Transcript analysis of D category phenotypes predicts hybrid RhD-CE-D proteins associated with alteration of D epitopes. Blood 1995;85:2.937-44Mouro I, Le Van Kim C, Rouillac C. Rearrangements of the blood group RhD gene associated with the DVI category phenotype . Blood 1994;83:1129-35Becker, E.A.M., Faas, B.H.W., Simser, S., Overbeeker, M.A.M., Van Rhernen, D.J., The genetic basic of a new partial D antigen: DDBT (1996) British Journal of Haematology, 93, pp. 720-727Wagner FF, Gassner C, Smuller TH, Schonitzer D, Schunter F, Flegel WA. Three molecular strutures cause Rhesus D category VI phenotypes with distinct immunohematological feature Blood 1998;91:2.157-68Hemker MB, Ligthart PC, Berger L, Van Rhenen DJ, Van Der Schoot CE, Maaskant-Van Wijk PA. DAR, a new RhD variant involving exon 4,5 and 7, often in linkage with ceAR, a new Rhce variant frequently found in African Black. Blood 1999;94:4.337-42Wagner, F.F., Frohmajer, A., Ladewig, B., Eicher, N.I., Lonicer, C.B., Muller, T.H., Weak D alleles express distinct phenotypes (2000) Blood, 95, pp. 2.699-2.708Müller, T.H., Wagner, F.F., Trockenbacher, A., Eicher, N.I., Flegel, W.A., Schönitzer, D., PCR screening for common weak D types shows different distributions in three Central Europeans populations (2001) Transfusion, 41, pp. 45-52Wagner, F.F., Flegel, W.A., The molecular basis of the Rh blood group phenotypes (review) (2004) Immunohematology, 20, pp. 23-36Flegel, W.A., Wagner, F.F., Molecular biology of partial D and weak D. Implications for blood bank practice (2002) Clin Lab, 48, pp. 53-59Castilho, L., Rios, M., Rodrigues, A., Pellegrino Jr, J., Saad, S.T.O., Costa, F.F., High frequency of partial DIIIa and DAR alleles found in sickle cell disease patients suggests increased risk of alloimmunization to RhD (2005) Transfusion Medicine, 15, pp. 49-55Flegel, W.A., Khull, S.R., Wagner, F.F., Primary anti-D immunization by weak D types 2 RBCs (2000) Transfusion, 40, pp. 428-434Mota, M., Fonseca, N.L., Rodrigues, A., Kutner, J.M., Castilho, L., Anti-D alloimmunization by weak D type 1 red blood cells with a very low antigen density (2005) Vox Sang, 88, pp. 130-135Ansart-Pirenne, H., Asso-Bonnet, M., Le Pennec, P.Y., Roussel, M., Patereau, C., Noizat-Pirenne, F., RhD variants in Caucasians: Consequences for checking clinically relevant alleles (2004) Transfusion, 44, pp. 1282-1286Rodrigues, A., Toledo, R., Zanelli, A.P., Oliveira, M.C.V., Ramos, R., Fujita, C., Molecular characterization of weak D in Brazilians: Impact for typing and transfusion strategy (2005) Transfusion, 45 (S), pp. SP340Reid, M.E., Rios, M., Powell, D., Charles-Pierre, D., Malavade, V., DNA from blood samples can be used to genotype patients who have recently received a transfusion (2000) Transfusion, 40, pp. 48-53Maaskant-Van Wijk, P.A., Faas, B.H., De Ruijter, J.A., Overbeeke, M.A., Von Dem Borne, A.E., Van Rhenen, D.J., Genotyping of RHD by multiplex polymerase chain reaction analysis of six RHD-specific exons (1998) Transfusion, 38, pp. 1015-1021Annex 2: Rh antibodies. Third International Workshop and Symposium on monoclonal antibodies against red blood cells and related antigens Transf Clin Biol 1996;6:525Flegel, W.A., Wagner, F.F., RHD epitope density profiles of RHD variant red cells analysed by flow cytometry (1996) Transf Clin Biol, 3, pp. 429-431Flegel, W.A., Curin-Serbec, V., Delamaire, M., Section 1B: Rh flow cytometry. Coordinator's report. Rhesus index and antigen density: an analysis of the reproducibility of flow cytometry determination (2002) Transf Clin Biol, 9, pp. 33-4

An Easy And Efficient Strategy For Kel Genotyping In A Multiethnic Population

Background: The Kell blood group system expresses high and low frequency antigens with the most important in relation to transfusion including the antithetic KEL1 and KEL2; KEL3 and KEL4; KEL6 and KEL7 antigens. Kell is a clinically relevant system, as it is highly immunogenic and anti-KEL antibodies are associated with hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. Although required in some situations, Kell antigen phenotyping is restricted due to technical limitations. In these cases, molecular approaches may be a solution. This study proposes three polymerase chain reaction genotyping protocols to analyze the single nucleotide polymorphisms responsible for six Kell antithetic antigens expressed in a Brazilian population.Methods: DNA was extracted from 800 blood donor samples and three polymerase chain reaction-restriction fragment length polymorphism protocols were used to genotype the KEL*1/KEL*2, KEL*3/KEL*4 and KEL*6/KEL*7 alleles. KEL*3/KEL*4 and KEL*6/KEL*7 genotyping was standardized using the NlalII and MnlI restriction enzymes and validated using sequencing. KEL*1/KEL*2 genotyping was performed using a previously reported assay. Results: KEL genotyping was successfully implemented in the service; the following distribution of KEL alleles was obtained for a population from southeastern Brazil: KEL*1 (22%), KEL*2 (97.8%), KEL*3 (0.69%), KEL*4 (99.31%), KEL*6 (2.69%o) and KEL*7 (9731%o). Additionally, two individuals with rare genotypes, KEL*1/KEL*1 and KEL*3/KEL*3, were identified. Conclusion: KEL allele genotyping using these methods proved to be reliable and applicable to predict Kell antigen expressions in a Brazilian cohort. This easy and efficient strategy can be employed to provide safer transfusions and to help in rare donor screening.35299102(2012), http://www.isbtweb.org, International Society of Blood Transfusion.[Internet].Amsterdam: ISBT, cited 2012 Jun 8Redman, C.M., Avellino, G., Pfeffer, S.R., Mukherjee, T.K., Nichols, M., Rubinstein, P., Kell blood group antigens are part of a 93,000-dalton red cell membrane protein (1986) J Biol Chem, 261 (20), pp. 9521-9525Lee, S., The value of DNA analysis for antigens of the Kell and Kx blood group systems (2007) Transfusion, 47 (SUPPL.1), pp. 32S-39SDaniels, G.L., Anstee, D.J., Cartron, J.P., Dahr, W., Henry, S., Issitt, P.D., Terminology for red cell surface antigens. Makuhari Report (1996) Vox Sang, 71 (4), pp. 246-248Daniels, G.L., Anstee, D.J., Cartron, J.P., Dahr, W., Issitt, P.D., Jorgensen, J., Blood group terminology 1995. ISBT Working Party on terminology for red cell surface antigens (1995) Vox Sang, 69 (3), pp. 265-279Lee, S., Molecular basis of Kell blood group phenotypes (1997) Vox Sang, 73 (1), pp. 1-11. , Erratum in: Vox Sang. 1998;74(1):58Daniels, G., The molecular genetics of blood group polymorphism (2009) Hum Genet, 126 (6), pp. 729-742Westhoff, C.M., Reid, M.E., Review: The Kell, Duffy, and Kidd blood group systems (2004) Immunohematology, 20 (1), pp. 37-49Russo, D., Redman, C., Lee, S., Association of XK and Kell blood group proteins (1998) J Biol Chem, 273 (22), pp. 13950-13956Lee, S., Zambas, E., Green, E.D., Redman, C., Organization of the gene encoding the human Kell blood group protein (1995) Blood, 85 (5), pp. 1364-1370. , . Erratum in: Blood. 1996;87(11):4922Lee, S., Russo, D., Redman, C.M., The Kell blood group system: Kell and XK membrane proteins (2000) Semin Hematol, 37 (2), pp. 113-121Vaughan, J.I., Manning, M., Warwick, R.M., Letsky, E.A., Murray, N.A., Roberts, I.A., Inhibition of erythroid progenitor cells by anti-Kell antibodies in fetal alloimmune anemia (1998) N Engl J Med, 338 (12), pp. 798-803. , Comment in: N Engl J Med. 2000;343(1):72N Engl J Med. 1998;338(12):830-831Duguid, J.K., Bromilow, I.M., Haemolytic disease of the newborn due to anti-k (1990) Vox Sang, 58 (1), p. 69Smoleniec, J., Anderson, N., Poole, G., Hydrops fetalis caused by a blood group antibody usually undetected in routine screening (1994) Arch Dis Child Fetal Neonatal Ed, 71 (3), pp. F216-F217Gorlin, J.B., Kelly, L., Alloimmunisation via previous transfusion places female Kpb-negative recipients at risk for having children with clinically significant hemolytic disease of the newborn (1994) Vox Sang, 66 (1), pp. 46-48Donovan, L.M., Tripp, K.L., Zuckerman, J.E., Konugres, A.A., (1973), 13 (3), p. 153. , Daniels G. Hemolytic disease of the newborn due to anti-Js a. TransfusionStanworth, S., Fleetwood, P., de Silva, M., Severe haemolytic disease of the newborn due to anti-Js(b) (2001) Vox Sang, 81 (2), pp. 134-135Daniels, G., Kell and Kx blood group systems (1995) Human Blood Groups, pp. 385-420. , Oxford: Blackwell ScienceRace, R.R., Sanger, R., (1975) Blood Groups In Man, , 6th ed. Oxford: Blackwell ScientificJungbauer, C., Routine use of DNA testing for red cell antigens in blood centres (2011) Transfus Apher Sci, 45 (1), pp. 61-68Hillyer, C.D., Shaz, B.H., Winkler, A.M., Reid, M., Integrating molecular technologies for red blood cell typing and compatibility testing into blood centers and transfusion services (2008) Transfus Med Rev, 22 (2), pp. 117-132Reid, M.E., Rios, M., Powell, V.I., Charles-Pierre, D., Malavade, V., DNA from blood samples can be used to genotype patients who have recently received a transfusion (2000) Transfusion, 40 (1), pp. 48-53Vallone, P.M., Butler, J.M., AutoDimer: A screening tool for primer-dimer and hairpin structures (2004) Biotechniques, 37 (2), pp. 226-231Santos, N.P., Ribeiro-Rodrigues, E.M., Ribeiro-Dos-Santos, A.K., Pereira, R., Gusmao, L., Amorim, A., Assessing individual interethnic admixture and population substructure using a 48-insertion-deletion (INSEL) ancestry- informative marker (AIM) panel (2010) Hum Mutat, 31 (2), pp. 184-190Renoud, K.J., Barracchini, K., Byrne, K.M., Adams, S., Pickett, A., Caruccio, L., KEL6 and KEL7 genotyping with sequence-specific primers (2006) Transfusion, 46 (9), pp. 1510-151