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

Rhce Variants Inherited With Altered Rhd Alleles In Brazilian Blood Donors

The high homology and opposite orientation of RH genes promote rearrangements between them and generate a large number of RHD and RHCE variants which can be inherited together. Searching of RHD-CE genotypes predicting partial antigens in donors is of interest in order to find more closely matched donors for African descent patients. This study aimed to evaluate a molecular approach to search for RhCE variants in a cohort of individuals with altered expression of D antigen and determine the association of RH variant alleles in Brazilian blood donors. METHODSFrom 80,961 blood samples tested, 421 with atypical D typing results were studied. The samples were phenotyped for C, c, E, e antigens. Rh variants were identified using molecular techniques. RESULTSAll 421 samples had altered RHD alleles, being 563% of them partial D. Among them, 949% presented variant RHCE*ce and the most common associations were: RHD*weak D type 4.2.2 with RHCE*ceAR; RHD*DAR linked to RHCE*ceVS.02; RHD*weak D type 4.0 linked to RHCE*ceVS.02 and RHCE*ce (c.48C, c.105T, c.733G, c.744C, c.1025T). Among the samples with RhCE variants, 106% predict partial c, partial e, hr(B)- and/or hr(S)- and 100% express low prevalence antigens. CONCLUSIONTargeting individuals with altered expression of D antigen can be a good strategy for finding donors with RhCE variants. In our study 949% of the partial D samples revealed altered RHCE variant alleles and 57% of the samples with altered RHD allele predicted partial c, partial e and the lack of the high prevalence hr(B)and hr(S)antigens.26428529

Avaliação sensorial de carne de capivaras adultas e jovens submetidas a diferentes dietas.

A carne de capivara ainda e uma incógnita quanto aos fatores que afetam o sabor e o aroma, os quais frequentemente podem ser indesejaveis para o consumidor. Hipóteses da idade e da alimentação tem sido sugeridas. O objetivo do presente trabalho foi estudar o efeito da alimentção e da categoria animal sobre o perfil sensorial de diferentes cortes de carne de capivaras

Two Novel Kel Alleles Encoding K0 Phenotypes In Brazilians

[No abstract available]54821282129Lee, S., Russo, D., Redman, C., Functional and structural aspects of the Kell blood group system (2000) Transfus Med Rev, 14, pp. 93-103Nunn, H.D., Giles, C.M., Dormandy, K.M., A second example of anti-Ku in a patient who has the rare Kell phenotype, Ko (1966) Vox Sang, 11, pp. 611-619Chown, B., Lewis, M., Kaita, K., A new Kell blood-group phenotype (1957) Nature, 180, p. 711Yu, L.C., Twu, Y.C., Chang, C.Y., Molecular basis of the Kell-null phenotype: A mutation at the splice site of human KEL gene abolishes the expression of Kell blood group antigens (2001) J Biol Chem, 276, pp. 10247-10252Lee, S., Russo, D.C., Reiner, A.P., Molecular defects underlying the Kell null phenotype (2001) J Biol Chem, 276, pp. 27281-27289Yang, M.H., Li, L., Kuo, Y.F., Genetic and functional analyses describe a novel 730delG mutation in the KEL gene causing K0 phenotype in a Taiwanese blood donor (2011) Transfus Med, 21, pp. 318-32