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

Population Genetics of GYPB and Association Study between GYPB*S/s Polymorphism and Susceptibility to P. falciparum Infection in the Brazilian Amazon

Merozoites of Plasmodium falciparum invade through several pathways using different RBC receptors. Field isolates appear to use a greater variability of these receptors than laboratory isolates. Brazilian field isolates were shown to mostly utilize glycophorin A-independent invasion pathways via glycophorin B (GPB) and/or other receptors. The Brazilian population exhibits extensive polymorphism in blood group antigens, however, no studies have been done to relate the prevalence of the antigens that function as receptors for P. falciparum and the ability of the parasite to invade. Our study aimed to establish whether variation in the GYPB*S/s alleles influences susceptibility to infection with P. falciparum in the admixed population of Brazil.Two groups of Brazilian Amazonians from Porto Velho were studied: P. falciparum infected individuals (cases); and uninfected individuals who were born and/or have lived in the same endemic region for over ten years, were exposed to infection but have not had malaria over the study period (controls). The GPB Ss phenotype and GYPB*S/s alleles were determined by standard methods. Sixty two Ancestry Informative Markers were genotyped on each individual to estimate admixture and control its potential effect on the association between frequency of GYPB*S and malaria infection.GYPB*S is associated with host susceptibility to infection with P. falciparum; GYPB*S/GYPB*S and GYPB*S/GYPB*s were significantly more prevalent in the in the P. falciparum infected individuals than in the controls (69.87% vs. 49.75%; P<0.02). Moreover, population genetics tests applied on the GYPB exon sequencing data suggest that natural selection shaped the observed pattern of nucleotide diversity.Epidemiological and evolutionary approaches suggest an important role for the GPB receptor in RBC invasion by P. falciparum in Brazilian Amazons. Moreover, an increased susceptibility to infection by this parasite is associated with the GPB S+ variant in this population

A PCR-Based Strategy for Dombrock Screening in Brazilian Blood Donors Reveals a Novel Allele: The DO*A-WL

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Background: Determination of the molecular basis underlying the antigens in the Dombrock blood group system has shown various rearrangements between the alleles associated with DO*A and DO*B. Based on this, we employed a PCR-based strategy to screen DO alleles (DO*A, DO*B, HY*1, HY*2 and JO) in Brazilians. Methods: We tested DNA of 278 Brazilian blood donors by PCR-RFLP on plates of 96 wells to determine the 793A/G (DO*A/DO*B), 323G/T (HY), 350C/T (JO) and 898C/G (HY*1/HY*2) single nucletide polymorphisms. In order to confirm the results sequence analysis was also performed. Results: When samples of these donors were analyzed, a novel allele combination, the DO*A allele (793A and 323G) associated with 898G was identified and designated as DO*A-WL allele. This new allele encoding 300Val is the same as HY*1 at nucleotide 898 on the molecular background of DO*A. Among the 556 alleles analyzed by PCR-RFLP, 3 were DO*A-WL and 78 were DO*B-WL. This represents an overall frequency of 0.5% for DO*A-WL and 14% for DO*B-WL across the population studied. Conclusion: Molecular screening of Brazilians revealed one novel allele, the DO*A-WL. Our data highlight the importance of testing a cohort of different populations to determine DO haplotypes and to establish reliable genotyping tests for predicting Do(a)/Do(b) status. J. Clin. Lab. Anal. 25:79-82, 2011. (C) 2011 Wiley-Liss, Inc.2527982Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [07/59546-1

A Pcr-based Strategy For Dombrock Screening In Brazilian Blood Donors Reveals A Novel Allele: The Do Z.ast;a-wl

Determination of the molecular basis underlying the antigens in the Dombrock blood group system has shown various rearrangements between the alleles associated with DO z.ast;A and DO z.ast;B. Based on this, we employed a PCR-based strategy to screen DO alleles (DO z.ast;A, DO z.ast;B, HY z.ast;1, HY z.ast;2 and JO) in Brazilians. Methods: We tested DNA of 278 Brazilian blood donors by PCR-RFLP on plates of 96 wells to determine the 793A/G (DO z.ast;A/DO z.ast;B), 323G/T (HY), 350C/T (JO) and 898C/G (HY z.ast;1/HY z.ast;2) single nucletide polymorphisms. In order to confirm the results sequence analysis was also performed. Results: When samples of these donors were analyzed, a novel allele combination, the DO z.ast;A allele (793A and 323G) associated with 898G was identified and designated as DO z.ast;A-WL allele. This new allele encoding 300Val is the same as HY z.ast;1 at nucleotide 898 on the molecular background of DO z.ast;A. Among the 556 alleles analyzed by PCR-RFLP, 3 were DO z.ast;A-WL and 78 were DO z.ast;B-WL. This represents an overall frequency of 0.5% for DO z.ast;A-WL and 14% for DO z.ast;B-WL across the population studied. Conclusion: Molecular screening of Brazilians revealed one novel allele, the DO z.ast;A-WL. Our data highlight the importance of testing a cohort of different populations to determine DO haplotypes and to establish reliable genotyping tests for predicting Do a/Do b status. © 2011 Wiley-Liss, Inc.2527982Banks, J.A., Hemming, N., Poole, J., Evidence that the Gy a Hy and Jo a antigens belong to the Dombrock blood group system (1995) Vox Sang, 68, pp. 177-182Rios, M., Hue-Roye, K., Oyen, R., Miller, J., Reid, M.E., Insights into the Holley- and Joseph-phenotypes (2002) Transfusion, 42, pp. 52-58Spring, F.A., Reid, M.E., Nicholson, G., Evidence for expression of the Joa blood group antigen on the Gya/Hy-active glycoprotein (1994) Vox Sang, 66, pp. 72-77Gubin, A.N., Njoroge, J.M., Wojda, U., Identification of the Dombrock blood group glycoprotein as a polymorphic member of the ADP-ribosyltransferase gene family (2000) Blood, 96, pp. 2621-2627Scofield, T.L., Miller, J.P., Storry, J.R., Rios, M., Reid, M.E., Evidence that Hy- RBCs express weak Jo a antigen (2004) Transfusion, 44, pp. 170-172Strupp, A., Cash, K., Uehlinger, J., Difficulties in identifying antibodies in the Dombrock blood group system in multiply alloimmunized patients (1998) Transfusion, 38, pp. 1022-1025Shirey, R.S., Boyd, J.S., King, K.E., Caturegli, P.P., Montgomery Jr, W.M., Ness, P.M., Assessment of the clinical significance of anti-Dob (1998) Transfusion, 38, pp. 1026-1029Moulds, J.J., Polesky, H.F., Reid, M., Ellisor, S.S., Observations on the Gy-a and Hy antigens and the antibodies that define them (1975) Transfusion, 15, pp. 270-274Rios, M., Hue-Roye, K., Lee, A.H., Chiofolo, J.T., Miller, J.L., Reid, M.E., DNA analysis for the Dombrock polymorphism (2001) Transfusion, 41, pp. 1143-1146Storry, J.R., Westhoff, C.M., Charles-Pierre, D., DNA analysis for donor screening of Dombrock blood group antigens (2003) Immunohematology, 19, pp. 73-76Rios, M., Hue-Roye, K., Storry, J.R., Lee, T., Miller, J.L., Reid, M.E., Molecular basis of the Dombrock null phenotype (2001) Transfusion, 4, pp. 1405-1407Rios, M., Storry, J.R., Hue-Roye, K., Chung, A., Reid, M.E., Two new molecular bases for the Dombrock null phenotype (2002) Br J Haematol, 117, pp. 765-767Lucien, N., Celton, J.L., Le Pennec, P.Y., Cartron, J.P., Bailly, P., Short deletion within the blood group Dombrock locus causing a Do(null)phenotype (2002) Blood, 100, pp. 1063-1064Baleotti Jr, W., Rios, M., Reid, M.E., Dombrock gene analysis in Brazilian people reveals novel alleles (2006) Vox Sang, 91, pp. 81-87Hashmi, G., Shariff, T., Seul, M., A flexible array format for large-scale, rapid blood group DNA typing (2005) Transfusion, 45, pp. 680-688Chapel-Fernandes, S., Callebaut, I., Halverson, G.R., Reid, M.E., Bailly, P., Chiaroni, J., Dombrock genotyping in a native Congolese cohort reveals two novel alleles (2009) Transfusion, 49, pp. 1661-1761Castilho, L., Baleotti Jr, W., Tossas, E., Molecular studies of DO alleles reveal that JO is more prevalent than HY in Brazil, while HY is more prevalent in New York (2008) Immunohematology, 24, pp. 135-13

Dombrock gene analysis in Brazilian people reveals novel alleles

Background and Objectives The Do(a) and Do(b) polymorphisms are associated with three single nucleotide polymorphisms (SNPs) in exon 2 of the DO gene: 378C/T, 624T/C and 793A/G for the DOA and DOB alleles, respectively. The SNPs 350C/T (JO allele) and 323G/T (HY allele) are associated with the Jo(a-) and Hy-negative phenotypes. Recently, two new DO alleles [DOB-SH (378C, 624C, 793G) and DOA-HA (378T, 624T, 793A)] were identified using microarray technology. Although the molecular background of Dombrock alleles is well defined, no studies have been conducted in the Brazilian population. Materials and Methods We employed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based assays and a microarray assay to determine the frequency of the DO alleles (DOA, DOB, HY1, HY2 and JO) in Brazilians. We tested DNA of 288 Brazilians from three different ethnic groups by PCR-RFLP to determine the 793A/G (DOA/DOB), 323G/T (HY), 350C/T (JO) and 898C/G (HY1/HY2) SNPs. We also tested DNA from 162 blood donors by using the HEA Beadchip (TM) assay to determine the 378C/T, 624T/C, 793A/G (DOA/DOB), 350C/T (JO allele) and 323G/T (HY) SNPs. Results Two novel allele combinations were found in our samples: the DOB allele (793G and 323G) associated with 898G (DOB-WL); and an allele carrying the nucleotides 378C, 624C, 793A and 323G (DOA-SH). We also found the DOB-SH and DOA-HA.alleles recently reported. Conclusions Our data demonstrate high heterogeneity of DO alleles in the Brazilian population. Our study also highlights the importance of testing a cohort of different populations to determine DO haplotypes and of establishing reliable genotyping tests for predicting Do(a)/Do(b) status.911818

Hla-drb1∗07:01 Allele Is Primarily Associated With The Diego A Alloimmunization In A Brazilian Population

Background The Diego blood group presents a major polymorphic site at Residue 854, causing a proline (Dib antigen) to leucine (Dia antigen) substitution. Dia alloimmunization has been observed among Asian and Native South American populations. Considering that Brazilians represent a genetically diverse population, and considering that we have observed a high incidence of Dia alloimmunization, we typed HLA-DRB1 alleles in these patients and performed in silico studies to investigate the possible associated mechanisms.Study Design and Methods We studied 212 alloimmunized patients, of whom 24 presented immunoglobulin G anti-Dia, 15 received Di(a+) red blood cells and were not immunized, and 1008 were healthy donors. HLA typing was performed using commercial kits. In silico analyses were performed using the TEPITOPEpan software to identify Diego-derived anchor peptide binding to HLA-DRB1 molecules. Residue alignment was performed using the IMGT/HLA for amino acid identity and homology analyses.Results HLA-DRB1∗07:01 allele was overrepresented in Dia-alloimmunized patients compared to nonimmunized patients and to healthy donors. Two motifs were predicted to be potential epitopes for Dia alloimmunization, the WVVKSTLAS motif was predicted to bind several HLA-DR molecules, and the FVLILTVPL motif exhibited highest affinity for the HLA-DRB1∗07:01 molecule. Pocket 4 of the DRB1∗07:01 molecule contained specific residues not found in other HLA-DRB1 molecules, particularly those at Positions 13(Y), 74(Q), and 78(V).Conclusion Individuals carrying the HLA-DRB1∗07:01 allele present an increased risk for Dia alloimmunization. The identification of susceptible individuals and the knowledge of potential sensitization peptides are relevant approaches for transfusion care, diagnostic purposes, and desensitization therapies.541024682476Jay, D., Cantley, L., Structural aspects of the red cell anion exchange protein (1986) Annu Rev Biochem, 55, pp. 511-538Lux, S.E., John, K.M., Kopito, R.R., Cloning and characterization of band 3, the human erythrocyte anion-exchange protein (AE1) (1989) Proc Natl Acad Sci U S A, 86, pp. 9089-9093Tanner, M.J., Martin, P.G., High, S., The complete amino acid sequence of the human erythrocyte membrane anion-transport protein deduced from the cDNA sequence (1988) Biochem J, 256, pp. 703-712Tanner, M.J., The structure and function of band 3 (AE1): Recent developments (review) (1997) Mol Membr Biol, 14, pp. 155-165Bruce, L.J., Ring, S.M., Anstee, D.J., Changes in the blood group Wright antigens are associated with a mutation at amino acid 658 in human erythrocyte band 3: A site of interaction between band 3 and glycophorin A under certain conditions (1995) Blood, 85, pp. 541-547Bruce, L.J., Zelinski, T., Ridgwell, K., The low-incidence blood group antigen, Wda, is associated with the substitution Val557 - >met in human erythrocyte band 3 (AE1) (1996) Vox Sang, 71, pp. 118-120Jarolim, P., Murray, J.L., Rubin, H.L., A Thr552 - >ile substitution in erythroid band 3 gives rise to the Warrior blood group antigen (1997) Transfusion, 37, pp. 398-405Jarolim, P., Murray, J.L., Rubin, H.L., Blood group antigens Rb(a), Tr(a), and Wd(a) are located in the third ectoplasmic loop of erythroid band 3 (1997) Transfusion, 37, pp. 607-615Jarolim, P., Rubin, H.L., Zakova, D., Characterization of seven low incidence blood group antigens carried by erythrocyte band 3 protein (1998) Blood, 92, pp. 4836-4843Daniels, G.L., Anstee, D.J., Cartron, J.P., International Society of Blood Transfusion Working Party on terminology for red cell surface antigens (2001) Vox Sang, 80, pp. 193-197Storry, J.R., Castilho, L., Daniels, G., International Society of Blood Transfusion Working Party on red cell immunogenetics and blood group terminology: Berlin report (2011) Vox Sang, 101, pp. 77-82Baleotti, W., Jr., Rios, M., Reid, M.E., A novel DI∗A allele without the Band 3-Memphis mutation in Amazonian Indians (2003) Vox Sang, 84, pp. 326-330Layrisse, M., Arends, T., The Diego systemSteps in the investigation of a new blood group systemFurther studies (1957) Blood, 12, pp. 115-122Bruce, L.J., Anstee, D.J., Spring, F.A., Band 3 Memphis variant II. Altered stilbene disulfonate binding and the Diego (Dia) blood group antigen are associated with the human erythrocyte band 3 mutation Pro854 - >leu (1994) J Biol Chem, 269, pp. 16155-16158Treml, A., King, K.E., Red blood cell alloimmunization: Lessons from sickle cell disease (2013) Transfusion, 53, pp. 692-695Shariatmadar, S., Pyrsopoulos, N.T., Vincek, V., Alloimmunization to red cell antigens in liver and multivisceral transplant patients (2007) Transplantation, 84, pp. 527-531Moise, K.J., Red blood cell alloimmunization in pregnancy (2005) Semin Hematol, 42, pp. 169-178Nurse, G.T., Jenkins, T., Genetically determined hazards of blood transfusion within and between races (1973) S Afr Med J, 47, pp. 56-61Tormey, C.A., Stack, G., Immunogenicity of blood group antigens: A mathematical model corrected for antibody evanescence with exclusion of naturally occurring and pregnancy-related antibodies (2009) Blood, 114, pp. 4279-4282Giblett, E.R., A critique of the theoretical hazard of inter vs intra-racial transfusion (1961) Transfusion, 1, pp. 233-238Rammensee, H.G., Chemistry of peptides associated with MHC class i and class II molecules (1995) Curr Opin Immunol, 7, pp. 85-96Menconi, F., Osman, R., Monti, M.C., Shared molecular amino acid signature in the HLA-DR peptide binding pocket predisposes to both autoimmune diabetes and thyroiditis (2010) Proc Natl Acad Sci U S A, 107, pp. 16899-16903Singh, A., Sharma, P., Kar, H.K., HLA alleles and amino-acid signatures of the peptide-binding pockets of HLA molecules in vitiligo (2012) J Invest Dermatol, 132, pp. 124-134Picard, C., Frassati, C., Basire, A., Positive association of DRB1 04 and DRB1 15 alleles with Fya immunization in a Southern European population (2009) Transfusion, 49, pp. 2412-2417Noizat-Pirenne, F., Tournamille, C., Bierling, P., Relative immunogenicity of Fya and K antigens in a Caucasian population, based on HLA class II restriction analysis (2006) Transfusion, 46, pp. 1328-1333Zhang, L., Chen, Y., Wong, H.S., TEPITOPEpan: Extending TEPITOPE for peptide binding prediction covering over 700 HLA-DR molecules (2012) Plos One, 7, p. e30483Green, A., The epidemiologic approach to studies of association between HLA and disease. II. Estimation of absolute risks, etiologic and preventive fraction (1982) Tissue Antigens, 19, pp. 259-268Reviron, D., Dettori, I., Ferrera, V., HLA-DRB1 alleles and Jk(a) immunization (2005) Transfusion, 45, pp. 956-959Arnold, P.Y., La Gruta, N.L., Miller, T., The majority of immunogenic epitopes generate CD4+ T cells that are dependent on MHC class II-bound peptide-flanking residues (2002) J Immunol, 169, pp. 739-749Vignali, D.A., Strominger, J.L., Amino acid residues that flank core peptide epitopes and the extracellular domains of CD4 modulate differential signaling through the T cell receptor (1994) J Exp Med, 179, pp. 1945-1956Chiaroni, J., Dettori, I., Ferrera, V., HLA-DRB1 polymorphism is associated with Kell immunisation (2006) Br J Haematol, 132, pp. 374-37

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

A novel DI*A allele without the band 3-Memphis mutation in Amazonian Indians

Background and Objectives The blood-group antigens Di(a) and Di(b) are carried on erythrocyte band 3 and are defined by a single amino acid substitution at position 854 (Leu for Di(a) and Pro for Di(b) ). The Band 3-Memphis variant has a point mutation (166A>G) in the SLC4A1 gene, which encodes the amino acid substitution Lys56Glu. Two types of Band 3-Memphis, variants I and II, are distinguished by their susceptibility to covalent labelling with 4,4'-diisothiocyanato-1,2-diphenylethane-2,2'-disulphonic acid (H-2 DIDS). Memphis II is more readily labelled than Memphis I or normal band 3. It is reported that Memphis II is associated with Di(a) . In a study designed to determine the frequency of the DI *A /DI *B and 166A>G polymorphisms in different populations in Brazil, we found a new DI *A allele. Materials and Methods We studied DNA samples from 70 Amazonian Indians, 71 individuals of Japanese descent, 93 random Brazilian blood donors and 84 blacks with sickle cell disease. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses were performed on all samples, using Msp I for DI *A/DI *B (exon 19) and Mnl I for 166A>G (exon 4). Exon 4 and exon 19 from four outliers were sequenced. Results Among Amazonian Indians, DI *A and 166G mutations both had a high frequency (0.57 and 0.54, respectively). In individuals of Japanese descent, these alleles were moderately frequent (0.07 and 0.19, respectively). We identified a new allele with DI *A and 166A (56Lys) in four Amazonian Indians. Conclusions Our results revealed that DI *A does not have a strict association with 166G . They also show the relevance of testing a cohort of different populations.84432633