Genetic diversity of Gerbich alleles in Brazilians reveals an unexpected prevalence of the GE:-2,-3,4 phenotype.

BACKGROUND AND OBJECTIVES: Gerbich (GE) blood group system carries high-frequency antigens and the absence of them leads to rare phenotypes: GE:-2,3,4, GE:-2,-3,4 and GE:-2,-3,-4. Their serological differentiation is limited and misclassification of Gerbich phenotypes may occur, but this can be avoided by molecular characterization. This study aimed to characterize the molecular background responsible for rare Gerbich phenotypes in Brazilian population. MATERIALS AND METHODS: We selected eight samples from patients with anti-Ge, six from their relatives and nine samples with normal expression of Gerbich antigens. Serological tests were performed in gel and red blood cells (RBCs) were tested with anti-Ge2 and anti-Ge3. Monocyte monolayer assay (MMA) was performed. Molecular investigation was performed with allele-specific polymerase chain reaction and DNA sequencing. RESULTS: Patient plasma samples reacted with all commercial RBCs. Patient RBCs showed negative results with anti-Ge2 and anti-Ge3. Using MMA two of eight antibodies were clinically significant. Exon 3 was not amplified in any of the patient samples and in two samples from relatives, suggesting the presence of GE*01.-03/GE*01.-03. By sequencing, we identified the genetic variability that interferes with the definition of deletion breakpoints, thus two options of genetic structure were suggested to be responsible for the GE:-2,-3,4 phenotype. CONCLUSION: This study showed for the first time the genetic diversity of GYPC alleles for carriers of Gerbich-negative phenotypes in a Brazilian population and showed an unexpected prevalence of the GE:-2,-3,4 phenotype. It also demonstrated the importance of using molecular tools to correctly classify Gerbich phenotypes for selection of variants in antigen-matched transfusions.

RHCE diversity among Brazilian patients with sickle cell disease (SCD) and selected groups of blood donors.

BACKGROUND: Several centers have selected Black donors to prevent Rh alloimmunization of patients with sickle cell disease (SCD). As the Brazilian population is considered very admixed and race definition by self-declaration is questionable, this study aimed to compare RHCE diversity among patients with SCD and selected groups of Brazilian blood donors to define which group of donors would be the adequate red cell supply for patients with SCD. METHOD: We compared RHCE allele frequencies between patients with SCD and four groups of Brazilian blood donors: self-declared Black donors (SDB), donors with predominant African genetic markers (AAM), donors with weak D expression (WDD), and random donors (RDs). Variant RHCE alleles were identified using molecular protocols. RESULTS: Among patients with SCD, 47% had at least one variant RHCE, in SDB and WDD this frequency was higher, 53% and 58.6%, respectively. In AAM and in RD the frequencies were 32% and 27.6%, respectively. In patients with SCD and SDB, the most common alleles were RHCE*ce.01, RHCE*ceVS.01, and RHCE*ceVS.02. WDD had a high frequency of RHCE*ceAR and highest frequency of variant RHCE in both alleles, followed by patients with SCD and SDB. CONCLUSION: This study showed that even in an admixed population the selection of SDB donors is the best choice of matching for transfusion support in patients with SCD. For specific RHCE alleles, selection of donors with weak D expression could be a good option.

Antigen matching for transfusion support in Brazilian female patients with sickle cell disease to reduce RBC alloimmunization.

BACKGROUND: Red blood cell (RBC) alloimmunization is a complication of patients with sickle cell disease (SCD) and it has a greater impact on pregnancy, leading to a risk of hemolytic disease of the newborn and reducing blood availability for pregnant women. This study proposed to evaluate antigen matching transfusion protocols, aiming to reduce RBC alloimmunization in Brazilian female patients with SCD. METHODS: Samples from female patients with SCD (153) and self-declared Afro-Brazilian donors (307) were genotyped for RBC antigens and RH variants were investigated. The transfusion needs of patients during 1-year period and the number of compatible donors were assessed using three antigen-matching transfusion protocols: prophylactic CEK antigen-matched RBCs, prophylactic extended antigen-matched RBCs, and extended-matched red blood cells (RBCs) only for alloimmunized patients. In addition, RH molecular matching has been proposed for patients carrying variant RHCE. RESULTS: Provision of CEK antigen-matched donors would have been possible in 92.4% of transfusion events while provision of prophylactic extended antigen-matched RBCs would cover 88.7% of the transfusion events. Extended antigen matching for alloimmunized patients would be efficient in 99% of the cases. The presence of partial D in 10 patients increased the need of D-negative donors. Compatible donors could be enough for four of the five patients with altered RHCE genotypes in both alleles. CONCLUSION: In Brazilians, screening African descent donors allows the implementation of prophylactic CEK and extended antigen-matching transfusion protocols to female patients with SCD to reduce RBC alloimmunization; however, the supply of compatible blood can be impaired for patients with Rh variants.

Serologic strategy in detecting RHD altered alleles in Brazilian blood donors

ABSTRACT Background: We evaluated different technological approaches and anti-D clones to propose the most appropriate serologic strategy in detecting the largest numbers of D variants in blood donors. Methods: We selected 101 samples from Brazilian blood donors with different expressions of D in our donor routine. The tests were performed in immediate spin (IS) with eleven commercially available anti-D reagents in a tube and microplate. The D confirmatory tests for the presence of weak D included the indirect antiglobulin test (IAT) in a tube, gel and solid-phase red blood cell adherence (SPRCA). All DNA samples were extracted from peripheral blood and the D variants were classified using different molecular assays. Results: The RHD variants identified by molecular analysis included weak D types (1, 2, 3, 11 and 38) and partial Ds (DAR1.2, DAR1, DAR3.1, DAU0, DAU2, DAU4, DAU5, DAU6, DMH and DVII). The monoclonal-monoclonal blend RUM-1/MS26 was the best anti-D reagent used in detecting the D antigen in the IS phase in a tube, reacting with 83.2% of the D variants, while the anti-D blend D175 + 415 was the best monoclonal antibody (MoAb) used in a microplate to minimize the need for an IAT, reacting with 83.2% of the D variants. The D confirmatory tests using SPRCA showed a reactivity (3 - 4+) with 100% of the D variant samples tested. Conclusion: Our results show that, even using sensitive methods and MoAbs to ensure the accurate assignment of the D antigen, at least 17% of our donor samples need a confirmatory D test in order to avoid alloimmunization in D-negative patients.

Serologic strategy in detecting RHD altered alleles in Brazilian blood donors.

BACKGROUND: We evaluated different technological approaches and anti-D clones to propose the most appropriate serologic strategy in detecting the largest numbers of D variants in blood donors. METHODS: We selected 101 samples from Brazilian blood donors with different expressions of D in our donor routine. The tests were performed in immediate spin (IS) with eleven commercially available anti-D reagents in a tube and microplate. The D confirmatory tests for the presence of weak D included the indirect antiglobulin test (IAT) in a tube, gel and solid-phase red blood cell adherence (SPRCA). All DNA samples were extracted from peripheral blood and the D variants were classified using different molecular assays. RESULTS: The RHD variants identified by molecular analysis included weak D types (1, 2, 3, 11 and 38) and partial Ds (DAR1.2, DAR1, DAR3.1, DAU0, DAU2, DAU4, DAU5, DAU6, DMH and DVII). The monoclonal-monoclonal blend RUM-1/MS26 was the best anti-D reagent used in detecting the D antigen in the IS phase in a tube, reacting with 83.2% of the D variants, while the anti-D blend D175 + 415 was the best monoclonal antibody (MoAb) used in a microplate to minimize the need for an IAT, reacting with 83.2% of the D variants. The D confirmatory tests using SPRCA showed a reactivity (3 - 4+) with 100% of the D variant samples tested. CONCLUSION: Our results show that, even using sensitive methods and MoAbs to ensure the accurate assignment of the D antigen, at least 17% of our donor samples need a confirmatory D test in order to avoid alloimmunization in D-negative patients.

Characterization of RHD alleles present in serologically RHD-negative donors determined by a sensitive microplate technique.

BACKGROUND AND OBJECTIVES: Weak D phenotypes with very low antigen densities and DEL phenotype may not be detected in RhD typing routine and could be typed as D-negative, leading to D alloimmunization of D-negative recipients. The present study aimed to investigate the presence of RHD-positive genotypes in blood donors typed as D-negative by an automated system using the solid-phase methodology as a confirmatory test. METHODS: Two screenings were performed in different selected donor populations. For the first screening, we selected 1403 blood donor samples typed as D-negative regardless of the CE status, and in the second screening, we selected 517 donor samples typed as D-negative C+ and/or E+. RhD typing was performed by microplate in an automated equipment (Neo-Immucor®), and the confirmatory test was performed by solid-phase technique using Capture R® technology. A multiplex PCR specific to RHD and RHDψ was performed in a pool of 6 DNA samples. Sequencing of RHD exons was performed in all RHD-positive samples, and a specific PCR was used to identify the D-CE(4-7)-D hybrid gene. RESULTS AND CONCLUSION: No weak D type was found in either screening populations. Additionally, 353 (18·4%) D-negative samples presented previously reported non-functional RHD genes, 2 samples had a DEL allele, and 6 samples demonstrated new alleles, including one novel DEL allele. Our study identified six new RHD alleles and showed that the inclusion of a confirmatory test using serological methodology with high sensitivity can reduce the frequency of weak D samples typed as D-negative.

Correlation among automated scores of agglutination, antigen density by flow cytometry and genetics of D variants.

BACKGROUND: Laboratory testing to identify the molecular basis of serologic weak D phenotypes is recommended to determine whether a pregnant woman or potential transfusion recipient should be managed as RhD-positive or RhD-negative. The variation in D antigen expression on RBCs, different potencies of anti-D typing reagents, lack of standardized test methods, and the subjectivity of interpreting agglutination reactions complicate the detection of D variants. We evaluated the correlation of agglutination scores by an automated immunoassay analyzer with D antigen densities determined by flow cytometry, and D variant types identified by molecular analysis. MATERIALS AND METHODS: We selected 273 blood donor samples with agglutination scores of less than 92 (4+), measured by an automated analyzer (NEO®, Immucor, Norcross, GA, USA). D antigen densities were measured by flow cytometry for 89 samples. Samples were classified as molecularly-determined weak D or partial D variants by multiplex PCR, PCR RFLP and DNA sequencing. RESULTS: All samples with a D antigen density ≥15% had an agglutination score >80 (4+). Agglutination scores for weak D types varied from 10 to 90. Agglutination scores for partial D antigens were graded with scores varying from 60 to 99. D antigen densities varied from 0.55% to 10.67% for weak Ds and 4.1% to 30.5% for partial Ds. DISCUSSION: Our results showed that score values follow a pattern among D variants that could be related to antigen density and to the RhD variant classification.

Celiac Disease in Brazilian First-degree Relatives: The Odds Are Five Times Greater for HLA DQ2 Homozygous.

First-degree relatives (FDRs) of 47 outpatients with celiac disease (CD) answered a questionnaire about symptoms related to CD and were investigated for human leukocyte antigen (HLA)-DQ2, DQB102 homozygosis, and DQ8 alleles. Genetically susceptible individuals were tested for antitransglutaminase antibody immunoglobulin A. Seropositive FDR underwent small bowel biopsies.From 114 FDR, 74.5% (n = 85) were positive for DQ2, DQ8, or both haplotypes. Homozygosity of DQB102 was found in 11.4% (n = 13) individuals. Three FDR were previously diagnosed with CD. Among the genetically susceptible individuals, 67.1% had at least 1 symptom related to CD. Seropositivity was 8/82 (9.8%), and 4/8 biopsies were compatible with CD. Therefore, the total number of FDR with CD was 6.1% (7/114), 95% confidence interval (1.71, 10.49). Three out of 7 FDR with CD were HLA DQB102 homozygous. The odds of being CD is 5 times, 95% confidence interval (0.99, 26.23), greater for HLA DQ B102 homozygous in FDR.

SMIM1 polymorphisms in a donor population from southeast Brazil and their correlation with VEL expression.

BACKGROUND: Vel is a high frequency blood group antigen and its alloantibody is involved in haemolytic transfusion reactions. After elucidation of the molecular basis of the Vel-negative phenotype defined by a 17-base pair deletion in SMIM1, genotyping has been the technique of choice to identify the Vel-negative phenotype, and molecular investigations have contributed to explain Vel expression variability. The present study was aimed at screening for Vel negative blood donors and characterising the genetic changes found in Brazilian donors with altered Vel expression. MATERIALS AND METHODS: Molecular screening for the SMIM1*64_80del allele was performed in 1,595 blood donor samples using a SNaPshot protocol previously standardised in our laboratory. Four hundred donor samples were also submitted to serological screening using a polyclonal anti-Vel from our inventory. Samples with variability in antigen strength were selected for SMIM1 sequencing. RESULTS: No homozygous SMIM1*64_80del allele was found and the SMIM1*64_80del allele frequency was 1.01%. Different patterns of reactivity were observed in serological testing varying from negative to 3+. Through sequencing analysis we highlighted two polymorphisms: rs1175550 and rs6673829. The minor G allele of rs1175550 was found in 16/20 samples reacting 3+, while the major A allele was found in 21/23 samples reacting 2+. Regarding rs6673829, the minor A allele was present in 14/23 and 3/20 samples reacting 2+ and 3+ respectively. DISCUSSION: We included molecular VEL screening in a previously standardised SNaPshot protocol, which besides enabling detection of Vel-negative donors, also searches for eight other rare blood types. Additionally, the present study demonstrated that although the SMIM1*64_80del allele is responsible for some variation of Vel phenotype in this donor population, Vel expression is also controlled by molecular changes in SMIM1 intron 2.

Prevalence of genetic susceptibility for celiac disease in blood donors in são paulo, brazil / Prevalência da predisposição genética para doença celíaca nos doadores de sangue em São Paulo, Brasil

ABSTRACT Background Celiac disease is a permanent intolerance induced by gluten, which is expressed by T-cell mediated enteropathy, and has a high prevalence in the general population. There is evidence of a strong genetic predisposition to celiac disease. Objective To determine the prevalence of genetic markers HLA-DQ2 and HLA-DQ8 in blood donors from São Paulo and measure human recombinant tissue transglutaminase antibody IgA class in HLA-DQ2 and HLA-DQ8 positive donors. Methods A total of 404 blood donors from São Paulo city and Jundiaí were included in the study and signed the informed consent form. Information regarding diarrhea, constipation and abdominal pain in the last 3 months was collected. Determination of HLADQ2 and HLADQ8 alleles was performed in all participants and human recombinant tissue transglutaminase antibody class IgA was measured only in blood donors who presentedDQ2 and/or DQ8. Results HLADQ2 and/or HLADQ8 were positive in 49% (198/404) of subjects. Positive samples were associated with alleles DR3, DR4, DR7, DR11 and DR12. The most frequent genotype was DR4-DQ8, which was present in 13.6% of samples, followed by genotypes DR3-DQ2 and DR7-DQ2 with DQB1*02 in heterozygous, which were present in 10.4% and 8.7%, respectively. Eleven out of 198 positive donors (5%) were positive to human tissue transglutaminase test. Conclusion We observed a high prevalence of genetic markers for celiac disease, HLA-DQ2 and HLA-DQ8, in blood donors from São Paulo, similar to prevalence described in Europe. These findings show that the prevalence of celiac disease should not be rare in our country, but underdiagnosed.

RESUMO Contexto A doença celíaca é uma enteropatia imuno mediada causada pela intolerância permanente induzida pelo glúten, que se expressa por enteropatia mediada por linfócitos T, e possui uma alta prevalência na população geral. Há evidências de forte predisposição genética para doença celíaca. Objetivo Determinar a prevalência dos marcadores genéticos HLA-DQ2 e HLA-DQ8 em doadores de sangue da cidade de São Paulo e realizar rastreamento sorológico para doença celíaca com anticorpo antitransglutaminase tissular recombinante humana de classe IgA naqueles doadores de sangue com genotipagem HLA-DQ2 e HLA-DQ8 positivos. Métodos Estudo transversal prospectivo em que participaram 404 doadores de sangue, residentes na cidade de São Paulo e Jundiaí. A determinação dos alelos HLADQ2 e HLADQ8 foi realizada por PCR multiplex e alelo específico em todos os participantes do estudo e o anticorpo antitransglutaminase tissular recombinante humana de classe IgA e dosagem sérica de IgA foi realizada apenas nos doadores de sangue que possuíam DQ2 e/ou DQ8 positivo. Resultados O HLADQ2 e/ou DQ8 foi positivo em 49% (198/404) dos indivíduos, destes, 11 (5%) apresentaram anticorpo antitransglutaminase tissular humana positivo. Conclusão Podemos concluir que a prevalência dos marcadores genéticos para doença celíaca, HLA-DQ2 e DQ8 em São Paulo, mostrou-se elevada e similar à encontrada na Europa, assim como foi elevada a soroprevalênca para doença celíaca nos doadores de sangue com presença HLA-DQ2 e DQ8. Estes achados permitem afirmar que a prevalência da doença celíaca não deve ser rara em São Paulo, mas sim subdiagnosticada.

PREVALENCE OF GENETIC SUSCEPTIBILITY FOR CELIAC DISEASE IN BLOOD DONORS IN SÃO PAULO, BRAZIL.

BACKGROUND: Celiac disease is a permanent intolerance induced by gluten, which is expressed by T-cell mediated enteropathy, and has a high prevalence in the general population. There is evidence of a strong genetic predisposition to celiac disease. OBJECTIVE: To determine the prevalence of genetic markers HLA-DQ2 and HLA-DQ8 in blood donors from São Paulo and measure human recombinant tissue transglutaminase antibody IgA class in HLA-DQ2 and HLA-DQ8 positive donors. METHODS: A total of 404 blood donors from São Paulo city and Jundiaí were included in the study and signed the informed consent form. Information regarding diarrhea, constipation and abdominal pain in the last 3 months was collected. Determination of HLADQ2 and HLADQ8 alleles was performed in all participants and human recombinant tissue transglutaminase antibody class IgA was measured only in blood donors who presentedDQ2 and/or DQ8. RESULTS: HLADQ2 and/or HLADQ8 were positive in 49% (198/404) of subjects. Positive samples were associated with alleles DR3, DR4, DR7, DR11 and DR12. The most frequent genotype was DR4-DQ8, which was present in 13.6% of samples, followed by genotypes DR3-DQ2 and DR7-DQ2 with DQB1*02 in heterozygous, which were present in 10.4% and 8.7%, respectively. Eleven out of 198 positive donors (5%) were positive to human tissue transglutaminase test. CONCLUSION: We observed a high prevalence of genetic markers for celiac disease, HLA-DQ2 and HLA-DQ8, in blood donors from São Paulo, similar to prevalence described in Europe. These findings show that the prevalence of celiac disease should not be rare in our country, but underdiagnosed.

Frequency of Wra antigen and anti-Wra in Brazilian blood donors

BACKGROUND: Wra is a low-incidence antigen, which is antithetical to the high prevalence red blood cell antigen, Wrb. Anti-Wra is a naturally occurring antibody that is found in approximately 1-2% of blood donors. The aim of this study was to determine the frequency of Wra and anti-Wra in Brazilian blood donors.METHODS: A total of 1662 Brazilian blood donors were molecularly analyzed using the SNaPshot methodology to determine the WR*A/B alleles and to predict the frequency of the Wra antigen. To detect the anti-Wra, samples from 1049 blood donors were analyzed using a gel test with Wr(a+) red blood cells. The serum was treated with dithiothreitol (DTT) to determine the immunoglobulin classes. Immunoglobulin (Ig)-G isotype classification was performed in a gel test using the IgG1/IgG3 card. A monocyte monolayer assay was employed to predict the clinical significance of IgG anti-Wra.RESULTS: Of the 1662 donors, only one sample had the DI*02.03 allele in heterozygous predicting the Wr(a+b+) phenotype. Anti-Wra was detected in 34 (3.24%) samples, 64.7% in females and 35.3% in males. Regarding the immunoglobulin class, eight (23.5%) cases of anti-Wra were classified as IgG and 26 (76.5%) as IgM. Of the eight cases of IgG anti-Wra, four were IgG1, two were IgG3 and three anti-Wra were not IgG3 or IgG1, and thus probably IgG2 or IgG4. The results of the monocyte monolayer assay showed that IgG anti-Wra might be of clinical significance.CONCLUSION: This study shows a very low frequency (0.06%) of the Wra antigen in Brazilian blood donors. Additionally, it shows that the frequency of anti-Wra in this population is higher than previously reported.

Frequency of Wr(a) antigen and anti-Wr(a) in Brazilian blood donors.

BACKGROUND: Wr(a) is a low-incidence antigen, which is antithetical to the high prevalence red blood cell antigen, Wr(b). Anti-Wr(a) is a naturally occurring antibody that is found in approximately 1-2% of blood donors. The aim of this study was to determine the frequency of Wr(a) and anti-Wr(a) in Brazilian blood donors. METHODS: A total of 1662 Brazilian blood donors were molecularly analyzed using the SNaPshot methodology to determine the WR*A/B alleles and to predict the frequency of the Wr(a) antigen. To detect the anti-Wr(a), samples from 1049 blood donors were analyzed using a gel test with Wr(a+) red blood cells. The serum was treated with dithiothreitol (DTT) to determine the immunoglobulin classes. Immunoglobulin (Ig)-G isotype classification was performed in a gel test using the IgG1/IgG3 card. A monocyte monolayer assay was employed to predict the clinical significance of IgG anti-Wr(a). RESULTS: Of the 1662 donors, only one sample had the DI*02.03 allele in heterozygous predicting the Wr(a+b+) phenotype. Anti-Wr(a) was detected in 34 (3.24%) samples, 64.7% in females and 35.3% in males. Regarding the immunoglobulin class, eight (23.5%) cases of anti-Wr(a) were classified as IgG and 26 (76.5%) as IgM. Of the eight cases of IgG anti-Wr(a), four were IgG1, two were IgG3 and three anti-Wr(a) were not IgG3 or IgG1, and thus probably IgG2 or IgG4. The results of the monocyte monolayer assay showed that IgG anti-Wr(a) might be of clinical significance. CONCLUSION: This study shows a very low frequency (0.06%) of the Wr(a) antigen in Brazilian blood donors. Additionally, it shows that the frequency of anti-Wr(a) in this population is higher than previously reported.

A simple approach to screen rare donors in Brazil.

Providing blood units for patients with an antibody to a high-prevalence antigen or with multiple common antibodies is a constant challenge to the blood banks. Finding a compatible donor requires extensive screening, with incurs a large amount of investment. In this article, we share our experience of organizing a rare donor inventory with limited resources, we include the strategy used for finding rare donors, and we share the difficulties found during the implementation of the approach and the results obtained.

Novel RHAG allele encoding the Rh(null) phenotype in Brazil.

Rhnull is a rare phenotype characterized by the loss of Rh antigen expression. This phenotype can be related to several molecular backgrounds. In this study, we show a novel allele in a Brazilian pregnant woman encoding the Rhnull phenotype due to a change in RHAG exon2 c.310C>T, which leads to a premature stop codon (Gln104Stop).

General aspects of physalis cultivation / Aspectos gerais da cultura da physalis

The Physalis peruviana L. is an exotic fruit that belongs to the Solanaceae family with recent production in Brazil. This is presented as an alternative production with nutritional potential and economic value. The cape gooseberry plant has a high added value that can be used from its root to the fruit itself. The roots and leaves are rich in medicinal properties that are used in pharmacology. The fruit is sweet and have good content of vitamins A, C, iron, phosphorus and fiber, often used in making jellies, jams, juices and ice creams. To improve the plant canopy, but also contribute to the quality and appearance of the fruit produced some cultural practices such as staking, training systems, pruning and thinning are used. The aim of this review is to provide technical and scientific data about the culture of physalis.

A Physalis peruviana L. é uma fruta exótica pertencente à família Solanaceae com produção recente no Brasil. Esta se apresenta como uma alternativa de produção com potencial valor nutricional e econômico. A planta da fisalis possui um alto valor agregado, podendo ser utilizada desde sua raiz até o fruto propriamente dito. As raízes e as folhas são ricas em propriedades medicinais que são utilizadas na farmacologia. O fruto é açucarado e com bom conteúdo de vitaminas A, C, ferro, fósforo e fibras, muito utilizado na fabricação de geleias, doces, sucos e sorvetes. Para melhorar o dossel vegetativo da planta, como também contribuir para a qualidade e aparência do fruto produzido, utiliza-se, durante seu cultivo, algumas práticas culturais, como tutoramento, condução, poda e desbrote. O objetivo desta revisão é fornecer dados técnico-científicos sobre a Cultura da Physalis.

How do we identify RHD variants using a practical molecular approach?

Serologic resolution of Rh discrepancies due to partial D or weak D phenotypes is a frequent problem encountered during routine typing that can be solved by RHD genotyping because it provides better characterization of these variants. The objective of the current study was to develop algorithms for identification of D variants in multiethnic populations based on a logic sequence of molecular tests using a large number of atypical RhD specimens. Thus, a total of 360 blood samples with atypical D antigen expression were analyzed. A previously published multiplex polymerase chain reaction (PCR) procedure was performed and depending on multiplex PCR analysis, the associated RHCE allele, and D variant frequency in our population, an algorithm was developed composed of six flow charts using specific PCR-restriction fragment length polymorphism and/or specific exon sequencing. This strategy allowed the identification of 22 different variants with few assays and a much reduced cost. This study describes a simple and practical algorithm that we use to determine RHD genotypes in samples with unknown RHD. This strategy is relatively easy to implement and the algorithm can be adapted to populations with various ethnic backgrounds after an initial assessment of the type and frequency of D variants. Essentially, we demonstrate that sequencing of all RHD exons is not necessary for the identification of the majority of known D variants.