Dombrock genotyping in Brazilian blood donors reveals different regional frequencies of the HY allele.

BACKGROUND: Dombrock blood group system genotyping has revealed various rearrangements of the Dombrock gene and identified new variant alleles in Brazil (i.e., DO*A-SH, DO*A-WL and DO*B-WL). Because of the high heterogeneity of the Brazilian population, interregional differences are expected during the investigation of Dombrock genotypes. OBJECTIVE: The present study aims to determine the frequencies of Dombrock genotypes in blood donors from Minas Gerais and compare the frequencies of the HY and JO alleles to those of another population in Brazil. METHODS: The frequencies of the DO alleles in Minas Gerais, a southeastern state of Brazil, were determined from the genotyping of 270 blood donors. Genotyping involved polymerase chain reaction and restriction fragment length polymorphism analysis to identify the 323G>T, 350C>T, 793A>G, and 898C>G mutations, which are related to the HY, JO, DO*A/DO*B, and DO*A-WL/DO*B-WL alleles, respectively. Moreover, the frequencies of rare HY and JO alleles were statistically compared using the chi-square test with data from another Brazilian region. RESULTS: The HY allele frequency in Minas Gerais (2.4%) was almost twice that of the JO allele (1.5%). The frequency of the HY allele was significantly higher (p-value = 0.001) than that in another Brazilian population and includes a rare homozygous donor with the Hy- phenotype. In addition, the DO*A-WL and DO*B-WL alleles, which were first identified in Brazil, were found in the state of Minas Gerais. CONCLUSIONS: The data confirm that the frequencies of DO alleles differ between regions in Brazil. The population of Minas Gerais could be targeted in a screening strategy to identify the Hy- phenotype in order to develop a rare blood bank.

HLA among Brazilian patients with spontaneous chronic urticaria and positive autologous serum skin test.

BACKGROUND: An association between class I and II alleles of the major histocompatibility complex and idiopathic chronic urticaria has previously been observed in different populations, but there are still no studies on Brazilian populations in this regard. OBJECTIVE: The involvement of the major histocompatibility complex classes I and II (loci A, B and DR) in Brazilian patients with idiopathic chronic urticaria and a positive autologous serum skin test was investigated and compared with a healthy population group. METHODS: DNA was extracted from the blood of 42 patients with idiopathic chronic urticaria and major histocompatibility complex classes I and II alleles were determined using the polymerase chain reaction and a laboratory test for oligonucleotide hybridization using a single-filament probe. The frequencies of these alleles in patients with chronic urticaria were compared with the frequencies in 1000 genetically unrelated voluntary blood donors from the same region of Brazil. The diagnosis of idiopathic chronic urticaria was based on the patients' clinical history and routine laboratory tests. Only the patients with positive autologous serum skin test were selected. The allele distribution resulted from the patient and control groups were analyzed using odds ratios and 95% confidence intervals. RESULTS: No statistically significant differences were found between the positive autologous serum skin test patients with chronic urticaria and the control group. CONCLUSIONS: We found that in this population group, there was no specific association between the HLA alleles studied and chronic urticaria. We believe that further population studies are needed in order to investigate the possible existence of this association.

Dombrock genotyping in Brazilian blood donors reveals different regional frequencies of the HY allele

Background: Dombrock blood group system genotyping has revealed various rearrangements of the Dombrock gene and identified new variant alleles in Brazil (i.e., DO*A-SH, DO*A-WL and DO*B-WL). Because of the high heterogeneity of the Brazilian population, interregional differences are expected during the investigation of Dombrock genotypes. Objective: The present study aims to determine the frequencies of Dombrock genotypes in blood donors from Minas Gerais and compare the frequencies of the HY and JO alleles to those of another population in Brazil. Methods: The frequencies of the DO alleles in Minas Gerais, a southeastern state of Brazil, were determined from the genotyping of 270 blood donors. Genotyping involved polymerase chain reaction and restriction fragment length polymorphism analysis to identify the 323G>T, 350C>T, 793A>G, and 898C>G mutations, which are related to the HY, JO, DO*A/DO*B, and DO*A-WL/DO*B-WL alleles, respectively. Moreover, the frequencies of rare HY and JO alleles were statistically compared using the chi-square test with data from another Brazilian region. Results: The HY allele frequency in Minas Gerais (2.4%) was almost twice that of the JO allele (1.5%). The frequency of the HY allele was significantly higher (p-value = 0.001) than that in another Brazilian population and includes a rare homozygous donor with the Hy- phenotype. In addition, the DO*A-WL and DO*B-WL alleles, which were first identified in Brazil, were found in the state of Minas Gerais. Conclusions: The data confirm that the frequencies of DO alleles differ between regions in Brazil. The population of Minas Gerais could be targeted in a screening strategy to identify the Hy- phenotype in order to develop a rare blood bank. .

CD63 e CD123 expressão, autoanticorpos IgG e acurácia do teste do soro autólogo em pacientes com urticária crônica / CD63 and CD123 expression, IgG autoantibody and accuracy of autologous serum test in patients with chronic urticaria

INTRODUÇÃO: Na urticária crônica (UC), o teste cutâneo do soro autólogo (TCSA) pode sugerir a etiologia autoimune. Recentemente, uma nova técnica laboratorial denominada teste de ativação de basófilos (TAB) vem sendo utilizada para esse diagnóstico. OBJETIVOS: Analisar o TCSA em relação ao TAB, assim como avaliar os receptores da interleucina 3 (IL3) (CD123) e a presença de autoanticorpos da classe de imunoglobulina G (IgG) inespecíficos ligados aos basófilos de pacientes com UC. MÉTODOS: Estudamos 33 adultos com UC espontânea com idade média de 42,5 + 14 anos. Por meio da citometria de fluxo foi feita a análise da expressão das moléculas CD63 em basófilos de um doador atópico após o estímulo pelo soro dos pacientes com UC. Também realizamos a pesquisa da expressão da molécula CD123 e de autoanticorpos IgG inespecíficos. RESULTADOS: O odds ratio (OR) entre o TCSA e o TAB foi de 1 (intervalo de confiança [IC] 95 por cento: 0,22-4,5). O TCSA para o diagnóstico da UC autoimune mostrou acurácia de 54,5 por cento, sensibilidade de 66 por cento, especificidade de 33 por cento, valor preditivo positivo de 63 por cento e valor preditivo negativo de 36 por cento. Não houve diferença estatística entre os grupos estudados quanto à média de expressão dos anticorpos IgG inespecíficos e das moléculas CD123 (para um p < 0,05). DISCUSSÃO: Este estudo demonstrou baixa precisão do TCSA no diagnóstico da UC autoimune; o grupo de pacientes com TCSA positivo não mostrou diferença estatística em relação ao grupo com TCSA negativo nos demais aspectos analisados. CONCLUSÃO: Pelos poucos estudos existentes e pela relevância do assunto, acreditamos na necessidade de mais estudos abordando esses aspectos.

INTRODUCTION: The autologous serum skin test (ASST) may suggest an autoimmune etiology in chronic urticaria (CU). A new laboratory technique called basophil activation test (BAT) has been currently employed for its diagnosis. OBJECTIVE: To analyze ASST in relation to BAT as well as to evaluate interleukin 3 (IL3) receptors (CD123) and non-specific immunoglobulin G (IgG) autoantibodies bound to basophils in patients with chronic urticaria. METHODS: We studied 33 adults with CU and mean age of 42.5 + 14 years. After stimulation by serum from patients with CU, CD63 expression on basophils from one atopic donor was analyzed by flow cytometry. Furthermore, we investigated CD123 and IgG autoantibody expressions. RESULTS: The odds ratio (OR) between ASST and BAT was 1.00 (95 percent confidence interval [CI]: 0.22 to 4.5). The ASST for autoimmune CU diagnosis showed an accuracy of 54.5 percent, sensitivity of 66 percent, specificity of 33 percent, positive predictive value of 63 percent, and negative predictive value of 36 percent. There was no statistical difference between the studied groups as to mean non-specific IgG and CD123 expressions (for a p < 0.05). DISCUSSION: This study demonstrated that ASST has low accuracy in the diagnosis of autoimmune CU. Concerning other analyzed aspects, there was no statistical difference between positive ASST and negative ASST. CONCLUSIONS: Due to insufficient studies in this area and the relevance of this issue, further investigation is required.

HLA among Brazilian patients with spontaneous chronic urticaria and positive autologous serum skin test / HLA em brasileiros com urticária crônica espontânea e teste cutâneo do soro autólogo positivo

BACKGROUND: An association between class I and II alleles of the major histocompatibility complex and idiopathic chronic urticaria has previously been observed in different populations, but there are still no studies on Brazilian populations in this regard. OBJECTIVE: The involvement of the major histocompatibility complex classes I and II (loci A, B and DR) in Brazilian patients with idiopathic chronic urticaria and a positive autologous serum skin test was investigated and compared with a healthy population group. METHODS: DNA was extracted from the blood of 42 patients with idiopathic chronic urticaria and major histocompatibility complex classes I and II alleles were determined using the polymerase chain reaction and a laboratory test for oligonucleotide hybridization using a single-filament probe. The frequencies of these alleles in patients with chronic urticaria were compared with the frequencies in 1000 genetically unrelated voluntary blood donors from the same region of Brazil. The diagnosis of idiopathic chronic urticaria was based on the patients' clinical history and routine laboratory tests. Only the patients with positive autologous serum skin test were selected. The allele distribution resulted from the patient and control groups were analyzed using odds ratios and 95% confidence intervals. RESULTS: No statistically significant differences were found between the positive autologous serum skin test patients with chronic urticaria and the control group. CONCLUSIONS: We found that in this population group, there was no specific association between the HLA alleles studied and chronic urticaria. We believe that further population studies are needed in order to investigate the possible existence of this association.

FUNDAMENTOS: A associação entre os alelos do MHC classe I e II e a urticária crônica idiopática tem sido previamente constatada em diferentes populações, sendo que na população brasileira ainda não existem estudos a este respeito. OBJETIVOS: Foi estudado o envolvimento do MHC classe I e II (locci A, B e DR) em pacientes brasileiros com urticária crônica idiopática e teste cutâneo do soro autólogo positivo, comparando-se com um grupo populacional saudável. MÉTODOS: O DNA foi extraído do sangue de 42 pacientes com urticária crônica idiopática e o MHC classe I e II determinado por reação em cadeia da polimerase e teste laboratorial de hibridização de oligonucleotídeo com sonda de filamento único. A freqüência destes alelos em pacientes com urticária crônica idiopática foi comparada com a de 1000 doadores de sangue voluntários e geneticamente não relacionados, da mesma região do Brasil. O diagnóstico de urticária crônica idiopática foi baseado na história clínica do paciente e exames laboratoriais de rotina; foram selecionados apenas os pacientes com teste cutâneo do soro autólogo positivo. O resultado da distribuição alélica entre o grupo de pacientes e o grupo controle foi analisado através do odds rate com o cálculo do intervalo de confiança de 95% (95% IC). RESULTADOS: Não foram encontradas diferenças com significância estatística entre os pacientes com urticária crônica teste cutâneo do soro autólogo positivos e o grupo controle. CONCLUSÕES: Verificamos que neste grupo populacional estudado não houve associação específica entre os alelos HLA estudados e a urticária crônica; acreditamos na necessidade de outros estudos populacionais, para podermos verificar a possível existência desta associação.

Avaliação de reagentes anti-D na detecção dos antígenos D fraco e D parcial / Evaluation of anti-D reagents in the detection of weak D and partial D antigens

Anti-soros monoclonais anti-D IgG e IgM têm sido produzidos para substituir os policlonais na determinação do antígeno D. No entanto, pouco se conhece a respeito da utilização destes reagentes na detecção dos antígenos RhD fraco e RhD parcial. Estudos moleculares e sorológicos que possam esclarecer a expressão do antígeno D são importantes para a seleção adequada dos reagentes anti-D utilizados na fenotipagem RhD. Foram analisados anti-soros anti-D monoclonais IgG e IgM quanto à capacidade de detecção dos antígenos D fraco de alta e baixa densidade antigênica e dos antígenos D parciais que reagem como D fraco em 56 amostras de sangue caracterizadas como D fraco. O anti-D IgM foi analisado pela reatividade de aglutinação à temperatura ambiente enquanto o anti-D IgG foi analisado pela reatividade pelo teste da antiglobulina humana. Os tipos de D fraco foram caracterizados molecularmente, e a densidade antigênica dos antígenos RhD identificados foi determinada por citometria de fluxo. Todas as amostras que continham os tipos de D fracos mais freqüentes (DF1, DF3 e DF4) foram detectadas à temperatura ambiente (TA) com anti-D IgM enquanto as amostras DF2 e D parcial foram detectadas com anti-D IgG reativo pelo teste da antiglobulina humana (AGH). Nossos resultados demonstram que o anti-D IgM não detecta antígenos D parciais reativos como D fraco e antígenos D fraco de baixa densidade antigênica e, portanto, pode ser utilizado na rotina de pacientes. O anti-D IgG detecta a maioria dos tipos de D fraco e associações D fraco parcial e deve ser utilizado na rotina de doadores.

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.