RHCE variant alleles and risk of alloimmunization in Brazilians.

Variant RHCE alleles are found mainly in Afro-descendant individuals, as well as in patients with sickle cell disease (SCD). The most common variants are related to the RHCE*ce allele, which can generate partial e and c antigens. Although RHCE variant alleles have been extensively studied, defining their clinical significance is a difficult task. We evaluated the risk of RhCE alloimmunization as a consequence of partial antigens in patients with a positive phenotype transfused with red blood cell (RBC) units with the corresponding antigen. A retrospective study was performed with Brazilian patients, evaluating the number of antigen-positive transfused RBC units (incompatible due to partial antigen) in 27 patients with SCD carrying RHCE variant alleles who did not develop antibodies as well as evaluating the variants present in 12 patients with partial phenotype and correlated antibody (one patient with SCD and 11 patients with other pathologies). Two patients showed variant alleles with molecular changes that had not yet been described. Variant RHCE alleles were identified in a previous study using molecular methods. RHCE*ceVS.01 was the most frequent allele found among the patients without antibodies. Six patients with partial c antigen had a mean of 3.8 c+ RBC units transfused, and 10 patients with partial e antigen were exposed for a mean of 7.2 e+ RBC units. Among the variant alleles found in alloimmunized patients, the most frequent was RHCE*ceAR, which was found in five patients; the antibodies developed were anti-hrS and/or anti-c. Our results showed that RHCE*ceVS.01 is indeed the most frequent variant allele in our cohort of patients with SCD, but the partial antigens that were identified have low risk of alloimmunization. RHCE*ceAR is the most impactful variant in the Brazilian population with high risk of alloimmunization and clinically significant anti-hrS formation.

Challenges in providing compatible blood with Rh genotype-matching in Brazilian patients with sickle cell disease.

OBJECTIVES: The aim of this study was to verify the possibility of performing prophylactic Rh genotype-matching in Brazilian patients with sickle cell disease (SCD) and identify the genotypes that are lacking or insufficient in our donor cohort. BACKGROUND: Rh alloimmunisation is still a challenge in transfused patients with SCD. Rh genotype-matching may mitigate Rh alloimmunisation. METHODS/MATERIALS: We examined the transfusion requests for antigen-matched donor units in SCD patients with Rh variants and compared the Rh altered alleles in the patients to the Rh allele frequency in a selected donor population. For each patient and donor, RBC antigen genotyping was performed using HEA, RHD and RHCE BeadChip arrays. Sequencing was used to clarify inconclusive results. Twenty-one patients and 956 Brazilian blood donors were genotyped. RESULTS: According to our matching strategies, 47·6% of patients filled most of their unit requests, but 52·4% of patients had insufficient donors to fill their annual transfusion needs. We found different combinations of RHCE variant alleles in patients and donors, but the most frequent genotypes that are lacking or insufficient in our donor cohort are those associated with the lack of hrB and hrS high prevalence antigens and those co-inherited with altered RHD alleles. CONCLUSION: Our study shows that the provision of compatible blood with Rh genotype-matching in Brazilian patients with SCD can be feasible but challenging and, efficient strategies of recruitment of African-Brazilian donors must be developed.

HLA polymorphisms and risk of red blood cell alloimmunisation in polytransfused patients with sickle cell anaemia.

BACKGROUND: Red blood cell (RBC) alloimmunisation is an event that may occur due to factors such as numerous blood transfusions, age, gender and genetic factors such as human leukocyte antigen (HLA). AIMS/OBJECTIVES: The aim of the present study was to investigate the possibility of alloimmunisation to red blood cell group antigens associated with the HLA of individuals and to relate alloimmunisation to risk factors. METHODS: A total of 172 polytransfused patients with sickle cell anaemia (SCA) (44 alloimmunised, 128 non-alloimmunised) participated in this study. Blood group genotyping was performed by the DNA microarray method and HLA genotyping by polymerase chain reaction - specific sequence of oligonucleotides. RESULTS: The number of transfusions received directly influenced the incidence of alloimmunisation, and the most common alloantibodies were against Rh (48·8%) and Kell (17%) systems. The HLA-C*06 and HLA-DQB1*03 variants were significantly higher in alloimmunised patients. The HLA-DRB1*04 and HLA-DRB1*11 were more often found in individuals who developed the alloantibodies anti-Fya and anti-K, respectively. CONCLUSION: This study suggests that polytransfused patients with SCA possessing the HLA-DQB1*03 and HLA-C*06 allele variants are more susceptible to alloimmunisation. In addition, HLA-DRB1*04 and HLA-DRB1*11 alleles were seen to be associated with the production of anti-Fya and anti-K antibodies, respectively.

RHCE variants inherited with altered RHD alleles in Brazilian blood donors.

BACKGROUND: 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. METHODS: From 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. RESULTS: All 421 samples had altered RHD alleles, being 56·3% of them partial D. Among them, 94·9% 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, 10·6% predict partial c, partial e, hr(B) - and/or hr(S) - and 100% express low prevalence antigens. CONCLUSION: Targeting individuals with altered expression of D antigen can be a good strategy for finding donors with RhCE variants. In our study 94·9% of the partial D samples revealed altered RHCE variant alleles and 5·7% 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.

International Society of Blood Transfusion Working Party on red cell immunogenetics and blood group terminology: Cancun report (2012).

The International Society of Blood Transfusion Working Party on red cell immunogenetics and blood group terminology convened during the International congress in Cancun, July 2012. This report details the newly identified antigens in existing blood group systems and presents three new blood group systems.

RHD allelic identification among D-Brazilian blood donors as a routine test using pools of DNA.

BACKGROUND: RHD alleles leading to a reduced expression of D antigen of the red blood cell (RBC) surface may be erroneously typed as D- by serology and may cause anti-D immunizations when transfused to recipients. METHODS: To determine the occurrence of such alleles among apparent D- blood donors, molecular typing was implemented as a routine test using a pool of DNA. A total of 2,450 pretyped D- samples were tested in pools of 10 for the RHD-specific polymorphism in intron 4 and exon 7. Samples in polymer chain reaction (PCR) positive pools were individually reevaluated by exon-specific PCRs, sequencing, and serologic methods. RESULTS: Among 2,450 serologically D- blood donor samples tested, 101 (4.1%) carried the RHD gene. Nonfunctional RHD (RHDψ, RHD*CE(2-9)-D, and RHD*CE(3-7)-D), different weak D alleles such as RHD*weak D type 1, RHD*weak D type 4.3, RHD*weak D type 5, RHD*weak D type 38, and RHD*DEL were identified. CONCLUSION: We employed a PCR-based assay for RHD as a routine test using pools of ten DNA blood donor samples. The integration of RHD genotyping into the routine screening program using pools of DNA samples was straightforward. As a consequence, 19 (0.8%) blood donors carrying a weak D and Del phenotypes with the potential of causing anti-D immunizations in recipients were reclassified as D+. For each population, it would be necessary to adapt the RHD genotyping strategy to the spectrum of prevalent alleles.

Consortium for Blood Group Genes (CBGG): 2009 report.

Consortium for Blood Group Genes is a worldwide organization whose goal is to have a vehicle to interact, establish guidelines, operate a proficiency program, and provide education for laboratories involved in DNA and RNA testing for the prediction of blood group, platelet, and neutrophil antigens. Currently, the consortium operates with representatives from Brazil, Canada, and the United States. Membership is voluntary with the expectation that members actively contribute to discussions involving blood group genetics. This year witnessed a change in the standing committee membership and the institution of a representative for the human platelet antigens group. Looking forward, the consortium sees challenges for the nomenclature of blood group alleles and user-required specifications for laboratory information systems to store genotype information.

DNA array analysis for red blood cell antigens facilitates the transfusion support with antigen-matched blood in patients with sickle cell disease.

BACKGROUND: Blood samples from patients with sickle cell disease (SCD) present to transfusion service with numerous antibodies, making the searching for compatible red blood cells (RBC) a challenge. To overcome this problem we developed an effective strategy to meet needs of supplying RBC-compatible units to SCD patients using DNA arrays. METHODS: We selected DNA samples from 144 SCD patients with multiple (receiving > 5 units) transfusions previously phenotyped for ABO, Rh(D, C, c, E, e), K1, Fy(a) and Jk(a). We also selected DNA samples from 948 Brazilian blood donors whose ABO/RhD phenotype matched that of the patients. All samples were analysed by DNA array analysis (HEA Beadchip(TM), Bioarray Solutions) to determine polymorphisms associated with antigen expression for 11 blood group systems (Rh, Kell, Kidd, Duffy, MNS, Dombrock, Lutheran, Landsteiner-Wiener, Diego, Colton, Scianna); and one mutation associated with haemoglobinopathies. RESULTS: Based on genotype results we were able to predict phenotype-compatible donors needed in order to provide compatible units to this group of patients. Based on their ABO/Rh phenotype we were able to find in this pool of donors compatible units for 134 SCD patients. CONCLUSION: Blood group genotyping by DNA array contributes to the management of transfusions in SCD patients by facilitating the transfusion support with antigen-matched blood. It has the potential to improve the life of thousands of SCD-transfused patients by reducing mortality due to transfusion reactions and immunization.

Consortium for Blood Group Genes (CBGG): 2008 report.

The Consortium for Blood Group Genes is a worldwide organization whose goal is to have a vehicle to interact, establish guidelines, operate a proficiency program, and provide education for laboratories involved in DNA and RNA testing for the prediction of blood group, platelet, and neutrophil antigens.

Molecular analyses of GYPB in African Brazilians.

The molecular background of variant forms of GYPB is not well studied in Brazilians of African descent. The present study was carried out to determine the molecular bases of the S-s- phenotype and the frequency of GYPB*S silent gene for the S-s+ phenotype in a blood donor population of African Brazilians. In this study, 165 blood samples from African Brazilians (Northeastern Brazil) who phenotyped as S-s- (n = 17) and S-s+ (n = 148) by hemagglutination were selected. Allele-specific (AS)-PCR and PCR-restriction fragment length polymorphism (RFLP) were used to identify the variant forms of GYPB. In 13 of 17 S-s- samples (76.5%), both GYPB were deleted. In 137 of the 148 S-s+ samples (92.6%), the AS-PCR was consistent with the S-s+ phenotype. In 4 of the S-s- samples (23.5%) and 11 of the S-s+ samples (7.4%), the AS-PCR showed the presence of a GYPB*S allele associated with silencing of S. In the 4 donors with the S-s- phenotype, there was homozygosity (or hemizygosity) for the GYP(P2) allele (n = 2), homozygosity (or hemizygosity) for the GYP(NY) allele (n = 1), and heterozygosity for the GYP(P2) and GYP(NY) alleles (n = 1). In the 11 donors with the S-s+ phenotype, there was heterozygosity for GYP(P2) allele (n = 8) and heterozygosity for GYP(NY) allele (n = 3). This study reports for the first time the molecular mechanisms responsible for the S-s- phenotype in a population of African Brazilians and provides new information about the frequency and molecular bases of the GYPB*S silent gene (7.4%) in this population.

Molecular studies of DO alleles reveal that JO is more prevalent than HY in Brazil, whereas HY is more prevalent in New York.

Because of the scarcity of anti-Hy and anti-Jo(a), hemagglutination typing for the Dombrock blood group system antigens, Hy and Jo(a), is not feasible. The molecular bases associated with these antigens have been determined, making it possible to distinguish HY and JO from wild-type DO. This provides a tool to predict the probable phenotype of patients and to screen for antigen-negative donors. PCR-RFLP assays and a microchip assay were used to determine the frequency of HY and JO alleles in donors from Brazil and New York. DNA from random Brazilian donors, 288 by PCR-RFLP and 599 by the bead array method (BeadChip, BioArray Solutions, Warren, NJ), was tested to determine 323G/T (HY+/HY-) and 350C>T (JO+/JO-) single-nucleotide polymorphisms. In New York, 27,226 donors who self-identified as being African American were tested by hemagglutination with anti-Gy(a). Nonreactive and weakly reactive samples were tested by PCR-RFLP for the same alleles as listed above. In Brazil, 30 (3.4%) of the samples were JO/DO and 13 (1.4%) were HY/DO. In New York, of the samples that had HY or JO alleles, 14 were homozygous HY/HY 132 were heterozygous HY/DO, 13 were heterozygous HY/JO, 14 were heterozygous JO/DO, and 3 were homozygous JO/JO. These results show that in donors from Brazil, JO (30 alleles) is more than twice as prevalent as HY (13 alleles), whereas in donors from New York, HY (173 alleles) was more than five times more common than JO (33 alleles).

Opposite lipemic response of Wistar rats and C57BL/6 mice to dietary glucose or fructose supplementation.

The metabolic effects of carbohydrate supplementation in mice have not been extensively studied. In rats, glucose- and fructose-rich diets induce hypertriacylglycerolemia. In the present study, we compared the metabolic responses to two monosaccharide supplementations in two murine models. Adult male Wistar rats (N = 80) and C57BL/6 mice (N = 60), after 3 weeks on a standardized diet, were submitted to dietary supplementation by gavage with glucose (G) or fructose (F) solutions (500 g/L), 8 g/kg body weight for 21 days. Glycemia was significantly higher in rats after fructose treatment (F: 7.9 vs 9.3 mM) and in mice (G: 6.5 vs 10 and F: 6.6 vs 8.9 mM) after both carbohydrate treatments. Triacylglycerolemia increased significantly 1.5 times in rats after G or F supplementation. Total cholesterol did not change with G treatment in rats, but did decrease after F supplementation (1.5 vs 1.4 mM, P < 0.05). Both supplementations in rats induced insulin resistance, as suggested by the higher Homeostasis Model Assessment Index. In contrast, mice showed significant decreases in triacylglycerol (G: 1.8 vs 1.4 and F: 1.9 vs 1.4 mM, P < 0.01) and total cholesterol levels (G and F: 2.7 vs 2.5 mM, P < 0.05) after both monosaccharide supplementations. Wistar rats and C57BL/6 mice, although belonging to the same family (Muridae), presented opposite responses to glucose and fructose supplementation regarding serum triacylglycerol, free fatty acids, and insulin levels after monosaccharide treatment. Thus, while Wistar rats developed features of plurimetabolic syndrome, C57BL/6 mice presented changes in serum biochemical profile considered to be healthier for the cardiovascular system.

Consortium for Blood Group Genes (CBGG): 2007 report.

The Consortium for Blood Group Genes is a worldwide organization whose goal is to have a vehicle to interact, establish guidelines, operate a proficiency program, and provide education for laboratories involved in DNA and RNA testing for the prediction of blood group, platelet, and neutrophil antigens.