Genetic risk factors and COVID-19 severity in Brazil: results from BRACOVID study.

The coronavirus disease 2019 (COVID-19) pandemic has changed the paradigms for disease surveillance and rapid deployment of scientific-based evidence for understanding disease biology, susceptibility and treatment. We have organized a large-scale genome-wide association study (GWAS) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected individuals in Sao Paulo, Brazil, one of the most affected areas of the pandemic in the country, itself one of the most affected in the world. Here, we present the results of the initial analysis in the first 5233 participants of the BRACOVID study. We have conducted a GWAS for COVID-19 hospitalization enrolling 3533 cases (hospitalized COVID-19 participants) and 1700 controls (non-hospitalized COVID-19 participants). Models were adjusted by age, sex and the 4 first principal components. A meta-analysis was also conducted merging BRACOVID hospitalization data with the Human Genetic Initiative (HGI) Consortia results. BRACOVID results validated most loci previously identified in the HGI meta-analysis. In addition, no significant heterogeneity according to ancestral group within the Brazilian population was observed for the two most important COVID-19 severity associated loci: 3p21.31 and Chr21 near IFNAR2. Using only data provided by BRACOVID, a new genome-wide significant locus was identified on Chr1 near the genes DSTYK and RBBP5. The associated haplotype has also been previously associated with a number of blood cell related traits and might play a role in modulating the immune response in COVID-19 cases.

The future of blood services amid a tight balance between the supply and demand of blood products: Perspectives from the ISBT Young Professional Council.

BACKGROUND AND OBJECTIVES: Blood services manage the increasingly tight balance between the supply and demand of blood products, and their role in health research is expanding. This review explores the themes that may define the future of blood banking. MATERIALS AND METHODS: We reviewed the PubMed database for articles on emerging/new blood-derived products and the utilization of blood donors in health research. RESULTS: In high-income countries (HICs), blood services may consider offering these products: whole blood, cold-stored platelets, synthetic blood components, convalescent plasma, lyophilized plasma and cryopreserved/lyophilized platelets. Many low- and middle-income countries (LMICs) aim to establish a pool of volunteer, non-remunerated blood donors and wean themselves off family replacement donors; and many HICs are relaxing the deferral criteria targeting racial and sexual minorities. Blood services in HICs could achieve plasma self-sufficiency by building plasma-dedicated centres, in collaboration with the private sector. Lastly, blood services should expand their involvement in health research by establishing donor cohorts, conducting serosurveys, studying non-infectious diseases and participating in clinical trials. CONCLUSION: This article provides a vision of the future for blood services. The introduction of some of these changes will be slower in LMICs, where addressing key operational challenges will likely be prioritized.

Genome-wide association study of early ischaemic stroke risk in Brazilian individuals with sickle cell disease implicates ADAMTS2 and CDK18 and uncovers novel loci.

Ischaemic stroke is a common complication of sickle cell disease (SCD) and without intervention can affect 11% of children with SCD before the age of 20. Within the Trans-Omics for Precision Medicine (TOPMed), a genome-wide association study (GWAS) of ischaemic stroke was performed on 1333 individuals with SCD from Brazil (178 cases, 1155 controls). Via a novel Cox proportional-hazards analysis, we searched for variants associated with ischaemic stroke occurring at younger ages. Variants at genome-wide significance (p < 5 × 10-8 ) include two near genes previously linked to non-SCD early-onset stroke (<65 years): ADAMTS2 (rs147625068, p = 3.70 × 10-9 ) and CDK18 (rs12144136, p = 2.38 × 10-9 ). Meta-analysis, which included the independent SCD cohorts Walk-PHaSST and PUSH, exhibited consistent association for variants rs1209987 near gene TBC1D32 (p = 3.36 × 10-10 ), rs188599171 near CUX1 (p = 5.89 × 10-11 ), rs77900855 near BTG1 (p = 4.66 × 10-8 ), and rs141674494 near VPS13C (1.68 × 10-9 ). Findings from this study support a multivariant model of early ischaemic stroke risk and possibly a shared genetic architecture between SCD individuals and non-SCD individuals younger than 65 years.

Estimated glomerular filtration rate in Brazilian adults with sickle cell disease: results from the REDS-III multicenter cohort study.

Chronic kidney disease (CKD) has a significant impact on sickle cell disease (SCD) morbidity and mortality. Early identification of individuals at highest risk of developing CKD may allow therapeutic intervention to prevent worse outcomes. This study aimed to evaluate the prevalence and risk factors for reduced estimated glomerular filtration rate (eGFR) among adults with SCD in Brazil. Participants in the REDS-III multicenter SCD cohort with more severe genotypes aged ≥ 18 years with at least two serum creatinine values were analyzed. The eGFR was calculated using the Jamaica Sickle Cell Cohort Study GFR equation. The eGFR categories were defined according to the K/DOQI. Participants with eGFR ≥ 90 were compared to those with those with eGFR < 90. Among the 870 participants, 647 (74.4%) had eGFR ≥ 90, 211 (24.3%) had eGFR 60 to 89, six (0.7%) had eGFR 30 to 59, and six (0.7%) had ESRD. Male sex (OR: 37.3; 95%CI: 22.4-65.1), higher age (OR: 1.04; 95%CI: 1.02-1.06), higher diastolic blood pressure (OR: 1.03; 95%CI: 1.009-1.06), lower Hb (OR: 0.80; 95%CI: 0.68-0.93), and lower reticulocytes (OR: 0.94; 95%CI: 0.89-0.99) levels were independently associated with eGFR < 90. There was a trend towards higher odds of death in participants with eGFR < 90 (OR: 1.8; 95%CI: 0.95-3.32; p = 0.065). In turn, participants with eGFR < 60 had a 12.2 (95%CI: 2.1-96.9) times higher odds for death when compared to those with eGFR ≥ 60. In this study, eGFR < 90 was observed in one-quarter of adults. Older age, male sex, higher diastolic blood pressure, lower hemoglobin, and lower reticulocyte levels were associated with occurrence of eGFR < 90. Estimated GFR < 60 increased the risk of mortality.

The Recipient Epidemiology and Donor Evaluation Study-IV-Pediatric (REDS-IV-P): A research program striving to improve blood donor safety and optimize transfusion outcomes across the lifespan.

BACKGROUND: The Recipient Epidemiology and Donor Evaluation Study-IV-Pediatric (REDS-IV-P) is a new iteration of prior National Heart, Lung, and Blood Institute (NHLBI) REDS programs that focus on improving transfusion recipient outcomes across the lifespan as well as the safety and availability of the blood supply. STUDY DESIGN AND METHODS: The US program includes blood centers and hospitals (22 including 6 free-standing Children's hospitals) in four geographic regions. The Brazilian program has 5 participating hemocenters. A Center for Transfusion Laboratory Studies (CTLS) and a Data Coordinating Center (DCC) support synergistic studies and activities over the 7-year REDS-IV-P program. RESULTS: The US is building a centralized, vein-to-vein (V2V) database, linking information collected from blood donors, their donations, the resulting manufactured components, and data extracts from hospital electronic medical records of transfused and non-transfused patients. Simultaneously, the Brazilian program is building a donor, donation, and component database. The databases will serve as the backbone for retrospective and prospective observational studies in transfusion epidemiology, transfusion recipient outcomes, blood component quality, and emerging blood safety issues. Special focus will be on preterm infants, patients with sickle cell disease, thalassemia or cancer, and the effect of donor biologic variability and component manufacturing on recipient outcomes. A rapid response capability to emerging safety threats has resulted in timely studies related to Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2). CONCLUSIONS: The REDS-IV-P program endeavors to improve donor-recipient-linked research with a focus on children and special populations while also maintaining the flexibility to address emerging blood safety issues.

Psychological impact of the COVID-19 pandemic on young professionals in blood banks and transfusion services: A global cross-sectional survey.

BACKGROUND AND OBJECTIVES: The COVID-19 pandemic brought about changes to daily life as measures to contain the spread of the virus increased across the world. The aim of this survey was to assess the psychological impact of the pandemic on young professionals (YPs) in transfusion medicine. MATERIALS AND METHODS: A cross-sectional web-based survey was distributed electronically to ISBT members inviting YPs (≤40 years) to participate. Statistical analysis was performed using SPSS software. RESULTS: Two hundred and fifty-nine YPs completed the survey, including 107 clinicians/physicians and/or nurses. Almost half of the YPs (52.5%) indicated increased stress levels and 15.4% indicated symptoms of depression. YPs highlighted the loss of social engagement (59.1%) and increased pressure from information seen on media (35.5%) as factors negatively impacting their psychological wellbeing. Further, 20.8% expressed increased economic stress resulting from concerns about job security. Almost half of the YPs indicated that their organization provided moderate/occasional holistic support to them and their families. Sixty percent and 74.4% of YPs reported increased workload and staff absence due to COVID-19 infection, respectively. Only half of clinicians/physicians and/or nurses indicated that they often had sufficient personal protective equipment. The majority of these (76.6%) had family/household members living with them, and 61% indicated that they were significantly worried about infecting them because of the nature of their work. CONCLUSION: COVID-19 had a major impact on the well-being of YPs working in transfusion medicine. Measures are required to ensure that YPs are protected and mentally supported while undertaking their duties in current and future pandemics.

Impact of HLA-G +3142C>G on the development of antibodies to blood group systems other than the Rh and Kell among sensitized patients with sickle cell disease.

BACKGROUND: Patients' inflammatory history is an important factor underlying red blood cell (RBC) alloimmunization, which is a frequent transfusion complication among individuals with sickle cell disease (SCD). HLA-G has been associated with different inflammatory and auto - immune diseases. Our goal was to verify whether the HLA-G + 3142 C>G and 14-bp Ins/Del variations are associated with RBC antibody development among SCD patients. METHODS: This was a single-center case-control study. SCD patients were randomly selected for the study and divided into two groups: 'Alloimmunized' and 'Nonalloimmunized' depending on the presence of irregular antibodies. The 'Alloimmunized'group was further divided into two subgroups according to the presence of only antibodies against the Rh and Kell blood group systems or the existence of antibodies to antigens of the other blood group systems. RESULTS: A total of 213 patients were included in the study (110 alloimmunized and 103 non-alloimmunized). The 'Alloimmunized' and 'Non-alloimmunized' groups did not differ statistically regarding the HLA-G + 14 bp Ins/Del ( p = 0.494) and + 3142 C>G ( p = 0.334). Individuals who had only antibodies against the Rh and Kell antigens had a frequency of HLA-G + 3142GG genotype almost twice as high compared to the groupwith antibodies against less immunogenic antigens ( p = 0.043). CONCLUSIONS: The genotype frequency of HLA-G + 3142 C>G differs among alloimmunized SCD patients, depending on the presence of antibodies against low immunogenic RBC antigens. This highlights a possible role played by the HLA-G molecule in the RBC alloimmunization process.

Variant genotypes associated with reduced expression of RhCE antigens among Brazilian blood donors.

BACKGROUND: The genetic diversity of the RHCE gene locus has been explored in diverse populations of different racial backgrounds. Data referring to the diversity of RHCE encoding weakened expression of C, c, E, and e in multiethnic populations is still incomplete. METHODS: Samples from Brazilian blood donors presenting reduced expression of C, c, E, or e on gel method were selected for the study. All exons and flanking introns of RHCE were genotyped though direct Sanger sequencing for the included donors. RESULTS: Sixty-six donors were included: 23 with weak C, 22 with weak c, 6 with weak E, 14 with weak e, and 1 with weak c and E. Among the samples with weak C, the following altered RH*C were encountered: RHCE*CeMA (n = 3), RHCE*Ce941C (n = 1), and RHCE*CeVA (n = 1). RHD*D-CE(4-7)-D was detected in six cases, RHCE*CE was presumably present in five cases, and seven cases were unexplained. Two altered alleles underlay the weak c phenotype: RHCE*ceJAL (n = 20) and RHCE*ce340T (n = 2), and two altered RHCE justified weak e: RHCE*ceMO (n = 6) and RHCE*ceJAL (n = 8). Three variant RHCE were associated with weak E: RHCE*cEJU (n = 4), RHCE*cE382C (n = 1), and RHCE*cEIV (n = 1). The RHCE*cE905A justified one case of weak c and E. CONCLUSION: We describe the distribution of RHCE variants found in association with weak expression of C, c, E, and e in blood donors of multiethnic origin, which differs in comparison to that previously reported for people of African or Caucasian descent.

Correlation between SARS-COV-2 antibody screening by immunoassay and neutralizing antibody testing.

BACKGROUND: The efficacy of convalescent plasma (CP), an alternative for the treatment of COVID-19, depends on high titers of neutralizing antibodies (nAbs), but assays for quantifying nAbs are not widely available. Our goal was to develop a strategy to predict high titers of nAbs based on the results of anti-SARS-CoV-2 immunoassays and the clinical characteristics of CP donors. STUDY DESIGN AND METHODS: A total of 214 CP donors were enrolled and tested for the presence of anti-SARS-CoV-2 antibodies (IgG) using two commercial immunoassays: EUROIMMUN (ELISA) and Abbott (Chemiluminescence). Quantification of nAbs was performed using the Cytopathic Effect-based Virus Neutralization test. Three criteria for identifying donors with nAbs ≥ 1:160 were tested: - C1: Curve ROC; - C2: Conditional decision tree considering only the IA results and - C3: Conditional decision tree including both the IA results and the clinical variables. RESULTS: The performance of the immunoassays was similar referring to both S/CO and predictive value for identifying nAbs titers ≥1:160. Regarding the studied criteria for identifying CP donors with high nAbs titers: (a) C1 showed 76.1% accuracy if S/CO = 4.65, (b) C2 presented 76.1% accuracy if S/CO ≥4.57 and (c) C3 had 71.6% accuracy if S/CO was ≥4.57 or if S/CO was between 2.68-4.57 and the last COVID-19-related symptoms were recent (within 19 days). CONCLUSION: SARS-CoV-2 IgG immunoassays (S/CO) can be used to predict high anti-SARS-CoV-2 nAbs titers. This study has proposed different criteria for identifying donors with ≥1:160 nAbs titers, all with high efficacy.

Diversity of variant alleles encoding Kidd, Duffy, and Kell antigens in individuals with sickle cell disease using whole genome sequencing data from the NHLBI TOPMed Program.

BACKGROUND: Genetic variants in the SLC14A1, ACKR1, and KEL genes, which encode Kidd, Duffy, and Kell red blood cell antigens, respectively, may result in weakened expression of antigens or a null phenotype. These variants are of particular interest to individuals with sickle cell disease (SCD), who frequently undergo chronic transfusion therapy with antigen-matched units. The goal was to describe the diversity and the frequency of variants in SLC14A1, ACKR1, and KEL genes among individuals with SCD using whole genome sequencing (WGS) data. STUDY DESIGN AND METHODS: Two large SCD cohorts were studied: the Recipient Epidemiology and Donor Evaluation Study III (REDS-III) (n = 2634) and the Outcome Modifying Gene in SCD (OMG) (n = 640). Most of the studied individuals were of mixed origin. WGS was performed as part of the National Heart, Lung, and Blood Institute's Trans-Omics for Precision Medicine (TOPMed) program. RESULTS: In SLC14A1, variants included four encoding a weak Jka phenotype and five null alleles (JKnull ). JKA*01N.09 was the most common JKnull . One possible JKnull mutation was novel: c.812G>T. In ACKR1, identified variants included two that predicted Fyx (FY*X) and one corresponding to the c.-67T>C GATA mutation. The c.-67T>C mutation was associated with FY*A (FY*01N.01) in four participants. FY*X was identified in 49 individuals. In KEL, identified variants included three null alleles (KEL*02N.17, KEL*02N.26, and KEL*02N.04) and one allele predicting Kmod phenotype, all in heterozygosity. CONCLUSIONS: We described the diversity and distribution of SLC14A1, ACKR1, and KEL variants in two large SCD cohorts, comprising mostly individuals of mixed ancestry. This information may be useful for planning the transfusion support of patients with SCD.

Reduced volume of red blood cell priming is safe for pediatric patients undergoing therapeutic plasma exchange.

RATIONAL: Therapeutic Plasma Exchange (TPE) procedures in pediatric patients are challenging due to the large extracorporeal volume of the cell separators, which were designed for adults. Red blood cell (RBC) priming is an alternative for overpassing the risks of hypovolemia, but data referring to the volume of packed RBCs to be infused are yet incomplete. Restricting the volume of RBC priming may potentially be associated with less transfusion reactions. GOAL: To determine the safety of administering a reduced volume of RBC priming for pediatric patients undergoing TPE, in comparison to the standard volume recommended by the cell separators' manufacturers. METHODS: This was a case-control study which enrolled 15 pediatric patients undergoing TPE and weighting more than 10Kg. The TPE procedures (n = 406) were divided in two groups: 1) Group1: TPE with ≤150 mL of packed RBC priming and 2) Group2: TPE with 150-250 mL of RBC priming. Groups were compared in terms of hemoglobin / hematocrit and occurrence of adverse reactions. RESULTS: Group1 and Group2 did not differ significantly in relation to pre- and post-TPE hemoglobin (Hb) levels (p = 0.19 and p = 0.18, respectively). The Δ Hb (Hb pre-TPE - Hb post-TPE) was also not statistically different between the groups. The number of adverse reactions was significantly higher in Group 2 in relation to Group 1 (p = 0.01). The number of allergic reactions was also higher in Group 2 (p = 0.06). CONCLUSIONS: Restricting the volume of RBC priming to less than 150 mL is safe for pediatric patients weighting more than 10Kg and associated with lower rates of transfusion-related adverse reactions.

Novel mutations in the bone morphogenetic protein 6 gene in patients with iron overload and non-homozygous genotype for the HFE p.Cys282Tyr mutation.

BACKGROUND: Five main genes are associated with hemochromatosis; however, current studies show that, in addition to these genes, others may be associated with primary iron overload (IO). One of these is the bone morphogenetic protein 6 (BMP6), which encodes a protein that modulates hepcidin synthesis and, consequently, iron homeostasis. AIM: To identify BMP6 gene pathogenic variants in patients with IO and non-homozygous genotype for the HFE p.Cys282Tyr mutation. MATERIALS AND METHODS: Fifty-three patients with primary IO and non-homozygous genotype for the HFE p.Cys282Tyr were selected. Subsequent bidirectional DNA sequencing of BMP6 exons was performed. RESULTS: Two novel variants were found. One at homozygous state p.Gln158Ter (c.472C>T) was pathogenic, the other one at heterozygous state p.Val394Met (c.1180G>A) was of uncertain significance (VUS); the third variant at heterozygous state p.Arg257His (c.770G>A) has already been described and associated with IO. No BMP6 pathogenic variants that would explain iron overload phenotypes were detected in 94% of the studied patients. CONCLUSION: Identification of the BMP6 pathogenic variants in Brazilian patients with primary IO might contribute to the genetic understanding of this phenotype.

FcγR2B B2.4 haplotype predicts increased risk of red blood cell alloimmunization in sickle cell disease patients.

BACKGROUND: Red blood cell (RBC) alloimmunization is an important transfusion complication which is prevalent among sickle cell disease (SCD) patients. Autoimmune diseases are a known risk factor for RBC alloimmunization, suggesting that autoimmunity and post-transfusion alloantibody development occur through similar physiopathological pathways. Polymorphisms in the FcγR2B gene have already been associated with several autoimmune disorders and hypothetically could be associated with RBC alloimmunization. Our goal was to evaluate if important polymorphisms of FcγR2B have an impact on the risk of RBC alloimmunization among SCD patients. STUDY DESIGN AND METHODS: This was a case-control study in which alloimmunized and non-alloimmunized SCD patients were compared in terms of the genotype frequency of the FcγR2B polymorphisms -386G/C, -120 T/A, and 695C/T, genotyped through direct Sanger sequencing. RESULTS: A total of 237 patients met the eligibility criteria, 120 cases (alloimmunized) and 117 controls (non-alloimmunized). RBC alloimmunization was associated with female sex (p < 0.001), lifetime number of RBC units transfused (p = 0.002) and 120 T/A FcγR2B genotype (p = 0.031). The FcγR2B promoter region haplotype 2B.4 (386C120A) was positively associated with RBC alloimunization (p = 0.045). The logistic regression (LR) model identified female sex (OR 10.03, CI 95% 5.16-19.49; p < 0.001) and FcγR2B 2B.4 haplotype (OR 4.55, CI95% 1.1118.65; p = 0.035) as independent predictors of RBC alloimmunization in SCD patients. CONCLUSION: SCD patients with the FcγR2B 2B.4 haplotype had over a fourfold higher risk for RBC alloimmunization. This highlights the role played by FcγR2B on RBC alloimmunization and may be helpful in identifying the immune responders.

Blood utilization and characteristics of patients treated with chronic transfusion therapy in a large cohort of Brazilian patients with sickle cell disease.

BACKGROUND: Red blood cell (RBC) transfusions are used in sickle cell disease (SCD) to treat acute complications or as chronic transfusion therapy (CTT) to prevent severe manifestations. The objectives of this study were to describe blood utilization and adverse events (AEs) associated with RBCs in the Brazilian SCD population and compare characteristics of patients treated or not with CTT. STUDY DESIGN AND METHODS: A SCD cohort was established at six Brazilian centers. Medical and blood bank records were abstracted for clinical and transfusion history. Two controls not treated with CTT matched on center, SCD genotype, sex, and age were selected for each CTT case within the cohort to compare characteristics between the two groups. RESULTS: Most of the 2794-member cohort had received a transfusion (75.0% of children and 89.2% of adults) with 29.2% of patients receiving transfusion in the prior year. There were 170 (10.6%) children and 115 (9.2%) adults treated with CTT. Children not treated with CTT were more likely to have pain and acute chest hospitalizations in the prior year (25.3% vs. 11.9%, p = 0.0003; and 22.0% vs. 10.7%, p = 0.002, respectively). Both iron overload and alloimmunization were more common in CTT cases compared to controls (65.6% vs. 17.0% and 36.2% vs. 15.9%, respectively). A higher proportion of adults treated with CTT demonstrated oxygen saturation of greater than 95% compared to controls not treated (51.1% vs. 39.2%), while there was no difference in oxygenation between children treated or not. Of 4501 transfusion episodes, 28 (0.62%) AEs were reported. There was no difference in AEs associated with transfusions for acute indications versus CTT. CONCLUSION: Red blood cell transfusion was common in Brazilian SCD patients, with utilization driven by CTT. Transfusion reactions were not common; however, alloimmunization and iron overload were frequent among those on CTT, highlighting the need for novel clinical strategies to mitigate these risks.

Using droplet digital PCR to screen for rare blood donors: Proof of principle.

BACKGROUND: Digital droplet PCR (ddPCR) is a very sensitive high throughput genotyping methodology. To date, the use of ddPCR in immunohematology is restricted to fetal genotyping of red blood cell antigens. Our hypothesis is that this technology could be applied to screen for rare red blood cell genotypes, such as Di(b-). METHODS: Nucleic acid of 3168 donors was extracted for viral screening routine in pools of 6, which were converted into three types of 48-donor pools: control pools (only DI*B/*B samples), pools with varying amount of DI*A/*B samples (n = 1-5) and a pool with one rare DI*A/*A sample. Pools were genotyped using ddPCR to detect and quantify DI*A and DI*B alleles. RESULTS: DI*A allele was accurately detected in all pools containing Di(a + b+) samples and in the pool containing one Di(a + b-) sample. No copies were detected in the control pools (n = 60). The ratio between the number of DI*A and DI*B copies varied significantly between the pools and the triplicates. CONCLUSION: The proposed ddPCR assay was accurate in identifying the rare DI*A allele in large pools of donors and can be applied to screen for Di(b-) phenotype. The strategy can potentially be extended to search for other rare RBC phenotypes.

Effectiveness of strategies to screen for blood donors with RH variants in a mixed population.

INTRODUCTION: Patients with RH variants presenting antibodies directed to RH high frequency antigens or multiple RH antibodies might, in some occasions, be better served with RH genotype-matched units, requiring screening for RH variants among blood donors. To date, strategies to identify donors with RH variants were restricted to selecting individuals of African descent based on self-reported race, what can be inaccurate in racially mixed population. Our goal was to: 1) Screen for donors with RH variants in a mixed population using self-declared race and Rh phenotype as selection criteria; and 2) Verify if including the Duffy null genotype in the screening algorithm increases its effectiveness. METHODS: Brazilian donors were included if self-declared as black and phenotyped as R0r or R1r. All individuals were genotyped for RHCE exons 1, 5, 6 and 7 and for the FY*B c.-67 T > C polymorphism in order to determine the Duffy null genotype. RHD variants were searched for in cases of altered RHCE. RESULTS: Among 2500 blood donors, 217 fulfilled the inclusion criteria and were enrolled. Fifty-three (24.4 %) had a predicted clinically relevant Rh phenotype (partial antigens or lack of high frequency antigens). Twelve donors (5.5 %) had a predicted RhCE phenotype lacking either hrB or hrS. Most cases with predicted lack of high frequency antigens (66.7 %) occurred in donors with the Duffy null genotype. CONCLUSION: Selecting donors based on self-declared race, Rh phenotype and Duffy null genotype is feasible and effective in identifying RH variants lacking Rh high frequency antigens among racially mixed donors.

Weak D types 38 and 11: determination of frequencies in a Brazilian population and validation of an easy molecular assay for detection.

CONCLUSIONS: Recent evidence shows that, among Brazilians, the distribution of weak D types significantly differs from that represented in people of European descent, with a high percentage of weak D types 38 and 11. Our goal was to determine the population frequencies of weak D types 38 and 11 in a Brazilian population and to validate a molecular approach to identify these two variants. Blood donors were sequentially enrolled in the study in a 5-year period. Donors with serologic weak D phenotype had the RHD coding region sequenced. The frequencies of weak D type 38 and weak D type 11 (CDe-associated) were calculated. Two allele-specific-polymerase chain reaction (AS-PCR) assays were designed to detect RHD*weak D type 38 and RHD*weak partial 11 and were validated with samples positive and negative for these two variants, respectively. A total of 618,542 donors were enrolled, of which 265 presented with a serologic weak D phenotype. When considering all donors evaluated, the frequencies of weak D types 38 and 11 were 0.013 and 0.002 percent, respectively. In the subgroup of donors with a serologic weak D phenotype, the frequencies of weak D types 38 and 11 were 30.2 and 4.9 percent, respectively. The two proposed AS-PCR assays for detection of RHD*weak D type 38 and RHD*weak partial 11 showed 100 percent accuracy. The frequencies of weak D types 38 and 11 among Brazilians are high compared to that previously described for other populations. The AS-PCR assays to detect RHD*weak D type 38 and RHD*weak partial 11 represent potentially helpful tools for investigating Brazilian individuals with these weak D phenotypes.

A Toll-like receptor 2 genetic variant modulates occurrence of bacterial infections in patients with sickle cell disease.

Despite adequate immunization and penicillin prophylaxis, bacterial infections remain a leading cause of morbidity and mortality in patients with sickle cell disease (SCD). Besides hyposplenism, inflammatory and genetic factors might modulate their susceptibility to bacterial infections. We performed a candidate gene association of single nucleotide polymorphisms (SNPs) located in Toll-like receptor (TLR) genes, encoding prominent molecules for innate immune responses, with the occurrence of bacterial infections in patients with SCD. A cohort followed in centres in Brazil, France and Senegal (n = 430) was divided in two groups: patients who presented at least one episode of bacterial infection (n = 235) and patients who never had bacterial infections (n = 195). There were no differences in gender or age distribution among the groups. The frequency of the TLR2 rs4696480 TA genotype was significantly lower in the infected group (50% vs. 67%, odds ratio [OR] = 0·50, 95% confidence interval [CI] 0·34-0·75, P < 0·001), and the TT genotype was significantly higher in the infected group (15% vs. 5%, OR = 3·18, 95% CI 1·53-6·61, P < 0·001). Previous reports demonstrated higher secretion of inflammatory factors in cells from AA individuals, lower occurrence and severity of immune diseases in T carriers. The rs4696480 TA genotype might stand between deleterious effects of over inflammatory response (AA genotype) and inefficient responses (TT genotype) to infectious agents in SCD settings.

SMIM1 intron 2 gene variations leading to variability in Vel antigen expression among Brazilian blood donors.

BACKGROUND: There is a significant inter-individual heterogeneity of Vel antigen expression which can lead to inaccuracies on Vel phenotyping of blood donors and, potentially, to hemolytic post-transfusion reactions. Our aim was to evaluate the impact of genetic variants in the SMIM1 intron 2 on the expression of Vel antigen among Brazilian blood donors harboring the c.64_80del17 deletion in heterozygosity. METHODS: Donors presenting the SMIM1 c.64_80del17 in heterozygosity were included in the study and subjected to SMIM1 intron 2 direct sequencing aiming to genotype the following polymorphisms: rs143702418, rs1181893, rs191041962, rs6673829, rs1175550 and rs9424296. RESULTS: SMIM1 intron 2 sequencing was performed on two hundred donors presenting one c.64_80del17 allele. The rs1175550 polymorphism significantly impacted on Vel antigen expression. Variations in the strength of agglutination on Vel phenotyping were also observed according to the rs6673829 genotype, but this difference did not persist with statistical relevance after multivariate analysis. CONCLUSION: The presence of the rs1175550A allele of SMIM1 is significantly and independently associated with a decrease in Vel antigen expression. Even though the population in Brazil is intensely mixed, the allele frequencies obtained in the current study were very similar to that reported for Europeans.

Diversity of RH and transfusion support in Brazilian sickle cell disease patients with unexplained Rh antibodies.

BACKGROUND: Genetic diversity in the RH genes among sickle cell disease (SCD) patients is well described but not yet extensively explored in populations of racially diverse origin. Transfusion support is complicated in patients who develop unexpected Rh antibodies. Our goal was to describe RH variation in a large cohort of Brazilian SCD patients exhibiting unexpected Rh antibodies (antibodies against RH antigens to which the patient is phenotypically positive) and to evaluate the impact of using the patient's RH genotype to guide transfusion support. STUDY DESIGN AND METHODS: Patients within the Recipient Epidemiology and Evaluation Donor Study (REDS)-III Brazil SCD cohort with unexpected Rh antibodies were selected for study. RHD and RHCE exons and flanking introns were sequenced by targeted next-generation sequencing. RESULTS: Fifty-four patients with 64 unexplained Rh antibodies were studied. The majority could not be definitively classified as auto- or alloantibodies using serologic methods. The most common altered RH were RHD*DIIIa and RHD*DAR (RHD locus) and RHCE*ce48C, RHCE*ce733G, and RHCE*ceS (RHCE locus). In 53.1% of the cases (34/64), patients demonstrated only conventional alleles encoding the target antigen: five of 12 anti-D (41.7%), 10 of 12 anti-C (83.3%), 18 of 38 anti-e (47.4%), and one of one anti-E (100%). CONCLUSION: RHD variation in this SCD cohort differs from that reported for African Americans, with increased prevalence of RHD*DAR and underrepresentation of the DAU cluster. Many unexplained Rh antibodies were found in patients with conventional RH allele(s) only. RH genotyping was useful to guide transfusion to determine which patients could potentially benefit from receiving RH genotyped donor units.