HLA-DRB1*07:01 allele is primarily associated with the Diego a alloimmunization in a Brazilian population.

BACKGROUND: The Diego blood group presents a major polymorphic site at Residue 854, causing a proline (Di(b) antigen) to leucine (Di(a) antigen) substitution. Di(a) alloimmunization has been observed among Asian and Native South American populations. Considering that Brazilians represent a genetically diverse population, and considering that we have observed a high incidence of Di(a) alloimmunization, we typed HLA-DRB1 alleles in these patients and performed in silico studies to investigate the possible associated mechanisms. STUDY DESIGN AND METHODS: We studied 212 alloimmunized patients, of whom 24 presented immunoglobulin G anti-Di(a) , 15 received Di(a+) red blood cells and were not immunized, and 1008 were healthy donors. HLA typing was performed using commercial kits. In silico analyses were performed using the TEPITOPEpan software to identify Diego-derived anchor peptide binding to HLA-DRB1 molecules. Residue alignment was performed using the IMGT/HLA for amino acid identity and homology analyses. RESULTS: HLA-DRB1*07:01 allele was overrepresented in Di(a) -alloimmunized patients compared to nonimmunized patients and to healthy donors. Two motifs were predicted to be potential epitopes for Di(a) alloimmunization, the WVVKSTLAS motif was predicted to bind several HLA-DR molecules, and the FVLILTVPL motif exhibited highest affinity for the HLA-DRB1*07:01 molecule. Pocket 4 of the DRB1*07:01 molecule contained specific residues not found in other HLA-DRB1 molecules, particularly those at Positions 13(Y), 74(Q), and 78(V). CONCLUSION: Individuals carrying the HLA-DRB1*07:01 allele present an increased risk for Di(a) alloimmunization. The identification of susceptible individuals and the knowledge of potential sensitization peptides are relevant approaches for transfusion care, diagnostic purposes, and desensitization therapies.

HNA-3 gene frequencies in Brazilians and a new polymerase chain reaction-restriction fragment length polymorphism method for HNA-3a/3b genotyping.

BACKGROUND: HNA-3 antigens are the result of a rs2288904 single-nucleotide polymorphism (SNP) in the CTL2, and the HNA-3a and HNA-3b variants are encoded by a guanine and adenine at Nucleotide Position 461. Anti-HNA-3 are involved in severe transfusion-related acute lung injury reactions and in neonatal alloimmune neutropenia. Since the distribution of the HNA-3 system was unknown in South Americans, in this study we determined the frequency of the HNA-3 alleles in Brazilians. STUDY DESIGN AND METHODS: DNA of 500 blood donors, 120 Xikrin Amerindians, 74 Japanese individuals, and 124 African Brazilians were genotyped for rs2288904 by a polymerase chain reaction (PCR)-restriction fragment length polymorphism assay. The PCR product was digested with enzyme Taq(α) 1, specific to nucleotide guanine (HNA-3a). RESULTS: The results showed that the frequencies of the HNA-3a/HNA-3b alleles were 0.81/0.19 in blood donors, 1.00/0.00 in Amerindians, 0.63/0.37 in Japanese, and 0.85/0.15 in African Brazilians. All 81 individuals genotyped as HNA-3a/a did not present the SNP c.457T by molecular sequencing. CONCLUSION: The frequencies of HNA-3 genotypes in Brazilian blood donors is similar to that described in Caucasians; however, all Amerindians were HNA-3a/a, African Brazilians showed a lower frequency of HNA-3b/b, and Japanese had a higher prevalence of HNA-3b/b, suggesting that they may be at risk for developing anti-HNA-3a alloantibodies.

A PCR-based strategy for Dombrock screening in Brazilian blood donors reveals a novel allele: the DO* A-WL.

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

Impact of using different laboratory assays to detect human leukocyte antigen antibodies in female blood donors.

BACKGROUND: HLA antibodies passively transferred to transfused recipients may cause transfusion reactions such as transfusion-related acute lung injury (TRALI), but in many of the reported TRALI incidents, no white blood cell antibodies have been identified. We investigated whether a higher number of anti-HLA would be detected in donor's plasma by using a method with potential higher sensitivity rate. STUDY DESIGN AND METHODS: Sera from 300 previously pregnant female blood donors were screened for anti-HLA using a solid-phase mixed-antigen assay (enzyme-linked immunosorbent assay [ELISA]). Samples from 60 women with three or more pregnancies with a negative ELISA were further tested using microbead-flow assays (LABScreen mixed, panel-reactive antibodies [PRA], and single antigen). RESULTS: Anti-HLA Class I and/or Class II were detected by ELISA in 26.7% (80/300) of all women and in 37.0% (37/100) of women with three or more pregnancies. The LABScreen assays detected additional anti-HLA specificities (44 Class I and 17 Class II) in 28.3% (17/60) of ELISA-negative donors with three or more pregnancies. HLA antibodies were detected in 8.3% (5/60), 18.3% (11/60), and 21.7% (13/60) of ELISA-negative women by LABScreen mixed, PRA, or single antigen, respectively. CONCLUSION: Our data showed that the microbead-flow detected more HLA antibodies than ELISA, but the clinical significance of these antibodies is currently unknown. Detecting anti-HLA is useful for donor management and could contribute to the decision to definitively defer blood donors involved in TRALI incidents. However, further studies are necessary to better determinate the relative risk of TRALI induced by anti-HLA detected only by techniques with higher sensitivity rate.

Transfusion-related acute lung injury.

Transfusion-related acute lung injury (TRALI) is a serious clinical syndrome associated with the transfusion of plasma-containing blood components. Recently, TRALI has come to be recognized as the leading cause of transfusion-related death in the United States and United Kingdom. This complication typically presents as shortness of breath, hypoxemia, hypotension, fever and noncardiogeneic pulmonary edema, all occurring during or within 6 h after transfusion. Although the mechanism of TRALI has not been fully elucidated, it has been associated with human leukocyte antigen antibodies (class I, class II or neutrophil alloantigens) and with biologically active mediators in stored cellular blood components. Most of the donors implicated in cases of TRALI are multiparous women. Rarely diagnosed, TRALI can be confused with other causes of acute respiratory failure. Greater knowledge regarding TRALI on the part of clinicians could be crucial in preventing and treating this severe complication of blood transfusion.

Anti-KEL7 (anti-Js(b)) alloimmunization diagnostic supported by molecular KEL*6,7 typing in a pregnant woman with previous intrauterine deaths.

Anti-KEL7 (anti-Js(b)) is a rare antibody that has been related to haemolytic transfusion reactions and HDN. We report a case of anti-KEL7 alloimmunization detected in a pregnant woman who had an obstetric previous history of four miscarriages and one stillborn. Employing classical immunohematological techniques, we studied the propositus and her available relatives. Due to the unavailability of commercial anti-KEL6 and anti-KEL7 reagents, we used a KEL*6,7 genotyping method as an alternative tool to contribute with the identification of the alloantibody origin. The results of KEL genotyping showed that the propositus was KEL*6/6 homozygous, while her second partner was KEL*7/7 homozygous.

Application of noninvasive phagocytic cellular assays using autologous monocytes to assess red cell alloantibodies in sickle cell patients.

We investigated red cell (RBC) alloantibodies in 125 sickle cell anemia (SCA) patients using tube indirect antiglobulin test (PEG, LISS or enzyme) and gel centrifugation test (LISS or enzyme). Prediction of clinical significance of alloantibodies was evaluated by the monocyte monolayer assay (MMA) and the chemiluminescence test (CLT) using autologous monocytes. The alloimmunization rate was 20.8% and the gel test detected a higher number of alloantibodies than the tube test (26 v 21, p = 0.02). We observed 58.3% and 69.2% positive MMA and CLT results, respectively. Eighteen (69.2%) antibodies exhibited clinical relevance, 14 (58.3%) antibodies reacted by both MMA and CLT, while 4 (15.4%) antibodies reacted only by CLT. In conclusion, the application of phagocytic cellular assays using autologous monocytes defined clinical significance of about 70% of RBC alloantibodies detected in SCA patients. The data also suggest that the CLT may be more valuable than the MMA as a noninvasive test for predicting hemolysis after transfusion of incompatible blood in SCA patients.