Análise do perfil de Rh e Kell em doadores de sangue em Porto Alegre, Rio Grande do Sul / Phenotypic profile analysis of Rh and Kell blood group systems among donors in Porto Alegre, Rio Grande do Sul

Introdução: A hemoterapia é uma prática terapêutica pelo meio de transfusão sanguínea. Devido ao baixo estoque de bolsas de sangue e o aumento de pacientes crônicos e emergenciais, se faz necessária a realização de testes imuno-hematológicos para minimizar os riscos de reações transfusionais e aloimunizações em doadores e receptores de sangue. Deste modo, no estudo foi avaliada a prevalência dos antígenos dos sistemas Rh e Kell em doadores de sangue de Porto Alegre ­ RS.Métodos: Estudo quantitativo, transversal e retrospectivo que foi realizado através da análise das informações dos doadores de sangue contidas no banco de dados do Hemocentro do Estado do Rio Grande do Sul, nos anos de 2018 e 2019.Resultados: Das 6.479 amostras fenotipadas, quanto ao sistema Rh, 44,6% são Rh positivo e 55,4% são Rh negativo. As frequências dos antígenos encontradas foram de, CC 10,1%, Cc 27%, cc 62,9%, EE 1,2%, Ee 13,9%, ee 84,9%. E, para o sistema Kell, K1 positivo 7,1% e K1 negativo 92,9%.Conclusões: Antígenos do sistema Rh e Kell exibem um grande nível de imunogenicidade e uma forte ligação com a Doença Hemolítica do Recém-nascido, podendo ocorrer a sensibilização em pacientes caso não haja a compatibilidade sanguínea. Este estudo ressalta a importância da implementação da fenotipagem eritrocitária em doadores de sangue, sugere-se mais estudos com períodos distintos para a pesquisa de resultados satisfatórios.

Introduction: Hemotherapy is a therapeutic practice consisting of blood transfusion. Low blood supply and an increase in chronic and emergency patients have made it necessary to conduct immunohematology tests to minimize the risks of adverse reactions and alloimmunization in donors and recipients. Therefore, this study aimed to assess the prevalence of Rh and Kell blood group antigens among blood donors in Porto Alegre, Rio Grande do Sul, Brazil.Methods: We conducted a quantitative, cross-sectional, retrospective study. Information from blood donors included in the Rio Grande do Sul's Blood Center database from 2018 to 2019 were analyzed.Results: A total of 6,479 samples were phenotyped, of which 44.6% were Rh-positive and 55.4% were Rh-negative. Antigen prevalence was CC (10.1%), Cc (27%), cc (62.9%), EE (1.2%), Ee (13.9%), and ee (84.9%). As for the Kell group, 7.1% were K1-positive and 92.9% were K1-negative.Conclusions: The Rh and Kell antigens are highly immunogenic and have a strong link with the hemolytic disease of the newborn. Sensitization may occur in patients if there is no blood compatibility. This study highlights the importance of implementing erythrocyte phenotyping in blood donors. Further studies should be conducted in different time frames to achieve satisfactory results.

Reduction of anti-K-mediated hemolytic disease of newborns after the introduction of a matched transfusion policy: A nation-wide policy change evaluation study in the Netherlands.

BACKGROUND: During pregnancy, maternal red blood cell (RBC) antibodies can lead to life-threatening fetal hemolysis and anemia. Women can become immunized by a pregnancy or an unmatched transfusion. Our aim was to quantify the effect of a nationwide K-matched transfusion policy for women of childbearing age potential to prevent K-immunization in pregnancy. STUDY DESIGN AND METHODS: In this nation-wide policy change evaluation study we determined the occurrence of RBC antibodies before and after introduction of a K-matched transfusion policy and evaluated the cause K alloimmunization 10 years after introduction of this measure. K-matched transfusion for females under 45 years of age is advised in the Dutch transfusion guideline since 2004. We used laboratory data from pregnancies with RBC antibodies identified in the period 1999-2018 obtained as part of a population-based screening program in the Netherlands. RESULTS: Tests of 36 286 pregnancies produced a positive antibody screening result which concerned anti-K in 1550 pregnancies. The occurrence of anti-K decreased from 67.9 to 20.2 per 100 000 pregnancies. The relative risk reduction was 0.70 which largely exceeded the relative risk reduction of 0.27 for antibodies against RBC antigens for which no preventive matching is required. The number of pregnancies at risk for anti-K-mediated disease decreased from 9.7 to 4.2 per 100 000 pregnancies. CONCLUSIONS: A K-matched transfusion policy is associated with a major decrease in a number of pregnant women with anti-K and pregnancies at risk for anti-K-mediated disease. A relatively simple measure is now shown to impact prevention of hemolytic disease in the fetus and newborn.

Poly(I:C) causes failure of immunoprophylaxis to red blood cells expressing the KEL glycoprotein in mice.

Polyclonal anti-D (Rh immune globulin [RhIg]) therapy has mitigated hemolytic disease of the newborn over the past half century, although breakthrough anti-D alloimmunization still occurs in some treated females. We hypothesized that antiviral responses may impact the efficacy of immunoprophylaxis therapy in a type 1 interferon (IFN)-dependent manner and tested this hypothesis in a murine model of KEL alloimmunization. Polyclonal anti-KEL immunoprophylaxis (KELIg) was administered to wild-type or knockout mice in the presence or absence of polyinosinic-polycytidilic acid (poly[I:C]), followed by the transfusion of murine red blood cells (RBCs) expressing the human KEL glycoprotein. Anti-KEL alloimmunization, serum cytokines, and consumption of the transfused RBCs were evaluated longitudinally. In some experiments, recipients were treated with type 1 IFN (IFN-α/ß). Recipient treatment with poly(I:C) led to breakthrough anti-KEL alloimmunization despite KELIg administration. Recipient CD4+ T cells were not required for immunoprophylaxis efficacy at baseline, and modulation of the KEL glycoprotein antigen occurred to the same extent in the presence or absence of recipient inflammation. Under conditions where breakthrough anti-KEL alloimmunization occurred, KEL RBC consumption by inflammatory monocytes and serum monocyte chemoattractant protein-1 and interleukin-6 were significantly increased. Poly(I:C) or type I IFN administration was sufficient to cause breakthrough alloimmunization, with poly(I:C) inducing alloimmunization even in the absence of recipient type I IFN receptors. A better understanding of how recipient antiviral responses lead to breakthrough alloimmunization despite immunoprophylaxis may have translational relevance to instances of RhIg failure that occur in humans.

Resolving Blocked Antigen Phenomenon in Hemolytic Disease of the Fetus and Newborn Due to Anti-K.

High-titer antibodies are a cause of false-negative reactions in red blood cell antigen phenotyping, an event referred to as blocked antigen phenomenon (BAP). In hemolytic disease of the fetus and newborn, BAP complicates laboratory workups as fetal phenotype is helpful in confirming the responsible antibody. Acid elution techniques, techniques using ethylenediaminetetraacetic glycine acid, as well as those using chloroquine diphosphate have been used to resolve BAP; however, ethylenediaminetetraacetic glycine acid destroys K-antigen expression and chloroquine diphosphate is not always effective. We report a case of severe hemolytic disease of the fetus and newborn from anti-K where a modified gentle heat elution resolved BAP. Although infrequently considered with isolated reports in the literature, heat elution is simple, is effective, and involves readily available materials in most blood banks.

Comparative study of alloimmunization against red cell antigens in sickle cell disease & thalassaemia major patients on regular red cell transfusion.

BACKGROUND & OBJECTIVES: : Sickle cell disease (SCD) patients require red cell transfusion during different clinical complications of the disease. Such patients are at a high risk for developing alloantibody against red cell antigens. From India, there are limited data available on alloantibody formation in multiply transfused SCD patients. The present study was thus undertaken to fill up this lacunae by looking at the development of red cell alloantibodies in SCD and ß-thalassaemia patients on regular transfusion. METHODS: : All sickle cell disease patients undergoing red cell transfusion between 2008 and 2016, were included. During this period, a large number of ß-thalassaemia major patients also underwent regular red cell transfusion. These thalassaemia patients were also included to compare the tendency of antibody formation between SCD and ß-thalassaemia major patients. All patients before regular transfusion were regularly assessed for the development of red cell antibody. Red cell antigen, antibody screen crossmatch and antibody identification were done using the standard technique. RESULTS: : A total of 138 patients with SCD aged between 4 and 53 yr (mean 17.6 yr) consisting of 83 males and 55 females (male:female, 1.5:1) along with 333 transfusion-dependent ß-thalassaemia patients were studied. Over the last eight years, 15 patients with SCD and four patients with thalassaemia developed alloantibody (P <0.001). Antibody specificity of their alloantibodies was against Rhc, RhE, Kell, Fya and Fyb only. Sickle cell disease patients with and without alloantibody required on the average 11.8 and 8.6 units of red cell concentrate, respectively (P <0.05). INTERPRETATION & CONCLUSIONS: : About 11 per cent of the transfused sickle cells patients developed alloantibodies. The antibody specificity was restricted to Rh, Kell and Duffy blood group systems. Extended antigen matching involving Rh, Kell and Duffy antigens may prevent alloantibody in such patients.

Transfusion practices and complications in thalassemia.

BACKGROUND: The severe forms of thalassemia are the most common inherited anemias managed with regular blood transfusion therapy. Transfusion policies and complications are critical to quality of life and survival, but there is a lack of standardized care. STUDY DESIGN AND METHODS: A survey of 58 items was completed in 2016 by 11 centers in California, Washington, Oregon, Nevada, and Arizona providing long-term care for thalassemia. The questionnaire addressed demographic information, transfusion practices and complications, and educational needs. RESULTS: The centers followed 717 patients with ß-thalassemia (314, 43.8%) or α-thalassemia (394, 55%). One-third (34.7%) of patients were transfusion-dependent. Indications and goals of transfusion therapy differed between centers. Prestorage leukoreduction was universal, while routine irradiation of units was limited to one site. Red blood cell antigen phenotype was determined before the first transfusion and patients received Rh/Kell-matched units. However, more than half of the transfused patients had received blood at multiple hospitals within or outside the United States. Alloantibodies were seen in 16.9% of transfused group, but management of such patients was variable. Unusual or emerging transfusion-transmitted pathogens were not observed. Multiple educational needs were recognized, with iron overload as the biggest challenge; the approach to iron chelation varied within the group. CONCLUSION: This study identified many patients not included in earlier surveys limited to major national centers, suggesting that the thalassemia population in the United States is vastly underestimated. Lack of evidence-based guidelines is a barrier to optimal care, which should be addressed through regional consortia of thalassemia centers.

SOME CLINICALLY IMPORTANT ERYTHROCYTE BLOOD GROUP ANTIGENS IN DONORS.

The distribution of erythrocyte blood group antigens was evaluated among 656 donors; samples were provided by the diagnostic laboratory "Medina" Ltd Health Centre of Batumi. Lab analysis of the sample was conducted by the immunogenetics laboratory at Batumi Shota Rustaveli State University. The frequency of the ABO allele system in donors was as follows: r (0.70), q (0.23), p (0.07). The distribution of Rhesus (Rh) factor in the donor population was as follows: Rh(-) was found among 16.3±1.43% of investigated donors; the Rh(+) phenotype was found in 83.7±1.43% of donors. Additionally, the CcDee phenotype frequency was 29.9±1.78%; CCD-ee was 17.2±1.47%; ccddee was 14.9±1.38%; and CcD-Ee was 13.9±1.34%; ccD-Ee phenotype was 11.1±1.22%; ccD-ee was 5.5±0.88%; same phenotype indicators -2.1±0.55 were observed for CcD-EE and ccD-EE; CCD-Ee was 1.4±0.45%, CCD-EE was 0.4±0.26%; and finally, the frequency of Ccddee phenotype amounts was 1.1±0.40%, ccddEe and CCddee phenotypes were both 0.2±0.17%. The analysis of the Kell system allele revealed a low frequency for the p allele at 0.05, whereas the frequency of the q allele was 0.95. This large epidemiologic analysis of donor blood provides valuable information for hematological and transfusion centers to inform the preparation of blood components for transfusion.

Alloimmunization against platelets, granulocytes and erythrocytes in multi-transfused patients in Iranian population.

BACKGROUND: This study aims at alloantibody screening, determination of the types of these antibodies in multiple-transfused patients with chronic hematologic diseases. PATIENTS AND METHODS: This descriptive study was performed on 240 patients with chronic hematological diseases referred to public hospitals in Iran. Single blood sample was taken and tested for the presence of antibodies. In case of a positive antibody screening, antibody identification was performed using granulocyte agglutination test (GAT), granulocyte indirect immunofluorescence test (GIIFT), platelet indirect immunofluorescence test (PIIFT), monoclonal antibody-specific immobilization of platelet antigen (MAIPA) and panel cells. RESULTS: Out of 240 patients, 105 patients (43.75 %) had been alloimmunized. The incidence of alloantibodies against red blood cells (RBCs) in positive alloantibodies patients were 84.76% (89/105). The most common alloantibody was against antigens of the kell (anti-K) and Rh (anti-E) and (anti D) systems (46.66%, 18.09% and 11.43% respectively). The overall incidence of anti- human leukocyte antigen (HLA) antibodies were 65.7% (69/105). Polymorphonuclear (PMN)-specific antibodies were found in 6.66% (7/105). Also from 105 patients, 14 patients had alloantibodies against platelet. DISCUSSION: In general, it is recommend that to decrease the rate of alloantibody synthesis, the packed cells should be cross matched for minor blood groups especially for Rh (E) and kell. In addition, the use of leukodepleted blood products can decrease the frequency of alloimmunization against platelet (PLT), PMN and HLA antigens.

Phenotyping Rh/Kell and risk of alloimmunization in haematological patients.

BACKGROUND: One of the biggest concerns in transfusion medicine is to avoid red blood cell alloimmunization. We evaluated the rate of alloimmunization in two groups of chronically transfused patients (A - not phenotyped and B - phenotyped for Rh/K antigens before the first transfusion) with primary haematological disorders and its distribution among the main haematological diseases, in order to adopt an efficient transfusional strategy. STUDY DESIGN AND METHODS: As methodology, we used the SIBAS and SAM databases for the retrospective study of all patients with primary haematological disorder between January 2011 and April 2013. RESULTS: A statistical difference in the rate of alloimmunization comparing groups A and B was found (P <0·0001). We also observed that alloimmunization was not homogeneously distributed in all primary haematological diseases. CONCLUSIONS: The Rh/K phenotype should be performed on all patients diagnosed with bone marrow failure, plasma cell dyscrasia and myelodysplastic syndrome in order to avoid alloimmunization. In patients with acute leukaemia and lymphoma it seems not necessary to perform it. In patients with haemoglobinopathy and myeloproliferative disorders, a larger group of patients is needed to decide its efficacy.

Next-Generation Sequencing for Antenatal Prediction of KEL1 Blood Group Status.

The KEL1 antigen can give rise to immunization of KEL2 mothers. Maternal antibodies can be transferred to the fetus and destroy fetal red blood cells and their stem cell precursors and give rise to serious fetal disease. It is important to be able to predict the fetal KEL status in order to intervene in those pregnancies where the fetus is at risk, and to ascertain when the fetus is not at risk. Technically it can be demanding to predict KEL1 status from a maternal blood sample. The KEL1 allele is based on a single SNP present in about 1-10 % of cell-free maternal DNA after gestation week 10. Here we describe our protocol for antenatal prediction of fetal KEL1 status by NGS analysis of maternal DNA on a MiSeq instrument.

Frequency and specificity of red cell antibodies in thalassemia patients in Albania.

INTRODUCTION: Thalassemia major is a common hemoglobinopathy in Albania. However, there are no data available on the frequency of RBC alloimmunization and autoimmunization in transfusion-dependent Albanian patients with thalassemia. METHODS: A total of 118 patients with thalassemia receiving regular transfusions were studied during 5 years with antibody screening. During this period, they were exclusively transfused with blood matched for ABO, Rhesus and Kell system. These patients were previously exposed to unmatched blood because of blood shortages. RESULTS: Fourteen of 118 (11, 8%) patients developed alloantibodies. Twelve (10, 1%) were already present at the start of the study. Only 2 (1, 7%) were formed after the application of a strict Rh and Kell matching policy. The most common antibody was anti-K, followed by anti-D, anti-C, anti-E, anti-c, anti-e, anti-Jk(b) , and anti-C(w) . Three patients developed anti-D plus anti-C. Anti-K was combined with Rh antibodies in two of five cases. Anti-c was combined with anti-E in two of three cases. The majority of antibodies (10/14) belonged to the Rh blood group system. With the exception of the anti-Jk(b) and the anti-C(w) , all antibodies were already present at the beginning of the follow-up period. During our follow-up, 27 patients (22.8%) developed autoantibodies. A strong coincidence was found between the presence of alloantibodies and autoantibodies. Eleven of 14 (78%) of the patients with alloantibodies had also autoantibodies, whereas autoantibodies were found in 16 of 104 (15%) of patients with thalassemia without autoantibodies. The rate of alloantibody formation dropped from 10.1% to 1.7% after application of a strict Rh and Kell matching policy. CONCLUSION: A policy of Rhesus and Kell matching without occasional exceptions greatly reduced the development of new alloantibodies and autoantibodies. Self-sufficiency through regular blood donation is necessary for the full implementation of an extended match policy and the prevention of antibody formation in our patients.

Three uncommon KEL alleles in one family with unusual Kell phenotypes explain a 35-year old conundrum.

BACKGROUND: Kell is a complex blood group system comprising 35 antigens. Kell antigens are absent from rare red cells of the Ko (null) phenotype and expressed only weakly in the Kmod phenotype. Molecular analysis of three uncommon KEL alleles elucidated the obscure pattern of inheritance of Kell antigens in one family. MATERIALS AND METHODS: Standard serological methods were employed. All exons, flanking intronic sequence and introns 15 and 16 of KEL were sequenced from genomic DNA. cDNA was obtained from erythroid cells cultured from progenitor cells isolated from peripheral blood. RESULTS: The Kmod propositus was heterozygous for two KEL mutations: c.2107G>A, p.Gly703Arg and a synonymous mutation, c.1719C>T, in the codon for p.573Gly. Sequencing of cDNA revealed that c.1719C>T caused skipping of exon 16, resulting in a silent allele. Her KEL:3,-4 brother was heterozygous for KEL*03/04 and c.1719C/T. CONCLUSION: A synonymous mutation caused complete exon skipping, despite being located 16 bases downstream of the 3' splice site, resulting in a null KEL allele. The combined effects of two mod alleles, one responsible for KEL3 expression and the other for p.Gly703Arg, were probably responsible for an unexpected KEL:3,-4 phenotype.

Next-generation sequencing: proof of concept for antenatal prediction of the fetal Kell blood group phenotype from cell-free fetal DNA in maternal plasma.

BACKGROUND: Maternal immunization against KEL1 of the Kell blood group system can have serious adverse consequences for the fetus as well as the newborn baby. Therefore, it is important to determine the phenotype of the fetus to predict whether it is at risk. We present data that show the feasibility of predicting the fetal KEL1 phenotype using next-generation sequencing (NGS) technology. STUDY DESIGN AND METHODS: The KEL1/2 single-nucleotide polymorphism was polymerase chain reaction (PCR) amplified with one adjoining base, and the PCR product was sequenced using a genome analyzer (GAIIx, Illumina); several millions of PCR sequences were analyzed. RESULTS: The results demonstrated the feasibility of diagnosing the fetal KEL1 or KEL2 blood group from cell-free DNA purified from maternal plasma. CONCLUSION: This method requires only one primer pair, and the large amount of sequence information obtained allows well for statistical analysis of the data. This general approach can be integrated into current laboratory practice and has numerous applications. Besides DNA-based predictions of blood group phenotypes, platelet phenotypes, or sickle cell anemia, and the determination of zygosity, various conditions of chimerism could also be examined using this approach. To our knowledge, this is the first report focused on antenatal blood group determination using NGS.

Blueberry muffin rash, hyperbilirubinemia, and hypoglycemia: a case of hemolytic disease of the fetus and newborn due to anti-Kp(a).

Hemolytic disease of the fetus and newborn occurs when maternal IgG antibodies cross the placenta and cause hemolysis of fetal red blood cells. Kp(a) is a low frequency red blood cell antigen that has rarely been implicated in hemolytic disease of the fetus and newborn. The few reported cases attributed to anti-Kp(a) have typically had minimal clinical consequences. We report a critically ill neonate who presented with purpura, respiratory failure, severe liver dysfunction, hyperbilirubinemia, hypoglycemia and anemia. This case report broadens the spectrum of neonatal disease associated with anti-Kp(a), addresses the evaluation of hemolysis with liver failure in a neonate, and emphasizes the importance of screening for antibodies to low frequency red blood cell antigens in suspected hemolytic disease of the fetus and newborn.

Should we be screening for anti-Js(a)?

We analyzed our historic patient database at North Shore University Hospital and determined both the overall frequency of anti-Js(a) and the frequency at which it was detected in combination with other alloantibodies to red blood cell (RBC) antigens. Screening cells used currently are negative for Js(a). Our data suggest that anti-Js(a) would not be detected in 30 to 40 percent of patients in which it is the sole antibody present. Since 1996 the antibody was only detected when other antibodies were found in the screening process. We are exposing 1.7 percent of our patients (90 patients/year) to Js(a). The clinical significance of anti-Js(a) is apparent with previous literature, and our conclusion is that additional studies should be performed to determine whether Js(a) should be included in current antibody screening cells.

Benefits of blood group genotyping in multi-transfused patients from the south of Brazil.

We evaluated the usefulness of blood group genotyping as a supplement to hemagglutination to determine the red blood cell (RBC) antigen profile of polytransfused patients with hematological diseases and renal failure. Seventy-nine patients were selected. They all received more than three units of blood and eight (10%) had already clinical significant alloantibodies occurring alone or in combination against Rh, K, Fya, and Di antigens. DNA was prepared from blood samples and RHCE*E/e, KEL*01/KEL*02, FY*01/FY*02 and JK*01/JK*02 alleles were determined by using PCR-RFLP. RHD*/RHD*Ψ and RHCE*C/c were tested using multiplex PCR. Discrepancies for Rh, Kell, Duffy, and Kidd systems were found between the phenotype and genotype-derived phenotype in 16 of the 38 chronically transfused patients. The genotypes of these patients were confirmed by DNA array analysis (HEA Beadchip(™); Bioarray Solutions, Warren, NJ). Genotyping was very important for the determination of the true blood groups of the polytransfused patients, helped in the identification of suspected alloantibodies and in the selection of antigen-negative RBCs for transfusion.