Duffy phenotyping and FY*B-67T/C genotyping as screening test for benign constitutional neutropenia

ABSTRACT Objective: Low levels of neutrophils can be an intrinsic condition, with no clinical consequences or immunity impairment. This condition is the benign constitutional neutropenia (BCN), defined as an absolute neutrophils count (ANC) ≤2000 cells/mm. Diagnosis of BCN is of exclusion where patients are submitted to blood tests and possibly to invasive diagnostic search until secondary causes of neutropenia are ruled out. The natural history of the disease suggests benign evolution and Brazilian study showed an overall frequency of 2.59%. The main mechanisms include reduced neutrophil production, increased marginalization, extravasation to the tissues and immune destruction. Genetic studies showed strong association between the single nucleotide variant rs2814778 located on chromosome 1q23.2 in the promoter region of the atypical chemokine receptor 1 (Duffy blood group system) gene (ACKR1, also termed DARC) and BCN. The aim of this study is to evaluate FY phenotypes and genotypes including the analysis of the rs2814778 SNP in Brazilian patients with BCN in order to determine an effective diagnostic tool, allowing reassurance of the patient and cost reduction in their care. Methods: Case control study, with 94 individuals (18 patients and 76 controls). Phenotyping was performed by gel test and genotyping was performed by PCR-RFLP. Results: White blood cell (WBC) and absolute neutrophils (AN) counts showed lower levels in patients compared to controls. In the patient group 83.3% were genotyped as FY*B/FY*B. The SNP rs2814778 (-67T > C) was identified in 77.8% of the patients genotyped as FY*B-67C/FY*B-67C. In the control group, 72.7% were homozygous for the wild type and 23.3% were heterozygous. Conclusion: This study reinforces that FY phenotyping and genotyping can be used to detect most people with BCN, avoiding excessive diagnostic investigation. Besides, this procedure may reduce health costs and be reproductible in clinical practice.

Serologic strategy in detecting RHD altered alleles in Brazilian blood donors

ABSTRACT Background: We evaluated different technological approaches and anti-D clones to propose the most appropriate serologic strategy in detecting the largest numbers of D variants in blood donors. Methods: We selected 101 samples from Brazilian blood donors with different expressions of D in our donor routine. The tests were performed in immediate spin (IS) with eleven commercially available anti-D reagents in a tube and microplate. The D confirmatory tests for the presence of weak D included the indirect antiglobulin test (IAT) in a tube, gel and solid-phase red blood cell adherence (SPRCA). All DNA samples were extracted from peripheral blood and the D variants were classified using different molecular assays. Results: The RHD variants identified by molecular analysis included weak D types (1, 2, 3, 11 and 38) and partial Ds (DAR1.2, DAR1, DAR3.1, DAU0, DAU2, DAU4, DAU5, DAU6, DMH and DVII). The monoclonal-monoclonal blend RUM-1/MS26 was the best anti-D reagent used in detecting the D antigen in the IS phase in a tube, reacting with 83.2% of the D variants, while the anti-D blend D175 + 415 was the best monoclonal antibody (MoAb) used in a microplate to minimize the need for an IAT, reacting with 83.2% of the D variants. The D confirmatory tests using SPRCA showed a reactivity (3 - 4+) with 100% of the D variant samples tested. Conclusion: Our results show that, even using sensitive methods and MoAbs to ensure the accurate assignment of the D antigen, at least 17% of our donor samples need a confirmatory D test in order to avoid alloimmunization in D-negative patients.

The rare holley antibody associated with a severe hemolytic transfusion reaction: the importance of this antibody identification to find a compatible blood unit / Raro anticorpo associado à reação transfusional hemolítica grave: a importância de sua identificação para encontrar uma unidade sanguínea compatível

ABSTRACT The correct identification of erythrocyte antibodies is fundamental for the searching for compatible blood and haemolytic transfusion reactions prevention. Antibodies against antigens of high prevalence are difficult to identify because of the rarity of their occurrence and unavailability of negative red cells for confirmation. We report a case of 46-years-old woman, diagnosed with hemoglobinopathy, and who had symptomatic fall in hemoglobin levels (5.3g/dL) after blood transfusion suggestive of transfusion reaction. The patient's blood type was O RhD-positive. Irregular antibody screening was positive and demonstrated a panreaction against all erythrocytes tested, but this result was not reactive with dithiothreitol. Using negative red cells for antigens of high prevalence of our inventory we could identify in the serum of the same erythrocytes an anti-Holley antibody associated with anti-E. Molecular analysis confirmed that the patient was negative for E and Holley antigens. The crossmath with compatible units confirmed the results. Holley is a high prevalence antigen of the Dombrock blood system whose negative phenotype is extremely rare in all populations and is associated with hemolytic transfusion reactions. This is an antibody that is difficult to identify because laboratories need to have experience in solving complex cases, and have available a large stock of rare sera and erythrocytes, as well other tools such as enzymes, thiol reagents and molecular tests. The correct identification of a rare antibody is initial and mandatory for searching of compatible donors, and to guarantee a satisfactory transfusional support.

RESUMO A correta identificação dos anticorpos eritrocitários é fundamental na busca de sangue compatível e na prevenção das reações transfusionais hemolíticas. Anticorpos contra antígenos de alta prevalência são de difícil identificação, devido à raridade de sua ocorrência e à indisponibilidade de hemácias negativas para sua confirmação. Apresentamos aqui o caso de uma paciente do sexo feminino, 46 anos, com diagnóstico de hemoglobinopatia, que apresentou queda sintomática dos níveis de hemoglobina (5,3g/dL) após transfusão sanguínea, sugestiva de reação transfusional. O tipo sanguíneo da paciente era O RhD-positivo. A pesquisa de anticorpos irregulares foi positiva, demonstrando panreação contra todos os eritrócitos testados, mas não reativo ao ditiotreitol. Utilizando hemácias selecionadas negativas para antígenos de alta prevalência do nosso inventário, foi possível identificar no soro da mesma um anticorpo anti-Holley associado a um anti-E. A análise molecular confirmou que a paciente era negativa para os antígenos E e Holley, e as provas de compatibilidade com unidades fenotipadas confirmaram os resultados. Holley é um antígeno de alta prevalência do sistema sanguíneo Dombrock, cujo fenótipo negativo é extremamente raro em todas as populações e está associado a reações transfusionais hemolíticas. Trata-se de anticorpo de difícil identificação, pois os laboratórios precisam ter experiência na resolução de casos complexos, grande estoque de soros e eritrócitos raros, além de outras ferramentas, como enzimas, reagentes tiol e testes moleculares. A identificação correta de um anticorpo raro é inicial e obrigatória para a busca de doadores compatíveis, garantindo um suporte transfusional satisfatório.

Transfusion management for patients taking an anti-CD38 monoclonal antibody

Abstract Introduction: Pre-transfusion tests, essential for the release of blood components, may be affected by drugs. Monoclonal antibodies represent a class of medications increasingly used in the clinical practice, with anti-CD38 monoclonal antibodies (daratumumab) being a promising resource in the treatment of refractory myeloma. This monoclonal antibody recognizes CD38 in myeloma cells and interferes with pre-transfusion tests by causing panreactivity in indirect antiglobulin tests thereby clinically masking alloantibodies. Dithiothreitol is a reagent that breaks disulfide bonds and effectively destroys antigenic sites for CD38 on red blood cells. This study reports the immunohematological findings of pre-transfusion tests of patients with multiple myeloma receiving daratumumab and on solutions to prevent the interference of this monoclonal antibody. Methods: Serum samples from five patients on anti-CD38 monoclonal antibody treatment were evaluated. Tests performed included ABO/RhD typing, indirect antiglobulin test, direct antiglobulin test and eluate test. A daily evaluation was performed to determine the shelf life of dithiothreitol-treated red blood cells when stored in Alsever's solution. Results: No interference in the ABO/RhD typing results was noted but in all samples, a panreactivity was observed in indirect antiglobulin tests. Regarding the direct antiglobulin test, two samples presented positive results but negative eluates. In all samples, treatment of reagent red blood cells with 0.2 M dithiothreitol offset interference by anti-CD38 monoclonal antibodies. Dithiothreitol-treated red blood cells stored in Alsever's solution were stable for up to 15 days. Conclusion: Treatment of reagent red blood cells with dithiothreitol can be efficient and accessible to offset the interference of the anti-CD38 drug in pre-transfusion tests. The number of costly serological workups can be reduced by having stored dithiothreitol red blood cells with this proving to be a useful reagent for investigating anti-CD38.

Impact of a confirmatory RhD test on the correct serologic typing of blood donors

BACKGROUND: The RHD gene is highly polymorphic, which results in a large number of RhD variant phenotypes. Discrepancies in RhD typing are still a problem in blood banks and increase the risk of alloimmunization. In this study, the RhD typing strategy at a blood bank in Brazil was evaluated.METHODS: One-hundred and fifty-two samples typed as RhD negative and C or E positive by routine tests (automated system and indirect antiglobulin test using the tube technique) were reevaluated for RhD status by three methods. The method with the best performance was implemented and evaluated for a period of one year (n = 4897 samples). Samples that were D positive exclusively in the confirmatory test were submitted to molecular analysis.RESULTS: The gel test for indirect antiglobulin testing with anti-D immunoglobulin G (clone ESD1) presented the best results. Seventy samples (1.43%) previously typed as RhD negative showed reactivity in the gel test for indirect antiglobulin testing and were reclassified as D positive. D variants that may cause alloimmunization, such as weak D type 2 and partial DVI, were detected.CONCLUSION: The confirmatory RhD test using the gel test for indirect antiglobulin testing represents a breakthrough in transfusion safety in this blood center. Our results emphasize the importance of assessing the blood group typing strategy in blood banks.

Frequency of Wra antigen and anti-Wra in Brazilian blood donors

BACKGROUND: Wra is a low-incidence antigen, which is antithetical to the high prevalence red blood cell antigen, Wrb. Anti-Wra is a naturally occurring antibody that is found in approximately 1-2% of blood donors. The aim of this study was to determine the frequency of Wra and anti-Wra in Brazilian blood donors.METHODS: A total of 1662 Brazilian blood donors were molecularly analyzed using the SNaPshot methodology to determine the WR*A/B alleles and to predict the frequency of the Wra antigen. To detect the anti-Wra, samples from 1049 blood donors were analyzed using a gel test with Wr(a+) red blood cells. The serum was treated with dithiothreitol (DTT) to determine the immunoglobulin classes. Immunoglobulin (Ig)-G isotype classification was performed in a gel test using the IgG1/IgG3 card. A monocyte monolayer assay was employed to predict the clinical significance of IgG anti-Wra.RESULTS: Of the 1662 donors, only one sample had the DI*02.03 allele in heterozygous predicting the Wr(a+b+) phenotype. Anti-Wra was detected in 34 (3.24%) samples, 64.7% in females and 35.3% in males. Regarding the immunoglobulin class, eight (23.5%) cases of anti-Wra were classified as IgG and 26 (76.5%) as IgM. Of the eight cases of IgG anti-Wra, four were IgG1, two were IgG3 and three anti-Wra were not IgG3 or IgG1, and thus probably IgG2 or IgG4. The results of the monocyte monolayer assay showed that IgG anti-Wra might be of clinical significance.CONCLUSION: This study shows a very low frequency (0.06%) of the Wra antigen in Brazilian blood donors. Additionally, it shows that the frequency of anti-Wra in this population is higher than previously reported.

Molecular matching of red blood cells is superior to serological matching in sickle cell disease patients

OBJECTIVE: To evaluate the usefulness of DNA methods to provide a means to precisely genotypically match donor blood units for the antigen-negative type of 35 sickle cell disease patients<. METHODS: Red blood cell units were investigated for ABO, D, C, c, E, e, K, Fyª, Fy b, Jkª, Jk b, S, s, Diª and RH variants by performing a molecular array (Human Erythrocyte Antigen BeadChipTM, BioArray Solutions), polymerase chain reaction followed by restriction fragment length polymorphism analysis and sequencing of patient samples and donor units that had been serologically matched based on the ABO, Rh and K phenotypes and the presence of antibodies. RESULTS: Matches for 21 of 35 sickle cell disease patients presented discrepancies or mismatches for multiple antigens between the genotype profile and the antigen profile of their serologically-matched blood units. The main discrepancies or mismatches occurred in the RH, FY, JK and MNS systems. Eight Rh alloimmunized patients presented RHD and RHCE variants that had not been serologically identified. According to these results better matches were found for the patients with genotyped units and the patients benefited as shown by better in vivo red blood cell survival. CONCLUSION: Molecular matching is superior to serological matching in sickle cell disease patients, decreasing the risk of transfusion reactions, especially delayed transfusion reactions to existing alloantibodies and preventing alloimmunization.

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. .

An easy and efficient strategy for KEL genotyping in a multiethnic population

BACKGROUND: The Kell blood group system expresses high and low frequency antigens with the most important in relation to transfusion including the antithetic KEL1 and KEL2; KEL3 and KEL4; KEL6 and KEL7 antigens. Kell is a clinically relevant system, as it is highly immunogenic and anti-KEL antibodies are associated with hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. Although required in some situations, Kell antigen phenotyping is restricted due to technical limitations. In these cases, molecular approaches maybe a solution. This study proposes three polymerase chain reaction genotyping protocols to analyze the single nucleotide polymorphisms responsible for six Kell antithetic antigens expressed in a Brazilian population. METHODS: DNA was extracted from 800 blood donor samples and three polymerase chain reaction-restriction fragment length polymorphism protocols were used to genotype the KEL*1/KEL*2, KEL*3/KEL*4 and KEL*6/KEL*7 alleles. KEL*3/KEL*4 and KEL*6/KEL*7 genotyping was standardized using the NlaIII and MnlI restriction enzymes and validated using sequencing. KEL*1/KEL*2 genotyping was performed using a previously reported assay. RESULTS: KEL genotyping was successfully implemented in the service; the following distribution of KEL alleles was obtained for a population from southeastern Brazil: KEL*1 (2.2%), KEL*2 (97.8%), KEL*3 (0.69%), KEL*4 (99.31%), KEL*6 (2.69%) and KEL*7 (97.31%). Additionally, two individuals with rare genotypes, KEL*1/KEL*1 and KEL*3/KEL*3, were identified. CONCLUSION: KEL allele genotyping using these methods proved to be reliable and applicable to predict Kell antigen expressions in a Brazilian cohort. This easy and efficient strategy can be employed to provide safer transfusions and to help in rare donor screening.

Incidência de aloimunização eritrocitária em pacientes poli transfundidos / Prevalence of erythrocyte alloimmunization in polytransfused patients

Objective: To determine the incidence and the rate of red blood cell alloimmunization in polytransfused patients. Methods: A polytransfused patient was defined as having received at least 6 units of red cell concentrates during a 3-month period. The recordsof all patients (n = 12,904) who had received red blood cell units were examined retrospectively by searching the computer database at Hospital Israelita Albert Einstein in São Paulo, Brazil, over a 6-year period, between 2003 and 2009. Results: During this time, 77,049 red cell concentrate transfusions were performed in 12,904 patients. There were 3,044 polytransfused patients, 227 of whom (7.5%) presented with irregular erythrocyte antibodies. The prevalence of alloantibody specificity was: Anti-E>anti-D>anti-K>anti-C>anti-Dia>anti-c>anti-Jka>anti-S in 227 polytransfused patients. We found combinations of alloantibodies in 79 patients (34.8%), and the most commonspecificities were against the Rh and/or Kell systems. These antibodies show clinical significance, as they can cause delayed hemolytic transfusion reactions and perinatal hemolytic disease. About 20% of the patients showed an IgG autoantibody isolatedor combined with alloantibodies. Interestingly, a high incidence of antibodies against low frequency antigens was detected in this study, mainly anti-Dia. Conclusion: Polytransfused patients have a high probability of developing alloantibodies whetheralone or combined with autoantibodies and antibodies against low frequency antigens. Transfusion of red blood cells with a phenotype-compatible with RH (C, E, c), K, Fya, and Jka antigens is recommended for polytransfused patients in order to preventalloimmunization and hemolytic transfusion reactions.

Objetivo: Determinar a incidência e a taxa de aloimunização eritrocitária em pacientes politransfundidos. Métodos: Foram classificados como politransfundidos todos os pacientes que receberam no mínimo 6 unidades de concentrado de hemácias no período de 3 meses. Foram examinados retrospectivamente os prontuários de todos os pacientes(n = 12.904) que receberam transfusões de unidades de hemácias procurados nas bases de dados computadorizados do Hospital Israelita Albert Einstein, em São Paulo (SP), no período de 6 anos, entre 2003 e 2009. Resultados: Nesse período foram realizadas 77.049 transfusões de concentrado de hemácias em 12.904 pacientes. Os pacientes politransfundidos totalizaram 3.044, sendo que 227 (7,5%) apresentam anticorpos eritrocitários irregulares. A prevalência da especificidade dos aloanticorpos encontrados nos 227 pacientes politransfundidos foi: Anti-E>anti-D>anti-K>anti-C>anti-Dia>antic>anti-Jka>anti-S. Em 79 pacientes (34,8%) foram encontradas associações de aloanticorpos e as combinações mais freqüentes foram dos anticorpos dos sistemas Rh e/ou Kell. Esses anticorpos têm importância clínica, pois podem causar reações transfusionais hemolíticas tardias e doença hemolítica perinatal. Cerca de 20% dos pacientes apresentavam autoanticorpo IgG isolado ou em associação com aloanticorpos. Um achado interessante neste estudo foi a alta incidência de anticorpos contra antígenos de baixa frequência, com predomínio anti-Dia. Conclusão: Pacientes politransfundidos têm alta probabilidade de desenvolver aloanticorpos isolados ou em associação com autoanticorpos e anticorpos contra antígenos de baixa frequência. A transfusão de concentrado de hemácias com fenótipo compatível para os antígenos RH (C, E, c), K, Fya, e Jka deve ser recomendada para o grupo de pacientes politransfundidos, com objetivo de evitar a aloimunização e a reação transfusional hemolítica.

Genetic polymorphisms of Rh, Kell, Duffy and Kidd systems in a population from the State of Paraná, southern Brazil

BACKGROUND: Red blood group genes are highly polymorphic and the distribution of alleles varies among different populations and ethnic groups. AIM: To evaluate allele polymorphisms of the Rh, Kell, Duffy and Kidd blood group systems in a population of the State of Paraná METHODS: Rh, Kell, Duffy and Kidd blood group polymorphisms were evaluated in 400 unrelated blood or bone marrow donors from the northwestern region of Paraná State between September 2008 and October 2009. The following techniques were used: multiplex-polymerase chain reaction genotyping for the identification of the RHD gene and RHCE*C/c genotype; allele-specific polymerase chain reaction for the RHDΨ and restriction fragment length polymorphism polymerase chain reaction for the RHCE*E/e, KEL, FY-GATA and JK alleles. RESULTS: These techniques enabled the evaluation of the frequencies of Rh, Kell, Duffy and Kidd polymorphisms in the population studied, which were compared to frequencies in two populations from the eastern region of São Paulo State. CONCLUSION: The RHCE*c/c, FY*A/FY*B, GATA-33 T/T, JK*B/JK*B genotypes were more prevalent in the population from Paraná, while RHCE*C/c, FY*B/FY*B, GATA-33 C/C, JK*A/JK*B genotypes were more common in the populations from São Paulo.

Infecções de corrente sanguínea relacionadas ao cateter venoso central em terapia intensiva: ensaio clínico randomizado aberto / Bloodstream infections related to the central venous catheter in intensive care: opened randomized clinical article

Trata-se de um ensaio clínico randomizado aberto, que analisa o impacto da implantação de um protocolo de Inserção, Manutenção e Retirada de cateter venoso central, nas infecções de corrente sanguínea. Utilizou-se um grupo controle e um grupo experimental configurados de maneira aleatória, por meio de um programa de computador. Foram analisados os procedimentos e a densidade de infecção correspondente em cento e quarenta e quatro pacientes, distribuídos nos dois grupos. Os resultados apontaram uma diminuição de 23,1 infecções/1000 cateteres/dia na densidade de infecção a favor do grupo em que se utilizou o protocolo, mostrando efetiva diminuição das infecções nas correntes sanguíneas.

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.