Immune-mediated neutropenias comprise a heterogeneous group of disorders characterized by antibody-mediated destruction of neutrophils, in which the detection of anti-neutrophil antibodies remains a significant diagnostic challenge. Human neutrophil antigens (HNAs) are key targets in both autoimmune and alloimmune conditions, and their identification requires an integrated laboratory approach combining serological assays, HNA genotyping, and clinical evaluation. However, variability in assay sensitivity, the presence of low-titer or conformationally dependent antibodies, and interference from anti-HLA antibodies may lead to inconclusive or misleading results. This review summarizes the immunological mechanisms underlying anti-HNA antibody-mediated neutropenia and critically evaluates current laboratory methods, including cell-based and bead-based assays. The role of HNA genotyping in supporting antibody identification and improving diagnostic accuracy is also discussed. In addition, we highlight the importance of interpreting serological findings according to antibody specificity and clinical context. An integrated and multidisciplinary diagnostic approach is essential to ensure accurate diagnosis and appropriate clinical management, while emerging technologies may further improve antibody detection in the future.

BACKGROUND:Thrombocytopenia arises from heterogeneous inherited and acquired disorders, and identifying the underlying platelet clearance mechanisms remains challenging. Platelet desialylation, characterised by loss of sialic acid and consequent exposure of terminal β-galactose residues recognised by the Ashwell-Morell receptor, represents an alternative pathway of hepatic platelet clearance. This study aimed to validate a modified platelet desialylation test (PDT) and assess its applicability in multitransfused, alloimmunized, and hereditary thrombocytopenia patients as a functional in vitro assay to detect plasma-induced platelet desialylation and explore its potential as a complementary biomarker of platelet clearance mechanisms. METHODS:PDT performance was evaluated using plasma from 20 healthy donor pools, 10 antibody-positive refractory patients, and 15 individuals with hereditary thrombocytopenias by FITC-RCA-I flow cytometry. Analytical validation included assessment of reproducibility, incubation time, lectin concentration, and ratio-based result interpretation. RESULTS:β-Lactose showed significantly lower expression mean fluorescence intensity (MFI) than both untreated reference control and neuraminidase-treated conditions (p < 0.00001), whereas patient plasma exhibited higher fluorescence than the reference control (p < 0.0001). A 1 h incubation using 0.3 μL per reaction RCA-I-FITC stock solution (5 mg active conjugate/mL) provided stable and reproducible discrimination. In inherited thrombocytopenias, 66.7% of patients were PDT-positive despite HLA/HPA antibodies being detected in only 13.3%. PDT-positive samples showed increased desialylation-associated MFI (p = 0.002) with a strong correlation between MFI and ratio (ρ = 0.877). The assay required approximately 4 h and cost US$3.62 per test. CONCLUSION:The PDT is a practical, reproducible, and cost-effective assay capable of detecting physiologic, enzymatic, and immune-mediated platelet desialylation, providing a complementary biomarker for investigating thrombocytopenic disorders.

BACKGROUND:Platelet transfusion refractoriness (PTR) is a major challenge in transfusion medicine and may result from both immune and non-immune mechanisms. Although alloantibodies are well-established contributors, Fc-independent pathways such as platelet desialylation have emerged as alternative mechanisms of clearance. STUDY DESIGN AND METHODS:In this prospective diagnostic study, 81 patients with suspected PTR were evaluated using an integrated approach combining the platelet immunofluorescence test (PIFT) to detect antibody-mediated refractoriness and a modified platelet desialylation test (PDT) to assess Fc-independent clearance. Demographic, clinical, and laboratory variables were analyzed using non-parametric tests, including Mann-Whitney U, Kruskal-Wallis, and binomial tests. RESULTS:The cohort exhibited severe thrombocytopenia and diverse clinical profiles. PIFT detected platelet-bound antibodies in 63/81 cases (78%), while PDT identified desialylation in 41/81 patients (50%). PIFT positivity was similar between PDT-positive (80.5%) and PDT-negative (76.9%) groups, with no significant association (χ2 = 0.15, p = .69). Although mean fluorescence intensity (MFI) varied across diagnostic categories, no significant differences were observed (p = .3095). Fever was the only clinical variable significantly associated with reduced desialylation (median MFI 401 vs. 1328.3; p = .0109), while splenomegaly, infection, antifungal use, and bleeding had no significant effects. PDT status was not significantly associated with the number of pooled platelet transfusions (p = .391) or apheresis procedures (p = .515), indicating that desialylation alone does not predict transfusion demand. DISCUSSION:PTR occurs both independently and in parallel with antibody-mediated pathways. The combined PIFT-modified PDT approach improves mechanistic characterization and enhances diagnostic accuracy, in cases of platelet refractoriness of immune and non-immune etiology.

INTRODUCTION:Autoimmune neutropenia (AIN), a common cause of chronic neutropenia, has been categorized as primary (pAIN) and secondary (sAIN). This study investigates the specificity of anti-HNA autoantibodies in both types. MATERIALS AND METHODS:A prospective cohort study of 85 chronic neutropenia patients included those with detectable anti-HNA autoantibodies. Anti-HNA was assessed using granulocyte agglutination (GAT), granulocyte immunofluorescence (GIFT), and LABScreen Multi Kit (LSM). Molecular analysis was performed to assess HNA expression. RESULTS:Of 85 patients, 7 had pAIN and 8 had sAIN. All pAIN patients exhibited only anti-HNA-1, while anti-HNA-3 was found in 3/8 sAIN patients. All patients with anti-HNA-3 tested positive in LSM; one was positive in both GAT and GIFT. DISCUSSION:While anti-HNA-3 has been described exclusively as an alloantibody, this is the first report of anti-HNA-3 as an autoantibody in AIN. CONCLUSION:Anti-HNA-3 autoantibody is associated only with sAIN, suggesting distinct mechanisms in pAIN and sAIN.

BACKGROUND:There is an increasing number of biopsies with histological features of antibody mediated rejection but lacking the detection of anti-human leucocyte antigen (HLA) antibodies or C4d deposition. Anti-Human neutrophil antigens-3 (HNA-3) have been reported in severe and fatal cases of Transfusion-Related Acute Lung Injury and Neutropenia Alloimmune Neonatal, a phenomenon mediated by antibody endothelial activation. This study investigated the association between the presence of pre-formed anti-HNA-3 antibodies and the development of a recurrent episode of acute rejection (AR) after kidney transplantation. METHODS:Patients who experienced the first biopsy confirmed acute rejection (BCAR) between 2011-2019 were screened for the presence of anti-HNA-3 antibodies using pretransplant serum samples. Three techniques were used, Granulocyte Agglutination Test (GAT), Granulocyte Immunofluorescence Test (Flow-GIFT), and microsphere-based technique (LABSCreen Multi kit, One Lambda) to detect and determine the specificity of the antibodies. HNA-3 genotyping of recipients and donors was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). A matched control group included patients with first BCAR but without pretransplant anti-HNA-3 antibodies. RESULTS:Of 1256 patients with first BCAR, 33 (2.6 %) had anti-HNA-3 antibodies. Compared to the matched control, survival-free of a second BCAR was lower in patients with any anti-HNA-3 antibody (p = 0.014) and in patients with donor specific anti-HNA-3 antibodies (p = 0.015). No differences were observed in histological phenotypes of the rejection episodes comparing patients with and without anti-HNA-3 antibodies. CONCLUSIONS:This matched cohort study suggests that preformed anti-HNA-3 antibodies are associated with an increased risk of recurrent BCAR. The involved mechanisms remain to be elucidated.

BACKGROUND:Fetal/neonatal alloimmune thrombocytopenia (FNAIT) and neonatal alloimmune neutropenia (NAIN) result from maternal alloantibodies targeting fetal human platelet antigen (HPA) and human neutrophil antigen (HNA) antigens, respectively. However, increasing evidence supports the pathogenic role of HLA class I alloantibodies in these conditions. Since the simultaneous occurrence of FNAIT and NAIN has not been systematically investigated, this study aimed to determine its prevalence, characterize the specificity and strength of associated alloantibodies, and correlate findings with neonatal cell counts. STUDY DESIGN AND METHODS:In this cross-sectional study, 10,000 umbilical cord blood samples were analyzed for platelet and neutrophil counts. Neonates with thrombocytopenia and neutropenia were selected. Genotyping for HPA, HNA, and HLA class I was performed in mother-infant pairs to assess incompatibilities. Maternal sera were tested for anti-HPA, anti-HNA, and anti-HLA antibodies. RESULTS:Ten cases (0.1%) of concurrent cytopenias were identified. Alloantibodies were detected in four cases: one with combined anti-HPA-5b, HNA-2, and HLA-A2 antibodies; and three with isolated high-mean fluorescence intensity (MFI) HLA antibodies (anti-HLA-A2, HLA-A3, HLA-B7). Anti-HLA-A2 was linked to the lowest neutrophil counts, and anti-HLA-B7 to severe thrombocytopenia. The estimated prevalence of simultaneous FNAIT and NAIN was 0.04% (1 in 2500 neonates). DISCUSSION:This is the first large-scale study to document the co-occurrence of FNAIT and NAIN. Our findings explore the serological and molecular features of these immune syndromes and underscore the potential pathogenic role of maternal anti-HLA class I antibodies, even in the absence of detectable anti-HPA or anti-HNA, and support including HLA testing in the diagnostic workup of neonatal cytopenias.

British Journal of HaematologyEarly View LETTER TO THE EDITOR Long-lasting autoimmune neutropenia and GFI1 variant: A case of familial inheritance Renato Cerqueira, Corresponding Author Renato Cerqueira [email protected] orcid.org/0000-0002-5064-8484 Department of Clinical and Experimental Oncology, Universidade Federal de São Paulo, Sao Paulo, Brazil Correspondence Renato Cerqueira, Department of Clinical and Experimental Oncology, Universidade Federal de São Paulo, Sao Paulo, Brazil. Email: [email protected]Search for more papers by this authorJosefina A. P. Braga, Josefina A. P. Braga Department of Pediatrics, Universidade Federal de São Paulo, Sao Paulo, BrazilSearch for more papers by this authorElyse Moritz, Elyse Moritz orcid.org/0000-0002-0636-4556 Department of Clinical and Experimental Oncology, Universidade Federal de São Paulo, Sao Paulo, BrazilSearch for more papers by this authorJoão B. Pesquero, João B. Pesquero Department of Biophysics, Universidade Federal de São Paulo, Sao Paulo, BrazilSearch for more papers by this authorJosé O. Bordin, José O. Bordin Department of Clinical and Experimental Oncology, Universidade Federal de São Paulo, Sao Paulo, BrazilSearch for more papers by this author Renato Cerqueira, Corresponding Author Renato Cerqueira [email protected] orcid.org/0000-0002-5064-8484 Department of Clinical and Experimental Oncology, Universidade Federal de São Paulo, Sao Paulo, Brazil Correspondence Renato Cerqueira, Department of Clinical and Experimental Oncology, Universidade Federal de São Paulo, Sao Paulo, Brazil. Email: [email protected]Search for more papers by this authorJosefina A. P. Braga, Josefina A. P. Braga Department of Pediatrics, Universidade Federal de São Paulo, Sao Paulo, BrazilSearch for more papers by this authorElyse Moritz, Elyse Moritz orcid.org/0000-0002-0636-4556 Department of Clinical and Experimental Oncology, Universidade Federal de São Paulo, Sao Paulo, BrazilSearch for more papers by this authorJoão B. Pesquero, João B. Pesquero Department of Biophysics, Universidade Federal de São Paulo, Sao Paulo, BrazilSearch for more papers by this authorJosé O. Bordin, José O. Bordin Department of Clinical and Experimental Oncology, Universidade Federal de São Paulo, Sao Paulo, BrazilSearch for more papers by this author First published: 20 June 2024 https://doi.org/10.1111/bjh.19606Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. REFERENCES 1Fioredda F, Skokowa J, Tamary H, Spanoudakis M, Farruggia P, Almeida A, et al. 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BackgroundHemolysis due to ABO incompatibility is an important differential diagnosis in newborns presenting with jaundice. Clinical studies evaluating ABO hemolytic disease of fetus and newborn (ABO-HDFN) question the diagnostic value of the direct antiglobulin test (DAT) in this situation.GoalsTo determine the clinical and laboratorial findings associated with the occurrence of ABO-HDFN and to evaluate the accuracy of DAT as a diagnostic tool.MethodsThis was a nested case control study with a cohort of 4122 newborns. Clinical and immunohematological data were retrieved from medical files including clinical and laboratorial factors associated with ABO-HDFN. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of positive DAT were calculated.ResultsAmong the 4122 newborns, 44 had the diagnosis of ABO-HDFN. Positive DAT, group O mother and group A newborn were significantly associated with the occurrence of neonatal jaundice and this association persisted in a multivariable model (p-value <0.001). DAT presented 65.85 % sensitivity, 96.28 % specificity, 16.9 % PPV and 99.6 % NPV for the diagnosis of ABO-HDFN. There were no cases of positive DAT in cases other than O/A and O/B incompatibilities. The newborn hemoglobin was significantly lower in O/A incompatibility (p-value <0.001).ConclusionPositive DAT, mother of group O and newborn of group A are independent risk factors associated with ABO-HDFN. DAT exhibited high NPV for the diagnosis of this complication. Thus, performing DAT in newborns with O/A and O/B incompatibilities is a cost-effective strategy that can be applied as routine by blood banks.

Introdução O Antígeno Neutrofílico Humano-2 (HNA-2, CD177) é uma glicoproteína presente na membrana de uma subpopulação de neutrófilos, com uma porcentagem variável de neutrófilos positivos. A superexpressão do HNA-2 está relacionada a condições inflamatórias graves, como a Covid-19 e sepse, entretanto não se sabe se esta expressão é primária ou secundária ao evento inflamatório. A pré-eclâmpsia (PE) é uma síndrome sistêmica caracterizada por intenso estado inflamatório e antiangiogênico, que acomete gestantes após a 20ªsemana, com hipertensão associada a proteinúria. Objetivos 1) Determinar se o grau de expressão do HNA-2 é maior em gestantes com PE em comparação com gestantes saudáveis e mulheres não gestantes; 2) Avaliar se a expressão do HNA-2 se mantém estável ao longo da vida, através da recoleta de indivíduos saudáveis com fenótipo HNA-2 conhecido. Materiais e métodos : Um estudo transversal incluiu 46 gestantes com PE, 75 gestantes saudáveis e 83 não-gestantes. Amostras de sangue das gestantes foram coletadas em dois hospitais obstétricos durante o segundo trimestre de gestação. A parte prospectiva do estudo incluiu 17 voluntários saudáveis, com a primeira coleta de sangue realizada entre 2006 e 2015 e a última em 2024 (9-18 anos). A expressão do HNA-2 foi determinada por citometria de fluxo (BD FACSCanto II, BD Biosciences) usando o anticorpo monoclonal MEM166/PE (Abcam, Cambridge, UK) e software Infynicyt V1.7.0.d (Cytognos S.L). Neutrófilos não marcados foram utilizados como controle negativo da reação. A análise estatística foi executada pelo programa SigmaPlot®. Resultados : Gestantes com PE apresentaram uma mediana de neutrófilos expressando HNA-2 significativamente maior (88%) comparada com gestantes saudáveis (70%) e não gravidas (72%) (p < 0.0001; ANOVA). A maioria das mulheres com PE [32/46 (69,6%)] apresentaram alta expressão (≥ 80%) quando comparadas a 28/75 (37,3%) grávidas saudáveis e 24/83 (28,9%) mulheres não grávidas (p = 0,0007 e p < 0,0001, respectivamente). A expressão do HNA-2 em indivíduos saudáveis mostrou-se estável ao longo da vida, sem diferença significativa nos dois momentos da coleta (p = 0,94; Teste de Wilcoxon). Discussão Nossos achados demonstraram que a maioria das gestantes com PE apresentou alta expressão do HNA-2 (≥80%), comparada com uma parcela muito menor das mulheres não gestantes e gestantes saudáveis, evidenciando uma relação entre a superexpressão do HNA-2 e a síndrome inflamatória da pré-eclâmpsia. Além disso, a expressão do HNA-2 parece ser estável em indivíduos saudáveis ao longo da vida, o que sugere que a alta expressão do antígeno não seja secundária à condição inflamatória, e que, em gestantes, possa ser considerada um biomarcador de risco para o desenvolvimento da PE. Conclusão A identificação de novos marcadores associados à pré-eclâmpsia e possíveis alvos terapêuticos abre espaço para o desenvolvimento de estratégias preventivas, diagnósticas e de manejo mais eficazes para essa condição grave.

Background and Objectives: The isolation of neutrophils and subsequent detection of anti-human neutrophil antigens (HNA) antibodies are crucial in clinical medicine for the diagnosis of autoimmune neutropenia, neonatal alloimmune neutropenia (NAIN) and transfusion-related acute lung injury (TRALI). This study reports two cases of maternal anti-Fc-gamma-receptor-IIIb (Fc gamma RIIIb) isoimmunization without NAIN symptoms and compares the efficiency of immunomagnetic negative selection (IMNS) with traditional dextran/Ficoll for neutrophil isolation in HNA serological assays. Materials and Methods: Investigating two cases of maternal anti-Fc gamma RIIIb isoimmunization, neutrophils from three donors were isolated from 8 mL of whole blood using IMNS and dextran/Ficoll. Serological assays included the granulocyte agglutination and immunofluorescence test, monoclonal antibody immobilization of granulocyte antigens and the LABScreen Multi (One Lambda). IMNS and dextran/Ficoll were compared in terms of cell yield, viability, time, cost and purity. Results: Maternal anti-Fc gamma RIIIb isoantibodies with FCGR3B gene deletion were detected in both cases. Newborns and fathers exhibited specific gene combinations: FCGR3B*02/FCGR3B*02 (Case 1) and FCGR3B*02/FCGR3B*03 (Case 2). IMNS outperformed dextran/Ficoll, yielding four times more neutrophils (average neutrophil counts: 18.5 x 10(3)/mu L vs. 4.5 x 10(3)/mu L), efficiently removing non-neutrophil cells and reducing processing time (30-40 min vs. 70-90 min), although it incurred a higher cost (2.7 times). Conclusion: Two cases of maternal anti-Fc gamma RIIIb isoantibodies, unrelated to NAIN, were identified. Although neutropenia has not been described in these cases, we emphasize the importance of identifying asymptomatic cases with the potential for severe neutropenia. Additionally, IMNS is introduced as a rapid, high-yield, high-purity neutrophil isolation technique, beneficial for serological assays detecting anti-HNA antibodies.

Aims: To evaluate the expression of neutrophilic cathelicidin pro-LL-37 (hCAP18) and the expression of CAMP gene in patients with chronic primary neutropenia (CPN), and its correlation with the etiology of the disease. Methods: Prospective study, carried out from 2021 to 2023, including children and adult patients treated at a neutropenia reference service. We analyzed 4 patients with severe congenital neutropenia (SCN), 2 with cyclic neutropenia (CN), 3 with autoimmune neutropenia (AIN), 3 with chronic idiopathic neutropenia (CIN) and 2 patients with glycogen storage disease 1B (DDG1B). The hCAP18 protein expression was evaluated in the plasma of patients with different types of neutropenia and healthy control individuals, using the Western-Blot technique. The CAMP gene expression assay was performed using the real-time PCR (qPCR) technique, using RNA extracted from whole blood. Results: The expression of hCAP18 was not observed in patients with SCN, even after increasing the neutrophil count using filgrastim. In individuals with CN, the expression of hCAP18 was also not observed during the neutropenic phase, but the protein was detected in the phase of normalization of the neutrophil count, showing a positive correlation of the protein expression with the number of neutrophils in this etiology, different from that of SCN. Expression of hCAP18 was also detected in patients with AIN, CIN and DDG1B, despite pronounced neutropenia in some patients, and in the control group. The qPCR showed a low expression of CAMP in patients with SCN, while the other etiologies showed variations in gene expression, being positive in some individuals and negative in others with the same etiology. However, using this method, it was possible to notice a positive correlation (r = 0.732 and p < 0.05 – Spearman test) between the expression of the CAMP gene and the expression of hCAP18. Discussion: hCAP18, encoded by the CAMP gene, is an important component of the secondary granules of neutrophils, with immunological and bactericidal activity. The results of this work corroborate the data by Karlsson et al., who showed absence or significant decrease in the expression of hCAP18 in individuals with SCN and its expression in individuals with CIN and AIN. In addition, we also demonstrated a positive correlation between the expression of the CAMP gene and hCAP18 and a decrease in the expression of the CAMP gene in patients with SCN. Therefore, plasma hCAP18 assessment is suggested as a differential CPN screening method, aiding in the diagnosis of SCN due to low or undetectable hCAP18 levels. Conclusion: There is a correlation between CAMP gene expression and hCAP18 expression with CPN. In addition, we also demonstrated that hCAP18 is not expressed in patients with SCN and in the neutropenic phase of CN. Thus, the use of this technique is suggested as a new screening routine for the investigation of patients with CPN, especially in patients with SCN and CN, differentiating them from the others and avoiding more invasive tests.

Abstract Background Current blood-matching practices are challenging for patients with autoimmune hemolytic anemia (AIHA) because autoantibodies may interfere in hemagglutination assays. Blood group genotyping methods are an important complement to serology and allow the prediction of the phenotype from deoxyribonucleic acid (DNA) with high accuracy. Furthermore, technology for genotyping of red blood cell antigens in donor-recipient matching for blood group polymorphisms is moving into practice, mainly for patient groups with hemoglobinopathies. However, the routine use of antigen molecular matching for AIHA patients remains to be better determined. We hypothesized that performing the molecular matching between AIHA patients and blood donors could improve the selection of antigen-matched red blood cell units. Methods Using the blood-MLPA assay, we determined the blood group genotype from 198 donors and 24 AIHA patients and performed the RBC molecular matching between the two groups. Besides, using the hemagglutination technique we performed the RBC phenotyping for major blood group antigens of AIHA patients and compare the phenotyping with the genotyping results. Results: We were able to find RBC genotype-matched donors for 20/24 (83.3%) of our AIHA patients. From 118 phenotyping results, we found three (2.5%) discrepancies between the phenotype predicted by genotyping and the phenotype determined by serology. Conclusions In this study, our data indicate the real benefits of RBC genotyping for AIHA patients who usually present problematic immunohematological serologic reactions. Such a strategy may present information about additional RBC alloantibodies and reduce the potential risk of transfusion reactions.

BACKGROUND AND OBJECTIVES:Human neutrophil antigens (HNAs) are categorized into five systems: HNA-1 to HNA-5. Given the importance of neutrophils in immunity, we sought to create awareness of the role of HNA diagnostic services in managing immune neutropenia and transfusion-related acute lung injury. To provide health communities all around the world with access to these services, we conducted a survey to create a directory of these HNA diagnostic services.MATERIALS AND METHODS:An Excel table-based survey was created to capture information on the laboratory's location and was emailed to 55 individuals with known or possible HNA investigation activity. The collected data were then summarized and analysed.RESULTS:Of contacted laboratories, the surveys were returned from 23 (38.2%) laboratories; 17 have already established HNA diagnostic (of them 12 were regular participants of the International Granulocyte Immunobiology Workshop [ISBT-IGIW]), 4 laboratories were in the process of establishing their HNA investigation and the remaining 2 responder laboratories, did not conduct HNA investigations. In established laboratories, investigation for autoimmune neutropenia (infancies and adults) was the most frequently requested, and antibodies against HNA-1a and HNA-1b were the most commonly detected.CONCLUSION:The directory of survey respondents provides a resource for health professionals wanting to access HNA diagnostic services. The present study offers a comprehensive picture of HNA diagnostics (typing and serology), identifying weak points and areas for improvement for the first time. Identifying more laboratories involved in HNA diagnostics with limited access to international societies in the field will globally improve HNA diagnostics.

Introdução: O sistema de Antígenos de Neutrófilos Humanos-3 (HNA-3) está localizado na proteína Transportadora de Colinta-2 (CTL2), e é codificado pelo gene SLC44A2 e possui duas variantes: HNA-3a (SLC44A2*01) e HNA-3b (SLC44A2*02). Os antígenos HNA-3 e são expressos em neutrófilos, células endoteliais microvasculares dos pulmões, células tubulares renais e diversos tecidos humanos. Os anticorpos direcionados contra esses antígenos podem ser formados após a gestação ou transfusão sanguínea e têm sido associados à complicações como neutropenias aloimunes neonatais e reações transfusionais graves. A formação desses anticorpos após o transplante renal e sua relação com a rejeição e perda do enxerto são pouco compreendidas. Relato de caso: paciente do sexo masculino, 22 anos, DRC de etiologia indeterminada, sem histórico de transfusão sanguínea e com histórico de perda do transplante renal por rejeição aguda celular tardia. As amostras testadas foram coletadas antes da realização do segundo transplante renal e, a pesquisa de anticorpos anti-HNA-3 foi realizada através das seguintes técnicas: Teste de Aglutinação de Granulócitos (GAT), teste de Imunofluorescência de Granulócitos (Flow-GIFT) e técnica baseada em microesferas (kit LABSCreen Multi, One Lambda). A genotipagem HNA-3 dos receptores e dos doadores do transplante renal foi realizada pela técnica de polimorfismo de fragmentos de restrição utilizando a reação em cadeia da polimerase (PCR–RFLP). Testes laboratoriais realizados após a perda do primeiro transplante revelaram a presença de anticorpos anti-HLA específicos contra o doador (DSA) de especificidade DPB1*13 e *17, além de revelarem a presença de anticorpos anit-HNA-3b. A análise molecular demonstrou que o doador do transplante apresentava a genotipagem SLC44A2*01/SLC44A2*02 (HNA-3ab), enquanto o paciente apresentava genotipagem SLC44A2*01/SLC44A2*01 (HNA-3aa), corroborando a possível aloimunização induzida pelo transplante renal. Conclusão: Esse caso clínico sugere que a formação de anticorpos anti-HNA-3 pode ser induzida pelo transplante renal. A presença desse anticorpo em sinergia com anticorpos anti-HLA, pode ter contribuído ou exacerbado a rejeição do transplante. A compreensão dos mecanismos subjacentes a formação desse anticorpo e a sua determinação sistemática podem auxiliar na estratificação de risco de perda do enxerto renal.

Introdução: O sistema de antígenos de Neutrófilos Humanos-3 (HNA-3) é codificado pelo gene SLC44A2 e possui duas variantes: HNA-3a e HNA-3b. São expressos em neutrófilos, células endoteliais microvasculares dos pulmões, células tubulares renais e outros tecidos humanos. Os aloanticorpos direcionados contra esses antígenos são formados a partir da exposição alogênica e estão associados principalmente à casos graves de Lesão pulmonar Aguda relacionada à Transfusão (TRALI) e Neutropenia Aloimune Neonatal (NAN). Devido a presença do HNA-3 no tecido renal, existe a suspeita de que a aloimunização neutrofílica também possa estar envolvida no desenvolvimento da rejeição aguda após o transplante renal. Objetivo: Determinar a influência dos anticorpos anti-HNA-3 pré-formados no contexto da rejeição aguda do transplante renal. Materiais e métodos: Trata-se de um estudo retrospectivo onde foram analisados 66 pacientes que apresentaram rejeição após o transplante renal, divididos igualmente entre pacientes que possuem anticorpos anti-HNA-3 (n = 33) e pacientes sem anticorpos (grupo controle; n = 33). As amostras testadas foram coletadas antes da realização do transplante renal e, a pesquisa de anticorpos anti-HNA-3 foi realizada através das seguintes técnicas: Teste de Aglutinação de Granulócitos (GAT), teste de Imunofluorescência de Granulócitos (Flow-GIFT) e técnica baseada em microesferas (kit LABSCreen Multi, One Lambda). A genotipagem HNA-3 dos receptores e dos doadores do transplante renal foi realizada pela técnica de polimorfismo de fragmentos de restrição utilizando a reação em cadeia da polimerase (PCR–RFLP). A influência dos anticorpos anti-HNA-3 no contexto do transplante renal foi avaliada durante 12 meses após o evento da primeira rejeição e foi analisada de acordo com o número de episódios de rejeição, perda e/ou óbito do paciente durante esse período. Resultados: O segundo episódio de rejeição do transplante renal esteve presente em 11/33 (33,3%) pacientes com anticorpos anti-HNA-3 vs. 3/33 (9,1%) pacientes do grupo controle (p = 0,033). O método de Kaplan-Meier foi empregado para analisar a evolução do enxerto renal nos dois grupos estudados, a análise demonstrou que, pacientes com anticorpos anti-HNA-3 apresentaram rejeição significativamente mais precoce em comparação com o grupo controle durante o segundo episódio de rejeição do enxerto renal (teste log-rank, p = 0,014). Durante o período analisado, não foi observada a influência do anticorpo anti-HNA-3 na frequência aumentada de óbito e/ou perda no enxerto nos grupos estudados. Conclusão: Este estudo sugere que a presença de anticorpos anti-HNA-3 pré-formados é mais um fator de risco para o desenvolvimento de rejeições recorrentes do enxerto renal, além disso, pacientes com anticorpos anti-HNA-3 apresentaram o segundo episódio de rejeição significativamente mais precoce em comparação com o grupo controle.

The FcγRIIIb is the most immunogenic glycoprotein of the neutrophil membrane and anti-FcγRIIIb isoantibodies are formed after allogeneic exposure when the individual has a deletion of the glycoprotein and consequently has the human neutrophil antigen-1 null (HNA-1 null) phenotype, which is uncommon in the population. We report two cases of maternal alloimmunization against fetal FcγRIIIb glycoprotein using immunomagnetic negative selection for neutrophil isolation. We report two cases of mothers that have deletion of the FCGR3B gene (FCGR3B*null), and consequently the HNA-1-null phenotype. Neutropenia was not detected during hospitalization in both cases, however in a second case there was a considerable reduction in the number of neutrophils on the third day of the neonate's life. The isolation of neutrophils used to perform the granulocyte agglutination test (GAT) and granulocyte immunofluorescence test (Flow-GIFT) was tested by EasySep™Direct Human Neutrophil Isolation Kit (StemCell Technologies, Inc.) and confirmed by Dextran and Ficoll (conventional technique). Furthermore, monoclonal antibody immobilization of granulocyte antigens (MAIGA) and bead-based assay also were performed. In both cases, HNA-1 genotyping of the father, mother and newborn was performed by PCR-SSP. Sera from both women contained anti-FcγRIIIb alloantibodies detected by the all applied methods. In both cases the HNA genotyping of the mothers showed absence of epitopes of the FCGR3B gene leading to the HNA-1-null phenotype. In the first case the newborn and father were genotyped as HNA-1b/1b and in the second case the newborn and father were genotyped as HNA-1b/1c, confirming the incompatibility responsible for the mothers' alloimmunization. The isolation of neutrophils from the immunomagnetic negative selection has similar sensitivity and specificity when compared to conventional technique, however, the immunomagnetic negative selection technique is faster to be performed and has a higher cell yield. The molecular and serologic results confirm the findings of these two rare cases of HNA-1-null leading to the formation of specific FcγRIIIb alloantibodies. The isolation of neutrophils from the immunomagnetic negative selection can be used to perform the GAT and Flow-GIFT tests which are considered gold standard techniques for detecting anti-HNA antibodies.

Introduction: Human neutrophil antigens 3 (HNA-3a and HNA-3b) are located on choline transporter protein 2 (CTL2) and expressed on neutrophils, lungs, kidneys and other human tissues. Alloantibodies directed against these antigens are formed from allogeneic exposure and are mainly associated with transfusion-related acute lung injury (TRALI) and immune neutropenias, however due to the presence of this antigen in renal tissue and the doubt that HNA-3 alloimmunization may also be involved in cases of kidney transplant rejection, the study of the frequency of anti-HNA-3 antibodies in this context becomes a focus of great clinical relevance. Objective: Investigate the presence of anti-HNA-3 antibodies in the serum of patients who had kidney transplant rejection. Materials and Methods: A total of 604 patients with kidney transplant rejection were included in the study. The Granulocyte Agglutination Test (GAT) was performed as a screening test in all individuals included in the present study with a panel of granulocytes from at least three individuals previously genotyped for all HNA systems. Positive samples in GAT were tested using the microsphere-based technique (LABSCreen Multi kit, One Lambda); we analyzed the normalized background values ≥ 10 and immunofluorescence ≥ 1000 as a positive result for HNA-3 antibodies. HNA-3 genotyping by PCR-RFLP was performed only in individuals who had a positive result in serological techniques. Results: We detected 85/604 (14.1%) positive samples in GAT and 11/85 positive samples in both GAT and LSM multi for anti-HNA antibodies. Regarding the specificity of these 11 samples, 6 (54.5%) individuals presented an anti-HNA-3b antibody confirmed by genotyping (HNA- 3a/HNA-3a). In addition to the anti-HNA-3b antibodies, other specificities of anti-HNA antibodies were also identified: 1/11 (9.1%) anti-HNA-1a, 2/11 (18.2%) anti-HNA-1b, 1/11 (9.1%) anti-HNA-FCγRIIIb and 1/11 (9.1%) anti-HNA-2. Furthermore, 14/85 (16.5%) individuals had anti-HLA antibodies detected by LSM multi: 8/14 (57.1%) class I, 3/14 (21.4%) class II and 3/14 (21.4) class I and II. No patient presented simultaneously anti-HNA and HLA antibodies. Conclusion: Our data show that HNA-3b alloimmunization has a greater tendency in patients who rejected kidney transplantation compared to healthy blood donors (1.0% vs. 0.2% respectively, p=0.05). These findings indicate that the detection of anti-HNA-3b antibodies may allow a better understanding of patients’ immune response against renal allograft when no HLA antibody is identified supporting evidence of immunological risk. The authors would like to thank the recipients who participated in the study and the researchers at the Granulocyte Immunohaematology Research Laboratory for donating blood to perform the GAT and f low-WIFT. We would also like to thank the Coordination for the Improvement of Higher Education Staff [Personnel Coordenação de Aperfeiçoamento de Pessoal de Nível Superior; CAPES] for granting the scholarship and financial aid that made this study possible and Biometrix for the technical support.

To describe 2 cases of autoimmune neutropenia (AIN) patients infected with Sars-Cov-2. Two subjects case report. Case report 1: A girl with primary AIN since 1 year and 10 months old, maintaining severe neutropenia and mild recurrent infections. Presented to the emergency department in June/2020, at 3 years and 8 months old, with flu-like symptoms, afebrile, in good general condition. Physical examination was normal. The absolute neutrophil count (ANC) was 0.279 × 109/L. At hospital admission, Sars-Cov-2 (RT-PCR) tested positive and filgrastim (G-CSF) 5 μg/kg/day was initiated. Chest X-ray was also normal and blood culture resulted negative. She remained in great general condition, afebrile, and was discharged on the 2nd day of hospitalization, with clarithromycin (15 mg/kg/day). After G-CSF, ANC: 0.494 × 109/L (1st dose), 1.431 × 109/L (2nd dose). On outpatient follow-up, she had no long-term complications from Covid-19. Case Report 2: A man with chronic immune thrombocytopenia purpura (ITP) since 2008, autoimmune hemolytic anemia since 2013, evolved with AIN on May/2020, at 42 years old, with ANC lower than 0.5 × 109/L. On 6/1/2020, he had ANC 0.170 × 109/L. On 6/25/2020, he started flu-like symptoms, had ANC of 5.118 × 109/L, and tested positive for Sars-Cov-2 (RT-PCR). He kept high fever (102,2°F) and was hospitalized for 10 days without use of G-CSF. After discharge, on outpatient follow-up, he had no long-term complications from Covid-19, and presented ANC 0.338 × 109/L (Aug/2020). At beginning of Covid-19 pandemic, severity infection in children was unknown. Today is known that most of them have milder clinical course, regardless of chronic diseases. In adults, in contrast, the inflammatory response tends to exacerbation, with more severe clinical conditions. Furthermore, many case reports of patients infected by SARS-CoV-2 with comorbidities literature are published. However, to date there are no reports on the impact of COVID-19 in AIN patients. Increased neutrophil counts during infectious episodes are common in AIN, which appears to be related to the benign course of most infections. We reported 2 cases of AIN patients diagnosed with Covid-19, both with favorable clinical outcomes despite heterogenic clinical course. On the first case, she presented few symptoms and ANC increased only after using G-CSF. On the second reported patient, there was a spontaneous increase of ANC and greater inflammatory response than the first case. It could suggest a correlation between inflammatory response to COVID-19 and ANC in cases of autoimmune neutropenia. In the reported cases, clinical course of disease and neutrophil count were different between adult and pediatric patients. It is not possible to state whether this difference is due to age group, individual response to infection or other variables. It is important to assess other cases of AIN infected by COVID 19 to better understand correlation between severity of infection and neutrophil count response.