Neutrophil Antigens Research Articles

Flow cytometric measurement of respiratory burst activity and surface expression of neutrophils for septic patient prognosis

The hyperinflammation that begins with sepsis is essential for eradicating infection but also causes hypoperfusion and organ failure. To understand the innate immune status of septic patients, the functional and phenotypic changes of neutrophils during sepsis and their clinical implication were studied. Seventy-four patients who were admitted to intensive care unit due to severe sepsis or septic shock were enrolled. Surface antigens of neutrophils (CD64, CD10, and CD16) were detected by flow cytometry. Respiratory burst activity (RBA) was measured by flow cytometry using 2',7'-dichlorofluorescein diacetate and phorbol-12-myristate-13-acetate. The parameters were serially examined at Days 1 and 8 in septic shock patients. High CD64 and low CD10 and CD16 on Day 1 was associated with sepsis severity (P = 0.003, 0.017, and 0.007, respectively). On Day 1, RBA and CD64 were higher in survivors than in nonsurvivors of septic shock patients (P = 0.012 and 0.027, respectively), and on Day 8, CD10 and CD16 were higher in survivors than in nonsurvivors (P = 0.019 and 0.036, respectively). High RBA and high CD64 on Day 1 showed low 28-day mortality in univariate analysis (P = 0.018 and 0.034, respectively). In multivariate analysis, RBA maintained statistical significance (P = 0.042) but CD64 revealed only a tendency (P = 0.064). Neutrophil surface antigen (CD64, CD10, and CD16) could reflect sepsis severity. High CD64 expression and high RBA at early phase of sepsis might be associated with better prognosis, whereas high expression of CD10 and CD16 at late phase of sepsis might be associated with better prognosis. © 2015 International Clinical Cytometry Society.

Genetic mechanism of human neutrophil antigen 2 deficiency and expression variations.

Human neutrophil antigen 2 (HNA-2) deficiency is a common phenotype as 3–5% humans do not express HNA-2. HNA-2 is coded by CD177 gene that associates with human myeloproliferative disorders. HNA-2 deficient individuals are prone to produce HNA-2 alloantibodies that cause a number of disorders including transfusion-related acute lung injury and immune neutropenia. In addition, the percentages of HNA-2 positive neutrophils vary significantly among individuals and HNA-2 expression variations play a role in human diseases such as myelodysplastic syndrome, chronic myelogenous leukemia, and gastric cancer. The underlying genetic mechanism of HNA-2 deficiency and expression variations has remained a mystery. In this study, we identified a novel CD177 nonsense single nucleotide polymorphism (SNP 829A>T) that creates a stop codon within the CD177 coding region. We found that all 829TT homozygous individuals were HNA-2 deficient. In addition, the SNP 829A>T genotypes were significantly associated with the percentage of HNA-2 positive neutrophils. Transfection experiments confirmed that HNA-2 expression was absent on cells expressing the CD177 SNP 829T allele. Our data clearly demonstrate that the CD177 SNP 829A>T is the primary genetic determinant for HNA-2 deficiency and expression variations. The mechanistic delineation of HNA-2 genetics will enable the development of genetic tests for diagnosis and prognosis of HNA-2-related human diseases.

Involvement of high mobility group box 1 in the activation of C5a-primed neutrophils induced by ANCA

C5a plays a central role in antineutrophil cytoplasmic antibody (ANCA)-mediated neutrophil recruitment and activation. A previous study showed that C5a played a crucial role in the regulation of high mobility group box 1 (HMGB1) release from human neutrophils. The current study further investigated the interaction between C5a and HMGB1 in ANCA-induced neutrophil activation. The effects of HMGB1 inhibitors on the translocation of ANCA antigens, ANCA-induced respiratory burst and degranulation of C5a-primed neutrophils were tested. We found that blocking HMGB1 decreased C5a-mediated translocation of ANCA antigens, as well as ANCA-induced respiratory burst and degranulation of C5a-primed neutrophils. Further study showed that supernatant of C5a-primed neutrophils, which contained HMGB1, also caused translocation of ANCA antigens of primary neutrophils, whereas blocking HMGB1 decreased the translocation. In conclusion, blocking HMGB1 may attenuate ANCA-induced activation of C5a-primed neutrophils. The interaction between HMGB1 and C5a might play an important role in ANCA-induced neutrophil activation.

Antibody-mediated transfusion-related acute lung injury; from discovery to prevention.

Transfusion-related acute lung injury (TRALI), a syndrome of respiratory distress caused by blood transfusion, is the leading cause of transfusion-related mortality. The majority of TRALI cases have been related to passive infusion of human leucocyte antigen (HLA) and human neutrophil antigen (HNA) antibodies in donor blood. In vitro, exvivo and invivo animal models have provided insight in TRALI pathogenesis. The various classes of antibodies implicated in TRALI appear to have different pathophysiological mechanisms for the induction of TRALI involving endothelial cells, neutrophils, monocytes and, as very recently has been discovered, lymphocytes. The HLA and HNA-antibodies are found mainly in blood from multiparous women as they have become sensitized during pregnancy. The incidence of TRALI has decreased rapidly following the introduction of a male-only strategy for plasma donation. This review focuses on pre-clinical and clinical studies investigating the pathophysiology of antibody-mediated TRALI.

Human neutrophil antigen profiles in Banjar, Bugis, Champa, Jawa and Kelantan Malays in Peninsular Malaysia.

Human neutrophil antigens (HNA) are polymorphic and immunogenic proteins involved in the pathogenesis of neonatal alloimmune neutropenia, transfusion-related acute lung injury (TRALI) and transfusion-related alloimmune neutropenia. The characterisation of HNA at a population level is important for predicting the risk of alloimmunisation associated with blood transfusion and gestation and for anthropological studies. Blood samples from 192 healthy, unrelated Malays were collected and genotyped using polymerase chain reaction-sequence specific primers (HNA-1, -3, -4) and polymerase chain reaction-restriction fragment length polymorphisms (HNA-5). The group comprised 30 Banjar, 37 Bugis, 51 Champa, 39 Jawa and 35 Kelantan Malays. The most common HNA alleles in the Malays studied were HNA-1a (0.641-0.765), -3a (0.676-0.867), -4a (0.943-1.000) and -5a (0.529-0.910). According to principal coordinate plots constructed using HNA allele frequencies, the Malay sub-ethnic groups are closely related and grouped together with other Asian populations. The risks of TRALI or neonatal neutropenia were not increased for subjects with HNA-1, -3 and -4 loci even for donor and recipient or pairs from different Malay sub-ethnic groups. Nonetheless, our estimates showed significantly higher risks of HNA alloimmunisation during pregnancy and transfusion between Malays and other genetically differentiated populations such as Africans and Europeans. This study reports HNA allele and genotype frequencies for the five Malay sub-ethnic groups living in Peninsular Malaysia for the first time. These Malay sub-ethnic groups show closer genetic relationships with other Asian populations than with Europeans and Africans. The distributions of HNA alleles in other lineages of people living in Malaysia (e.g. Chinese, Indian and Orang Asli) would be an interesting subject for future study.

Human neutrophil antigens: a nomenclature update based on new alleles and new antigens

In 1998, the ISBT Granulocyte Antigen Working Party established a new nomenclature for the human neutrophil antigens (HNA) which included five antigen systems and a total of seven antigens. In the meantime, new antigens and new alleles have been described and these now make it necessary for the ISBT HNA nomenclature subcommittee to present a proposal for an HNA nomenclature update. HNA‐1 includes four antigens, HNA‐1a, HNA‐1b, HNA‐1c and HNA‐1d, which can result from five different Fcγ receptor IIIb (FcγRIIIb)‐encoding alleles, FCGR3B*01, *02, *03, *04 and *05. Due to copy number variation, individuals may exhibit between zero and four alleles leading to either the HNA‐1 null type or combinations of the known HNA‐1 antigens. Current data indicate that HNA‐2 is an isoantigen without allelic variation, encoded by the CD177 gene. Individuals without any HNA‐2 expression on their neutrophils are addressed as HNA‐2 null. The two antithetical antigens of the choline‐transporter‐like protein 2 (CTL2), HNA‐3a and HNA‐3b, are encoded by the three different alleles SLC44A2*01, *02 and *03, which discriminate in one or two nucleotide substitutions. HNA‐4a, which is encoded by the ITGAM*01 allele, is an antigen of the αMβ2‐integrin. The αLβ2‐integrin contains HNA‐5a as the only antigen which is encoded by the ITGAL*01 allele. Antigens and alleles other than those listed have to be approved by the ISBT Granulocyte Immunobiology Working Party (GIWP) before they can be assigned to the official nomenclature.

Induction of immune tolerance to RBC, platelet, and neutrophil antigens and IgA

Antibodies to red blood cell (RBC), platelet, and neutrophil antigens, and IgA may cause serious clinical problems. With a few exceptions, preventing these conditions is a matter of limiting exposure to the foreign antigen while treatment consists of managing the consequences. Might immune tolerance induction (ITI) be possible and beneficial in these situations?Neonatal exposure to antigens is known to induce central tolerance. However central tolerance may not be absolute. Factors that determine whether an antibody will be produced in response to an antigen are not well understood but include the appropriate expression of major histocompatibility complex-class II and/or co-stimulatory molecules on dendritic cells, the presence or absence of adjuvants and whether or not the antigen is presented together with agonists for the toll-like receptor. Modifying these may prevent alloimmunization. Peripheral tolerance, in sensitized individuals, as routinely used in patients with allergic/anaphylactic reactions, those with haemophilia A or B with inhibitors and acquired haemophilia, may also be possible. Briefly, monitored, graded, increasing exposure to the antigen of interest with or without additional immunosuppression is used.Neither central nor peripheral ITI has been tried or suggested for individuals sensitizable or sensitised to RBC, platelet, and neutrophil antigens, or IgA. Theoretically, this is possible and may be of benefit.

Estrogen modulates 7/4 antigen distribution within eccentrically contracted injured skeletal muscle

Eccentric contractions are skeletal muscle stretches with concurrent active force production; these contractions commonly occur during dynamic sports activities and can cause acute muscle injury. Recovery from this injury depends in part on pro-inflammatory processes, such as neutrophil infiltration at the injured site, which is affected by estrogen. This estrogen effect has been examined broadly, but without distinguishing between major compartments within muscle in which neutrophil infiltration can occur. Therefore, we compared neutrophil antigen expression in two compartments of eccentrically contracted muscle of ovariectomized mice with or without estrogen. To quantify neutrophil antigen expression, serial cross sections of muscle were immunolabeled with antibodies that recognize 7/4 or Ly6C/G, then quantified using computer-assisted image analysis. At 48 h post injury, estrogen-positive (E+) mice had more 7/4-positive and Ly6C/G-positive myofibers, increased 7/4 area percentage, and more 7/4-positive cells in the connective tissue. In addition, E+ mice showed more 7/4-positive myofibers that were Ly6C/G-negative and more Ly6C/G-positive myofibers that were 7/4-negative. These data suggest that in injured muscle, estrogen increases 7/4 antigen in connective tissue and myofibers and is associated with more Ly6C/G-positive myofibers when the 7/4 antigen is absent from these myofibers.

Evaluation of HNA-expressing cell line-based antigen capture systems and a solid-phase system for detecting HNA-1a antibodies.

Granulocyte immunofluorescence and granulocyte agglutination tests are standard methods for detecting human neutrophil antigen (HNA) antibodies (Abs); however, these require a typed panel of neutrophils, which can be time-consuming to develop, and it remains difficult to determine antibody specificity in some cases. We established and evaluated four detection systems for HNA-1a Abs based on an HNA-1a-expressing cell line (KY cells) and antigen capture. We additionally evaluated a commercial solid-phase system. Eleven HNA-1a antibody-positive samples, including the World Health Organization Reference Reagent, and 40 serum samples derived from male blood donors were used as positive and negative control samples, respectively. Although specificity was >0.90 in all systems evaluated, the sensitivity varied among the systems. The KY cell-based monoclonal antibody specific immobilisation of granulocyte antigens (KY-MAIGA) system using certain, but not all, monoclonal Abs, and the solid-phase system revealed higher sensitivity than other systems. In conclusion, the KY-MAIGA and commercial solid-phase systems were superior in terms of specific and sensitive detection of HNA-1a Abs.

FCGR3B gene frequencies among ethnic Thai blood donors from a regional hospital in Eastern Thailand.

The FCGR3B gene encodes three human neutrophil antigens which consist of HNA-1a, HNA-1b, and HNA-1c. These antigens are encoded by three alleles in the FCGR3B locus: FCGR3B*01, FCGR3B*02, and FCGR3B*03 alleles, respectively. The frequencies of FCGR3B alleles have been reported in different ethnic populations. This study compared the FCGR3B gene frequencies among 230 unrelated healthy Eastern Thai blood donors in Rayong hospital with the previously published studies. The polymerase chain reaction-sequence-specific primers method was performed to determine FCGR3B genotypes. The results showed that the allele frequencies of FCGR3B*01, FCGR3B*02, and FCGR3B*03 were 0.722, 0.274, and 0.009, respectively. The FCGR3B*01 and FCGR3B*02 frequencies found in the Eastern Thais were similar to the previous reports investigating in Northern Thais, Chinese Han, Taiwanese, and Japanese populations. Interestingly, our data showed statistically significant difference (P < 0.05) to Central Thais, Korean, Indian, Turkish, Australian, Tunisian, American, German, and Italian populations. In addition, one FCGR3Bnull , which represents a gene deletion, was also found in this study. This information is important not only for the assessment of neutrophil antibody-mediated clinical conditions and for disease association studies but also for anthropological studies.

Risk estimation of HNA-3 incompatibility and alloimmunization in Thai populations.

Severe transfusion-related acute lung injury (TRALI) is often due to antibodies in blood components directed against human neutrophil antigen (HNA)-3a. This study aimed to report the genotype frequencies of the HNA-3 system and to estimate the potential risk of HNA-3 incompatibility and alloimmunization in two Thai populations. Eight hundred DNA samples obtained from 500 unrelated healthy blood donors at the National Blood Centre, Thai Red Cross Society, Bangkok and 300 samples from the Blood Bank, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand were included. HNA-3 genotyping was performed using an in-house polymerase chain reaction with sequence-specific primer (PCR-SSP) technique. The observed frequencies of the HNA-3a/3a, HNA-3a/3b, and HNA-3b/3b genotypes were 0.528, 0.380, and 0.092 in central Thais and 0.600, 0.350, and 0.050 in northern Thais, respectively. The frequencies were used to estimate HNA-3 incompatibility and risk of HNA-3a alloimmunization. The HNA-3 incompatibility in central Thais (33.28%) was higher than northern Thais (28.75%), corresponding to a significantly higher probability of HNA-3a alloimmunization (P<0.05) similar to Japanese and Chinese populations. This study showed the high risk of HNA-3 incompatibility and alloimmunization, especially in central Thai blood donors. A molecular-based identification of the HNA-3 genotype of female donors is suggested to reduce the risk of TRALI following plasma and whole blood allogeneic transfusion.

Allelic Discrimination by TaqMan-PCR for Genotyping of Human Neutrophil Antigens.

Neutrophil antigens are implicated in a variety of clinical conditions, including neonatal immune neutropenia, transfusion-related acute lung injury, refractoriness to granulocyte transfusions, febrile transfusion reactions, and autoimmune neutropenia. In this report, we describe simultaneous genotyping of human neutrophil antigens (HNA)-1, -3, -4, and -5 using PCR with allele-specific TaqMan probes and end-point fluorescence detection, which is a robust, rapid, and reproducible method, allowing for high-throughput genotyping.

High-resolution melting analysis as an alternative method for human neutrophil antigen genotyping

Abstract Human neutrophil antigen (HNA)-typed granulocyte panels are widely used to screen for the presence of HNA antibodies and to determine antibody specificity. Many laboratories screen donors for HNA genotypes using low-throughput methods such as allele-specific polymerase chain reaction (PCR), PCR–restriction fragment–length polymorphism, and multiplex PCR. In the present study, we used a high-resolution melting (HRM) analysis to determine HNA genotypes. For the HRM analysis, purified genomic DNA samples were amplified via PCR with HNA-specific primers. Nucleotide substitutions in genes encoding HNAs were differentiated on the basis of the HRM curves, and the results of HRM and DNA sequencing analyses were determined to be in complete agreement. The gene frequency of HNA-1a, -1b, -1c, -3a, -3b, -4a, -4b, -5a, and -5b in the Japanese population was consistent with the previous reports. Our results suggest that HRM analysis can be used for genotyping HNA antigens determined by single nucleotide substitutions. Immunohematology 2015;31:7–13.

Geno- and phenotyping of human neutrophil antigens.

For typing of human neutrophil antigens (HNA) usually genotyping techniques are used, except for HNA-2, which-due to a gene expression defect-requires phenotyping. For genotyping, several techniques have been described. Most reference laboratories use variations of the polymerase chain reaction (PCR) for antigen typing which showed good results in international quality assessment exercises. The granulocyte immunofluorescence test has been the gold standard technique for phenotyping for all HNA antigens except for HNA-3a and -3b phenotyping. The expression of the latter antigens on neutrophils is often better shown by the use of the granulocyte agglutination test.

Frequencies of human neutrophil antigen-4 and human neutrophil antigen-5 among Thai blood donors.

Context:Antibodies against human neutrophil antigens (HNAs) are implicated in immune-mediated neutropenia, transfusion-related acute lung injury and febrile transfusion reactions.Aims:This study aimed to determine HNA gene frequencies of the HNA-4 and HNA-5 systems among Thai populations and compare these frequencies with those previously reported for other populations.Materials and Methods:800 DNA samples obtained from 500 unrelated healthy blood donors from Bangkok and 300 samples from Chiang Mai, Thailand were included. Samples were typed for each HNA allele including HNA-4a, HNA-4b, HNA-5a, and HNA-5b using an in-house polymerase chain reaction with sequence-specific primer technique.Results:The frequencies of HNA-4a and HNA-4b alleles in central Thais were 0.975 and 0.025, respectively and for Northern Thais, their frequencies were 0.965 and 0.035, respectively. For HNA-5a and HNA-5b alleles, their frequencies were 0.771 and 0.229; 0.748, and 0.252 in central and Northern Thais, respectively. The frequencies of HNA-4 and HNA-5 systems in central Thais are closely related to those in Northern Thais (P > 0.05). However, their frequencies were different from other populations (P < 0.001), except HNA-5a and HNA-5b gene frequencies in Thais were similar to Caucasians (P > 0.05).Conclusion:This study could contribute to predict the risk of alloimmunization to HNA-4 and HNA-5 systems, especially in feto-maternal incompatibility in Thais.

Detection of granulocyte‐reactive antibodies: a comparison of different methods

Granulocyte-reactive antibodies can cause autoimmune and neonatal immune neutropenias as well as transfusion-related acute lung injury. The classical antibody-detection methods granulocyte aggregation test (GAT), granulocyte immunofluorescence test (GIFT) and monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) are time-consuming and technically challenging. In recent years, flow cytometric white blood cell immunofluorescence test (Flow-WIFT) and the microbeads assay LabScreen® Multi have emerged and are still subject of evaluation. These serological tests were compared on a screening and specification level. For screening, the combination of GAT/GIFT was compared to Flow-WIFT testing 333 samples. Positive samples were further analysed with MAIGA and LabScreen® Multi. Granulocyte aggregation test/GIFT detected 77 positive samples, Flow-WIFT found 108 granulocyte-reactive samples. Six Samples were only positive in GAT/GIFT, and 37 samples were only positive in Flow-WIFT (κ = 0.682). Antibody specification with MAIGA and the microbeads assay confirmed granulocyte-reactivity in 83 cases with 70 matching results (κ = 0.742). However, out of six detected human neutrophil antigen (HNA) reactivities only two specificities matched in both assays. Flow-WIFT may be a valuable addition to GIFT for granulocyte-reactive antibody screening. MAIGA remains the most reliable laboratory method for antibody specification.

Diagnosis and management of primary autoimmune neutropenia in children: insights for clinicians.

Autoimmune neutropenia of infancy (AIN), also called primary autoimmune neutropenia, is a disease in which antibodies recognize membrane antigens of neutrophils, mostly located on immunoglobulin G (IgG) Fc receptor type 3b (FcγIIIb receptor), causing their peripheral destruction. It is the most frequent type of neutropenia in children under 3-4 years of age and in most cases shows a benign, self-limited course. The diagnosis is based on evidence of indirect antineutrophil antibodies, whose detection frequently remains difficult. In this review we have analyzed the literature regarding AIN and present our personal experience in diagnosis and management.

Simultaneous Occurrence of Neonatal Alloimmune Thrombocytopenia and Neutropenia Associated with Multiple Maternal Antibodies

Background: Neonatal alloimmune thrombocytopenia (NAIT) results from feto-maternal platelet antigen incompatibility leading to the production of maternal antibodies and destruction of fetal platelets during pregnancy. Similarly neonatal alloimmune neutropenia (NAN) is caused by maternal alloimmunization to incompatible fetal neutrophil antigens followed by transplacental transfer of the maternal anti-neutrophil antibody causing fetal or neonatal neutropenia. Although there are many reports in the literature describing these rare disorders, the simultaneous occurrence of NAIT and NAN has not been systematically evaluated. In this study we report a case of NAIT associated with NAN.Material and Methods: A healthy 46-year-old primiparous woman gave birth to triplets in the 30.9th gestational week, by C-section, after a fertility treatment using an egg donation banking. The first neonate, a 1,600g female newborn with a first/fifth minute Apgar score of 8/9, healthy at physical examination, showed severe thrombocytopenia (platelet count = 30x109/L) and a neutrophil count of 1.6X109/L at birth. The second neonate, a 1,275g male newborn had a first/fifth minute Apgar score of 7/7, was healthy at physical examination and showed thrombocytopenia (platelet count = 82x109/L) and neutropenia (neutrophil count = 0.66x109/L). Intracranial bleeding, perinatal infections, genetic and hematologic disorders were ruled out and the neonates showed a spontaneously normalization of the platelets and neutrophils counts. Unfortunately, blood sample from the third neonate, a 1,525g female newborn with first/fifth minute Apgar score of 9/9, was not available. Mother and neonates were genotyped for platelets antigens (HPA-1-11,-15) and neutrophil antigens (HNA-1,-3). HPA genotyping was performed by PCR-SSP, PCR-RFPL and xMAP technology (IDHPAXT, Progenika-Grifols, Spain). HNA genotyping was performed by PCR-SSP and PCR-RFPL. Platelets antibodies were investigated in maternal serum by platelet enzyme-linked immunosorbent assay (PaK12G, Immucor GTI Diagnostic Inc, USA); neutrophil antibodies were investigated by granulocyte agglutination test (GAT) using a panel of donors previously genotyped for HNA-1,-3 systems, while anti-HLA antibodies were investigated by ELISA (LAT Mixed, One Lambda Inc., USA and PaK12G, Immucor GTI Diagnostic Inc, USA).Results: Genotyping studies revealed mismatches for HPA-1,-5 and HNA-1,-3 systems between mother and neonates (Table1). Serologic antibody screening tests showed presence of anti-HPA-5b, anti-HNA-3b and anti-HLA in maternal serum. TABLE 1GENOTYPING RESULTSHPA-1HPA-5HNA-1HNA-3MOTHERHPA-1a1aHPA-5a5aHNA-1a1aHNA-3a3aNEONATE 1HPA-1b1bHPA-5a5bHNA-1b1bHNA-3a3bNEONATE 2HPA-1b1bHPA-5a5bHNA-1b1bHNA-3a3aConclusion: We described the occurrence of NAIT associated with NAN in preterm neonates with important thrombocytopenia and neutropenia due a multiple maternal sensitization to incompatible fetal antigens. As these syndromes pose a risk to the neonate’s life, the early and correct diagnosis is very important in order to apply appropriate treatment to the neonates and allow an effective prenatal therapy for future pregnancies. DisclosuresNo relevant conflicts of interest to declare.

Anti-Granulocyte Antibodies in Transfusion Medicine

Granulocyte antibodies, in contrast to antibodies recognizing red blood cells and platelets, have been widely neglected in transfusion medicine for several decades, although their clinical relevance is well-described. Autoimmune neutropenia of infancy and neonatal alloimmune neutropenia are probably the most well-known granulocyte antibody-induced disorders. The facts that antibody-induced neutropenias are rare disorders with a favorable prognosis and that sophisticated, labor-intensive in-house techniques are required for granulocyte antibody detection may both have contributed to this negligence. When transfusion-related acute lung injury (TRALI) was delineated as the leading cause of transfusion-associated mortality and, subsequently, granulocytes were identified as key players in TRALI, granulocyte antibodies received progressively higher attention. It took only a short period of time to unravel that transfusion of granulocyte antibodies can lead to activation of the transfusion recipient’s own granulocytes with subsequent breakdown of the pulmonary endothelial barrier and lung edema. Clinical and regulatory pressure has prompted the identification of new target antigens on granulocytes, the development of better diagnostic approaches for antibody identification, and drastic changes in blood component production in order to reduce the risk of TRALI. There were two major fields of controversy in this rather straight-forward process. First, there are two groups of antibodies associated with TRALI: 1) granulocyte antibodies in sensu strictu, i.e., those which recognize human neutrophil antigens (HNA); and 2) human leukocyte antigen (HLA) antibodies which can bind to granulocytes, but also to other cells. These observations led to a debate on whether granulocytes are primary target cells or, at least in some cases, “auxiliary” cells in TRALI. Evidence from animal studies supports the idea that HLA class I antibodies may bind to endothelial cells and recruit granulocytes to the vessel wall, where they get activated; and that HLA class II antibodies, since their target antigen is not expressed on quiescent granulocytes, may bind to monocytes which then activate granulocytes. Recent data on HNA antibodies which cause TRALI by direct binding to pulmonary endothelium in the absence of granulocytes have added to the complexity, as has the observation from prospective clinical trials that HLA class I antibodies are of less relevance than previously anticipated. Today, there is consensus that different antibodies can cause TRALI by different pathways which do partially overlap. Second, the presence of granulocyte antibodies in blood components as the only causative factor for TRALI appeared partially doubtful: a minority of components produced from the same donor and transfused to different recipients led to TRALI. These doubts were further substantiated by the fact that in some TRALI cases, no antibodies whatsoever could be identified. Other substances often referred to as biological response modifiers (BMR) were demonstrated causative for TRALI, but only in animals pre-exposed to priming agents. This observation was crucial in understanding that not only factors present in a blood component (antibody or BMR) need to be considered, but also factors present in the recipient; this led to the “two-hit model” or “threshold model” of TRALI. Nowadays, there is consensus that antibody-mediated TRALI and non-antibody-mediated TRALI exist, with the latter one posing new challenges for transfusion medicine. This overview will start from pre-TRALI knowledge of granulocyte antibodies, consider antibody-mediated TRALI in detail, and will finally return to non-TRALI disorders in the light of relevant new findings in the field. DisclosuresNo relevant conflicts of interest to declare.

Neonatal Alloimmune Neutropenia Due to Maternal Alloimmunization Against Human Neutrophil Alloantigen-1 and -3

Introduction: Neonatal alloimmune neutropenia (NAN) is a potentially lethal disorder that results from maternal alloimmunization to human neutrophil antigens (HNAs) present in the fetus. The alloantibodies more frequently involved in NAN are against the HNA-1 and -2 systems; however HNA-3, HNA-4 and HNA-5 systems have also been associated with cases of NAN. In this study we examined the frequency of neonatal neutropenia and investigated the presence of maternal anti-HNA-1, anti-HNA-3 and anti-HLA alloantibodies when neonatal neutropenia and maternal-fetal incompatibility to HNA-1 and HNA-3 systems were observed. Furthermore, we investigated the association between neutrophil alloimmunization and HLA-DRB1 alleles.Methods: A cross sectional study included samples from 10,000 unselected neonates born in 4 obstetric units in Sao Paulo City (Brazil). Neonatal neutropenia was defined as neutrophil count <1.5x109/L in cord blood. Genotyping studies were performed in 88 neutropenic newborns and their mothers (83 mothers with 3 pairs of twins and 1 triplet) by PCR-SSP technique for the detection of HNA-1a, -1b and -1c alleles and by PCR-RFLP technique for the detection of HNA-3a and -3b alleles; the PCR-RFLP technique amplifies the sequence for rs2288904 and the amplified product was digested with enzyme Taqα1 specific to nucleotide guanine that codifies the HNA-3a antigen (Lopes et al., Transfusion 2014;54(6):1619-21.). Serologic studies for detecting maternal HNA alloantibodies were performed in maternal-fetal incompatibility cases by granulocyte agglutination test (GAT) that was done in duplicate using a specific panel of donors previously genotyped for HNA-1 and HNA-3 systems. Anti-HLA-I/II antibodies were investigated in maternal serum, and all 83 mothers were genotyped for the HLA-DRB1 gene. Two control groups included mothers with maternal-fetal incompatibility but without alloantibodies, and healthy individuals.Results: Neonatal neutropenia was identified in 88 of 10,000 (0.9%) newborns in the studied population. Genotyping studies revealed 39/88 (44.3%) maternal-fetal HNA incompatibilities corresponding to 28/88 (31.8%) for HNA-1 and 13/88 (14.8%) for HNA-3. In 12/28 (42.9%) cases we found incompatibility for the HNA-1a allele; in 9/28 (32.1%) for HNA-1b; in 4/28 (14.3%) for HNA-1c; in 1/28 (3.6%) for both HNA-1a and -1c; and in 2/28 (7.1%) for both HNA-1b and -1c alleles. In all neutropenic cases related to HNA-3 system mothers were typed as HNA-3a/a and neonates as HNA-3a/b. Using the GAT, HNA alloantibodies were found in 21/39 (53.8%) mothers including 16/28 (57.2%) anti-HNA-1 and 5/13 (38.5%) anti-HNA-3b. The specificity of HNA-1 alloantibodies was confirmed in 9 cases for anti-HNA-1a and in 7 for anti-HNA-1b. Anti-HLA antibodies were detected in 6/16 GAT(+) maternal serum containing anti-HNA-1 and in 2/5 GAT(+) with anti-HNA-3. Two maternal-fetal incompatibility cases occurred concomitantly for HNA-1 and HNA-3 systems; however, only anti-HNA-3b alloantibodies could be identified using the panel of donors. HLA-DRB1 genotyping showed an increased frequency of HLA-DRB1*01 and HLA-DRB1*10 alleles in mothers with HNA incompatibility when anti-HNA were not found. A statistically significant difference was observed between these mothers with HNA incompatibility not alloimmunized and the healthy individuals (HLA-DRB1*01 p=0.0433; HLA-DRB1*10 p=0.0208).Conclusions: The observed frequency of neonatal neutropenia in Brazilians of 0.9% is similar to those described in North Americans and Europeans with a comparable positive serology rate of 53.8% more frequently due to the presence of anti-HNA-1 (76.2%). As for the best of our knowledge this is the first study reporting the presence of anti-HNA-3b alloantibodies in newborns with NAN (23.8%). The frequencies of the HLA-DRB1*01 and HLA-DRB1*10 alleles were increased in mothers with HNA incompatibility without HNA alloantibodies suggesting that such HLA-DRB1 alleles may confer protection against neutrophil alloimmunization in cases of maternal-fetal incompatibility for HNA systems involved with NAN. DisclosuresNo relevant conflicts of interest to declare.