Human Neutrophil Antigens Antibodies Research Articles

Neutrophil antigen antibodies affect engraftment and secondary graft failure in hematopoietic progenitor cell transplantation.

Research is limited on the role of antibodies against human neutrophil antigen (HNA) in hematopoietic progenitor cell (HPC) transplantation outcomes. A retrospective review was conducted on medical records of patients at the NIH Clinical Center enrolled in six research protocols. This case-control study included 21 patients tested for HNA antibodies from January 2010 to March 2022 who underwent HPC transplantation. In addition, 42 patients following the same research protocols were randomly selected as a control group. The cumulative incidence of time to neutrophil engraftment was significantly impacted by the patients' anti-HNA status (p = .042), with the patients with anti-HNA experiencing delayed engraftment. Secondary graft failure occurred in 4 out of 42 patients (9.52%; 95% confidence interval [CI]: 3.7-22.1) of the control group, while 5 out of 9 patients (55.5%; 95% CI: 26.7-81.1) with anti-HNA experienced secondary graft failure (p = .005). Furthermore, patients with anti-HNA had a lower proportion (p = .008 for full and p = .002 for partial chimerism) and cumulative incidence (p = .016 for full and p = .010 for partial chimerism) of achieving donor chimerism compared to the control group. The study reveals a potential link between anti-HNA and HPC transplantation outcomes not previously reported. Patients with anti-HNA had a lower proportion and cumulative incidence of achieving donor chimerism. Additionally, anti-HNA status affected the time for neutrophil engraftment, with a slower rate of neutrophil engraftment and increased risk of secondary failure in patients with anti-HNA.

A novel immunocomplex capture fluorescence assay (ICFA) using fluorescent beads and transfected cells for specific identification of human neutrophil antigen (HNA)-1a and -1b antibodies.

To identify specific human neutrophil antigen (HNA) antibodies, assays using neutrophils such as monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) are recommended. However, these assays are limited by labor-intensive neutrophil preparation and varying antigen expression levels. We evaluated a newly developed immunocomplex capture fluorescence assay (ICFA) for identifying HNA-1 antibodies and compared it to MAIGA and LABScreen Multi (LABM), which utilizes recombinant HNA-coated Luminex beads. For ICFA, HNA-1a or HNA-1b transfected cells replaced neutrophils. Cells incubated with serum were lysed, and immune complexes were captured using five CD16 monoclonal antibody-conjugated Luminex beads. Nine antisera with known specificity and 26 samples suspected of containing HNA antibodies were analyzed by ICFA and MAIGA using neutrophils or transfected cells (ICFA-N or ICFA-T, and MAIGA-N or MAIGA-T, respectively). ICFA-T and MAIGA-N accurately determined the specificity of all antibodies in the nine antiserum samples. The ICFA-T detection limit was 2048-fold for anti-HNA-1a and 256-fold for anti-HNA-1b; the limits of MAIGA-T, MAIGA-N, and LABM were 32-, 4 ~ 64-, and 128-fold for anti-HNA-1a and 64-, 16 ~ 64-, and 32-fold for anti-HNA-1b, respectively. Twelve and 7 of the remaining 26 samples tested negative and positive, respectively, in both ICFA-T and MAIGA-N. Antibody specificity against HNA-1a or HNA-1b determined using ICFA-T agreed with that determined using MAIGA-N and LABM. Another seven samples tested positive in ICFA-T but negative in MAIGA-N. The novel ICFA is highly sensitive and exhibits specificity similar to MAIGA and LABM for detecting HNA-1 antibodies.

HLA and HNA Antibodies Are More Prevalent in Multiparous Women with Red Blood Cell Alloimmunization

Background: Leukocyte antibodies against HLA class I, class II and human neutrophil antigens (HNA) are formed from exposure to these antigens by transfusion, pregnancy or transplant, and may be associated with transfusion-related acute lung injury (TRALI) and immune neutropenias. Risk factors for acquisition of leukocyte antibodies are yet to be determined and present great importance for the prevention of severe and fatal transfusion reactions.Aims: To compare the prevalence of leukocyte antibodies in women with or without red blood cell (RBC) alloimmunization.Methods: This transversal study included women whose only stimulus to alloimmunization was pregnancy, with no transfusion history. We analyzed 147 blood samples from women with RBC alloimmunization, confirmed by the investigation and identification of RBC antibodies by the DG Gel-Card technique (Grifols-Spain). The control group consisted of 563 blood donors women with pregnancy history but without RBC alloimmunization. The identification of leukocyte antibodies was performed by: 1) granulocyte agglutination test (GAT), 2) white cell immunofluorescence test (Flow-WIFT), both tests using a panel of neutrophils obtained from three donors with previous HNA genotyping, and 3) bead-based assay - LABScreen Multi (LSM) (One Lambda), capable of detecting antibodies against HNA-1a, -1b, -1c, -2, -3a, -3b, -4a, -5a, -5b and HLA class I and II antigens. The identified HNA antibodies were confirmed by genotyping the corresponding antigen.Results: In the cohort of women with RBC alloimmunization we found 179 RBC antibodies: anti-D (30.7%), anti-Lea (21.3%), anti-C (15.1%), anti-E (8.9%), anti-K (5.6%), anti-M (5.6%) and other antibodies (12.8%). We identified 74/147 (50.3%) samples with anti-HLA antibodies and 11/147 (7.5%) samples with anti-HNA antibodies with the following specificities: 5/11 anti-HNA-1a, 2/11 anti-FCγRIIIb and 4/11 anti-HNA-3b. In the control group, we found 238/563 (42.3%) samples with anti-HLA and 24/563 (4.3%) samples with anti-HNA: 8/24 anti-HNA-1a, 01/24 anti-HNA-1c, 03/24 anti-HNA-2, 02/24 anti-HNA-3a, 5/24 anti-HNA-3b, 03/24 anti-HNA-5a and 02/24 anti-HNA-5b. Regarding the gestational history, 82/147 (55.8%) women in the group with RBC alloimmunization and 493/563 (87.6%) in control group had two or more pregnancies. The statistical analysis was performed in total samples, regardless the number of pregnancies, however no significant difference was observed in the prevalence rate of anti-HNA and anti-HLA between the two groups (p=0.10 and p=0.07, respectively). When the analysis was stratified including only multiparous women (two or more pregnancies) the prevalence of HNA alloimmunization was statistically significant in the group of women with RBC alloimmunization: 9/82 (11%) versus 23/493 (4.7%) blood donors, p=0.02, OR=2.52 (95% CI=1.15-3.79). Similarly a higher prevalence of HLA antibodies in the group of multiparous women with RBC alloimmunization was also observed: 51/82 (62.2%) versus 224/493 (45.4%), p=0.004, OR=1.97 (95% CI=1.18-2.71).Conclusions: The data show that RBC alloimmunization was significantly associated with the development of antibodies against leukocytes (anti-HNA and anti-HLA). A higher frequency of HNA alloimmunization (11%) observed in the group of multiparous women with RBC alloimmunization compared to the control group (4.7%), suggest that they are better immune responders and that they react strongly to allogeneic exposure. DisclosuresNo relevant conflicts of interest to declare.

Serologic and molecular studies to identify neonatal alloimmune neutropenia in a cohort of 10,000 neonates.

Neonatal alloimmune neutropenia (NAIN) is caused by maternal alloimmunisation to fetal human neutrophil antigens (HNAs). This study investigated maternal HNA/HLA alloantibodies involved with NAIN and identified the frequency of NAIN in Brazilian neonates. Neonatal neutropenia (neutrophil count<1.5×109 /L) was investigated in samples from 10,000 unselected neonates, resulting in 88 neutropenic newborns (NBs) and their 83 mothers. Genotyping was performed by PCR-SSP (HNA-1/-4) and PCR-RFLP (HNA-3/-5). Serologic studies were performed by GAT (granulocyte agglutination test), Flow-WIFT (white blood cells immunofluorescence test) and LABScreen-Multi-HNA-Kit (OneLambda®) (LSM). Neonatal neutropenia was identified in 88/10,000 (0·9%) NBs. Genotyping revealed 60·2% maternal-fetal HNA incompatibilities (31·8% for HNA-1; 14·8% for HNA-3; 15·9% for HNA-4; 21·6% for HNA-5). Serologic studies revealed 37·3% of mothers with positive results with at least one technique. The detected anti-HNA specificities were confirmed in eight positive cases related to HNA-1/-3 systems. In cases with maternal-fetal HNA-4/-5 incompatibility, no specific neutrophil alloantibodies were found but anti-HLA I/II were present. Anti-HNA-2 was not identified. This is a large Brazilian study which involved the investigation of antibodies against all five HNA systems in neutropenia cases and showed a frequency of NAIN in 8/10,000 neonates. Among the HNA antibodies identified, we highlight the anti-HNA-1d and anti-HNA-3b, antibodies unusual in alloimmunised women, and rarely related to NAIN cases.

Frequency and specificity of alloantibodies to HNA system antigens in donors of blood and blood components

Aim. To study the rate of human neutrophil antigens (HNA) alloimmunization in donors of blood and blood components.&#x0D; Methods. The study included blood samples of 1,127 donors who donated blood and its components in the Russian Scientific and Research Institute of Hematology and Transfusiology, Saint Petersburg, 635 male and 492 female. 36.18% of female donors were aged between 18 and 30 (n=178) and 63.82% were aged between 30 and 60 (n=314). The medical histories of all donors did not contain the records about the transfusion of blood components. Screening and identification of HNA alloantibodies were performed using the flow cytometric granulocyte immunofluorescence test with monoclonal antibodies CD16-PE, CD45-PC7, CD19-APC, Goat F(ab')2 Anti-Human IgG-FITC and the panel of donor neutrophils with detected HNA genotypes.&#x0D; Results. The frequency of HNA alloantibodies in blood donors was 0.35% (n=4). No HNA alloantibodies were found in the male donors. Alloantibodies were detected only in female blood donors aged between 30 and 60. HNA antibody frequency in female donors was 0.81%. It were detected anti-HNA-1a, anti-HNA-3b and anti-HNA-4b antibodies of the IgG immunoglobulin class. In one donor, the specificity of antibodies could not be established. To establish whether detected antibodies in immunized donors were alloantibodies, HNA genotypes were determined.&#x0D; Сonclusion. HNA alloantibodies occur with low frequency and are determined in 0.35% of blood donors. Alloantibodies were detected only in female donors and were directed against HNA-1a, HNA-3b and HNA-4b. The frequency of HNA antibodies in female donors was 0.81%. HNA genotyping confirmed that detected antibodies were alloantibodies.

Analysis of Transfusion-Related Acute Lung Injury and Possible Transfusion-Related Acute Lung Injury Reported to the French Hemovigilance Network From 2007 to 2013.

Using the French Hemovigilance Network database from 2007 to 2013, we provide information on demographics, incidence, and risk factors of reported transfusion-related acute lung injury (TRALI) and possible TRALI, analyze TRALI mitigation efforts for fresh frozen plasma and platelet concentrates, and consider the impact of platelet additive solutions on TRALI incidence. We applied the Toronto consensus conference definitions for TRALI and possible TRALI. Two TRALI subgroups were considered: "antibody positive" when a donor has human leukocyte antigen (class I or II) and/or human neutrophil antigen antibodies and the recipient has cognate antigen, and "antibody negative" when immunological investigation is negative or not done. The analysis targeted 378 cases, divided into antibody-positive TRALI (n=75), antibody-negative TRALI (n=100), and possible TRALI (n=203). TRALI patients were younger and received more blood components than the general population of transfused patients. Moreover, we identified the following clinical conditions where patients seemed to be at higher risk to develop TRALI: postpartum hemorrhage, acute myeloid leukemia, liver transplantation, allogeneic and autologous hematopoietic stem cells transplantation, polytrauma, and thrombotic microangiopathy. Policy measures intended to reduce antibody-positive TRALI were found effective for apheresis platelet concentrates and fresh frozen plasma but not for whole blood-derived platelet concentrates. The use of platelet additive solutions was associated with a significant reduction in the incidence of TRALI following transfusion of buffy coat-derived platelet concentrates but not following transfusion of apheresis platelets. Our data reinforce the concept that possible TRALI and TRALI, as defined in the Canadian consensus conference, share many characteristics. No specific policy measures are currently directed at mitigation of possible TRALI despite its impact on transfusion safety. Despite TRALI mitigation measures, the overall incidence of TRALI cases reported to the French Hemovigilance system was not significantly reduced. Therefore, additional research is needed to reduce, if not eradicate, all TRALI categories.

Interaction of leucocyte antibodies with endothelial cells in transfusion‐related acute lung injury

Transfusion‐related acute lung injury (TRALI) is a severe pulmonary reaction characterized by pulmonary oedema, breathing distress and consequently hypoxia temporally associated with a blood transfusion event. In antibody‐mediated TRALI, alloantibodies reactive with recipient's neutrophils are mainly considered as responsible for lung injury. These antibodies target either human leucocyte antigens (HLAs) class I or II or human neutrophil antigens (HNAs). Antibody interaction with the cognate antigen is believed to induce neutrophil activation and sequestration, which consequently leads to endothelial damage and lung injury. Analysing the effects of these antibodies on neutrophil‐depleted animals however has challenged this dogma. Previous studies have documented that among anti‐HNA antibodies, anti‐HNA‐3a are implicated in severe and fatal TRALI. Epitopes for anti‐HNA‐3 antibodies reside on choline transporter‐like protein 2 (CTL2) which has a broad expression pattern on different cells including neutrophils and endothelial cells. In vitro as well as in vivo research has identified the binding of anti‐HNA‐3a to endothelial cells as initiator of lung injury in anti‐HNA‐3a‐mediated TRALI. Studies have also demonstrated that binding of HLA antibodies to cognate antigens expressed on endothelial cells, by triggering multiple signalling pathways, influences endothelial functions. This presentation will summarize findings on the effects of HNA and HLA antibodies binding to endothelial cells and highlight the contribution of endothelial‐reactive antibodies in the mechanism of antibody‐mediated TRALI.

Clinical Features of Alloimmune Neutropenia: Composite of 2 Case Series and 39 Case Reports

▪Introduction: Alloimmune neutropenia (AIN) is a rare condition resulting from transplacental passage of maternal alloantibodies against paternal human neutrophil antigens (HNA). The resulting neutropenia may persist for months and predisposes neonates to bacterial and fungal infections, in particular sepsis. Despite the danger posed to neonates, little is known about the clinical features of AIN; no reviews incorporating more than 10 cases have comprehensive clinical information. The aim of this summary is to assess clinical data describing cases of AIN.Methods: A Pubmed search was performed for cases of AIN. In addition, a search was performed from the reference lists of all identified articles. Only English language articles describing serologically confirmed cases of AIN were included in our review. Analysis was primarily descriptive. By including case reports, a bias is incorporated to more severe and unusual cases but this was the only approach to obtaining information on AIN cases.Twenty-seven studies were identified - two case series[Tomicic Am J Reprod Immunol. 2014 (15 cases) and van den Tooren-de-Groot Acta Paediatr. 2014. (35 cases)] and 25 case reports that described 39 cases of AIN, a total of 89 cases (Table 1). The two case series relied on antibody testing to identify confirmed cases of AIN whereas the 25 articles described neutropenic neonates that were eventually diagnosed with AIN.Results: Analysis revealed that AIN was first suspected due to an infection or as a part of routine blood work completed for preterm infants. Neonates either presented with no infection (29%), omphalitis (25%) or other infections; sepsis was identified in 14% (only 1 death). The mean gestational age was 35.6-36 weeks and mean birth weight was 2500-2700 grams. The HNA antibodies most commonly identified in AIN were anti-HNA-1b (25%), anti-HNA-1a (16%), and anti-HNA-1c (16%) with anti-HNA-2 (18%) and anti-FcgammaRIII (11%) next. Differences in course between different anti-HNA antibodies were not identified. Antibiotics were most commonly used in treatment (>65%), most of the time (52%) on their own; G-CSF was used in 11% of cases. Neutropenia resolved within 6 months. In very few cases was information provided on subsequent pregnancies.Conclusions: Together, the studies provided information on why neonates were tested for AIN, HNA-antibody target, treatment of infections, weight and age of neonates and the average duration of neutropenia. In comparison to fetal and neonatal disorders hemolytic disease and alloimmune thrombocytopenia, there is still much to be learned about AIN. Analysis of the 89 cases suggests that AIN should be thought of earlier in infected neutropenic neonates, especially those with omphalitis and more consideration given to G-CSF and/or IVIG. The low incidence rate, self-limiting course, and current practice not to routinely obtain CBCs with differentials in neonates all contribute to the low rate of identifying AIN. Furthermore, serologic testing is not rapidly available in most clinical settings. To obtain a better understanding of the clinical features of AIN, a comprehensive prospective study evaluating full clinical data in real time of neonates serologically tested and found to have AIN should be conducted.Table 1Comparison of Cases of Alloimmune Neutropenia (AIN)van den Tooren-de Groot et al. 2014(n = 35)Tomicic et al. 2014 (n = 15)Case Reports(n = 39)Total(n = 89)Average Birth Weight (g)2776-2556.6Average Gestational Age (Weeks)35.6-36.0Average Absolute Neutrophil Count (/mm3)430631197HNA Antibodiesanti-HNA-1a71412 (16%)anti-HNA-1b12-719 (25%)anti-HNA-1c21912 (16%)anti-HNA-283314 (18%)anti-HNA-3a1--1 (1%)anti-HNA-5a1-23 (4%)anti-FcgammaRIIIb4239 (11%)anti-HLA-426 (8%)TreatmentNo Treatment-639 (12%)Antibiotics257739 (52%)Antibiotics and IVIG-1-1 (1%)Antibiotics and G-CSF-189 (12%)G-CSF1--1 (1%)Other--1616 (21%)Clinical DataNo Infection14-923 (29%)UTI1--1 (1%)Omphalitis67720 (25%)Candida Mucositis1--1 (1%)FUO6-17 (8%)Sepsis72514 (18%)Other--1212 (15%)Death0011 (1%)Average Time for Neutropenia to Resolve (Weeks)-7.79.8 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.

The fundamentals of neutrophil antigen and antibody investigations

Background The neutrophil population makes up the largest proportion (40–70%) of white blood cells and is crucial to our innate immune response. An unexpectedly significant proportion of neutrophils (∼28%) is located in the pulmonary circulation. This is an immune system front line wherein neutrophils guard an important interface between the external environment and the body. Neutrophils migrate to sites of infection and/or inflammation, where they phagocytose and destroy foreign microorganisms and mediate inflammation and eventual healing. Therefore, a decrease in neutrophil numbers often results in increased susceptibility to infection. Sustained or transient immune neutropenia occurs when neutrophil‐reactive alloantibodies or autoantibodies destroy neutrophils, or significantly compromise their function. Neutrophil‐reactive antibodies are capable of activating the neutrophil’s microbicidal arsenal with resultant damage to the surrounding tissue.Antigens and Antibodies There are currently eight well characterized human neutrophil antigens (HNA): HNA‐1a, HNA‐1b, HNA‐1c, HNA‐2a, HNA‐3a, HNA‐3b, HNA‐4a and HNA‐5a. Only HNA‐1a, HNA‐1b and HNA‐1c are specific to neutrophils, whilst the others are also expressed on other cell types. It is important to remember that neutrophils also express HLA Class I antigens, and that there is some evidence that adequately activated neutrophils may express HLA Class II antigens. This provides an interface between the innate and adaptive immune response. Therefore, both HNA and HLA Class I and II antibodies are capable of reacting with neutrophils in vivo and in vitro.Routine Antigen and Antibody Investigation Antigen genotyping protocols are now available for HNA‐1a, HNA‐1b, HNA‐1c, HNA‐3a, HNA‐3b, HNA‐4a and HNA‐5a. The serological examination of neutrophils is reliant on harvesting a representative, pure population of neutrophils, which is difficult because neutrophils are short lived (6–8 h) and fragile. The International Granulocyte Immunobiology Workshop recommends the use of both the granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT), complemented by the monoclonal antibody immobilization of granulocyte antigen (MAIGA) assay. The GIFT uses paraformaldehyde‐fixed neutrophils and detects the binding of antibodies to surface HNA and HLA antigens. Serological reaction in the GAT is reliant on viable neutrophils. It demonstrates the ability of antibodies to sensitize neutrophils and stimulate them to undergo chemotaxis towards other sensitized neutrophils, thus forming microscopic agglutinates. The MAIGA is useful in confirming antibody specificities as it employs monoclonal antibodies to known HNA epitopes. To screen for neutrophil‐reactive antibodies, serum or plasma is exposed to a screening panel of neutrophils in the GIFT and GAT. To differentiate between HNA and HLA antibodies, the simple strategy of using platelet pools to absorb out HLA antibodies can be used. Specificity is determined using the GIFT and GAT, and a phenotyped and genotyped neutrophil panel and confirmed with the MAIGA.Application The combination of the GIFT, GAT and MAIGA was originally developed for the reliable investigation of immune neutropenias. However, in the last decade these techniques have become fundamental to the investigation of immune mediated TRALI where neutrophils are a key target and effector cell.

TRALI – new challenges for histocompatibility and immunogenetics in transfusion medicine

Antibodies against human leukocyte antigens (HLAs) have long been associated with transfusion-related acute lung injury (TRALI). In contrast to febrile transfusion reactions and refractoriness to platelet transfusions in immunized patients, the causative antibodies in TRALI are present in the transfused blood component, i.e. they are formed by the blood donor and not by the recipient. Consequently, blood components with high plasma volume are particularly associated with TRALI. In addition to antibodies against HLAs, antibodies directed against human neutrophil antigens (HNAs) present in the plasma of predominantly multiparous female blood donors can induce severe TRALI reactions. Especially, antibodies to HLA class II and HNA-3a antigens can induce severe or even fatal ALI in critically ill patients. Over the last decade, the clinical importance of TRALI as major cause for severe transfusion-related morbidities has led to the establishment of new guidelines aimed at preventing this condition, including routine testing for HLA and -HNA antibodies for plasma donors with a history of allogeneic sensitization. This, in turn, poses new challenges for close collaboration between blood transfusion centers and histocompatibility and immunogenetics laboratories, for sensitive and specific detection of the relevant antibodies.

Molecular testing for platelet and granulocyte antigens

Human platelet antigen (HPA) genotyping is a valuable tool in confirming platelet antigen specificities of alloantibodies detected in patient sera, complementing the clinical history in the diagnosis of alloimmune platelet disorders such as neonatal alloimmune thrombocytopenia (NAIT), post‐transfusion purpura (PTP) and platelet transfusion refractoriness (PR). Likewise, human neutrophil antigen (HNA) typing also plays a critical role in the investigation and diagnosis of some clinical conditions induced by granulocyte alloantibodies, like neonatal alloimmune neutropenia (NAIN) and transfusion‐related acute lung injury (TRALI).Up to now, 27 HPA alloantigens have been described, including the 6 most clinically relevant biallelic systems and a significant number of low‐frequency or rare HPAs, which continues to increase with new reports. The genetic polymorphisms underlying these antigens have been characterized and in all cases but one, are single nucleotide polymorphisms (SNPs) in the coding sequence of the different membrane proteins where the HPA antigens are expressed. In contrast, the molecular basis of the HNA antigens are a little bit more heterogeneous. Within the 5 recognized HNA systems, the first alloantigens to be described, HNA‐1a and HNA‐1b differ in their coding sequence by 5 nucleotides, which result in 4 aminoacid changes in the protein sequence of the FcγRIIIb. Another alloantigen, the HNA‐2a, has been identified on the CD177 glycoprotein but cannot be genotyped, despite knowing the corresponding coding sequence. The HNA‐2a negative phenotype is due to splicing defects in the mRNA and no mutations have been detected in the gene sequence. The remaining HNA alloantigens result from single nucleotide changes, including the clinically relevant HNA‐3a, which has recently been characterized as the result of a single base change in the choline transporter‐like protein (CTL) 2 gene.Currently, genotyping methods are routinely applied to HPA and HNA testing in all laboratories performing platelet and granulocyte immunohematology studies. Many different techniques have been developed for typing these polymorphisms, including PCR with sequence‐specific primers, which is still the most widely used, 5′‐nuclease assays and high‐throughput SNP typing platforms. These latter ones are based on specific hybridization to oligonucleotide probes immobilized in a solid suport, usually a glass slide or beads. Among these, the newest platform developed for HPA typing, ID‐HPA, is based on the xMAP® technology and the beads are analyzed with the Luminex 100/200™ System, which is a robust and convenient system, already implemented in most HLA laboratories. Interestingly, the same technology can recently be used for the detection of HPA and HNA antibodies, which offers the possibility of multiple applications with a unique platform. These features may foster the introduction of such flexible assay systems in the near future. In any case, already at present, different high‐throughput genotyping platforms analyzing HPA polymorphisms are already being used for large‐scale blood or platelet apheresis donor typing, and further automation will probably lead to their final implementation in centralized National Blood Centers, facilitating the provision of antigen‐negative platelets for alloimmunized patients.Finally, it is worth to remark the novel strategies that have been recently developed for the non‐invasive fetal HPA‐1 typing from the maternal plasma DNA. These highly sensitive approaches may be applied in the future to other clinically significant HPA specificities, and will represent a major improvement in the management of pregnancies at risk of NAIT.

Is it time to reconsider neutrophil antibody testing of platelet donors?

Is it time to reconsider neutrophil antibody testing of platelet donors?

The frequency and specificity of human neutrophil antigen antibodies in a blood donor population

Transfusion-related acute lung injury (TRALI) has been associated with both human leukocyte antigen (HLA) and human neutrophil antigen (HNA) antibodies. HNA antibody frequency, specificity, and demographic associations have not been well defined in the blood donor population. A subset of 1171 donors (388 nontransfused males, 390 HLA antibody-negative females with three or more pregnancies, and 393 HLA antibody-positive females with three or more pregnancies) from a larger Leukocyte Antibody Prevalence Study was tested for immunoglobulin (Ig)G and IgM HNA antibody using a granulocyte immunofluorescence flow cytometry assay. Additional testing on selected samples included monoclonal antibody immobilization of granulocyte antigen-flow cytometry and granulocyte genotyping. Eight samples were HNA antibody positive (prevalence, 0.7%; 95% confidence interval [CI], 0.3%-1.3%]). Three HNA antibodies (one IgG and two IgM) were found in nontransfused males (prevalence, 0.8%; 95% CI, 0.2%-2.2%); all were panreactive or nonspecific. One HLA antibody-negative previously pregnant female had an IgG HNA antibody with HNA-1a specificity (prevalence, 0.3%; 95% CI, 0.01%-1.4%). Four HLA antibody-positive previously pregnant females demonstrated HNA antibodies, three IgG and one IgM (prevalence, 1%; 95% CI, 0.3%-2.6%). Two of these were HNA-1a specific, one HNA-4a specific, and one nonspecific. HNA antibodies occur with low frequency in the donor population and are present in both male and female donors. Despite the implementation of TRALI reduction strategies, HNA antibodies are still present in donor blood products. Although our data do not create a case for urgent implementation of donor HNA antibody testing, future new developments for high-throughput HNA antibody screening, including for HNA-3a, may warrant reconsideration.

Mechanism of Transfusion-Related Acute Lung Injury Induced by HLA Class II Antibodies.

Abstract 19 Introduction:Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-associated fatalities in developed countries. It has been demonstrated that antibodies to human neutrophil antigens (HNA) present in a blood product can precipitate a TRALI reaction in the blood recipient. In the majority of TRALI cases, however, HLA class II antibodies rather than HNA antibodies are detected in the implicated donor. HLA class II antigens are not present on neutrophils or endothelial cells, the cellular key players in TRALI pathophysiology. Whether or not HLA class II antibodies are capable of inducing TRALI is thus under discussion. Here, we investigated a possible mechanism of HLA class II antibody-induced TRALI via monocytes. Methods and Results: Plasma from blood donors containing HLA class II antibodies (H2+plasma) is capable of activating monocytes expressing cognate antigen(s) as demonstrated by the release of IL-8, GRO-alpha, and TNF-alpha in culture supernatants. H2+plasma does not activate monocytes that do not express the cognate HLA class II antigen(s). Culture supernatants from matched pairs, but not H2+plasma itself, induce a respiratory burst reaction in neutrophils. In Boyden chamber experiments, FITC-albumin transendothelial flux is significantly increased in the presence of H2+plasma, matched monocytes, and neutrophils, but unchanged in the presence of non-matched monocytes. Diphenyleneiodonium chloride inhibits this barrier breakdown. In the ex vivo rat lung model, supernatants from matched pairs, but not from non-matched pairs, are capable of inducing TRALI in the presence, but not in the absence, of neutrophils. H2+plasma precipitates a TRALI in this model if neutrophils and matched, but not non-matched, monocytes are present. IgG-depletion of H2+plasma abolishes this effect. Conclusions: HLA class II antibodies present in donor plasma can induce TRALI in a transfusion recipient via monocyte activation. Once activated, monocytes stimulate neutrophils to produce reactive oxygen species, which lead to an increase in endothelial permeability and subsequent lung edema. This cascade depends on a match between the donor's antibodies and the recipient's HLA class II antigens and can be reproduced in a rat lung model. Our findings have important impact on current strategies that aim to prevent TRALI by donor testing and/or exclusion. Disclosures:No relevant conflicts of interest to declare.

Index of Suspicion

Index of Suspicion

Neonatal Alloimmune Neutropenia Due to Anti-HNA-1b

Neonatal Alloimmune Neutropenia Due to Anti-HNA-1b