Human Neutrophil Antigens Research Articles

Development of a Multiplex PCR for Human Neutrophil Antigens-1, -3, -4, and -5 Genotyping Among Thai Blood Donors.

Different polymerase chain reaction (PCR) techniques for human neutrophil antigens (HNA) genotyping have been implemented to diagnose the clinical conditions of patients with alloimmune neutropenia, febrile non-hemolytic transfusion reactions, and transfusion-related acute lung injury and to provide effective HNAmatched granulocyte transfusions. The present study aimed to develop an in-house multiplex-PCR for HNA-1, -3, -4, and -5 genotyping in the Thai population. Altogether, 500 DNA samples obtained from unrelated, healthy Thai blood donors at the National Blood Centre, Thai Red Cross Society, Bangkok, Thailand were included. Three hundred DNA samples of known HNA genotyping based on PCR with sequence specific primers (PCR-SSP), as previously described, were tested with the newly developed multiplex PCR. Additionally, 200 DNA samples of unknown HNA genotyped donors were tested for HNA-1, -3, -4, and -5 genotyping using multiplex-PCR. Validity of HNA-1, -3, -4, and -5 genotyping by multiplex PCR using known DNA controls and the comparison of the genotyping results between PCR-SSP and multiplex PCR were in agreement. Interestingly, the rare genotype HNA-4b4b was not found in this study, similar to previous studies in Thai and other populations. Moreover, 30 samples were randomly tested twice for HNA genotyping using the multiplex-PCR and demonstrated reproducible results and were confirmed by DNA sequencing. This study shows that the newly developed multiplex-PCR is cost effective and less time consuming compared with PCR-SSP. The multiplex PCR can be used as an alternative technique for HNA-1, -3, -4, and -5 genotyping for routine testing, especially in other developing countries due to its simplicity and accuracy.

Re‐evaluation of HNA‐1 antibodies for their reactivity to different recombinant variants of FcγRIIIb

Background and ObjectivesThe Fcgamma receptor IIIb (FcγRIIIb) carries the human neutrophil antigen (HNA)‐1 system. The three FCGR3B alleles encode four antigens: one allele encodes HNA‐1a, and two alleles encode two antigens each, HNA‐1b and HNA‐1d and HNA‐1b and HNA‐1c, respectively. Several other non‐synonymous substitutions have also been reported. Little is known about the properties of HNA‐1d antibodies because HNA‐1d was recently discovered, and HNA‐1d antibodies have been reported only in a couple of cases. These findings raise the possibility that the polymorphisms in the HNA‐1 system and spectrum of the specificity of resulting antibodies are more complex than previously thought. We address this possibility in this work.Materials and MethodsSeveral HNA‐1 antibodies, which had been classified as HNA‐1a or HNA‐1b before HNA‐1d was discovered, were re‐evaluated for their reactivity to different recombinant variants of FcγRIIIb.ResultsThe HNA‐1a antibody, but not the reference HNA‐1a antibody, cross‐reacted to HNA‐1d. Further, nucleotide replacement in FCGR3B alleles *02 at 193G→A (AA position 54 E→K) resulted in the disappearance of HNA‐1d, but not HNA‐1b, antigenicity.ConclusionThe HNA‐1 system may be more complex than previously understood.

C-reactive protein enhances murine antibody–mediated transfusion-related acute lung injury

AbstractTransfusion-related acute lung injury (TRALI) is a syndrome of respiratory distress triggered by blood transfusions and is the leading cause of transfusion-related mortality. TRALI has primarily been attributed to passive infusion of HLA and/or human neutrophil antigen antibodies present in transfused blood products, and predisposing factors such as inflammation are known to be important for TRALI initiation. Because the acute-phase protein C-reactive protein (CRP) is highly upregulated during infections and inflammation and can also enhance antibody-mediated responses such as in vitro phagocytosis, respiratory burst, and in vivo thrombocytopenia, we investigated whether CRP affects murine antibody–mediated TRALI induced by the anti–major histocompatibility complex antibody 34-1-2s. We found that BALB/c mice injected with 34-1-2s or CRP alone were resistant to TRALI, however mice injected with 34-1-2s together with CRP had significantly enhanced lung damage and pulmonary edema. Mechanistically, 34-1-2s injection with CRP resulted in a significant synergistic increase in plasma levels of the neutrophil chemoattractant macrophage inflammatory protein-2 (MIP-2) and pulmonary neutrophil accumulation. Importantly, murine MIP-2 is the functional homolog of human interleukin-8, a known risk factor for human TRALI. These results suggest that elevated in vivo CRP levels, like those observed during infections, may significantly predispose recipients to antibody-mediated TRALI reactions and support the notion that modulating CRP levels is an effective therapeutic strategy to reduce TRALI severity.

C-Reactive Protein (CRP) Enhances Murine Antibody-Mediated Transfusion Related Acute Lung Injury (TRALI)

Transfusion-related acute lung injury (TRALI), a syndrome characterized by respiratory distress triggered by blood transfusions, is the leading cause of transfusion-related mortality. Mostly, TRALI has been attributed to passive infusion of human leucocyte antigen (HLA) and human neutrophil antigen (HNA) antibodies present in the transfused blood product. Several animal models have been developed to study the pathogenesis of antibody-mediated TRALI and various mechanisms for TRALI induction have been suggested, including involvement of endothelial cells, neutrophils and monocytes. In 2006, a murine of model of antibody-mediated TRALI was developed using a monoclonal MHC class I antibody (clone 34-1-2s). This antibody was shown to cause significant lung damage (excess lung water: pulmonary edema) within 2 hours of administration into BALB/c mice, which in follow-up studies was only reproducible after initial priming with the gram-negative endotoxin lipopolysaccharide (LPS). 34-1-2s was also shown to cause severe lung damage in severe combined immunodeficient (SCID) mice. We investigated 34-1-2s mediated TRALI in BALB/c mice, without LPS priming, and found no difference in TRALI severity when compared with injection with an control isotype antibody for 34-1-2s (Isotype Mouse IgG2a antibody), as examined by lung wet-to-dry ratios, a measure for pulmonary edema. Recently it was described that the acute phase protein C-reactive protein (CRP), heavily up-regulated during acute infections and also present at lower levels in healthy individuals, was able to enhance antibody-mediated platelet destruction both in vitro and in vivo via Fc-receptor mediated phagocytic responses. Considering the fact that TRALI has been shown to be mainly antibody-mediated, plus the fact that it has been suggested to be an Fc-dependent process as well, we investigated the effect of CRP in a murine antibody-mediated TRALI. We tested if CRP would be able to enhance antibody-mediated TRALI in the murine 34-1-2s based BALB/c TRALI model. For that purpose, we co-injected CRP together with 34-1-2s and compared that to co-injection of CRP together with control isotype mouse IgG2a or to injection with CRP alone. We found that CRP+34-1-2s injection resulted in significantly higher lung damage than CRP+isotype antibody, as well as than CRP alone, with at least 43% of the mice in the CRP+34-1-2s group having a lung wet-to-dry ratio of higher than 5, which is considered to represent severe lung damage. As the monocyte-derived neutrophil chemoattractant macrophage inflammatory protein 2 (MIP-2: murine equivalent of human IL-8) was recently shown to play a central role in murine (SCID) 34-1-2s-mediated TRALI induction, we measured MIP-2 values in our BALB/c TRALI model and found that CRP alone was capable of producing high levels of MIP-2, which were found to be even more increased when 34-1-2s was co-injected with CRP. We propose a mechanism in which CRP plays a synergistic role with 34-1-2s antibody to significantly increase the induction of antibody-mediated TRALI via enhanced stimulation of monocyte-derived MIP-2 secretion. DisclosuresNo relevant conflicts of interest to declare.

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.

Genotype frequency of human neutrophil antigen-3 polymorphisms in the Yi, Han, and Tibetan populations of China.

Human neutrophil antigen-3 (HNA-3) alloantibodies can cause fatal transfusion-related acute lung injury (TRALI). Most frequencies of SLC44A2 alleles encoding the HNA-3a/b antigens have been established in Han individuals by polymerase chain reaction with sequence-specific priming (PCR-SSP). We sequenced SLC44A2 gene fragments and determined allele frequencies in three ethnicities of China. Genomic DNA was extracted from 448 samples of 100 blood donors of Yi ethnicity in Xichang, Liangshan; 248 Han in Nanjing, Jiangsu; and 100 Tibetan in Lhasa, Tibet. A PCR-SSP was applied to determine the phase of two single-nucleotide polymorphisms (SNPs); SLC44A2 haplotypes were constructed. In the 567 nucleotides of the SLC44A2 gene covered by our sequencing approach in Han individuals, we detected the known 331-44G>A (rs12972963) and 461G>A (rs2288904) polymorphisms. In the 243 nucleotides sequenced in Yi and Tibetan populations, we detected the known 461G>A and 503-15T>C (rs1560711) polymorphisms. A PCR-SSP for the common HNA-3a/b SNP was 100% concordant. The frequencies of the HNA-3a allele were 0.58, 0.66, and 0.69 in Yi, Han (Nanjing), and Tibetan, respectively (0.42, 0.34, and 0.31 for HNA-3b). The Yi population of China had the highest frequency of blood donors at risk of harboring anti-HNA-3a compared to any population studied so far. We confirmed that the underlying SLC44A2*2 allele is more common in China than in any European or African populations.

Full-length nucleotide sequences of 30 common SLC44A2 alleles encoding human neutrophil antigen-3.

Human neutrophil antigen-3a (HNA-3a) alloantibodies can cause severe transfusion-related acute lung injury. The frequencies of the single-nucleotide polymorphisms (SNPs) indicative of the two clinically relevant HNA-3a/b antigens are known in many populations. In this study, we determined the full-length nucleotide sequence of common SLC44A2 alleles encoding the choline transporter-like protein-2 that harbors HNA-3a/b antigens. A method was devised to determine the full-length coding sequence (CDS) and adjacent intron sequences from genomic DNA by eight polymerase chain reaction amplifications covering all 22 SLC44A2 exons. Samples from 200 African American, 96 Caucasian, two Hispanic, and four Asian blood donors were analyzed. We developed a decision tree to determine alleles (confirmed haplotypes) from the genotype data. A total of 10 SNPs were detected in the SLC44A2 CDS. The noncoding sequences harbored an additional 28 SNPs (one in the 5'-untranslated region [UTR]; 23 in the introns; and four in the 3'-UTR). No SNP indicative of a nonfunctional allele was detected. The nucleotide sequences for 30 SLC44A2 alleles (haplotypes) were confirmed. There may be 66 haplotypes among the 604 chromosomes screened. We found 38 SNPs, including one novel SNP, in 8192 nucleotides covering the CDS of the SLC44A2 gene among 302 blood donors. Population frequencies of these SNPs were established for African Americans and Caucasians. Because alleles encoding HNA-3b are more common than non-functional SLC44A2 alleles, we confirmed our previous postulate that African American donors are less likely to form HNA-3a antibodies compared to Caucasians.

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.

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.

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.

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.

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.

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.

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.

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.