The frequencies of human neutrophil alloantigens in the Chinese Han population of Guangzhou

BACKGROUND: Antibodies against polymorphic structures on human neutrophil antigens (HNAs) play a role in alloimmune‐mediated neutropenia and are the leading cause of antibody‐mediated transfusion‐related acute lung injury (TRALI). This study aimed to determine the frequencies of HNAs in the major Han ethnic group living in Guangdong Province, Southern China. STUDY DESIGN AND METHODS: A total of 493 healthy Chinese Han blood donors from Guangzhou were recruited. DNA samples were isolated and typed for all five HNA‐1, ‐2, ‐3, ‐4, and ‐5 systems using allele‐specific polymerase chain reaction approaches. Results were compared with available data from other Chinese cohorts and other Asian and Caucasian populations. RESULTS: In this cohort, the gene frequency for HNA‐1a (0.667) was approximately twice that of HNA‐1b (0.333). In contrast to Caucasian populations, HNA‐1a represents the most frequent allele in the Chinese population. HNA‐3 system genotyping revealed comparable frequencies for HNA‐3a (0.738) and ‐3b (0.262) in Chinese and Caucasian populations. Homozygous HNA‐3bb individuals were found in 5.64% of our cohort. HNA‐4 genotyping revealed no HNA‐4bb homozygous individuals. In contrast, HNA‐5bb homozygous individuals represented 2.43% of the population. Typing the HNA‐2 system for the single‐nucleotide polymorphism C42G showed that the C‐allele (69%) is overrepresented and is associated with an increased number of HNA‐2a–positive neutrophil subpopulations. CONCLUSION: This study describes for the first time the frequencies of all HNA systems, including the newly identified HNA‐3, within one cohort of Chinese Han population. Comparison with Caucasian populations may allow assessment of anti‐HNA alloimmunization and estimation of alloimmune neutropenia and TRALI incidence in Chinese populations.     

Genetic variation of the HNA-3a encoding gene

BACKGROUND: Antibodies against the human neutrophil alloantigen‐3a (HNA‐3a) play an important role in transfusion‐related acute lung injury. The HNA‐3a and ‐3b alloantigens result from a single‐nucleotide exchange in the choline transporter‐like protein 2 gene (CTL2). We sought for additional polymorphisms that might impair antibody binding to or genotyping of the HNA‐3a or ‐3b antigens. STUDY DESIGN AND METHODS: CTL2‐specific complementary DNA (cDNA) fragments were generated from 67 unrelated blood donors followed by DNA sequencing. Polymerase chain reaction with sequence‐specific primers (PCR‐SSP) was used to test a higher number of donors for relevant new single‐nucleotide polymorphisms (SNPs). The granulocyte agglutination test recommended for HNA‐3a antibody detection was performed to check HNA‐3a antibody binding to the products of the CTL‐2 gene variants. RESULTS: Two new missense mutations were demonstrated in the CTL2 cDNA: a 537C>T * exchange leading to a Leu153Phe amino acid substitution and 988C>T variation predicting Thr301Met change. The inherited 537T variant is located in HNA‐3a allele results impaired granulocyte agglutination by four of 14 antibodies tested while 988T remains nearly unaffected. CONCLUSIONS: The Leu153Phe exchange next to the HNA‐3a/b defining amino acid position can impede the binding of HNA‐3a alloantibodies. The HNA‐3a genotyping by PCR‐SSP might produce misleading results in HNA‐3ab heterozygous individuals with the additional CTL2‐537T variation of the HNA‐3a antigen. These findings must account for the development of new screening assays.     

Rapid enzyme‐linked immunosorbent assay for the detection of antibodies against human neutrophil antigens ‐1a, ‐1b,and ‐1c

BACKGROUND: Several methods exist for the detection of neutrophil antibodies; most of them, however, require fresh neutrophils. In this study, an enzyme‐linked immunosorbent assay (ELISA) using recombinant HNA‐1 antigens (rHNAs) was developed to detect HNA‐1a, ‐1b, and ‐1c alloantibodies in serum samples. STUDY DESIGN AND METHODS: Soluble rHNA‐1a, ‐1b, and ‐1c were isolated from culture supernatant of transfected insect cells. Purified rHNA antigens were immobilized on microtiter wells using antibody against V5‐Tag protein. Sera were added, and bound antibodies were detected by enzyme‐labeled secondary antibodies. In parallel, monoclonal antibody–immobilized granulocyte antigen (MAIGA) was performed with two different monoclonal antibodies (MoAbs) against FcγRIIIb (3G8 and BW209). RESULTS: Fifteen MAIGA‐positive sera containing HNA‐1a alloantibodies were tested in ELISA. Thirteen of 15 (86.7%) MAIGA‐positive sera captured by MoAbs 3G8 and/or BW209 reacted specifically with rHNA‐1a. Four (26.7%) HNA‐1a sera showed additional reaction with rHNA‐1c. When anti‐HNA‐1b alloantibodies were analyzed in ELISA, 13 of 15 (86.7%) showed specific positive reaction with rHNA‐1b, and 12 of 15 (80.0%) cross‐reacted with rHNA‐1c. Two HNA‐1c sera reacted specifically with rHNA‐1c. Immunoprecipitation analysis of all ELISA‐negative HNA‐1a and ‐1b sera did not show any specific band indicating false‐positive reaction of these sera in MAIGA assay. CONCLUSIONS: These results suggested that rapid ELISA using recombinant neutrophil antigens may provide a valuable method for rapid screening of human alloantibodies against HNA‐1a, ‐1b, and ‐1c in patients with neutropenia and in blood donors.     

Determination of neutrophil antigen HNA‐3a and HNA‐3b genotype frequencies in six racial groups by high‐throughput 5′ exonuclease assay

BACKGROUND: People with the human neutrophil antigen (HNA)‐3b/3b type can make HNA‐3a antibodies, which have been reported to cause immune neutropenia disorders and are especially prone to cause severe cases of transfusion‐related acute lung injury. However, knowledge of HNA‐3 allele frequencies outside Caucasian populations is limited. We developed a high‐throughput genotyping assay and determined the HNA‐3a/3b genotype frequencies in six different racial and ethnic groups. STUDY DESIGN AND METHODS: Genotyping utilized TaqMan 5′ exonuclease chemistry and real‐time polymerase chain reaction. A total of 742 DNA samples from six different racial and ethnic groups were genotyped for HNA‐3a and HNA‐3b. RESULTS: The genotyping assay showed 100% sensitivity and specificity compared to sequencing and phenotyping and had high throughput. A significant percentage of Caucasians (6.5%), Han Chinese (16%), and Asian Indians (6%) typed HNA‐3b/3b, but only a small percentage of Hispanics (1%) and no African or Native Americans. CONCLUSIONS: The HNA‐3 genotyping assay had high sensitivity, specificity, and sample throughput. HNA‐3b/b genotype results determined for 742 individuals representing six different racial and ethnic groups showed that there could be a significant risk of producing anti‐HNA‐3a in Chinese, as well as in Caucasian and Asian Indian blood donor populations, but a very low risk in Hispanic, African, or Native American populations.     

Mass-scale high-throughput multiplex polymerase chain reaction for human platelet antigen single-nucleotide polymorphisms screening of apheresis platelet donors

BACKGROUND: Treatment with human platelet antigen (HPA)‐matched platelets (PLTs) is the optimal therapy for bleeding secondary to neonatal alloimmune thrombocytopenia. Recent advances in high‐throughput DNA‐based blood group and PLT antigen genotyping have made it possible to screen plateletpheresis donors for potential HPA‐matched PLT transfusion. STUDY DESIGN AND METHODS: This prospective study evaluated genomic DNA from plateletpheresis donors for single‐nucleotide polymorphisms (SNPs) associated with HPA‐1, ‐2, ‐3, ‐4, ‐5, and ‐15 to determine whether high‐throughput multiplex genomic DNA PCR and oligonucleotide extension technology can be used for mass‐scale PLT antigen genotyping. Genotyping using SNP technology was confirmed using sequence‐specific polymerase chain reaction (SSP‐PCR). RESULTS: Of the 748 donors screened, 277 were found to be negative for antigens implicated in alloimmune thrombocytopenia. In addition, two donors were homozygous for HPA‐1b/b and ‐2b/b, six donors for HPA‐1b/b and ‐3b/b, one for HPA‐2b/b and ‐3b/b, one for HPA‐1b/b and ‐5b/b, 10 for HPA‐1b/b and ‐15 b/b, four for HPA‐5b/b and ‐15b/b, and one for HPA‐2b/b and ‐15b/b. Retesting using SSP‐PCR was conducted for 60 donors. Discrepant results occurred between SNP and SSP‐PCR in less than 20% of samples for HPA‐1b/1b/HPA‐3b/3b, HPA‐5b/5b, and HPA‐15b/b. DISCUSSION: High‐throughput multiplex PCR SNP and confirmatory molecular genotyping are useful for mass‐scale screening of apheresis PLT donors to provide antigen‐negative genotypes. Refinements to mass‐scale multiplex analysis technology would reduce further the confirmatory testing needed.     

Granulocyte antibody screening: evaluation of a bead‐based assay in comparison with classical methods

BACKGROUND: Granulocyte antibodies have been implicated in allo‐ and autoimmune neutropenia and in transfusion reactions. STUDY DESIGN AND METHODS: Fifty‐one sera from suspected alloimmune neutropenia or transfusion‐related acute lung injury (TRALI) and 40 sera from suspected autoimmune neutropenia were tested for granulocyte antibodies using LABScreen MULTI (One Lambda, Inc.), compared with classical tests (flow cytometry [FC] and granulocyte agglutination [GAT] followed by monoclonal antibody–specific immobilization of granulocyte antigens [MAIGA]). RESULTS: In alloimmune situations, 48 sera were concordant (94%), two sera positive for HNA with LABScreen MULTI were negative by FC/GAT and/or MAIGA, and one serum sample negative for HNA with LABScreen MULTI was positive by classical tests. In autoimmune neutropenia, 30 sera were concordant (75%), four sera positive for HNA with LABScreen MULTI were negative by FC/GAT and/or MAIGA, and six sera negative for HNA with LABScreen MULTI were positive by FC/GAT and/or MAIGA. For detection of autoantibodies, the LABScreen MULTI was less concordant. However, with the exception of one case, the discrepancies were observed in sera that did not show a clear specificity. CONCLUSIONS: LABScreen MULTI correlated well with our classical methods for HNA‐1 and HNA‐2a antibody screening. It can be used for screening blood donors or patients suspected of TRALI, but GAT is still needed for HNA‐3a antibody screening.     

The allele frequencies of HPA 1‐16 determined by PCR‐SSP in Chinese Cantonese donors

Background: Typing of human platelet antigens (HPA) has proven to be useful in some clinical situations related to platelet alloimmunization. Objective: The objective of this study was to investigate HPA 1–16 and to determine genotype and allele frequencies by polymerase chain reaction‐sequence specific primer (PCR‐SSP) in apheresis platelet donors in Guangzhou Blood Center. Methods: A total of 200 random samples from donors were involved in the study. Genotype and allele frequencies of HPA 1 to 16 were detected by PCR‐SSP method. Results: The frequencies obtained from these donors were 99·50 and 0·50% for HPA‐1a and ‐1b; 96·25 and 3·75% for HPA‐2a and ‐2b; 54·25 and 45·75% for HPA‐3a and ‐3b; 99·50 and 0·50% for HPA‐4a and ‐4b; 99·00 and 1·00% for HPA‐5a and ‐5b; 97·00 and 3·00% for HPA‐6a and ‐6b and 42·25 and 57·75% for HPA‐15a and ‐15b. There is only a/a homozygosis detected in HPA‐7, ‐8, ‐9, ‐10, ‐11, ‐12, ‐13, ‐14 and ‐16. In this study, none of HPA‐1b/ 1b, ‐2b/2b, ‐5b/5b homozygosis were detected which were found in other racial groups. One homozygosis of HPA‐6b/6b in 200 individuals was detected which was not found in a study involving 1000 Chinese ( Feng et al., 2006 ). Conclusion: The HPA‐3 and ‐15 appear to the highest priority and HPA‐2, ‐6, ‐5 ‐1 and ‐4 to be the second priority in Chinese Cantonese when it comes to the diagnosis of neonatal alloimmune thrombocytopenia and to provide the HPA‐matched platelet for patients with platelet transfusion refractoriness. The PCR‐SSP method makes it possible to detect genotype of HPA‐1 to ‐16 in less than 4 h and to establish a donor database for HPA genotype in a blood bank.     

Neonatal alloimmune neutropenia due to immunoglobulin G antibodies against human neutrophil antigen‐5a

BACKGROUND: Neonatal alloimmune‐mediated neutropenia (NAIN) due to maternal alloantibodies directed against one of the human neutrophil antigens (HNAs) can cause severe infections. NAIN has been described as caused by antibodies against HNA‐1a, ‐b, ‐c, ‐2a, ‐3a, or ‐4a, but not by antibodies against HNA‐5a. RESULTS: Blood from a 3‐week‐old newborn and from his parents was sent to our laboratory because of suspicion of NAIN. Granulocyte‐specific antibodies were present in the maternal antiserum and reactive with the paternal granulocytes. The specificity of the maternal alloantibodies was shown to be anti‐HNA‐5a by the monoclonal antibody immobilization of granulocyte antigens assay. The mother was genotyped HNA‐5a negative, and the father was genotyped homozygous HNA‐5a positive. CONCLUSION: We identified a first case of NAIN due to maternal alloantibodies against HNA‐5a.     

Implication of transfected cell lines for the detection of alloantibodies against human neutrophil antigen-3

BACKGROUND: Alloantibodies against human neutrophil antigen‐3 (HNA‐3) are responsible for the fatalities reported in transfusion‐related acute lung injury. Consequently, reliable detection of these alloantibodies is mandatory to improve blood transfusion safety. In this study, we developed stable cell lines for the detection of HNA‐3 antibodies. STUDY DESIGN AND METHODS: HEK293T were transfected with HNA‐3a or HNA‐3b constructs and sorted by flow cytometry according to high surface expression. Transfected cells were tested with sera containing HNA‐3 antibodies in flow cytometry and antibody capture assay (ACA). The results were compared with granulocyte agglutination test and granulocyte immunofluorescence test. RESULTS: In flow cytometry, 12 of 14 HNA‐3a sera reacted specifically with HNA‐3aa cells. One serum sample showed positive reaction with HNA‐3bb cells. All HNA‐3b sera recognized HNA‐3bb cells. No reaction was observed with broad reactive antibodies against HLA Class I. In ACA, all HNA‐3a sera (12/12) showed positive reactivity with HNA‐3aa cells with no cross‐reactivity with HNA‐3bb cells. Again, all HNA‐3b sera reacted with HNA‐3bb cells only. Furthermore, genotyping of 249 individuals detected a new HNA‐3 allele caused by a nucleotide substitution C>T at Position 457 leading to L₁₅₃F mutation in choline transporter‐like protein‐2. This mutation impairs polymerase chain reaction with sequence‐specific primers based HNA‐3a typing. However, analysis with cells expressing F₁₅₃ isoform showed that this mutation did not alter the binding of HNA‐3 antibodies. CONCLUSIONS: This study demonstrated that HEK293T cells expressing stable recombinant HNA‐3 are suitable for the detection of HNA‐3 alloantibodies allowing reliable screening of blood products.     

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

BACKGROUND: 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. STUDY DESIGN AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

Detection of HNA-3a and -3b antibodies using transfected cell lines and recombinant proteins

BACKGROUND: HNA‐3 is a diallellic system located on choline transporter‐like protein 2 (CTL2), defined by a polymorphism at Amino Acid 154. HNA‐3a antibodies are of clinical importance in transfusion‐related acute lung injury but antibody detection requires labor‐intensive granulocyte isolation from HNA‐typed donors and the use of techniques such as the granulocyte agglutination test or granulocyte immunofluorescence test. Also, there is no commercial test for detection of HNA‐3 antibodies. STUDY DESIGN AND METHODS: HEK293 cells were transfected to generate stable cell lines expressing CTL2 fragments (Amino Acids 55‐230) and full‐length membrane bound CTL2 with HNA‐3a and ‐3b epitopes. Soluble fragments were used in enzyme‐linked immunosorbent assays to detect HNA‐3 antibodies. The cell lines expressing full‐length proteins were trypsin treated to remove HLA antigens and frozen at ?80°C. Thawed cells were then used to detect HNA‐3 antibodies by flow cytometry. RESULTS: Glycosylated and soluble CTL2 fragments were correctly recognized by 15 of 31 anti‐HNA‐3a sera and by both available anti‐HNA‐3b sera. Twenty‐one anti‐HLA sera reacted variably with untreated cell lines expressing full‐length CTL2. After trypsin treatment of the cell lines, reactivity with HLA antisera was abrogated and all 31 anti‐HNA‐3a and two anti‐HNA‐3b sera bound to the corresponding cell line. CONCLUSION: Whereas soluble, glycosylated CTL2 fragments cannot be used for the detection of HNA‐3 antibodies, the HEK293 cells expressing full‐length CTL2 proteins were useful in the detection of HNA‐3 antibodies even in the presence of HLA antibodies. Moreover, the cell lines can be stored for at least 6 months before use.     

HNA-3a-specific antibodies recognize choline transporter-like protein-2 peptides containing arginine, but not glutamine at Position 154

BACKGROUND: Antibodies specific for the neutrophil antigen HNA‐3a cause severe, sometimes fatal transfusion‐related acute lung disease (TRALI) when transfused, but it has not been possible to screen blood donors for anti‐HNA‐3a because using neutrophils as targets was impractical and molecular properties of the antigen were unknown. Recently it was shown that HNA‐3a is carried on choline transporter–like protein‐2 (CTL2) and that the HNA‐3a/b phenotype is closely correlated with an R154Q amino acid polymorphism in CTL2. However, it has not been shown by direct experiment that R154 is essential for the HNA‐3a epitope. STUDY DESIGN AND METHODS: Preliminary attempts to express recombinant full‐length CTL2 (R154) recognized by anti‐HNA‐3a were unsuccessful. We therefore tested HNA‐3a–specific antibodies from donors implicated in TRALI reactions for reactivity against chemically synthesized linear and cyclic CTL2 peptides containing R154 or Q154. RESULTS: Nine of 20 HNA‐3a antibodies recognized the R154, but not the Q154 version of a cyclic 36‐residue CTL2 peptide (D131‐K166). However, 11 others failed to distinguish between the two versions of this peptide. CONCLUSION: The findings provide direct evidence that R154 in the context of CTL2 D131‐K166 is necessary to create the HNA‐3a epitope but, in the context of cyclic CTL2 peptide D131‐K166, is sufficient to detect only about one‐half of the HNA‐3a–specific antibodies implicated in TRALI. It is likely that fragments of CTL2 longer than can be made on a large scale with an automated synthesizer will be needed to produce a target capable of detecting all examples of anti‐HNA‐3a in donated blood.     

Transfusion‐related alloimmune neutropenia with no pulmonary complications: one donor—five cases

BACKGROUND: Neutrophil alloantibodies are well‐known triggers of transfusion‐related acute lung injury (TRALI) and also cause immune neutropenia. Alloimmune neutropenia due to transfusion is an isolated phenomenon that is only rarely identified. Its incidence is specified in the literature as being less than one in 10,000 transfused plasma‐containing units. We expect that this phenomenon is underreported. STUDY DESIGN AND METHODS: We observed five cases of alloimmune neutropenia with no respiratory complications with only one case initially reported as a suspected transfusion reaction. The other four cases were detected in the course of the subsequent lookback investigation. RESULTS: The first case was reported as a potential transfusion reaction when a female patient showed a decrease in the white blood cell count after a platelet (PLT) transfusion. Examinations of the donor blood revealed an antibody against the human neutrophil antigen HNA‐1b; the recipient was typed HNA‐1b positive and HNA‐1a negative. After examining the blood counts of other patients who previously received PLT concentrates from the same donor, we identified four other patients with an unreported decrease in the leukocyte and/or granulocyte count of more than approximately 50% after transfusion. CONCLUSION: HNA antibodies are generally regarded as potential triggers of TRALI. Here we describe an HNA antibody that reproducibly caused transfusion‐related neutropenia only without pulmonary complications. Factors predisposing patients to TRALI development are widely discussed. Our case suggests that antibody characteristics are also relevant in the development of TRALI. Current measures to prevent TRALI should also prevent transfusion‐related alloimmune neutropenia.     

Genotyping of HPA-1 to -7 and -15 in the Thai population using multiplex PCR

Background : Different polymerase chain reaction (PCR) techniques for human platelet antigens (HPA) genotyping have been implemented, in order to diagnose the clinical syndromes of patients with thrombocytopaenia and provide effective HPA‐matched platelet donors. Objectives : The aim of this study is to develop an in‐house multiplex PCR for HPA‐1 to ‐7 and ‐15 genotyping in the Thai population. Methods : One hundred DNA samples of known HPA genotyping by the PCR with sequence‐specific primers (PCR‐SSP), as previously described, were tested with the multiplex PCR. Additionally, 300 DNA samples of group O donors were tested for HPA‐1 to ‐7 and ‐15 genotyping using multiplex PCR. Results : The comparison of HPA‐1 to ‐7 and ‐15 genotype results between multiplex PCR and PCR‐SSP technique was in 100% concordance. Interestingly, HPA‐2b2b genotype was found in two samples; however, other low‐incidence genotypes such as HPA‐1b1b, HPA‐5b5b, HPA‐6b6b and HPA‐7b7b were not found in this study. Moreover, 30 samples were randomly tested twice for HPA genotyping using the multiplex PCR and demonstrated reproducible results. Conclusions : This study shows that the in‐house multiplex PCR is simple, cost‐effective and suitable for HPA genotyping for routine laboratories in other developing countries. Nevertheless, a large‐scale evaluation of this technique through multicentre analysis is suggested.     

IMMUNOHEMATOLOGY: Rapid screening of granulocyte antibodies with a novel assay: flow cytometric granulocyte immunofluorescence test

BACKGROUND: White blood cell (WBC)‐associated antibodies can lead to severe pulmonary transfusion reactions (transfusion‐related acute lung injury [TRALI]). Investigation of a large number of blood donor samples using the standard granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT) proved to be difficult to perform due to the time‐consuming process and the large quantity of test cells required. This study describes the novel flow cytometric GIFT (Flow‐GIFT) method for a rapid detection of granulocyte antibodies by flow cytometric analysis. STUDY DESIGN AND METHODS: A total of 141 sera were analyzed for the presence of granulocyte antibodies that were previously associated with suspected TRALI. As test cells whole blood samples from human neutrophil antigen (HNA)‐typed donors were isolated using cell sedimentation in a ficoll density gradient. WBCs were incubated with the respective serum and binding of antibodies to the test cells was detected using fluorescein isothiocyanate–conjugated anti‐human antibody. Standard GIFT and GAT were performed as reference methods. RESULTS: Seven sera containing anti‐HNA‐3a, CD16, and HLA Class I were negative in the standard GIFT and eight sera containing anti‐HNA‐2a, anti‐CD16, and anti‐HLA Class I were not detected in the GAT. The novel Flow‐GIFT was able to detect all granulocyte antibodies, which were only detectable in a combination of standard GIFT and GAT. In serial dilution tests, the Flow‐GIFT detected the antibodies at higher dilutions than the reference methods GIFT and GAT. CONCLUSION: The Flow‐GIFT method permits rapid detection of granulocyte antibodies requiring fewer donor test cells. This method is ideal for automation and will potentially open the way for screening of granulocyte antibodies in a large donor population.     

The risk of antibody formation against HNA1a and HNA1b granulocyte antigens during pregnancy and its relation to neonatal neutropenia

The evaluation of immunization by the HNA1a and 1b antigens during pregnancy was based on (i) their genotyping in 1038 unselected mothers and newborns of homozygous mothers, (ii) granulocyte counting in all born infants and (iii) examination of granulocyte antibodies in maternal sera if an HNA1 incompatibile child was born. A total of 548 (52.8%) mothers were heterozygous--thus further examinations were not done. Four hundred and ninety (47.2%) were homozygous, of whom 203 (41.3%) delivered an incompatible child, i.e. 19.6% of all the infants. Among available sera from 195 mothers with feto-maternal incompatibility, the granulocyte-specific antibodies were found in nine (4.5%); six of these (3%) were HNA1 (four anti-1a, two anti-1b), and in three others the specificity was not determined. In the remaining 28 sera, the only antibodies detected were HLA. Hence, six out of 1000 pregnant women can be expected to develop anti-HNA1. In none of the newborns was the cord neutrophil count below 1.5 x 109 L-1 and signs of infection found, thus the incidence of NAIN seems to be lower than 1 per 1000 infants. A comparison with our previous, unpublished data suggests that the incidence of severe NAIN is roughly 1 per 6000 (four cases among 24101 newborns).     

Full-length recombinant choline transporter-like protein 2 containing arginine 154 reconstitutes the epitope recognized by HNA-3a antibodies

BACKGROUND: Recent reports have shown that the HNA‐3a leukocyte antigen, a target for antibodies that cause severe transfusion‐related acute lung injury, correlates with an arginine 154 (rather than glutamine) polymorphism in choline transporter–like protein 2 (CTL2) but did not show directly that R154 determines HNA‐3a. CTL2 peptides containing R154 are recognized by only half of HNA‐3a antibodies studied to date. Constructs that react with all HNA‐3a antibodies are needed to fully define the HNA‐3a epitope. STUDY DESIGN AND METHODS: HEK293 cells were transfected with cDNA encoding full‐length CTL2 linked to green fluorescent protein (GFP). Transfectants were selected for GFP expression and tested with antibodies specific for HNA‐3a and ‐3b. RESULTS: Each of 20 HNA‐3a antibodies reacted preferentially with HEK293 cells expressing the R154 CTL2 construct. An HNA‐3b antibody reacted only with CTL2 (Q154). CONCLUSIONS: These findings provide direct evidence that R154 in the context of full‐length CTL2 is both necessary and sufficient to create the HNA‐3a epitope but suggest that posttranslational modifications of the protein, for example, S‐S bonds or addition of glycans, are necessary for recognition of HNA‐3a by many antibodies. This could complicate development of an assay for large‐scale screening of blood donors to detect anti‐HNA‐3a.     

Screening of multiparous women to avoid transfusion‐related acute lung injury: a single centre experience

summary The aim of this study was to investigate which approach for serological testing of multiparous donors might be feasible and effective to reduce the risk of transfusion‐related acute lung injury (TRALI). TRALI is a serious adverse event of blood transfusion. Antibodies to granulocytes and human leucocyte antigens (HLAs) are frequently detected in sera of implicated donors. These donors are often multiparous women. A general deferral of female plasma or screening strategies for leucocyte antibodies has been proposed to increase blood safety. A prospective study was initiated in 2003. Until 2006, serum samples from all female donors reporting three or more pregnancies (n = 229) were screened for the presence of antibodies against granulocytes and HLAs by immunofluorescence and agglutination tests as well as by a commercial HLA enzyme immunoassay. In total, 40% of all multiparous women were reactive in one of the assays. Twenty‐nine percent of the reactive sera contained antibodies to granulocytes but not to HLAs. During the observation period, three TRALI reactions occurred in our hospital, two of which would have been prevented if the screening program had been extended to all previously pregnant donors. We conclude from these data that, not unexpectedly, the number of previous pregnancies is not a reliable indicator for the likelihood of inducing TRALI. More importantly, screening strategies for antibodies that might induce TRALI should probably not be reduced to HLA antibody screening. This finding awaits further research.     

Frequencies of SLC44A2 alleles encoding human neutrophil antigen-3 variants in the African American population

BACKGROUND: The human neutrophil antigen‐3 (HNA‐3) epitopes reside on the choline transporter‐like protein‐2 (CTL2). A single‐nucleotide substitution (461G>A; Arg154Gln) on the CTL2 gene (SLC44A2) defines the allele SLC44A2*1, which expresses HNA‐3a, and SLC44A2*2, which expresses HNA‐3b; an additional substitution (457C>T; Leu153Phe) in SLC44A2*1:2 may impact genotyping systems. People who only express HNA‐3b may develop anti‐HNA‐3a. These alloantibodies have been linked to severe transfusion‐related acute lung injury, which may be a reason to screen blood donors for SLC44A2*2 homozygosity. For Caucasian and Asian populations, SLC44A2 allele frequencies are known. Our primary objective was to determine the SLC44A2 allele frequencies in the African American population. STUDY DESIGN AND METHODS: Purified DNA from 334 individuals (202 male, 132 female; 241 African American, 93 Caucasian) was collected. Two real‐time polymerase chain reaction assays were developed to genotype all samples; results were confirmed by nucleotide sequencing. RESULTS: In 241 African American donors, the allele frequency of SLC44A2*1 was 93% (85%‐<100%; 95% confidence intervals, Poisson distribution) while SLC44A2*2 was 7% (5%‐10%). In 93 Caucasian donors, the allele frequency of SLC44A2*1 was 83% (71%‐98%) and SLC44A2*2 was 17% (11%‐24%), matching previously reported data for Caucasians but differing from African Americans (p < 0.001, Fisher's exact test). CONCLUSIONS: This study describes the allele frequencies of the three known HNA‐3 variants in an African American population. We found that African Americans have a significantly lower probability of possessing the SLC44A2*2 allele and may thus be less likely to form the clinically relevant anti‐HNA‐3a.     

Human platelet antigen polymorphisms (HPA‐1, ‐2, ‐3, ‐4, ‐5 and ‐15) in major ethnic groups of Pakistan

Objectives: Gene frequencies of human platelet antigens (HPA) determine the magnitude of platelet immunological disorders like neonatal alloimmune thrombocytopenia, platelet refractoriness and ease of availability of particular HPA‐typed platelet donors in a given community. Background: However, the pattern of HPA in Pakistani population is not known. Aim: The aim of present study was to determine the gene frequencies of HPA (HPA‐1 to ‐5 and ‐15) in individuals belonging to major ethnic groups and castes of Pakistani population. Materials and Methods: HPA genotyping was done in 593 individuals belonging to all ethnic groups of Pakistan, by polymerase chain reaction‐sequence specific primers with detection on polyacrylamide electrophoresis. Results: The gene frequencies of the ‘a’ and ‘b’ alleles of HPA‐1 to ‐5 and ‐15 in Pakistanis were as follows: HPA‐1a/b, 0·885/0·115; HPA‐2a/b, 0·92/0·08; HPA‐3a/b, 0·69/0·31; HPA‐4a/b, 1/0; HPA‐5a/b, 0·9/0·1; HPA‐15a/b, 0·59/0·41. Except for significant difference regarding gene frequency of HPA‐3 between Pathans and Sindhis, there was no significant difference of HPA‐1 to ‐5 and ‐15 between major ethnic groups of Pakistan. The estimated mismatch probability regarding platelet antigens 1–5 and 15 in Pakistanis, after transfusion of random donor platelets, is from 14 to 37%. The expected incidence of neonatal alloimmune thrombocytopenia due to anti‐HPA‐1a in Pakistani pregnant females is < 1 of 1000 pregnancies and 8–12 of 1000 in case of anti‐HPA‐5b. Homozygosity of HPA‐1b, ‐2b and ‐5b genotypes ranged from 1 to 2% in the Pakistani population, whereas homozygosity of HPA‐3b and ‐15b was 11 and 18%. Conclusions: There is a need to establish donor registries typed for HPA in the transfusion centres of the country.