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.     

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.     

Epitope mapping of antibodies directed against the human neutrophil alloantigen 3a

BACKGROUND: Severe transfusion‐related acute lung injury is often caused by antibodies directed against the human neutrophil alloantigen (HNA)‐3a. HNA‐3a results from an amino acid exchange (Arg154Gln) in the first extracellular loop of the choline transporter‐like protein 2 (CTL2). The characteristics of the binding domain(s) of HNA‐3a antibodies are unknown. STUDY DESIGN AND METHODS: For epitope mapping, a library of 23 different HNA‐3a (R₁₅₄) and three HNA‐3b (Q₁₅₄) peptides covering different parts of the first extracellular loop of CTL2 (aa_55‐231) was synthesized in Escherichia coli and tested by Western blot with two HNA‐3a alloantibody–containing plasma samples and by enzyme immunoassay (EIA) with different HNA‐3a‐ (n = 21) and HNA‐3b‐ (n = 1) positive plasma samples. RESULTS: Despite promising Western blot results using highly reactive plasma samples, we found widely varying reactivities of different HNA‐3a plasmas in the EIA, with only 11 of 21 HNA‐3a antibodies binding to any of the tested HNA‐3a peptides and with no peptide recognized by more than nine of the 21 antibodies. The HNA‐3b plasma did not react with R₁₅₄ peptides in the EIA nor with R₁₅₄ or Q₁₅₄ peptides in Western blot experiments. Plasma reactivity profiles with the peptides did not correlate with those observed using granulocyte agglutination and granulocyte immunofluorescence tests. CONCLUSION: Binding of HNA‐3a alloantibodies depends on the conformation of the intact CTL2 protein and their binding sites may differ substantially. Peptide‐based assays for detection of HNA‐3a antibodies bear the risk to be insensitive and require systematic validation with a large panel of antibodies.     

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.     

Geno‐ and phenotyping and immunogenicity of HNA‐3

BACKGROUND: Alloantibodies directed against the human neutrophil alloantigen (HNA)‐3a are frequently implicated in severe and fatal transfusion‐related acute lung injury (TRALI). The HNA‐3a/3b system results from a single‐nucleotide exchange (461G>A; Arg154Gln) in the choline transporter‐like protein 2 gene. Genotyping may allow identification of blood donors at risk to develop HNA‐3a antibodies. STUDY DESIGN AND METHODS: A polymerase chain reaction using sequence‐specific primers (PCR‐SSP) for genotyping of HNA‐3a and ‐3b alleles was designed. Results of genotyping and phenotyping were compared in 40 randomly selected individuals and in blood donors and recipients of six TRALI cases associated with HNA‐3a antibodies. Phenotyping was performed by granulocyte immunofluorescence and granulocyte agglutination using typing sera for HNA‐3a and two recently found HNA‐3b–reactive sera. Immunogenicity of HNA‐3a was determined by the rate of HNA‐3a alloantibodies in HNA‐3b homozygous parous women. RESULTS: Genotyping and phenotyping results correlated to 100% and were in accordance with alloantibody formation and binding in HNA‐3a antibody associated TRALI cases. Gene frequencies of HNA‐3a and ‐3b were 0.792 and 0.207 in the German population with 64.1% homozygous individuals for the HNA‐3a allele, 5.5% for the HNA‐3b allele, and 30.4% heterozygous individuals, in accordance with the Hardy‐Weinberg equilibrium and the gene frequencies of 0.819 and 0.181 reported in 1964. Immunization rates were estimated to be of 7% for HNA‐3a and 0.5% for HNA‐3b. CONCLUSION: The PCR‐SSP method allows reliable determination of the HNA‐3a and ‐3b genotypes; approximately 7% of HNA‐3b homozygous women develop antibodies when exposed to the HNA‐3a antigen during pregnancy.     

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.     

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.     

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.     

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.     

Homozygous type 2N R854W von Willebrand factor is poorly secreted and causes a severe von Willebrand disease phenotype

Summary. Background: von Willebrand disease (VWD) type Normandy (VWD 2N) is caused by mutations at the factor (F)VIII‐binding site of von Willebrand factor (VWF), located in the D′and D3 domains on the N‐terminus of mature VWF. The R854Q mutation is the most frequent cause of this phenotype. Objectives: We report the characterization of a homozygous VWD 2N mutation, R854W, detected in a patient with a severe VWD phenotype. Methods: The plasma VWF phenotype was studied, transient expression of recombinant mutant full‐length VWF in 293 EBNA cells was performed, and the results were compared with those obtained with wild‐type (WT) VWF. Furthermore, expression was also examined in HEK293 cells, which form Weibel–Palade body‐like granules when transfected with WT VWF. Results: The multimer analysis of plasma VWF showed the lack of the typical triplet structure, with the presence of the central band only, and a relative decrease in the high molecular mass multimers. Homozygous expression of recombinant R854W VWF resulted in normal amounts of cellular VWF, but with a severe reduction in secretion into the medium. Severe reductions in FVIII binding to R854W VWF, glycoprotein Ib binding activity and collagen binding of secreted W854 VWF was observed, and reproduced the phenotypic parameters of plasma VWF. In HEK293 cells, homozygous R854W VWF failed to form Weibel–Palade body‐like granules. Conclusions: Our results demonstrate that a homozygous R854W mutation in the D′ domain of VWF induces impaired secretion and activity of the protein, thereby explaining the severe phenotype of the patient.     

Reduced von Willebrand factor secretion is associated with loss of Weibel–Palade body formation

Background:  von Willebrand disease (VWD) is caused by mutations in von Willebrand factor (VWF) that have different pathophysiologic effect in causing low plasma VWF levels. Type 1 VWD includes quantitative plasma VWF deficiency with normal VWF structure and function. Objectives:  We report three novel type 1 VWF mutations (A1716P, C2190Y and R2663C) located in different VWF domains that are associated with reduced secretion and reduced formation of elongated Weibel–Palade body (WPB)‐like granules. Methods:  Transient expression of recombinant mutant full‐length VWF in 293 EBNA cells was performed and secretion, collagen binding and GpIb binding assessed in comparison with wild‐type VWF. Expression was also examined in HEK293 cells that form WPB‐like granules when transfected with wild‐type VWF. Results:  Laboratory results and multimer analysis of plasma VWF was compatible with type 1 VWD. Expression experiments demonstrated slightly reduced VWF synthesis and drastically impaired secretion upon homozygous expression. In HEK293 cells, homozygous expression of A1716P and C2190Y VWF variants failed to form elongated WPB‐like granules, while R2663C was capable of WPB‐like granules. Heterozygous expression of VWF variants had a negative impact on wild‐type VWF with a reduction in elongated WPB‐like granules in co‐transfected cells. Conclusions:  Our results demonstrate that homozygous and heterozygous quantitative VWF deficiency caused by missense VWF mutations in different VWF domains can be associated with inability to form endothelial WPB‐like granules.     

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.     

某地区无偿献血者中性粒细胞特异性抗体筛查分析

目的探讨河南地区无偿献血者中性粒细胞特异性(HNA)抗体的分布和特异性,分析HNA抗体引起的免疫性输血不良反应。方法随机收集女性标本156例,男性标本80例,采用LABScreen Multi试剂盒对标本HNA抗体进行检测。结果 236例无偿献血者中,女性HNA抗体检出阳性率为5.77%(9/156),男性为5.00%(4/80)。其中HNA 1A者7例,HNA4A者4例,同时检出HNA 1A和HNA 4A、HNA 1A和HNA 1C各1例。结论 HNA抗体分布无性别差异,研究HNA抗体的分布和特异性,可为指导临床用血安全和相关政策的制定提供有力的理论基础。     

New K103 β3 allele identified in a context of severe neonatal thrombocytopenia

BACKGROUND: A new β3 allele was identified in a severe case of neonatal alloimmune thrombocytopenia (<7 × 10⁹/L). STUDY DESIGN AND METHODS: Diagnosis was done by use of monoclonal antibody–specific immobilization of platelet (PLT) antigen for serologic analyses and polymerase chain reaction (PCR)–sequence‐specific primers (SSP) and PCR–restriction fragment length polymorphism (RFLP) for genotyping. Direct sequencing of PCR product was done and mutant αIIbβ3 expressed in HEK‐293 cells. RESULTS: Serologic analysis revealed in the maternal serum an anti‐human PLT alloantigen (HPA)‐1a alloantibody associated to an anti‐α2β1. Anti‐HPA‐1a alloimmunization diagnosis was confirmed by genotyping showing maternofetal incompatibility. However, investigation of rare HPA polymorphisms revealed discrepant HPA‐16b assignation between PCR‐RFLP and PCR‐SSP. Sequencing revealed a new c.385C>A mutation in the β3 coding sequence resulting in a false assignation of the HPA‐16b allele by PCR‐RFLP. This mutation leads to a Q103K substitution in mature β3. The K103‐β3 form of the complex was expressed in HEK‐293 cells but did not react with the maternal serum. CONCLUSION: We have characterized a new rare allele (frequency < 1%) of β3 that yields false HPA‐16b genotyping in PCR‐RFLP. This new case of false typing assignation emphasizes the necessity to use two genotyping techniques in diagnosis. This particularly applies for rare HPA polymorphisms when PLT phenotyping cannot be used.     

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.     

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.     

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.     

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.     

Cotesia vestalis parasitization suppresses expression of a Plutella xylostella thioredoxin

Thioredoxins (Trxs) are a family of small, highly conserved and ubiquitous proteins involved in protecting organisms against toxic reactive oxygen species. In this study, a typical thioredoxin gene, PxTrx, was isolated from Plutella xylostella. The full‐length cDNA sequence is composed of 959 bp containing a 321 bp open reading frame that encodes a predicted protein of 106 amino acids, a predicted molecular weight of 11.7 kDa and an isoelectric point of 5.03. PxTrx was mainly expressed in larval Malpighian tubules and the fat body. An enriched recombinant PxTrx had insulin disulphide reductase activity and stimulated Human Embryonic Kidney 293 (HEK293) cell proliferation. It also protected supercoiled DNA and living HEK293 cells from H₂O₂‐induced damage. Parasitization by Cotesia vestalis and injections of 0.05 and 0.01 equivalents of C. vestalis Bracovirus (CvBv), the symbiotic virus carried by the parasitoid, led to down‐regulation of PxTrx expression in host fat body. Taken together, our results indicate that PxTrx contributes to the maintenance of P. xylostella cellular haemostasis. Host fat body expression of PxTrx is strongly attenuated by parasitization and by injections of CvBv.