HNA antibody-mediated neutrophil aggregation is dependent on serine protease activity.

BACKGROUND AND OBJECTIVES: Transfusion-related acute lung injury (TRALI) is often caused by antibodies against human neutrophil alloantigen-2 (HNA-2) and HNA-3a. Neutrophil aggregation is considered as a major cause of TRALI, but little is known about how HNA antibodies initiate this process. We explored mechanisms involved in neutrophil aggregation induced by HNA-2 and HNA-3a antibodies. MATERIALS AND METHODS: Isolated neutrophils were pretreated with broad-spectrum or specific inhibitors against different cell functions or proteases. Granulocyte agglutination test (GAT) was performed with serially diluted anti-HNA-2 and anti-HNA-3a plasmas or control plasma, and reactivity was evaluated microscopically. Reactive oxygen species (ROS) production in neutrophils was investigated using a lucigenin-based chemiluminescence assay. RESULTS: HNA-2 and HNA-3a antibody-mediated neutrophil aggregation was inhibited by pretreatment with formaldehyde, iodoacetamide and the serine protease inhibitors Pefabloc-SC, N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and Nα-tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK). In contrast, inhibition of actin polymerization, respiratory burst, cysteine proteases, metalloproteases or aspartic proteases did not affect neutrophil aggregation. Furthermore, HNA-3a antibodies did not directly cause ROS production in neutrophils. CONCLUSION: Aggregation of neutrophils induced by HNA-2 and HNA-3a antibodies is an active process and depends on trypsin- or chymotrypsin-like serine proteases but is not dependent on the production of ROS. These findings may open new prospects for the pharmacologic prevention of neutrophil-associated acute lung injury.

External quality assessment of human neutrophil antigen (HNA)-specific antibody detection and HNA genotyping from 2000 to 2012.

Since 2000, Quality Assurance (QA) exercises for the detection and identification of granulocyte antibodies and DNA typing for human neutrophil antigens (HNA) have been distributed within the International Granulocyte Immunobiology Workshops, which are linked to International Society of Blood Transfusion. The exercises were standardised at the outset to enable laboratory performance to be monitored. Between 2000 and 2012, nine exercises were distributed to 20 laboratories. Overall, 45 examples of 42 unique samples containing defined granulocyte reactive antibodies were distributed for serological analysis together with 20 samples for HNA genotyping. The level of satisfactory serological performance was initially set at 50% and later increased to 70%, while the 'cut-off' for HNA genotyping was set at 100% after 2008. Failure to achieve the minimum score in the QA exercises in consecutive years resulted in temporary exclusion. In 2000, the 15 participating laboratories had a mean score of 56.1% for serological analysis and 13 laboratories attempted HNA-1a and -1b genotyping, while 11 attempted HNA-1c typing. Steady improvements in proficiency for serological testing and HNA typing occurred in subsequent exercises. In 2012, the mean score for serology was 88.5% and 12/13 laboratories scored 100% for HNA-1a, -1b, -1c, -3a, -3b, -4a, -4bw, -5a and -5bw genotyping. These QA exercises have provided an invaluable tool to monitor and improve the standard of granulocyte immunology investigations for participating laboratories, thereby enhancing performance for both clinical investigations and donor screening programmes to reduce the incidence of TRALI.

Frequency and severity of transfusion-related acute lung injury--German haemovigilance data (2006-2007).

OBJECTIVE: In an observational cohort study (2006-2007) the Paul-Ehrlich-Institut collected epidemiological data to investigate the frequency and causes of TRALI. METHODS: Diagnosis of TRALI was confirmed according to criteria of the European Haemovigilance Network. Subsequent testing of white blood cell antibodies (WBC-Ab) against HLA or human neutrophil alloantigens was performed. RESULTS: Of a total of 187 reported TRALI cases, 44 could be confirmed consisting of 35 cases of antibody-mediated TRALI and nine cases of non-immune-mediated TRALI. Eight of 44 affected patients (18%) had a fatal outcome, seven cases with WBC-Ab positive plasma donors and one case with red blood cell donors. WBC antibodies were found in one male and 39 female donors. In 34 female donors, a history of pregnancy was confirmed. WBC-Ab positive donors presented four HLA class I antibodies, 15 HLA class II antibodies, 13 HLA class I and class II antibodies, one HNA-2a, and seven HNA-3a antibodies. WBC antibodies matching with recipient antigens were found exclusively in 28 female donors; 26 FFP donors, one platelet donor and one red blood cell donor. Reporting frequency of immune-mediated TRALI was 1:66,000 for fresh frozen plasma, 1:2.86 million for red blood cell concentrates and 1:420,000 for platelet concentrates. Reporting frequency of TRALI-related fatalities was 1:285,000 for FFP. SUMMARY: Haemovigilance data show the significance of female donors with a history of pregnancy for the development of antibody-mediated TRALI. Manufacturing of FFP from male plasma and female donor screening for WBC-Ab could represent preventive measures.

Recommendations of the ISBT Working Party on Granulocyte Immunobiology for leucocyte antibody screening in the investigation and prevention of antibody-mediated transfusion-related acute lung injury.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is currently one of the most common causes of transfusion-related major morbidity and death. Among the many TRALI mediators, leucocyte antibodies have been identified as important triggers of severe TRALI. STUDY DESIGN AND METHODS: These recommendations were compiled by experts of the ISBT Working Party on Granulocyte Immunobiology, based on the results obtained in eight international granulocyte immunology workshops, their personal experiences and on published study results. RESULTS: Leucocyte antibody screening has to include the detection of human leucocyte antigen (HLA) class I, class II and human neutrophil alloantigen antibodies using established and validated techniques. HLA class I antibody detection should be restricted to antibodies clinically relevant for TRALI. To avoid unnecessary workload, TRALI diagnosis should be assessed by consultation with the reporting clinician and thorough exclusion of transfusion-associated circulatory overload/cardiac insufficiency. In patients diagnosed with TRALI having donors with detectable leucocyte antibodies, evidence of leucocyte incompatibility should be provided by either cross-matching or typing of patient for cognate antigen. CONCLUSION: Leucocyte antibody screening for the immunological clarification of TRALI cases as well as for identification of potentially alloimmunized blood donors is feasible and can be performed in a reasonable and quality assured manner. This practice can contribute to the prevention of antibody-mediated TRALI.

Specificities of leucocyte alloantibodies in transfusion-related acute lung injury and results of leucocyte antibody screening of blood donors.

BACKGROUND: Antibody-mediated transfusion-related acute lung injury (TRALI) is an important cause of transfusion-associated morbidity and death. Preventive strategies are currently a matter of debate. METHODS: Specificities of leucocyte antibodies implicated in previous severe TRALI reactions were determined using standard techniques. Based on these results, a leucocyte antibody screening strategy for the testing of parous female donors was introduced. RESULTS: Of 36 TRALI cases, 17, 12, four and three were due to human leucocyte antigen (HLA) class II, human neutrophil alloantigen (HNA), HLA class I, and mixtures of HLA class I and II antibodies, respectively. HNA-3a antibodies accounted for 10 of 12 HNA antibody-mediated reactions and 6 of 10 fatalities including one after transfusion of red blood cells. Investigation 5332 parous female donors showed leucocyte antibodies in 473 samples, resulting in an alloimmunization rate of 8.9%. Sixty-one per cent of these donors presented HLA class I, 19% class II, 12% HLA class I and II antibodies and 5% HNA antibodies. Additional HLA class I antibodies were found in 39% of HLA class II and in 17% of HNA antibodies containing sera. Our restrictive plasma strategy did not result in a shortage of plasma or platelets. No antibody-mediated TRALI case was observed since introduction of the policy of plasma from male, nulliparous or tested multiparous donors. CONCLUSION: Compared to HLA class I antibodies, those directed against HLA class II and HNA-3a were of greater clinical relevance. Isolated HLA class I antibody screening was found to be insufficient for leucocyte antibody screening.

Renal effects of gadopentetate dimeglumine in patients with normal and impaired renal function.

Gadolinium chelates are widely used in magnetic resonance imaging as contrast medium in patients with nephropathy. However, only few studies have investigated the effect of gadolinium on serum creatinine concentration and estimated GFR as surrogate markers of renal function. This study was performed to evaluate the effect of gadopentetate dimeglumine in a dose sufficient for diagnostic and interventional purposes on renal function in a large sample of patients. We analyzed serum creatinine and serum-urea levels before and after the administration of gadopentetate dimeglumine in patients with normal and patients with pre-existing impaired renal function. Age, height, body mass, sex, medication and preexisting illnesses such as diabetes, renal artery stenosis and heart disease were monitored. In 181 patients with normal renal function, there was no statistically significant change in serum creatinine concentration after the administration of gadopentetate dimeglumine (at baseline: 0.72 +/- 0.18 mg/dl, after gadolinium: 0.73 +/- 0.22 mg/dl). In contrary, serum creatinine levels decreased significantly after the administration of gadolinium in 198 patients with pre-existing renal impairment (1.82 +/- 1.03 mg/dl before and 1.72 +/- 1.03 mg/dl after gadolinium) (p < 0.01). According to this surrogate marker of renal function, the change of estimated GFR in patients with normal baseline renal function was not significant, while in patients with impaired renal function, GFR increased after the administration of gadolinium (p < 0.001). The high diagnostic value of gadolinium contrast media is associated with a very small risk of adverse reactions. Our findings show that the administration of gadolinium even is associated with a decrease of serum creatinine in patients with pre-existing renal impairment. In conclusion, the use of gadolinium-based contrast media may be considered as a safe alternative in patients with impaired renal function for whom use of iodine-based contrast agents is prone to a high rate of radiocontrast-induced nephropathy.

Genotyping of human neutrophil antigen-5a (Ond).

Antibodies to human neutrophil alloantigens (HNA) can cause immune-mediated neutropenias and transfusion complications. The diagnosis of antibodies to the HNA-5a (Ond) isoform of the alphaLbeta2 integrin is hampered by the lack of reliable methods for HNA-5a antigen typing. We have devised a polymerase chain reaction sequence-specific primer method (PCR-SSP) and used it to determine the HNA-5a gene frequencies in 320 individuals from different ethnicities. 15.3% were found to be HNA-5a negative, with no significant deviation between the populations. Results of HNA-5a genotyping were in accordance with phenotyping. Availability of HNA-5a PCR-SSP will facilitate the diagnosis of Ond antibody-mediated clinical conditions.

High-resolution DNA typing in immunoglobulin A deficiency confirms a positive association with DRB1*0301, DQB1*02 haplotypes.

Selective immunoglobulin A (IgA) deficiency, the most common form of primary immunodeficiency, is related to the HLA genes. Previous studies demonstrated associations with particular HLA-DR-DQ haplotypes and a neutral amino acid at position 57 of the DQbeta chain was implicated in the susceptibility to selective IgA deficiency. In this study we reanalyzed the reported findings by high-resolution DNA typing of the loci DRB1, DQB1 and DQA1. We compared the typing results of 74 IgA-deficient individuals, detected by screening of blood donors, with those taken from 111 healthy controls. Results confirmed a strong positive association with DRB1*0301, DQB1*02 and a negative association with DRB1*1501, DQB1*0602. Considering the molecular interactions between HLA class II alleles and the peptides bound we conclude that the amino acid at position 57 of the DQbeta chain may contribute to the susceptibility to selective IgA deficiency, but not determine it. An extended statistical analysis strengthened the hypothesis that selective IgA deficiency might be communicated by the distinct haplotype DRB1*0301, DQB1*02.

Polymorphisms in the tumor necrosis factor genes in Wegener's granulomatosis.

Two known di-allelic polymorphisms in the tumor necrosis factor (TNF) genes were investigated in 35 patients suffering from Wegener's granulomatosis (WG) in comparison to 111 healthy controls. The first polymorphism lay in the promotor region of the TNF-alpha gene at position -308, the second in the first intron of the TNF-beta gene. For both polymorphisms we could not find any statistically significant differences in allele frequencies between patients and healthy individuals. In the patient group the TNF 2/2 phenotype was slightly elevated, however (WG 5.7% vs. normal 1.8%). Investigating the clinical course of the disease according to TNF polymorphisms, TNF 1/1 patients were found to have a higher mean disease extension index (DEI) than TNF 1/2 individuals (TNF 1/1 10.5 vs. TNF 1/2 9.4).