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.

Blood donor screening with cobas s 201/cobas TaqScreen MPX under routine conditions at German Red Cross institutes.

BACKGROUND: In 1997 the German Red Cross (GRC) blood donor services introduced mini-pool nucleic acid testing (NAT) for human immunodeficiency virus (HIV)-1, hepatitis C virus (HCV) and hepatitis B virus (HBV) to increase blood safety. With the new cobas s 201/cobas TaqScreen MPX, a fully automated extraction method and a multiplex amplification system specifically adapted to the needs of blood donation services is available. METHODS: The cobas s 201 system was evaluated at the GRC BTS locations Hagen, Springe and Frankfurt. In phase A, the analytical sensitivity for the detection of HBV, HCV and HIV-1 was investigated and in phase B, at least 60,000 samples at each test site were screened in parallel with the MPX test on s 201 system and the existing routine mini-pool NAT system to compare the diagnostic specificity and the diagnostic sensitivity. RESULTS: Comparable analytical sensitivities in a range of 1.6-3.6 IU/ml, 4.9-10.9 IU/ml and 14.7-26.6 IU/ml for HBV, HCV HIV, respectively, for the MPX test on s 201 system (95% probability based on probit analysis) were determined at all test sites. The diagnostic sensitivity was 99.8% and the diagnostic specificity was 99.85%. CONCLUSIONS: The MPX test on s 201 system is a fully automated NAT system suitable for routine blood donor screening. The analytical sensitivity as well as the diagnostic sensitivity fulfilled all requirements of the Paul Ehrlich Institute for blood donor screening in mini-pools up to 96 donations per pool. A major benefit of the automated NAT system is the reduced personnel time and the extensive complete barcode-controlled process documentation.

[Autoimmune thrombocytopenia, neutropenia and hemolysis]. / Autoimmune Thrombozytopenie, Neutropenie und Hämolyse.

Autoantibodies reduce the life span of platelets, granulocytes, and red blood cells. This may result in thrombocytopenia with bleeding, in neutropenia with infection, and in anemia, respectively. Immune-hemocytopenias can manifest as primary disease without another cause, or they are associated with other underlying morbidities such as autoimmune diseases, lymphoproliferative diseases, immune defects, or viral infections. Diagnosis is confirmed by laboratory tests showing autoantibodies against the respective blood cells. Indication for treatment is the clinical manifestation of symptoms: bleeding in autoimmune thrombocytopenia, infections in neutropenia, and symptomatic anemia, respectively. Especially in case of thrombocytopenia patients should not be treated because of abnormal laboratory values, only. To date steroids are the basic treatment in autoimmune thrombocytopenia and hemolytic anemia, while prophylactic antibiotics are the main treatment in autoimmune neutropenia. Growth factors like the new thrombopoietin receptor agnonists in autoimmune thrombocytopenia or G-CSF in autoimmune neutropenia, and anti-CD20 antibodies are new options for treatment.

Neutrophil CD177 (NB1 gp, HNA-2a) expression is increased in severe bacterial infections and polycythaemia vera.

The NB1 glycoprotein (CD177, HNA-2a antigen) is exclusively expressed on human neutrophils. As the clinical significance of CD177 expression is unknown, we investigated its expression in healthy individuals before and after stimulation with granulocyte colony-stimulating factor (G-CSF), in patients with rheumatoid arthritis, viral hepatitis, severe bacterial infections and polycythaemia vera. Expression was quantitatively determined by flow cytometry and by real time polymerase chain reaction. Only G-CSF-stimulated individuals and patients with severe bacterial infections and polycythaemia showed a significantly (P < 0.001) increased CD177 expression compared with healthy individuals, indicating that neutrophil CD177 expression can increase significantly in certain clinical conditions.

TRALI due to granulocyte-agglutinating human neutrophil antigen-3a (5b) alloantibodies in donor plasma: a report of 2 fatalities.

BACKGROUND: TRALI is usually an immunologic reaction to WBC antibodies in infused plasma and ranks second only to ABO mismatch as a cause of transfusion-associated death. Implicated donors are usually multiparous women (>/=3 pregnancies). STUDY DESIGN AND METHODS: Two fatal cases of TRALI were evaluated by reviewing clinical and laboratory findings and characterizing alloantibodies present in donor plasma. Investigation for WBC antibodies was by lymphocytotoxicity (LCT), FlowPRA (FlowPRA, One Lambda, Inc.) and granulocyte immunofluorescence and agglutination assays. Patient 1 was a 62-year-old man with chronic T-cell lymphocytic leukemia, and Patient 2 was a 54-year-old woman undergoing a cadaveric kidney transplant. Both patients developed acute respiratory distress and hypotension during (Patient 1) and approximately 30 minutes after (Patient 2) transfusion. Fulminant pulmonary edema ensued in both cases necessitating mechanical ventilation and both patients died within 24 hours of the onset of respiratory complications. RESULTS: The donors of the implicated blood components were women with a history of two pregnancies but no blood transfusions. Weak apparently panreactive granulocyte antibodies were detected with flow cytometry. However, in the granulocyte agglutination test, strong antibodies specific for human neutrophil antigen (HNA)-3a (5b) were identified in both donors. CONCLUSION: It is concluded that female blood donors with only two previous pregnancies can form clinically important granulocyte-reactive alloantibodies leading to fatal TRALI reactions in recipients. The sometimes devastating consequences of TRALI should prompt the development of strategies to prevent or reduce its incidence. Further research is warranted to investigate recipient and donor factors responsible for TRALI, including whether 5b (HNA-3a) alloantibodies are especially prone to cause severe reactions, and to better characterize the HNA-3a (5b) antigen, particularly at the molecular level.

Molecular basis of the neutrophil glycoprotein NB1 (CD177) involved in the pathogenesis of immune neutropenias and transfusion reactions.

The human granulocyte alloantigen NB1, recently clustered as CD177, is heterogenously expressed on neutrophils of 88-97% of healthy individuals. Since its molecular nature has remained unknown, we isolated NB1 glycoprotein from granulocyte lysate by immunoaffinity chromatography. MALDI-TOF mass spectrometry identified a 50,556 Da glycoprotein which was reduced to 43,069 Da after removal of N-linked carbohydrates. Following N-terminal amino acid sequencing and NB1-specific primer construction, rapid amplification of cDNA ends PCR yielded a 1,614-bp cDNA for NB1. COS-7 cells transfected with the cDNA expressed immunoreactive NB1 glycoprotein. A 1,311-bp sequence was identified to be the entire coding region. The 5' and 3' untranslated regions consist of 27 bp and 276 bp, respectively. The open reading frame codes for 437 amino acids of which the first 21 form the signal peptide. The remaining 416 residues form a N-terminal extracellular protein with two cysteine-rich domains, three N-linked glycosylation sites and short transmembrane and cytoplasmic segments including a glycosyl-phosphatidylinositol attachment (omega) site. Database searches revealed homology to Ly-6 (uPAR) domain, suggesting that NB1 belongs to urokinase plasminogen activator receptor/CD59/Ly-6 snake toxin superfamily.

[The TRALI syndrome--a life-threatening transfusion reaction]. / Das TRALI-Syndrom. Eine lebensbedrohliche Transfusionsreaktion.

Transfusion related acute lung injury (TRALI) is a serious complication of blood transfusion, characterized by non-cardiogenic lung oedema. We describe a case of TRALI due to granulocyte-specific antibodies. The 58-year-old patient received 2 units of fresh frozen plasma following colon surgery and within 30 min the patient developed an acute respiratory distress syndrome. Granulocyte-specific antibodies were found in one of the transfused plasma of a female blood donor who most likely became immunized against granulocyte alloantigens during her three pregnancies.

Serum granulocyte colony-stimulating factor levels are not increased in patients with autoimmune neutropenia of infancy.

OBJECTIVE: To assess the role of granulocyte colony-stimulating factor (G-CSF) in autoimmune neutropenia (AIN). DESIGN: Serum G-CSF levels were measured in 57 children with AIN. Two different G-CSF-dependent assays were used: a solid-phase "sandwich" enzyme-linked immunosorbent assay and a proliferation assay. Sera from healthy persons and from patients with severe congenital neutropenia were used for negative and positive controls. RESULTS: The median G-CSF level in healthy persons (n = 13) was low, 45.6 pg/mL (range <39 to 141 pg/mL). The median G-CSF level in patients with AIN (n = 57) was very similar, 45.5 pg/mL (range <39 to 2500 pg/mL). Forty-five (79%) of 57 patients with AIN had levels within the range of the control group. Seven (12%) had marginally increased G-CSF levels (141 to 400 pg/mL), and only 5 (9%) had levels higher than 400 pg/mL. The G-CSF levels measured by enzyme-linked immunosorbent assay correlated well with levels measured by the proliferation assay, thus demonstrating that antibodies present in patient sera did not affect the biologic activity of G-CSF. CONCLUSION: G-CSF production in AIN is not increased despite the low neutrophil count, similar to thrombopoietin in immune thrombocytopenic purpura.

Phase I/II trial of neutrophil transfusions from donors stimulated with G-CSF and dexamethasone for treatment of patients with infections in hematopoietic stem cell transplantation.

We examined the feasibility of a community blood bank granulocyte transfusion program utilizing community donors stimulated with a single-dose regimen of subcutaneous granulocyte colony-stimulating factor (G-CSF) plus oral dexamethasone. The recipients of these transfusions were neutropenic stem cell transplantation patients with severe bacterial or fungal infection. Nineteen patients received 165 transfusions (mean 8.6 transfusions/patient, range 1-25). Community donors provided 94% of the transfusions; relatives accounted for only 6% of the transfusions. Sixty percent of the community donors initially contacted agreed to participate, and 98% of these individuals indicated willingness to participate again. Transfusion of 81.9 +/- 2.3 x 10(9) neutrophils (mean +/- SD) resulted in a mean 1-hour posttransfusion neutrophil increment of 2. 6 +/- 2.6 x 10(3)/microL and restored the peripheral neutrophil count to the normal range in 17 of the 19 patients. The buccal neutrophil response, a measure of the capacity of neutrophils to migrate to tissue sites in vivo, was restored to normal in most patients following the transfusion. Chills, fever, and arterial oxygen desaturation of >/= 3% occurred in 7% of the transfusions, but these changes were not sufficient to limit therapy. Infection resolved in 8 of 11 patients with invasive bacterial infections or candidemia. These studies indicate that transfusion of neutrophils from donors stimulated with G-CSF plus dexamethasone can restore a severely neutropenic patient's blood neutrophil supply and neutrophil inflammation response. Further studies are needed to evaluate the clinical efficacy of this therapy.

Diagnosis and clinical course of autoimmune neutropenia in infancy: analysis of 240 cases.

Primary autoimmune neutropenia (AIN) is caused by granulocyte-specific autoantibodies and occurs predominantly in infancy. Clinical presentation and diagnosis have not been well established, resulting in burdening diagnostic investigations and unnecessary treatment with granulocyte colony-stimulating factor (G-CSF). In the present study, clinical, laboratory, and immunologic data of 240 infants with primary AIN were evaluated. Suspected association with parvovirus B19 infection was investigated using serologic and DNA-based methods. Primary AIN was mainly diagnosed at the age of 5 to 15 months but was observed as early as day 33 of life. In 90% of the cases, AIN was associated with benign infections despite severe neutropenia. Spontaneous remission, shown by 95% of the patients, usually occurred within 7 to 24 months. Autoantibodies in the patient's sera were not always present, and screening had to be repeated several times until antibody detection succeeded. About 35% of the autoantibodies showed preferential binding to granulocytes from NA1 and NA2 homozygous donors. Bone marrow was typically normocellular or hypercellular, with a variably diminished number of segmented granulocytes. A significant association with parvovirus B19 infection was not found. Symptomatic treatment with antibiotics was sufficient in most patients. Eighty-nine percent of the patients received antibiotics (cotrimoxazole) for prophylaxis of infections. For severe infections or for surgical preparation, G-CSF, corticosteroids, and intravenous IgG were administered, resulting in increased neutrophil counts in 100%, 75%, and 50% of the patients treated, respectively. In combination with the detection of granulocyte-specific antibodies, the typical clinical picture allowed diagnosis of AIN without burdening investigations. Treatment with G-CSF was found to be a reliable alternative to temporarily increase the neutrophil count.

Quantitation of granulocyte antibodies in sera and determination of their binding sites.

An enzyme immunoassay using eluates was developed for the quantitation of granulocyte antibodies in sera. Incubation of donor granulocytes with sera containing granulocyte alloantibodies (NA1, NA2, NB1, 5b, HLA) or autoantibodies resulted in a significantly higher amount of immunoglobulin G (IgG) per cell than did incubation with control sera. Ultracentrifugation of the sera prior to testing reduced unspecific binding of IgG to granulocytes. In eight of 10 assessed sera from patients with Felty's syndrome eluted IgG decreased from elevated to normal levels. Ultracentrifugation further allowed determination of the binding sites of granulocyte-reactive alloantibodies. Using sera containing NA1- and NA2-specific alloantibodies 173,000-188,000 binding sites on homozygous and 84,000-110,000 on heterozygous cells were determined. A similar number was found using a Fc gamma receptor III (FcRIII)-specific monoclonal antibody. Granulocytes from patients with paroxysmal nocturnal haemoglobinuria showed greatly reduced binding sites for NA-specific alloantibodies. The binding sites for the human alloantibody NB1 ranged from 36,000 to 318,000 and for the 5b alloantibody from 44,000 to 65,000. Mean values of 139,000 binding sites for HLA antigens and 27,000 binding sites for the FcRII were determined using monoclonal antibodies.

[Quality of thrombocyte preparations from buffy coat or platelet-rich plasma]. / Untersuchungen zur Qualität von Thrombozytenpräparaten aus Buffy-coat oder plättchenreichem Plasma.

One hundred platelet concentrates, fifty prepared from buffy coat and fifty prepared from platelet-rich plasma were examined. In platelet concentrates from buffy coat a lower leukocyte contamination (1.4 +/- 1.2 x 10(7) vs 13.2 +/- 15 x 10(7], a significant higher ATP content (6.3 +/- 1.7 mmol/10(14) platelets vs 3.9 +/- 1.9 mmol/10(14) platelets) and a better response in ADP- or collagen-induced aggregation could be determined during storage of five days. Our results indicate that preparation of platelet concentrates from buffy coat is preferable to their preparation from platelet-rich plasma.