Neutrophil antibody diagnostics and screening: review of the classical versus the emerging.

BACKGROUND AND OBJECTIVES Severe transfusion-related acute lung injury (TRALI) events have been linked to donor-derived neutrophil antibodies. The journey to developing mass donor neutrophil antibody screening platforms is challenged by the fragility of neutrophils and their unique-specific antigenic characteristics. MATERIAL AND METHODS This article critically evaluates the capabilities and potential of five emerging antibody screening platforms designed to detect neutrophil reactive antibodies relevant to TRALI. They are compared with established neutrophil serological methods. RESULTS Data from two recombinant antigen platforms and a method using human neutrophil antigens-expressing KY cells indicated high specificity. Two mixed cellular flow cytometric assays have the advantage of presenting native conformation of the human polymorphonuclear neutrophil antigenic epitopes. CONCLUSIONS To date, the number and specificity of test sera applied to each platform is small. This needs to be substantially increased and further rigorous serological evaluation is yet needed to compare the sensitivity and specificity limits of each new platform with classical methods. With a limited world supply of TRALI-relevant test sera, a collaborative effort of laboratories with neutrophil and TRALI investigation expertise is required.

Heteroligomeric forms of codon 54 mannose binding lectin (MBL) in circulation demonstrate reduced in vitro function.

Mannose binding lectin (MBL) is a pattern recognition molecule that plays a pivotal role in innate immunity. This liver derived, circulating plasma protein binds organisms displaying high-density carbohydrate structures and flags them for destruction via opsonisation and initiation of the lectin pathway of the complement cascade. The present study reveals native, oligomeric forms of human MBL in plasma from healthy blood donors of differing genotypes and correlates the relative abundance of observed molecular weight species with mannan binding activity and C4 deposition in vitro. Wild type (A/A) individuals demonstrate predominately high molecular weight MBL that correlated with high mannan binding capacity and C4 deposition. A/C individuals demonstrated predominantly low molecular weight MBL with decreased mannan binding and C4 deposition activity. A/D individuals demonstrated both high molecular weight and low molecular weight MBL with reduced mannan binding and C4 deposition predominantly seen in combination with LX promoter. We identified A/B individuals as a unique group with large variation in MBL level, mannan binding activity and C4 deposition and propose a model for C4 deposition based on differential binding of MASP.

Mannose binding lectin acute phase activity in patients with severe infection.

Mannose Binding Lectin (MBL) is a liver derived, circulating plasma protein that plays a pivotal role in innate immunity. MBL functions as a pathogen recognition molecule, opsonising organisms and initiating the complement cascade. MBL deficiency arising from mutations and promoter polymorphisms in the MBL2 gene is common and has been associated with risk, severity, and frequency of infection in a number of clinical settings. With MBL therapy on the horizon, the usefulness of replacement MBL therapy has been challenged by the notion, that as an acute phase protein, MBL levels may rise under stress to sufficient levels, in individuals who are usually deficient. This report demonstrates that in patients with sepsis and septic shock, the majority of patients do not display an MBL acute phase response: 41.4% of individuals maintained consistent MBL levels throughout hospital stay, 31.3% of individuals demonstrated a positive acute phase response, and a negative acute phase response was observed in 27.3% of individuals studied. Importantly, a positive acute phase response was generally observed in individuals with wild-type MBL2 genes. When a positive acute phase response was observed in individuals with coding mutation, these individuals demonstrated a normal MBL level on admission to hospital. Furthermore, no individual, regardless of genotype who was MBL deficient at admission was able to demonstrate a positive acute phase response into the normal MBL range. These findings indicate MBL demonstrates a variable acute phase response in the clinical setting of sepsis and septic shock.

Managing passively acquired autoimmune neonatal neutropenia: a case study.

Pregnant women with autoimmune neutropenia (AIN) and circulating neutrophil-specific autoantibodies can deliver neutropenic neonates at risk of sepsis. We report the case of a woman who had two such pregnancies. The woman had been on prophylactic granulocyte colony-stimulating factor (G-CSF) treatment, but this was ceased prior to conception in both pregnancies. In the first pregnancy, there was no monitoring or interventions, and the neonate was neutropenic and required intensive care treatment. In the second pregnancy, the maternal neutrophil autoantibody level was monitored, and G-CSF treatment was introduced in the third trimester. The second infant had no neutropenia at delivery and an excellent Apgar score. We discuss the management strategy in the second pregnancy that included monitoring of serial titres of the maternal autoantibody and the introduction of G-CSF in the third trimester, which may have contributed to a more favourable clinical outcome. This may assist other clinicians faced with similar dilemmas in the future.

Alternatives to conventional vaccines--mediators of innate immunity.

Vaccines have been described as "weapons of mass protection". The eradication of many diseases is testament to their utility and effectiveness. Nevertheless, many vaccine preventable diseases remain prevalent because of political and economic barriers. Additionally, the effects of immaturity and old age, therapies that incapacitate the adaptive immune system and the multitude of strategies evolved by pathogens to evade immediate or sustained recognition by the mammalian immune system are barriers to the effectiveness of existing vaccines or development of new vaccines. In the front line of defence against the pervasiness of infection are the elements of the innate immune system. Innate immunity is under studied and poorly appreciated. However, in the first days after entry of a pathogen into the body, our entire protective response is dependant upon the various elements of our innate immune repertoire. In spite of its place as our initial defence against infection, attention is only now turning to strategies which enhance or supplement innate immunity. This review examines the need for and potential of innate immune therapies.

Investigating transfusion-related acute lung injury (TRALI).

AIM: Transfusion-related acute lung injury (TRALI) can be a life-threatening transfusion complication and should be considered whenever respiratory distress occurs during a transfusion. Management of donors implicated in TRALI is a n important haemovigilance responsibility for blood services. To enable this, it is imperative to develop an effective strategy for investigating TRALI. The present paper describes an effective approach. METHODS: Cases of suspected TRALI we re referred to the Platelet and Granulocyte Immunobiology Laboratory at the Australian Red Cross Blood Service-Queensland; a reference neutrophil testing service. Recipient and donor samples were tested for the presence of leucocyte antibodies. Where possible, compatibility testing was performed between donor and recipient samples. RESULTS: From March 1999 to June 2001 , leucocyte antibodies directed against neutrophil-specific or human leucocyte antigens (HIA) were detected in at least one donor in seven of the nine cases investigated. Incompatibility with patient antigens (HNA-2a, non-specific HLA and HLA B5, B16, B35) was confirmed by cross matching in three cases. CONCLUSION: TRALI is a serious non-infectious hazard of transfusion that must be reported and investigated promptly. Prompt investigations allow appropriate management of implicated donations and donors so as to minimize the incidence of TRALI. Therefore, the role of clinicians in reporting such cases and the hospital blood banks in collecting appropriate samples is critical. We suggest that hospital blood banks retain transfused donation units for at least 24 h after transfusion to expedite TRALI investigations. Due to the specialized nature of investigation, it is necessary to direct such investigations to specialist reference neutrophil testing services. In cases where the recipient has the leucocyte antibody, the use of white cell filters in future transfusions should be beneficial, because there is little evidence to substantiate the use of phenotyped blood products.

Human neutrophil antigen-4a gene frequencies in an Australian population, determined by a new polymerase chain reaction method using sequence-specific primers.

Human neutrophil antigen-4a (HNA-4a) is a high-frequency (99% in the USA) neutrophil antigen, which has recently been linked to a case of alloimmune neonatal neutropenia. We have devised a new polymerase chain reaction sequence-specific primer (PCR-SSP) method to assess HNA-4a genotype, and used it to determine the HNA-4a gene frequencies in an Australian population. The gene frequency was found to be 0.906, which is the same as in the American population. The PCR-SSP genotyping method perfectly correlates with serological phenotyping and is efficient for screening large numbers of samples.

Alloimmune neonatal neutropenia linked to anti-HNA-4a.

This is a novel case report of alloimmune neonatal neutropenia (ANN) linked to the neutrophil antibody anti-HNA-4a (MART). Since its discovery, the HNA-4a antigen has never been associated with any clinical neutropenia. A first-born neonate with respiratory distress was found to be severely neutropenic, because of ANN. The broad reactivity of the antibody together with its capture by CD11b and CD18 in monoclonal antibody immobilization of granulocyte antigen test suggested HNA-4a specificity. DNA sequencing confirmed that the father is HNA-4a-positive and that the mother is HNA-4a-negative, supporting the diagnosis of ANN linked to MART.

Analysis of the relationship between mannose-binding lectin (MBL) genotype, MBL levels and function in an Australian blood donor population.

The mannose-binding lectin (MBL) pathway of complement activation is an important component of innate host defence. Numerous studies have described associations between the MBL genotype, MBL levels and disease susceptibility. However, genotyping and quantitative assays used in these studies have frequently been limited, and comprehensive data examining the interaction between structural and coding MBL genetic variants, MBL antigenic levels and MBL functional activity are lacking. Such data may be important for accurate planning and interpretation of studies of MBL and disease. This study has examined MBL in a cohort of 236 Australian blood donors. Five MBL promoter and coding single nucleotide polymorphisms were genotyped using polymerase chain reaction-sequence-specific priming (PCR-SSP). Plasma levels of MBL antigen were quantified using a double-antibody enzyme-linked immunosorbent assay (ELISA), and functional MBL levels were quantified using a mannan-binding assay. Activation of the complement pathway by MBL was measured in a C4-deposition assay. Significant associations were found between both coding and promoter polymorphisms and MBL antigenic and functional levels. There was significant correlation between the results of MBL double-antibody, mannan-binding and C4-deposition assays. Comprehensive MBL genotyping and functional MBL quantitation using mannan-binding and C4-deposition assays have the potential to be highly informative in MBL disease association studies.

Investigating severe neutropenia with a simple bone marrow immunofluorescence test.

Severely neutropenic patients who are suspected of having circulating autoantibodies to neutrophils and/or myeloid precursors present a diagnostic problem. This report describes the use of a simple bone marrow immunofluorescence test (BMIFT) to demonstrate autoantibodies reactive with the patient's own bands, neutrophils and myeloid precursors represented on fresh-frozen bone marrow aspirate smears. Positive results were seen in two of 13 evaluable marrows from children with primary neutropenia and in eight of 44 evaluable marrows from adults with primary or secondary neutropenia. The BMIFT is simple enough to perform in most laboratory environments and provides serological information to support a diagnosis of immune neutropenia when other means of investigation are precluded.

Maternal immunization to Gov system alloantigens on human platelets.

BACKGROUND: Immunization to platelet alloantigens can occur during pregnancy or after the transfusion of blood components. Platelet alloantibodies can cause neonatal alloimmune thrombocytopenia and posttransfusion purpura. Transfusion-induced alloimmunization to a novel platelet alloantigen system, Gov, expressed on the 175-kDa glycosyl phosphatidylinositol-anchored platelet glycoprotein, CD109, was previously described. This report describes three unrelated patients who were alloimmunized to Gov(a) or Gov(b) during pregnancy. STUDY DESIGN AND METHODS: Platelets were typed by using radioimmunoprecipitation for HPA-1a, -3a, -5a, -5b, Gov(a), and Gov(b) and by polymerase chain reaction-restriction fragment length polymorphism for HPA-1a, -1b, -3a, and -3b. Maternal sera were screened for platelet antibodies by using radioimmunoprecipitation and the antigen capture assay. RESULTS: Patients 1 and 2 were investigated after the diagnosis of neonatal alloimmune thrombocytopenia in their children, and alloantibodies specific for Gov(b) and Gov(a), respectively, were detected in maternal serum. Serum from patient 3, who had mild idiopathic thrombocytopenia purpura with no detectable autoantibody, was found to contain alloantibodies to Gov(b) and to HPA-5b, presumably as a result of immunization during pregnancy. Platelet typings confirmed that the patients were at risk for alloimmunization to the respective antigen. CONCLUSION: This report of three cases of maternal alloimmunization to antigens in the Gov system indicates that immunization can occur via placental transfer of antigen and that Gov system alloantibodies may be associated with neonatal alloimmune thrombocytopenia.

Examining technical aspects of the monoclonal antibody immobilisation of granulocyte antigen assay.

BACKGROUND AND OBJECTIVES: This study was an attempt to improve detection and characterization of alloantibodies to neutrophil antigens, using the monoclonal antibody immobilisation of granulocyte antigens (MAIGA) assay. MATERIALS AND METHODS: We explored the effect of different detergent concentrations, detergent class, stabilising additives, combinations of detergents, and use of a broad range of monoclonal antibodies on the results of the MAIGA assay. RESULTS: Non-ionic detergents, Triton X and Nonidet, were confirmed as the most suitable for solubilisation of the recognised, neutrophil-associated alloantigens. Anionic and zwitterionic detergents did not assist the demonstration and characterisation of new neutrophil alloantigens in the MAIGA assay. Some reproducible positive MAIGA assay results were obtained for antisera 01, 08, anti-RED, anti-NB2, and anti-NB1, using particular monoclonal antibodies. CONCLUSIONS: Although we are reluctant to suggest association of the targeted antigens with the particular glycoproteins identified by the capture monoclonal antibodies, NB1 glycoprotein may be closely associated, before or after antibody binding, with CD46, CD11b, CD50, and/or CD66b.

Granulocyte colony stimulating factor treatment for alloimmune neonatal neutropenia.

Granulocyte colony stimulating factor (G-CSF) treatment was successfully used in three preterm infants with alloimmune neonatal neutropenia (AINN). Two infants had persistent neutropenia despite treatment with intravenous immunoglobulin and random donor granulocyte transfusions for presumed sepsis. Neutrophil counts returned to normal with G-CSF treatment; the response was least convincing in one infant with fulminant necrotising enterocolits. It is suggested that treatment with G-CSF be considered early for the treatment of infants with AINN.

Abnormal neutrophil phenotype and neutrophil FcRIII deficiency corrected by bone marrow transplantation.

BACKGROUND: Neutrophils from a patient in first remission of acute myeloid leukemia were found to lack NA1 and NA2 alloantigens. This NA null phenotype was converted to the normal phenotype of NA1, NB2 by the transplantation of bone marrow from an HLA-identical sibling. To investigate the inherited or acquired nature of this rare phenotype, a combination of conventional neutrophil serology and recently developed restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) assays was used. STUDY DESIGN AND METHODS: Diagnosis, remission, and posttransplant patient peripheral blood samples were used for neutrophil phenotyping by granulocyte agglutination and immunofluorescence tests. The presence and dose of the gene for neutrophil Fc gamma RIIIb (Fc gamma RIIIB) were tested for with RFLP and Southern analysis and PCR-based RFLP tests. Plasma levels of circulating soluble Fc gamma RIII (sFc gamma RIII) were measured with radioimmunoassay. The sibling bone marrow donor and the patient's parents were also studied. RESULTS: RFLP analysis of DNA obtained from the patient at the time of diagnosis showed that she lacked the Fc gamma RIIIB gene for neutrophil Fc gamma RIII (i.e., Fc gamma RIIIb), but that, in DNA prepared from posttransplant samples, the Fc gamma RIIIB gene was present. Quantitation of plasma levels of soluble FcRIII (sFcRIII) demonstrated a complete absence of sFcRIII in the patient's pretransplant plasma. However, 20 units of sFcRIII were detected in the patient's plasma by 160 days after graft. Hair samples from the patient provided sufficient nonhematopoietic, genomic DNA to confirm that her genotype was NA0NA0. DNA prepared from lymphocytes of both parents and the sibling marrow donor was used to quantitate their Fc gamma RIIIB gene dose. The mother and brother had only one Fc gamma RIIIB gene each, while the father apparently had a normal complement of two Fc gamma RIIIB genes. CONCLUSION: In this case, an inherited absence of Fc gamma RIIIB gene in a patient with acute myeloid leukemia was unintentionally corrected by the transplantation of bone marrow from a sibling donor who himself carried only one Fc gamma RIIIB gene.