The fundamentals of neutrophil antigen and antibody investigations

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Abstract

Background

The neutrophil population makes up the largest proportion (40–70%) of white blood cells and is crucial to our innate immune response. An unexpectedly significant proportion of neutrophils (˜28%) is located in the pulmonary circulation. This is an immune system front line wherein neutrophils guard an important interface between the external environment and the body. Neutrophils migrate to sites of infection and/or inflammation, where they phagocytose and destroy foreign microorganisms and mediate inflammation and eventual healing. Therefore, a decrease in neutrophil numbers often results in increased susceptibility to infection. Sustained or transient immune neutropenia occurs when neutrophil-reactive alloantibodies or autoantibodies destroy neutrophils, or significantly compromise their function. Neutrophil-reactive antibodies are capable of activating the neutrophil's microbicidal arsenal with resultant damage to the surrounding tissue.

Antigens and Antibodies

There are currently eight well characterized human neutrophil antigens (HNA): HNA-1a, HNA-1b, HNA-1c, HNA-2a, HNA-3a, HNA-3b, HNA-4a and HNA-5a. Only HNA-1a, HNA-1b and HNA-1c are specific to neutrophils, whilst the others are also expressed on other cell types. It is important to remember that neutrophils also express HLA Class I antigens, and that there is some evidence that adequately activated neutrophils may express HLA Class II antigens. This provides an interface between the innate and adaptive immune response. Therefore, both HNA and HLA Class I and II antibodies are capable of reacting with neutrophils in vivo and in vitro.

Routine Antigen and Antibody Investigation

Antigen genotyping protocols are now available for HNA-1a, HNA-1b, HNA-1c, HNA-3a, HNA-3b, HNA-4a and HNA-5a. The serological examination of neutrophils is reliant on harvesting a representative, pure population of neutrophils, which is difficult because neutrophils are short lived (6–8 h) and fragile. The International Granulocyte Immunobiology Workshop recommends the use of both the granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT), complemented by the monoclonal antibody immobilization of granulocyte antigen (MAIGA) assay. The GIFT uses paraformaldehyde-fixed neutrophils and detects the binding of antibodies to surface HNA and HLA antigens. Serological reaction in the GAT is reliant on viable neutrophils. It demonstrates the ability of antibodies to sensitize neutrophils and stimulate them to undergo chemotaxis towards other sensitized neutrophils, thus forming microscopic agglutinates. The MAIGA is useful in confirming antibody specificities as it employs monoclonal antibodies to known HNA epitopes. To screen for neutrophil-reactive antibodies, serum or plasma is exposed to a screening panel of neutrophils in the GIFT and GAT. To differentiate between HNA and HLA antibodies, the simple strategy of using platelet pools to absorb out HLA antibodies can be used. Specificity is determined using the GIFT and GAT, and a phenotyped and genotyped neutrophil panel and confirmed with the MAIGA.

Application

The combination of the GIFT, GAT and MAIGA was originally developed for the reliable investigation of immune neutropenias. However, in the last decade these techniques have become fundamental to the investigation of immune mediated TRALI where neutrophils are a key target and effector cell.

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