Specialized Methods


Methodologies / Instrumentation


 

The International Society of Blood Transfusion (ISBT) - Granulocyte Immunobiology Working Party (GIWP) has strongly recommended the use of both the granulocyte immunofluorescence test (GIFT) AND the granulocyte agglutination test (GAT) techniques in the detection of granulocyte (neutrophil) antibodies. It is of vital importance to use both the GAT and GIFT methods when detecting HNA antibodies. International Granulocyte Immunology Workshop (IGIW) external quality assessments have demonstrated that HNA antibodies detected by GAT have NOT BEEN DECTECTED by GIFT. Two IGIW assessments have included HNA-3a antibodies where laboratories using GIFT alone failed to detect antibody to HNA-3a samples involved in TRALI fatalities.

Both of these laboratory assays require viable intact granulocytes for detection and identification of human neutrophil antigen (HNA) antibodies. Granulocytes are intrinsically designed to respond to physiologic priming signals, so they must be handled very carefully. Once activated it is impossible to interpret any serologic test result involving these cells, as false positive results abound. Granulocytes are also extremely labile and must be used within 24 hours following collection. This necessitates ready access to panel cell donors with known HNA types. Finally, the presence of HLA antibodies in the test sera can make identifying HNA antibodies difficult as granulocytes also express HLA class I antigens on their cellular membrane. This obstacle can be overcome with the use of the monoclonal antibody immobilization of neutrophil antigens (MAINA) technique.

The isolation of a pure granulocyte suspension from peripheral blood is accomplished using the Ficoll-Hypaque discontinuous gradient technique. The double-density gradient centrifugation of leukocyte rich plasma yields a granulocyte suspension with a purity of 90 - 99%. Preparing a panel of donors to include all known HNA currently defined by ISBT using a suspension of pure granulocytes will aid in the detection and identification of HNA antibodies.

 


Laboratory techniques used in the American Red Cross National Neutrophil Immunology Laboratory to detect HNA and HLA Class I antibodies.


 

Granulocyte Aggulation Test (GAT): This test is biphasic and is based upon the instrinsic response of granulocytes to aggregate when stimulated by antibodies reacting to cognate cell surface antigens. The resulting aggregation is the consequence of the granulocyte activation that occurs during the sensitization phase, which induces the granulocytes to form pseudopods and slowly migrate toward one another during the aggregation phase, until membrane contact is established. GAT provides the best observation as to how the antibodies affect PMNs in vivo, as the observed aggregation is the result of viable PMNs becoming sensitized by antibodies that lead to chemotaxis and producing homotypic PMN:PMN agglutinates that are distinct from passive cross-linking via IgM. Serum or plasma from the specimen being analyzed is incubated with a granulocyte suspension. The reactions are evaluated using an inverted-phase microscope. These reactions are graded based upon the percentage of granulocytes involved in the aggregates. Both IgG and IgM antibodies are detected by this method.

 

Granulocyte Immunofluorescence Test (GIFT): Normally, immunoglobulins are present on neutrophil surfaces but these proteins are nonspecifically attached via the granulocyte Fc receptor and can be removed by the use of paraformaldehyde. Paraformaldehyde is also used to stabilize the cell membrane. Serum or plasma is incubated with an optimized concentration of granulocytes. During this incubation HNA reactive antibodies will bind to their cognate antigen epitopes. After a wash step that removes unbound antibodies, the granulocytes are then incubated with F(ab')2 fragments of a fluorescent conjugated anti-human antibody. The assay's performance is optimized with the use of a fluorescent secondary probe that can detect IgG, IgM and IgA antibody isotypes (to ensure the detection of both primary and secondary immune responses) and the use of F(ab')2 lg fragments to prevent the probe from binding high concentrations of Fc receptors that may remain on the granulocyte surface membrane following paraformaldehyde treatment. The granulocyte go through another wash cycle and analyzed for HNA antibodies using a flow cytometer

 

MAINA: The monoclonal antibody immobilization of neutrophil antigens (MAINA) assay allows the detection of antibodies to specific neutrophil membrane glycoproteins. MAINA relies on the capture of neutrophil-specific antigen - antibody (ag-ab) complexes by a murine monoclonal antibody onto a solid-phase surface. The benefits of this test are twofold; first, this is currently the most sensitive assay available for the detection of granulocyte antibodies, and second, the assay is designed to detect only HNA antibodies even when HLA antibodies are present in the test specimen. The disadvantage of this procedure is its' complexity.

Granulocytes are incubated with test serum. This granulocyte suspension is then washed to remove any unbound immunoglobulins and incubated with a murine monoclonal antibody conjugated to a specific neutrophil glycoprotein. After another wash step, the granulocyte membranes are disrupted in a mild detergent and centrifuged. The resulting lysate is then transferred to polystyrene microwells coated with anti-mouse immunoglobulins and incubated. The subsequent tri-molecular complex of neutrophil antigen - patient HNA antibody - murine monoclonal antibody (ag-ab-ab) present in the lysate is captured onto the solid phase, whereas any HLA antigen- antibody (ag-ab) complexes (if present) are remove with a subsequent wash step. Remaining complexes are then detected by the addition of anti-human IgG conjugated to horseradish peroxidase followed by incubation with substrate resulting in a color change for specimens positive for HNA antibody. The reaction is then analyzed with a spectrophotometer.

 

HLA Antibody Screen, Class I, IgG: The presence of HLA antibodies in a patient's specimen can make identifying HNA antibodies difficult as granulocytes also express HLA class I antigens on their surface membrane. If no HLA Class I antibodies are detected by this method it suggests that the antibody identified in GAT and/or GIFT is granulocyte specific. If HLA Class I antibody is detected, utilizing the MAINA technique will verify if HNA antibody is also present.

This test is a qualitative solid phase enzyme linked immunosorbent assay (ELISA) that uses microwells coated with affinity purified HLA Class I glycoproteins. During the incubation of the patient's sample HLA Class I antibody, if present, will bind with its' cognate antigen epitope. A wash step will remove unbound antibodies from the microwells and anti-human IgG conjugated to an enzyme is added followed by another incubation step. This anti-human conjugate reagent will attach to the solid phase matrix if antibody to HLA Class I was bound during the initial incubation and will be detected with the addition of a substrate reagent. The reaction is then analyzed with a spectrophotometer.

 

Neutrophil Typing: Typing HNA antigens or alleles can be ascertained by using serologic techniques (GAT and GIFT) for the former or molecular methods for the later. Neutrophil typing can be valuable when confirming HNA antibody specificity previously detected in patient samples, in cases of alloimmune neonatal neutropenia (ANN) where parental HNA types provide information on the probable risk of affected neonates, and in identifying blood donors that are at high risk of producing HNA-3 antibodies which have been implicated in severe or fatal cases of TRALI.

DNA based genotyping is performed using sequence specific primer- polymerase chain reaction (SSP-PCR) techniques. These methods can determine the genotype of patients for the following neutrophil antigen systems; HNA-1, HNA-3, HNA-4, and HNA-5. Advantages of using genotyping methods over serologic methods are related to the specimen tested, as sample age, handling, storage and shipping are less stringent. Genotyping is also widely used and is considered the gold standard technique in neutrophil typing.

  • HLA Antibody Screen for Transplantation, Class I & II
  • HLA Antibody Identification, Class I, lgG
  • HLA Antibody Identification, Class II, lgG: These assays are used for the detection and identification of HLA Class I and HLA Class II antibodies associated with transfusion-related acute lung injury (TRALI). These assays are performed by the American Red Cross National HLA laboratory using the LABScreen® Mixed and LABScreen® Single Antigen test systems.
 

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