Nelson H. Tsuno, Department of Transfusion Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan E-mail: email@example.com
Human neutrophil antigens (HNA) are involved in the pathogenesis of a variety of clinical conditions, such as neonatal immune neutropenia (NIN), refractoriness to granulocyte transfusions, alloimune neutropenia after bone marrow transplantation, febrile transfusion reactions, and transfusion-related acute lung injury (TRALI). The accurate detection of granulocyte antibodies is essential for the diagnosis and prevention of the clinical conditions in which these antibodies are involved, especially TRALI. The international granulocyte immunology workshop recommends the use of granulocyte agglutination test (GAT) and granulocyte immunofluorescence test (GIFT) for the screening of granulocyte antibody, and the use of monoclonal antibody-specific immobilization of granulocyte antigen (MAIGA) assay for the determination of the antibody specificity. Presently, however, no single technique available is enough to detect all clinically relevant granulocyte antibodies. Here, we describe the advantages and disadvantages of the mixed-passive hemagglutination (MPHA) assay in the general context of granulocyte serology.
Human neutrophil antigens (HNA) are involved in the pathogenesis of a variety of clinical conditions, such as neonatal immune neutropenia (NIN), refractoriness to granulocyte transfusions, alloimune neutropenia after bone marrow transplantation, febrile transfusion reactions, and more recently, its involvement in the pathogenesis of transfusion-related acute lung injury (TRALI) was also confirmed.
HNA-1 locates on the Fcγ receptor IIIb, a member of the immunoglobulin superfamily, which is expressed specifically on neutrophils. HNA-2 has been denominated CD177, which is exclusively expressed on neutrophils, among the blood cells. HNA-3 antigen arises from a single-nucleotide exchange on the choline transporter-like protein-2. It is expressed on neutrophils and lymphocytes, but the expression on platelets is controversial. Alloantibodies to HNA-3a were associated with severe cases of TRALI. HNA-4 is located on the CD11b subunit of Mac-1 (CD11b/CD18), which is expressed on neutrophils, monocytes and natural killer cells. HNA-5 locates on the CD11a chain of LFA-1, expressed on all leukocytes.
The accurate detection of granulocyte antibodies is essential for the diagnosis and prevention of the clinical conditions in which these antibodies are involved, especially TRALI. However, presently no single technique available is enough to detect all clinically relevant granulocyte antibodies. The international granulocyte immunology workshop recommends the use of granulocyte agglutination test (GAT) and granulocyte immunofluorescence test (GIFT) for the screening of granulocyte antibody, and the use of monoclonal antibody-specific immobilization of granulocyte antigen (MAIGA) assay for the determination of the antibody specificity . GIFT is generally more sensitive than GAT, but a few antibodies such as anti-HNA-3a are best detectable by GAT. All these techniques need the use of intact and preferably fresh granulocytes, and for the preparation of panel cells, granulocytes with known specificities are required. Thus, the major obstacle for the screening and detection of anti-HNA antibodies is the absence of a methodology in which granulocyte panels can be preserved, and suspected sera can be tested in large scale.
In our lab, the mixed-passive hemagglutination (MPHA) assay, developed by Prof. Yoichi Shibata, has been used as the platelet serology method for a long time, and much knowledge is accumulated about this method. We have been attempting to adapt MPHA for the granulocyte serology, and here, our experience together with the related reports will be described, in the general context of granulocyte serology.
The MPHA assay
The method is based on the spontaneous sedimentation of indicator cells, consisting of sheep red cells coated with anti-human IgG, over a monolayer of platelets coated to the bottom of U-bottomed 96-well plates.
The MPHA assay is few-stepped and very simple, allowing the testing of a great number of samples in a single microtitre plate. Also, it allows the cryopreservation of platelet panels for periods longer than 1 year, without loss of antigenicity. Therefore, the collection of blood from volunteer donor every time the assay is to be performed can be avoided, and the panel of platelets can be prepared in advance to testing.
Some expertise is necessary for the judgment of the results, which is based on the comparison with positive- and negative-control wells, and the assay is affected by the co-existing anti-HLA antibodies, which recognize the antigenic determinant present on the target cells (platelets or granulocytes). The HLA antigenicity can be removed by the treatment of cells with chloroquine solution, as described below, but this treatment may result in the loss of other antigenic determinants, such as HNA-4 (CD11b).
Preparation of microtitre plates
Fresh granulocytes isolated from peripheral blood are coated to the bottom of U-bottomed 96-well microtitre plates. The cells are fixed by the glutaric fixative solution (glutaraldehyde/PBS solution (pH 6·0) (1:500 dilution)), and can be frozen-preserved until use in cryopreservation solution, consisting of 5% sucrose (pH 6·0).
As shown in Fig. 1, test sera are added to the wells of the 96-well microtitre plate, and antibodies are allowed to react with the respective antigens. After sera is washed-out, the indicator cells, consisting of sheep red cells coated with anti-human IgG (or anti-mouse IgG, in case of testing of monoclonal antibodies), are added to the wells. Indicator cells spontaneous sediment to the bottom of the U-bottomed plates, giving a clear button of indicator cells, which can be seen by the naked-eye. However, in case antibodies reactive with granulocyte antigens are present, antibodies coated to the surface of indicator cells react with them, and indicator cells do not sediment, and thus, the clear button of indicator cells is not seen. Usually, the results are judged in comparison with the positive and negative control wells, as shown in Fig. 2.
To remove the HLA antigenicity, which in most cases interfere in the determination of anti-HNA antibodies, granulocytes are treated with chloroquine solution for 2 h at room temperature. As shown in Fig. 3, HLA are removed from the surface of granulocytes and, theoretically, HNA remain.
Variations of MPHA
Some modifications can be applied to the MPHA in an attempt to improve its sensitivity/specificity. For example:
1 Use of extracted granulocyte antigens instead of intact cells: it was previously reported by Araki et al. . By this procedure, human antibodies specific for NA1 (now HNA-1a), NA2 (now HNA-1b), NB1 (now HNA-2a), 5b (now HNA-3a) and Sar-a could be determined, as well as monoclonal antibodies against CD11a, CD11b, CD13, CD16, CD18 and HLA class I. The extracted granulocyte antigen panels can be stored frozen for at least 1 year at −80°C.
2 Use of Terasaki plates instead of 96-well plates: it was also previously reported by Araki et al.  Granulocyte specific antibodies could be determined in the MPHA assay using Terasaki plate. The advantage of this plate is the need of small volumes of the samples for testing.
3 Use of magnetic beads instead of sheep red cells as the indicator cells: the assay using magnetic beads as indicator cells has been developed , and called magnetic (M)-MPHA. It is being applied to the field of platelet immunology. The great advantage of using these indicators is that the period of the assay can be shortened significantly. Indicator magnetic beads are pulled down to the U-bottomed plates, using a magnetic plate. Thus, the results can be judged in approximately 3 min, whereas the sheep red cells need more than 4 h to spontaneously sediment.
The MPHA assay is able to detect antibodies against HNA-1, HNA-2, HNA-4 and HNA-5. In the present condition, the sensitivity of the HNA3 system is quite low. The ISBT Working Party on Granulocyte Immunobiology  recommends the use of GAT and GIFT for the detection of HNA-3 antibodies, and both methods are very sensitive for their detection. Whereas in GAT, unfixed granulocytes are tested, and in GIFT, granulocytes are fixed with paraformaldehyde, in MPHA, cells are fixed with glutaric fixative. It may be one explanation for the low sensitivity of HNA-3 antibodies in MPHA, and there is need to test other fixatives or unfixed cells in MPHA. The sensitivity of MPHA for the detection of antibodies against HNA-1, HNA-2, HNA-4 and HNA-5 was comparable to that of GIFT.
The MPHA method is a sensitive method for the detection of platelet antibodies, and is largely applied in Japan and some Asian countries. We and others  are applying this method for the testing of granulocyte antibodies, but presently, it is not fully developed nor largely applied even in Japan. Modifications should be introduced to improve the detection ability of antibodies against HNA-3. Since Araki et al.  were able to detect the antibody against 5b (presently, HNA-3a), and they have used extracted granulocyte antigens, without fixatives, it is possible that the fixative agent affects HNA-3 antigenicity. Also, some antigenicities, such as HNA-4, can be lost by the treatment of granulocytes with chloroquine to remove HLA antigenicity. Therefore, the determination of HNA-4 antibodies may be disturbed in case of co-existing anti-HLA antibody.
Since the sensitivity of MPHA was quite similar to that of GIFT related to HNA-1, -2, -4 and -5, in case we are able to improve the sensitivity related to HNA-3, MPHA may be a promising method for the screening of anti-HNA antibodies. Use of magnetic beads in MPHA (M-MPHA) seems to improve sensitivity of anti-HNA-3 (unpublished data). In comparison with GIFT, the great advantage of MPHA is that it is very simple and allows the testing of a large number of samples in a single microtitre plate. Also, MPHA plates can be frozen-preserved for long periods of time, and especially those prepared with extracted granulocyte antigens  seem to be promising. Another issue to be addressed is the time necessary for the assay. The conventional MPHA needs approximately half day, whereas in GIFT, the results are obtained in a few hours. Use of Terasaki plates instead of 96-well plates or magnetic beads indicators instead of the sheep red cells significantly shortens the assay time.
The ISBT Working Party on Granulocyte Immunobiology  recommends the confirmation of the HNA specificity of the detected antibodies in an antigen-specific assay, including monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) assay. From our experience with the granulocyte immunology assays, we believe that, after appropriate modifications are introduced to MPHA, it will have an important role as a screening assay, similar to GIFT and GAT, in the granulocyte immunology field.
Recently, Yasui et al.  reported the establishment of gene transfected K562 cell lines that express HNA-1a, -1b, or -2a antigens. Later on, they established new cell lines that selectively express HNA-1c, -4a, -4b, -5a, or -5b antigens . These cell lines seem to have a low background, and lack the expression of HLA class I, HLA class II and HNAs. Therefore, they would be the ideal tool for the testing of anti-HNA antibodies, not only in flow-cytometry, but also in MPHA. In MPHA, the availability of cells without expression of HLA will importantly improve the method. In addition, when these transfectants become available, there will be no need to collect blood every time an assay is to be performed. Presently, undoubtedly MAIGA is the first option for the determination of the antibody specificity in granulocyte serology. In case platelet panels can be prepared using such transfectant cells, in addition to MAIGA, MPHA may be developed as an antigen-specific assay for the antigen determination, and we hope it will occur.