Clinical applications may be divided into those which are cellular and those which are humoral.

Cellular Applications

Cellular studies with flow cytometry have not to date produced a direct in vitro assay of acute or chronic rejection. The major use of immunophenotyping has been to monitor the administration of Anti Thymocyte Globulin (ATG) to transplant patients. The use of dual platform assays as shown in the figure allows live gating, elimination of monocytes, and the determination of the T cell (CD3+) cells within the lymphocyte pool. By a total white blood cell and lymphocyte count the number of CD3+ T cells may be determined. A level of 50 cells per ul is used as a threshold for treatment.

With the availability of polyfluorescent beads absolute counting technology has expanded and single platform analyses have been introduced. Beads provide an absolute control of cell numbers and are now used for T cell monitoring in transplantation.

Humoural Antibodies

Humoral immunity in transplantation has been shown to have particular relevance to graft survival. There are a number of flow cytometric techniques which have been used. In antibody screening before transplantation, panel reactivity is used in a flow cytometric patient IgG binding assay against EBV transformed B cell lines or more recently in a PRA flow system where specificity to Class I or Class II antigens is defined. With the interest in determination of the specificity of antibodies in pretransplant sera multiplex bead kits are now available for identifying the specificity of antibodies both before and after transplantation.

Whilst ATG therapy is able to control most rejection episodes, some patients lose their grafts and require further therapy for control of rejection. We have developed a DUPLEX assay where antibodies to ATG or ALG are detected. This assay uses two different sizes of beads with either ATG (rabbit) or ALG (horse) bound to their surface. After incubation with test sera the beads are washed and an anti human IgG FITC conjugate is added to detect the antibodies. 6 AB control sera are used as negative controls in the assay.

Flow Cytometric Crossmatch

This assay is used in all almost all transplant centres for determining the presence of IgG antibodies to donor cells. The test is performed immediately before transplantation and involves the incubation of donor cells with the sera from potential graft recipients. After the washing of the cell / sera mixtures anti human IgG which is FITC conjugated is added. After washing anti CD3 PE is added to identify T cells , or anti CD19 (CD20) PE to identify B cells. Dual colour flow cytometric analysis allows the idenfication of T or B cells with the FL2 channel and the degree of FITC binding of the anti-IgG to any IgG antibodies from the test sera. Normally 6AB control sera are used as neagtive controls and a high reacting serum is added as a positive control. Comparative studies with the binding assay for IgM have shown that the conventional cytotoxic crossmatch is more sensitive. Considerable care in performing the test is required particularly with regard to the washing of the cells.

Developments of the test include 1. Three colour crossmatching with CD3PE, CD19(20) PERCP(ECD), and AntiIgG FITC. 2. The use of pooled lymphoblastoid cell lines to examine panel reactivity.

Research Applications

It is impossible to list the contribution that flow cytometry can make to research in transplantation. Immunophenotyping for activation of lymphocytes and expression of cytokines and their receptors (to define Th1 and Th2 cells) have been reported. In studies with cultured cells for in vitro studes purity of transfectants, adhesion and migration assays using flow cytometry have been reported. Immunosuppressive drug induced apoptosis has also been reported.