Histocompatibility testing seeks to identify appropriate donor-recipient pairs that will result in successful transplantation. Immune considerations for renal transplantation include ABO compatibility, HLA matching, and nondeleterious recipient antidonor immunity. Besides ABO compatibility, the pretransplant crossmatch result has been thought to be the most important procedure performed in the histocompatibility laboratory. However, it is also important to know whether the recipient serum immunoglobulin, responsible for causing the positive crossmatch, is an immunoglobulin G human leukocyte antigen (IgG HLA) antibody. Many crossmatch test results may be positive, but because they have been shown to be clinically irrelevant, they should not contraindicate the donor-recipient pairing for transplantation. Therefore, careful evaluation of the crossmatch test result and its clinical relevance (the presence of HLA antibody) is very important. Thus, ABO blood group compatibility and clinically irrelevant crossmatch results are absolute requirements for successful transplant outcome.

HLA class I and class II antigens serve as the primary targets for immune reactivity in solid organ transplantation. The transplant recipient’s immune response to donor (HLA) antigens is critical in determining the postoperative fate of the allograft. Exposure to HLA antigens from pregnancy, blood transfusions, or loss of a previous transplant could lead to a cellular or antibody-mediated immune response against these HLA antigens and subsequently to graft rejection and loss. Some patients display HLA antibodies in the absence of pregnancy, transfusions, or loss of a previous transplant. These antibodies are believed to be due to cross-reactivity between microbial epitopes and HLA antigens. No test is currently performed to measure pretransplant cellular immune reactivity that is used as a contraindication to transplant. Therefore, procedures are used to identify the presence of HLA or other alloantibodies in patient sera that could cause a clinically relevant positive crossmatch and would contraindicate the transplant.

It should be noted that the detection of (clinically) relevant antibodies causing positive crossmatches is technique dependent. During the past 35 years, test procedures have become more sensitive at detecting antibody reactivity and our understanding of the clinical relevance of a crossmatch has always been a work in progress. The premise that all positive crossmatches or reactive sera are correlated with poor graft outcome and all negative crossmatches and nonreactive sera are correlated with good graft outcome was not true. While many positive crossmatch patients did have poor graft outcomes, some also had good long-term survivals. Similarly, while many negatively reactive patients had good long-term outcomes, some also experienced early rejections and graft losses. Those results led to improvements in antibody detection and crossmatch procedures that resulted in a significant reduction in early rejections and graft losses.

Advances in immunosuppressive therapies and control of acute rejection episodes have led clinicians to ask whether detection of HLA antibodies or positive crossmatches are clinically relevant and/or represent a contraindication to transplant. At the end of this chapter the reader should appreciate that pretransplant patient sera that only presents with flow cytometry-detected IgG HLA antibody is at risk for rejection and that patients with donor-specific IgG HLA antibody, and a positive donor-specific flow cytometry
crossmatch, are at risk for rejection and a high frequency of graft loss (1). There is some preliminary evidence that the strength (titer) of the antibody present in the sera plays a role in the timing and severity of rejection and graft loss (2). Patients with either no or very little IgG HLA antibody may experience a small frequency of reversible rejections with little or no graft loss.

Methods used to detect the presence and donor-target reactivity of antibodies include membrane-dependent and membrane independent assays (Table 2.1). In membrane-dependent assays detected immunoglobulins and/or other serum materials bind to cell surface membrane receptors (including HLA). Membrane-dependent assays include the standard NIH complement-dependent cytotoxicity (CDC) assay, the Amos-modified CDC, the antihuman globulin-(AHG-)enhanced CDC, and the flow cytometry panel-reactive antibody (PRA) and crossmatch assay (3, 4, 5, 6, 7). These assays differ in their degree of sensitivity, temperatures, and incubation times for target cells with serum and/or complement, wash steps, and target cells tested (unseparated peripheral blood lymphocytes [PBL] or separated T or B cells), as well as the delineation of IgM or IgG reactivity. In contrast, membrane-independent assays utilize solubilized HLA antigens, rather than cell membranes. The HLA antigen targets are derived following platelet extraction of class I HLA, lymphocyte culture supernatants, or column-purified HLA class I and class II antigens. Solid phase assays detecting specific HLA antibodies include an enzyme-linked immunosorbent assay (ELISA) and microparticle-based technologies measured by flow cytometry and flow PRA (8, 9, 10). Both of these assays are more sensitive and specific than the cytotoxicity assays (CDC, AHG) in identifying antibodies to class I and class II HLA antigens (11). Finally, the interpretation of any crossmatch result must be made in concert with clinical patient information (primary disease, sensitizing events, primary or retransplant, etc.).

The standard microlymphocytotoxicity-CDC assay utilizes potential donor target PBL mixed in the wells of microtest trays with undiluted and 1:2, 1:4, and 1:8 dilutions of transplant candidate serum samples. The serum dilutions are used to test for the possibility of prozone or excessive antibody that could block cytotoxicity. The trays are incubated for 30 minutes to allow antibody binding to the cell-surface membrane antigen (if present in the serum). A volume of 0.005 mL of complement is added and the mixture incubated for 60 minutes to allow complement fixation to antigen-antibody complexes which results in cell-membrane lysis. To visualize dead and live cells under phase contrast microscopy, a vital dye, eosin or trypan blue, is added. Live cells exclude the dye and appear bright, while dead cells take up the dye and appear dark. A test is considered positive when there is at least a 20% increase in the percentage of dead cells in the test wells compared with control wells (3).

To be evaluated as a positive test, the standard CDC procedure requires that a lot of antibodies are present in the serum to bind target-cell antigens so that the antigen-antibody complex can fix complement resulting in cell lysis. If the test result is evaluated as negative, it could mean that either there were no antibodies present or that too few antibodies were present to lead to cell-membrane lysis. The standard CDC is, therefore, relatively insensitive in detecting subliminal antibodies and can result in a phenomenon of cytotoxicity negative but (antibody) adsorption positive (CYNAP) (12).

The simplest modification to the standard CDC assay was to extend the incubation time of cells and serum to 60 minutes and the incubation time for complement to 120 minutes. The binding of low avidity antibodies is then enhanced as well as the fixation of complement and cell death. This method detects alloantibodies but can also detect clinically irrelevant autoantibody activity. Amos et al (4) described a modification of the standard CDC assay which included adding a wash step (or several wash steps) after incubation of cells and serum and before addition of complement, which allows for elimination (washing away) of weakly bound antibodies (auto or allo) to target cells, as well as elimination of anticomplementary factors that could result in false positive CDC crossmatches.

The detection of CYNAP and other antibodies may be accomplished by the addition of a developing second-step antibody, an antihuman kappa light chain antibody. This AHG-enhanced cytotoxicity assay was first described by Johnson et al (5). The AHG crosslinks antibodies (IgM or IgG) for efficient (bivalent) C1q binding and initiation of complement-mediated lympholysis (13). This AHG technique also incorporates wash steps to rid the test wells of weakly bound serum factors before the addition of the AHG and complement. The AHG-enhanced CDC increases antibody detection two- to threefold. The AHG procedure has been shown to detect HLA antibodies and can detect noncomplement-fixing
antibodies (5,14). The AHG assay is usually performed using T-cell targets. However, the AHG assay can be performed by a two-color immunofluorescence technique wherein B cells are first labeled with fluorochrome-conjugated antiimmunoglobulin, thereby preventing the AHG from binding to surface immunoglobulin and permits B cells to be used as targets (15).

Garovoy (6) introduced the flow cytometry assay for detection of antidonor antibody reactivity. Flow cytometry does not rely on complement fixation or cytotoxicity and is more sensitive than either the standard CDC or AHG procedures (6,16). Flow cytometry is the measurement of cellular and fluorescent properties of stained cells in a fluid stream as they move past a set of fixed detectors. A flow cytometer can provide information concerning cell size (forward scatter), granularity or internal complexity (side scatter), and relative fluorescence intensity. Combining the two light scatter parameters separates cells into three distinct populations based on size and granularity (lymphocytes, monocytes, and granulocytes). Commonly used fluorochromes, such as fluorescein isothiocyanate (FITC) and phycoerythrin (PE) are used to label cells of interest. The flow cytometry detection of antibody bound to target cells is performed by incubating donor cells with potential recipient serum followed by addition of a fluoresceinated (FITC) goat, antihuman IgG or IgM reagent. In addition, a PE-labeled monoclonal antibody that detects B cells (CD20) and a peridinin chlorophyll protein-(PerCP-) conjugated monoclonal antibody that detects T cells (CD3) are added. This three-color combination simultaneously detects alloantibodies reacting with T cells and B cells and eliminates background binding due to natural killer cells and monocytes. Results are then analyzed by flow cytometry and expressed as positive or negative based on a shift in median channel fluorescence intensity of the test serum with respect to negative control or autologous serum.

The standard CDC, AHG-CDC, and flow cytometry assays are dependent on immunoglobulins binding to surface receptors. The read-out results may not always reflect HLA antibody reactivity unless specificity studies are performed to confirm that reactivity is HLA specific (17). Methodologies have recently been introduced that use specific soluble HLA antigens as targets immobilized in the wells of microtest trays, and immunoglobulin binding is assessed by ELISA technology, or as targets immobilized on polystyrene beads (flow beads), and immunoglobulin binding is assessed by flow cytometry (9,10). These methods are based on the binding of serum immunoglobulins (IgG, IgM, IgA) to soluble class I and class II antigens. They offer increased objectivity and reproducibility, and do not require viable target cells or target cell separations into T and B cells. Most important they allow for the identification of clinically relevant patient HLA antibody reactivity (11). They also allow for the accurate identification of pretransplant HLA antibody in patient sera (ELISA-PRA, flow-PRA). This will then allow for delineation of the appropriate clinical meaning of a crossmatch positive serum in the presence or absence of HLA antibody. Each laboratory will need to confirm the clinical significance of having patient sera that are AHG-CDC antibody and crossmatch negative in the presence of flow-PRA detected HLA antibodies and a positive flow cytometry crossmatch.

Serum-screening procedures serially test a patient’s sera for the presence of antibody reactivity. Patients who experienced hyperacute rejections and/or early graft loss, but did not display a pretransplant positive crossmatch, may have undergone an anamnestic (memory) response (18). Therefore, older serum samples should be tested in the crossmatch to insure a more complete evaluation of a patient’s serum antibody reactivity. With the appreciation that a patient’s antibody response could fluctuate over time (due to various sensitizing events), it is important that evaluation of a patient’s serum for reactivity be performed at various time points. Thus, sera collected from potential transplant recipients are tested against a panel of cells (NIH-CDC, AHG-CDC, flow cytometry) or a soluble antigen display (ELISA or flow beads) to identify (HLA) antibody reactivities. Results of the serum-screening tests are reported as the percentage of reactivity against the panel of cells (panel reactive antibody) or soluble HLA antigens (% PRA). The most clinically informative sera (the peak or historically highest PRA, a current and the pretransplant sera) should be tested during the crossmatch against specific donor HLA targets. This author believes that ELISA or flow-PRA detection of HLA antibody suggests a risk. The crossmatch procedure should be at the same level of sensitivity as the antibody detection assay. Therefore, if HLA antibodies are detected by ELISA or flow-PRA, the crossmatch method must also be flow cytometry.

When identifying antibody reactivity, it is important to be aware of clinically irrelevant results. A common cause of these irrelevant, but positive reactions are auto-lymphocytotoxic antibodies, that is antibodies present in a patient’s serum that are reactive with that same patient’s lymphocytes (19). These antibodies occur under various conditions that do not involve alloimmunization, such as in patients with infectious mononucleosis, autoimmune diseases, viral diseases, and in patients treated with antiarrhythmia drugs (procainamide) or the antihypertensive medication hydralazine. These antibodies are reactive to autologous lymphocytes; are mostly IgM in nature and of low avidity; could often be removed by a wash step; usually reacted more strongly in the cold or at room temperature; and reacted more frequently with B than T cells (17,18). The easiest test for the presence of autoantibody is to incubate patient sera with diothiothreitol (DTT) or dithioerythritol (DTE), which reduces disulfide bonds of the pentameric IgM complex but leaves the complement-mediated cytotoxicity of IgG intact (18). Since autoantibodies are often of the IgM isotype, their inactivation by the procedure is suggestive, but not proof of
non-HLA antibody activity. There is overwhelming evidence to suggest that autoantibodies, whether they react with B cells alone or with T and B cells, are not damaging to renal allografts even when they cause a positive crossmatch and in fact may be beneficial (17, 18, 19, 20, 21).