Circulating HLA antibodies may form in a sensitization process through prior exposures to blood transfusions, pregnancy, organ transplantation, or use of a ventricular assist device. Transplant candidates with preexisting HLA antibodies not only are at risk of hyperacute and acute antibody-mediated rejection very early after transplantation, but also have lower rates of longer-term survival as well. Sensitive new bead methods now allow detection of circulatory antibodies and use of those antibody specificities to define "unacceptable" donor HLA antigens. The role of non-HLA antibodies in acute and chronic rejection and treatment strategies before transplant (desensitization), or when antibodies are detected after transplantation, is becoming better understood but remains in evolution.
The techniques used for identification of HLA antibodies have evolved significantly in recent years. The serological lymphocytotoxic assay for antibody screening is based on mixing patient serum (unknown) with a panel of cells whose HLA typing is known and adding complement (complement-dependent cytotoxicity or CDC assay). If antibodies are present in the patient serum that react with the cell's HLA molecules, cell death occurs because of complement activation. This method is limited to detection of antibodies represented by the panel of antigens tested and by issues with sensitivity and accuracy. The specificity of the antigen to which the antibody is reacting (HLA or non-HLA antibody) is sometimes not clear because of the presence of other proteins in the patient serum, or the antibody isotype (IgG or IgM, etc; some antibody isotypes do not fix complement), and the titer (low, high) is not determined. Several enhancements of this assay were developed to address these issues: (1) heat or chemical treatment of the patient serum to inactivate IgM antibodies and identify IgG antibodies, which were felt to be more important to outcomes; (2) additional washes of the target cells following incubation of the patients serum to "wash off" the nonspecific reactants; and (3) addition of antihuman globulin (AHG) to the reaction to increase the detection of low titer IgG antibody (CDC-AHG method). There are other subvariations of these serologic enhancements.
The antibody-screening assay consists of a panel of known HLA-typed cells, with each cell representing a unique set of antigen targets for the serum to react. The assay generates the panel reactive antibody titer (PRA), which is reported out as a percent of the prospective donor pool that would likely be killed by the patient's serum. The result of this analysis is termed percent calculated PRA or %cPRA. The %cPRA gives a better indication of the patient's likelihood of having a compatible offer of a UNOS deceased donor organ because it is based on actual UNOS-typed donors. The panel may consist of any number of cells; however, minimally, cells from at least 30 carefully selected individuals are needed to cover the most common HLA antigen targets. In addition to the %PRA, the specificity of the antigen targets could be identified based on the individual cell reactions. The more different HLA antigens to which the patient is sensitized, the higher the %PRA and the less likely the patient is to have a compatible donor identified. Using conventional matching criteria, %PRA greater than 10% and greater than 25% have been associated with incrementally lower survival large registry reports.
Despite the use of enhancing techniques, serological antibody screening and methods of identification are not able reliably to detect low levels of HLA antibody, and are relatively poor for characterizing Class II antibody.
An ELISA plate method was the first solid-phase assay developed. In this assay, solubilized known HLA antigen protein targets are captured onto the surface of the wells of a microtiter plate. Patient serum is reacted with these proteins in each respective well. A secondary antihuman IgG antibody conjugated to a colorimetric reporter molecule is then added to the wells. This allows quantitative spectrophotometric determinations with a threshold absorbance used to distinguish a positive versus negative antigen–antibody reaction. Two additional hallmarks of the ELISA assay are the ability to: (1) objectively evaluate and quantify HLA antibody reactivity; and (2) eliminate the need for dependence on complement fixing for antibody detection, making ELISA a complement-independent assay. The colorimetric ELISA assay was able to evaluate better the presence of HLA Classes I and II antibody than the CDC-AHG method. Key faults of the ELISA assay are: (1) sensitivity of the colorimetric detection methods; and (2) purity and reliability of the solubilized HLA antigen captured in the well, because HLA can change its shape and thus antibody reactivity when bound to the plate.
The development of solid-phase microbead-based flow cytometry assays represents a monumental improvement from the initial solid-phase ELISA antibody screening and identification techniques. The use of fluorescent dyes is foremost as report molecules, allowing detection of the antigen–antibody reactions using a flow cytometer. Fluorescent dye light emissions are severalfold more sensitive than colorimetric dyes. As with the ELISA assay, the detection of the antibody–antigen reaction is made using beads coated with HLA molecules. Each HLA molecule-specific bead is identified by its unique mixture of two fluorescent dye colors incorporated into the bead. A secondary antihuman antibody is conjugated to a reporter molecule, in this case, a fluorescent dye of another color. The higher the titer of the antibody in the patient's serum, the more antibody is available to react to the antigen conjugated to the bead, and the more intense is binding of the secondary flouresceinated antibody. The more reporter dye becomes bound to the bead complex, the more fluorescent emission is produced from the bead when analyzed by the flow cytometer. The fluorescent emission signal of each bead type is then averaged and the normalized value is reported as the mean fluorescence intensity (MFI) of the bead. For most laboratories, the MFI value greater than or equal to 1000 is considered to be a positive reaction for the presence of the HLA antibody. This cutoff was derived in a similar fashion to that for the ELISA assay, namely, twice the MFI value for the negative control serum.
This resultant value is called the shift median channel value (sMCV). The sMCV cutoff for a positive versus negative flow crossmatch is statistically determined by each HLA laboratory initially and periodically by crossmatch of up to 100 individuals with serum that does not contain HLA antibody (negative). Shift values of less than or equal to two standard deviations from the mean of these histograms median channel value is considered to be negative, between two and three standard deviations to be equivocal, and three or more standard deviations to be positive.
The HLA antigen is conjugated directly to the microbead, not simply captured, and the source of which is recombinant cell lines. This allowed manufacturing of beads coated not only with the antigens of a single individual mimicking a cell (multi-antigen beads), but also with a single HLA antigen (single antigen beads). These single antigens can be further described to the exact HLA allele of that antigen. Because individuals become sensitized to the amino acid epitopes encoded by the antigen allele, microbead-based antibody analysis opened a completely new level of insight into characterizing antibodies contained in patient serum. This process is called epitope mapping. In combination with allele level typing of patients and potential donors, epitope mapping of antibodies can better predict outcomes of transplants in highly sensitized patients. The flow cytometer instrument may be either a larger instrument, which can acquire data from cells or beads, or a mini-flow cytometer, which only acquires data from beads, such as a Luminexx instrument (Fig. 63-11).
Solid-phase microbead-based assays for HLA antibodies have improved sensitivity and specificity of detection. Single HLA antigens from recombinant cell lines are conjugated to a colored microsphere. The bead is reacted against candidate or recipient serum, resulting in the specific adherence of circulating antibody to the selected HLA antigen. The microsphere HLA–serum antibody is reacted to an antihuman IgG antibody conjugated to a fluorescent dye. The microsphere complex is channeled through a flow cytometer and mean fluorescence intensity (MFI) for the combined emitting dye is measured. (Adapted with permission from Luminexx Corporation, Austin, TX.)
Using solid-phase HLA assays (SPA) or flow cytometric assays, the laboratory can easily and reliably test for patient antibodies to HLA-A, -B, C, DRB1, DRB3, DRB4, DRB5, DQB1, DQA1, and DPB1.
The results of the serum antibody test and the donor crossmatch are interpreted together as a final assessment of recipient and donor compatibility. Composite MFI values of 4000 or greater are generally predictive of a positive flow crossmatch. This composite MFI value is derived from the sum of MFI values and the single antigen–antibody analysis for each donor target antigen present, either Class I or II. This is a rule of thumb, as cellular expression of these target antigens varies. Most centers post unacceptable antigens in UNOS for those single antigens with MFI values of 4000 or greater, because these single antigens alone may result in a positive flow crossmatch with donors. Based on these MFI values, a virtual or paper crossmatch can be reliable in predicting the actual cellular crossmatch, especially with high MFI values. In addition, unacceptable antigens can be identified by virtual crossmatch (discussed in the following), excluding a particular donor that expresses certain donor antigens from consideration for a recipient who is sensitized against one or more of those antigens.
Crossmatch of patient serum with potential donors is no different than performing antibody testing.
Practical realities of matching sensitized candidates with nonlocal donors resulted in the acceptance of the virtual crossmatch (VXM). The VXM is a comparison of the donor HLA genotype determined by organ procurement organizations routinely as part of the donor (heart, lung) organ distribution process to the gene families represented by beads that bound antibody from the sensitized candidate recipient. If gene families are shared between donor and the bead, the VXM is positive. For example, a candidate with antibodies against A1, A11, and B7 by Luminexx single antigen beads would be incompatible with a donor typed as A11, A25, B55, and B57.15 The anti-A1 antibody determined by SPA corresponds to the donor typed A1. Although currently there is no method to determine the functional characteristics of the antibodies, recently it was estimated that the positive predictive value of an incompatible VXM compared with cytotoxic crossmatch was nearly 80%. Most centers refuse nonlocal donors based on an incompatible VXM and insist on a prospective CDC-AHG crossmatch for sensitized patients when possible.
It has become increasingly clear that non-HLA antibodies that can cause injury in thoracic organ transplants.16,17 About 16% of HLA antibody-negative heart recipients may lose their graft to the mixed diagnosis of primary failure within 30 days of transplant.18 SPA does not detect non-HLA antibodies, but flow cytometry methods can detect MICA/B. Antibodies-to-donor endothelial antigens are the largest poorly discussed group of clinically important non-HLA antibodies. These include endothelial, autoantibodies and those to MHC Class I chain A (MICA) and B (MICB). Endothelial antigen targets may exist, constitutively or as induced autoantigens because of activation of the endothelium. MICA and MICB are polymorphic antigens expressed on epithelial and, to an unknown extent, on endothelium. MICA antibodies may occur in up to 20% of candidates and have been associated with poorer survival but not increased rejection.18
Autoantibodies against conserved (non-polymorphic) proteins are frequently found in the blood of thoracic transplant recipients in association with chronic rejection. Vimentin (an intracellular cytoskeletal protein found in vessel walls and activated lymphocytes), cardiac proteins (cardiac myosin), and collagen type 5 (expressed mainly in the lung) are among the antigens against which autoantibodies have been described. Antivimentin antibodies form earlier than anti-HLA after transplantation and are a response in up to 30% of heart recipients to exposure of antigens mounted on the surface of damaged and activated cells.19 Antivimentin antibodies reflect tissue injury but might also activate platelets and neutrophils.20 Antiheart antibodies exist preoperatively in some because of their primary cardiac disease. At present, it is difficult to know their true significance. Finally, IgM non-HLA antibodies are cytotoxic and can react to all leukocytes, even the patient's own. Antigen specificity is unknown, as is the clinical relevance.
Once unacceptable antigens are determined from SPA, they can be entered into the UNOS website (http://optn.transplant.hrsa.gov/resources/professionalResources.asp?index=10). This will provide the cPRA. If the percentage chance of any donor will not be acceptable is greater than 50%, then it is reasonable to initiate a desensitization protocol. An optimal protocol has not been established. High-dose IVIG (2 g/kg/over 2 days q 2-4 weeks), plasmapheresis (1.5 volume exchange ×5 days), monoclonal anti-CD20 B-cell therapy (rituximab 1 g IV weekly ×4), and in the past cyclophosphamide (1 mg/kg/day) have been used in various combinations.
At UCLA, plasmapheresis, IVIG, and rituximab reduced circulatory antibody levels from 70.5 to 30.2%. Heart transplantation of these candidates after negative CDC resulted in similar 5-year outcomes to control patients and untreated but high PRA patients (81.1%, 75.7%, 71.4%). Of interest, the freedom from CAV was 74.3, 72.7, and 76.2%, respectively.21
It has been agreed that patients waiting for transplantation with circulating HLA antibodies should be studied every 3 months, and those desensitized every 2 weeks after therapy. Patients on VADs or who receive blood transfusions or with infections should be closely monitored as well. After transplantation donor-specific antibody monitoring is recommended at regular intervals and when a humoral rejection event is suspected. Donor-specific titers should be measured daily for 1 to 2 weeks and frequently thereafter in desensitized patients and those considered high risk for antibody response. This higher-risk group of recipients is generally treated with thymoglobulin plus IVIG, plasmapheresis and/or rituximab. Maintenance immunosuppressive therapy that has best controlled cellular and antibody-mediated rejection includes tacrolimus, mycophenolate mofetil, and prednisone.22
Hyperacute antibody-mediated rejection (AMR) became extremely rare with improving crossmatch techniques, but clinical interest in AMR was renewed in 1990 by Halloran and associates who described features of pure AMR in a few renal transplant recipients.23 Later, C4d became established as an important histologic associate.24 The incidence of AMR after cardiac transplantation is uncertain because of a lack of universal screening in asymptomatic recipients. Symptomatic pure AMR without a component of acute cellular rejection occurs in 10 to 15% of cardiac transplant recipients, but AMR features have been reported in up to 40% of patients with acute cellular rejection (mixed rejection).25 Clinical symptoms of cardiac AMR are those common in heart failure using echo. A greater than 25% decrease in ejection fraction and an increase in left ventricular mass distinguished antibody-mediated rejection from cell-mediated rejection.26 Reduced R-wave voltage-conduction abnormalities, including bundle branch block, are electrocardiographic associates. As discussed, presensitization to HLA Class I or II antibodies predisposes to AMR.27 Those recipients who develop presensitized antibodies, especially donor-specific, risk AMR.28 Non- HLA antibodies, including those against cardiac myosin, vimentin, and endothelial cells have been less often associated with AMR.29–31 Cardiac AMR occurs early (weeks to months) after transplant and, if avoided early, rarely occurs later.32 Classically, histologic diagnosis of cardiac AMR has included evidence of endothelial swelling and activation macrophages in the graft plus confirming immunofluorescence or immunoperoxidase staining for immunoglobulin (IgG or IgM) (Figs. 63-12 and 63-13). Although absence of immunofluorescence seems reasonable to rule out AMR, capillary swelling (63%) and macrophage vascular adherence (30%) do not33 and complement (C3d and C4d). Non-AMR causes of C4d staining include organ reperfusion injury, immunosuppression, treatment with monoclonal antibodies, and viral infections.34 To date, the distribution (diffuse versus localized) and intensity of the stains has not found a standardization for grading. The suggested algorithm for AMR includes: (1) Present clinical evidence of cardiac graft dysfunction and histologic evidence with or without interstitial edema and hemorrhage; and (2) positive immunofluorescence or immunoperoxidase for donor-specific antibodies. This creates further questions about the importance of asymptomatic (allograft function conserved) AMR-associated histopathology. It is likely that AMR is a pathologic continuum with clinical involvement in later or more aggressive forms. Asymptomatic AMR has been related to worse cardiovascular outcomes and the presence of cardiac allograft vasculopathy (CAV).35,36
Histologic evidence of AMR-associated endothelial swelling, inflammation, and thrombosis accompanied by interstitial edema and hemorrhage (H&E staining). (Reproduced with permission from SA Webber.)
Specific C4d deposition on a lung transplant allograft in a recipient with circulating HLA antibody is continuous and subendothelial in capillaries and small arterioles (left). Specific C4d has been seen on 31% of transbronchial biopsies in recipients with HLA-Ab (middle). (Ionescu DN: Transplant Immunol 2005; 15:63-68.) In contrast, nonspecific staining of the interstitium and elastic layer is depicted (right). (Reproduced with permission from KR McCurry.)
Although a clinical phenotype for AMR after lung transplantation and its response to anti-antibody regimens has been discussed, unfortunately, no strong consensus exists regarding its diagnostic characteristics.37 Recently, the Washington University program evaluated the effect on donor-specific HLA antibodies (DSA) and bronchiolitis obliterans syndrome (BOS) in 65/116 recipients developed DSA. Antibody-depleting therapy with IVIG, with or without rituximab, resulted in no differences in the incidence of acute rejection, lymphocytic bronchiolitis, and BOS between those with and those without antibody.38
Gene Expression Profiling
A DNA microarray-based real-time polymerase chain reaction–derived bio signature for cardiac rejection has been developed by XDx, the maker of allomap molecular expression testing (XDx, Brisbane, CA).39 Peripheral blood mononuclear cells provided a panel of 11 informative genes that were associated with acute cellular rejection.40 Several pathways were identified that participate in regulation of effector cell activation, trafficking and morphology, platelet activation, plus corticosteroid sensitivity. These included PDCD1 and ITGA4 for T-cell activation and migration, ILIR2 steroid-responsive gene, the decoy for IL-2, and WDR40A plus CMIR of the micro-RNA gene family. Peripheral blood samples are assigned a score with higher numbers associated with progressive risk of lack of immunologic quiescence. This approach was recently randomized against surveillance endomyocardial biopsy in a multicenter trial in low-risk rejection cardiac recipients, 6 months to 5 years after transplantation.41 The evaluation excluded recipients with a significant history of rejection, CAV, or allograft dysfunction. Results indicated 14.5% of patients profiled versus 15.3% of those surveilled by endomyocardial biopsy reached a composite endpoint of rejection with hemodynamic compromise, graft dysfunction owing to other causes, death, or retransplantation. The low risk for rejection of the enrolled patients made interpretation to earlier and higher-risk recipients problematic.42 Other reports found that the profiled genes regulatory T-cell homeostasis and corticosteroid sensitivity can distinguish mild from moderate and severe rejection and are evident before histologic, detectable rejection.43,44
Functional Activity of Immune System
A recipient's individual immune responses are affected by their susceptibility to immunosuppression, their clinical state, genetic background, age, gender, and diet. An assay of cell function would be useful to quantitate the dynamic positive and negative regulatory responses of the immune system. Clinicians have struggled, often in relative ignorance, to titrate doses of immunosuppressive drugs to achieve levels that deter the alloresponse while also minimizing the risk of infection. A long-imagined goal of selective treatment based on a quantitative assessment of the net state of immunosuppression has been approached clinically with some success. An assay has been developed (Cylex, ImmuKnow, Columbia, MD) to measure the intracellular concentration of adenosine triphosphate (ATP) of CD4+ T cells. ImmuKnow measures T-cell responses by quantifying ATP activity45 to phytohemagglutinin, a T-cell mitogen. In general, the collective studies suggest that ATP levels less than 100 ng/mL correlate with an increased risk of infection.46 In a study in 296 heart recipients spanning 2 weeks to 10 years posttransplant, infection in 39 recipients occurred with an average 187 + 126 ng/ATP/mL versus a steady state of 280 + 126 ng/ATP/mL (Fig. 63-14). Rejection scores in eight recipients averaged 328 ng/ATP/mL and did not differ from baseline. However, 3 of 8 with antibody-mediated rejection scored 49/I-12/ng/ATP.46 This assay opens the field to personalized immunosuppression that might help balance risks of infection and rejection.
Immune monitoring scores are calculated from adenosine triphosphatase (ATP) released from activated lymphocytes (ImmuKnow; Cylex, Columbia, MD). The hazard curves above are plotted for risk of infection versus odds for rejection in 296 heart transplant recipients (864 assays). In theory, the crossing point defines a score that minimizes infection and rejection. (Adapted with permission from Kobashigawa JA, Kiyosaki KK, Patel JK, et al: Benefit of immune monitoring in heart transplant patients using ATP production in activated lymphocytes. J Heart Lung Transplant 2010; 29:504-508.)