A predictive test for the efficacy of intravenous immunoglobulin in the inhibition of alloantibodies

Preformed recipient HLA-specific antibodies can cause hyperacute rejection of a transplanted kidney if they are directed against mismatched donor HLA antigens. To avoid hyperacute rejection it is essential that recipient antibodies be identified during patient workup for transplantation and HLA antigens to which a patient is sensitized then be avoided when selecting a kidney donor for them. Absence of donor-specific reactivity is then confirmed by a pretransplantation crossmatch test.     

Hyperacute and acute kidney graft rejection due to antibodies against B cells.

Because of the perception of its uncertain clinical significance, the B cell crossmatch is not universally performed before renal transplantation. Even though sporadic cases of hyperacute rejection associated with B cell antibodies have been reported, doubts remain in light of other studies suggesting no effect on graft survival. This report describes 4 cases of graft rejection (3 hyperacute and 1 acute) that occurred in patients with anti-B-cell antibodies specific against donor HLA-DR or DQ antigens. Absence of anti-donor class I antibodies was confirmed in all cases by 2-color flow cytometry. Strong evidence for an antibody-mediated mechanism was found in one patient with anti-class I and anti-class II antibodies in serum transplanted with a class II mismatched kidney. In this case, only anti-class II antibodies were recovered in the eluate of the nephrectomy specimen. These four cases were compiled from three different institutions over a four-year period, which confirms the infrequent occurrence of these events. While anti-class II antibodies may not always be detrimental for graft survival, these results also confirm that they have the potential to cause hyperacute or acute graft loss. We conclude that the information provided by the B cell crossmatch should be available at the time that a decision to proceed with a renal transplant is made.     

Successful renal allografts in recipients with crossmatch-positive, dithioerythritol-treated negative sera. Race, transplant history, and HLA-DR1 phenotype

Graft survival was examined in 15 renal allograft recipients from a group of 20 patients with IgM autolymphocytotoxic antibody that could be removed in a crossmatch assay using a reducing agent, dithioerythritol (DTE). The significant differences in this group of 20 patients compared with end-stage renal disease (ESRD) patients lacking autolymphocytotoxic antibodies included an increased frequency of black patients (P = 0.002), a lack of previous transplants (P = 0.003), and an increased frequency of the HLA-DR1 phenotype (P = 0.0001). Sex and the number of transfusions did not appear significant, whereas the cause of ESRD was primarily systemic lupus erythematosus. Fifteen of the 20 patients were transplanted against a positive donor crossmatch. Eleven were recipients of cadaveric kidneys, nine of which are still functioning for periods ranging from 0.5 to 40 months. Two fo the cadaveric recipients died with functional grafts. Four received living-related donor transplants, one of which was lost to acute rejection one month posttransplant, while the remaining three have survived 1.5, 9, and 21 months, respectively. Fourteen patients had immediate allograft function with no hyperacute rejection and only one case of acute tubular necrosis (ATN) was found. In summary, a negative crossmatch using DTE-treated, autologous reactive recipient sera may identify a group of patients who can be transplanted with minimal concern for hyperacute rejection or ATN. In addition to cause of ESRD, race, transplant history, and HLA-DR phenotype may further define this group of transplant candidates having IgM autolymphocytotoxic antibody. Extrapolation of these conclusions to transplant candidates lacking autolymphocytotoxic antibodies is not warranted.     

Hyperacute rejection in ABO-incompatible kidney transplantation: Significance of isoagglutinin subclass

IntroductionABO-incompatible transplantation has expanded the limited donor pool for kidney transplantation. Despite the successful desensitization protocols and immunosuppression, undesirable cases of hyperacute rejection occurs.ObjectiveFlow cytometry was used to measure isoagglutinin titer and its IgG subclasses in assessment of the cause of hyperacute rejection in ABO-incompatible kidney transplantation.Materials and methodsThe recipient was admitted for kidney transplantation due to end-stage renal disease. Pre-transplantation work-up for ABO-incompatible kidney transplantation included blood group typing, HLA DNA typing and HLA antibody analyses. HLA crossmatch analysis was conducted using donor lymphocytes and anti-HLA antibody assay using Luminex panel reactive antibody test (One Lambda, Inc., Canoga Park, CA). Desensitization protocol was composed of therapeutic plasma exchange sessions and rituximab.ResultsDespite negative HLA crossmatch results, a case of hyperacute rejection occurred after living donor kidney transplantation. Rejection resulted in immediate removal of graft, and the patient later received a second kidney transplantation. Retrospective evaluation of isoagglutinin titer and its subclasses using flow cytometry identified the cause of rejection to increased IgG1 subclass. Desensitization protocol for ABO-incompatible kidney transplantation now implements further caution for blood group O recipients.DiscussionHyperacute rejection resulting from increased IgG1 isoagglutinin subclass has not been previously confirmed using flow cytometry. Unfortunate outcome of this rejection case provides insight to how we should approach and ensure successful ABO-incompatible kidney transplantation.     

Alloantibodies against donor epidermis and early kidney transplant rejection

We have previously observed that transplant recipient sera with endothelial antibodies are bound to epidermal cells, as shown by immunofluorescence on sections of skin. It was also reported that early kidney failures that occurred despite negative T cell crossmatches were associated with, and could have been predicted by, a crossmatch with donor skin. Ninety patients undergoing kidney transplantation have now been evaluated using this technique. A few other patients were excluded from the analysis because of the presence of autoantibodies staining autologous skin. In 12 the crossmatch with donor skin was positive, and 9 of them had severe rejection within the first 10 days after transplantation. The three patients with a positive skin crossmatch and a benign course had not been tested with autologous skin and therefore autoantibodies could not be excluded. Only 7 of the 78 patients with negative skin crossmatches had early rejection. The correlation between skin crossmatch and early rejection was statistically highly significant (P less than 0.0001). Studies by flow cytometry have shown that these antigens are found on the surface of epidermal and endothelial cells, and are modulated by gamma interferon. When tested against a panel of skin donors, skin alloantibodies gave different patterns of positive and negative reactions, suggesting polymorphism.     

Hyperacute graft dysfunction in an orthotopic heart transplant in the presence of non‐HLA antibodies

Antibody‐mediated rejection (AMR) in heart transplants in the absence of anti‐HLA donor‐specific antibody (DSA) is not well studied or documented. This case reviews hyperacute fulminant graft dysfunction suspected to be mediated by non‐HLA antibodies. After cross clamp removal, the patient developed severe pulmonary edema, profound coagulopathy, and biventricular failure. The patient's presumed AMR, cardiogenic shock, and coagulopathy were treated with extracorporeal membrane oxygenation (ECMO), plasmapheresis, intravenous immunoglobulin (IVIG), multiple blood products, and prothrombin complex concentrate. The recipient was 0% panel‐reactive antibody (PRA), ABO, and crossmatch compatible. Intraoperative biopsy sample revealed a thrombotic process suggestive of a coagulation pathway activated by AMR; however, no C4d deposition was detected. Postmortem biopsies also suggested AMR. Retrospective testing of the patient's pretransplant serum revealed strong antiangiotensin II type 1 receptor (AT1R) antibodies and a strongly positive endothelial cell crossmatch. Anti‐AT1R antibodies are known to be AT1 receptor agonists and may trigger inflammation and activate the extrinsic coagulation pathway. Given the potential effects of signaling through the AT1R, the patient's preexisting anti‐AT1R antibodies and procoagulant therapy may have adversely affected the patient's clinical course.     

Overcoming a Positive Crossmatch in Living-Donor Kidney Transplantation

Many patients who have an otherwise acceptable living-kidney donor do not undergo transplantation because of the presence of antibodies against the donor cells resulting in a positive crossmatch. In the current study, 14 patients with a positive cytotoxic crossmatch (titer 相似文献   

The association of antivascular endothelial cell antibody with hyperacute rejection: a case report

Early graft loss almost always occurs when recipients of a renal allograft develop antibody directed against antigens specific for donor vascular endothelial cells (VECs) and peripheral blood monocytes. In studies involving recipients of human leukocyte antigen identical, living-related grafts exhibiting preformed antibody to the VEC antigens of their donors, the median onset of rejection was 3 days after transplantation. Although preformed antibody to VEC antigens has been related in numerous articles to early graft loss, there has never been a published report of anti-VEC antibody leading to hyperacute rejection. We report a patient who hyperacutely rejected a renal allograft after undergoing a donor-specific transfusion protocol with her mother in which the kidney was removed in less than 24 hours. Nine months later the patient had a retransplantation with an allograft from a cadaveric donor. The cadaveric graft was again hyperacutely rejected, and this kidney was removed immediately. Anti-VEC/monocyte antibody directed against both donors was detected in the patient's pretransplant sera. With the exception of a positive B-lymphocyte crossmatch with her mother, all the standard crossmatches were negative.     

For Anti-HLA-Specific Donor Antibodies Detection By Flow Cytometry Cytotoxic Crossmatches Comparison of Methods

Anti-HLA-specific donor antibodies induce rapid, irreversible destruction of the transplant (hyperacute rejection) that today happens rarely due to immunologic studies—prospective crossmatch—of patients awaiting the kidney graft. The usual approach for pretransplant donor/recipient evaluation is based on 2 methods: (1) the cytotoxic complement crossmatch (CDC) and (2) the flow cytometric crossmatch (FCX). The CDC crossmatch is positive when complement-fixing antibodies are present, an absolute contraindication to kidney transplantation. The more sensitive FCX-positive crossmatch detects low concentrations of unable to fix performed antibodies complement. It is an “index” of possible damage due to accelerated rejection. The target of our study was to develop a cytotoxic flow cytometry crossmatch (cFCX) that detected cytotoxic antibodies move sensitively than the traditional CDC method and also was less subjective and more standardized for interpretation studying sera from 23 patients; the cFCX showed the requested efficiency characteristics even in an emergency. In addition, the new method permited one to calculate a cutoff for positivity (average value of the negative control + 2 standard deviations), assuring an “objective” interpretation of the results that agreed with the CDC but was more sensitive and accurate allowing solution of ambiguous results for cases of “doubt”-positive CDC crossmatch. Furthermore, our aim was to correlate the effect of the strength of the anti-HLA antibodies determined by mean fluorescence intensity value of LabScreen Single Antigen beads with results of CDC, cFCX, and FCX methods.     

Role of the antibody to vascular endothelial cells in hyperacute rejection in patients undergoing cardiac transplantation

Traditionally, the human lymphocyte antigens have been considered to be the major barrier to successful transplantation, and lymphocytes have been used as the target cell in evaluating histocompatibility. The presence in the serum of recipients of preformed antibodies, cytotoxic to donors lymphocytes, is associated with a high probability of hyperacute rejection. We identified 11 patients in whom, despite a compatible direct lymphocytotoxic cross-match, acute failure of the cardiac homograft was associated with histologic and immunologic findings consistent with hyperacute rejection. Direct immunofluorescence and immunohistochemical staining showed the presence of antibodies on the surface of vascular endothelial cells in each of these 11 patients. The serum of these recipients was found to contain antibodies against a panel of endothelial cells. In contrast, cytotoxic antibodies to vascular endothelial cells were not present in a control group of 18 heart transplant recipients who did not experience hyperacute rejection. Thus the presence of antibodies against vascular endothelial cells seems to be related to hyperacute rejection of the cardiac allograft.     

The role of natural IgM in the hyperacute rejection of discordant heart xenografts.

The mechanism of xenograft hyperacute rejection in discordant species combinations remains controversial. The purpose of this work was to study the role of natural antibodies in the hyperacute rejection of guinea pig hearts transplanted into rats, a highly discordant combination. This study was conducted in vitro, ex vivo, and in vivo. The endothelial cells of the graft being the first targets damaged in the process of hyperacute rejection, the binding of rat natural antibodies to guinea pig endothelial cells was studied by immunofluorescence. The study was carried out in vitro on guinea pig endothelial cells in culture, and ex vivo on isolated guinea pig hearts perfused with either rat serum or immunoglobulins or immunoglobulin fragments bearing the antigen-binding site. In vitro and ex vivo, rat natural IgM were found to bind specifically to guinea pig endothelial cells, since IgM fragments bearing the antigen-binding site (Fab mu and Fab' mu) could be detected on these cells. IgM fragments were able to inhibit the fixation of native IgM molecules. In contrast, rat IgG only bound to endothelial cells through Fc portions. Thus rat natural IgM might play a role in hyperacute rejection by binding to the graft endothelial cells and triggering the complement cascade activation. In order to test the role of natural IgM in vivo, isolated guinea pig hearts were first perfused with rat Fab' mu, which inhibit the binding of IgM and are unable to activate the complement cascade. These hearts were then transplanted into Lewis rats. The rejection time of Fab' mu-perfused guinea pig hearts was prolonged compared with hearts perfused with buffer or IgG F(ab')2. Therefore, in the guinea pig to rat combination, preventing the binding of the recipient's natural IgM to the graft endothelium delays the hyperacute rejection. In addition, natural IgM are likely to play a greater role than natural IgG.     

XM-ONE detection of endothelium cell antibodies identifies a subgroup of HLA-antibody negative patients undergoing acute rejection

Background

Kidney transplant recipients exhibiting antibodies (Ab) against either HLA or non-HLA antigens undergo frequent episodes of rejection and exhibit decreased long-term graft survival. The novel flow cytometry crossmatch kit XM-ONE, detects Abs to HLA antigens plus those directed to Tie-2-positive precursor endothelial cells (anti-endothelial cell antibodies, AECA). We studied the clinical importance of these lesser known antibodies.

Methods

We retrospectively analyzed 208 sera from 160 recipients of deceased donor grafts for AECA using non-donor peripheral blood endothelial progenitor cells as targets and Luminex methodology for HLA antibodies.

Results

AECA were detected in 64 patients (40%). A significantly higher proportion of patients showing a positive endothelial crossmatch experienced rejection (31 AECA-positive among 43 rejection cases, 72%) compared with those without rejection (33/117, 28.2%). Immunoglobulin M(IgM) predominated (66%) over IgG (14%) and IgG plus IgM (20%). HLA antibodies positively and significantly associated with rejection as expected. Of special interest were the 19 patients who presented with acute rejection episodes along with restricted AECA positivity. The relative-risk for an acute rejection episode with either AECA or HLA—13.87 and 2.43, respectively—was significant. When HLA was already positive, the relative risk for AECA was 1.24, a non-significant increase.

Conclusions

Our data identified AECA-positive patients that showed an increased risk to develop an acute rejection episode early after transplantation. Moreover, restricted AECA-positive patients with acute rejection are an important subgroup which otherwise may be wrongly labeled as non-humoral rejection. Among HLA-negative cases, AECA conferred a significantly greater risk for rejection.     

Detection of alloantibodies against non-HLA antigens in kidney transplantation by flow cytometry

The presence of alloantibodies against human leucocyte antigen (HLA)-I and HLA-II antigens has been associated with hyperacute and accelerated graft rejections. However, occasionally these rejections occur in patients without donor-specific anti-HLA antibodies, suggesting the presence of other antigenic complexes that are shared by the graft and other cell populations. Usually, these antibodies are not routinely studied and their role in graft rejection is poorly understood. For this reason, we tested, by flow cytometry, the presence of panel-reactive alloantibodies (PRA) using different cell populations in 30 pre-transplant sera of kidney graft recipients. The patients studied had or had not hyperacute and accelerated rejection episodes (HARE) and did not have alloantibodies against HLA of their specific donors. We found that IgG and IgM alloantibodies directed against HLA-I antigens, different to the HLA-I antigens of the specific donors, as well as IgG against endothelium/monocyte antigens, IgM against platelets, and IgM against T cells are significantly associated with HARE, independently of the percentage of PRA. Our findings suggest that the detection of antibodies by flow cytometry against non-major histocompatibilty complex antigens may be useful as a pre-transplant crossmatch in living related donor kidney transplants to diminish the incidence of HARE.     

Liver transplantation and the lymphocyte crossmatch

Hyperacute rejection related to donor-specific antibodies rarely occurs in liver transplantation, probably because of the role of Kupffer cells, sinusoidal cells, and the dual blood supply. However, the long-term outcome of liver grafts, transplanted in the context of a positive lymphocytotoxic crossmatch, is approximately 20% lower than that of grafts with a negative crossmatch. Without recipient selection based on the crossmatch, which is currently the generally accepted policy, 11% to 24% of the liver transplants will be performed with a positive crossmatch. Using the level of panel reactive antibodies, (eg > 30%) as an indicator for sensitization, a lymphocytotoxic crossmatch can be performed before transplantation on a limited number of patients, to avoid a positive crossmatch. Sometimes the medical urgency does not allow the patient to wait for a negative crossmatch. Therefore, plasmapheresis should be performed before transplantation to diminish the antibody level and the posttransplant use of prostaglandin-E and high-dose steroids should be considered to avoid the short-term immunologic complications. These measures require more study before they can become standard practices. Possibly, Mycophenolate Mofetil might be effective in preventing chronic rejection. The introduction of the flowcytometry crossmatch procedure will not only provide a more sensitive, but also a faster procedure (~4 hours), allowing a wider use of the pretransplant crossmatch test in liver transplantation. However, the increased sensitivity and speed are balanced by the much lower specificity of the positive flowcytometric crossmatch test. The most appropriate way to use flow techniques for lymphocyte crossmatching has not yet been clearly determined because of the specificity problem. In the setting of other solid organ transplantation, the presence of preformed donor-specific antibodies is linked with occurrence of hyperacute or accelerated rejection, and is likely to result in graft failure in a short period of time. Therefore, a positive crossmatch directed against donor HLA antigens is a concern in organ transplantation, especially in kidney and heart transplantation. A positive IgG crossmatch test precludes transplantation in most cases. In liver transplantation, the picture is not so clear, but probably represents a relative contraindication to transplantation.     

The significance of the anti-class I antibody response. I. Clinical and pathologic features of anti-class I-mediated rejection

In renal transplantation, preformed cytotoxic antibody against donor HLA class I antigens causes hyperacute rejection of renal allografts, but its pathogenic significance when it develops in the posttransplant period is unknown. In the present studies we describe the clinical and pathologic features of patients with rejection associated with anti-class I. In the course of 400 consecutive cadaveric renal transplants, 7 patients were identified who had antibody against donor class I HLA antigens in association with atypical but distinctive patterns of rejection. All 7 were presensitized. In 3 patients, the transplant had been inadvertently performed with a positive donor-specific T cell crossmatch. In the remaining 4, the T cell crossmatch on current sera was negative but became positive posttransplant. The clinical picture was deterioration of graft function with rapid onset of oliguria, apparently due to acute tubular necrosis, but with persistence of blood flow demonstrable by radioisotope scan studies. Renal histology showed that the typical lesions observed in cell-mediated rejection, such as tubulitis and interstitial infiltration, were absent. Granular complement deposition (6), polymorphonuclear infiltration (6), and endothelial injury in the microvasculature (6) were common, and mononuclear infiltrates were absent (2) or not prominent (4). In 3 patients the glomerular changes resembled a picture of hemolytic uremic syndrome, with capillary fibrin thrombi and widening of the subendothelial space. IgG staining was negative. The pathologic features suggest that anti-class I antibody appearing or persisting in the early posttransplant period injures the endothelium of the microvasculature, with the clinical presentation different from that of hyperacute rejection. Particularly in sensitized patients, rapid deterioration in function, leading to a picture of acute tubular necrosis, with pathologic features of endothelial injury in the microcirculation, should suggest the diagnosis of anti-class I-mediated rejection.     

Formation of donor-specific human leukocyte antigen antibodies after kidney transplantation: correlation with acute rejection and tapering of immunosuppression

BACKGROUND: Before kidney transplantation, a serological crossmatch is routinely performed between donor and recipient to prevent hyperacute rejection by donor-specific anti-human leukocyte antigen (HLA) antibodies. After transplantation, the presence of these antibodies is not routinely monitored. We wanted to know whether donor-specific anti-HLA antibodies are detectable during acute rejection (AR), before or after reduction of immunosuppression in kidney transplant recipients who were converted from cyclosporine A (CsA) to the less nephrotoxic azathioprine (AZA) or mycophenolate mofetil (MMF) at 1 year after transplantation. METHODS: Plasma samples were collected before transplantation, at several time points after transplantation, and during AR. Antibodies were measured in 29 patients: 5 patients with AR during the first year after transplantation (before conversion), 14 patients with AR after conversion or dose-reduction of AZA or MMF, and a control group of 10 patients without AR during a follow-up of 2 years (1 year before and 1 year after conversion of immunosuppression). Antibodies were measured by complement-dependent cytotoxicity assay, enzyme-linked immunosorbent assay (ELISA), and flow-cytometry in a crossmatch with donor spleen cells. RESULTS: Donor-specific antibodies were not detectable after transplantation in the control group without AR, nor in patients with AR shortly after transplantation during CsA therapy. After conversion from CsA to AZA or MMF, antibodies appeared only in one patient after graft failure followed by transplantectomy and in patients during AR on AZA but not on MMF therapy. CONCLUSION: In this patient group, we could not detect donor-specific antibodies during CsA treatment, not even at the time of AR using three different techniques. Donor-specific antibodies were primarily present during AR in patients converted from CsA to AZA and were not found in the sera from patients converted to MMF.     

Successful living donor renal transplantation despite ABO incompatibility and a positive crossmatch

Potential live kidney donors have been rejected when the prospective recipients are blood type or crossmatch incompatible. By utilizing plasmapheresis combined with intravenous immune globulin (PP/IVIg) prior to surgery, donor-specific antibodies against blood group or human leukocyte antigens (HLA) have been removed, thereby allowing successful renal transplantation. A 26-yr-old male with a panel reactive antibody level of 100% and repeated positive crossmatches against deceased donor kidney offers, including zero HLA mismatched donors, successfully underwent ABO-incompatible kidney transplantation from his HLA-identical but nevertheless crossmatch-incompatible sister. The initial anti-A blood group isoagglutinin titers were 128, 256, and 1024 at room temperature, 37 degrees C, and 37 degrees C anti-IgG enhanced, respectively. With an individualized PP/IVIg regimen based on donor-specific antibody titer, however, the relevant antibodies were adequately reduced and hyperacute rejection avoided. Subsequent antibody-mediated rejection, likely directed against a minor histocompatibility antigen, was diagnosed on postoperative day 7 and successfully treated. Neither ABO, or crossmatch incompatibility, or both in combination prohibit kidney transplantation.     

Hyperacute rejections of two consecutive renal allografts and early loss of the third transplant caused by non-HLA antibodies specific for endothelial cells

The immunological rejection of HLA-identical kidney transplants indicates that non-HLA antigens may also be targets for transplant rejection. Interest in the possible role of endothelial specific antigens has grown steadily over the years. Most of the studies published, regarding the association of such antibodies with rejection, have demonstrated the reactivity of endothelial antibodies also with monocytes and keratinocytes, but not with lymphocytes. Such antibodies escape detection in conventional crossmatch tests.In this paper, we present a case report of a 10-year-old girl, whose two consecutive kidney allografts, (one living and one cadaveric donor) were hyperacutely rejected in spite of the fact that she had neither been alloimmunized, nor had any HLA-specific antibodies. Endothelial cell specific antibodies were detected in vivo and in vitro after transplantation only 11 days apart, which were considered to be responsible for rejection. The third cadaveric kidney was lost within 1 week post-transplant. Immunopathological investigation of the three rejected grafts revealed deposition of IgM in the endothelium of arteries and in some glomeruli. No deposition of IgG antibodies was found. Antibodies from this patient did not react with lymphocytes, monocytes or keratinocytes. Patient serum had IgM antibodies that were specifically reactive with cultured endothelial cells, demonstrated by binding in vitro and by complement-dependent cytotoxicity of IL-β stimulated endothelial cells. No HLA antibodies were found following the first two transplantations, but were demonstrated 1 week after the third transplantation, at the time of an acute irreversible rejection. Western blots of proteins solubilized from endothelial cell membranes, indicated that the antibodies reacted with a 97–110 kD protein. Endothelial cell antigen preparations were made from several different umbilical cord veins. Some primary cell cultures, but not all, reacted with the patient's serum. Therefore, we suggest that the target determinant might be polymorphic.These findings imply that the non-HLA endothelial cell specific molecules may function as target(s) for hyperacute antibody-mediated destruction of kidney allografts.     

The role of natural antibodies in the activation of xenogenic endothelial cells.

Hyperacute rejection of organ xenografts is thought to be mediated by the reaction of naturally occurring antibodies and complement of the recipient with blood vessels in the donor organ. We have suggested previously that the pathogenesis of hyperacute rejection might involve the activation of endothelial cells in the graft. To evaluate the potential role of natural antibodies and complement in hyperacute xenograft rejection, sixteen human sera were tested for variation in the ability to activate porcine endothelial cells as manifested by the release of biosynthetically labeled heparan sulfate from the cells. It was then asked to which extent such variation might reflect differences in natural antibody titer and/or complement activity. The sera mediated release of 3.6-57% of endothelial cell-associated heparan sulfate. Heparan sulfate release correlated significantly with the titer, in the sera, of IgM antibodies that bound to cultured endothelial cells (P = 0.0008) or to a triad of glycoproteins believed to represent the major targets of natural antibodies in porcine to primate xenografts (P = 0.001); correlation was also observed with the total concentration of IgM (P = 0.0046). The release of heparan sulfate did not correlate with corresponding properties of serum IgG, with anti-swine hemagglutination or with isohemagglutination titers. Heparan sulfate release correlated with deposition on endothelial cells of iC3b (P = 0.0095), but not with serum complement activity. Our findings indicate that in the reaction between human serum and xenogeneic endothelial cells, it is the concentration of xenoreactive IgM and not differences in complement activity that limits the ensuing pathophysiologic events.