Histocompatibility and Immune Evaluation
The HLA Laboratory
The Histocompatibility and Immune Evaluation Laboratories, under the direction of Dr. Ronald H. Kerman, Professor of Surgery, provide comprehensive pre- and post-transplant immune evaluation testing for kidney, heart, liver, and pancreas transplant recipients. The laboratories serve The University of Texas Medical School at Houston, The Memorial-Hermann Hospital, The St. Luke’s Episcopal Hospital, The Texas Heart Institute, and Texas Children’s Hospital and also function as a regional and national reference lab for transplantation immunology and immune evaluation of patients with immunologically related diseases. In Years 26-27, the lab performed an average of 23,815 tests/year (range, 21,139–26,281 tests/year).
Flow Panel Reactive Antibody (PRA) Identified HLA Antibodies
Pre-transplant histocompatibility testing seeks to determine compatible donor-recipient pairs using sera from prospective transplant recipients that have been selected to reflect the patient’s anti-HLA reactivity (including the historically most reactive or highest PRA) against a recent sample and the pre-transplant sera. Our goal has always been to transplant the donor organ into the most compatible recipient. A corollary to that goal is not to incorrectly deny a recipient a donor organ that can be successfully transplanted.
In the past, we have looked for recipient anti-HLA reactivity by testing pre-transplant serum samples against target lymphocyte panels representing all HLA antigens. The end-point read-outs of these assays were either cytotoxicity (cell death) or positive fluorescence. These assays depended upon immunoglobulin binding to cell surface receptors (presumably HLA antigens), and our interpretation was that the percent cytotoxicity or fluorescence represented HLA reactivity. These reactive sera were then used in the donor-specific crossmatch test, and patients with a positive anti-human globulin crossmatch (AHG-XM) or flow cytometry crossmatch (FCXM) would be ruled out of being transplanted while the negative crossmatch recipient received the donor organ.
This practice fulfilled the rule of transplanting the donor organ into a compatible recipient. However, we presumed anti-HLA reactivity when we denied the positive crossmatch recipient and never really knew whether we were correct in doing so. The methods used did not identify HLA antibodies, but rather measured binding to cell membranes (which might have been non-HLA receptors) and correlated this to the presence of HLA antibodies and rejection.
There now are assays available that utilize soluble HLA antigens as targets, in the absence of other membrane receptors. These can be performed using either ELISA or flow cytometry bead methodologies. The flow PRA (bead) assay is more sensitive than the ELISA and uses micro-particle beads coated with soluble HLA antigens and flow cytometry to detect HLA antibody reactivity.
Relevance of Flow PRA Identified HLA Antibodies and Flow Cytometry Crossmatching
We previously suggested that renal allograft recipients with flow PRA-identified pre-transplant IgG HLA antibodies were at significant risk for rejection and recipients with both HLA Ab and a positive donor-specific FCXM experienced both rejection and graft loss. However, not all of the Ab(+) FCXM(+) grafts were lost. To understand why all of these grafts were not lost to rejection, sera from Ab(+) and FCXM(+) recipients were evaluated for HLA Ab specificity and titer (strength of Ab), with results compared to rejection and graft loss. We studied 16 sirolimus-treated recipients of cadaveric donor renal allografts who were transplanted following negative IgG anti-human globulin (AHG) crossmatches (testing the most reactive historical and pre-transplant sera). All 16 recipients experienced acute humoral rejection between days 7 and 21 post-transplant. Retrospective testing of pre-transplant sera revealed that all 16 recipients had flow PRA-identified donor-specific IgG HLA Ab and donor-specific FCXM reactivity. Rejections were treated with plasmapheresis and OKT-3, thymoglobulin and/or intravenous immunoglobulin (IVIg). Reversal of these acute humoral rejections was achieved in 69% (11/16) of the recipients as evidenced by a mean decrease in serum creatinine (SCr) from 6.4±2.9 to 1.4±0.5 mg/dL. However, 31% (5/16) of the grafts were lost to rejection.
To determine whether there were any immune differences between successful and failed grafts, we studied the pre-transplant sera from 12 of the 16 recipients for HLA Ab titer. Pre-transplant sera from four recipients who lost their grafts did not reach their end point titers until 1:256, 1:256, 1:512, and 1:1024. In contrast, pre-transplant sera from eight recipients who kept their grafts lost Ab reactivity at titers of 1:8, 1:8, 1:8, 1:8, 1:16, 1:16, 1:16, and 1:32, respectively. While all 16 recipients presented with what may be considered immune contraindications (+ donor-specific IgG HLA Ab and +donor-specific FCXM), those patients with lower antibody titers responded to rejection therapy. These data suggest that the strength of Ab reactivity (Ab titer) may be important in identifying high-risk recipients who may be amenable to desensitization protocols and/or therapeutic intervention resulting in prevention or successful reversal of antibody-mediated rejections.
The presence of flow PRA-identified IgG HLA antibodies in the pre-transplant sera of renal allograft recipients is significantly correlated with the occurrence of acute rejections. Little is known, however, about the clinical significance of post-transplant-detected HLA antibodies. Moreover, since different immunosuppressive regimens are used among transplant centers, it would be important to understand the impact of specific immunosuppressants on the presence of post-transplant HLA antibodies. We therefore studied the pre- and post-transplant sera of 133 sirolimus-treated primary recipients of a deceased donor renal allograft for the presence of HLA Ab. Results were correlated with the occurrence of acute and chronic rejection episodes. The follow-up time was 12 to 55 months post-transplant. Pre-transplant, 58% (77/133) of the recipients presented with no HLA Ab, and 8% experienced acute rejection episodes within 12 months post-transplant, 15.6% (12/71) of these recipients presented with de novo HLA Ab.
In contrast, 18% (24/133) of recipients presented with Class I HLA Ab only and experienced significantly more acute rejection episodes (29% vs. 8%; p<0.02) than the 0% HLA Ab recipients. Post-transplant, 50% (12/24) of these recipients continued presenting with Class I HLA Ab, 8% (2/24) now presented with Class II HLA Ab; however, 42% (10/24) converted to 0% HLA Ab presentation. Only 8% (11/113) of the recipients presented with Class II HLA Ab only and experienced 18% (2/11) acute rejection episodes (18% vs. 8%; p<0.05). Post-transplant, 45% (5/11) remained the same, one was now positive for Class I HLA Ab, and five recipients converted to 0% HLA Ab. Finally, 16% (21/133) of the recipients presented with both Class I and II HLA Ab, three were in Class II only and three lost Class I and four lost both Class I and II HLA Ab. The mean time to chronic rejection for recipients with pre-transplant 0% HLA Ab was 32±14 months; for recipients with Class I, II, or both pre-transplant, but who lost HLA Ab post-transplant, the time was similar, 30±11 months. In contrast, recipients with Class I, II, or both HLA Ab pre- and/or post-transplant experienced the occurrence of chronic rejection within 11±8 months (32 ± 14 vs 11 ± 8, p<0.02). Therefore, the presence of HLA Ab pre- and/or post-transplant was a significant risk factor for both acute and chronic rejections.
Finally, at our transplant center, cardiac allograft recipients undergo transplantation following a negative IgG AHG-XM. Recent data from renal transplant studies suggested that recipients with pre-transplant sera containing flow PRA-identified IgG HLA Ab and displaying positive IgG donor-specific FCXM experienced a high rejection frequency and graft loss. Therefore, we retrospectively evaluated the pre-transplant sera of 127 cardiac allograft recipients for the presence of HLA Ab and a positive or negative IgG donor-specific FCXM. The results were correlated to post-transplant rejection and patient survival.
Recipients with little or no (<5%) IgG HLA Ab experienced significantly fewer rejections (10%, 9/86 vs. 56%, 23/41; p<0.001) and better graft survival (92%, 65/71 vs. 78%, 32/41; p<0.01) than recipients with >5% (39 ± 19%) IgG HLA Ab. More importantly, those recipients with pre-transplant IgG HLA Ab (four Class I only, three Class II only, and three Class I and II) and a positive IgG donor-specific FCXM experienced a 90% (9/10) rejection frequency and only had a 70% (7/10) 1-year graft survival, compared with an 11% (7/64) rejection frequency and a 92% (59/64) survival for recipients with no HLA Ab and a negative FCXM (p<0.01 for rejection and survival, respectively). Recipients presenting with no HLA Ab and positive IgG FCXM experienced an 8% (1/13) rejection frequency and a 92% (12/13) survival. These data suggest that the pre-transplant presence of flow PRA-detected IgG HLA Ab in recipient sera correlated with a significant risk for rejection. Recipients presenting pre-transplant with both IgG HLA Ab and a positive IgG donor-specific FCXM are at risk for both rejection and graft loss. The presence of IgM may be beneficial to graft outcome. Having pre-transplant IgG HLA Ab information may influence clinicians to desensitize patients prior to transplantation and/or to choose different immunosuppressive regimens for those sensitized vs. non-sensitized recipients.
Location & Contact
6431 Fannin Street
MSB 6.240
Houston, Texas 77030
Tel: 713-500-7400
Fax: 713-500-0785
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