Clinical identification of transplant antigens

Clinical Identification of Transplant Antigens R. Emirog˘lu, H. Karakayalı, S. Sevmis¸, M. Turan, N. Bilgin, and R. Erdal

I

N THE 1940s, Peter Medawar’s classic studies of skingrafted rabbits heralded the beginning of modern transplant immunology. Today, our understanding of the immunologic aspects of transplantation is much greater, but graft rejection continues to be an important issue for all those involved in this field of medicine. Although a complex variety of factors affect the success of renal transplants, about 40% of first-year graft failures are immunologic in nature, acute rejection being the major cause of allograft failure in this period.1 There is no doubt that matching recipients and donors with regard to their major histocompatibility genes reduces the risk of acute rejection and improves graft survival. In addition, the presence of autoantibodies can also influence the survival of transplants. Various crossmatching techniques are used to detect HLA antibodies, and a negative class 1 complement-dependent cytotoxicity crossmatch (CDC-XM) is a prerequisite for renal transplantation. However, many centers have also found flow cytometry crossmatching (FC-XM) to be a very sensitive method for detecting immunoglobulin G (IgG) antibodies in the sera of pretransplantation patients. These antibodies are known to have a significant effect on subsequent renal graft function, and FC-XM is increasingly regarded as a better predictive crossmatch for renal graft survival. In this study we investigated the influence of plasmapheresis on positive FC-XM results and compared various aspects of the CDC-XM and FC-XM tests. METHOD When patients with end-stage renal disease (ESRD) are admitted to the transplantation unit at Bas¸kent University Hospital, Ankara, Turkey, both recipient and donor (living or cadaveric) undergo testing for blood type and HLA phenotype. The next step is to check the recipient for the presence of antidonor antibody using the standard lymphotoxicity cross-match (CDC-XM) and FC-XM. All cytotoxic testing was performed using a modification of a standard National Institutes of Health technique. FC-XM results were then expressed as a mean fluorescence index (FI):

Mean FITC fluorescence of the patient’s serum ⫹Mean FITC fluorescence of the negative control FI ⫽ Mean FITC fluorescence of the positive control ⫹ Mean FITC fluorescence of the negative control

A FI of 20% was considered a positive flow-cytometry crossmatch. © 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 32, 547–549 (2000)

All patients in the study were treated with induction immunosuppression involving the simultaneous use of methylprednisolone (1 mg/kg) and azathioprine (2 to 3 mg/kg). Cyclosporine A (CyA) was started 4 to 6 hours postoperatively at a dose of 5 mg/kg. Azathioprine was continued after surgery, with dosage adjustments made according to white cell counts and clinical picture, whereas prednisolone was tapered at the end of 1 month to 10 to 15 mg/d. Patients with negative CDC-XM but positive FC-XM were put on a plasmapheresis program until they tested negative. We separated plasma from blood cells by hollow fiber module with two or three sessions per week. A total of approximately 36 sessions was required for 12 patients to convert the crossmatch results to negative. RESULTS

Since 1997 we have been using standard CDC-XM combined with FC-XM to choose suitable donors for ESRD transplant recipients. During this time, of 257 potential donors, 21 (8%) cadaver and 236 (92%) (first- or seconddegree relatives) underwent 512 tests in assessment for renal transplantation. Although they had negative crossmatch results, it was necessary to eliminate some candidates on the basis of malignancy, infection, hypertension, proteinuria, reduced renal function, and other medical problems. For cadaver grafts, we tested until an ESRD patient with negative crossmatch results was found. By July 1999, 96 ESRD patients had received transplants. In our system, the most suitable HLA matches are selected as donor-recipient pairs, and then both CD-XM and FC-XM are used to detect anti-HLA antibodies. The 96 recipients included 27 women (28%) and 69 men (72%) with a mean age of 28 years. Donor kidneys were cadaveric in 31 cases (32%) and living in 65 cases (68%). After eliminating 224 tests performed for cadavers and for the same donor-recipient pairs, the other 288 donor test results are shown in Table 1. A total of 26 potential recipients with positive crossmatch results were treated with plasmapheresis. Twelve of these From the Bas¸kent University Faculty of Medicine, Department of General Surgery, Ankara, Turkey. Address reprint requests to Dr Emirog˘lu, Bas¸kent University Faculty of Medicine, Department of General Surgery, 1. Cadde No: 77 Bahcelievler, Ankara, Turkey. 0041-1345/00/$–see front matter PII S0041-1345(00)00884-8 547

EMIROG˘LU, KARAKAYALI, SEVMIS¸ ET AL

548 Table 1. The Results of CDC XM and FCXM in Renal Transplant Candidates Test No.

CDC XM

FCXM T cell

FCXM B cell

211 43 23 8 3

⫺ ⫹ ⫺ ⫺ ⫹

⫺ ⫹ ⫺ ⫹ ⫺

⫺ ⫹ ⫹ ⫹ ⫹

individuals converted to negative after an average of three sessions of plasmapheresis. The other 14 patients are still positive and remain on the waiting list for a suitable cadaveric donor. We decided to operate on the 12 initially positive patients who, after plasmapheresis, produced a minimum of three negative results. As of July 1999, 7 of 12 had received transplants. Among 96 transplant recipients, 3 patients (3.1%) had originally been positive on both tests and 4 (4.1%) had been positive on the FC-XM B cell test only. Of these 96 patients, 1 cross-positive and 4 cross-negative patients lost their grafts after experiencing two acute rejection episodes. In both cases, the diagnosis of rejection was confirmed by biopsy and the patients had received 5 g corticosteroid and 14 doses of OKT3 at 5 mg/d. Two patient were lost because of aspergillosis in the cross-negative group. DISCUSSION

It is hoped that the identification of risk factors influencing graft survival will lead to the development of models for predicting graft outcome. Such models could provide guidance for immunosuppressive therapy, help measure posttransplantation outcome, and eventually improve graft survival in high-risk patients. HLA typing is performed routinely on all living and cadaveric donors, but acute cellular rejection continues to be a serious problem in kidney transplantation. This suggests that undetected presensitization may be a factor. HLA match is correlated with short-term and long-term graft survival; thus, HLA antibody testing allows us to predict whether a graft is at risk. Such transplants may not function immediately and there may be an accelerated increase in creatinine level because early graft rejection is the largest contributing factor to the development of chronic rejection. Knowledge of this risk is very useful in terms of recipient monitoring. CDC-XM has long been accepted as the sole test for histocompatibility, but FC-XM is being used more and more as an additional crossmatching procedure. FC-XM offers advantages over the standard CDC-XM in that it can detect noncomplement activating antibodies and more sensitive for detection of low levels of HLA specific antibodies. FC-XM also allows a reliable distinction to be made between IgG and IgM antibodies, and anti-T cell and anti-B cell antibodies.2 Results indicate that FC-XM is up to five times more sensitive for measuring IgG alloantibodies, whereas the opposite is true for IgM. IgG antibodies can be

detected in serum that exhibits no IgM activity.3 Because it is now widely accepted that many antibodies of the IgG class are more detrimental to graft survival, FC-XM has the potential to become the main crossmatching technique for renal transplantation.4 IgA and monocyte reactive antibodies can be detected only by using FC-XM analysis. These antibodies have been shown to bind to the kidney vascular endothelial cells, and are therefore considered to be important.5 In addition, a high proportion of patients had a weakly positive cytotoxic B cell crossmatch caused by antibodies with HLA class I specifities, despite a negative cytotoxic T cell crossmatch, thus confirming that positive B cell cytotoxicity crossmatches due to HLA class I antibodies should not be ignored. However, there are some centers that continue to evaluate the relationship of positive/or negative cytotoxic crossmatches to either graft or rejection using only T cells as targets, although it has been well established that B cells express higher amounts of both class 1 and 2 HLA antigens.6 Some investigators have claimed there is no statistically significant difference between FC-XM–positive and negative results relative to graft survival,7 but most believe that the incidence of acute rejection is lower in patients who have a negative FC-XM before transplantation.2,8 –10 Because FC-XM–positive patients are considered to be at high risk for acute rejection, some experts recommend that OKT3 be administered during induction therapy.11 Others suggest the use of immunoadsorbtion technique.12 But our strategy is to obtain negative FC-XM before transplantation. Both FC-XM and CDC-XM have certain disadvantages, such as detecting antibodies against donor HLA as well as non-HLA antigens. Flow cytometric tests are more sensitive than complement dependent tests, but they show 10% false-positive reactions that can deny some recipients the chance for transplantation.13 To improve the specificity, we find effective plasmapheresis to be sufficient. The mode of action here is primarily rapid depletion of specific disease-associated factors (antibodies, immune complexes, or alloantigens), a process which occurs through plasmapheresis.14 CONCLUSION

We believe that FC-XM should be part of the routine work-up of potential transplant donors and recipients. There were 31 (10%) patients with negative CDC-XM but positive FC-XM in our group. The FC-XM test results prevented the recipients to take kidneys from these donor candidates. To accept individuals for renal transplantation without any effort to eliminate crossmatch positivity is not wise, and only serves to increase the risk of allograft loss. On the other hand, nearly 10% of donor-recipient pairs have a positive crossmatch when they are tested. Adopting a policy of excluding such cases will deny many patients the opportunity to receive kidney transplants.5,15 Plasmapheresis offers one possible solution for those with crossmatch positivity. We conclude that flow cytometric crossmatches

TRANSPLANT ANTIGENS

should be used in combination with the cytotoxicity assay to aid in better recipient-donor pair selection and plasmapheresis may help to decreases false-positive results. REFERENCES 1. Cecka M, et al: Surg Clin North Am 78:133, 1998 2. Bittencourt MC, Rebibou J-M, et al: Nephrol Dial Transplant 13:2059, 1998 3. Sutton PM, Harmer AH, Bayne AM, et al: Transpl Int 8:360, 1995 4. Sutton PM, Harmer AH, Bayne A, et al: Transpl Int 8:360, 1995 5. Karruppan SS, Lindholm A, Moller E, et al: Transplantation 53:666, 1992 6. Pellegrino MA, Belvedere M, Pellegrino AG, et al: Transplantation 25:93, 1978

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