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Eur J Cardiothorac Surg 1998;14:197-200
© 1998 Elsevier Science NL

Vascular rejection post heart transplantation is associated with positive flow cytometric cross-matching¹

^a Department of Thoracic and Cardiovascular Surgery, Section of Cardiac Transplantation and Mechanical Circulatory Assist Program, Cleveland Clinic Foundation, Cleveland, Ohio, USA
^b Histocompatibility Laboratory Transplant Center, Cleveland Clinic Foundation, Cleveland, Ohio, USA
^c The Department of Pathology, Cleveland Clinic Foundation, Cleveland, Ohio, USA
^d Department of Cardiology, Section of Heart Failure, Cleveland Clinic Foundation, Cleveland, Ohio, USA

Received 6 October 1997; received in revised form 14 April 1998; accepted 12 May 1998.

Corresponding author. Transplant Center Histocompatibility Laboratory, Desk L12, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195, USA. Tel.: +1 216 444 2805; fax: +1 216 444 8261; e-mail djc@tt.ccf.org

	Abstract

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Objective: Use of flow cytometry cross-matching for measurement of donor-specific alloreactivity and monitoring anti-donor antibodies is well established. This study was performed to determine (1) its accuracy as a marker of vascular rejection, (2) its correlation with post-transplant outcome and (3) its ability to monitor highly sensitized patients requiring antibody removal with plasma exchange. Methods: Serial serum samples from 99 heart transplant recipients were examined for the presence of anti-donor antibodies of the IgG class that were reactive with T and/or B cryopreserved donor lymphocytes. A sub-group of 20 HLA sensitized patients required plasma exchange to remove the anti-HLA antibodies and were monitored with flow cytometry cross-matching to assess the degree of antibody removal. Results: Positive T-cell reactions were observed in 26 patients and positive B-cell reactions in 54. Twenty patients had vascular rejection. A significantly larger number of patients with a positive flow cytometry cross-match had vascular rejection (42% versus 12% for T-cell reactions, and 32% versus 7% for B-cell reactions; P=0.002 each). Of the patients who had vascular rejection, 11 had a positive T-cell reaction (flow cytometry cross-match sensitivity of 55%), and 17 had a positive B-cell reaction (sensitivity of 85%). Of the 79 patients who did not develop vascular rejection, 64 had a negative T-cell reaction (specificity of 81%), and 42 had a negative B-cell reaction (specificity of 53%). The actuarial 2-year survival estimates were significantly higher in patients with negative T-cell reactions (90% versus 75%; P=0.04), and B-cell reactions (95% versus 78%; P=0.02). In the highly sensitized subgroup (n=20) the effectiveness of plasma exchange to decrease anti-HLA antibody reactivity was a strong predictor of outcome. For patients in whom plasma exchange (PE) reduced anti-donor reactivity, 1-year survival was 87% compared to 25% in those whom PE did not reduce the level of antibody binding as assessed with flow cytometry cross-matching (P<0.0001). Conclusions: Flow cytometry cross-matching provides a valuable marker for the detection of vascular rejection after cardiac transplantation. Quantitative measurements may allow evaluation of the efficacy of treatment modalities employed in the management of vascular rejection in an attempt to improve outcome.

Key Words: Flow cytometry cross-match • Vascular rejection • Cardiac transplant

	Introduction

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The acute rejection of cardiac allografts has previously been thought to be primarily mediated by the cellular arm of the immune response, specifically T lymphocytes, as evidenced by the current internationally accepted rejection grading system that scores biopsy specimens by the quantity and extent of lymphocytic infiltration [1]. However, a type of rejection has recently been recognized in which interstitial infiltrates are absent but in which light microscopic evidence of endothelial cell activation exists. This form of rejection, called vascular rejection (VR), is thought to be an antibody mediated phenomenon and is becoming recognized as a cause of cardiac dysfunction with a significant mortality rate in the early post-transplantation period, and appears to be a risk factor for subsequent development of graft vascular disease [2] [3] [4] [5] [6].

The diagnosis of vascular rejection requires light microscopic and immunofluorescence examination of endomyocardial biopsy specimens [7]. While this continues to be the `gold standard', any non-invasive marker of VR would be welcomed. One tube of blood from the recipient is all that is required to perform a flow cytometric cross-match (FCXM). This study was performed to determine (1) the accuracy of utilization of post-transplant FCXM as a marker of vascular rejection, (2) its correlation with post-transplant outcome and (3) its ability to monitor highly sensitized patients requiring antibody removal with plasma exchange.

	Materials and methods

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Patient characteristics
This retrospective study comprised 99 patients randomly selected from a group of 500 primary adult cardiac transplant recipients at the Cleveland Clinic Foundation from 1984 to 1997. Maintenance immunosuppression consisted of a three-drug combination of cyclosporin, azathioprine and prednisone. Patients with compromised renal function were selectively treated with OKT3 monoclonal antibody for induction, followed by conversion to cyclosporin-based immunosuppression when renal function improved. Episodes of acute rejection were initially treated with intravenous methylprednisolone for three days. Recurrent or refractory rejection was treated with steroids and OKT3. Other treatment modalities, such as plasmapheresis, immunoglobulin and cytolytic therapy, were used in cases of acute rejection where a significant humoral component was suspected.

Serologic testing and specimen analysis
The technique of FCXM has been described in detail in previous publications [8] [9] [10]. Serum samples from 99 cardiac transplant recipients were collected at the time of transplant and post-transplant and examined for the presence of anti-donor antibodies of the IgG class that were reactive with T and/or B cryopreserved lymphocytes. Reactions were developed using a fluoresceinated anti-IgG reagent, and T- and B-cell reactions were analyzed using appropriate fluorochrome-labelled monoclonal antibodies. The diagnosis of vascular rejection was based on the demonstration of immunoglobulin and complement on the coronary vascular endothelium by immunofluorescent staining, according to the criteria defined by Hammond and colleagues, in addition to evidence of endothelial cell swelling and activation on light microscopy [11]. Immunofluorescent staining was performed on all endomyocardial biopsy specimens from patients who showed persistent findings of vascular rejection or hemodynamic compromise during follow up.

Statistical analysis
Univariant analysis was performed using Fisher's exact test. The Pearson ² test was used to compare groups. To determine actuarial survival estimates, the Kaplan Meier statistic was used. The log-rank test was used for equality of vascular rejection and survival functions among patients with T- and B-cell reactions. A P-value less than 0.05 was considered significant.

	Results

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Positive T-cell reactions were observed in 26 patients and positive B-cell reactions in 54. Twenty patients had vascular rejection. A significantly larger number of patients with a positive T-cell FCXM (P=0.002) and/or B-cell FCXM (P=0.002) had vascular rejection compared with those with a negative FCXM ( Fig. 1 ). Of the patients who had vascular rejection, 11 had a positive T-cell reaction (FCXM sensitivity of 55%), and 17 had a positive B-cell reaction (FCXM sensitivity of 85%). Of the 79 patients who did not develop vascular rejection, 64 had a negative T-cell reaction (FCXM specificity of 81%), and 42 had a negative B-cell reaction (FCXM specificity of 53%). Correlation between FCXM and survival is shown in Fig. 2 Fig. 3 . The actuarial 2-year survival estimates were significantly higher in patients with negative T-cell reactions (90% versus 75%; P=0.04, log-rank), and B-cell reactions (95% versus 78%; P=0.02, log-rank).

View larger version (31K): [in this window] [in a new window]   Fig. 1. The relationship between percentage of patients experiencing vascular rejection and positive and negative post-transplant T-cell (light shade) and B-cell (dark shade) FCXM reactions.

View larger version (12K): [in this window] [in a new window]   Fig. 2. The relationship between post-transplant positive and negative T-cell FCXM and survival.

View larger version (11K): [in this window] [in a new window]   Fig. 3. The relationship between post-transplant positive and negative B-cell FCXM and survival.

	Discussion

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The role of humoral immunity in acute cardiac allograft rejection has only recently been recognized. There have been reports of decreased survival rates and development of vascular rejection and graft atherosclerosis in patients with circulating antibodies to HLA antigens [2] [3] [4] [5] [6]. Vascular rejection is being increasingly recognized as a significant cause of cardiac dysfunction and mortality after heart transplantation [2] [3] [4] [5] [6]. Hammond and coauthors have described the endothelial swelling on light microscopy and immunoglobulin and complement deposition on the coronary endothelium of the allograft by immunofluorescent staining that is characteristic of vascular rejection [2] [11].

Vascular rejection has also been associated with OKT3 induction and the consequent development of human antimouse antibodies [12]. However exposure to OKT3 is not necessary for the occurrence of vascular rejection. Ratliff and colleagues described a population, who had vascular rejection, but had not received OKT3 [13]. This population had a high subset of multiparous females (83%) in a transplant population that was predominantly males (75%), which is consistent with the thesis that pre-formed antibodies are important in vascular rejection. Also consistent with this is that vascular rejection is more likely to occur in patients with high levels of panel reactive antibody or positive donor-specific lymphocyte cross-match [14] [15].

Flow cytometric cross-matching (FCXM), utilizing donor cells as targets, provides a more precise method for detection of donor-directed antibodies. It is relatively non-invasive and requires only a tube of blood from the patient. It is both sensitive and discriminating, which allows both the target antigens and the antibody class or sub-class to be determined in a quantitative fashion, and also allows measurement of the response to the mismatched antigens of the identified donor. The FCXM predicts a high risk group who should be followed more closely. Furthermore, of the patients who develop VR (and are thus in a high risk group), those with a positive FCXM have a worse prognosis compared with those with a negative FCXM, and they should therefore be followed more closely both clinically and with serial endomyocardial biopsies.

Plasma exchange is utilized pre-transplant and/or post-transplant. It is commenced pre-transplant in patients who are very unlikely to get a cross-match compatible transplant due to the high level of sensitization seen pre-transplant. This is typical of patients on left ventricular assist devices. Plasma exchange is considered post-transplant in those with a positive cross-match or in those who develop antidonor antibodies measured by FCXM that correlate with endomyocardial biopsy results of severe cellular and/or vascular rejection.

Being quantitative, the FCXM allows longitudinal surveillance of changes in humoral immunity over time and with different interventions. The present study shows that FCXM is a helpful surveillance tool to quantitate the response to plasma exchange or alternative therapies. It identifies a high risk group of patients who do not demonstrate a significant decrease in FCXM antibody level with plasma exchange. This particular group has a poor prognosis and additional interventions will be required in the future.

Much of the research in solid organ transplantation has focused on cellular aspects of the immune response. Current modalities of immunosuppressive therapy are thus more potent inhibitors of cellular than of humoral immunity. However, humoral immunity is undoubtedly important as vascular rejection, when compared with cellular rejection, is more resistant to immunosuppressive augmentation, causes more allograft dysfunction and is associated with a higher frequency of allograft loss [16] [17]. Further investigation of humoral responses to cardiac allografts coupled with research into agents with greater potency in suppressing humoral immune responses is required.

In conclusion, quantitative flow cytometry cross-matching provides a valuable marker for detection of vascular rejection after cardiac transplantation. Quantitative measurements may allow evaluation of the efficacy of treatment modalities employed in the management of vascular rejection in an attempt to improve outcome.

	Footnotes

 
Presented at the 11th Annual Meeting of the European Association for Cardio-thoracic Surgery, Copenhagen, Denmark, September 28 – October 1, 1997.

	Appendix A. Conference discussion

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Dr A. Murday (London, UK): I believe you did say that there is an association between the response to plasma exchange and survival. I have just a comment before I ask a question. My comment would be that plasma exchange is always only going to be temporary, and presumably what we're seeing there is just the patients that carry on producing large amounts of antibodies, of course those that are going to carry on having humoral rejection, and the plasma exchange is just one small event that reduces the antibody levels for a short period of time. We have had some experience which we have reported on a couple of occasions with total lymphoid irradiation, basically using it in a clinical context rather than with flow cytometry measurement of antibody levels, and we have been pleased with the response. Out of 14 patients we have only really had one serious complication, and certainly clinically the response of these patients who have every appearance of rejection apart from the histological appearance of rejection has been very gratifying. Do you have any experience with that treatment modality for these patients that have a poor response to plasma exchange?

Dr McCarthy: No. We have tried various options including monoclonal antibodies, cyclophosphamide, methotrexate and standard immunosuppressive treatments. But this is a very difficult group of patients to manage. We have not a large experience with total lymphoid irradiation but I'm sure that it sometimes works. I think it's all about options, because when you get into a difficult situation, one option may work and another may not.

I agree with your comment on the plasma exchange. While it's running it removes antibodies, and then you stop it and you can't expect that you have halted the immune response, or the underlying problem. However, when you do get a severe episode of vascular rejection, that in itself may kill the patient. The plasma exchange may allow you to get through that difficult period until the immune response subsides. I believe our results show some success with this strategy.

Dr W. Klepetko (Vienna, Austria): Could you give us some information as to what time point the vascular rejection occurred, and about the other therapeutical strategies you employed in those patients?

Dr McCarthy: The vascular rejection in these patients occurred early. It was generally seen in the first 6 weeks.

This slide depicts the clinical course of a young female requiring a cardiac transplant for giant cell myocarditis. Pre-transplant she required LVAD support. This was associated with substantial blood loss and transfusion. She also required dialysis for ATN. She had a PRA of 27%, directed against B7. The donor was B7 positive and we proceeded with the transplant because she was deteriorating. She had induction OKT3. Plasma exchange was also commenced as we judged that the flow cytometric cross-match would be positive. On day 4 post-operatively she had severe vascular rejection. Her flow cytometric cross-matches were very high. Cyclosporin was commenced, as her renal function improved, azathioprine was substituted with mycophenolate, and cyclophosphamide was also introduced. Over the next week the vascular rejection resolved and the flow cytometric cross-match decreased right down, and only in America, the patient got married in hospital on day 13 post-transplant.

Dr Klepetko: I think it points to the difficulties in treatment of those really very difficult patients.

	References

Top Abstract Introduction Materials and methods Results Discussion Appendix A. Conference... References

 

1. Billingham M.E., Cary N.R., Hammond M.E., Kemnitz J., Marboe C., McCallister H.A., Snovar D.C., Winters G.L., Zerbe A. A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: heart rejection study group. J Heart Transplant 1990;9:587-593.[Medline]
2. Hammond E.H., Yowell R.I., Nunoda S., Menlove R.L., Renlund D.G., Bristow M.R., Gay W.A., Jones K.W., O'Connell J.B. Vascular (humoral) rejection in heart transplantation: pathologic observations and clinical implications. J Heart Lung Transplant 1989;8:430-443.
3. Hammond E.H., Ensley R.D., Yowell R.L., Craven C.M., Bristow M.R., Renlund D.G., O'Connell J.B. Vascular rejection of human cardiac allografts and the role of humoral immunity in chronic allograft rejection. Transplant Proc 1991;23:26-30.[Medline]
4. Cherry R., Nielsen H., Reed E., Reemtsma K., Suciu-Foca N., Marboe C.C. Vascular (humoral) rejection in human cardiac allograft biopsies: relation to circulating anti-HLA antibodies. J Heart Lung Transplant 1992;11:24-30.[Medline]
5. Hammond E.H., Yowell R.L., Price G.D., Menlove R.L., Olsen S.L., O'Connell J.B., Bristow M.R., Doty D.B., Millar R.C., Karwande S.V., Jones K.W., Gay W.A., Renlund D.G. Vascular rejection and its relationship to allograft coronary artery disease. J Heart Lung Transplant 1992;11:S111-S119.[Medline]
6. Ratliff N.B., McMahon J.T. Activation of intravascular macrophages with myocardial small vessels is a feature of acute vascular rejection in human heart transplants. J Heart Lung Transplant 1995;14:338-345.[Medline]
7. Hammond E.H., Hansen J.K., Spenser L.S., Jensen A., Yowell R.L. Immunofluorescence of endomyocardial biopsy specimens: methods and interpretation. J Heart Lung Transplant 1993;12:S113-S124.[Medline]
8. Hurley J.P., Cook D.J., McCarthy P.M., Hobbs R.E., Koo A.P., Ratliff N.B., Klingman L.L., Mrzena K., Stewart R.W. Flow cytometry cross-matching: a method for monitoring antidonor antibodies in heart transplant recipients. Transplant Proc 1995;27(1):1301-1302.[Medline]
9. Cook D.J., Klingman L.L., Koo A.P., Goldfarb D., Denis V.W., Hodge E.E. Quantitative flow cytometry cross-matching for precise measurement of donor-specific alloreactivity. Transplant Proc 1994;26(5):2866-2867.[Medline]
10. Iwaki Y., Cook D.J., Terasaki P.I., Lau M., Terashita G.Y., Danovitch G., Fine R., Ettenger R., Mendez R., Kavalich A., Martin D., Soderblom R., Ward H., Berne T., Lieberman E., Strauss F. Flow cytometry cross-matching in human cadaver kidney transplantation. Transplant Proc 1987;19:764-766.[Medline]
11. Hammond E.H., Hansen J.K., Spencer L.S., Jensen A., Rieddell D., Craven C.M., Yowell R.L. Vascular rejection in cardiac transplantation: histologic, immunopathologic, and ultrastructural features. Cardiovasc Pathol 1993;2:21-34.
12. Hammond E.H., Wittwer C.T., Greenwood J., Knape W.A., Yowell R.L., Menlove R.L., Craven C., Renlund D.G., Bristow M.R., DeWitt C.W. Relationship of OKT3 sensitization and vascular rejection in cardiac transplant patients receiving OKT3 rejection prophylaxis. Transplantation 1990;50:776-782.[Medline]
13. Caple J.F., Cook D.J., McMahon J.T., Myles J.L., Hook S., Ratliff N.B. Acute vascular (humoral) rejection in non-OKT3 treated cardiac transplants. Cardiovasc Pathol 1995;4:13-18.
14. Endsley R.D., Hammond E.H., Renlund D.G., Yowell R.L., Bristoe M.R., DeWitt C.W., Menlove R.L., Ratkovec R.M., O'Connell J.B. Clinical manifestations of vascular rejection in cardiac transplantation. Transplant Proc 1991;23:1130-1132.[Medline]
15. Ma H., Hammond E.H., Taylor D.O., Yowell R.L., Bristow M.R., O'Connell J.B., Renlund D.G. The repetitive histologic pattern of vascular cardiac allograft rejection. Transplantation 1996;62:205-210.[Medline]
16. Olsen S.L., Wagoner L.E., Hammond E.H., Taylor D.O., Yowell R.L., Ensley R.D., Bristow M.R., O'Connell J.B., Renlund D.G. Vascular rejection in heart transplantation: Clinical correlation, treatment options and future considerations. J Heart Lung Transplant 1993;12:S135-S142.[Medline]
17. Miller L.W., Wesp A., Jennison S.H., Graham M.A., Martin T.W., McBride L.R., Pennington D.G., Peigh P. Vascular rejection in heart transplant recipients. J Heart Lung Transplant 1993;12:S147-S152.[Medline]

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