Clinical Relevance of Low Levels of Preformed Alloantibodies Detected by Flow Cytometry in the First Year Post–Kidney Transplantation

Clinical Relevance of Low Levels of Preformed Alloantibodies Detected by Flow Cytometry in the First Year Post–Kidney Transplantation

OUTCOMES Clinical Relevance of Low Levels of Preformed Alloantibodies Detected by Flow Cytometry in the First Year Post–Kidney Transplantation T. Mich...

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OUTCOMES Clinical Relevance of Low Levels of Preformed Alloantibodies Detected by Flow Cytometry in the First Year Post–Kidney Transplantation T. Michelon, R. Schroeder, I. Fagundes, R. Canabarro, H. Sporleder, H. Rodrigues, J. Silveira, J. Montagner, V. Garcia, J. Neumann, and M. Graudenz ABSTRACT Objective. To determine the prevalence of transplants performed with a false-negative cytotoxicity cross-match and to analyze the clinical relevance of alloantibodies (Ab) detected only by flow cytometry (flow). Methods. We studied 66 patients undergoing kidney transplantation from a cadaveric donor. All patients had a simultaneous negative T⫹AHG⫹DTT and B⫹DTT. Pretransplant sera were retrospectively analyzed by flow cytometry according to an Emory University protocol: (1) T⫹ and B⫺: Ab anti-class I; (2) T⫺ and B⫹: anti-class II; (3) T⫹B⫹: anti-class I ⫹ II. Chi-square, Fisher exact, Student t test, and Kaplan Meier analyses were employed with significance assigned at P ⱕ .05. Results. The overall incidence of false-negative cytotoxicity was 33.3% (22/66), namely, 6.1% (n ⫽ 4) anti-class I; 9.1% (n ⫽ 6) anti-class II; and 18.2% (n ⫽ 12) anti-class I ⫹ II. Primary nonfunctioning grafts occurred in 6.8% (3/44) and 13.6% (3/22) negative and positive flow patients (two anti-class I ⫹ II and one class II; P ⫽ .39). The incidence of graft loss in the first year was respectively, 13.6% (6/44) and 18.2% (4/22; two anti-class II and two anti-class I ⫹ II; P ⫽ .72). Compared to flow-negative grafts, creatinine levels were significantly higher among flow-positive patients at 8 and 12 weeks. One-year graft survivals were 86.4% among negative versus 81.8% for the positive group (P ⫽ .67). Conclusions. We observed that 33% of kidney transplant recipients had low levels of alloantibodies detected only by flow. This single factor was associated with the worst graft function in the first trimester with a suggestion of a higher risk for non-functioning graft.


NOWLEDGE ABOUT THE antigenicity of HLA molecules expressed on cellular membranes has shed some light on the paradigm of acceptance versus rejection of a transplanted organ. Based on this concept, the routine use of a pretransplant cross-match has reduced hyperacute rejection, making transplantation a less empiric practice.1 If preformed alloantibodies acquired from pregnancies, blood transfusions, or previous transplants can immediately destroy a graft,2 it is probable that lower titers of alloantibodies produced during graft exposure might also have a pathogenic role,3 including low levels of antibodies undiagnosed by the traditional techniques.4 The kidney expresses both class I and II human leukocyte antigen (HLA) mole-

cules. It becomes even more antigenic after ischemiareperfusion damage. The presence of antibodies directed against donor HLA molecules can trigger an antigenantibody reaction and the classic complement cascade with activation of polymorphonuclear cells and thrombocytes

From the Pathology Department Graduate Studies, FFFCMPA and Transplant Immunology Laboratory, Santa Casa Hospital, Porto Alegre, Brazil. Address reprint requests to T. Michelon, Av Independencia 75, 7th floor, Porto Alegre, RS, Brazil 90035-074. E-mail: [email protected]

0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.05.040

© 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710


Transplantation Proceedings, 37, 2750 –2752 (2005)


leading to irreversible tissue damage that becomes microscopically visible too late.5,6 Nowadays we have some clinical evidence that these alloantibodies are deleterious to the graft during the entire transplant period and in every type of organ.7 Alloantibodies are associated with acute tubular necrosis,8 severe and early acute rejection episodes in kidney,9 kidney-pancreas, and heart transplants,10 as well as obliterans bronchiolitis in lung recipients11 and chronic nephropathy12,13 with kidney graft loss.14 We sought to identify low levels of alloantibody, which were detected only by flow cytometry at the pretransplantation time, and to verify their clinical relevance during the short term after a cadaveric kidney transplant.

2751 Table 1. Clinical Outcomes According to Pretransplant Flow Cross-Match Outcome

Group I (n ⫽ 44)

Group II (n ⫽ 22)


Primary nonfunctioning graft (n ⫽ 6; %) Delayed graft function (n ⫽ 51; %) Acute cellular rejection (n ⫽ 37; %) Graft loss (n ⫽ 10; %) Death (n ⫽ 5; %)

6.8 75.0 59.1 13.6 9.1

13.6 81.8 50.0 18.2 4.6

.39 .74 .66 .72 .65

Results were described as mean values and standard deviations or standard errors. The analysis included chi-squared or Fisher exact test, Student t test, and Kaplan-Meier, they were considered significant when P values ⱕ .05.

PATIENTS AND METHODS We studied our 66 cadaveric kidney transplants performed during 2002. All patients received a kidney graft after a negative cytotoxicity plus anti-human globulin (CDC⫹AHG) cross-match against T cells and a simultaneous negative dithiotreitol (DTT) reaction against B lymphocytes immediately before transplantation. Immunosuppression was chosen according to clinical characteristics of the donor-recipient pair, employing cyclosporine, azathioprine, mofetil micophenolate, rapamycin, tacrolimus, and/or prednisone. Patients were asked to sign an informed consent at the time of surgery. They were submitted to elective graft biopsies in order to diagnose a hidden rejection episode at the end of the first week, the first month, and the third month posttransplantation. Serum creatinine value was estimated weekly until the third month, and, for purposes of this study, at 6 and 12 months posttransplantation. A retrospective cross-match was performed by flow cytometry using the pretransplant serum. Patients divided according the flow cross-match results— group I (flow-negative, n ⫽ 44) versus group II (flow-positive, n ⫽ 22)—were not different regarding gender, age, number of grafts, cold ischemia time, pretransplant panel reactivity, initial immunosuppression, induction therapy, and type of cadaveric donor (traditional or marginal). The 31.8% (n ⫽ 21) of donors considered marginal for graft function were divided as 29.5% (n ⫽ 13) in group I and 36.4% (n ⫽ 8) in group II (P ⫽ .77). We applied a flow cross-match protocol published by Emory University,15 for both T and B lymphocytes. Quantitative flow cross-match was performed using a three-color technique including a phycoerythrin-labeled anti-immunoglobulin G (IgG) reagent to identify anti-donor IgG, and fluorescein isothiocyanate-labeled monoclonal antibodies against both T (anti-CD3) and B (antiCD20) cells. All reactions were compared to a positive and a negative control serum. A shift of ⬎40 mean channels above the negative control was considered to be a positive T test. B-positive tests were classified regarding the T-test result for this same serum, being positive with: (1) a mean channel shift ⬎60 when T was negative, and (2) a mean channel shift ⬎100 when T was positive. For the purpose of this study, positive reactions against T cells were defined as anti-class I antibodies and positive reactions against B cells were defined as anti-class II antibodies. Concomitant T- and B-cell-positive reactions were indicative of the presence of both anti-class I and II antibodies. Clinical outcomes analyzed according to the pretransplant flow cross-match included primary nonfunction, delayed graft function, acute cellular rejection episode, serum creatinine, death, graft loss, and graft survival during the first year after transplantation. Acute cellular rejection was defined according to Banff 97 criteria.16


Among 66 patients, 22 underwent kidney transplant with both a negative T-cell CDC⫹AHG and a negative B-cell⫹ DTT test, but positive flow cytometry results. This 33% of false-negative CDC⫹AHG cross-match technique results compared to the flow tests included: four patients with antibodies against HLA class I; six against class II; and 12 against class I and II concomitantly. A previous positive panel reactivity antibody test (⬎10%) by flow or ELISA was observed in 15.9% (n ⫽ 7) of group I and 9.1% of group II (n ⫽ 2), and hypersensitivity (⬎75%) was present in 2.3% (n ⫽ 1) and 9.1% (n ⫽ 2), respectively. The outcomes according to flow cross-match results are described in Table 1. Patients transplanted with a positive flow cross-match who had primary nonfunctioning grafts showed class II (n ⫽ 1) or class I ⫹ II (n ⫽ 2) anti-donor antibodies. Those that displayed delayed graft function had class I (2/4), class II (5/6), or class I ⫹ II (11/12). Among the group who developed acute cellular rejection episodes, we observed class I (2/4), class II (4/6), or class I ⫹ II (5/12). Only one of these patients died (with anti-class II antibodies), and four lost their grafts (two with anti-class II and two with anti-class I ⫹ II) during the first year posttransplantation. Serum creatinine was significantly higher in group II at 8 and 12 weeks compared to group I (1.7 ⫾ 0.8 ⫻ 2.5 ⫾ 0.5 and 1.6 ⫾ 0.7 ⫻ 2.8 ⫾ 0.8; P ⫽ .05, respectively). When there were low levels of anti-class II antibodies, patients showed significantly higher serum creatinine values from 4 to 12 weeks. When there were anti-class I ⫹ II antibodies, it occurred at 4 to 8 or 12 weeks compared to negative patients. Even though graft survival did not change between the groups, the rates at the end of the first year were 86.4% in negative and 81.8% in positive groups (P ⫽ .67), being 87.5% when there was anti-class I and 77.8% when there were anti-class II and 83.3% with anti-class I ⫹ II (P ⫽ .88, .44, and .82, respectively). We did not find any difference in graft survivals among the marginal cadaveric donor recipients regardless of pretransplant flow cross-match or antibody class.



Among the 33% of kidney transplants performed despite the presence of low levels of alloantibodies detected only by flow were 6% of anti-class I, 9% of anti-class II, and 18% of anti-class I ⫹ II using T⫹AHG⫹DTT and B⫹DTT crossmatch. Regarding the higher sensitivity of flow for alloantibody detection,17,18 our results are consistent with other published studies. Some groups describe between 6%19 and 20%20,21 disagreement rate between these both methods. A positive flow cross-match showed a single association with worse graft function in the first trimester. We also found a suggestion of a higher risk of early graft loss, mainly due to a primary nonfunctioning graft. It has been reported since 1987 that a flow cross-match can predict a primary nonfunctioning graft and reduces graft survival than can T⫹AHG.22 Then, even when detected only by flow, low levels of preformed antibodies are important prognostic markers, which are associated with undesirable early outcomes23 and reduced graft survival at one year.24 Even though we found some evidence suggesting an association between anti-class II or I ⫹ II antibodies and decreased graft function or never-functioning grafts, the exact role of different classes of alloantibody for specific clinical syndromes remains a concern. Some reports have noted preformed anti-class I antibodies with both early and chronic events, other workers only with chronic losses.21,25 In conclusion, although a larger study is necessary before a clear definition of any specific association between flow cross-match and graft outcome is reached, this study has focused our attention on this problem. It is possible that a larger database may reveal that patients at high immunological risk may have low levels of preformed antibody and thus offer them an individualized immunosuppressive management. REFERENCES 1. Ting A, Terasaki PI: Lymphocyte-dependent antibody crossmatching for transplant recipient. Lancet 1:304, 1975 2. Terasaki PI: Humoral theory of transplantation. Am J Transpl 3:665, 2003 3. Park MS, Terasaki PI, Lau M, et al: Sensitization after transplantation. Clin Transpl 393, 1987 4. Gebel HM, Bray RA, Ruth JA, et al: Flow PRA to detect clinically relevant HLA antibodies. Transpl Procc 33:477, 2001 5. Mauiyyedi S, Crespo M, Collins AB, et al: Acute humoral rejection in kidney transplantation: II. Morphology, immunopathology, and pathologic classification. J Am Soc Nephrol 13:779, 2002

MICHELON, SHROEDER, FAGUNDES ET AL 6. Mauiyyedi S, Colvin RB: Humoral rejection in kidney transplantation: new concepts in diagnosis and treatment. Curr Opin Nephrol Hypertens 11:609, 2002 7. McKenna RM, Takemoto SK, Terasaki PI: Anti-HLA antibodies after solid organ transplantation. Transplantation 69:319, 2000 8. Feucht HE, Lederer SR, Kluth B: Humoral alloreactivity in recipients of renal allografts as a risk factor for the development of delayed graft function. Transplantation 65:757, 1998 9. Halloran P, Schlaut J, Solez K, et al: The significance of the anti-class I response, clinical and pathologic features of renal transplants. Transplantation 53:550, 1992 10. Michaels PJ, Espejo ML, Kobashigawa J, et al: Humoral rejection in cardiac transplantation: risk factors, hemodynamic consequences and relationship to transplant coronary artery disease. J Heart Lung Transplant 22:58, 2003 11. Palmer SM, Davis RD, Hadjiliadis D, et al: Development of an antibody specific to major histocompatibility antigens detectable by flow cytometry after lung transplant is associated with bronchiolitis obliterans syndrome. Transplantation 74:799, 2002 12. Racusen LC, Solez K, Colvin RC: Fibrosis and atrophy in the renal allograft: interim report and new directions. Am J Transplant 2:203, 2002 13. Lee PC, Tersaki PI, Takemoto SK, et al: All chronic rejection failures of kidney transplants were preceded by the development of HLA antibodies. Transplantation 74:1192, 2002 14. El-Awar N, Terasaki PI, Lazda V, et al: Most patients who reject a kidney transplant have anti-HLA antibodies. Tissue Antigens 60:553, 2002 15. Bray R: Flow cytometry crossmatching for solid organ transplantation. In Methods in Cell Biology, Vol 41. Academic Press: 1994, p 103 16. Racusen LC, Solez L, Colvin RB, et al: The Banff 97 working classification of renal allograft pathology. Kidney Int 55:713, 1999 17. Sumitran-Holgersson S: HLA-specific alloantibodies and renal graft outcome. Nephrol Dial Transpl 16:897, 2001 18. Gebel HM, Bray RA: Sensitization and sensitivity: defining the unsensitized patient. Transplantation 69:1370, 2000 19. Cinti P, Bachetoni A, Trovati A, et al: Clinical relevance of donor-specific IgG determination by FACS analysis in renal transplantation. Transplant Proc 23:1297, 1991 20. Iwaki Y, Terasaki PI: Sensitization effect. Clin Transplant 257, 1986 21. El Fettouh HA, Cook DJ, Flecner S, et al: Early and late impact of a positive flow cytometry crossmatch on graft outcome in primary renal transplant. Transplant Proc 33:2968, 2001 22. Cook DJ, Terasaki PI, Iwaki Y, et al: The flow cytometry crossmatch in kidney transplantation. Clin Transpl 409, 1987 23. Gebel HM, Bray RA, Ruth JÁ, et al: Flow PRA to detect clinically relevant HLA antibodies. Transplant Proc 33:477, 2001 24. Ogura K, Terasaki PI, Johson C, et al: The significance of a positive flow cytometry crossmatch test in primary kidney transplantation. Transplantation 56:294, 1993 25. Worthington JE, Martin S, Al-Husseini DM, et al: Posttransplantation production of donor HLA-specific antibodies as a predictor of renal transplant outcome. Transplantation 75:1034, 2003