FLOW CYTOMETRY CROSS-MATCH

A Method for Predicting Graft Rejection

Abstract

Long-term graft survival is mainly influenced by early graft rejection and posttransplant graft function. The ability of both complement-dependent cytotoxicity cross-match (CDC) and flow cytometry cross-match (FCXM) to predict acute rejection episodes has been evaluated by cross-matching 40 patients who received cadaveric kidney transplants, before(current serum) and after transplantation (on days 1, 7, 14, 21, 28, 60, and 90).

Of the 40 patients, all of whom had a negative CDC before transplant, seven patients had a positive FCXM before transplant: five of them(5/7=71.4%) experienced severe rejection within 2 months after transplantation. In patients with a negative FCXM before transplant, the incidence of acute rejection was lower (25.8%). Pre-transplant FCXM recipients who had a positive FCXM after transplant, experienced more frequent rejection(38.5%) than those pre-transplant FCXM recipients who never had a positive FCXM (15.8%). With respect to the incidence of acute graft rejection, no difference was found between patients who had a positive CDC after transplant and those who had a negative CDC after transplant.

Patients who had a positive FCXM before transplant had significantly higher creatinine levels within the first month after transplant. Immediate onset of function and accelerated lowering of the creatinine level were found to be more frequent in patients who had a negative FCXM before transplant.

As early graft rejection is the largest contributing factor for the development of chronic rejection and, therefore, of graft loss, we regard FCXM as a sensitive method for predicting long-term prognosis and graft survival, due to its competence in predicting both restricted graft function and early acute rejection, in particular.

The most important factor for the development of chronic rejection and decreased long-term survival is acute graft rejection^(1-3). A procedure that is able to predict such episodes before onset or even before transplantation should, therefore, improve long-term prognosis and graft survival. Even if some authors have observed lower 1-year graft survival and higher incidence of rejection in transplant recipients with positive flow cytometry cross-match(FCXM^*) before transplant^(4-10), consensus about the importance and clinical relevance of FCXM has not yet been reached. The aim of this study was to investigate the ability of both Terasaki's standard complement-dependent cytotoxicity cross-match (CDC)⁽¹¹⁾ and FCXM ⁽⁴⁾ to predict early acute graft rejection ⁽⁴⁾. For this purpose, cross-matches were performed, before and after transplant, on 40 patients who received cadaveric kidney transplants.

MATERIALS AND METHODS

Patients. Sera of 40 patients who received cadaveric kidney transplants (32 first graft, 8 regrafts) were taken before transplant (current sera) and after transplant (days 1, 7, 14, 21, 28, 60, and 90). The samples were stored at -80°C and cross-matched retrospectively, using both CDC and FCXM.

A positive CDC during recipient selection had been considered as contraindication for kidney transplantation. All patients, therefore, had a negative CDC before transplant.

Autoantibodies. All sera were checked for autoantibodies by CDC.

Definition of rejection episode. Rejection episodes were diagnosed on the basis of renal biopsy.

Cell preparation. After storage of donor spleen lymphocytes in liquid nitrogen, fractionation into T and B lymphocytes was performed immediately to cross-match, using nylon wool columns (Du-Pont, Boston, MA). Because of poor cell viability, CDC was performed after transplant on only 36 patients. FCXM was performed before transplant on only 38 recipients.

CDC. One microliter of undiluted sera was reacted with 1 μl of the separated T cells (2000 cells/μl) and incubated for 30 min. After the addition of 5 μl of rabbit complement (Behring, D-Marburg, Germany), the cells were incubated again (60 min), stained with eosin, and the cell viability was assessed. A positive CDC match was defined as a lysis of more than 10% of the incubated cells.

FCXM. Separated donor T cells (3×10⁵) and 50 μl of undiluted serum were incubated at 37°C for 30 min and washed twice with phosphate-buffered saline containing 0.1% NaN₃ (PBS-Az). The lymphocytes then were incubated for 30 min at 4°C in the dark, with 25μl of fluorescein isothiocyanate-conjugated goat F(ab′)₂ antihuman immunoglobulin (1/100 diluted; Immunotech, Hamburg, Germany), washed twice with PBS-Az, and resuspended in 200 μl of PBS-Az. Thereafter, samples were analyzed using a FACScan (Becton Dickinson, D-Heidelberg, Germany) flow cytometer and Lysis II software (Becton Dickinson). A channel shift of more than 2 SD between control samples containing only cells and fluorescein isothiocyanate-conjugated antihuman immunoglobulin and test samples was considered to be a positive FCXM.

Data analysis. The chi-square test and Student's t test were used for statistical analysis of data.

RESULTS

Association of FCXM/CDC with rejection episode. Of 40 patients who received kidney transplants and who were cross-matched before and after transplant, 21 had at least one positive FCXM (52.5%). Eleven patients experienced severe rejection (52.4%). In contrast, among 19 patients with a negative FCXM (before and after transplant), three rejection episodes occurred(15.8%; P<0.05). Previous sensitization (i.e., one or more pregnancies in 12 female recipients, pretransplant panel reactive antibodies>10% in 3 of 40 recipients), and transfusion favored neither the development of positive FCXM nor the occurrence of rejection episodes.

When comparing patients who had a positive CDC after transplant with patients who had a negative CDC after transplant, no difference was noted with respect to the occurrence of rejection episodes (30% vs. 34.6%). CDC was false-positive in 7 of 10 patients who had a positive CDC after transplant. ATG yielded none of the false-positive results.

Positive FCXM results, onset of rejection episode, and effect of therapy. As seen in Figure 1, CDC was positive before onset of rejection in only 1 of 12 episodes (8.25%). In 3 of 12 rejections(25%), CDC was able to detect antibodies after clinical diagnosis and biopsy. All but three patients with biopsy-proven rejection developed a positive FCXM(78.6%, n=14), nine of them (64.3%) on the day of biopsy or several days earlier (i.e., 2 days before rejection or before transplant). Due to immunosuppression in patients who developed a positive FCXM after transplant rejection, FCXM became negative approximately 3 weeks after therapy. In patients who received a positive FCXM before transplant, immunosuppressive regimen had no effect on FCXM. Even if rejections were reversible, FCXM continued to be positive in these patients.

Correlation of rejection episode and positive FCXM before transplant. Although all patients had a negative CDC before transplant, 7 of 38 (18.4%) were found to be positive by FCXM: five of them (71.4%) experienced severe rejection within the first 2 months(Table 1). Moreover, posttransplant serum creatinine levels were higher (Fig. 2), achieving statistical significance on days 7, 14, and 21. However, neither 1-year survival nor time of onset of the rejection episode were different between the patients who had a positive FCXM before transplant and patients who had a negative FCXM before transplant. Four of five rejection episodes in patients who had a positive FCXM before transplant occurred within the first 14 days after transplantation, compared to six of eight in patients with a negative FCXM before transplant. Rejection occurred in 8 of 31 patients (25.8%) who had a negative cross-match before transplant by both CDC and FCXM. While patients developing a positive FCXM after transplant showed a higher incidence of graft rejections (38.5%, n=13) than those having a negative FCXM at all times(16.7%, n=18), there was no correlation between the degree of positivity and the time of onset. Moreover, no significant differences in serum creatinine levels and 1-year survival were observed.

DISCUSSION

FCXM is undoubtedly more sensitive than the standard CDC assay^{(4, 12-14)}. Nevertheless, the relevance of a positive FCXM before or after transplantation has been unclear. Some studies have shown a decreased 3-month, 6-month, or 1-year survival in general ^(5-9), others restricted poor out-come to sensitized patients (regrafts) and patients with preformed antibodies (panel-reactive antibodies >10%) ⁽¹⁵⁾. Talbot et al. ⁽¹⁶⁾ and Wahlberg et al.⁽¹⁰⁾ described a higher incidence of postoperative complications (delayed graft function, higher serum creatinine levels, longer hospitalization) in patients who had a positive FCXM before transplant. In addition, rejection episodes were likely to be more frequent^{(4, 10, 17-20)}. Our data confirm a higher incidence of rejection episodes in patients with a positive FCXM. Both patients with a positive FCXM before transplant (current sera) (71.43% vs. 25.8%), and patients who developed antibodies after transplantation (38.5% vs. 25.8%), experienced rejection more often than those without any positive FCXM (i.e., false-negative FCXM). Although frequency of rejection was higher in patients with a positive FCXM versus patients with a negative FCXM, no difference in the severity of rejection episodes has been achieved. Graft loss and 1-year survival were similar in FCXM-positive and FCXM-negative patients. These data suggest that FCXM is a useful tool for predicting acute rejection episodes, even if FCXM and degree of rejection do not correlate.

Nevertheless, postoperative complications, i.e., primary nonfunction and increased serum creatinine levels, were more common in patients who had a positive FCXM before transplant: graft function was restricted within the first month after transplantation, leading to significantly higher serum creatinine levels at days 7, 14, and 21 (P<.05). In agreement with other studies ^{(10, 16)}, no difference in serum creatinine was found at 3, 6, and-in contrast to the report of Stefoni et al. ⁽²¹⁾-at 12 months after transplant.

In accordance with Scornik et al. ⁽²²⁾ and Sherlock et al. ⁽²³⁾, posttransplant proliferation of donor-specific antibodies is associated with rejection. Thus, patients developing antibodies after transplantation suffered more often from rejection than patients with a negative FCXM (42.9% vs. 15.8%, not significant). In contrast to some reports ^{(24, 25)}, degree of positivity, incidence of positive FCXM, and onset of rejection episode did not correlate. Moreover, as FCXM is more sensitive than CDC, in patients with a negative FCXM before transplant, donor-specific antibodies are detected earlier by FCXM than by CDC. Three of eight FCXM (37.5%) performed with sera taken at the onset of rejection have been positive, whereas CDC has been positive in only 8.25%. With respect to rejection episode, in patients who had a negative FCXM before transplant, sensitivity of posttransplant FCXM is 62.5%. Antibodies were detected before rejection (i.e., a few days before biopsy) in 60% of patients with a negative FCXM before transplant and a positive FCXM after transplant. This is in accordance with some former reports. Both Scornik et al. ⁽²²⁾ and Daniel et al.⁽¹⁸⁾ found positive FCXM several days before rejection episode.

Incidence of chronic graft rejection and long-term viability are strongly influenced by early graft rejection: Matas ⁽²⁾ described acute rejection as the most important risk factor for the development of chronic rejection. Ferguson ⁽¹⁾ and Cole et al.⁽³⁾ observed a higher risk of graft loss and, thus, a significant difference in 5-year graft survival. Therefore, the emphasis of investigations on long-term graft survival should be placed on methods that evaluate, before transplantation, the patient's risk of developing early acute rejection. Lower incidence of early graft rejection will then improve long-term survival.

Higher incidence of acute rejection and restricted graft function within the first month after transplantation in patients with a positive FCXM and in patients with a positive FCXM before transplant, in particular, suggest that the FCXM detects patients with higher risk. Moreover, as FCXM is able to predict most rejection episodes before transplantation or before onset(Fig. 1), we regard FCXM as a tool that meets the above requirement. It allows investigators to identify recipients with higher risk of early rejection before transplantation, as well as to predict graft rejection after transplant. Because of its abilities, we consider FCXM (in addition to the standard pretransplant CDC) as a method for predicting long-term viability for improving the results of kidney transplantation, by adapting the immunosuppressive treatment.

Figure 1: Relationship between the onset of rejection episode and positive cross-match result in patients with rejection.

Figure 2: Comparison of serum creatinine levels for seven patients with positive FCXM before transplant (○) and 31 patients with negative cross-match before transplant (•).