Flow Cytometric Crossmatching in Primary Renal Transplant Recipients with a Negative Anti-Human Globulin Enhanced Cytotoxicity Crossmatch

Study Population

Between June 1992 and June 2000, 249 HLA nonidentical primary renal transplants were performed at our center. Retrospective FCXM and flow-cytometric PRA determinations were performed in all patients in whom donor cells and/or recipient sera were available. We were able to study 143 of 249 individuals with FCXM and a further 60 (total = 203) with flow-cytometric PRA. Baseline demographic characteristics for these two cohorts and for the entire patient population are presented in Table 1. Before transplantation, all patients were AHG-CDC T cell and CDC B cell crossmatch negative with current sera, and only one patient exhibited a positive AHG-CDC T cell crossmatch and CDC B cell crossmatch with historical sera. During the study period, 26 recipients experienced early graft loss (≤2 wk posttransplant). We were able to perform a retrospective FCXM in 20 of these individuals. The remaining 123 of 143 FCXM study patients all maintained their grafts for a minimum of 3 mo.

Immunosuppressive protocols varied in the 143 FCXM study patients. All patients received cyclosporine and prednisone, as well as azathioprine (n = 71), mycophenolate mofetil (n = 42), basiliximab with mycophenolate mofetil (n = 23), and sirolimus (n = 7). Six patients who received cyclosporine, azathioprine, and prednisone also received induction therapy with OKT3 for either high PRA values (n = 4) or as part of a pediatric protocol (n = 2). The 20 patients who experienced early graft loss received cyclosporine, prednisone, and either azathioprine (n = 10), mycophenolate mofetil (n = 6), or basiliximab and mycophenolate mofetil (n = 4). Only one individual with early graft loss received OKT3 induction.

Acute rejection episodes were diagnosed by core needle biopsy or when the serum creatinine increased by ≥10% in the absence of any other etiologies (e.g., obstruction), returning to baseline with pulse corticosteroid ± OKT3 therapy. Protocol biopsies were performed at 1, 2, and 3 mo posttransplant in 69 of the FCXM patients. Biopsies were performed after obtaining informed patient consent and with the approval of the University of Manitoba Research Ethics Committee. Subclinical rejection was diagnosed when the biopsy displayed histologic rejection (Banff 97 ≥ Type Ia) and the serum creatinine was within 10% of baseline values. Other FCXM patients did not undergo protocol biopsies because they were transplanted in an era before protocol biopsies, participating in a randomized protocol biopsy study, or declined to consent to biopsies.

Explant histology was available for review in 23 of the 26 patients who experienced early graft loss during the study period, and we were able to perform a retrospective FCXM for 18 of these 23 cases. Biopsies were recut and examined with a Leder stain, to highlight polymorphonuclear leukocytes in allograft capillaries and by immunohistochemistry for the deposition of the C4d product of complement activation (13,14). C4d staining was performed on deparaffinized sections with a polyclonal anti-C4d antibody (15). An analysis of structure limited to non-necrotic areas of graft tissue was performed, and the cause of graft failure was categorized by a pathologist (S.D.) blinded to all clinical data. Antibody-mediated rejection was identified when a biopsy exhibited the following three features: arteriolar polymorphonuclear leukocytes aggregation; peritubular capillary polymorphonuclear leukocytes accumulation; and linear staining of C4d in small arteries, arterioles, and peritubular capillaries.

AHG-CDC Crossmatch and AHG-CDC PRA

Since 1992, our center has performed AHG-CDC PRA screening and AHG-CDC T cell and extended incubation CDC B cell crossmatching. The following sera have been considered mandatory for final crossmatching: (1) a current sample within 1 mo of transplantation, (2) historical sera including the peak PRA sample and all historically high PRA sera, and (3) all posttransfusion sera. AHG-CDC and CDC crossmatch techniques were identical to those described elsewhere (16). A positive crossmatch was identified when cell lysis detected by uptake of eosin dye with phase contrast microscopy was ≥20% of that seen in negative control samples.

PRA screening for anti-HLA antibodies pretransplant was performed monthly and after any potential sensitizing event (i.e., transfusion). PRA trays that contained peripheral blood T cells of known HLA specificity were generated from 50 local donors. The HLA class I antigen frequency was representative of the HLA allelic frequencies within our donor population. The percent PRA was determined by the proportion of wells exhibiting a positive AHG-CDC T cell crossmatch. An AHG-CDC PRA value ≥10% was deemed significant.

FCXM and FlowPRA

Retrospective T and B cell FCXM was performed for all recipients in whom donor cells and recipient sera were available. Splenic lymphocytes were available from 129 cadaveric donors and peripheral blood lymphocytes were obtained from 14 living related donors (Table 1). As in the AHG-CDC crossmatch, the recipient sera used for FCXM included (1) a pretransplant sample, (2) historical high PRA samples and the peak PRA sample, and (3) all posttransfusion sera. All sera used in the FCXM had been represented in the pretransplant AHG-CDC crossmatch.

For the FCXM, 100 μl of a 2.5 × 10⁶ cells/ml donor cell suspension was mixed with 20 μl of appropriate test and control sera. Samples were incubated for 20 min at 4°C then centrifuged and washed three times with cold phosphate-buffered saline. Fluorescence-labeled antibodies (3 μl anti-CD3 PerCP, 3 μl anti-CD19 phycoerythrin, and 20 μl of a working dilution of anti-human IgG F[ab]′ FITC) were then added. After a 20-min dark incubation, two wash steps with phosphate-buffered saline were performed, and lymphocytes were resuspended in 500 μl phosphate-buffered saline with 0.05% sodium azide and transferred into tubes for analysis. Three-color flow cytometric analysis was performed with a FACSCalibur instrument (BD Biosciences, NJ). Lymphocytes were gated on the basis of their forward and side-scatter characteristics. With a scale that expressed staining intensity as a linear channel value (0 to 1024), median channel fluorescence for anti-human IgG F(ab)′ FITC was quantified on CD3⁺ T cells and CD19⁺ B cells. A positive crossmatch was identified when the sample median fluorescence intensity exceeded that of negative control values by 3 SD. SD were derived by performing negative control FCXM with sera from 12 AB-negative nontransfused males and lymphocytes from 25 healthy donors (data not shown). A positive T cell FCXM and a positive B cell FCXM represented median channel shift values of ≥40 and ≥100, respectively.

Pretransplant serum samples were analyzed by both the AHG-CDC PRA technique and by a flow-cytometric technique that used microbeads coated with purified HLA antigens (FlowPRA, One Lambda, Canoga Park, CA) (2,17). These beads consist of a pool of 30 HLA class I– and 30 HLA class II–coated beads, representing the common HLA antigens. Five microliters of class I and II beads were incubated with 20 μl of recipient sera for 30 min. After two washes, 1 μl of anti-human IgG F(ab)′ FITC was added and allowed to incubate for 30 min. After two further washes, the sample was analyzed by flow cytometry. Class I and II beads were gated on the basis of their differing light-scatter characteristics. Anti-HLA antibodies captured on the bead surfaces were identified by comparing fluorescence intensity in patient sera to positive and negative control sera.

Statistical Analyses

Values reported are mean ± SEM or, where indicated, as medians and ranges. P ≤ 0.05 was considered to be significant, and values ≥0.10 were reported as NS. All statistical analyses were performed by use of Statview 5.0 software (SAS Institute, Inc., Cary, NC). Tests of association (χ² and Fisher’s exact test) were used for comparison of categorical variables, whereas the t test was applied to comparisons of continuous variables.

A Positive T cell FCXM is Associated with Early Graft Loss Due to Antibody-Mediated Rejection

Of the 18 positive T cell FCXM patients, 6 (33%) experienced graft loss within 2 wk of transplantation (Table 3). Five of these patients (three current positive T cell FCXM and two historical positive T cell FCXM) exhibited histologic features of antibody-mediated rejection (Table 4). The sixth patient experienced an extensive iliofemoral arterial thrombosis on the side of the transplant anastomosis that became clinically apparent upon recovery from anesthesia. The infarcted graft was removed within 8 h of implantation, and no features of antibody-mediated rejection were seen.

Histology was available in 12 patients with early graft loss and a negative T cell FCXM. Only one of these individuals exhibited features of antibody-mediated rejection (Table 4). Histologic features of acute cellular rejection were also prominent in this case (i.e., tubulitis, interstitial mononuclear cell infiltrates, and endothelialitis), and no anti-HLA antibodies were detected pretransplant with either AHG-CDC PRA or FlowPRA (vide infra). The remaining 11 patients with a negative T cell FCXM all appeared to have lost their grafts because of either a venous or an arterial thrombosis.

Explant histology was reviewed in an additional five patients who had a negative AHG-CDC crossmatch and early graft loss. Because of a lack of donor cells or recipient sera, however, we were unable to perform either a retrospective FCXM or FlowPRA in these cases. Four of these patients also exhibited features of antibody-mediated rejection (Table 4).

A Positive T Cell FCXM is Associated with Early, Severe, and Recurrent Rejection

One hundred and twenty-three patients who underwent a retrospective FCXM kept their grafts beyond the early posttransplant period and could be evaluated for the incidence of rejection in the first 3 mo posttransplant. Of these, 12 individuals exhibited a positive retrospective T cell FCXM, and 111 were negative. The incidence of clinical events in these two groups is presented in Table 3. The 12 patients with a positive T cell FCXM experienced more adverse events, including early rejection episodes, steroid-resistant rejection that required OKT3 rescue, and multiple acute rejections. Indeed, all but 1 of the 12 positive T cell FCXM patients experienced at least one acute rejection during the first 3 mo posttransplant, and the majority experienced either multiple acute rejections, steroid-resistant rejection, or both. Maintenance immunosuppression in these 12 patients included cyclosporine, prednisone, and either azathioprine (n = 9) or mycophenolate mofetil (n = 3). Adverse events occurred with similar frequency in the azathioprine-treated patients and those treated with mycophenolate mofetil (data not shown).

Subclinical Rejection and a Positive T cell FCXM

Sixty-nine FCXM study patients, including 9 of the 12 positive T cell FCXM patients who kept their grafts, underwent protocol biopsies at 1, 2, and 3 mo posttransplant (Table 5). The biopsy rates at these time periods were 91% (63/69), 94% (65/69), and 89% (62/69), respectively. A positive T cell FCXM was associated with a greater prevalence of subclinical rejection at 1 mo and with ≥2 subclinical rejections in the first 3 mo posttransplant.

A significant number of patients with subclinical rejection at 1 mo (6/17) had experienced an acute rejection during the preceding weeks. Notably, an acute rejection episode preceded a subclinical rejection at 1 mo in 4 of 6 positive T cell FCXM patients but only in 2 of 11 negative T cell FCXM patients(P = 0.10).

FlowPRA Identifies Sensitized Primary Renal Transplant Recipients Undetected by AHG-CDC PRA

A current (i.e., pretransplant) serum sample for AHG-CDC PRA and FlowPRA determinations was available in 203 of 249 primary transplant recipients. There was a marked difference in the sensitivity of anti-HLA antibody detection between the two techniques (Table 6). AHG-CDC PRA detected antibodies pretransplant in only 9 individuals (4%), whereas FlowPRA detected antibodies in 41 individuals (20%). Eighteen of the patients positive by FlowPRA exhibited isolated anti-HLA class I antibodies, 10 exhibited isolated anti-HLA class II antibodies, and 13 exhibited both class I and class II anti-HLA antibodies. As in patients with a positive T cell FCXM, patients with a positive FlowPRA pretransplant were more likely to be female, have a history of pregnancy, and were more likely to have received transfusions in the past (Table 7).

A positive AHG-CDC PRA did not correlate with clinical events posttransplant. In contrast, a positive FlowPRA (anti-HLA class I and/or II) was associated with an increased rate of early graft loss (27% versus 7%, P = 0.0002) and with acute rejection during the first 2 wk posttransplant (50% versus 30%, P = 0.03).

There was a notable difference in anti-HLA antibody detection by the two PRA techniques in the 18 patients with a positive T cell FCXM. Examining both current and historical sera, AHG-CDC PRA identified anti-HLA antibodies in only 8 of 18 patients, whereas FlowPRA detected anti-HLA antibodies in all 18. When the significance of anti-HLA antibodies detected with FlowPRA was examined in patients that lacked donor-specific antibodies (i.e., positive FlowPRA but negative T cell FCXM), a trend toward more early rejection was observed, but this did not reach statistical significance (Table 8). Therefore, the association of a positive FlowPRA with clinical events posttransplant was attributed to the presence of donor-specific antibodies in most patients.

An Isolated Positive B cell FCXM Did Not Correlate with Clinical Events

Twenty-two of 143 (15%) patients were found to have an isolated positive B cell FCXM (i.e., negative T cell FCXM). There was no correlation between an isolated positive B cell FCXM and adverse clinical events. FlowPRA was positive in only 5 of these 22 individuals, which suggests that, in the majority, non-HLA antibodies were detected with the B cell FCXM (e.g., autoantibodies). Recipient cells for autologous FCXM were not available in these individuals. Four of the five sensitized patients with an isolated positive B cell FCXM possessed isolated anti-HLA class II antibodies detected with FlowPRA, whereas the fifth patient possessed both anti-HLA class I and class II antibodies. One of the four patients with an isolated anti-HLA class II antibody experienced steroid-resistant rejection on day 6 and required OKT3 rescue. The others had uneventful clinical courses.