Detection Of Cell Mediated Immunity In Renal Transplantation By Cylex ImmuKnow Assay

Background:Cylex ImmuKnow (ImmuKnow) assay is the only FDA-approved method that can be applied to test cell-mediated immunity (CMI) function in transplant recipients. Quantified results of cell immunity can be obtained within 24 hours. Therefore, it provides doctors with a useful index to monitor the immune status of post-transplant patients, and in turn enables effective and individualized immunosuppression therapy. As a novel indication of immunological assessment, ImmuKnow assay should be verified in large-scale clinical trials. It also should be clarified whether such assay fits the Chinese population. Furthermore, Alemtuzumab, known as a depletion antibody, is widely used in renal transplant induction; however, there were few studies on the impact of CMI in renal transplant by Alemtuzumab.Objective:1. To better understand CMI after renal transplant and to evaluate the role of ImmuKnow assay in prediction of rejection and infection.2. To explore the feasibility of the clinical application of this assay and its role in assessing CMI, guiding clinical treatment and individualizing post-transplant immunosuppression therapy.3. To establish immune response zones in line with Chinese population.4. To study the impact of Alemtuzumab on CMI.Methods:By using Cylex ImmuKnow assay, immune cell function was assessed by measuring ATP content of CD4+T cells after PHA stimulation to monitor the status of renal transplant recipients pre-and post-transplant. ATP values of stability, rejection and infection were also measured in transplant patients. Between October 2008 and March 2010,259 patients with uremia or renal transplant were studied; 881 blood samples were collected at the Department of Urology, Fuzhou General Hospital of Nanjing Military Region. Routine triple immunosuppressant was applied, while some patients were treated with Alemtuzumab induction. ImmuKnow test was performed either on a protocol timing (pretransplant,15,30,60,90,180days post-transplant) or exceptional timing (on adverse events, or re-hospitalization). Patients were divided into 5 groups by the following criteria: 1, based on different post-transplant immune status:stable group (conventional stable group; CAMPATH stable group; general stable group), Od group, infection group, rejection group, MP group.2, repeated measurements (using the repeated measurement design,14, 30,60,90,180 days post-transplant, clinically stable):the conventional therapy group, CAMPATH group. ImmuKnow test procedures were as follow:1, cell stimulation,2, CD4 cell selection and ATP release,3, ATP measurement,4, data analysis and results calculation. Statistical analysis:Kruskal-Wallis test was used to analyze differences between groups, Nemenyi test was used to compare differences between each group, ROC analysis was used to evaluate diagnostic accuracy. Repeated measurement data was analyzed by ANOVA.Results:For those groups based on different status:1, Either by patient number or the sample number, no statistical difference was found between conventional stable group and Od group, general stable group and Od group (P>0.05); the ATP value of infection group is lower than that of conventional stable group (P<0.001) or general stable group (P<0.001); the ATP value of rejection group is higher than that of conventional stable group(P<0.001) or general stable group (P<0.001); the ATP value of Od group is higher than that of infection group (P<0.001), lower than that of rejection group (P<0.001); the ATP value of infection group is lower than that of rejection group (P<0.001).2, By patient number:the ATP value of CAMPATH stable group is lower than that of conventional stable group or Od group (P<0.001); no statistical difference was observed between CAMPATH stable group and infection group (P= 0.079); the ATP value of CAMPATH stable group is lower than that of rejection group (P<0.001); no statistical difference was found between CAMPATH stable group and MP group (P=0.82); the ATP value of MP group is lower than that of conventional stable group and general stable group, respectively (P<0.001); the ATP value of MP group is lower than that of Od group (P=0.002); the ATP value of MP group is higher than that of infection group (P=0.011); the ATP value of MP group is lower than that of rejection group (P<0.001).ROC analysis results (either by patient number or sample number, regardless of whether the stable group included CAMPATH stable group, regardless of clinical diagnosis of infection or rejection):1, The ROC areas under the curve were> 0.95 (P<0.001).2, The points with highest Youden's indexes were used as the diagnostic cut-off points, of which the indexes were higher than FDA recommended values. As to conventional stable group, based on number of patients,150ng/mL (the highest specificity),225ng/mL (FDA recommended),238ng/mL (the highest Youden's index, highest sensitivity) were used as infection cut-off points,482ng/mL (the highest Youden's index, highest sensitivity), 525ng/mL (FDA recommended),594ng/mL (the highest specificity) were used as rejection cut-off points. By comparing the diagnostic value of the cut-off points, especially the Youden's index,238ng/mL is proposed to be the infection cut-off point while 482ng/mL as the rejection cut-off point. Recommended renal transplant immune response zone is 239-481ng/mL for Chinese population, which is more accurate compared to the FDA recommended immune response zone (226-524ng/mL). Furthermore, this zone is likely to be more in accordance with the genetic background of Chinese population, and organ (kidney) specificity.Results of repeated measurements group are:a total of 66 cases, including 55 cases in conventional therapy group and 11 cases in CAMPATH group were tested. Sphericity test results:P= 0.355, showed there was no correlation between ATP levels at various detection timing. Tests of within-subjects effects esults:there is no interaction between time and group, the impact of time on the ATP levels was of no statistical significance. Tests of between-subjects effects results:differences among groups were statistically significant in ATP values (P<0.001). The ATP value of conventional group is significantly higher than those of CAMPATH group at various time points:14,30,60,90,180 days post-transplant. As shown on the profile plot:the slope of the estimated marginal mean was similar between 14-30 days and 90-180 days, opposite to that of 30-60 days; the slope of CAMPATH group slope as significantly larger than the conventional group in 60-90 days. Different from conventional group, ATP levels of CAMPATH group in 30-60 days were still decreasing while that in 60-90 days increased significantly.Conclusion:1. Cylex ImmuKnow assay may be applied to monitor CMI status among post-transplant recipients with various clinical conditions (stability, rejection, infection).2. This study provides a recommend renal transplant immune response zone for Chinese population, which is 239-481ng/mL. This zone is more accurate compared with the FDA recommended immune response zone (226-524ng/mL), and more in accordance with the genetic background of Chinese population and organ (kidney) specificity.3. For clinically stable patients, no matter with uremia or renal transplant, there is no difference in CMI. But the CMI status is lower in those renal recipients who received Alemtuzumab induction. 4. CMI status does not change over time in clinically stable renal recipients (no matter Alemtuzumab depletion administered or not), within 180 days post-transplant.5. The CMI status of clinically stable renal recipients who received Alemtuzumab induction is similar to that of recipients with infection or acute rejection who received steroid pulse therapy, which can not be distinguished by ImmuKnow assay. However, the CMI status of infection recipients is different from that of acute rejection recipients who received high doses of hormone therapy, which can be distinguished by the ImmuKnow assay.6. The effect of Alemtuzumab induction on the CMI status lasts for at least 180 days. The negative impact of Alemtuzumab induction on CMI status still existed within 30-60 days, and started to dissipate at 60-90 day.7. Cylex ImmuKnow assay may enable the possibility to establish a clinical immune monitoring laboratory. Such laboratory may provide a tool for assessment of high risk in infection and rejection and results in individualized immunosuppression therapy.