LOW T CELL RESPONSES CORRELATE WITH INFECTION IN PANCREAS TRANSPLANTATION: CASE REPORT FOR MONITORING POST TRANSPLANT LYMPHOPROLIFERATIVE DISORDER (PTLD) USING IMMUKNOW™ ASSAY BY CYLEX Carol Bentlejewski,1 Ngoc Thai,2 Gina Zdanowicz,1 Deanna Blisard,2 Amit Basu,2 Kusum Tom,2 Henkie Tan,2 Janice Glidewater,2 Ronald Shapiro,2 Judith Britz,3 Richard Kowalski,3 Diana Post,3 Adriana Zeevi.1 1Pathology, University of Pittsburgh, Pittsburgh, PA, USA; 2 University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 3Cylex, Inc, Columbia, MD, USA Aim: Campath-1H induction with tacrolimus maintenance therapy has been shown to be an effective immunosuppressive regimen for pancreas transplant patients. Despite the minimal immunosuppression in this steroid free protocol infection remains a significant morbidity. In this case study we report on the correlation of immune status as assessed by Cylex test and the clinical management of PTLD. Methods: ImmuKnowTM Cylex Assay was applied to monitor the immunological status of pancreas transplant recipients. Cylex assay was performed on 14 blood samples before and after the resolution of PTLD. The patients were also monitored for EBV titers by PCR. Scope: In stable patients (n⫽51), we detected a range of 100-300 ATP ng/ml while in patients with infection complications, the T-cell reactivity was suppressed (n⫽14, 38⫾26 ATP ng/ml). Of interest were two patients who developed EBV related complications. Patient 1 had a primary EBV infection and was diagnosed with PTLD on biopsy 80 days post-transplantation and his EBV viral load was 12,000-14,000 copies. Over the next 3 months, the immunosuppression was diminished: Cellcept was stopped and his tacrolimus levels were dropped from 8-10 ng/ml to ⬍ 2.5 ng/ml. The quantitative EBV-PCR showed diminished but still persistent viral load (1,100-1,500 copies). The Cylex assay was performed in the first month post-Campath depletion and the results were surprisingly high (day 7 - 360 ATP ng/ml; day 20 - 978 ATP ng/ml). However, on day 41, the T-cell reactivity dropped significantly to ⬍50 ATP ng/ml and stayed low for the following 3 months while the patient had evidence of PTLD and detectable EBV by PCR. T-cell immunity was recovered by 200 days post-Tx. (ImmuKnow-260 ATP ng/ml) and the EBV- PCR dropped to less than 120 copies. Patient 2 developed increased EBV-PCR levels at one year post-Tx and maintained these high levels (12,000-150,000) to over 3 months while the T-cell reactivity dropped from 202 ATP ng/ml to 60-100 ATP ng/ml. The CD3 T-cell reactivity stimulated by Con A also showed a diminished reactivity from 185 ATP ng/ml to 36-88 ATP ng/ml concomitantly with a loss of EBV-specific memory response. Conclusions: In summary, suppression of cell-mediated immunity was associated with EBV activation and required reduction of immunosuppression. Monitoring these patients for reconstitution of cell-mediated immunity by the Cylex assay together with quantitative PCR for EBV provides useful information for immunosuppression management.
QUANTITATIVE ANALYSIS OF CHIMERISM USING RT-PCR METHOD Youri A. Serov,1 Andrew Lobashevsky,1 A.P. Shah,2 B.K. Book,2 A.J. Tector.2 1Medicine, Indiana University, Indianapolis, IN, USA; 2Surgery, Indiana University, Indianapolis, IN, USA Aim: Microchimerism (MC) analysis is an important test in monitoring of stem cells ingraftment in bone marrow transplantation (BMT). It is also critical in controlling of status of tolerance in solid organ transplantation. Donor-derived leukocyte migration to the central lymphoid organs occurs within the hours after liver transplantation. Sensitivity of the method is a major consideration in detecting of donor specific signal. Purpose. In this study we present sensitivity data on real-time (RT) quantitative PCR methodology using amplification of donor specific HLA class I and class II alleles in mixtures of peripheral blood mononuclear cells (PBMC). Methods: PBMC from two individuals were mixed at different ratios. High resolution class I and class II HLA typing was done by PCR-SSP method. DNA and RNA were extracted from each cell mixture using QUAIGEN and High Pure RNA isolation kits accordingly. To estimate donor-specific signal intensity produced by alive cells we used the same primer set for cDNA amplification. Scope: RT-PCR DNA amplification of donor specific class I and class II alleles showed 0.0064% and 0.16% level of sensitivity respectively. However, when cDNA was used as a template the sensitivity obtained was 0.8%. Linearity was found between proportions of DNA/cDNA mixed and donor specific signal intensity. Conclusions: RT-PCR method for in vitro MC analysis represents a highly sensitive assay. The use of cDNA as a template is particularly important in detecting of a donor specific signal produced by alive cells. This approach seems to be a useful prognostic indicator of rejection, relapse, GVHD and stable tolerance in BMT and solid organ transplantation accordingly.