Abstracts S56 217 218 PHARMACODYNAMIC MONITORING OF IMMUNO- SIMULTANEOUS HEPATO-RENAL TRANSPLANTATION SUPPRESSIVE THERAPY WITH CALCINEURIN INHIBITORS WITH A POSITIVE CROSSMATCH IN SENSITIZED PATIENTS. Jaume Martorell 1,Olga Millán1; Merce Brunet2; Josep-Maria Campistol3; Ana Jaume Martorell, Marta Crespo, Vicente Torregrosa, Alejandro Faura3; Isabel Rojo1; Elena Vidal2; Olga Jiménez2; Federic. Oppenheimer3; Gutierrez, Federic Openheimer, Jordi Vives. Institut Clinic Jordi Vives1; Institut Clinic d’Infeccions i Immunologia (ICII)1, Servei de Toxicologia2, Unitat de Transplantament Renal3, IDIBAPS, Hospital Clinic, d’Infeccions i Immunologia (ICII), Unitat de Transplantament Renal, 08036 Barcelona, Spain IDIBAPS, Hospital Clinic, 08036 Barcelona, Spain Background: Graft survival depends on immunosuppressors efficacy. Introduction: A positive crossmatch (CM+) is a formal contraindication for Pharmacodynamic parameters are needed to evaluate individual biological effect and isolated renal transplantation but is not a contraindication for liver to complement pharmacokinetics, specially in multidrug low toxicity transplantation. It has been said that in simultaneous Hepato-Renal immunosuppressive therapies which include Calcineurin Inhibitors (CNi). transplantation (SHRT) the liver protects the kidney from rejection. In our Methods: Patients: 65 stable renal transplant recipients treated with: Cyclosporine-A center 21 SHRT had been performed since 1993, 2 of them with CM+. (CsA)(n=16), Tacrolimus (TRL)(n=10); Cyclosporine-A plus Mycophenolate-Mofetil Aim of the study: Retrospective analysis of two cases of SHRT with CM+. (CsA+MMF)(n=14); Tacrolimus plus Mycophenolate-Mofetil (TRL+MMF)(n=13) Patients and methods: 2 women (30 & 52 years) with hepatic cirrhosis VHC in and Mycophenolate-Mofetil (MMF)(n=12). Non-treated normal healthy controls hemodialysis after a first renal transplant failure, with Panel Reacting (NHC)(n=12). Parameters: Calcineurin activity (CNa) in the PBMCs measured by Antibodies (PRA) of 81 and 99 % (by Complement Dependent Cytotoxicity using 32P labeled peptide. IL-2 and IFN- production in PHA activated whole blood. NIH (CDC)) on the day of transplant day received a SHRT. Crossmatch was Results: In CNi treated groups CNa at 0 and 2hr post-dose was lower than that found performed pre-transplant, and 24, 48 hr post transplant by CDC and by Flow in NHC or MMF alone group (p<0.01). A good correlation was found between CNa at Cytometry with double labeling with CD3-PE and anti-human IgG-FITC. 0 and 2hr. IL-2 production was reduced in all CNi groups 2hr post-dose. No Patients received ATG, Cyclosporine and Prednisone. differences were found in CNa between groups receiving CNi alone or with MMF, but Results: CM was positive pre transplant by CDC and Cytometry. At 48 hr differences were evident concerning IL-2 production (p<0.001). Correlation between CDC became almost negative (10-20% mortality) and Cytometry became CNi concentration and CNa or IL-2 were better at 2hr than at 0hr. The IFN- negative 48 hr post transplant. One of the patients presented an episode of acute production was highly variable, with periods of non-inhibition during dose interval. rejection 10 days post-transplant that was resolved with a bolus of Conclusions: The measurement of CNa might be a good predictory parameter of the methilprednisolone. In both patients the grafts were functioning 20 and 23 biological efficacy of the monotherapy with CsA or TRL, whereas the IL-2 production moths post-transplant. Creatinin: 1.1, 1.5; GOT: 34, 14; GTP: 29, 12; GGT: 9, will be more useful to monitor combined therapies with calcineurin inhibitors and 66. MMF. CNa and IL-2 production at 2hr seems to be more informative than at 0hr. CNa Conclusions: Our experience suggests that a positive crossmatch is not an maintains inhibition in all the dosage interval, whereas IFN- production crossed absolute contraindication in simultaneous Hepato-Renal transplantation. A periods of not inhibition. good graft and patient survival is possible at least in some patients. Poster Group 12 - Technology and Typing Methods 219 220 RETROSPECTIVE ANALYSIS FOR 68 HAEMATOPOIETIC SEQUENCE ANALYSIS AND QUALITY CONTROL USING STEM CELL TRANSPLANTATIONS BY A FRENCH HOSPITAL ASSIGN 2.0TM : IMPLICATIONS FOR HIGH THROUGHPUT IN 2001 AND 2002 SEQUENCING BASED TYPING. C.ANDRE-BOTTE1, P.PERRIER1, L.CLEMENT2, D.BENSOUSSAN3, Damian M Goodridge and David C Sayer. Conexio Genomics, East F.WITZ2, P.BORDIGONI2; 1.HLA laboratory – 2.Transplant Center – 3. Fremantle, Western Australia Cellular Therapy Products Unit, CHU, rue du Morvan - 54511 VANDOEUVRE-les-Nancy Cedex, FRANCE We have developed a software package called Assign 2.0TM that dramatically reduces the sequence editing and allele assignment time for DNA SBT and also 68 allogeneic haematopoietic stem cell transplantations (HSCT) have been performed by provides valuable and informative quality control data. These issues are the NANCY universitary hospital in 2001 and 2002 : impediments to the use of SBT as a high throughput protocol for HLA typing. 38 with a familial donor (the average age for recipients was 28 years with 17 patients less Sequence analysis using Assign 2.0TM is performed on files that contain the than 18 years old); 22 with an unrelated donor (the average age for recipients was 14 years with 13 patients less than 18 years old); 8 with an unrelated cord blood unit (the average age nucleotide sequence and individual base quality values (QV). Large batches of for recipients was 11 years with 6 patients less than 18 years old). HLA class I and class II sequence (thousands) can be analysed in a short period of time (seconds). matching and the degree of polymorphism are presented in the three groups. Editing time is drastically reduced as only the base calls that do not meet QV For the 60 HSCT from a related or unrelated donor, donors and recipients were specification are required for manual review. An interactive editor facilitates the systematically typed by lymphocytotoxicity from a blood sample and by DNA-based PCR- editing process if required. Heterozygous allele ambiguities are reported as SSP from another blood sample (at two digit level in the familial genoidentical cases where allele combinations or as NMDP codes. Assign 2.0TM also enables the the 4 parental haplotypes could be identified : 26 cases, and at four digit level in the 34 other resolution of ambiguities by DNA sequencing. cases). The inclusion of base quality values enables a novel and highly effective Among the 38 HSCT from a familial donor, 36 patient/donor pairs were HLA- means of SBT quality control. The mean and standard deviation of QV of genoidentical, 1 pair presented a generic HLA-A mismatch after crossing over on the homozygous and heterozygous base calls is a sensitive indicator of sequence maternal haplotype and 1 pair was HLA-haploidentical. quality. Assign 2.0TM will calculate and plot the mean and standard deviation Among the 22 HSCT from an unrelated donor, 19 patient/donor pairs were fully matched of QV as histograms or longitudinal plots for a) individual sequence positions b) for the loci A, B, C, DRB1, DQB1, and 3 pairs had one mismatch (2 pairs with one A all positions for the sequence of a locus c) all samples on a sequencing run/gel allelic mismatch and 1 pair with one C generic mismatch). HLA- DPB1 matching was also d) all sequencing runs/gels between two timepoints. Additional features of analyzed. Assign 2.0TM include the ability to longitudinally monitor the forward and We note a weak polymorphism for the analyzed loci: 12 different alleles for locus A, 17 reverse sequence base call discrepancies and failed sequences. These features for locus B, 13 for locus C, 15 for locus DRB1, 9 for locus DQB1 and 18 for locus DPB1. and the ease of use of Assign 2.0TM remove data handling as an impediment to For the 8 HSCT from a unrelated cord blood unit, the patient/cord blood unit pairs were high throughput SBT and SNP scoring. matched at the generic level for HLA-A and B and at the allelic level for HLA-DRB1 : only 1 pair was fully matched, 5 pairs presented one mismatch (3 pairs : one A or B generic mismatch; 1 pair : one DRB1 generic mismatch; 1 pair : one DRB1 allelic mismatch). 2 pairs presented two mismatch (1 pair: one A generic mismatch + one B generic mismatch; 1 pair : one B generic mismatch + one DRB1 allelic mismatch). Survival and graft versus host disease (GvHD) outcome were compared according the following criteria: diagnosis, age and conditioning regimen of the recipient; CMV status of the recipient and the donor; related or unrelated donor; source of the stem cells (peripheral blood, bone marrow or umbilical cord blood); quantity of stem cells; HLA matching. Genes and Immunity Abstracts S57 221 222 QUALITY CONTROL ANALYSIS OF HLA SBT DATA USING CLONING AND EXPRESSION OF A SOLUBLE HLA-A*0201- ASSIGN 2.0TM REVEALS A HIGH LEVEL OF VARIATION PEPTIDE- 2m-heavy CHAIN FUSION PROTEIN BETWEEN BATCHES OF COMMERCIAL SEQUENCING REACTION KITS Britta Eiz-Vesper, Ute Holtkamp, Rainer Blasczyk. Department of Transfusion Medicine, Hannover Medical School, Linda K Smith, Rebecca S Whidborne, Laila S Gizzarelli, Alison Hannover, Germany. S Castley, Damian M Goodridge, David C Sayer. Dept. of Clin Recombinant soluble HLA molecules may be useful tools for the detection of Imm and Bioch Genetics, Royal Perth Hospital, Perth Western HLA-specific alloantibodies in the sera of patients awaiting transplantation or Australia 6000 peptide-specific T-cell recognition. The production of heterotrimeric HLA in procaryotic expression systems consists of several steps: expression of the The QC tool of the SBT software package, Assign 2.0TM allows the analysis of individual base call quality values (QV) for a) each base between different heavy chain, expression of 2-microglobulin ( 2M), isolation of the samples, b) all bases between samples and c) all samples between sequencing recombinant proteins, synthesis of specific peptide-ligands and a final refolding runs/gels.