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. In addition, Assign 2.0TM calculates the forward and reverse step to assemble the trimeric construct. In order to facilitate these expression sequence base call discrepancy rates, sequencing failure rates and the amount of and isolation procedures a peptide- 2M-HLA fusion protein was constructed a sequence for an individual locus that has been performed in a single direction and expressed in Escherichia coli. The extracellular domains ( 1, 2 and 3 only. This data is easily generated and can be plotted in a longitudinal manner to domains) of a human leukocyte antigen class I, HLA-A*0201, were cloned and provide extensive and informative SBT quality control. We have retrospectively physically connected to the C-terminus of 2M by a glycine-rich linker. For the analysed all SBT data from our laboratory for 2002 to determine the effect of detection of the recombinant protein by western blot and direct ELISA a V5 tag automated sequencing kit batches (Big Dye Terminator v3.0 from Applied was connected to the C-terminus, followed by a histidine-rich sequence for Biosystems) on sequence quality. An initial analysis of 2,186,498 bases from isolation purposes. We varied this single-chain protein by linking its N-terminus 4000 samples over 371 runs for HLA-B revealed significant differences in to immunodominant HLA-A*0201-restricted peptides by glycine-rich linkers. sequence quality reflected in the mean and standard deviation of QV for The proteins were expressed in the inclusion body compartment of E. coli. After homozygous and heterozygous base calls. Changes correlated precisely with isolation by immobilized metal affinity chromatography via the 6xHis-tag, they changes in sequencing kit batches. These changes were also seen in sequence of were finally refolded. An immunological in vitro refold assay involving the all other class I and class II loci. This data demonstrates the effectiveness of monoclonal antibody W6/32 was used to gauge refolding yielding a similar using Assign 2.0TM for the quality control of SBT and allows the setting of refolding behaviour compared to the heterotrimeric protein.. Thus, this straight standards for the evaluation of SBT reagents and a means for improving SBT forward strategy has the potential to simplify considerably large scale data. Assign 2.0TM enables SBT to be used in a high throughput manner. production of soluble HLA.

223 224 RELEVANCE OF AMINO-ACIDS INSTEAD OF ALLELES IN GENOTYPING OF HUMAN PLATELET ALLOANTIGENS (HPA) DISEASE ASSOCIATION AND TRANSPLANTATION. BASED ON OLIGOARRAY HYBRIDIZATION

Erik H.Rozemuller, Marcel G.J. Tilanus Peter Bugert and Harald Klüter Pathology, UMC-Utrecht, P.O.Box 85500, 3508GA Utrecht. Institute of Transfusion Medicine and Immunology, Red Cross Blood Service of Baden-Württemberg – Hessen, University of Heidelberg, Peptide presentation through HLA molecules is a crucial factor in the adaptive Faculty of Clinical Medicine Mannheim, Germany immune response. Presentation of non-self peptides can initiate an immune response, which may occur after infection. Also after transplantation, Most alloantigens (HLA, blood group antigens or HPA) are based on single mismatched alleles can be presented as foreign peptides and may lead to graft nucleotide polymorphisms (SNPs) in the corresponding gene sequences. failure or graft-versus-host disease (GVHD). Techniques for rapid genotyping of SNPs such as PCR-SSP or PCR-RFLP are HLA alleles are grouped into antigens reflecting serological reactivity. commonly in use. The complexity, i. e., the capacity for simultaneous detection However, DNA typing defined many alleles which are considered to belong to of a number of alleles at different gene loci, of the methods is limited. The the same serological antigen group. Alleles within e.g. the B27 group can differ hybridization of microarrays with allele specific oligos may fullfill the criteria from 1 up to 11 amino-acids. Alleles from different serological groups also can of a technique with high complexity and specifity. Aim of this study was the share certain amino-acids. The relevance of these differences in homology is not optimization of conditions for sample generation, hybridization and data clear. Therefore, amino-acid based analysis should be performed to appreciate evaluation for SNP-typing by oligoarray hybridization. We used the SNPs for the effects of the amino-acids rather than alleles. platelet antigens HPA-1, -2, -3, -5 and GOV as a model system with low We developed software which considers amino-acids instead of individual complexity. Up to 9 oligonucleotides of various lengths specific for one allele alleles or allele groups. This tool compares the HLA-typing of two groups, e.g. were spotted onto specialized glass slides. The samples were generated in a patients and controls, or patients and potential donors. The alleles are converted multiplex PCR reaction for all 5 loci with each reverse primer Cy-3 labelled at into their amino-acid sequences. Allele distribution statistics is performed to the 5’ end. After hybridization and stringent washing the arrays were scanned by identify potential allele associations. In addition, for each codon, the amino-acid using a laser scanner (GMS418, Affimetrix). Computer assisted evaluation of distribution is analyzed for differences between the two groups, identifying signal intensities revealed expected results for HPA-1, -2, -3 and GOV typing of potential relevant amino-acids. This analysis can be extended to nucleotide the reference DNA samples. Typing of the HPA-5 polymorphism failed so far, i. sequences, to epitopes, or to combinations of different positions, e.g. different e. non of the tested oligonucleotides revealed hybridization signals, presumably, pockets in the peptide binding groove. due to the high A/T content of this locus. The conditions for SNP genotyping This analysis tools elucidates the effects of single amino-acids, triplets or based on microarray hybridization optimized in this study will be used for the epitopes in HLA disease association and in the evaluation of donors in development of typing systems with higher complexity. Hematopoietic Stem Cell transplantation.

Genes and Immunity Abstracts S58

225 226 PYROSEQUENCING-STRATEGIES FOR HLA-CLASS II HIGH HLA-CLASS I HIGH RESOLUTION TYPING BY RSCA RESOLUTION TYPING *A.Poles, P.Brookes, R.Sergeant, M.McCluskey & N.Davey Patricia Entz, Mohammad R. Toliat, Jochen Hampe, Stefan Jenisch. *Clinical Immunology, Hammersmith Hospital, DuCane Rd Peter Nürnberg, Marion Nagy; MDC Berlin, Gene Mapping Centre, London W12 0HS Robert-Roessle-Str. 10, 13125 Berlin We have implemented RSCA for HLA Class I high resolution typing for the selection of volunteer unrelated donors and confirmatory typing of patients with Pyrosequencing™ is a realtime sequencing-by-synthesis method for the analysis end stage renal failure. The technique was validated by direct comparison with of large numbers of short to medium length DNA sequences. Nucleotides are results of Class I PCR-SSP typing. To assess the accuracy of the high resolution added separately according to a predetermined dispensation order. Their typing results produced by RSCA, we compared our results with sequencing incorporation is detected by luminescence. The height of the light intensity peaks data of samples from the National External Quality Assessment Scheme is strictly proportional to the number of nucleotides incorporated. (NEQAS). In the present study, we used pyrosequencing for DNA typing of the alleles of 232 samples typed by PCR-SSP and RSCA resulted in 100% concordance for the HLA-class II-DQB1 and DRB1 gene regions. The majority of the DQB1 and HLA-A and HLA-B. For HLA-Cw there was 3.8% discrepancy. This was due to DRB1 alleles can be identified by sequencing exons 2 of both genes. As a first non-detected alleles by PCR-SSP. 64 NEQAS samples were typed by both step, pyrosequencing was optimized for genotyping exon 2 of the HLA-DQB1 PCR-SSP, and RSCA for HLA Class I. Sequencing data was available for 24 of gene. Performing six pyrosequencing reactions with this fragment, we were able the 64 samples typed. There was only one discrepant result between RSCA and to detect 55 of 60 polymorphic residues occurring in DQB1 exon 2. The reading SBT, where RSCA assigned HLA*0203 and the SBT result assigned A*0224. length of the different sequencing reactions ranged from 11 to 39 nucleotides. In Overall in the NEQAS scheme RSCA performed well with 100% accuracy at addition to sequencing of exon 2, we have also established sequencing of two the “two digit” level of resolution with no miss-assigned alleles. short sections of DQB1 exon 3. This allows us to resolve allele groups 02 and 03. One disadvantage with RSCA was lack of discrimination between some After assorting the samples to the different groups of allele families most of the alleles for E.g. HLA-A (A*0201/0209 A*2402/2409) and HLA-B known alleles can be typed. (B*1501/1507, B*5701-5704, B*4409/4413). A further disadvantage is that not The strategy for DRB1 typing is different. Because of the high number of all alleles have had their mobilities defined. This accounted for the mis- alleles, a preselection by allele group-specific PCR with eight different PCR assignment of A*0203. These disadvantages are far outweighed with the high reactions for exon 2 was established. This allows in most cases an allele-specific throughput and low cost of RSCA compared to SBT. This year RSCA provided sequencing procedure. Then we can assess the alleles within the known group by high resolution HLA class I typing for 700 samples. one to six pyrosequencing reactions, depending on the group. In particular pyrosequence-based typing is a powerful tool to analyse heterozygous individuals, better than the classic sequencing methods, and offers a unique combination of high accuracy and throughput.

227 228 LUMINEX 100 SYSTEM: FUTURE TECHNOLOGY FOR HLA- DEVELOPMENT OF A NEW HLA-DRB REAL-TIME PCR ANTIBODY SREENINING AND IDENTIFICATION? TYPING METHOD

Yvonne C.M.van Berkel, Wil Allebes Eduard Palou, Natàlia Casamitjana, Rosa Faner, Roger Colobran, Anna Ribera, Department of Bloodtransfusion and Transplantation Immunology, Ricardo Pujol, Manel Juan. U.M.C. St. Radboud Hospital, Nijmegen, Netherlands. Laboratori d'Immunologia. Centre de Transfusió i Banc de Teixits. Aim: We use ELISA-LAT-M pre-screening for IgG anti-HLA class I/II for sera Barcelona, Spain. of kidney transplantation candidates. Specificity tests based on CDC are used PCR-based typing methods routinely used in the laboratory, such as PCR-SSP for the detection of relevant antibodies. We wanted to know whether the and PCR-SSO, suffer from the drawbacks of time-consuming post-PCR steps, LUMINEX100 could to be an alternative method. low potentiality for automation and high possibility of post-PCR contamination. Method: LUMINEX technology for antibody detection is based on detection of The aim of this study was the development of a HLA-DRB1 and DRB3/4/5 low- reactivity of human antibodies with micro-beads, coated with HLA antigens medium resolution typing method able to eliminate these problems. A real-time isolated from individuals. PCR system was designed by the use of specific primers but also of specific Test: Reactivity of selected sera was compared with LUMINEX100 pre- probes for HLA-DRB alleles allowing the reduction of the number of reactions screening IgG–HLA class I/II and LATM pre-screening. The identification and the increase of the typing resolution. capacity of the LUMINEX100 for IgG-HLA class I and II was compared with This method consists of a set of 16 PCR reaction tubes. Two pairs of primers CDC. are used per reaction: one pair for the DRB genes and the other pair for the Results: Screening: concordance of LUMINEX with LAT-M class I: 94%; class GAPDH gene to provide an internal positive amplification control. In each tube II 96%. Discrepancies between LUMINEX and LAT-M largerly depend on 3 probes with a different fluorescent label are utilised: 2 DRB specific probes difference in criteria for pos/neg. Identification: If history is taken into account, and a GAPDH probe. results are comparable. For individual sera the discrepancies between Two hundred clinical samples that had been typed previously for HLA-DRB LUMINEX and CDC results are due to difference in sensitivity. LUMINEX is by a standard PCR-based method were analyzed by this technique. All samples more sensitive than CDC and LUMINEX detects separated HLA class I/ II gave results in concordance with HLA types obtained previously. Moreover, reactive antibodies and non complement binding IgG antibodies. In presence of less ambiguous results were observed with this method than with the use of the anti-class I , interpretation for class II in CDC is hard to review. CDC also standard PCR-SSP or PCR-SSO methods. detects IgM reactivity, HLA as well as non-HLA directed reactivity. In conclusion, we have developed a reliable HLA-DRB typing method based Conclusion: LUMINEX100 seems to be an excellent alternative HLA-screening in real-time PCR that reduces the steps, the number of tubes and the time and antibody identification technique with the sensitivity and flexibility of required for its completion and, in addition, less ambiguous results are obtained. flowcytometry and simplicity of the ELISA.

Genes and Immunity Abstracts S59

229 230 HLA-C ANTIBODY ANALYSIS: THE MISSING PIECE OF THE IMPROVEMENT OF RESOLUTION IN THE LABTYPE SSO PUZZLE? SYSTEM BY SPECIALTY PROBES TO ESTABLISH LINKAGE OF TWO POLYMORPHIC SEQENCES. Cook Daniel, Kahle D., Klingman L. Allogen LaboratoriesThe Cleveland Clinic Foundation, Cleveland, OH K. Saito, L, Blair, T. Nong, K. Neswald, A. Bedrossian, A. Liu, D. Berman, A. Manzo, and JH Lee, One Lambda, Inc. Canoga Park, CA. Understanding a patient’s antibody specificities can provide significant help in the prediction of crossmatches. We have been using beads with We continue to improve LABType SSO DNA typing system. The system consists single HLA antigens attached, (FlowPRA Single Antigen, One Lambda of oligonucleotide probes immobilized on unique sets of microspheres (up to 100 Inc, Canoga Park, CA), for some time now for analyzing HLA-A and –B per test) and biotinylated locus-specific PCR primers. Hybridization of amplified antibodies. If the donor’s antigens are known, this has allowed us to DNA to probe is detected by labeling with a streptavidin-PE conjugate. A flow predict with reasonable accuracy flow cytometric class I crossmatches. analyzer capable of classifying the microspheres and detecting the PE We have been concerned with the fact that we really did not know fluorescence signal generates mean fluorescence intensity (MFI) from 100~200 anything about antibodies against HLA-C that might be present as well. beads per probe per test. An analysis software adjusts MFI and scores reactions In this preliminary study we have examined the serum of 24 sensitized positive or negative by comparing the MFI to pre-determined cut-off values. The heart transplant candidates for the presence of HLA-C antibodies using software then assigns HLA allele(s) that best match the reaction patterns. single antigen beads obtained from One Lambda. Included are beads Currently, LABType SSO system includes probe sets for HLA-A, B, C, DRB1, DRB3, 4, 5, DQB1 loci at intermediate resolution. The bead-based SSO method with C1, C2, C4, C5, C6, C7, C8, C9, C10, C12, C14, C15, C16, C17 provides high throughput, flexibility to increase the number of probes, excellent AND C18 in two tubes. The patients were divided into groups based on signal to noise ratio and statistically reliable data over the conventional methods. their reactions against the HLA-A and B antigens (broad or specific) and An assay using 96 well microplate and software-based data analysis typically the antigens they were reactive against (Table). allows 200-400 samples per day per a technician. Group N A B C reactions In the SSO or SBT methods certain genotypes that contain identical Broad 9 X X 7 Broad, 2 Specific polymorphism, but differ only in their arrangement, cannot be resolved unless a Specific 2 X 2 Negative polymorphism linkage can be established. This is a typical ambiguity problem in Specific 5 X 3 Negative, 2 Specific HLA typing using SSO or SBT methods and usually requires SSP method to Specific 8 X X 1 Negative, 7 Specific resolve. We have developed specialty probes that detect a presence of two distant unique sequences on the same strand of DNA to establish linkage information. Of 9 patients with broad reactivity against A&B, 7 had broad reactivity Typical ambiguities that require an extra group-specific amplification in SSO against C, and 2 had specific reactions. Negative reactions were seen in method can now be resolved from a single generic amplification product. 5 of 7 patients with specific reactions against A or B. In 8 patients with Representative ambiguities that can be resolved by the specialty probes are: specific reactions that included a mix of A&B reactivity, 7 showed A*2501, 02 specific probe to distinguish A*03/A*25 and A*32/A*66, evidence of specific reactivity against HLA-C. We believe that this A*25/A*74 and A32*/A*66; probes specific for two polymorphisms to resolve preliminary study indicates that HLA-C antibodies are common in Bw4 associated ambiguities in B-locus; DRB1*0308/1107 specific probe to patients sensitized against HLA-A&B, and that this may have to be taken exclude DRB1*0308 from common DRB1*11 and DRB1*03 alleles; CW*0106 into account if the information is to be used to predict crossmatches. specific probe to exclude CW*0106 ambiguities. We plan to develop more specialty probes to improve the resolution of LABType SSO system.

231 232 SEQUENCING BASED TYPING OF THE COMPLETE CODING PRENATAL HPA-GENOTYPING FROM MATERNAL PLASMA REGION OF HLA CLASS I GENES OR SERUM

Ulrich Christ Peter Bugert, Andrea Lese, Jessica Meckies, Wolfgang Zieger and Harald Protrans, Ketsch, Germany Klüter Institute of Transfusion Medicine and Immunology, Red Cross Sequencing gives the most reliable and accurate information of the DNA sequence of a gene and is therefore of particular interest for the determination of Blood Service of Baden-Württemberg – Hessen, University of the full extent and complexity of the HLA polymorphism. Over the last years, Heidelberg, Faculty of Clinical Medicine Mannheim, Germany sequencing-based typing of HLA genes has progressed considerably and the Neonatal alloimmune thrombocytopenia (NAIT) is caused by alloimmunization improvements made allow to implement PCR-SBT for routine HLA typing. of the homozygote mother against the heterozygote fetus‘ platelet antigens, most However, also sequencing is suffering from ambiguities which are caused either often HPA-1a or –5b. Current methods of prenatal testing such as amniocentesis by undefined cis linkages of sequence motifs or by motifs located outside of the and chorionic villus sampling are invasive and pose risks to the unborn. sequenced region. Although those ambiguities caused by undefined cis linkages Postnatal identification of affected fetuses is often too late to prevent the most can be sorted out by haplotype-specific amplification strategies, its application dangerous complication, intracranial hemorrhage. We attempt to establish a is mainly restricted to exon 2 through exon 4 due to PCR limitations. In order to non-invasive prenatal testing method of genotyping HPA-1 and -5 using fetal sort out ambiguities caused by variations outside exons 2 - 4 a PCR and DNA found in maternal plasma or serum. sequencing strategy for the complete gene from the 5’ to the 3’ flanking region Using a commercial kit, DNA was isolated from maternal whole blood, fetal was developed covering exons 1 - 7 and 8, respectively. To obtain a high PCR umbilical cord blood (UCB), and maternal plasma or serum, all from samples reliability a multiplex approach was designed separating the gene in a 5’ and 3’ taken during the birthing process from unselected pregnancies (22 cases). PCR fragment with intron 3 as separating site. This strategy allowed solving all Standard PCR-SSP was used for genotyping of HPA-1 and -5 on DNA samples ambiguities caused by varations outside exons 2 - 4 (e.g. A*74 group, B*07 from maternal whole blood and UCB. In 9 of the cases a difference between group, Cw*07 and 17 group) and is in combination with the haplotype-specific maternal (homozygous) and fetal (heterozygous) HPA status was found. We approach capable of sorting out all ambiguities occuring in HLA class I typing. then performed SSP typing using fluorescently labeled primers to obtain genotyping results from maternal plasma or serum in these cases. Amplification products were analyzed using an automated DNA sequencer. Using this technique we were able to identify the fetal HPA status in maternal serum or plasma in two cases analyzed. The method now needs to be validated and standardized as a routine test for prenatal HPA genotyping.

Genes and Immunity Abstracts S60

233 234 CELL-LINE SPECIFIC DETECTION OF CHIMERISM AFTER IS ELISA-LATM IGM A RELIABLE METHOD FOR DETECTING BLOOD STEM CELL TRANSPLANTATION USING IGM ANTIBODIES AGAINST HLA? FLUORESCENCE DETECTION OF MICROSATELLITES. Yvonne C.M. van Berkel, , Judith van Luijk, Wil Allebes. Ulrike Koehl, Olaf Beck, Dirk Schwabe, Thomas Klingebiehl, Department of Bloodtransfusion and Transpantation Immunology, Erhard Seifried, Christian Seidl. Dep. of Pediatric Oncology, JW U.M.C.St. Radboud Hospital, Nijmegen, The Netherlands. Goethe University and Dep. of Transplantation Immunology and Aim: IgG anti-HLA antibodies are considered to cause graft rejection, in Immunogenetics, RCBDS, Frankfurt am Main, Germany contrast with IgM anti-HLA. However the presence of IgM anti-HLA antibodies Determination of donor host chimerism in PB or BM by microsatellite (STR) is an idication that immunization has occurred and therefore it is useful to know marker provides relevant clinical information about engraftment of donor cells whether IgM anti-HLA antibodies are present. In CDC also non-HLA reactive after allogeneic stem cell transplantation. Lineage-specific chimerism in highly IgM antibodies can be detected, so we looked for a simple and reliable IgM anti- purified leukocyte subsets seems to be even more informative for detection of HLA detection method. graft failure or relapse of the disease. We compared a singleplex in-house STR Method: We screened 296 sera in ELISA LATM-IgM and IgG for presence of set with two commercially available multiplex STR systems (Profiler and class I/II antibodies, of which 80 sera also screened for IgM (DTT-sensitivity) in Cofiler, ABI, Germany) in pediatric patients, two suffered from ALL, one from CDC. Sera known with IgM auto or allo antibodies in CDC and sera before and thalassemia and one from aplastic anemia. Chimerism was detected routinely in after blood transfusions (TF) were screened. Criteria for pos/neg and sensitivity PB, BM and CD3 selected T-cells. In some cases the cell populations of the tests were established and locally validated. investigated included granulocytes, monocytes, B-cells, stem cells, NK cells and Results: 212/296: Neither IgM and IgG anti-HLA-class I and/or II. Only IgM T-cell subsets (CD4+ and CD8+) (all purified using microbeads, AutoMacs, anti-HLA-class 1:6 anti-HLA class II: 0 . Only IgG anti-HLA class I: 28, anti- Myltenyi Biotec, Germany). Based on the in-house STR system only, HLA-class II: 9. IgM and IgG anti-HLA class I:3, anti-HLA-class II: 3 patients calculation of chimerism resulted in 3-9 informative STRs per patient. Standard CDC-DTT sensitive:15. 9/15 with detectable auto reactivity in CDC, 0/9 IgM deviation was < 4.2% in PB (n=297), <5.5% in BM (n=46) and 6.9% (n=39) in anti-HLA. 6/15 without auto reactivity in CDC, 2/6 IgM anti-HLA. the purified leukocyte subsets CD3+, CD4+CD3+, CD8+CD3+, CD56+CD3++, Sera 1 month after TF: CDC-DTT sensitive:19. 12/19 with detectable auto CD14+, CD34+ and CD19+. Using both, the in-house and the multiplex STR reactivity, 0/12 with IgM anti-HLA. 6/19 without auto reactivity, 3/6 with IgM system gave comparable quantitative results. Our results indicate that molecular anti-HLA, 2/3 also had pre TF IgM anti-HLA. assessment of chimerism can be performed with a wide variety of STR loci Conclusion: Using ELISA-LAT-M IgM it is possible to detect IgM anti-HLA either by single or multiplex PCR. Quantitative determination of cell-line in the presence of non-HLA reactive IgM antibodies. In this way detection of specific chimerism should facilitate therapeutic interventions. primary immunisation should be possible. Supported by "Hilfe für Krebskranke Kinder Frankfurt e.V.

235 236 EVALUATION OF ONE LAMBDA ABDR SSP KIT IN HLA FLOWPRA CLASS II SINGLE ANTIGEN BEADS TYPING OF CADAVER ORGAN DONORS DURING NIGHT TIME DUTY Jarhow Lee, Rui Pei, Remi Shih, and Mike Chen A panel of color-coded microbeads, which were coated with purified Katri Haimila, Irma Matinlauri, Jukka Partanen recombinant single HLA Class II antigens (Table 1), was produced for flow Dept Tissue Typing, FRC Blood Transfusion Service, Helsinki, cytometric detection of HLA Class II antibodies. These single Class II antigens Finland reacted specifically with serologically defined HLA-DR specific sera and monoclonal antibodies. The single Class II antigen panel provided higher We evaluated the use of One Lambda’s HLA A, B, DR 96-well format SSP kit resolution than antigens purified from the regular cell panel for Class II in typing of cadaver organ donors during the night time duty. Typically more antibody detection by uncovering the specificities that were masked by other than 100 cadaver organ donors are annually HLA typed by the Department. In broader epitopes. Single antigens also clearly resolve the serum specificities that the period of Jan – Nov 2002, 96 cadaver donors were obtained for matching. would not able to be assigned by the regular panel due to HLA allele-linkage Of these 81 were typed, in addition to the standard serology, also using the One problems. For example, the single antigen panel can distinguish a DR2 vs. Lambda kit. The PCR typings were performed by 12 technicians experienced DR51 serum by its reaction pattern to DRB1 vs. DRB5 gene products without with the standard PCR-based HLA methodology. being affected by the fact that DR2 always associated with DR51. By examining Thirty-three typings of the 81 (41%) typings succeeded without any problems. high PRA sera reacting to more than 80% of the Class II antigens purified from Forty-eight typings had problems. In 11 typings (14%) no accurate typing result regular cell panel and which specificities would have difficulty to be assigned could be obtained at all. Four (5%) of these were due to technical problems by the regular cell panel, we showed that the single antigen panel facilitates the related to casting the gel. The remaining 7 cases without the result were due to accurate assignment of HLA Class II antibody specificities. multiple unamplified PCR reactions. In 37 cases, 1 or 2 reactions showed no amplification, but it was, however, possible to deduce the alleles with a Table 1: Recombinant Single HLA Class II Antigens sufficient accuracy. In particular, tube 8H gave blur results and gave no amplification in some serologically positive HLA B35 cases (n= 4). DR0101 DR1201 DRB50101 DR0801 DR1501 DR0404 DR0102 DR1101 There was no clear differences in success rates between individual DR0103 DR1301 DRB30201 DR0901 DR1601 DR0405 DR0302 DR1502 technicians, showing that the problems were more due to the kit itself or due to DR0401 DR1303 DRB40103 DR1001 DR0301 DR1202 DR0701 DR1401 the busy labwork during the night time duty, than due to inexperience of technicians. We conclude that in our hands the A B DR SSP kit seems not sufficiently robust to be used as the only HLA typing method of cadaver organ donors during the night time duty; the standard serology is still often needed to ensure accurate results.

Genes and Immunity Abstracts S61

237 238 COMPARISON OF ANTIBODY SCREENING METHODS FOR USE OF ASSIGN SOFTWARE FOR QC PURPOSES IN RENAL AND CARDIAC TRANSPLANT PATIENTS SEQUENCING-BASED TYPING.

Gary Cavanagh, Vaughan Carter, John Goodwin, Robert Pengilly, Shireen McGinnis2, Goodridge1, Stein2, Krausa2, Sayer1 Calander, Andrea Harmer (1Conexio Genomics, East Fremantle Western Australia.) National Blood Service, Newcastle and Sheffield Centres, UK (2Forensic Analytical, Hayward, CA USA.) Currently the number of available antibody screening methods is greater than at Advances in automated instrumentation and DNA sequencing chemistry have any previous time. In this study the utility of different techniques and their enabled sequencing-based typing (SBT) to become a common method for high suitability for 2 patient groups has been investigated. resolution HLA typing. A key component in the process is data analysis and Samples from 56 renal and 37 cardiac patients were screened for HLA class I until recently, tools appropriate to high throughput analysis have been and II antibodies using CDC, ELISA (LAT-M and GTI kits) and Luminex unavailable. In addition to aiding in the actual allele typing results, it is also (LABScreen and LifeMatch kits). In cases where 4 of the 5 methods gave the important to be able to measure the data quality. This is important with respect same result the non-concordant result was classed as false positive or negative. to identifying variations within and between data sets and identifying the Concordant results for all 5 methods were found for 68.6% and 55.4% of renal sources of deviations in data quality; i.e. instrumentation, reagent quality, or patients for class I and class II respectively. For cardiac patients concordance operator error. AssignTM is a SBT software program that provides high was found in only 43.2% of samples for both class I and II. A number of throughput allele typing and comprehensive SBT quality control (QC) analysis differences were observed between results for the 2 patient groups. A high of DNA sequence. In the current study, we used AssignTM to retrospectively proportion of false positive results was found in the cardiac patients (51.4% for analyze multiple lots of commercially released AlleleSEQR HLA-C SBT class I and 46% for class II). These were primarily due to non-concordant reagents. Before formal release of these reagents, each PCR and sequencing LABScreen results. In the renal patients the proportion of false positive results reagent is tested against a panel of control DNA representing a range of allele was lower (25.5% class I and 21.4% class II) In addition a number of false types to test for allele identification, allele balance (to identify drop-out) and negative results were also observed, particularly for class II where these sequence data quality (measured as signal strength and errors at non-variant accounted for 10.7% overall. The highest proportion of discordant results for the positions). The results demonstrated quantitatively, the level of lot-to-lot renal patient group was found using CDC screening. Overall the lowest consistency in these formulations. Implementation of AssignTM in a routine proportion of non-concordant results were found using the LAT-M and typing laboratory can help to increase throughput, improve data management, LifeMatch kits (3.2% and 9.7% respectively). minimize systematic errors, and streamline overall quality control processes. These results emphasise the importance of comparing a number of methods in order to select the most appropriate method for specific patient groups.

239 240 RAPID DETECTION OF ALL HLA-B*27 ALLELES BY GROUP- HLA CLASS II ANTIBODY IDENTIFICATION; A SPECIFIC POLYMERASE CHAIN REACTION COMPARISON OF ELISA & LUMINEX TECHNOLOGY.

Elisabetta Zino, Simona Di Terlizzi, Luigi D’Amato, Carmen Carugo, Robert Whittle1, Liz Buckland1, Ary Frost1, Nicola Ashby2, Robert Lewis2, Anne. M. Elisabetta Sironi, Daniela Ceresa, Chiara Bonini, Silvano Rossini, Walters2, Sami Sadek2, Deborah Sage1. 1 H&I Dept, NBS-Tooting, London , UK Katharina Fleischhauer. 2 Wessex Renal & Transplant Unit, Queen Alexandra Hosp, Portsmouth HLA Laboratory, Immunohematology and Blood Bank, Istituto Scientifico H.S. Raffaele, via Olgettina 60,I-20132 Milan, Italy In renal transplantation the importance of avoiding organs bearing antigens to which the recipient has preformed HLA antibodies is well established. New technologies have led to HLA-B*27 is strongly associated with a number of rheumatic diseases, improvements in the identification of HLA-specific antibody. including ankylosing spondylitis and reactive arthritis. Targeted detection of the This study compared CII antibody identification between our current ELISA HLA-B*27 gene by molecular methods is hampered by the extreme identification kit and 2 new kits using Luminex technology. heterogeneity of the serological HLA-B*27 group, with 28 different subtypes 64 sera, from 46 patients, positive by GTITM B-screen ELISA were tested for described to date. Here we describe a simple and rapid Sequence Specific CII antibody specificity by GTI Quik ID II ELISA, LifeMATCH ID CII and Primer (SSP) -based method for detection of all 28 B*27 alleles defined to date, LABScreenTM PRA CII kits. Results showed 5/64 (8%) sera were negative by a minimum of two and a maximum of four sequence-specific PCR reactions. when tested by the identification kits. In a comparison with ELISA, 39/64 The protocol foresees an initial screening by two PCR reactions at the same (61%) sera gave additional specificities by Life MATCH & by LABScreen. amplification conditions, which amplify all known B*27 alleles in addition to 27/39 (69%) sera demonstrating extra specificities by LifeMATCH also showed B*4202 and B*7301 (SSP-B27W) or most but not all HLA-B*27 alleles (SSP- extra specificities by LABScreen. Antibody profiles detected by the Luminex- B27R). Samples resulting positive for both reactions are therefore unequivocally based kits were broadly concurrent and were identical in 5/64 (8%) sera. identified to carry HLA-B*27. Samples resulting positive for SSP-B27W but Results obtained were dependent on panel composition and in addition to negative for SSP-B27R have to be further tested by two additional PCR analysis using computer software, manual analysis was required to account for reactions in order to exclude the presence of HLA-B*4202 or –B*7301.The patient history. The use of DRB1/DQ homozygous beads (LifeMATCH), a described protocol should be useful for laboratories involved in diagnostics and larger panel size (LABScreen 33 beads, LifeMATCH 22), together with unusual research of rheumatoid diseases. DR/DQ associations aided antibody identification. In our laboratory Luminex technology is a more sensitive technique, detecting a greater number of antibody specificities compared with our current technique. Improved antibody identification will reduce the likelihood of positive crossmatches and influence organ allocation.

Genes and Immunity Abstracts S62

241 242 SCREENING AND IDENTIFICATION OF HLA ANTIBODIES HIGH RESOLUTION DRB TYPING USING LUMINEX WITH LUMINEX-LIFEMATCH™ TECHNOLOGY. Bryan L. Ray, Amy Wayland-Smith, Ivan Balazs, Orchid Diagnostics Christian Demanet, Marc Andrien, Greta Houbregs, Ilias Doxiadis. The LIFEMATCH™ HLA DRB typing kit includes DR2 and DR52 primers in HLA Laboratory, Academic Hospital-VUB, Brussels, Belgium. addition to DRB generic primers. In earlier studies, it was found that approximately 25% of samples required DR52 and 10% required DR2 group The Luminex-LabMAP™ technology uses color-coded specific amplification to resolve serological ambiguities. Because of these high microspheres that differ by the ratio of two internal fluorescent percentages, these reactions were included in the LIFEMATCH HLA DRB dyes. To each individual microsphere different proteins can be typing kit. For many samples, the kit provides near allele-level resolution for attached covalently and up to 100 different microbeads may be DRB1*03,09,10,12,13,14,15 & 16. This combination of primer sets resolves combined in one suspension for a single test. This technology was virtually all serological ambiguities with the exception of some DR52 homozygous combinations. To aid in resolving these ambiguities, a DR11 group used by Orchid Diagnostics to develop assays for screening specific product for DR11 was developed. Although the resolution of DR11 is (Lifematch-Lifescreen or LMSC) and identification (Lifematch-ID low, it does help resolve the ambiguities that arise in DR52 homozygotes. For or LMID) of anti-HLA antibodies. example, the DRB typing kit cannot resolve 03xx,11xx from 0308,1307. The A panel of fully characterized sera (n=92) was obtained from Eurotransplant DR11 group specific product can eliminate the latter alternative. Thus DR11, in (Leiden Univ. Medical Center) and tested on both LMSC and LMID and combination with the current kit, resolves virtually all serological ambiguities. compared to a conventional cytotoxicity (CDC) assay. A selected panel (n=20) To provide higher resolution tyings for the remaining allele groups, we have was also tested in another laboratory (Hôpital Erasme, Brussels) with the developed DR1, 4, 11 and DR52 associated (DRB1*03,08,11,12,13,14) group Lambda Antigen Tray High Definition Class I assay (LAT1HD) from One specific products for the Luminex platform. The DR1 & DR4 group specific Lambda. Further more, 166 follow-up samples from kidney recipients or products provide near allele-level resolution for their respective serological patients on the waiting list were tested with LMSC and LMID (if necessary) and groups, whereas, DR52 associated provides high resolution for DR8. Typically, the results compared with those from CDC. The results between different once samples have been analyzed with generic, DR52 and DR2 primers, they technologies were scored and reported as follows: complete match (CM), partial can be grouped for the additional testing with DR1, 4, 11, 52 associated. Thus, match (PM) or no match (NM). the Luminex xMAP platform, now can easily produce near allele-level The results between LMSC and CDC showed: CM 90% and NM 10%. resolution for almost all allele groups. Comparable results were obtained with LAT1HD. Between LMID and CDC: CM 69%, PM 14% and NM 16%. Our follow up samples tested with LMSC and CDC showed CM 92% and NM 8%. The discrepant results were always in the same direction (LMSC pos. and CDC neg.). These findings suggest a higher sensitivity and subsequent earlier detection of anti-HLA class I antibodies with the LMSC assay.

243 244 DNA ON FILTER PAPER: ITS EMPLOY FOR HLA TYPING WHOLE GENOMIC AMPLIFICATION APPLIED TO LOW V.J.LEON, A.J.LEON*. RESOLUTION TYPING FOR THE HLA CLASS II V.J.Leon, R. Corral Servicio de Bioquimica. Hospital Universitario Servicio de Bioquimica. Hospital Universitario de Salamanca, de Salamanca. *Laboratorio de Pediatria e inmunología, Universidad de The SSP method usually employed for the molecular typing of the HLA system Valladolid. requires about 50 ng/mcl per tube. As a result we need quantities of about 40 The creation of a registry of samples of genomic material, usually requires a mcg of DNA for a complete molecular typing of HLA class I and II. This painstaking process to obtain the DNA and to store it at -85ºC. All this implies a high requirement makes it more difficult to study from HLA of hipocelular samples cost in terms of staff, material, and storage, which limits the size and the number of such as medular aphasia and cells from the umbilical cord. archives. In order to overcome this difficulty, we have made a previous amplification of The conservation of blood samples (or previously extracted DNA) on filter paper the sample with the DOP-PCR technique, based on the amplification with allows a great number of samples to be held within a small space without any degenerated primers, creating a source of oligonucleotides that have as their maintenance, making it very easy to access the DNA of the sample and carry out hybridation target, fixed segments on the chains of genomic DNA. The DNA different genomic studies, especially those that use PCR technique. hold of the sample can be multiplied, obtaining enough genomic material to be Several samples of 25 mcl of blood, or DNA previously extracted, on Whatman nº 3 employed for the complete study of HLA class II. paper, and dried at room temperature or in a heater at 45ºC. Insert the filter paper of the The samples, from 50 ng to 1mcg of DNA, were amplified using the DOP- sample into an envelope and label it. Enclose several of these envelopes within, in a PCR kit from Roche, obtaining 100 mcl in a concentration higher from 500 plastic bag with hermetic closing and silica gel desiccant. mcg/ml, and carried to a concentration of 50 ng/ml are used as preamplifier The spot on the filter paper for study is cut and placed in an Eppendorf tube, 200 mcl samples. At the same time, DNA from the same patients has been used as a of a 4M solution of guanidine isotiocianate is added, and it is then heated at 55ºC for parallel control. 30 minutes. The DNA released from the paper can be extracted either with saline For the genomic study of the system HLA class II, we have employed the kits precipitation, phenol chloroform or cationic resins. The technique we have chosen was for generic and specific typing, Dynal DRB1*, DQA1*, DQB1* and DPB1*. the Kit Direct II of Dynal, due to its ease and rapidity; it is based on a resin with a The tests have had identical results in both parallel assays with “normal” DNA metallic core which permits the separation of the DNA-resin complex with an and DNA amplified by DOP-PCR. magnetic concentrator, and the complex is undone at 65ºC for 5 minutes. After The application of the previous amplification, for DOP-PCR, permits the use removing the imam with the resin, DNA in a liquid phase remains at a quantity of 1-4 of very small quantities of DNA from hipocelular samples, blood smears, and mcg, ready to be employed. We have applied the DOP-PCR technique, when the blood spots on paper. amount of DNA available in a blood spot is not enough for our porpoises, such as a complete HLA genomic study. We have stored our samples over filter paper for periods of over a year, using the DNA restored for the genomic typing of HLA class I and II, and also for coagulation factors, satisfactory results in both cases.

Genes and Immunity Abstracts S63

245 246 COMPARISON OF MICROPARTICLE-FLOWCYTOMETRY HLA A*, B*, DRB1* SSP- LOW RESOLUTION TYPING OF HLA-ANTIBODY SCREENING AND DIFFERENTIATION WITH CORD BLOOD SAMPLES USING DILUTED DNA PREPARED ELISA AND CDC. WITH THE GENOMTM-6 AUTOMATED SYSTEM Mira Mosebach, Kerstin Lehniger, Vikica Dumlijan, Christiane Seide, Silvia Ulrich, Ursula Posch, Gerhard Lanzer Erhard Seifried, Christian Seidl. Department of Transplantation University Clinic of Bloodgroupserology and Transfusionmedicine Immunology and Immunogenetics, Institute of Transfusion Medicine Graz, Austria and Immunohematology, RCBDS, Frankfurt am Main, Germany For HLA typing with SSP (Sequence Specific Primer) concentration and quality Antibody screening and differentiation is an important parameter for risk of DNA are critical variables. We tested the amplification ability of diluted assessment in solid organ transplantation. Besides the microlymphocytoxicity DNA isolated from 350µl cord blood samples (n=20) using the GenoMTM-6 test (CDC), there have been recently additional methods established based on Robotic Workstation with the GenoPrepTM Cartride B 350 and the 350µl the detection of soluble HLA-antigens to detect and specify HLA IgG protocol. The DNA was eluted in 200µl aqua. dest., the yield of genomic DNA antibodies. Out of our local waiting list of patients, we have compared the varied from 6µg to 14µg depending on the number of white blood cells in the results of anti-HLA-positive patients (N=21) tested either by ELISA sample. For HLA A*, B*, DRB1* low resolution typing with SSP-Typing Kits (LATM/LAT12-88, One Lambda, Canogen Park, CA, USA) or microparticle (GenovisionTM) 6µg of DNAis needed. In order to have sufficient DNA for flowcytometry (LABScreen PRA Class I and Class II , One Lambda). These further high resolution class I and class II typing we reduced the amount of patients have been also tested historically by CDC with DTT. We observed the DNA to 15ng for each primermix reaction instead of 60ng as recommended in following results: the protocol. As a control of these typing results each cord blood sample was also isolated by a salting out procedure used routinely in our lab and the SSP LABScreen CI + LABScreen CI – typing was performed with 60ng DNA for each primermix reaction according to ELISA CI + 18/20 ( 90%) 2/20 (10%) the SSP protocol. Both typing procedures gave interpretable results with comparably high quality of specific and internal control bands. The data show LABScreen CII + LABScreen CII – that DNA of cord blood samples isolated and purified using the GenoM6TM- ELISA CII - 3/12 (25%) 9/12 (75%) Robitic Workstation is of such high quality that SSP can also work with only ELISA CII + 8/9 (89%) 1/9 (11%) 25% of the amount of DNA that is recommended in the protocol. Concordant results in all three tests (CDC/ELISA/LABScreen) for anti-HLA Class I (CI+) positive sera were obtained in 75% (15/20). Discordant cases were one sera positive only in CDC/ELISA and one sera positive only in ELISA. Three sera were positive in ELISA/LABScreen but negative by CDC. For anti-HLA Class II positive sera concordant results in all three tests were found in 44% (5/9). Four CDC-negative sera were positive by ELISA/LABScreen.

247 248 DNA EXTRACTION FROM BLOOD USING THE ABBOTT USING CAPILLIARY ELECTROPHORESIS READINGS OF PCR- M1000 AUTOMATED SAMPLE PREPARATION SYSTEM. SSP AMPLIFICATIONS FOR THE GENOMIC STUDY OF HLA Gerard J. Gundling, Scott G. Safar, and John M. Robinson. V.J.LEON, B.GARCIA-BERROCAL,O.HERRAIZ. Abbott Laboratories, Abbott Park, IL. USA Servicio de Bioquímica, Hospital Universitario de Salamanca.(Spain) The Abbott m1000 is an automated system designed for the purification of nucleic acids from clinical specimens using magnetic microparticle processes. The reading of PCR-SSP amplifications for the study of the HLA system The processes used in sample preparation are conducted in a single tube on an is usually carried out using electrophoresis on agarose gels at 2%. This automated platform with no user intervention. Reagents and protocols have been technique presents a number of problems that arise from the size of the developed that allow for either RNA isolation from 1 ml plasma samples, or amplification itself since in order to guarantee detection of amplifications DNA isolation from 0.2 ml blood samples. The DNA extraction protocol was evaluated with the LiPA HLA-A kit from Innogenetics. Two hundred of around 100bp a 3% gel is required, and likewise, for amplifications of microliters of EDTA collected blood was added to the chaotrope-based lysis 400bp a 1.5% gel is required. Consequently, the use of 2% gels limits us to reagent and the DNA was non-specifically bound to the magnetic particles. The an optimum range of 120-360bp, requiring repetition of the process in particles were washed extensively after the binding step to remove specimen doubtful cases, with gels of a size more suited to the amplification. components and contaminants. The bound nucleic acids were eluted from the It should be underlined that the time taken to prepare the gels, to load magnetic particles by incubation at an elevated temperature, and the eluates them with the samples for assay and the duration of the electrophoresis (240 microliters) were transferred automatically to output tubes for storage. itself, implies a significant cost in terms of laboratory man/hours. Five microliters of each purified specimen was used in the amplification step in This has led us to analyse a possible alternative for automating the the LiPA HLA-A assay, then the amplified products were hybridized to probes analysis of PCR-SSP products through capillary electrophoresis and fixed on membrane based strips. Following hybridization, the bound material examining the possible advantages that it might offer such as automation was visualized and the pattern of hybridization was evaluated to identify the using samplers. HLA-A locus alleles. All blood samples extracted on the m1000 and tested MATERIAL AND METHODS: Ten samples of DNA from healthy with the HLA-A test gave interpretable allele results. The m1000 system, combined with the DNA purification reagents, is an effective method for the subjects were extracted by salting-out and taken to a concentration of automated purification of genomic DNA from blood specimens for use in 100ng/ul. A PCR-SSP typing kit DQB “Generic” was used (DYNAL,) genomic assays such as the Innogenetics LiPA HLA-A assay. and assays were carried out according to manufacturers’ specifications, and duplicated, the first analysed using electrophoresis on 2% agarose gel, and the second placed in an automatic sampler of a capillary electrophoresis PACE MDQ BECKMAN, grading the conditions using the e CAP ds DAN Beckman, at a range of 72-1300 bp, with a capillary of 47cm, at 20ºc and a voltage of 9.4 Kv. to produce a field of 200V/cm and 100 uA. RESULTS: The amplifications subjected to electrophoresis revealed amplifications of a similar size, and in the same tubes, as those revealed by electrophoresis on agarose gel in all cases studied. The use of electrophoresis capillary represented a significant saving both in time and in the reagents that would be used for the agarose gels.

Genes and Immunity Abstracts S64

249 250 HLA MATCHING RELIABILITY RESTRICTED BY SEROLOGY PROTEIN CONTAMINATION IN DNA EXTRACTION TYPING IN ALLOGENIC HSCT METHODS

Zorana Andric, Ruzica Simonovic, Dragana Stamatovic*, V.J.Leon, M.B.Gonzalez*, N.C.Gutierrez*, Servicio de Bioquimica. Milomir Malesevic*, Gordana Cuk, Ivanka Stolic Hospital Universitario de Salamanca. *Centro del Cancer. Salamanca Tissue Typing Center, National Blood Transfusion Institute; *Clinic (SPAIN) of Hematology, Military Medical Academy, Belgrade Currently, the extraction of DNA from blood samples for Molecular Biology studies Background: In our country selection of HLA matched pairs for hematopoetic into Histocompatibility, may be affected by the large number of methods whose stem cells transplantation (HSCT) is still achieved by serology assays in physical-chemical principles vary greatly. addition that not all the members of patient’s core family are available for They all consist in two clearly defined stages. Firstly, the cells must be destroyed in typing. The purpose of this study is to estimate the reliability of HLA Class I order to free and suspend the DNA chains that they contain. In order to achieve this, and Class II matching defined by serology typing tests by analyzing the HLA usually, either the protein structures are digested by protesases, or they are suspended matches in performed related HSCT. Material and methods: 46 patients (pts) using appropriate detergents. In the second stage, the DNA fragment present in the with malignant hematological diseases treated with related HSCT were mixture obtained is isolated either chemically, by extraction it with phenol-chloroform, analyzed. Apart from patient and donor, it was possible to type both parents for saline precipitation of the proteins, or physio-chemical method, using the anionic 9 pts or the other members of family for 21 pts while for 16 HCSTs only nature of DNA by fixing it to an appropriate matrix. In our study we have attempted to donor/recipient pair were available. Mixed lymphocyte reaction was no reactive compare the quality of the DNA obtained from different methods, all of them in all donor/patient pairs. Results: Patient/donor two haplotype matched was commonly used and easily set up within the laboratory, focussing especially on established for 16 pts (Group 1) according to the results of parents or patient’s possible protein contamination. core family typing, but for 26 phenotipicaly identical pairs there was no certain Material and methods: The techniques set up in our laboratory and applied to evidence of two haplotype match (Group 2). Single mismatch was determined nucleated blood cells are summarised below, including there principal characeteristics: for 4 HSCT pairs (Group 3). Group 2 revealed higher incidence of mild acute (1) Proteinase K digestion: Ether-phenol chloroform extraction.. (2) Proteinase K GvHD (grade I-II) compare to Group 1 (61,5% vs. 56,2%). The severe acute digestion: Saline precipitation of proteins followed by alcoholic precipitation of DNA. GvHD (grade III-IV) also occurred more frequently in Group 2 (19,2% vs. (3) Proteinase K digestion : DNA obtained with silica gel. (4) Guanidine Isocyanate 6,2%), whereas the most frequent was observed in Group 3 (25%). In Group 3, suspension : Saline precipitation of proteins followed by alcoholic precipitation of 50% of analyzed patients rejected graft. Conclusion: The evaluation of DNA. (5) Guanidine Isocyanate suspension: DNA obtained with silica gel. (6) serologically defined HLA matches in related donor/recipient HSCT implies the Guanidine Isocyanate suspension: DNA obtained through ionic exchange with DEAE decreased incidence of severe acute GvHD in HLA matches defined by Sephadex. inherited haplotypes as well. The quality of the DNA was controlled measuring 260/280 nm. Protein contamination was detected carrying out electrophoresis in Agarose gel at 1%, stained with bright blue Coomassie R 250. RESULTS: The results obtained indicate the superiority of the methods using K- protease in terms of its purity and absence of protein contamination. On comparing the methods of DNA isolation, fragmentation with ether-phenol-chloroform, followed by saline precipitation offered the best results.

251 252 HLA-CLASS I AND CLASS II TYPING USING REVERSE SSO HLA-G TYPING WITH SNAPSHOT™ DDNTP PRIMER METHODOLOGY IN COMBINATION WITH FLUORESCENT EXTENSION TECHNIQUE. MICROSPHERES AND FLOW ANALYZER (LUMINEX™) G.M. Bäckström Falko Heinemann, Stanislav Ferencik, Hans Grosse- National Board of Forensic Medicine, Department of Forensic Wilde Genetics, Linköping, Sweden Institute of Immunology, University Hospital of Essen, Germany The 15 HLA-G alleles described in the IMGT/HLA sequence data base differ by variation at codon 31(A/T), 54(A/G), 57(G/A/C), 69(C/T) in exon 2, at codon Introduction: The LABType™ SSO assay (One Lambda, Inc.) enables HLA- 93(C/T), 107(A/T), 110(C/A), 130(C/T) in exon 3, and at codon 188(C/T), and class I and class II typing using probes bound to colour coded microspheres. 258(C/T) in exon 4. Samples from 100 individuals from South East of Sweden Hybridisation is detected by labeling with a streptavidin-phycoerythrin (PE) were investigated. The most frequent alleles were *01011 (0.53), *01012(0.20), conjugate. A flow analyzer (LABScan™100/LUMINEX™) identifies the *01041/01043(0.08), and *0106(0.05). The following polymorphisms were not fluorescent intensity of PE on each microsphere. Interpretation software then found: G in codon 54 (* 0102), C in 57(*1042) and T in 69 (* 01014). analyzes reaction patterns and assigns best matching HLA-alleles. These SNPs were determined by three separate multiplex reactions, one for In our study we investigated unselected DNA samples and performed HLA-A each exon, using the ABI PRISM SnaPshot ™ Multiplex Kit and the ABI (n=109), HLA-B (n=107), and HLA-DRB1 (n=121) typing by LABType™ PRISM 310 Genetic Analyser (Applied Biosystems). methodology. Furthermore, we compared the results with PCR-SSP (two digits resolution level). Results: 101 (93%) of the DNA samples revealed to be concordant at HLA-A between the LABType™ assay and the PCR-SSP whereas at the HLA-B locus 97 samples (91%) were concordant. All HLA-B*15 and B*40 positive samples could not be resolved into the serological specificities B60, B61, B62, B71, B72, and B75, as it was possible by the PCR-SSP method. Finally, 119 (98%) of the samples were concordant at HLA-DRB1 between both assays and could be typed at the two digits level by the LABType™ SSO assay (e. g. the DRB1*15/16 group). Conclusion: Our study revealed the LABType™ methodology to be a reliable alternative to established HLA-class I and class II PCR-SSP based low resolution typing methods. Moreover, it provides high throughput in combination with little efforts in technical staff.

Genes and Immunity Abstracts S65

253 254 ELISA TECHNIQUE POSSIBLE USE IN ANTI-HLA ANTIBODY ADJUSTING THE MEDIAN AMPLIFICATION SIZE IN DOP- STUDY FOR PATIENTS WAITING FOR A FURTHER KIDNEY PCR TRANSPLANT. V.J.Leon,R.Corral. Servicio de Bioquimica. Hospital Universitario de Praticò Loredana, Uboldi de Capei Mariafederica, Oda Alice, Zilio Micaela, Salamanca., (Spain) Rosati Federica, Leone Ercolino, Dall’Omo Anna M., Curtoni E. Sergio. Transplantation Immunology Service, Dept. Genetics, University of With only small quantities of DNA, the use of DOP-PCR greatly increases the Torino, Italy quantity of genomic material available by employing primer strands which join up with fixed segments of the DNA chain. We have found that the application of In the present study we analysed anti-HLA class I and II antibodies in the sera this technique for conditions and components that exist in the kinetics of patients who still underwent an organ transplant and, after its failure, returned commonly described in the literature leads to an extremely high replication of in waiting list. Our aim was 1) to confirm the results obtained in routine using amplifications of approximately 500 bp, in comparison to larger amplifications, Complement Dependent Citotoxicity (CDC) technique anti-Class I antibodies which means that when we work with DNA segments of over 800 bp we have to and 2) to characterize anti-Class II antibodies (which are very difficult to increase the quantity of the amplified target in order to obtain the desired analyse by CDC), using an Enzyme Linked Immunosorbent Solubility Assay results. (E) technique. The analysis of the chemical components of the reaction mixture reveals its The study included 31 patients: 28/31 were in waiting list for a second and kinetic role: the salts’ lends the necessary osmotic strength to allow for the 2/31 for a third kidney transplant; 1/31 patient had been previously received an correct hybridisation of the DNA primer chain and together with the buffer, heart and now was waiting for a kidney transplant. We selected two different creates the optimum conditions for the ploymerase Taq enzyme to work. The One Lambda ELISA kits: LATM20x5 (pre-screening) and LAT1288 concentration of nucleotides and magnesium maximises their access to the (specificity search). enzyme. Pre-screening results: The programming of the physical conditions of the thermocycler ensures that E - E + E + cl.I E + cl. II E + cl.I/II the starting blocks and the optimum temperature necessary for DOP-PCR kinetics are obtained. 9/31 22/31 4/22 7/22 11/22 Material and Methods: Series of reaction mixtures were set up in which from Specificity analyses: in 12/22 patients E + the antibodies specificities were 100 ng of DNA, in each reaction, the concentrations of primers, magnesium, directed against HLA antigens of their first donor; in 5/22 cases were related to NTPs, and reaction pH values were varied. The physical conditions of the HLA antigens of their blood donor. In 4/22 patients we do not known HLA-DR thermocycler were standard, as recommended by the Roche DOP-PCR kit, typing of the kidney donor. Only in 1/22 patient we were not able to relate which we used as a parallel control to our own programmed series. antibodies specificity with immunologic events. Results: The analysis of the series of chemical conditions studies revealed that In conclusion the ELISA technique can be a CDC subsidiary technique the median size of the amplifications obtained is proportionate to the salt providing further immunologic information, especially in selected cases to concentration, thereby creating a tool which will allow us to widen the select the most suitable further donor. applications of DOP-PCR.

255 256 FLOW CYTOMETRY CROSS-MATCH IN EMERGENCY: 3 OPTIMIZATION OF PCR-SSP CONDITIONS FOR HLA YEARS EXPERIENCE GENOTYPING FOR SMALL SAMPLES OF DNA

D. Fizet, C. Hitte, G. Vezon V.J.Leon, J.M.Gonzalez-Buitrago, Servicio de Bioquímica. Hospital Histocompatibility laboratory, Etablissement français du Sang Universitario de Salamanca (Spain) Aquitaine- Limousin, Bordeaux, FRANCE The SSP method usually applied to HLA molecular typing requires Cross-match(XM) is the critical test performed before transplantation. Positivity quantities of DNA about 50 ng per tube, it makes necessary amounts of of serum from recepient towards donor cells, especially if IgG are present, can DNA about 15 mcg for a complete HLA class I and II molecular typing. be an absolute contre-indication. This requirement, added to the necessity of storaging enough DNA Classical lymphocytotoxicity LCT with T and B lymphocytes from blood, lymph nodes or spleen are incubated with recipient sera then rabbit complement samples for complementary studies, implies in the standard routine the and cell viability determinated by fluorescent microscopy, antibodies subclasses extraction of large quantities of blood. Our procedure permits using the been determined after DTT serum treatment. SSP method with about 1 mcg of DNA. Fow cytometry with anti CD3 and anti CD19 fluorescent antibodies for We took three aliquots of whole blood, 50-200mcg, from 20 different lymphocytes labelling and revelation of fixation of antibodies from patient sera control people. Blood spots on 3MM filter paper from the first aliquot with Fab’anti IgG and IgM FITC antibodies. Positivity is measured comparing were dried and stored at room temperature. The second one was mixed 1:1 MFI with negative sera and must be almost 2 fold superior to be considered with ethanol, shaked and stored on a freezer. From the thirth aliquot, DNA positive .Some cells, especially B lymphocytes present non specific antibodies was directly obtained and apropiately conserved. fixation, so non specific positivity, which is always observed with spleen cells.. DNA was stracted by two different procedures: salt precipitation and A first gating using CD45 APC and a threshold with this fluorochrom remove resin balls with methalic core (kit DNA Direct II from Dynal). We chosed this artefact. 188 XM were realised in emergency at the time and post-graft by both the last one for its easyness and rapidity, obtaining 1-4mcg of DNA which technics.165 were negative by both technics and 14 positive. Only 12 results was carried to a final volume of 1ml to prepare our working solution. were discordant : 2 with the”day” serum, 14 historic sera, 3 negative by LCT the In all the 20 people, it has previously studied the following HLA class II day of graft and controlled positive by FCM and with post graft sera. locus: DRB1*-5, DQA1*, DQB1* and DPA1*, with the usual Cross-match by flow cytometry allow : methodology in our laboratory. 1. confirmation of almost all results obtained by LCT We have changed some general conditions of the typing kits class I and 2. revelation, because more sensitive, of antibodies non detected in II from the Dynal SSP method. We have modified the amount of DNA per historical sera tube to 2-4 ng and the concentration of the Taq Polymerase to 0,1 U/tube, 3. detection of antibodies, little or non fixing complement. instead of the 100 ng of DNA and 0,4 U /tube of Taq Polymerase recommended by the kit instructions. The conditions of the PCR had been: 1) 94ºC, 2 min., 1 cycle. 2) 94ºC 10 sec., 65ºC 60 sec. 10 cycles. 3) 94ºC 10 sec., 61ºC 50 sec., 72ºC 30 sec. 30 cycles. 4) 72ºC 3 min. The tests have successfully allowed us to study the HLA class II genotypes, getting similar results to the one’s obtained with the quantities of DNA and the amplification conditions recommended by kits of Dynal.

Genes and Immunity Abstracts S66 Poster Group: 13 - Special/Other Topics

257 258 OPTIMIZATION OF PCR-SSP CONDITIONS APPLIED TO HLA EVIDENCE FOR AN APPARENT MHC CLASS I TRIPLET IN CLASS I TYPING RHESUS MACAQUES

V.J.Leon, A.J.Leon*. Servicio de Bioquimica. Hospital Universitario de Corrine M. C. Heijmans, Nel Otting, Riet Noort, Gaby G. M. Salamanca. *Laboratorio de Pediatria e inmunología, Universidad de Doxiadis, Natasja. G de Groot, Nanine de Groot, Ronald E. Valladolid. Bontrop. Low and high resolution techniques of typing SSP for the HLA system, Biomedical Primate Research Centre in Rijswijk the Netherlands. have the strength and sensibility required to make them essential for the Department Genetics and Refinement. studies of molecular biology by PCR. The polymorphic equivalent of the HLA-A and –B gene products in rhesus A decisive factor is the length of the amplification process of the PCR, monkeys are named Mamu-A and –B and can be detected bij means of allo- which limits the perfomance of the thermocycler, since the standard time antisera. In order to understand if a serotype correlates with Mamu-class I parameters usually employed are the ones provided by the makers of the sequences, a panel of well defined MHC serotyped animals was subjected to kit. sequence analysis. These studies illustrate that the Mamu-class I region is The kinetic of the PCR cycle is based on three steps: denaturation of incredibly complex, and that rhesus macaques express multiple HLA-A and –B the genomic DNA, primers annealing and extension. These steps are like molecules per haplotype. determined by the specific temperature for each one, and how the The serological typing studies indicated that one rhesus monkey expresses a optimum temperature is reached is conditioned by the volume of the triplet at its MHC -A locus as well as at its -B locus. This monkey was found sample and the heat ramp that makes the thermocycler change from one positive for the Mamu-A2, -A25, -A26 and –B10, -B23, -B28 antigens. temperature of the cycle to another. 10 mcl of sample, usually employed Therefore this female was made part of an intensive breading program to elucidate the segregation profiles of the haplotypes in question. Pedigree for the SSP, makes it possible to increase by 1ºC/sec, top speed for most analysis demonstrated that the Mamu-A2, -A26 and –B10, -B28 serotypes thermocyclers, and fast enough to reach the optimum temperature for segregated on one haplotype. This haplotype was inherited in a stable fashion in each step of the cycle, and therefore able to reduce the length of time of females whereas in male rhesus macaques a segment of the apparent duplication the steps. All this has allowed us to reduce the duration of the technique was inactivated in two independent occasions. The molecular analysis of the by 30%, maintaining the reliability and resolution of the procedure. MHC class I genes that are encoded by the haplotype with the apparent Mamu There have been analyzed in paralel 40 samples using both the class I triplet will be discussed. recommended times by the manufacturer and the optimized by us. We have employed for our study SSP Dynal kits for low resolution for the locus HLA A, B and C. Standard PCR: 94ºC 2min, 1 cycle; 94ºC 10 sec., 61ºC 60 sec., 10 cycles; 94ºC 10 sec., 65ºC 50 sec, 72ºC 30sec, 20 cycles. Optimised PCR: 94ºC 2min, 1 cycle; 94ºC 5 sec., 61ºC 25 sec., 10 cycles; 94ºC 5 sec., 65ºC 25 sec, 72ºC 10sec, 20 cycles. The duration of the standard PCR is 1 hour 34 minutes while the optimised PCR takes 1 hour 3 minutes. Results in both series offered identical typings.

259 260 DETECTION OF A BLAST-SPECIFIC SINGLE NUCLEOTIDE SPECIES-SPECIFIC REGULATION OF RAT AND MOUSE IL-4 INSERTION IN EXON 3 OF HLA-A BY DNA SEQUENCING RECEPTOR EXPRESSION BASED HLA TYPING Anette Bohnert, Christine Weth, Ulrike Kaiser, Holger Hackstein, Linda K Smith, Romano Krueger, Frank T Christiansen, David C Sayer, Ingrid Kloeting, Gregor Bein. Institute of Clinical Immunology and Malcolm Webb, Dept of Clinical Immunology and Biochemical Genetics, Royal Transfusion Medicine, University of Giessen, Germany Perth Hospital, Perth, WA 6000 Interleukin-4 (IL-4) plays an important role in shaping immune responses. It’s HLA-A SBT of a sample from a patient with B cell ALL revealed a sequence effects depend upon binding to and signalling through the IL-4 receptor electropherogram profile consistent with an indel polymorphism. Serological complex. Information on the expression and regulation of the IL4R gene is typing identified HLA-A3 and A24. Subsequent sequencing with allele specific limited. primers detected three alleles: A*030101, A*2402 and an additional allele The proximal promotor region of the rat IL4R gene was determined by which differed from A*2402 (A*24mut) by a single nucleotide insertion sequence alignment with corresponding genomic sequences of the mouse IL4R between nucleotides 363 and 364 in exon 3. HLA A*24mut was not found in the gene and by direct sequencing of genomic DNA from 7 rat strains. Rat spleen parents or siblings of this individual. Subsequent sequencing of DNA from blast cells were cultivated with and without IL-4, and IL-4 receptor expression was cell and non blast cell populations sorted by flow cytometry revealed A*24mut quantified by FACS analysis. Alignment of the proximal promoter of the rat IL4R gene with the to be present on blast cells only whilst A*2402 was present on the non blast cell corresponding sequence of the mouse revealed about 88% homology. Despite population. Serological typing on cells from the blast cellpopulation revealed this strong homology, the essential palindromic STAT6 binding site in the that A*24mut was not expressed. mouse gene (TTCATCTGAA) is absent in the corresponding rat sequence It is unlikely that A*24mut would have been identified by any other molecular (GTCATCTGGA). Rat IL-4 receptor expression was down-regulated following typing technique and it is possible that point mutations which result in the loss incubation of spleen cells with IL-4, whereas in murine B cells IL-4 receptor of expression of HLA may be more common than originally thought. Blast cells expression is up-regulated by IL-4. that lack HLA which present tumour peptide would escape immune killing and The IL4R genes of rat and mouse have been placed in a different regulatory proliferate as a consequence. context during evolution. The different regulation of IL4R genes may also influence the IL-4 driven immune response in a species-specific manner. These and other observations on species-specific regulation of immune response genes may question the value of genetic animal models for the study of multi factorial human immune disorders.

Genes and Immunity