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Direct sequencing on DRB allele-specific templates for the selection of unrelated bone marrow donors
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Abstracts
C-6.2 #119
C-6.2 #120
AN EVALUATION OF RECIPIENT-DONOR COMPATIBHATY USING CLASS I1 AI.I.EIJC TYPING AND CF.IJ.ULAR RESPONSIVENKSS. JD McAuley, FL Johnson, RA Larson, KM Daly, GM Troup, TM Williams, RC Williams, Histocompatibility Laboratory, Blood Systems, Inc., Scottsdale, AZ, Department of Pediatrics and Department of Medicine, University of Chicago Medical Center, Chicago, IL, Department of Pathology, University of New Mexico, Albuquerque, NM. The efficacy of the mixed leukocyte culture (MLC) in recipient-donor matching for unrelated bone marrow transplantation has been debated. HLA class II (DR,DQ) low resolution alleles and cellular responsiveness in the MLC test (RR> 15%) were evaluated in 90 unrelated recipient-donor pairs, (64 by serology, 26 by DNA). Standard 2x4 contingency table analysis (3 d.f.) was used to evaluate 180 independent reciprocal events. The results were stratified by compatible and incompatible at low resolution typing and recipient-donor response and nonresponse in MLC. The null hypothesis was a uniform distribution of recipient-donor responses between compatible and incompatible low resolution phenotypes. It was rejected at the p=0.004 level of significance, (chi sq 13.19). However, this alone does not indicated the direction of the effect. The observed number of low response events for compatible persons is consistently higher than expected (in brackets below) as is the reciprocal effect, high response events for incompatible pairs, (see Table 1). Thus the MLC test does provide additional information at the level of the functional molecule for unrelated recipient-donor pairs. Further testing is now underway to determine its effect on class II high resolution matching and clinical outcome. Table 1 Rocipient Recipient Donor Donor Response Nonresponse Response Nonresponsc Compatible 28 [35] 23 [16] 27 [31] 24 [20] Incompatible 34 [27] 5 [12] 27 [23] 12 [16] Observed [Expected]
DIRECT SEQUEMC[MGON DR8 ALLELE=SPECIFICTEMPLATESFOg THE SELECTIONOF UMRELATEDBONE MARROWDOHORS.M. Br~liani, V. Mantovsni, P. Selva, E. Collins, G. Martiaelii*, D.Bastiu, ?. Darboni. Tissue Typing Laboratory, Malpighi Hospital, *Institute of 6aematology 'L.e A. Seragnoli', University of Bologna, Bologna, Italy. The selection of unrelated donors (URD) in bone marrov transplantation needs u molecular characterization of HLA class II genes to test the donor-recipient Hatching. |e describe here a direct sequencing method to perform the BLA=DRB geuotyping, in order to achieve the most detailed infornutios on the matching. To avoid the aibiguous interpretation of the most complex DRB heterozygote combinations, our sequencing-based typing is performed on allele-specific templates obtained by PCR with sequence-specific prilers (PCR-SSPJ amplification. Our procedure includes a preliminary low resolution DRB typing by a set of ]8 PCR-SSP reaction to identify the two ullelic variants. Then, the sequencing reaction with a nested primer is separately performed on the PCR-SSP prodsct of one allele at a time. The sequencing primers are designed to give rise to the highest resolution of the polimorphic positions inside each allelic variants. The method gives sequencing ladders easily readable and allows the determination of known alleles as well as sew seqsences. UMA preparation, PCR amplification and sequence processing are completed in 3-4 days. Unlike the other DMA analysis methods, the sequence=based typing is highly iaforintive and well suited [or a small number of samples. It isn't either labor-intensive or time-consuming and it doesn't miss new alleles. Thus, the direct sequencing results the best approach to the donor-recipient matching in bone narrow transplantation.
Abstracts
C-6.2 #119
C-6.2 #120
AN EVALUATION OF RECIPIENT-DONOR COMPATIBHATY USING CLASS I1 AI.I.EIJC TYPING AND CF.IJ.ULAR RESPONSIVENKSS. JD McAuley, FL Johnson, RA Larson, KM Daly, GM Troup, TM Williams, RC Williams, Histocompatibility Laboratory, Blood Systems, Inc., Scottsdale, AZ, Department of Pediatrics and Department of Medicine, University of Chicago Medical Center, Chicago, IL, Department of Pathology, University of New Mexico, Albuquerque, NM. The efficacy of the mixed leukocyte culture (MLC) in recipient-donor matching for unrelated bone marrow transplantation has been debated. HLA class II (DR,DQ) low resolution alleles and cellular responsiveness in the MLC test (RR> 15%) were evaluated in 90 unrelated recipient-donor pairs, (64 by serology, 26 by DNA). Standard 2x4 contingency table analysis (3 d.f.) was used to evaluate 180 independent reciprocal events. The results were stratified by compatible and incompatible at low resolution typing and recipient-donor response and nonresponse in MLC. The null hypothesis was a uniform distribution of recipient-donor responses between compatible and incompatible low resolution phenotypes. It was rejected at the p=0.004 level of significance, (chi sq 13.19). However, this alone does not indicated the direction of the effect. The observed number of low response events for compatible persons is consistently higher than expected (in brackets below) as is the reciprocal effect, high response events for incompatible pairs, (see Table 1). Thus the MLC test does provide additional information at the level of the functional molecule for unrelated recipient-donor pairs. Further testing is now underway to determine its effect on class II high resolution matching and clinical outcome. Table 1 Rocipient Recipient Donor Donor Response Nonresponse Response Nonresponsc Compatible 28 [35] 23 [16] 27 [31] 24 [20] Incompatible 34 [27] 5 [12] 27 [23] 12 [16] Observed [Expected]
DIRECT SEQUEMC[MGON DR8 ALLELE=SPECIFICTEMPLATESFOg THE SELECTIONOF UMRELATEDBONE MARROWDOHORS.M. Br~liani, V. Mantovsni, P. Selva, E. Collins, G. Martiaelii*, D.Bastiu, ?. Darboni. Tissue Typing Laboratory, Malpighi Hospital, *Institute of 6aematology 'L.e A. Seragnoli', University of Bologna, Bologna, Italy. The selection of unrelated donors (URD) in bone marrov transplantation needs u molecular characterization of HLA class II genes to test the donor-recipient Hatching. |e describe here a direct sequencing method to perform the BLA=DRB geuotyping, in order to achieve the most detailed infornutios on the matching. To avoid the aibiguous interpretation of the most complex DRB heterozygote combinations, our sequencing-based typing is performed on allele-specific templates obtained by PCR with sequence-specific prilers (PCR-SSPJ amplification. Our procedure includes a preliminary low resolution DRB typing by a set of ]8 PCR-SSP reaction to identify the two ullelic variants. Then, the sequencing reaction with a nested primer is separately performed on the PCR-SSP prodsct of one allele at a time. The sequencing primers are designed to give rise to the highest resolution of the polimorphic positions inside each allelic variants. The method gives sequencing ladders easily readable and allows the determination of known alleles as well as sew seqsences. UMA preparation, PCR amplification and sequence processing are completed in 3-4 days. Unlike the other DMA analysis methods, the sequence=based typing is highly iaforintive and well suited [or a small number of samples. It isn't either labor-intensive or time-consuming and it doesn't miss new alleles. Thus, the direct sequencing results the best approach to the donor-recipient matching in bone narrow transplantation.