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DNA oligotyping of HLA class II — A Canadian perspective
110
Abstracts
#326 4.3
#327 5.1
DNA OLIGOTYPING OF HLA CLASS II - A CANADIAN PERSPECTIVE. KH Wong, T Bladon, CJ O'Hara and H Mervart. National Reference Laboratory. The Canadian Red Cross Society, Ottawa~ Ontario, Canada. The complete characterization of 200 members of our local cell panel for HLA class II speciflcltles was performed using DNA ollgotyping. This involved the isolation of DNA from the cells of individual donors and the selective amplification of the second exons of the HLA-DRB,-DQA,-DQB,-DPA and -DPB genes using the pelymerase chain reaction (PCR) technique. The amplified gene products were subsequently hybrldized with an appropriate selection of sequence specific oligonucleotide probes. The reagents employed in the study were synthesized from previously published sequences. This approach was extremely useful in the confirmation of the serological typing of our donors a s w e l l as in the identification of alleles for which sera are not readily available particularly for the HLA-DQB (i.e. DQw4 and DQwg) and -DPB locl. This technique was also helpful in demonstrating that several panel donors who appeared to be homozygous serologically actually expressed 2 closely related subtypes (i.e. DRB*I501 and *1502)~ In our sample of 200 donors it was possible to identify almost all of the DNA alleles for the DQA,DQB,DPA and DPB recognizable with the workshop reagents. Only the DQA*0312,DQB*05032,DQB*0504 and DPB*I801 alleles were not observed. In our Canadian panel, 34 of the possible 47 DRB alleles could be detected. Information regarding the frequency of the DNA alleles and genes as well as haplotypes observed in the Canadian population will be presented.
PRESCREENING OF POTENTIAL HLA REAGENT ANTISERAUSING FLOW CYTOMETRY. IG Wood and EL Milford, Tissue Typing Laboratory, Brigham and Women's Hospital, Boston~ MA. Sera from 84 multiparous women were screened by flow eytometry and results compared to standard cytotoxic P R A o n 40 T and i0 E lymphocytes. For flow screening, equal numbers of T cells from 16 individuals, and B cells from 9, were pooled into a single suspension. This pool represented 52 class I and 15 class II antigens. 800,000 cells from the pool were incubated with 30ul of each serum, then counterstained with fluoresceinated Goat anti-Human IgG, and anti-B1 phycoerythrin to permit selective gating on BI+ (B cells) or B1- (T cells). Flow cytometry was sufficiently sensitive to detect positivity with 1 individual in the pool. Sera were considered positive by flow when reacting w i t h ~ 2 % of the pool. These results were compared to cytotoxic panel screening (CDC), where positives were considered to be k5% or ~10% panel reactivity (PRA) for T and B enriched cells respectively. Sera found to be CDC+ on B cells were later platelet absorbed to remove Class I (parentheses below}. There was good correlation between flow screen results and CDC-PRA with 96% and 98% specificity for Class I and Class II respectively. 57% of sera were Flow negative on T and B cells. Flow prescreening would allow labs to double serum acquisition efforts with a minor increase in workload. T CELL: Plow B CELL: Plow + + + 20 2 + 22(12) 7(I} CDC CDC -8 54 p<.01 -12 43 p<.01
Abstracts
#326 4.3
#327 5.1
DNA OLIGOTYPING OF HLA CLASS II - A CANADIAN PERSPECTIVE. KH Wong, T Bladon, CJ O'Hara and H Mervart. National Reference Laboratory. The Canadian Red Cross Society, Ottawa~ Ontario, Canada. The complete characterization of 200 members of our local cell panel for HLA class II speciflcltles was performed using DNA ollgotyping. This involved the isolation of DNA from the cells of individual donors and the selective amplification of the second exons of the HLA-DRB,-DQA,-DQB,-DPA and -DPB genes using the pelymerase chain reaction (PCR) technique. The amplified gene products were subsequently hybrldized with an appropriate selection of sequence specific oligonucleotide probes. The reagents employed in the study were synthesized from previously published sequences. This approach was extremely useful in the confirmation of the serological typing of our donors a s w e l l as in the identification of alleles for which sera are not readily available particularly for the HLA-DQB (i.e. DQw4 and DQwg) and -DPB locl. This technique was also helpful in demonstrating that several panel donors who appeared to be homozygous serologically actually expressed 2 closely related subtypes (i.e. DRB*I501 and *1502)~ In our sample of 200 donors it was possible to identify almost all of the DNA alleles for the DQA,DQB,DPA and DPB recognizable with the workshop reagents. Only the DQA*0312,DQB*05032,DQB*0504 and DPB*I801 alleles were not observed. In our Canadian panel, 34 of the possible 47 DRB alleles could be detected. Information regarding the frequency of the DNA alleles and genes as well as haplotypes observed in the Canadian population will be presented.
PRESCREENING OF POTENTIAL HLA REAGENT ANTISERAUSING FLOW CYTOMETRY. IG Wood and EL Milford, Tissue Typing Laboratory, Brigham and Women's Hospital, Boston~ MA. Sera from 84 multiparous women were screened by flow eytometry and results compared to standard cytotoxic P R A o n 40 T and i0 E lymphocytes. For flow screening, equal numbers of T cells from 16 individuals, and B cells from 9, were pooled into a single suspension. This pool represented 52 class I and 15 class II antigens. 800,000 cells from the pool were incubated with 30ul of each serum, then counterstained with fluoresceinated Goat anti-Human IgG, and anti-B1 phycoerythrin to permit selective gating on BI+ (B cells) or B1- (T cells). Flow cytometry was sufficiently sensitive to detect positivity with 1 individual in the pool. Sera were considered positive by flow when reacting w i t h ~ 2 % of the pool. These results were compared to cytotoxic panel screening (CDC), where positives were considered to be k5% or ~10% panel reactivity (PRA) for T and B enriched cells respectively. Sera found to be CDC+ on B cells were later platelet absorbed to remove Class I (parentheses below}. There was good correlation between flow screen results and CDC-PRA with 96% and 98% specificity for Class I and Class II respectively. 57% of sera were Flow negative on T and B cells. Flow prescreening would allow labs to double serum acquisition efforts with a minor increase in workload. T CELL: Plow B CELL: Plow + + + 20 2 + 22(12) 7(I} CDC CDC -8 54 p<.01 -12 43 p<.01