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Confirmatory typing of cord blood unit and cord blood recipients using DNA-based methods
Abstracts
S39
1.1 #7
CONFIRMATORY TYPING OF CORD BLOOD UNIT AND CORD BLOOD RECIPIENTS USING DNA– BASED METHODS Wei Dong,1 Susan Flesch,2 Margaret Larsen,1 Toolsee S. Singh,1 Susan H. Hsu.1 1HLA, ARC, Philadelphia, PA, USA; 2NMDP, MN, USA The immature and tolerant nature of umbilical cord blood lymphocytes has lead to their usage as an alternative source of hematopoetic stem cells for immune reconstitution. In 1999 NMDP established a registry of cord blood units collected by independent cord blood banks. At present, the Registry list over 20,000 CBUs. Since March 1, 2001, our lab has performed DNA– based CT typing for NMDP CBUs and corresponding recipient samples. This report summarizes our first year (March 1, 2001 – February 28, 2002) experience with this project. Typing resolution options, chosen by NMDP transplant centers, included IR level HLA–A,B, DRB3/4/5 with HR level HLA–DRB1 as well as HR “ala carte” choices for HLA–A,B, Cw and DQB1. HR level typing was performed 100% of the time at HLA–DRB1, 33% at HLA–A,B, 20% at HLA– Cw and 37% at HLA–DQB1. TAT was either 3 (59% of requests) or 9 (41% of requests) business days. Of the 311 CBU and 96 recip samples typed, 37% were whole blood, 23% CB spotted filter paper, 21% buffy coat, 6% CBU segment, 11% red cell fraction, 2% extracted DNA and 0.2% buccal swab. The filter paper (FP) samples presented the greatest challenge. Ninety– eight percent were collected in blood bags containing 35 mL CPD which contributed to inconsistent satisfactory class I gene amplification. HR typing for these samples was achieved using SSP. Approaches to further meet the problems presented by FP are underway. None of the other sample types posed any difficulty except in instances of small sample volume. The discrepancy rate between our lab and results initially obtained by the CBB labs was 6.4%. In all instances, erroneous results were traced back to the original typings submitted to the NMDP by the CBBs. During the first year of our involvement with this project, the NMDP facilitated 55 CB transplants. Typings were requested by 41 domestic and 16 international NMDP transplant centers. With the reported benefits associated with CB transplantation (i.e. lower risk of acute and chronic GVHD and a reconstitution rate comparable to BMT), the number of CB transplants can be expected to increase greatly in the coming years.
1.1 #8
VALIDATION STUDIES ON THE USE OF SSOP DNA TYPING OF HLA–C ALLELES WITH THE LUMINEX FLOW CYTOMETER Masako Osada, Melissa D’Ambrose, Ivan Balazs. Research & Development, Orchid Diagnostics, Stamford, CT The objective of this study was the development and validation of a SSOP assay for HLA–C alleles using fluorescent micro spheres in combination with the Luminex–100 flow cytometer. Over 100 different probe sequences, homologous to polymorphic regions, were evaluated. 35 oligonucleotide–probes were selected and each conjugated to a different group of LabMap micro spheres. Samples were amplified for Exon 2 and 3 of HLA–C using an asymmetric amplification protocol. The amplification conditions were the same as those for LifeMatch HLA–A and B. One of the primers in the primer pair was labeled with biotin. Amplified samples were transferred to a microtiter plate and hybridized for 1 hr to a mixture of microspheres containing all 35 probes. Hybridized samples were diluted with running buffer containing PE–labeled streptavidin and placed in the Luminex–100 for analysis. The median fluorescent value for each probe of a sample was used to assign the probes as positive or negative and to determine the phenotype of the sample. For the validation of this experimental protocol, 360 samples, previously typed using an SSOP method with alkaline phosphatase labeled probes, were analyzed by both procedures and results compared. The interpretation of the probe hits generated by the 2 procedures had a correlation of ⬍95%.
S39
1.1 #7
CONFIRMATORY TYPING OF CORD BLOOD UNIT AND CORD BLOOD RECIPIENTS USING DNA– BASED METHODS Wei Dong,1 Susan Flesch,2 Margaret Larsen,1 Toolsee S. Singh,1 Susan H. Hsu.1 1HLA, ARC, Philadelphia, PA, USA; 2NMDP, MN, USA The immature and tolerant nature of umbilical cord blood lymphocytes has lead to their usage as an alternative source of hematopoetic stem cells for immune reconstitution. In 1999 NMDP established a registry of cord blood units collected by independent cord blood banks. At present, the Registry list over 20,000 CBUs. Since March 1, 2001, our lab has performed DNA– based CT typing for NMDP CBUs and corresponding recipient samples. This report summarizes our first year (March 1, 2001 – February 28, 2002) experience with this project. Typing resolution options, chosen by NMDP transplant centers, included IR level HLA–A,B, DRB3/4/5 with HR level HLA–DRB1 as well as HR “ala carte” choices for HLA–A,B, Cw and DQB1. HR level typing was performed 100% of the time at HLA–DRB1, 33% at HLA–A,B, 20% at HLA– Cw and 37% at HLA–DQB1. TAT was either 3 (59% of requests) or 9 (41% of requests) business days. Of the 311 CBU and 96 recip samples typed, 37% were whole blood, 23% CB spotted filter paper, 21% buffy coat, 6% CBU segment, 11% red cell fraction, 2% extracted DNA and 0.2% buccal swab. The filter paper (FP) samples presented the greatest challenge. Ninety– eight percent were collected in blood bags containing 35 mL CPD which contributed to inconsistent satisfactory class I gene amplification. HR typing for these samples was achieved using SSP. Approaches to further meet the problems presented by FP are underway. None of the other sample types posed any difficulty except in instances of small sample volume. The discrepancy rate between our lab and results initially obtained by the CBB labs was 6.4%. In all instances, erroneous results were traced back to the original typings submitted to the NMDP by the CBBs. During the first year of our involvement with this project, the NMDP facilitated 55 CB transplants. Typings were requested by 41 domestic and 16 international NMDP transplant centers. With the reported benefits associated with CB transplantation (i.e. lower risk of acute and chronic GVHD and a reconstitution rate comparable to BMT), the number of CB transplants can be expected to increase greatly in the coming years.
1.1 #8
VALIDATION STUDIES ON THE USE OF SSOP DNA TYPING OF HLA–C ALLELES WITH THE LUMINEX FLOW CYTOMETER Masako Osada, Melissa D’Ambrose, Ivan Balazs. Research & Development, Orchid Diagnostics, Stamford, CT The objective of this study was the development and validation of a SSOP assay for HLA–C alleles using fluorescent micro spheres in combination with the Luminex–100 flow cytometer. Over 100 different probe sequences, homologous to polymorphic regions, were evaluated. 35 oligonucleotide–probes were selected and each conjugated to a different group of LabMap micro spheres. Samples were amplified for Exon 2 and 3 of HLA–C using an asymmetric amplification protocol. The amplification conditions were the same as those for LifeMatch HLA–A and B. One of the primers in the primer pair was labeled with biotin. Amplified samples were transferred to a microtiter plate and hybridized for 1 hr to a mixture of microspheres containing all 35 probes. Hybridized samples were diluted with running buffer containing PE–labeled streptavidin and placed in the Luminex–100 for analysis. The median fluorescent value for each probe of a sample was used to assign the probes as positive or negative and to determine the phenotype of the sample. For the validation of this experimental protocol, 360 samples, previously typed using an SSOP method with alkaline phosphatase labeled probes, were analyzed by both procedures and results compared. The interpretation of the probe hits generated by the 2 procedures had a correlation of ⬍95%.