- Email: [email protected]
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Abstracts / Human Immunology 73 (2012) 49–167
189-P
167
AN XML EXPORT OF THE IMGT/HLA DATABASE. James Robinson 1, Jane Pollack 2, Adrienne Walts 2, Joel Schneider 2, Read Fritsch 2, Anthony Barber 2, John Freeman 2, Martin Maiers 2, Steven G.E. Marsh 1. 1 HLA Informatics Group, Anthony Nolan Research Institute, London, United Kingdom; 2 Bioinformatics, National Marrow Donor Program, Minneapolis, USA. Aim: The IMGT/HLA Database provides a specialist database for sequences of the human major histocompatibility complex, known as HLA and includes the official sequences for the WHO Nomenclature Committee For Factors of the HLA System. The database currently provides exports of the data in a variety of formats; this is been expanded to include Extensible Markup Language (XML). Methods: The XML format defines a set of rules for encoding documents in a format that is both human and machine-readable. XML is becoming a common format for exporting database contents for dissemination over the World Wide Web. A collaborative working group has provided an XML export of the data contained within the IMGT/HLA Database. This export format would be supported by tools for the utilisation of this dataset into different database formats. Results: The XML format combines the data included in the sequence alignments with the data available in the individual allele reports. This combination of data has not previously been available in a single format. The XML format will enable users to identify the regions within the DNA sequence, such as exons, as well as reconstruct the sequence alignments. This machine-readable format provides a standardised format for importing data from the reference database into a variety of programs. In addition the collaborative project has developed a suite of tools for importing the data into different database schema for allowing incorporation into different laboratory systems. Conclusions: The XML files will be regularly updated as part of the quarterly releases of the IMGT/HLA Database. A beta test version of the XML format and associated tools will shortly be available from the hla.alleles.org and the National Marrow Donor Program websites.
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EVALUATION OF CELL ISOLATION METHODS FOR FLOW CROSSMATCH OF DECEASED SOLID ORGAN TRANSPLANTATION. Giles T. Crews, Wendy E. Wegner, Gansuvd Balgansuren, Dong-Feng Chen. Department of Pathology and Clinical Laboratories, Duke University Medical Center, Durham, NC, USA. Aim: Crossmatch (XM) for deceased solid organ transplant is a critical and challenging process due to limited cold ischemia time, the need for adequate cell yield and purity of cell isolation, and the process must be quick. In this study we evaluated different cell separation methods to select the most efficient one for clinical transplant crossmatch. Methods: Lymphocytes were isolated following lab procedure and/or manufacturer instructions (STEMCELL Technologies Inc.) using Ficoll, RosetteSep, RosetteSep with SepMate Tube, EasySep magnetic selections. Flow T and B cell crossmatches were performed following lab procedure. Results: The purity, yield, and average time spent isolating lymphocytes from PB samples obtained from 6 volunteers are given in the following table (Table 1). Conclusions: EasySep custom positive selection delivered excellent yield and purity; however, the cells showed strong background binding in the flow XM, making them unacceptable for use in the flow XM. The Ficoll method gave the highest yield with the lowest purity, which may require additional centrifugations to remove red cells, platelets and granulocyte, thus adding time for isolation. Many lymphocytes were lost during the separation using the RosetteSep and RosetteSep with SepMate Tube. However, the methods offer excellent cell purity and no additional separation is needed. Overall, the use of RosetteSep with SepMate tubes could be a practical method for routine lymphocyte isolation for deceased solid organ transplantation.
Table 1 Comparison of cell separations for cell purity and yield Ficoll 1 2 3 4 5 6 Average Time Consumed ⁄
Purity/Yield.
⁄
72%/95% 73%/69% 87%/69% 34%/66% 65%/49% 68%/54% 67%/67% 1 hr 14 min
RosetteSep
RosetteSep + SepMate tube
EasySep Neg
EasySep Custom CD2/CD20 Pos.
94%/52% 96%/45% 97%/27% 95%/38% 94%/44% 96%/41% 95%/41% 1 hr 39 min
95%/31% 94%/22% 98%/27% 95%/35% 91%/39% 97%/39% 95%/32% 1 hr 11 min
NT NT NT 85%/30% 84%/37% 95%/26% 88%/31% 1 hr 26 min
NT NT NT 95%/60% 94%/56% 96%/70% 95%/62% 1 hr 30 min
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167
AN XML EXPORT OF THE IMGT/HLA DATABASE. James Robinson 1, Jane Pollack 2, Adrienne Walts 2, Joel Schneider 2, Read Fritsch 2, Anthony Barber 2, John Freeman 2, Martin Maiers 2, Steven G.E. Marsh 1. 1 HLA Informatics Group, Anthony Nolan Research Institute, London, United Kingdom; 2 Bioinformatics, National Marrow Donor Program, Minneapolis, USA. Aim: The IMGT/HLA Database provides a specialist database for sequences of the human major histocompatibility complex, known as HLA and includes the official sequences for the WHO Nomenclature Committee For Factors of the HLA System. The database currently provides exports of the data in a variety of formats; this is been expanded to include Extensible Markup Language (XML). Methods: The XML format defines a set of rules for encoding documents in a format that is both human and machine-readable. XML is becoming a common format for exporting database contents for dissemination over the World Wide Web. A collaborative working group has provided an XML export of the data contained within the IMGT/HLA Database. This export format would be supported by tools for the utilisation of this dataset into different database formats. Results: The XML format combines the data included in the sequence alignments with the data available in the individual allele reports. This combination of data has not previously been available in a single format. The XML format will enable users to identify the regions within the DNA sequence, such as exons, as well as reconstruct the sequence alignments. This machine-readable format provides a standardised format for importing data from the reference database into a variety of programs. In addition the collaborative project has developed a suite of tools for importing the data into different database schema for allowing incorporation into different laboratory systems. Conclusions: The XML files will be regularly updated as part of the quarterly releases of the IMGT/HLA Database. A beta test version of the XML format and associated tools will shortly be available from the hla.alleles.org and the National Marrow Donor Program websites.
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EVALUATION OF CELL ISOLATION METHODS FOR FLOW CROSSMATCH OF DECEASED SOLID ORGAN TRANSPLANTATION. Giles T. Crews, Wendy E. Wegner, Gansuvd Balgansuren, Dong-Feng Chen. Department of Pathology and Clinical Laboratories, Duke University Medical Center, Durham, NC, USA. Aim: Crossmatch (XM) for deceased solid organ transplant is a critical and challenging process due to limited cold ischemia time, the need for adequate cell yield and purity of cell isolation, and the process must be quick. In this study we evaluated different cell separation methods to select the most efficient one for clinical transplant crossmatch. Methods: Lymphocytes were isolated following lab procedure and/or manufacturer instructions (STEMCELL Technologies Inc.) using Ficoll, RosetteSep, RosetteSep with SepMate Tube, EasySep magnetic selections. Flow T and B cell crossmatches were performed following lab procedure. Results: The purity, yield, and average time spent isolating lymphocytes from PB samples obtained from 6 volunteers are given in the following table (Table 1). Conclusions: EasySep custom positive selection delivered excellent yield and purity; however, the cells showed strong background binding in the flow XM, making them unacceptable for use in the flow XM. The Ficoll method gave the highest yield with the lowest purity, which may require additional centrifugations to remove red cells, platelets and granulocyte, thus adding time for isolation. Many lymphocytes were lost during the separation using the RosetteSep and RosetteSep with SepMate Tube. However, the methods offer excellent cell purity and no additional separation is needed. Overall, the use of RosetteSep with SepMate tubes could be a practical method for routine lymphocyte isolation for deceased solid organ transplantation.
Table 1 Comparison of cell separations for cell purity and yield Ficoll 1 2 3 4 5 6 Average Time Consumed ⁄
Purity/Yield.
⁄
72%/95% 73%/69% 87%/69% 34%/66% 65%/49% 68%/54% 67%/67% 1 hr 14 min
RosetteSep
RosetteSep + SepMate tube
EasySep Neg
EasySep Custom CD2/CD20 Pos.
94%/52% 96%/45% 97%/27% 95%/38% 94%/44% 96%/41% 95%/41% 1 hr 39 min
95%/31% 94%/22% 98%/27% 95%/35% 91%/39% 97%/39% 95%/32% 1 hr 11 min
NT NT NT 85%/30% 84%/37% 95%/26% 88%/31% 1 hr 26 min
NT NT NT 95%/60% 94%/56% 96%/70% 95%/62% 1 hr 30 min