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Luminex technology is significantly faster and less expensive than SSP methodology
Abstracts
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S85
NOVEL HLA-B ALLELES REVEALED BY SSOP AND IDENTIFIED BY SINGLE-ALLELE SEQUENCING Dong-Feng Chen,1 Lingsheng Dong,1 Alan Howard,2 Arthur Rabson,1 Patricia Fraser1, 1CBR Institute for Biomedical Research, Harvard Medical School, Boston, MA, USA; 2The National Marrow Donor Program®, Minneapolis, MN, USA Recently, we observed unusual HLA-B SSOP hybridization patterns in five NMDP® donor samples indicating existence of potential novel HLA-B alleles. We performed selective PCR amplification using TA/ CG group-specific primers to separate two alleles of each sample into single alleles and then performed cycle sequencing on these PCR products. A novel B*39 allele was identified in sample 1 which differed from B*3914 at codon 114 where AAC changed to GAC, resulting in an amino acid change from Asn to Asp. In sample 2 a silent base substitution at codon 78 (CTG TTG) which distinguished the novel allele from B*4001. We made a similar observation in sample 3, where we observed a new allele that was identical to B*5301, with the exception of a synonymous substitution at codon 45 (ACG ACT ). We observed a novel B*38 allele in sample 4, for which the sequence differed from B*3801 at 2 codons: at codon 152 (GTG GAG) resulting in Val to Glu change, and at codon 158 (ACC GCC) resulting in Thr to Ala change. Furthermore, we characterized another B*39 variant in sample 5 that differed from B*3901 at codon 158 (ACC GCC; Thr Ala change). The identification of novel alleles explained the unusual SSOP patterns and further extends the diversity of the HLA-B gene. TABLE 1 Sample 1 2 3 4 5
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Ethnic Group Caucasian Caucasian African American Caucasian Caucasian
HLA-A 02xx, 30xx 01xx, 02xx 68xx, — 02xx, — 24xx, 25xx
HLA-B 1801/17N, 39new 070201, 40new 1503, 53new 4001, 38new 150101/01N, 39new
DRB1 03PJJ, 13JZD 15KCP, 1302 08BMU, 15KCM 13JUE, 13JZA 14AMR, 07MT
LUMINEX TECHNOLOGY IS SIGNIFICANTLY FASTER AND LESS EXPENSIVE THAN SSP METHODOLOGY Walter F. Herczyk, Barbara O. Burgess, Jean L. Rundquist, Adella R. Clark, Claire Li, Nancy L. Reinsmoen, Pathology, Duke University Medical Center, Durham, NC, USA With increasing workload coupled with reduction of staff, we looked for an easier and faster method of typing patients that requires less hands-on time for technologists. The Luminex methodology (LabType )™ for A, B, C, DRB1, DRB3/4/5 and DQB1 loci was compared with SSP methodology (ABC SSP Unitray ™ and Micro SSP™ Generic Class II tray). Luminex testing principles involves reverse SSO coupled with multiplexing platform of the Luminex instrument. Luminex has the ability to type for Class I and Class II loci in one operation with smaller quantity of DNA (12L for Luminex vs. 119 L for SSP). Variables of quality/ quantity of DNA, rate of repeats and turn around time were explored. Typings for 150 patients were compared with each method. SSP typing requires not only a larger total volume of sample, but much purer DNA (A260/ A280 ratio between 1.5-2.0) at a concentration no lower than 25ng/L. Whereas Luminex gives typings result with concentrations as low as 15ng/L having ratios between 1.3 to 2.1. Reasons for repeats by either method were non-amplification (9.5% for SSP, 2.5% for Luminex), gel problems (6.7% for SSP), ambiguous results (4.6% for SSP, 3.8% for Luminex), and clarifying serologic equivalents (1.9% for SSP, 5.2% for Luminex). The overall percentage of repeats were 18.0% (SSP) and 11.3 % (Luminex). Comparison of total hands-on test time for sixteen typings was two hours for Luminex vs. eight hours for SSP. Utilizing Luminex for testing resulted in a decreased turn around time of 64% for solid organ patients and 44% for bone marrow patients. This study shows that the Luminex platform is faster, easier and provides a patient typing result at less cost than SSP.
3 91
S85
NOVEL HLA-B ALLELES REVEALED BY SSOP AND IDENTIFIED BY SINGLE-ALLELE SEQUENCING Dong-Feng Chen,1 Lingsheng Dong,1 Alan Howard,2 Arthur Rabson,1 Patricia Fraser1, 1CBR Institute for Biomedical Research, Harvard Medical School, Boston, MA, USA; 2The National Marrow Donor Program®, Minneapolis, MN, USA Recently, we observed unusual HLA-B SSOP hybridization patterns in five NMDP® donor samples indicating existence of potential novel HLA-B alleles. We performed selective PCR amplification using TA/ CG group-specific primers to separate two alleles of each sample into single alleles and then performed cycle sequencing on these PCR products. A novel B*39 allele was identified in sample 1 which differed from B*3914 at codon 114 where AAC changed to GAC, resulting in an amino acid change from Asn to Asp. In sample 2 a silent base substitution at codon 78 (CTG TTG) which distinguished the novel allele from B*4001. We made a similar observation in sample 3, where we observed a new allele that was identical to B*5301, with the exception of a synonymous substitution at codon 45 (ACG ACT ). We observed a novel B*38 allele in sample 4, for which the sequence differed from B*3801 at 2 codons: at codon 152 (GTG GAG) resulting in Val to Glu change, and at codon 158 (ACC GCC) resulting in Thr to Ala change. Furthermore, we characterized another B*39 variant in sample 5 that differed from B*3901 at codon 158 (ACC GCC; Thr Ala change). The identification of novel alleles explained the unusual SSOP patterns and further extends the diversity of the HLA-B gene. TABLE 1 Sample 1 2 3 4 5
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Ethnic Group Caucasian Caucasian African American Caucasian Caucasian
HLA-A 02xx, 30xx 01xx, 02xx 68xx, — 02xx, — 24xx, 25xx
HLA-B 1801/17N, 39new 070201, 40new 1503, 53new 4001, 38new 150101/01N, 39new
DRB1 03PJJ, 13JZD 15KCP, 1302 08BMU, 15KCM 13JUE, 13JZA 14AMR, 07MT
LUMINEX TECHNOLOGY IS SIGNIFICANTLY FASTER AND LESS EXPENSIVE THAN SSP METHODOLOGY Walter F. Herczyk, Barbara O. Burgess, Jean L. Rundquist, Adella R. Clark, Claire Li, Nancy L. Reinsmoen, Pathology, Duke University Medical Center, Durham, NC, USA With increasing workload coupled with reduction of staff, we looked for an easier and faster method of typing patients that requires less hands-on time for technologists. The Luminex methodology (LabType )™ for A, B, C, DRB1, DRB3/4/5 and DQB1 loci was compared with SSP methodology (ABC SSP Unitray ™ and Micro SSP™ Generic Class II tray). Luminex testing principles involves reverse SSO coupled with multiplexing platform of the Luminex instrument. Luminex has the ability to type for Class I and Class II loci in one operation with smaller quantity of DNA (12L for Luminex vs. 119 L for SSP). Variables of quality/ quantity of DNA, rate of repeats and turn around time were explored. Typings for 150 patients were compared with each method. SSP typing requires not only a larger total volume of sample, but much purer DNA (A260/ A280 ratio between 1.5-2.0) at a concentration no lower than 25ng/L. Whereas Luminex gives typings result with concentrations as low as 15ng/L having ratios between 1.3 to 2.1. Reasons for repeats by either method were non-amplification (9.5% for SSP, 2.5% for Luminex), gel problems (6.7% for SSP), ambiguous results (4.6% for SSP, 3.8% for Luminex), and clarifying serologic equivalents (1.9% for SSP, 5.2% for Luminex). The overall percentage of repeats were 18.0% (SSP) and 11.3 % (Luminex). Comparison of total hands-on test time for sixteen typings was two hours for Luminex vs. eight hours for SSP. Utilizing Luminex for testing resulted in a decreased turn around time of 64% for solid organ patients and 44% for bone marrow patients. This study shows that the Luminex platform is faster, easier and provides a patient typing result at less cost than SSP.