BACKGROUND: PCR with sequence-specific priming using allele-specific fluorescent primers and analysis on a capillary sequencer is a standard technique for DNA typing. We aimed to adapt this method for donor typing in a medium-throughput setting. METHODS: Using the Extract-N-Amp PCR system, we devised a set of eight multiplex allele-specific PCR with fluorescent primers for Fya/Fyb, Jka/Jkb, M/N, and S/s. The alleles of a gene were discriminated by the fluorescent color; donor and polymorphism investigated were encoded by product length. Time, cost, and routine performance were collated. Discrepant samples were investigated by sequencing. The association of new alleles with the phenotype was evaluated by a step-wise statistical analysis. RESULTS: On validation using 312 samples, for 1.1% of antigens (in 5.4% of samples) no prediction was obtained. 99.96% of predictions were correct. Consumable cost per donor were below EUR 2.00. In routine use, 92.2% of samples were successfully predicted. Discrepancies were most frequently due to technical reasons. A total of 11 previously unknown alleles were detected in the Kell, Lutheran, and Colton blood group systems. In 2017, more than 20% of the RBC units prepared by our institution were from donors with predicted antigen status. A steady supply of Yt(a-), Co(a-) and Lu(b-) RBC units was ensured. CONCLUSIONS: Pooled capillary electrophoresis offers a suitable alternative to other methods for extended donor DNA typing. Establishing a large cohort of typed donors improved transfusion support for patients.
BACKGROUND: PCR with sequence-specific priming using allele-specific fluorescent primers and analysis on a capillary sequencer is a standard technique for DNA typing. We aimed to adapt this method for donor typing in a medium-throughput setting. METHODS: Using the Extract-N-Amp PCR system, we devised a set of eight multiplex allele-specific PCR with fluorescent primers for Fya/Fyb, Jka/Jkb, M/N, and S/s. The alleles of a gene were discriminated by the fluorescent color; donor and polymorphism investigated were encoded by product length. Time, cost, and routine performance were collated. Discrepant samples were investigated by sequencing. The association of new alleles with the phenotype was evaluated by a step-wise statistical analysis. RESULTS: On validation using 312 samples, for 1.1% of antigens (in 5.4% of samples) no prediction was obtained. 99.96% of predictions were correct. Consumable cost per donor were below EUR 2.00. In routine use, 92.2% of samples were successfully predicted. Discrepancies were most frequently due to technical reasons. A total of 11 previously unknown alleles were detected in the Kell, Lutheran, and Colton blood group systems. In 2017, more than 20% of the RBC units prepared by our institution were from donors with predicted antigen status. A steady supply of Yt(a-), Co(a-) and Lu(b-) RBC units was ensured. CONCLUSIONS: Pooled capillary electrophoresis offers a suitable alternative to other methods for extended donor DNA typing. Establishing a large cohort of typed donors improved transfusion support for patients.
Entities:
Keywords:
Blood donor typing; Colton blood group; Immunohematology; Kell blood group; Lutheran blood group; Molecular blood group typing; Null allele
Authors: J Perreault; J Lavoie; P Painchaud; M Côté; J Constanzo-Yanez; R Côté; G Delage; F Gendron; S Dubuc; B Caron; R Lemieux; M St-Louis Journal: Vox Sang Date: 2009-03-16 Impact factor: 2.144
Authors: H W Reesink; C P Engelfriet; H Schennach; C Gassner; S Wendel; R Fontão-Wendel; M A de Brito; P Sistonen; J Matilainen; T Peyrard; B N Pham; P Rouger; P Y Le Pennec; W A Flegel; I von Zabern; C K Lin; W C Tsoi; I Hoffer; K Barotine-Toth; S R Joshi; K Vasantha; V Yahalom; O Asher; C Levene; M A Villa; N Revelli; N Greppi; M Marconi; Y Tani; C C Folman; M de Haas; M M W Koopman; E Beckers; D S Gounder; P Flanagan; L Wall; E Aranburu Urtasun; H Hustinx; C Niederhauser; C Flickinger; S J Nance; G M Meny Journal: Vox Sang Date: 2008-10 Impact factor: 2.144
Authors: Yanli Ji; Barbera Veldhuisen; Peter Ligthart; Lonneke Haer-Wigman; John Jongerius; Mohamed Boujnan; Aicha Ait Soussan; Guangping Luo; Yongshui Fu; C Ellen van der Schoot; Masja de Haas Journal: Transfusion Date: 2014-08-25 Impact factor: 3.157
Authors: Franz F Wagner; Rita Bittner; Eduard K Petershofen; Andrea Doescher; Thomas H Müller Journal: Transfusion Date: 2008-04-15 Impact factor: 3.157
Authors: Jan Portegys; Gabi Rink; Pia Bloos; Erwin A Scharberg; Harald Klüter; Peter Bugert Journal: Transfus Med Hemother Date: 2018-09-24 Impact factor: 3.747