Extended Donor Typing by Pooled Capillary Electrophoresis: Impact in a Routine Setting

Abstract

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 Fy^a/Fy^b, Jk^a/Jk^b, 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.

Molecular Blood Group Screening in Donors from Arabian Countries and Iran Using High-Throughput MALDI-TOF Mass Spectrometry and PCR-SSP

“…During the last decade, different molecular strategies for extended blood group typing have been developed. These include multiplex PCR-SSP and TaqMan reactions, PCR oligonucleotide extension assays with fluorescent terminator nucleotides, and pooled capillary electrophoresis with fluorescent dyes [19,[42][43][44]. MALDI-TOF MS has been applied to type thousands of blood donors [23,24,34].…”

Discrepancies between red cell phenotyping and genotyping in daily immunohematology laboratory practice

“…Low-, medium-and high-throughput techniques have been developed for blood group genotyping and in the last decade we have seen a great expansion and evolution of the technologies available [6][7][8][9][10][11][12]. Thus, molecular testing is rapidly advancing and offers tremendous help as a powerful tool with potential advantages in the identification of rare RBC donors and finding antigen matches for chronically transfused patients [7,[13][14][15][16][17].…”

The screening of rare blood donors in a highly admixed population: A new approach for Holley and Diego genotyping and impact of genomic and self‐reported ancestry

“…However, there still remains a need for easy‐to‐execute techniques to identify rare blood carriers that provide rapid and precise results and can meet the needs of laboratories with different levels of complexity and access to resources. The sensitivity of the currently available genotyping methods has enabled the evaluation of pooled samples . The genotyping of pooled samples may allow the analysis of a greater number of samples to find rare blood donors, saving time and resources.…”