Introduction of a real‐time based blood group genotyping approach

Abstract

Background and Objective Genotyping may be applied for rare blood group polymorphisms in a high-throughput mode as well for the molecular determination of blood groups due to unclear serological results. Material and MethodsWe developed and validated a DNA typing method for the determination of KEL1/2, JK1/2, FY1/2, FY0, MNS1/2, MNS3/4, DO1/2, CO1/2 and LU1/2 alleles using a melting curve analysis downstream from a fully automated DNA extraction. All assays were validated in terms of specificity, sensitivity, assay variability and robustness. The usability was proven by a batch of 200 blood samples with partially known phenotype.Results Assays for all blood groups were within the range of specificity (100%), assay variability and robustness (coefficient of variance < 3%). Genotypes of 200 random platelet donors were fully consistent with existing phenotype data. The obtained genotype distribution is in complete concordance with existing data for the European population underlined by a complete absence of CO2 homozygous donors and the FY0 allele among the cohort. ConclusionWe introduce an approach for blood group genotyping of particular samples or gene loci in glass capillary format and for medium-throughput analysis in 96/384-well format. The advantages of this real-time polymerase chain reaction method are its automation potential, the flexibility regarding hardware and the rapid cycling time.

Future of Molecular Testing for Red Blood Cell Antigens

“…Detection of red cell blood group SNPs by melting-curve analysis has been used to genotype for KEL1/2, JK1/2, FY1/ 2, FY0, FYX, MNS1-4, DO1/2, CO1/2, LU1/2, YTA/B, and DIA/DIB. [6][7][8][9] Pyrosequencing is a method of DNA sequencing based on the sequencing-bysynthesis principle. It differs from the common Sanger sequencing relying on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides.…”

Molecular typing for blood group antigens within 40 min by direct polymerase chain reaction from plasma or serum

“…Either plasma or serum was used; we did not make use of any extracted DNA. Our capillary rapid-cycle PCR was an adaption of previously used methods (Polin et al , 2008) with the thermocycling conditions pushed to an extreme short time limit. We added 1 μl of serum or plasma to 9 μl of a master mix.…”

“…Currently the fastest available or published turnaround times, such as the conventional capillary cycling approaches (Polin et al , 2008) and the Flougene platform (Honger et al , 2015), approximate 90 min after DNA isolation. A turnaround time of 1 h is considered acceptable for red cell genotyping in the case of urgent blood need (Flegel et al , 2015a).…”

How to find, recruit and maintain rare blood donors

“…Methods have been described for semi-automated genotyping that could be applied to screening for rare donors but have been described primarily for use in prediction of common antigens [20][21][22][23][24][25]. In the selection of prediction of antigen types for each automated genotyping assay, the majority are common typings and a few are specific for high-prevalence types.…”