Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for genotyping of human platelet-specific antigens.
BACKGROUND
Genotyping of single-nucleotide polymorphisms (SNPs) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an emerging technique, where finally tools for end users have become available to design primers and analyze SNPs of their own interest. This study investigated the potential of this technique in platelet (PLT) genotyping and developed a validated method for genotyping of clinical relevant human PLT antigens (HPAs).


METHODS
A multiplex assay using MALDI-TOF MS to analyze six HPA loci (HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, and HPA-15) simultaneously in a single reaction was applied for the genotyping of 100 DNA samples from a cohort of plateletpheresis donors and a patient population (n = 20) enriched for rare alleles. The genotyping results using MALDI-TOF MS were validated by the comparison with the results from typing by polymerase chain reaction with sequence-specific primers and conventional DNA sequencing.


RESULTS
Both homozygous and heterozygous genotypes of HPA-1 to -5 and -15 of the 120 individuals were easily identified by a six-plexed assay on MALDI-TOF MS. The three approaches achieved a 100 percent concordance for the genotyping results of the six HPA loci.


CONCLUSIONS
Compared to conventional methods, the MALDI-TOF MS showed several advantages, such as a high velocity, the ability to perform multiplexed assays in a single reaction, and automated high-throughput analysis of samples. This enables cost-efficient large-scale PLT genotyping for clinical applications.
Garritsen
Henk S P
HS
Institute for Clinical Transfusion Medicine, Städtisches Klinikum Braunschweig gGmbH, Germany.
Fan
Alex Xiu-Cheng
AX
Bosse
Nicole
N
Hannig
Horst
H
Kelsch
Reinhard
R
Kroll
Hartmut
H
Holzgreve
Wolfgang
W
Zhong
Xiao Yan
XY
D000483:Alleles; D016824:Antigens, Human Platelet / Q000235:genetics*; D015331:Cohort Studies; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D005787:Gene Frequency; D006580:Genetic Carrier Screening; D005838:Genotype; D006720:Homozygote; D006801:Humans; D008832:Microchemistry / Q000379:methods*; D036103:Nanotechnology / Q000379:methods*; D010983:Plateletpheresis; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D016824:Antigens, Human Platelet; D017931:DNA Primers; D004247:DNA