Genotyping of human neutrophil antigens 1, 3, 4 and 5 using a novel multiplex polymerase chain reaction.
granulocyte-reactive antibodies
human neutrophil antigens
multiplex polymerase chain reaction
Journal
Transfusion medicine (Oxford, England)
ISSN: 1365-3148
Titre abrégé: Transfus Med
Pays: England
ID NLM: 9301182
Informations de publication
Date de publication:
Apr 2019
Apr 2019
Historique:
received:
22
11
2018
revised:
01
03
2019
accepted:
15
03
2019
entrez:
12
4
2019
pubmed:
12
4
2019
medline:
5
9
2019
Statut:
ppublish
Résumé
Our study aimed to establish a novel multiplex polymerase chain reaction (PCR) for rapid simultaneous detection of all relevant human neutrophil antigen (HNA)-1, -3, -4 and -5 alleles. Granulocyte-reactive antibodies are involved in several diseases, such as neonatal alloimmune neutropenia, autoimmune neutropenia and transfusion-related acute lung injury. A panel of well-defined test granulocytes is required for diagnostic antibody detection and prospective blood donor screening. Several genotyping methods for the detection of HNA alleles have been described, but most approaches require separate amplification of each HNA allele or at least a separate amplification of the HNA-1 alleles. The new method is based on simultaneous detection in one reaction tube, where each HNA-1 allele is amplified by two allele-specific primers, one primer of which is labelled with a fluorescent dye (HEX, FAM). Allelic polymorphisms for HNA-3, -4 and -5 were amplified with one common unlabelled primer and two fluorescence-labelled (HEX, FAM) allele-specific primers. DNA fragments of HNA alleles are analysed on a Genetic Analyser 3130xl by amplicon size and fluorescent dye. A total of 110 blood donors with known genotypes were studied. In the 110 DNA samples studied, all HNA-1, -3, -4 and -5 alleles could be detected precisely. All results matched perfectly with those from reference typing by PCR-sequence-specific primer. Amplification performed well even at low DNA concentrations (10 ng μL Our method enables fast and easy genotyping of all relevant HNA-alleles in one PCR reaction. Results are easy to analyse due to different amplicon sizes and fluorescent dyes. Furthermore, the method is suitable for high sample throughput.
Sections du résumé
OBJECTIVES
OBJECTIVE
Our study aimed to establish a novel multiplex polymerase chain reaction (PCR) for rapid simultaneous detection of all relevant human neutrophil antigen (HNA)-1, -3, -4 and -5 alleles.
BACKGROUND
BACKGROUND
Granulocyte-reactive antibodies are involved in several diseases, such as neonatal alloimmune neutropenia, autoimmune neutropenia and transfusion-related acute lung injury. A panel of well-defined test granulocytes is required for diagnostic antibody detection and prospective blood donor screening. Several genotyping methods for the detection of HNA alleles have been described, but most approaches require separate amplification of each HNA allele or at least a separate amplification of the HNA-1 alleles.
METHODS
METHODS
The new method is based on simultaneous detection in one reaction tube, where each HNA-1 allele is amplified by two allele-specific primers, one primer of which is labelled with a fluorescent dye (HEX, FAM). Allelic polymorphisms for HNA-3, -4 and -5 were amplified with one common unlabelled primer and two fluorescence-labelled (HEX, FAM) allele-specific primers. DNA fragments of HNA alleles are analysed on a Genetic Analyser 3130xl by amplicon size and fluorescent dye. A total of 110 blood donors with known genotypes were studied.
RESULTS
RESULTS
In the 110 DNA samples studied, all HNA-1, -3, -4 and -5 alleles could be detected precisely. All results matched perfectly with those from reference typing by PCR-sequence-specific primer. Amplification performed well even at low DNA concentrations (10 ng μL
CONCLUSION
CONCLUSIONS
Our method enables fast and easy genotyping of all relevant HNA-alleles in one PCR reaction. Results are easy to analyse due to different amplicon sizes and fluorescent dyes. Furthermore, the method is suitable for high sample throughput.
Substances chimiques
Isoantigens
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
110-115Informations de copyright
© 2019 British Blood Transfusion Society.