Human platelet antigen typing of neonatal alloimmune thrombocytopenia patients using whole genome amplified DNA and a 5'-nuclease assay.
BACKGROUND
A serious constraint in the investigation of the human platelet antigen (HPA) status of potential neonatal alloimmune thrombocytopenia (NAIT) cases is the limited amount of DNA available from the neonates. Whole genome amplification (WGA) of these DNA samples could overcome this problem, but requires validation to ensure that it is sufficiently sensitive and accurate before its application in a clinical diagnostic setting.


METHODS
This study has validated the use of WGA DNA for HPA-1, -2, -3, -4, -5, and -15 genotyping with a panel of six controls and 13 previously HPA-typed samples from neonates together with parental DNA, using a 5'-nuclease (TaqMan) assay. WGA was performed using titrated amounts of genomic and WGA DNA template. HPA typing was performed on genomic and amplified DNA using a 5'-nuclease assay or polymerase chain reaction with sequence-specific primers (PCR-SSP).


RESULTS
WGA DNA yields were in the suggested range of 400x to 800x, as assessed by spectrophotometry and gel analysis, and did not require further purification. HPA genotyping showed 100 percent concordance when using down to 5 ng of genomic or WGA template.


CONCLUSIONS
This study demonstrates that WGA can be used for HPA typing using PCR-SSP or plate-based 5'-nuclease assays. The use of WGA for HPA typing in clinical samples from NAIT patients was validated with 100 percent concordance, and it is suggested that this technology can be used for other analyses where DNA amounts are limited.
Lemnrau
Alina G
AG
Histocompatibility & Immunogenetics Department, NHSBT, London, UK.
Cardoso
Sandra
S
Creary
Lisa E
LE
Brown
Colin
C
Miretti
Marcos
M
Girdlestone
John
J
Navarrete
Cristina V
CV
D016824:Antigens, Human Platelet / Q000235:genetics*; D017931:DNA Primers; D015894:Genome, Human; D005838:Genotype; D006801:Humans; D007231:Infant, Newborn; D021141:Nucleic Acid Amplification Techniques / Q000379:methods / Q000592:standards*; D011933:Reagent Kits, Diagnostic; D054098:Thrombocytopenia, Neonatal Alloimmune / Q000175:diagnosis*
D016824:Antigens, Human Platelet; D017931:DNA Primers; D011933:Reagent Kits, Diagnostic