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HOW TO DO PLATELET AND NEUTROPHIL ANTIGEN GENOTYPINGM
Allele-specific PCR. Analysis is accomplished by subjecting
samples of patients genomic DNA to 3 PCR amplifications. One reaction
includes the NA1-specific 5 and 3, oligonucleotide primers, the
second reaction includes the Na2-specific 5, and 3 oligonucleotide
primers, and the third reaction includes a SH-specific 5 and 3
oligonucleotide. Types are assigned after PCR products are analyzed by
agarose gel electrophoresis (Hessner MJ, 1996. Determination of
neutrophil antigen NA gene frequencies by PCR).
HPA and HNA-phenotyped individuals were selected from blood
donors after screening of a large population. Phenotyping was performed
with assay of monoclonal antibody-specific immobilization of platelet
and granulocyte antigens, as described previously. Blood was obtained
from individuals carrying the rare alloantigens HPA-4a/4b and HPA-8bW,
-12bW, and 13bW and from fathers of newborns with neonatal alloimmune
thrombocytopenia. Blood samples from an HPA-4b-homozygous and an
HPA-6bW-homozygous donor and an HPA-6bW-homozygous donor were a gift
from Yoichi Shibata, MD (Tokyo, Japan). Riita Kekomaki, MD (Helsinki,
Finland), provided cells from an HPA-6bw-heterozygous individual. Blood
samples from individuals carrying the low-frequency alloantigens
HPA-7bW, -9bW, and -11bW were made available by A>EG>Kr. Von dem
Borne, MD (Amsterdam, the Netherlands). The phenotypes of all
individuals were confirmed by genotyping. ESTABLISHMENT
OF B-LYMPHOBLASTOID CELL LINES (B-LCLs) Heparin-anticoagulated
sterile blood (40 ml) from HPA-phenotyped donors was diluted with 80 ml
of phosphate-buffered saline and layered onto a density gradient (Fiocoll-Paque,
Pharmacia Biotech, Uppsala, Sweden). After centrifugation at 570 X g for
35 minutes, the cells at the interface were harvested and washed three
times with phosphate-buffered saline (once at 570 X g for 10 minutes and
twice at 120 X g for 20 minutes). A 1-ml aliquot of cells (2 X 10 7/ml)
in medium (RPMI) supplemented with 2% fetal calf serum was treated with
150 mL
of leucyl-leucin-methylester (0.66 M in methanol solution in 10 ml RPMI;
Bachem, Heidelberg, Germany) for 15 minutes at room temperature. The
reaction was stopped by the addition of 5 ml of 2 % fetal calf serum in
RPMI. After being washed three times (350 X g, 12 minutes), the cells
were incubated with 2 ml of EBV source in the disk (Petriperm, In Vitro
Systems, Osterode, Germany) for 2 hours at 37ēC. The virus source was
the supernatant of an aged (2-4 weeks) culture of the marmoset cell line
B95-8 (ATCC, Rockville, MD), which had been filtered through a 0.22-mm
filter and stored at -70°C. Three ml of Iscoves medium containing L-alanyl-L-glutamine
(Glutamax) and 4-% Minimum Essential Medium alpha (both: Gibro BRL,
Eggenstein, Germany), 15% fetal calf serum, 0.5 %
penicillin/streptomycin (Gibro BRL), 7.7 mM insulin (27 USP/mg; Sigma,
Dreieich, Germany), and 1.5 mM oxalacetic acid (Sigma) was added for 16
hours. The cells were transferred to a 25-mL tissue cultured flask
(Greiner, Frickenhausen, Germany), grown for 3-6 weeks, and fed as
necessary. The B-LCLs were either stored in a frozen state in liquid
nitrogen or genotyped directly. ISOLATION
OF GENOMIC DNA FROM WHOLE BLOOD AND B-LCLs
DNA of peripheral blood leukocytes was isolated from 3 ml of EDTA
blood by use of a DNA extraction kit (Pure Gene, Gentra Systems, Biozym,
and Oldendorf, Germany) and stored at -20°C until it was used. After
thawing, B-LCLs were grown for 2-6 weeks and harvested by centrifugation
at 570 X g for 10 minutes. Genomic DNA (50-100 mg)
was then isolated from 10 7/cells.
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