1. Art and Diseño

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Determination of Human Platelet Antigen Frequencies in the Dutch Population
by Immunophenotyping and DNA (allele-specific restriction enzyme) Analysis
By S . Simsek, N.M. Faber, P.M. Bleeker, A.B.J. Vlekke, E. Huiskes, R. Goldschmeding, and A.E.G.Kr. von dem Borne
Platelets from 200 random Dutch blood donors were typed
for the human platelet alloantigens HPA-1 to -5 recognized
at present and for Nak”. Nak” is an epitope on glycoprotein
IV, not expressed on the platelet of individuals with hereditary GP IV deficiency. Platelet immunofluorescence and
monoclonal antibody-specific immobilization of platelet
antigens (MAIPA) were applied for this purpose. The observed phenotype frequencies were 97.86% and 28.64%
for HPA-1a and -1b, 100%and 13.15% for HPA-2a and
-2b. 80.95% and 69.84% for HPA-3a and -3b. 100%and
0%for HPA-4a and -4b. 100%and 19.7% for HPA-5a and
HPA-5b, respectively. Plateletsfrom all donors reacted with
the anti-Nak” antibodies. To determine the gene frequencies
for the HPA-1, HPA-2 and HPA-3 systems directly, DNA
from 98 of these donors was isolated from peripheral blood
mononuclear leucocytes and specific fragments were amplified by polymerase chain reaction (PCR). The fragments
were analyzed using allele-specific restriction enzymes
(ASRA). In all amplified PCR products an ”internal control”
for each assay, ie, a restriction site for the applied enzyme
independent from the phenotype of the donor was present.
In all donors tested, phenotypes, as determined by serological methods and genotypes, directly determined by the
ASRA, were identical. Thus, the PCR-ASRA described in
this report is a practical and reliable technique for the determination of alleles that code for platelet antigen allotypes, at least in the Dutch population.
0 1993 by The American Society of Hematology.
H
Serological Characterization
UMAN PLATELET alloantigens are of importance in
neonatal alloimmune thrombocytopenia (NAITP),
refractoriness to platelet transfusion therapy, and post transfusion purpura (PTP).’ To date, five distinct biallelic platelet
“specific” alloantigen systems have been described.2“ In four,
it has been shown that a single base pair substitution leads
to a single amino acid difference in the respective glycoprotein
(GP).’-’O Table 1 summarizes the glycoprotein location and
the amino-acid polymorphisms of the HPA-1 (Zw), HPA-2
(KO),HPA-3 (Bak), and HPA-4 (Yuk) systems.
The mutations responsible for the HPA- 1, HPA-2, HPA3 and HPA-4 systems turned out to be associated with recognition sites for restriction enzymes in the DNA in one of
the two alleles. The genomic organization of the DNA coding
for GP IIIa (HPA-I and HPA-4), GP Iba (HPA-2), and GP
IIb (HPA-3) is known.”-13Thus, it is possible to determine
the genotype of an individual for the HPA- 1, HPA-2, HPA3 and HPA-4 systems by allele-specific restriction enzyme
analysis (ASRA) using genomic DNA. We did not apply this
technique for the HPA-4 system for reasons stated below.
We developed procedures for the amplification of gene fragments for the relevant glycoproteins, which could be performed on DNA from purified mononuclear blood cells. Only
restriction enzymes were applied that have a target site in the
amplified fragment independent of the allotype (internal
control). In this report we show that the genotypes for HPA1, HPA-2 and HPA-3, determined in this way, are strictly
concordant with the phenotypes established by serologic
methods. The ASRA analysis described is quick, reliable, and
easy to perform. Therefore, it may be of great value in typing
for alleles that encode platelet alloantigens when no platelets
are available, eg, in cases of suspected PTP and in fetuses at
risk for NAITP.’4s’5
MATERIALS AND METHODS
Blood
From 200 volunteer blood donors, 20 mL of blood was collected
in EDTA as an anticoagulant. All donors were white inhabitants of
four small Dutch villages. Platelets were isolated by differential centrifugation.16 Mononuclear leukocytes were purified by density centrifugation over a Ficoll Isopaque layer. Pelleted leukocyteswere stored
at -20°C until use.
Blood. Vol81, No 3 (February 11, 1993: pp 835-840
The platelet-specific allo antisera applied were: anti-HPA- 1a, antiHPA- I b, anti-HPA-2a, anti-HPA-2b, anti-HPA-3a, anti-HPA-3b,
anti-HPA4a, anti-HPA-Sa, and anti-HPA-Sb. Anti-HPA-3b and antiHPA-Sa sera were kindly provided by Dr C Mueller Eckhardt, Gies
sen, Germany. Anti-HPA-4a and anti-Nak” were gifts from Dr Y.
Shibata, Tokyo, Japan. The phenotype of the platelets was determined
in the platelet immunofluorescence testI6 and/or the monoclonal antibody-specific immobilization of platelet antigens (MAIPA).” The
monoclonal antibodies used in the MAIPA assay were MB45, against
the CD42b antigen (GPIba); CLB-thromb/I and Y2/51, both against
the CD61 antigen (GP IIIa); MB9, against the CD41 antigen (GP
Ilb); and CLB-thromb/4, against the CDw49b antigen (VLA-2).’*
The donors who were positive for the HPA-Sa antigen were also
tested for the HPA-Sb antigen.
Genetic Characterization
Genomic DNA was purified from leukocytes according to the
method described by Ciulla et aI.l9 Oligonucleotide primers selected
for the polymerase chain reaction (PCR) were used to amplify those
parts of the genomic DNA that contain the polymorphic sequences
corresponding to the HPA- I, HPA-2, and HPA-3 alleles respectively.
Sequence and genomic position of the oligonucleotide primers used
for the PCR are shown in Table 2.
PCR was performed on 0.7 p g genomic DNA template in a
total volume of 50 p L with 20 pmol of the appropriate primers
From the Department of Immunologic Haematology, Central
Laboratory ofthe Netherlands Red Cross Blood Transfusion Service
and Laboratory for Clinical and Experimental Immunology, University ofAmsterdam; and the Department of Haematology, Academic
Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Submitted April 13, 1992; accepted October 8, 1992.
Address reprint requests to A.E.G.Kr. von dem Borne, MD, c/o
Publication Secretariat, Central Laboratory of the Netherlands Red
Cross Blood TransfusionService, PO Box 9406, I006 AK Amsterdam,
The Netherlands.
The publication costs ofthis article were defiayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1993 by The American Society of Hematology.
0006-4971/93/8 103-0024$3.00/0
835
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SIMSEK ET AL
836
with I U Msp I, Sfa NI, and Fok I (Bethesda Research Laboratories,
Bethesda, MD) for the HPA-I, HPA-2 and HPA-3 systems respectively. Digests were analyzed on a 8% polyacrylamide (HPA1 and HPA-3) or a 2% agarose (HPA-2) gel. Fragments were visualized by U V illumination after ethidium bromide staining.
Table 1. Molecular Basis of the Human Platelet Antigens
System
Antigens
HPA-1
HPA-la
HPA-1b
HPA-2a
HPA-2b
HPA-3a
HPA-3b
HPA-4a
HPA-4b
HPA-2
HPA-3
HPA-4
Glycoprotein
Location
Amino Acid
Substitution
GPlla
References
Leucine33
Proline33
Methi~nine''~
Threonine'46
Isoleucinesu
Serinew3
GlutaminelArginine143
GP Ib
GPIlb
Gp 'la
7
ASRA Analysis of PCR Products
8
HPA-I sysfem. The C + T substitution at base number 12,548
ofthe complete genomic DNA sequencecreates an Nci I and an Msp
I restriction enzyme cleavage site in the HPA-lb GPllla DNA. We
have chosen for Msp I restriction analysis for reasons stated below.
The position of the Msp I restriction sites in the HPA-1 PCR product
is depicted in Fig I A.
HPA-2 sysfem. The C + T substitution at base 482 of the GP
Iba gene creates an Sfa NI restriction site in the HPA-2b allele. The
position of the Sfa NI restriction sites in the PCR product and the
length of the fragments generated after digestion are schematically
shown in Fig 2A.
HPA-3 sysfcm. The G + T substitution at base 13,962 of the
genomic GP Ilb DNA sequence is responsible for the loss of an
Fok I site in the HPA-3b allele. The position ofthe Fok I restriction
9
10
and 2 U of Taq DNA polymerase in the buffer recommended by
the manufacturer (Promega, Madison, WI). Thirty-three cycles
of amplification were performed, with denaturation for I minute
at 95°C. annealing for 2 minutes at 62"C, and extension for 2%
minutes at 72OC.
Restriction-enzyme digests of the amplification products were
performed under conditions recommended by the manufacturers
A
0
GP Ills:
2
1
I
I
111 I
1111 I I
0
111
111
I
I
I
I
'
3
I I
I I
40kb
I
4
L
2
Msp I(only in HPA-lb)
279
D *
4
106
-4
Msp I
..
-t
I
173
1
197
197
D 4
HPA-la
HPA-lb
40 2 4
27-
1974
1 7 b
10-
Fig 1. (A) Diagramatic representation of the genomic organization of glycoprotein Illa.
The PCR product has a length
of 482 bp. The Msp Irecognition
sites, which are present in both
alleles, are shown. Also the Msp
1, which is created by the C +
T transition in the HPA-1b allele
and the actual fragments after
digestion with Msp I are also
shown. (B) The 482-bp PCR
products in the HPA-1(a+,b-),
HPA-1(a+,b+),
and HPA1(a-,b+)
phenotypes were
digested with Msp 1 and electrophoresed through a 8% polyacrylamide gel. M is a marker.
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837
HPA FREQUENCIES IN THE DUTCH POPULATION
Table 2. Sequence of Oligonucleotide Primers Selected for the PCR
OlioonucleotideSeauences
1.
2.
3.
4.
5.
6.
Localizationtntl
References
12447
12923
57
765
13834
14281
11
5ATAAGCTTAGCTATTGGGAAGTGGTAGGGCCTG3’
5 CTTCTGACTCAAGTCCTAACG3’
5ATAAGCTTGCTGCTCCTGCTGCCAAGCC3
VTAGAATTCAGCATTGTCCTGCAGCCACG3
5 TGGAAGAAAGACCTGGGAAGG3’
5 CTCCTTAACGTACTGGGAAGC3
12
13
Primers 1 and 2 were used for the HPA-1 PCR. Primers 3 and 4 were used for HPA-2 PCR. Primers 5 and 6 were used for the HPA-3 PCR. The
position of the 5 end of the primers in the gene sequence is indicated.
sites in the HPA-3 PCR fragment and the length of the digestion
fragments are shown in Fig 3A.
in the PCR productsofall three systems
for the A S m analysis
an “internal control” for the efficiencyofthe enzyme is present, ie,
the enzyme used in the assay has another target site in the pCR
fragment, which is present in both alleles.
RESULTS
Plufelef phenolyping. Platelets from 200 donors were
phenotyped for the antigens of the HPA-I, HPA-2, HPA-3,
and HPA-4 SyStems in the platelet i~mUnOflUOreSCenCetest
and in the MAIPA. The HPA-5 system was tested in the
A
GPlb :
I
I
PCR-tragmonta:
192
.
627
192
279
(a+,b-)
H PA-2
(a+,b+)
HPA-2
(a-,b+)
-
-
-
H PA-2
B
SfaN1
+
bP
Fig 2. (A) The PCR strategy
to a m p l i i a region in the genomic glycoprotein Iba in which
the HPA-2 polymorphism is located and the fragments after
digestion with Sfa N1 are
shown. The Sfa N1 site, which
is created by the C + T substitution is located at base 471 in
this PCR product. (B) Ethidium
bromide staining after a 2%
agarose gel electrophoresis of
the digested samples in the
HPA-Z(a+,b-), HPA-S(a+,b+).
and HPA-P(a-,b+) phenotypes
isshown. M1 and M 2 are markers.
719
527 L
279
248
192
248
-
-1
2% agarose
+
+
.
M1
HPA-2.
HPA-20
M2
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838
SIMSEK ET AL
26
&//\
II
111
100
I
okl
4
Fokl
191
149
30
I
Fig 3. (A) The genomic organization of glycoprotein Ilb
and the 448-bp PCR product is
shown. The HPA-3 polymorphism is attributable to a G + T
substitution leading t o the loss
of a recognition site for Fok Ilocated at 108 bp in this PCR
product. (6)The PCR product
of the HPA-3(a+,b-),
HPA3(a+,b+), and HPA-B(a-,b+)
phenotypes were electrophoresed through a 8% polyacrylamide gel after digestion with
FokI.
448bp
PCR fragments:
149
299
B
Fok I
HPA-3(a+,b-)
-
+
HPA-3(a+,bt)
-
+
HPA-3(a-,b+)
-
+
be
- 448
- 299
-191
-1 4 9
- 108
MAIPA only. Results are shown in Table 3. No discrepancies
were observed between the results of the platelet immunofluorescence test and those of the MAIPA (Table 4).
Donor genofyping. ASRA analysis for the HPA- 1, HPA2, and HPA-3 system was performed on DNA derived from
98 of the 200 donors whose platelets were phenotyped. The
genotype of the donors could be directly determined by the
PCR-ASRA. The fragments obtained by Msp I (HPA-I), Sfa
NI (HPA-2), and Fok I (HPA-3) digestions are shown in Fig
IB, 2B, and 3B respectively. The digestion products are of
the expected size. The digested fragments by the ASRA and
the genotypes derived from these results are consistent with
the observed phenotypes as determined by the PIFT and/or
the MAIPA. Thus, the phenotype and genotype correspond
in all 98 cases tested (Table 4).
DISCUSSION
Phenotyping of platelet alloantigens has so far depended
on the availability of platelets and alloantigen-specific reagents. The technique most commonly used is the platelet
immunofluorescence test. For the detection of platelet alloantigens (eg, HPA-5) present in low concentration on the
platelet surface, a catching ELISA (MAIPA) has been developed."
The platelets of 200 Dutch blood donors were phenotyped
by these two serologic techniques. Table 3 shows the frequencies of the platelet alloantigens in the Dutch population.
The phenotype frequencies for most of the HPA systems is
comparable to the frequencies found in the white population
described previously? In contrast, the phenotype frequency
that is found for the HPA-3a antigen is lower than reported
previously." This discrepancy is probably attributable to the
presence of noncomplement fixing HLA antibodies, not detectable in the lymphocyte cytotoxicity test (LCT), in the
Table 3. Human Platelet Antigen Frequencies in the Dutch Population
Phenotype Frequency (%Y (n = 200)
System
HPA-1 [ZW, PIA]
HPA-2 [KO, Sib]
HPA-3 [Bak, Lek]
HPA-4 [Pen, Yuk]
HPA-5 [Br, Hc, Zav]
Calculated Gene Frequency' (n = 200)
Antigens
Mean
95% CI
Mean
95% CI
HPA-la
HPA-1b
HPA-2a
HPA-2b
HPA-3a
HPA-3b
HPA-4a
HPA-4b
HPA-5a
HPA-5b
97.86
28.84
100
13.15
80.95
69.84
100
0
100
19.7
(94.43-99.05)
(22.59-33.45)
(97.65-100)
(8.94-1 8.72)
(74.67-85.92)
(62.89-75.93)
(97.65-100)
(0-2.35)
(97.65-100)t
(14.56-25.95)
0.846
0.154
0.934
0.066
0.555
0.445
1,000
0,000
0.902
0.098
(0.787-0.891)
(0.108-0.212)
(0.888-0.96 1)
(0.037-0.1 11)
(0.483-0.624)
(0.37545 16)
(0.985-1 .000)
(0.000-0.015)
(0.849-0.936)
(0.063-0.150)
95% CI,95% confidence interval of the frequencies.
The calculated frequencies are based on immunofluorescence and MAIPA results.
t Only HPA-5b+individualswere tested for HPA-5a expression.
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HPA FREQUENCIES IN THE DUTCH POPULATION
839
Table 4. Concordance Between lmmunophenotyping
and DNA Analysis
~
~~
n
98
PIFT
MAIPA
ASRA
HPA-1 (a+, b-)
HPA-1 (a+, b+)
HPA-1 (a-, b+)
62
35
1
62
35
1
62
35
HPA-2 (a+, b-)
HPA-2 (a+, b+)
HPA-2 (a-, b+)
86
12
86
12
86
12
-
-
-
HPA-3 (a+, b-)
HPA-3 (a+, b+)
HPA-3 (a-, b+)
28
53
17
28
53
17
28
53
17
=
1
From the 98 donors from which DNA was purified to directly determine
the genotypes, the results of the PIFT, MAIPA, and the ASRA were
compared.
only anti-Bak" (anti-HPA-3a) serum available at the time of
the original study (unpublished observation). In the population studied in the present investigation, all donors were
positive for the HPA-4a antigen. Previously, we also noted
HPA-4b antigen negativity with all donors tested in the Dutch
population, using anti-HPA-4b antibodies, testing a different
group of 48 donors. Therefore, we decided not to perform
genotyping for HPA-4 by molecular biological methods.
However, this is also possible in ASRA by applying the restriction enzyme CviRI, which recognizes the -TGCA- sequence.20 Furthermore, there was no single person whose
platelets did not react with the anti-Nak" isoantibodies, indicating that GP IV-deficiency does not occur at a significant
rate in the Dutch population. The molecular biological basis
for the HPA-5 polymorphism has not yet been established,
and for this reason this system cannot be included in the
ASRA analysis.
Reagents for immunotyping (serotyping) of the HPA- 1b,
HPA-2a, HPA-2b, and HPA-3b antigens are rare and available only in a few specialized laboratories in small quantities.
Moreover, in some clinical situations no platelets are available
for phenotyping of the platelet alloantigens.I4,l5 In contrast,
genotyping might be performed on any material from which
DNA can be derived. We have designed PCR oligonucleotide
primers applicable on genomic DNA. This eliminates the
need for isolation of mRNA and reverse transcription into
cDNA. Our methodology for platelet antigen genotyping can
be applied and performed by anyone with basic experience
in molecular biological techniques. We have compared the
results of conventional phenotyping and of genotyping for
HPA-1, HPA-2, and HPA-3 in 98 random donors of the
Dutch population. Our results show that there is a perfect
correlation between the serologic results and those obtained
by the ASRA analysis. ASRA using Nci I to type for the
HPA-1 system has been described by Newman et al.' However, this enzyme may give false positive results because of
incomplete or failed digestion (unpublished observation). In
contrast to Nci I, Msp I has the advantage that the PCR
product used for the ASRA contains a restriction site for Msp
I in both alleles. This provides an internal control for complete
digestion in each assay. Similarly there are additional restric-
tion sites in both alleles for the Sfu NI-ASRA in the HPA-2
PCR fragment and for the Fok I in both of the HPA-3 alleles.
Other techniques for genotyping of allelic antigens to detect
just one base-pair difference, such as the competitive oligonucleotide priming (COP) assay2' and allele-specific oligonucleotide (ASO) dot-blot hybridization22 have been described. In comparison with the PCR-ASRA analysis
described in this report, the COP assay is time-consuming,
requires radioactivity, and depends on critical conditions
needed for allele-specific hybridization based on a single basepair difference. This last aspect also applies to the AS0 dotblot hybridization (radioactive or nonradioactive) technique.
For these techniques, the hybridization temperatures for the
respective oligonucleotide probes and the hybridization buffer
(ASO) should be carefully controlled. The results may be
affected by variations in sample condition or quality of the
PCR products. Moreover, incorporation of an internal control
in each AS0 or COP assay appears not to be feasable.
In conclusion, we have determined the phenotype and genotype frequencies of the most important platelet specific
alloantigens in the Dutch population. The PCR-ASRA analysis for genotyping of HPA alleles is a valuable tool to replace
platelet alloantigen typing, especially in cases in which platelets for phenotyping are not available or difficult to obtain.
Moreover, it eliminates the need for scarcely available antisera.
ACKNOWLEDGMENT
We thank B. Bossers, H. Bruinink, I. Damsma, L. Buddelmeijer,
J. Heeremans, C. van der Meer, and K. van der Kolk for technical
support. Furthermore, we thank Dr C.P. Engelfriet for critically
reading the manuscript and Dr H.Th.M. Cuijpers for useful discussions.
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SIMSEK ET AL
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From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1993 81: 835-840
Determination of human platelet antigen frequencies in the Dutch
population by immunophenotyping and DNA (allele-specific restriction
enzyme) analysis
S Simsek, NM Faber, PM Bleeker, AB Vlekke, E Huiskes, R Goldschmeding and AE von dem
Borne
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