THROMBOSIS RESEARCH Printed in the United
vol. States
8, PP* 319-327, Pergamon Press,
1976 Inc.
FACTOR VIII (WILLEBRAND) ANTIGEN AND RISTOCETIN-WILLEBRAND FACTOR IN PIGS WITH VON WILLEBRAND'S DISEASE D. N. Fass, W. J. Brockway, C. A. Owen, Jr. and E. J. W. Bowie Mayo Clinic and Mayo Foundation Section of Hematology Research Rochester, Minnesota, U.S.A. 55901
(Received
in revised form 19.1.1976. 11.12.1975; Accepted.by Editor K.M. Brinkhous)
ABSTRACT The Willebrand antigen and the ristocetin-Willebrand factor were virtually undetectable in pigs with a severe form of von Willebrand's disease. These findings establish that porcine von Willebrand's disease is essentially the same as the human counterpart. The Willebrand antigen and ristocetin-Willebrand factor were reduced approximately 60% in pigs which are heterozygous. Furthermore these two new tests can be done on capillary blood, permitting earlier identification of the disease.
INTRODUCTION
A bleeder strain of swine was first described by Hogan and colleagues in 1941 (1).
When these pigs were .found to have long bleeding times,
reduced levels of factor VIII, an overresponse of factor VIII to transfusion of plasma (2), and then reduced retention of platelets in glass bead columns, the similarity to human von Willebrand's disease became obvious (3, 4). In most patients with von Willebrand's disease, an antigen apparently related to factor VIII is decreased (5) and ristocetin fails to induce platelet aggregation (6) because an activity in the plasma (the ristocetinWillebrand factor) is decreased (7).
We have previously reported studies
on the factor VIII level, bleeding time and platelet retention in von Willebrand pigs (8) and we now report the lack of a plasmatic antigen in these animals.
The antigen was detected by a rabbit antibody raised 319
PIGS
320
against
a fraction
induced
platelet
tivity
and
without
of
the
rabbit
pigs.
human disease.
of
carrier
pigs
are
In
that
the
assays,
this
This
analyzed
(8).
factor
Griggs
the
swine
able
to
al
also
and absent
factor
were
measured.
similar
to
that
of
the
and ristocetinneonatal
porcine
possible
for
and platelet
identified
used
antigen
not
time,
ac-
this
antigen
is
bleeding
have
is
of
identify
identification
is
in normals level
Willebrand
been
VIII
fraction,
, present
in
the
VIII,
et
this
study,
deficiency using
disease.
from
ristocetin
no factor
ristocetin-Willebrand
we have
when only
“PAF” assay
the
promotes
contains
an antigen present
DISEASE
which
fraction
produced
the
Furthermore,
factor
carriers
plasma
This
and
indicate
Willebrand
porcine
antiserum,
antigen”)
The results
WILLEBRAXD
to quantitate
Willebrand
(“Willebrand
normal
VON
aggregation.
absorption
in von
WITH
many
retention
carriers
with
their
(9).
ANIMAJA A pig cy, of
with
a bleeding factor
time
VIII
severe
bleeder of
genetic
study
tests
and the were
static
which that
the
inbred
or
only
obtained
tests pigs
in
no bleeding
infinite),
a level
= 78-118 = >
were
U/dl),
70%]).
the
The
offspring
of
An extensive
trait,
and that
the
no clinical of
bleeding
tendency
and in
a normal
a bleeder
The bleeding and
abnormal.
these
with
carriers
a herd
of
platelet
were retention
By the
use
can
identified
be
of
a “hemo-
Yorkshire-Hampshire We have
tendency.
Yorkshire-Hampshire
the
times
previously
pigs
are
the
from
the
parent
pig
same as
(8). Pigs shown those
(4).
METHODS Thirty-seven Missouri,
were
pigs, studied.
which
were
pigs
homozygous.
activity
from
tenden-
pigs.
an autosomal
mildly
a bleeding
(normal
carrier
carrier.
50% of
(often
strain)
the mating
coagulant
has
O-30% [normal
are
have
of
VIII
30 U/dl,
with
is
a presumed
and have
hemostatic
Poland-China
pigs
normal
were
bleeder
disease
(4)
10 minutes
below
bleeding
with
factor
pigs
than
(usually
of
approximately
score”,
are
the
disease
Poland-China
offspring
pig
either
Normal
or
heterozygous
a bleeder
normal
swine
significant
are
more
activity
suggests
Presumed
of
is
(originally
bleeder
study
Willebrand’s
retention
pigs
clinically
this
that
platelet
matings
with
von
coagulant
and decreased
or
severe
descended
colony
in
in
Vo1.8,No.3
PIGS
Blood collection.
WITH
VON
WILLEBFUND
32
DISEASE
Large volumes of blood anticoagulated with 0.1
volume of 3.8% trisodium citrate were obtained from slaughter house swine or by jugular vein catheterization of single selected donors.
Small volumes
of blood similarly anticoagulated were withdrawn from superficial ear or leg veins through 18 gauge disposable needles into plastic syringes.
For
the platelet retention test, 4 units of heparin per ml (Panheprin, Abbott Laboratories) was used instead of the sodium citrate.
Platelet rich plasma
was obtained by centrifugation at 100 x g for 10 minutes at 22'.
Platelet
poor plasma resulted from 10 min. centrifugation at 20,000 x g also at room temperature. Normal donor pooled plasma was prepared from blood collected from 20 slaughter house animals into 0.1 volume of an anticoagulant
containing
3.8% trisodium citrate, 0.1 M benzamidine hydrochloride, 0.5 M EACA, and soybean trypsin inhibitor 50 mg per liter.
Aliquots of this plasma were
stored at -70'. Capillary blood was obtained from an incision made at the margin of the ear through its entire thickness using a #ll Bard-Parker blade.
A
portion of the blood was drawn into one or more 85 ~1 capillary tubes (capilets, Dade) coated with 4-6 USP units of ammonium heparin.
The
capilets were sealed with clay and centrifuged in an autocrit centrifuge (Adams) at 12,000 rpm for 5 minutes.
The red cell-containing portion of
the capilet was broken off and discarded while the cell-free plasma was expressed from the remainder of the capilet with a rubber bulb.
Twenty to
50 ul of plasma could be obtained in this fashion. Human Platelet concentrates were prepared from 450-500 ml of CPD (citrate-phosphate-dextrose) anticoagulated whole blood which was centrifuged at 2500 x g at 20-25'C for 3 min. to obtain platelet rich plasma. This plasma was expressed into a satellite bag and centrifuged at 5,000 x g for 10 min. to make a platelet button.
The platelet poor plasma was re-
turned to the packed erythrocytes leaving 20-30 ml in the platelet bag. When the platelet button was resuspended in the residual plasma, the count varied between 1 and 2 x 106/mm3. Gel filtration and washing of platelets.
Ten to 14 ml of platelet
concentrate was gently layered on a column of 2% agarose (Sepharose 2B) measuring 2.5 x 17 cm (siliconized glass column).
The buffer was a modifi-
cation of that described by Tangen and associates (LO), NaCl 0.145 M; Tris 0.05 M; CaC12 5 x 10w5M; NaN3 O.OZ%;‘pH 7.4.
The column was developed at
room temperature under continuous pressure of 3-5 cm water.
The flow rate
PLGS
322
was 30-40 the
platelet
greater at
ml/hr
and 3-4
peak
than
0.5
500 x g for
mately
K;TH VON WILLEBFUND
emerged, were
all
per
Ristocetin-Willebrand of
was carried
out
tinuous
in
stirring
the
test
(10
mg/ml)
were
sample.
the of
were
against of
centage
as
the
the
standard
buffer
to contain
assay
hundred
the
aggregation
log
the
the of
measured
the
ristocetin-Willebrand
curve.
activity
One unit
present
in
the
assay
was
normal
pig
the
quantitative
after
a normal
curve
donor
was plotted and 50% dilution and the
per-
interpolated factor
pooled
by
a standard
system
ristocetin-Willebrand
one ml of
solution
curve
from
present
of
was determined
Undiluted
same assay
con-
by 50~1
stock
dilution
plasma.
with
washed-gel-filtered
aggregation
each
(11)
platelets
followed
plasma
of
factor of
37’C
was performed, of
slope
in
of
of
a ristocetin
of
added
~1
aggregation
dilutions
of
at
cuvette
platelet
approxi-
by Olson’s
aggregation
fifty
of
part
day
dilution
were
50 ~1
of
out
aggregometer
Four
320 nm
platelets”).
was carried
channel
Serial
the
Tangen’s
The actual
steepest
Each
the
plasma
of
that
of
of
by centrifugation
The rate
and
log
test
to
follows.
tested
the
the
the
as
washed
30 seconds
ristocetin.
was prepared pool
After
slope
were
in
at
Once
The platelets
dual
rpm.
tubes.
an absorbance
method.
added
was added.
measuring addition
900
plastic
mm3 (“washed-gel-filtered
a Payton
at
human platelets
(12)
in
with
factor
Weiss’
collected
fractions
and resuspended
platelets
modification
were
the
pooled.
7 min.
250,000
ml fractions
DISEASE
is
plasma
from defined
(one
hundred
percent). Willebrand phoretic
antigen
technique
(5).
Rabbit
local
modifications
of
was
assayed
Laurel1
antiserum
at
(13)
a 1:30
by
as modified dilution
itmnunoelectro-
by Zimmerman and associates
is
added
to
the
agarose.
Our
include:
1.
The use
of
1% agarose
2.
Pouring
the
plates
(Seakem)
with
instead
a template
of
so
as
0.9%. to
achieve
uniform
1 mm
thickness. Increasing
3.
the
well
diameters
from
1 mm to
2 mm (sample
size
4 ul)* The addition
4. Results donor
are
as
M Na2 EDTA to
0.1
a percentage
of
all the
buffers antigen
used. found
in
pooled
normal
plasma. Factor
the
expressed
of
degree
activated porcine
VIII of
correction
partial plasma
coagulant
activity it
imparted
thromboplastin stored
at
-70’
was assessed
in
titrated
to human hemophilia
time was used
test.
for
A standard
daily
pig
A plasma normal
calibration.
plasma in
by
the
titrated
Values
for
Vo1.8,No.3
PIGS
WITH
VON
WILLEBW
DISEASE
323
normal pigs ranged from 80-110% of the standard plasma. Platelet retention in a glass bead column was measured by the method Normal values are more than 70% of platelets retained.
of Bowie et al (14).
The bleeding time was estimated by the ear-stab warm saline immersion method of Mertz (15).
Normal values are less than 4 minutes.
Antibody to porcine ristocetin-Willebrand factor.
Purified Willebrand
factor was prepared by precipitation, gel filtration and DEAE ion exchange chromatography as described by Olson et al (16).
Fractions containing
Willebrand factor activity but lacking measurable factor VIII coagulant activity were treated with l/100 volume of Al(OH)3 gel for 1 hour at room temperature.
The Al(OH)3 was removed by centrifugation and resuspended in
0.9% NaCl SO that each ml of suspension contained 2.5 units of adsorbed Willebrand factor.
A New Zealand white rabbit was injected intramuscularly
with this material for 11 weeks at the rate of 0.1 ml per week. obtained weekly for 7 months.
Serum was
Weekly subcutaneous booster immunizations
were carried out for six weeks, starting five months after the first series was completed. RESULTS Eighteen von Willebrand pigs and 19 obligate carriers of von Willebrand's disease were tested for levels of both Willebrand antigen and ristocetin-Willebrand factor.
The values obtained are expressed as a percentage
of normal which in turn is defined by a plasma standard pooled from at least 20 normal pigs.
The results of these assays are presented in Table 1.
All animals defined as having von Willebrand's disease were severely affected and none showed antigen levels higher than 3%. the lower reliable limit of the quantitative assay.
Three per cent is
These von Willebrand
animals also showed markedly reduced ristocetin-Willebrand factor activity. The results for 17 of 18 pigs were below the limits of detectability ("zero").
One animal had a trace value.
In the carrier animals the mean
of the antigen levels and the mean of the ristocetin-Willebrand factor levels were within one standard deviation of each other.
There was not,
however, significant correlation between antigen and factor activities in individual animals.
There were no obvious sexual differences in these two
assays. About 100~1
of capillary blood was collected from ear punctures from
23 normal pigs less than 7 days old.
Laurel1 immunoelectrophoresis was
performed to determine antigen levels.
One value was found to be greater
than 2.5 S.D. from the mean and was discarded.
Of the remaining 22, the
25
10
3.2 + 1.4
7 ->
-21
68f
21
19 f 14
__
40
25
18
19
14
84 + 18
28f
98 + 10
35
22
Pool of 26
33 f
vol. States
8, PP* 319-327, Pergamon Press,
1976 Inc.
FACTOR VIII (WILLEBRAND) ANTIGEN AND RISTOCETIN-WILLEBRAND FACTOR IN PIGS WITH VON WILLEBRAND'S DISEASE D. N. Fass, W. J. Brockway, C. A. Owen, Jr. and E. J. W. Bowie Mayo Clinic and Mayo Foundation Section of Hematology Research Rochester, Minnesota, U.S.A. 55901
(Received
in revised form 19.1.1976. 11.12.1975; Accepted.by Editor K.M. Brinkhous)
ABSTRACT The Willebrand antigen and the ristocetin-Willebrand factor were virtually undetectable in pigs with a severe form of von Willebrand's disease. These findings establish that porcine von Willebrand's disease is essentially the same as the human counterpart. The Willebrand antigen and ristocetin-Willebrand factor were reduced approximately 60% in pigs which are heterozygous. Furthermore these two new tests can be done on capillary blood, permitting earlier identification of the disease.
INTRODUCTION
A bleeder strain of swine was first described by Hogan and colleagues in 1941 (1).
When these pigs were .found to have long bleeding times,
reduced levels of factor VIII, an overresponse of factor VIII to transfusion of plasma (2), and then reduced retention of platelets in glass bead columns, the similarity to human von Willebrand's disease became obvious (3, 4). In most patients with von Willebrand's disease, an antigen apparently related to factor VIII is decreased (5) and ristocetin fails to induce platelet aggregation (6) because an activity in the plasma (the ristocetinWillebrand factor) is decreased (7).
We have previously reported studies
on the factor VIII level, bleeding time and platelet retention in von Willebrand pigs (8) and we now report the lack of a plasmatic antigen in these animals.
The antigen was detected by a rabbit antibody raised 319
PIGS
320
against
a fraction
induced
platelet
tivity
and
without
of
the
rabbit
pigs.
human disease.
of
carrier
pigs
are
In
that
the
assays,
this
This
analyzed
(8).
factor
Griggs
the
swine
able
to
al
also
and absent
factor
were
measured.
similar
to
that
of
the
and ristocetinneonatal
porcine
possible
for
and platelet
identified
used
antigen
not
time,
ac-
this
antigen
is
bleeding
have
is
of
identify
identification
is
in normals level
Willebrand
been
VIII
fraction,
, present
in
the
VIII,
et
this
study,
deficiency using
disease.
from
ristocetin
no factor
ristocetin-Willebrand
we have
when only
“PAF” assay
the
promotes
contains
an antigen present
DISEASE
which
fraction
produced
the
Furthermore,
factor
carriers
plasma
This
and
indicate
Willebrand
porcine
antiserum,
antigen”)
The results
WILLEBRAXD
to quantitate
Willebrand
(“Willebrand
normal
VON
aggregation.
absorption
in von
WITH
many
retention
carriers
with
their
(9).
ANIMAJA A pig cy, of
with
a bleeding factor
time
VIII
severe
bleeder of
genetic
study
tests
and the were
static
which that
the
inbred
or
only
obtained
tests pigs
in
no bleeding
infinite),
a level
= 78-118 = >
were
U/dl),
70%]).
the
The
offspring
of
An extensive
trait,
and that
the
no clinical of
bleeding
tendency
and in
a normal
a bleeder
The bleeding and
abnormal.
these
with
carriers
a herd
of
platelet
were retention
By the
use
can
identified
be
of
a “hemo-
Yorkshire-Hampshire We have
tendency.
Yorkshire-Hampshire
the
times
previously
pigs
are
the
from
the
parent
pig
same as
(8). Pigs shown those
(4).
METHODS Thirty-seven Missouri,
were
pigs, studied.
which
were
pigs
homozygous.
activity
from
tenden-
pigs.
an autosomal
mildly
a bleeding
(normal
carrier
carrier.
50% of
(often
strain)
the mating
coagulant
has
O-30% [normal
are
have
of
VIII
30 U/dl,
with
is
a presumed
and have
hemostatic
Poland-China
pigs
normal
were
bleeder
disease
(4)
10 minutes
below
bleeding
with
factor
pigs
than
(usually
of
approximately
score”,
are
the
disease
Poland-China
offspring
pig
either
Normal
or
heterozygous
a bleeder
normal
swine
significant
are
more
activity
suggests
Presumed
of
is
(originally
bleeder
study
Willebrand’s
retention
pigs
clinically
this
that
platelet
matings
with
von
coagulant
and decreased
or
severe
descended
colony
in
in
Vo1.8,No.3
PIGS
Blood collection.
WITH
VON
WILLEBFUND
32
DISEASE
Large volumes of blood anticoagulated with 0.1
volume of 3.8% trisodium citrate were obtained from slaughter house swine or by jugular vein catheterization of single selected donors.
Small volumes
of blood similarly anticoagulated were withdrawn from superficial ear or leg veins through 18 gauge disposable needles into plastic syringes.
For
the platelet retention test, 4 units of heparin per ml (Panheprin, Abbott Laboratories) was used instead of the sodium citrate.
Platelet rich plasma
was obtained by centrifugation at 100 x g for 10 minutes at 22'.
Platelet
poor plasma resulted from 10 min. centrifugation at 20,000 x g also at room temperature. Normal donor pooled plasma was prepared from blood collected from 20 slaughter house animals into 0.1 volume of an anticoagulant
containing
3.8% trisodium citrate, 0.1 M benzamidine hydrochloride, 0.5 M EACA, and soybean trypsin inhibitor 50 mg per liter.
Aliquots of this plasma were
stored at -70'. Capillary blood was obtained from an incision made at the margin of the ear through its entire thickness using a #ll Bard-Parker blade.
A
portion of the blood was drawn into one or more 85 ~1 capillary tubes (capilets, Dade) coated with 4-6 USP units of ammonium heparin.
The
capilets were sealed with clay and centrifuged in an autocrit centrifuge (Adams) at 12,000 rpm for 5 minutes.
The red cell-containing portion of
the capilet was broken off and discarded while the cell-free plasma was expressed from the remainder of the capilet with a rubber bulb.
Twenty to
50 ul of plasma could be obtained in this fashion. Human Platelet concentrates were prepared from 450-500 ml of CPD (citrate-phosphate-dextrose) anticoagulated whole blood which was centrifuged at 2500 x g at 20-25'C for 3 min. to obtain platelet rich plasma. This plasma was expressed into a satellite bag and centrifuged at 5,000 x g for 10 min. to make a platelet button.
The platelet poor plasma was re-
turned to the packed erythrocytes leaving 20-30 ml in the platelet bag. When the platelet button was resuspended in the residual plasma, the count varied between 1 and 2 x 106/mm3. Gel filtration and washing of platelets.
Ten to 14 ml of platelet
concentrate was gently layered on a column of 2% agarose (Sepharose 2B) measuring 2.5 x 17 cm (siliconized glass column).
The buffer was a modifi-
cation of that described by Tangen and associates (LO), NaCl 0.145 M; Tris 0.05 M; CaC12 5 x 10w5M; NaN3 O.OZ%;‘pH 7.4.
The column was developed at
room temperature under continuous pressure of 3-5 cm water.
The flow rate
PLGS
322
was 30-40 the
platelet
greater at
ml/hr
and 3-4
peak
than
0.5
500 x g for
mately
K;TH VON WILLEBFUND
emerged, were
all
per
Ristocetin-Willebrand of
was carried
out
tinuous
in
stirring
the
test
(10
mg/ml)
were
sample.
the of
were
against of
centage
as
the
the
standard
buffer
to contain
assay
hundred
the
aggregation
log
the
the of
measured
the
ristocetin-Willebrand
curve.
activity
One unit
present
in
the
assay
was
normal
pig
the
quantitative
after
a normal
curve
donor
was plotted and 50% dilution and the
per-
interpolated factor
pooled
by
a standard
system
ristocetin-Willebrand
one ml of
solution
curve
from
present
of
was determined
Undiluted
same assay
con-
by 50~1
stock
dilution
plasma.
with
washed-gel-filtered
aggregation
each
(11)
platelets
followed
plasma
of
factor of
37’C
was performed, of
slope
in
of
of
a ristocetin
of
added
~1
aggregation
dilutions
of
at
cuvette
platelet
approxi-
by Olson’s
aggregation
fifty
of
part
day
dilution
were
50 ~1
of
out
aggregometer
Four
320 nm
platelets”).
was carried
channel
Serial
the
Tangen’s
The actual
steepest
Each
the
plasma
of
that
of
of
by centrifugation
The rate
and
log
test
to
follows.
tested
the
the
the
as
washed
30 seconds
ristocetin.
was prepared pool
After
slope
were
in
at
Once
The platelets
dual
rpm.
tubes.
an absorbance
method.
added
was added.
measuring addition
900
plastic
mm3 (“washed-gel-filtered
a Payton
at
human platelets
(12)
in
with
factor
Weiss’
collected
fractions
and resuspended
platelets
modification
were
the
pooled.
7 min.
250,000
ml fractions
DISEASE
is
plasma
from defined
(one
hundred
percent). Willebrand phoretic
antigen
technique
(5).
Rabbit
local
modifications
of
was
assayed
Laurel1
antiserum
at
(13)
a 1:30
by
as modified dilution
itmnunoelectro-
by Zimmerman and associates
is
added
to
the
agarose.
Our
include:
1.
The use
of
1% agarose
2.
Pouring
the
plates
(Seakem)
with
instead
a template
of
so
as
0.9%. to
achieve
uniform
1 mm
thickness. Increasing
3.
the
well
diameters
from
1 mm to
2 mm (sample
size
4 ul)* The addition
4. Results donor
are
as
M Na2 EDTA to
0.1
a percentage
of
all the
buffers antigen
used. found
in
pooled
normal
plasma. Factor
the
expressed
of
degree
activated porcine
VIII of
correction
partial plasma
coagulant
activity it
imparted
thromboplastin stored
at
-70’
was assessed
in
titrated
to human hemophilia
time was used
test.
for
A standard
daily
pig
A plasma normal
calibration.
plasma in
by
the
titrated
Values
for
Vo1.8,No.3
PIGS
WITH
VON
WILLEBW
DISEASE
323
normal pigs ranged from 80-110% of the standard plasma. Platelet retention in a glass bead column was measured by the method Normal values are more than 70% of platelets retained.
of Bowie et al (14).
The bleeding time was estimated by the ear-stab warm saline immersion method of Mertz (15).
Normal values are less than 4 minutes.
Antibody to porcine ristocetin-Willebrand factor.
Purified Willebrand
factor was prepared by precipitation, gel filtration and DEAE ion exchange chromatography as described by Olson et al (16).
Fractions containing
Willebrand factor activity but lacking measurable factor VIII coagulant activity were treated with l/100 volume of Al(OH)3 gel for 1 hour at room temperature.
The Al(OH)3 was removed by centrifugation and resuspended in
0.9% NaCl SO that each ml of suspension contained 2.5 units of adsorbed Willebrand factor.
A New Zealand white rabbit was injected intramuscularly
with this material for 11 weeks at the rate of 0.1 ml per week. obtained weekly for 7 months.
Serum was
Weekly subcutaneous booster immunizations
were carried out for six weeks, starting five months after the first series was completed. RESULTS Eighteen von Willebrand pigs and 19 obligate carriers of von Willebrand's disease were tested for levels of both Willebrand antigen and ristocetin-Willebrand factor.
The values obtained are expressed as a percentage
of normal which in turn is defined by a plasma standard pooled from at least 20 normal pigs.
The results of these assays are presented in Table 1.
All animals defined as having von Willebrand's disease were severely affected and none showed antigen levels higher than 3%. the lower reliable limit of the quantitative assay.
Three per cent is
These von Willebrand
animals also showed markedly reduced ristocetin-Willebrand factor activity. The results for 17 of 18 pigs were below the limits of detectability ("zero").
One animal had a trace value.
In the carrier animals the mean
of the antigen levels and the mean of the ristocetin-Willebrand factor levels were within one standard deviation of each other.
There was not,
however, significant correlation between antigen and factor activities in individual animals.
There were no obvious sexual differences in these two
assays. About 100~1
of capillary blood was collected from ear punctures from
23 normal pigs less than 7 days old.
Laurel1 immunoelectrophoresis was
performed to determine antigen levels.
One value was found to be greater
than 2.5 S.D. from the mean and was discarded.
Of the remaining 22, the
25
10
3.2 + 1.4
7 ->
-21
68f
21
19 f 14
__
40
25
18
19
14
84 + 18
28f
98 + 10
35
22
Pool of 26
33 f
