Cerus Corporation
2550 Stanwell Drive, Concord, CA 94520 USA
www.cerus.com
1.925.288.6000
Tovo David
[email protected] T: 925-288-6060
Evaluation of In Vitro Platelet Functions of INTERCEPT™
Treated Platelet Components (PC) Using a
“Low Dose/Low Volume” Configuration
T. David, M.von Goetz, E. Evans, N. Doan, J. Crabtree, T. Diaz, and N. Mufti
Cerus Corporation, Concord, CA
Presented at the American Association of Blood Banks Annual Meeting (AABB)
Philadelphia, Pennsylvania • October 25th - 28th, 2014
Evaluation of In Vitro Platelet Functions of INTERCEPT™ Treated Platelet
Components (PC) Using a “Low Dose/Low Volume” Configuration
T. David, M.von Goetz, E. Evans, N. Doan, J. Crabtree, T. Diaz, and N. Mufti
Cerus Corporation, Concord, CA, US
Background
The INTERCEPT™ Blood System was developed to
reduce transfusion-transmitted infections by inactivating
pathogens in blood components. This system utilizes
amotosalen and low energy ultraviolet light A to crosslink
nucleic acids and prevent replication of pathogens and
leukocytes. INTERCEPT has CE Mark registration for
treatment of platelet concentrates (PC) ranging from 2.5
to 8 x 1011 platelets in additive solutions or 100% plasma
with volumes between 255-420 mL. In order to implement
INTERCEPT for platelets in Japan, a kit that can process
a 2 x 1011 platelet dose in a volume of 200 mL plasma
(±20%) is required (Figure 1).
Aims
Methods
The objective of this study was to
evaluate platelet in vitro function
over 6 days of storage following
treatment of 2 x 1011 platelets in
220 mL plasma using two prototype
configurations with either a full or
half-size Compound Adsorption
Device (CAD) and PO80 Kawasumi
bags as storage containers.
A pool and split strategy was used to generate an Untreated unit, a Half-CAD (HC) treated unit, and a
Full-CAD treated unit (FC) with a platelet dose of 2 x 1011 and a volume of 220 mL for each replicate
using platelet components in 100% plasma collected on a TRIMA platform. A total of 5 replicates were
performed. After treatment, the split units were stored and tested for in vitro platelet functions at day
3 and day 6. Function assays included platelet dose, pH, morphology score, Extent of Shape Change
(ESC), CD62P, ATP, glucose, platelet lysis and perfusion assay on collagen Platelet perfusion experiments
were performed using a Bioflux System. Plates were coated with 75 μg/ml collagen and blocked with 1%
PBS-BSA. Platelets were labelled with 10 μM Calcein AM. Thrombus formation was monitored real time
under a Zeiss AX10 inverted microscope and a 10X objective. Images were analyzed off-line for platelet
surface coverage using the Bioflux 200 software version 2.5.0.0. Statistical analyses were performed
with computer software (GraphPad Prism 6.0, GraphPad, San Diego, CA). Data were reported as mean ±
standard error and were analyzed by One-Way ANOVA with Tukey’s multiple comparisons test. A p value
less than 0.05 was considered to be significant.
A
B
pH day3 and 6
C
Glucose
2 x 1011 platelets
Platelet
in 100% plasma
Collection
(~220mL)
Illumination
3J/cm2 UVA
Half
or Full CAD*
Final Storage:
PO80 bags
*Compound Adsorption Device
ATP
5
5
2
2
2
0
0
0
0
0
0
1504
150
4
6.5
F
F
Extent of Shape
Change CD62P
100
20
80
* 400
60
350
15
ESC ( %)
100
F
CD62P CD62P
G
0
200
150
-1
Perfusion
on collagen
800s 800s -1
Perfusion G
on collagen
800s
G Perfusion
on collagen
-1
0
-1
-1
H Perfusion
on collagen
1600s1600s -1
H Perfusion
on
1600s
H collagen
Perfusion
on collagen
20000
20000
100
Morphology
Score20000 E20000Extent ofEShape
E Extent
of20000
Shape Change
20000
Morphology
Score D
Change
Morphology
Score
D
Extent
of Shape
Change
da
al
y
f -C
3
A
D
da
Fu
y
ll3
C
A
U Un D d
% CD62P Positive
nt tr
r e ea ay
3
at te
ed d
dada
H Ha
al l
f -Cf -C y 6y 3
AA
DD
dad
F
F
u
U
u
yay
l
nt
l -lCl 6 3Morphology score
C
re
ADA
at
D
edU
d ad
yay
dnatr e
H
63
U
al
% CD62P Positive
f -C ntr y a3te
e
d
A H at d
Da e a
dal f d d y
F
U ul l - Hal y-C3A ay 6
nt C Un f -C D 3 Morphology score
% CD62P Positive
re A t
U ate DFdruea A D day
nt d F al l -te da 6
H r ea Hdaul l -Cy C3dA da y 3
al te aly A D y
f - d f -3 D d 3
H CA Un da CA d ay
al D tr y D ay 6
Fu f -C Fdua eat6 da 3
l l - A D lyl -C3ed y 3
C
F A Hada AD da
U Unutl l - DUnd l f -Cy 6 d ay 6 Morphology score
nt r eC tar A y
r e a AD yea D 3
at teF d 3te d
ed dul a d d ay
H dl
H
H al daal fa-Cyy A6 ay 6
al f f -C CA y -6CA3D d 6
D ay
A D
da 6
D Fu d
F
a
y
Fu ul dal l -Cy
6
l
y
3
ll- C
A
C A 6 D
D
A
d
U
ay
Area Under the Curve
nt D Ud d a
6
r e nay y
t
at r e6 3
ed at
da ed
H
al
d
f -C Ha y 6 ay
Un
CD62P
Positive
3
A l f -%
tr e
C
D
A
at F
da D
ed u
d
dlal -C Ful y 6 ay
Ha
l
y
3 Area Under the Curve
A
lf C
U3 nD d AD
CAU
nDt
U
d
rde tnr et aay
ESC ( %)
Fu
r t 6 ay
aayt
3
e3d eaetd
lld
C
Area Under the Curve
AHD UHadl a ed ady
n fy
al
3
Un
a
dfa- Htrae-lC
y
Cy A af -tA3 D
tr e
6
3FD CedA d
at
edF
Huadl la- Ddaay 3
Ha U udlal - Fluf -CyA dya3y
l f - ntr yCU6A lCl -AC3D
ESC ( %)
Dd 6
CA ea nDt
UD te F rde ADdaay
nt d ul aat
3
y
d
Fu H rdea d l -Cyed ay3
l l - a ayte6H ay A3D d
C l f - Uda 3 day 6
AD C ntdl f ay 6
H A r eaC
adl D ay A
3
Fu fa-Cy Fda te6dD d
6
l l - AHDul ly 3 daay
C a Fu AD ldfa-CCA y 66
U
U ntl l - d yA6DD d
nt r eC a
ESC ( %)
d ay
r e a AFDu y
at te dl l - 3 ay 66
d
ed aCA
d
H Hal da ayy 6D d
al f f -C CA y 6 3 ay
6
A D
D d
FuFul da ay
l l - l -C y 6 3
C A
U AD D d
nt
Area
Under
theUnder
Curvethe Curve
y
r e dUanyaArea
Un
at tr 6 3
tr
ed ea
ea
te H
da ted
da
y
dal f
d
Ha
y-C Hal 6 ay
lf 3A f -C
3
C
D
AD
Area Under the Curve
Fu U da A D
dlal n F y
Fu
d
t
y
6
a
C3 ruela
lly
AD l -tC
CA
3
edA
D
d
Area Under the Curve
Un
Dda
a
da UHU
tr
y natnrl ft- y 6 dya 3
ea
3
e
y
r
C
aetaA
te
3
etdeD
d
da HF
ddda
Ha
ua
adyy
y H
lf ay33
6 llafl --lC
C
C
f -ACA
6
AD
D
D
A Ddd
da UFnt
Fu
a
adyy
y uFrlel
llay33
6 u-aCt
CA
l l -Aed
6
C
D
D
d
A da
da UHna
D
y
a
t
y rl ef -C
y
6
atA da63
y
edD
ddaa 6
HFua
yy
l fl66
CA
AD
ddaa
Fu
yy 6
ll6
CA
D
da
y
6
E
5
0
250
150
0
5
5
10
5
0
40
D
60
60
350
350
40
300
300
5000
0
400
20
250
20
20
250
250
0
200
0
2000
200
150
150
150
at
ed
20
20
15000 15000
CD62P
20
15
15
20000
10000
80
15
10000 10000
*
5000
10
10
60
10
5000
15000
5000
5000
5000
40
5
0
5
0
10000
0
0
0
20
0
0
5000
F
15000
100
10000
5
0
15000
G
0
15000 15000
Perfusion
on collagen 800s
10000 10000
0
U
H
nt
re
*
400
80
40
300
*
80
-1
da
al
y
f -C
3
A
D
da
Fu
y
ll3
C
AD
U
da
nt
y
re
3
at
ed
da
H
al
y
f -C
6
A
D
da
Fu
y
ll6
C
AD
da
y
6
H
at
ed
20
Platelet
components suspended
15
in 100%
plasma treated with
10
INTERCEPT
using low dose/low
5
volume configuration prototypes
0
(Half or Full-CAD) displayed
expected platelet loss, minimal
volume loss and acceptable in
vitro characteristics over 6 days
of storage when compared to
H Perfusion platelet
on collagen 1600s
untreated
components.
20000
These early results support the
15000
development of a new INTERCEPT
10000
low dose/low volume configuration
5000
kit, developed for treating smaller
0
platelet doses stored in plasma.
da
al
y
f -C
3
A
D
da
Fu
y
ll3
C
AD
U
da
nt
y
re
3
at
ed
d
H
ay
al
f -C
6
A
D
da
Fu
y
ll6
C
AD
da
y
6
5
10
350
5
300
10
H
10
4
10
-1
da
Ha
y
lf 3
C
AD
da
Fu
y
ll3
CA
D
Un
da
tr
y
ea
3
te
d
d
Ha
ay
lf 6
C
AD
da
Fu
y
ll6
CA
D
da
y
6
6
400
10
E Extent of Shape Change
Conclusions
at
ed
200
6
15
Morphology
Score
at
ed
200
250
ATPD
ESC ( %)
6
200
8
15
15
nt
re
250
300
10
2
7.0
0
15
8
250
8
ATP
C
U
15
15
7.54
20
C
300
300
CATP
Area Under the Curve
20
Glucose
350
tr
e
20
350
nt
re
20
350
Un
5
B
Glucose
B
U
20
20
nt e d a
r
y
Haeat da 3
l f - ed y 3
Ha CA da
D y
l
Fuf -C da 6
ll- A D y 3
Fu CA da
pH (22C)
UUnn l l -UC D d y 6
ttrr ee nAtr e ay
aat D a 3 Total ATP nmole/10^8 platelets
teed dtae
d yd
HHaa Ha ddaay 6da
ll ff --C l f - y 36 y 3
CAA CA
DD D
FFuu Fu ddaay da
ll ll --C l l -C y 36 y 3
CAA A
Un U DD d D d
tr e ntr e daayy ay
at at 36 3
ed ed
Ha Ha da da
lf - lf - y 6 y 6
CA CA
D D
F F d d
U ul l ul l - a y a y
nt -C C 6 6
r e A AD
Glucose mmole/L
at D
ed d a d a
y
da y 6 6
H
al
y
f -C
3
A
D
da
F
y
U ul l 3
nt C
r e AD
Glucose mmole/L
U ate d
nt
a
d
H r ea day 3
al te
f- d y 3
H CA da
al
D y
Fuf -C da 6
A
ll- D y 3
C d
A a
F
UUnutl l - D d y 6
ntr eC
Glucose mmole/L
r ea AD ay
atte d 3
edd a
dday
HHal
ay 6
al f f -CCA y 36
AD
Dd
daay
FFuul
ll l -C
y3
-CA
6
ADD
U
dda Un
nt
Morphology score
re
ayy 3 tr e
at
at
6
ed
ed
da
H
da
al
y Ha
y
f -C
6
lf 3
CA
A
D
D
da
Fu
da
y Fu
lly
ll6
C
3
C
AD
U A
U
n
Morphology score
nt
d a U tr e D d
re
nt a t a
y Total
ATP
nmole/10^8
platelets
at
y
r
e
6
e
d 3
ed
H ate da
d
U
H
ay nt al f - d d y 3
al
Morphology score
f -C
3 Hraela CA ay
f -tCe D 6
A
D
F d d
da Ha ul l A Dda ay
Fu
-C dy 3
l
y
U ll3 Fuf -lCA ADa3y
nt C
U l -C D d 6
r e AD
n
platelets
U ate d Total
d anmole/10^8
y
Fu tr e AATP
nt
a
atD ady 3
d
l
y
r
l
3
e
e
d
C
H a
al te ay 3 H AD d ady
f- d
3Un a d ay6
H CA da
tr e l f -C ay 6
al
D
y
at A D 3
f
Fu -C da 6
e
l l - A D y 3 H Fu d d da
C d
l
al l -C ay y 6
A a
F
f -C A 6
UUnutl l - D d y 6
A D
ntr eC
D d
r ea AD ay Total
platelets
da anmole/10^8
F
y
atte d 3 ul ATP
y 6
edd a
l -C
6
d
AD
daayy 6
HHal
al f da
f -CCA y 36
y
AD
6
D
dda
FFu
ayy
ull l 36
l -CCA
D
A
U
D
dda
Untr e
ayy 3 Un
nt a
r e te
6 tr e Morphology score
ESC ( %)
at d
at
e
H
ed
d da
a
y
d
H lf
da
al -C ay 6 Ha
f -CA
y
3
l
f
3
-C
AD d
D a
A
Fu
U
D
da y
Ful l
n
l l --CA y 36 Fu tr e da
ESC ( %)
C D
l l - at y 3
AD
C ed
d
U
d aay UUn H AD da
nt
y 6 tnr al f d y 3
re
ESC ( %)
3 tera -C ay
at
etae A
ed
3
D
d
t
da H F edd d
H
al
y Hal ul l ady ay
f -C
6 af -C -C a 3 3
l f -A A y 6
A
D
CD D
da F Un A Dda d a
Fu
y ul l tr e dy y
ll6 Fu-C at a 3 3
C
AD
l l -AD e d y 6
d a Un Ha CA d a da
tr l D y y
y
6 eatf -CA d a 3 6
ed D y
H Fu da da6
al l
f -C l -C y 6 y 6
A A
D D
da d a
Fu
y y
ll6 6
C
AD
da
y
6
8.0
6
B
Glucose
Total ATP nmole/10^8 platelets
U
at
ed
nt
re
U
da
Ha
y
lf 3
CA
D
da
Fu
y
ll3
CA
D
Un
da
tr e
y
Uan
pH (22C)
3
tet
rde
a
dtae
Ha
yd
l f -H
6da
CAa
y
lDf Un
pH (22C)
3
C
dAa D
Ftrue
y d
lal -tFe
6a
CudAl
y
lDd-aC
Ha
3
y
A
lf d a3D
C
UAn
y da
6 y
tDr e
daa
Fu
3
tye
lld3
CHA
da
aDl
y
Un
fd-C
6
aAy
tr e
D3
at
da
Feud
y
l ld-a
Ha
6
C
yA
lf 6D
CA
da
D
y
da
Fu
6
y
ll6
CA
D
da
y
U
6
nt
re
Glucose mmole/L
at
ed
da
H
al
Un
ypH (22C)
f
3
tr e
CA
at
D
ed
da
Fu
da
Ha
y
lly
3
lf UCA
3
CA
nt D
re d
U
Glucose mmole/L
D
nt
at a
da
Fu
re
edy
y
ll3d
at
3
H
CA
ay
U
aeld
n
D
f
3 Glucose mmole/L
d
-Ca
Un
Htr e
da
Ay
al a
tr e
y
D
f
t
6
-CFed
3
at
da
ed
uAlDda
H
y
l
a
y
3
da
Ha
Fluf -C CdAa3
y
lf D
y
l
A
6
U
l -Cn D
6d a
CA
tAr eda
y
Fu
D
D
3
y
a
l
da
Fu
l -C
dtea 3
y
d
llA
y
6
d
CA
HD
U
al d 6ay
nt
D
f -Cay
6
re
da
A3
at
y
D
e
6
da
Fud d
H
y
l a
al
6
f -C l -CyA6
D
A
D
da
da
Fu
y
y
6
ll6
C
AD
da
y
6
at
ed
pH (22C)
tr e
Un
400
400
Figure 2, A - H : Platelet Component In Vitro Characteristics Pre- And PostLow Dose/Low
400Volume INTERCEPT Process
0
Not approved for sale in the USA
Presented at the American
Association
of
Blood
Banks
Annual
Meeting
(AABB)
G
H
F
G
H
G Perfusion on collagen 800s
CD62P
th
th
H Perfusion on collagen 1600s
Philadelphia,
Pennsylvania
•
October
25
28
,
2014
*
15000
20000
15000
-1
-1
1600s
Perfusion on collagen 1600sPerfusion on collagen
-1
20000
15000
20000
e Curve
80
10000
20000
e Curve
*
20000
e Curve
100
15000
-1
Perfusion on collagen 800s Perfusion on-1collagen 800s
CD62P
Curve
F
20000
e Curve
CD62P
-1
Curve
H Perfusion on collagen 1600s
er the Curve
ositive
0
Amotosalen
2.25mM or
1.2 mM
20
8
8.0 and 6
GlucoseB
B
ATP
C
pH day3
A
Glucose
ATP
C
pH day3 and 6
A
20 exposure was observed
8
Table
1:
Platelet
Component
In
Vitro
Characteristics
Pre- And Post- Low Dose/Low Volume INTERCEPT Process
Following pathogen inactivation,
acceptable pH,
efficient
increased
CD62P
for
the
FC
8.0
20
8
8.0
15
6
S-59 removal and platelet/volume losses
configuration when compared to controls. The impact
7.5consistent with
Day 0
Day 1
15
6
15
6
those observed for our
were of this increase on platelet
viability
andand
hemostatic
7.5 current approved process
10
4 Glucose
B
ATP
C
pH day3
6
A
7.5
UnUnmeasured for the HC and FC configurations.
Comparable functions has yet10to be8.0
established. 10
Finally, in a platelet
204
Half-CAD
Full-CAD 8
Half-CAD
Full-CAD
7.0
4
treated
treated
5
2
metabolic indicators7.0(ATP and glucose levels) were
perfusion assay on collagen, comparable thrombus
7.0
count
152
5
906.6 ±237.3
837.3 ±24
838.6 ±14 6
888.9 ±18.1
763.9 ±27.7*** 772.2 ±13.9***
5
3
measured at day 3 and 6 for both configurations when
formations were detected
for the untreated,
HC and
7.5
/µL)
(x
10
6.5
0
0
compared to untreated
FC configurations0 at day 3 and 6 when using 800s-1
100
6.5 platelet components.6.5At day 3
pH (22)
7.404 ±0.04
7.398 ±0.02
7.390 ±0.034
7.492 ±0.029
7.464 ±002
7.438 ±0.01
0
0
and 6, HC and FC platelet components displayed similar and 1600s-1 shear rates.
7.0 The latter data set suggests
Volume (mL) 5
221.4 ±8.4
224.2 ±2.7
208.9 ±3.4 2
221.4 ±8.3
221.2 ±5.3
215.5 ±13.3
Extent of Shape Change and morphology scores when
that platelet components suspended in 100% plasma
compared to controls. These 2 in vitro assays are known treated using HC and 6.5
FC prototypes exhibit adequate
Dose (1011)
2.0 ±0.03
1.96 ±0.05
1.96 ±0.05 0
1.97 ±0.04
1.69 ±0.07****
1.67 ±0.1****
0
to correlate with platelet survival and recovery in vivo,
hemostatic function when compared to controls, at least
S-59 (µM)
n/a
0.8 ±0.2
0.2 ±0.0
thus suggesting adequate platelet viability for platelets ex vivo.
CAD: Compound adsorption device. ***: p<0.001. ****: p<0.0001. n/a: not applicable
processed with the 2 proposed prototypes. At day 3,
Morphology Score
D
E Extent of Shape Change
MorphologyDScoreMorphology Score
D
E Extent of Shape
Change
E Extent
of Shape Change
ATP
C
pH day3
and 6 pH day3 and 6
A
A
8
H day3 and 6
0
Integrated Container Set
**In Development
nt
r e Glucose mmole/L
Total ATP nmole/10^8 platelets
at
ed
da
H
d
H
ay
al
y
al
f -C
3
f -C
3
A
A
D
U
D
da
Fu
Fu ntr e da
y
lll l - at y 3 Total ATP nmole/10^8 platelets
3
C
e
C
AD
A dd
U
d a UUn Ha D
a
nt
tnr e l f - d a y 3
y
re
trae
CAyTotal ATP nmole/10^8 platelets
3
at
tae
ed
D3
td
da
Fuedda da
H
H
a
y
al
l dy
y
f -C
3
6 Hlafl-C l -Cay3
f -CA D AD6
A
D
UAd
Fu nt D a d a
da
Fu
y
r
y
llFlul -C eadtay 3
3
6
C
e
y
A
l
AD
U l -HC D d d 6da
A a
nt
da
y
y
r e al D
y
at f -CdAa 3 6
6
ed
yD
6d
d
H
al Ful ay ay
f -C l -C 6
6
A
D AD
da d a
Fu
y
y
ll6
6
C
AD
da
y
6
Results
Figure 1: Proposed Low Dose/Low Volume Configurations**
20000
15000
-1