Efficacy and Safety Metrics in Plasma-Based Products Philip C. Spinella, MD, FCCM Associate Professor of Pediatrics Washington University in St Louis THOR 2014 RDCR Symposium Solstrand Hotel 10 June 2014 1
Efficacy and Safety Metrics in Plasma-Based Products
Philip C. Spinella, MD, FCCM Associate Professor of Pediatrics Washington University in St Louis
THOR 2014 RDCR Symposium Solstrand Hotel
10 June 2014 1
Disclosures
• Consultant – US Army Blood Research Program – Norwegian Navy Blood Research Program – TerumoBCT, Entegrion, Octapharma
• Research Support – Haemonetics, Diapharma
• Financial support: – DoD/USAMRMC/NHLBI, 3 U01 HL0772268-09S1 – DoD/USAMRAA, W81XWH-10-1-0023 – DoD/USAMRAA, W23RYX0216N601-N602 – NIH/NHLBI, 5R01HL095470-02
2
Objectives
• Summarize processing differences between plasma-based products
• Present data on quality measure differences between plasma-based products
3
Plasma Collection Process*
Centrifuge 5000 g, 5 min Centrifuge 1200 g, 2.5 min
FP24 FFP
Platelet-rich plasma
Thaw slowly at 4°C, centrifuge at 4°C
Freeze at −18°C within 24 h
Freeze at −18°C within 8 h
Centrifuge
Thaw at 37°C
Cryoprecipitate
Platelet-poor plasma
Thawed plasma
Platelet concentrate
Red blood cells
Whole blood
*Plasmapheresis also provides FFP and platelets as stand-alone products 4
Available Plasma-Based Products in US
FFP1,2 FP241,2
Prepara+on • From whole blood or apheresis • Frozen at ≤-‐18° C within 8 h
• From whole blood or apheresis • Stored at 1°-‐6° C < 8 h from collec+on • Frozen at ≤-‐18° C within 24 h
Characteris+cs • Contains plasma proteins including all coagula+on factors
• Normal levels of FV and FVIII
• Reduced levels of FVIII and protein C • Levels of FV and other labile proteins variable vs FFP
• Differences are of UNKNOWN clinical relevance
1. Karam O et al. Transfusion. 2013 Aug 27. [Epub ahead of print] 2. FDA Guidance for Industry: An Acceptable Circular of Informa+on for the Use of Human Blood and Blood Components. www.fda.gov/biologicsbloodvaccines/ guidancecomplianceregulatoryinforma+on/guidances/blood/ucm364565.htm. Accessed 9/1/13.
5
Available Plasma-Based Products in US
Thawed Plasma*1-‐3 Liquid Plasma1-‐3 Solvent Detergent Plasma Prepara+on • FFP, FP24 derived
• Stored at 1°-‐6° C for 5 d in the US
• Derived from whole blood • Never frozen • Stored at 1°-‐6° C up to 26 d (if CPD whole blood)
• Pooled plasma deriva+ve • Stored frozen • Used within hours of thaw
Characteris+cs • Coagula+on factors and endothelial repair ability reduce over +me
• Unknown clinical relevance
• Coagula+on factors reduce over +me
• Unknown clinical relevance
• Pathogen reduced • Filtering and extrac+on phase reduces cells, micropar+cles
• Dilu+on of HLA/HNA Abs • Reduced TRALI reported • Unknown clinical relevance on TRIM
*Effec+ve July 2, 2012, FDA allowed infusion of thawed FFP for up to 24 h aier thawing. Aier 24 h, product should be labeled as “thawed plasma.”
6
Types of Plasma Transfused: 2008 & 2011
0
500
1000
1500
2000
2500
3000
FFP FP24 Ped Plasma
Jumbo Plasma
Cryo Reduced Plasma
Thawed Plasma
2008
2011
FFP, fresh frozen plasma; FP24, plasma frozen within 24 hours of phlebotomy
US Department of Health and Human Services. The 2011 National Blood Collection & Utilization Survey Report. ISBN #: 978-1-56395-843-03. www.hhs.gov/ash/bloodsafety/nbcus/. Accessed 8/14/13.
Units transfused
in th
ousand
s
7
Plasma Product Content
• Pro and anticoagulant factors
• Immunoglobulins, proteins (Albumin)
• Thousands of proteins and lipids
– Many unmeasured, unknown functions
8
FFP Contents
• Residual1 – WBCs: 50-950 cells/microliter
– RBCs: 600-11,400 cells/microliter – Platelets: 2000-38,000 cells/microliter
• Microparticles of these cellular elements – Biologically active
• Donor variability affects cell and MP counts
1. Lambrecht B. et al. Transfusion. 2009:49;1195-1204.
9
Assessment of Plasma Efficacy and Safety
• We oversimplify plasma therapeutic effects - INR reduction is narrow minded assessment
of efficacy • We misdirect our safety concerns - TRIM larger concern than TRALI
• Need to take broader more comprehensive approach to the evaluation of plasma efficacy and safety
10
Optimal Plasma Qualities
• Efficacy – Hemostasis – Endothelial integrity – Vasoregulation effects
• Impact on flow and oxygen delivery support is essential
• Safety – Immune effects
• Reduced TRALI and TRIM
11
Thawed Plasma Effect on Thrombin Generation
*FFP stored and aged at 4°C between days 0, 5, and 10
Pa+ S et al. J Trauma. 2010;69(suppl 1):S55-‐S63. Used by permission. 12
Pa+ S et al. J Trauma. 2010;69(suppl 1):S55-‐S63. 13
14
15
Methods
• 10 FFP and LP single donor units matched by sex and blood group analyzed
• FFP thawed for 5 days at 4C • LP stored for 26 days at 4C • Analyzed samples over time
– TEG, TG assay, platelet counts and microparticles, pro and anti-clotting factors
16
Results
Platelet MP phenotypes included: CD 41, P selectin, Tissue Factor, Annexin V
Platelet Count by flow cytometry with CD 41
17
Results
18
PotenLal mechanisms for sustained thrombin generaLon: platelet microparLcles, cold acLvaLon of factors, reduced protein S
Results
19
Study Conclusions
• LP has similar to better hemostatic effect than TP
• Freeze-thaw cycle decreases clot strength in TP
• PLT microparticle generation in LP may maintain hemostatic effect as coagulation factors decrease
• LP maintains efficacy and improves availability
20
Limitations
• Are data generalizable ? • Platelet count in LP is OMG high
– 80,000 cells/µl – Other reports from FFP is 2,000-38,000 cells/µl – Our FFP data is at most 2,000-8,000 cells/µl
• What is the cost of PLT microparticles? – Immune suppression effects?
21
Efficacy and Safety Metrics in Plasma-Based Products
In vitro study with 10 units each of – FFP derived thawed plasma
• Type AB – Liquid plasma (LP)
• Type AB – Solvent Detergent (SD) Plasma
• Type A (one donor pool) – Resusix –Spray Dried SD Plasma
• Type AB (one donor pool)
22
Hypothesis
• Cellular elements and microparticles are reduced in SD Plasma/Resusix compared to FFP/LP
• Platelet amount in LP lower than previously reported
• Thrombin generation would be improved in SD Plasma/Resusix compared to FFP/LP
• Vasoregulatory properties would be improved in SD Plasma/Resusix compared to FFP/LP
– Due to removal of RBC derived microparticles
23
Methods
• Blood Centers of Pacific supplied the FFP and LP
• Entegrion supplied SD Plasma and Resusix
• FFP, SD plasma
– Stored at 4C post thaw
• LP maintained at 4C
• Resusix reconstituted then stored at 4C
24
Methods
• Same plasma units used for all four groups of experiments • All lab methods performed at Wash U except for thrombin
generation assays done at Entegrion • Funded by US Office of Naval Research
– Via Entegrion
25
• Cell counts for RBC, WBC, and platelets – Sysmex XN-3000 device – Flow cytometry – Hemocytometer
Cell Counts in Plasma - Methods
26
Sysmex XN-3000 Automated Cell Counting Device
• Light scatter & fluorescence
• Body Fluid Mode – Larger volume to account
for rare events
• RBC • WBC • PLT
27
Platelet Counts using Sysmex Platelets
Resusix SD FFP LP
0
1000
2000
500
1500
4000
4500
Plasma Product
PLT
(cel
ls /
uL)
Median=200 Median= 0 Median=800 Median=1900
*significant compared to Resusix p<0.05 *
n=10 n=6 n=10 n=3
28
RBC Counts using Sysmex RBC
Resusix SD FFP LP
0
100
200
20
40
60
80
120
140
160
180
380
400
420
Plasma Product
RB
C (c
ells
/ uL
)
Median=0 Median= 0 Median=100 Median=100
n=10 n=6 n=10 n=3
p<0.05 * *significant compared to Resusix
29
WBC Counts using Sysmex WBC
Resusix SD FFP LP
0
20
5
10
15
25
30
35
80
75
85
Plasma Product
WBC
(cel
ls /
uL)
n=10 n=6 n=10 n=3
30
Median = 5.0 Median = 0.5 Median = 0.5 Median = 1.0
• Becton Dickinson Accuri C6 Flow Cytometer
• To identify cells, following monoclonal antibodies:
– Platelets (CD41a PerCP Cy5.5),
– RBC (CD235a APC)
– Leukocytes (CD45 PE Cy7)
Flow Cytometry Methods
31
Platelet Counts using FC Platelets
Resusix SD FFP LP
0
2000
4000
6000
8000
10000
Plasma Product
Pla
tele
ts (c
ells
/uL)
Median=294 Median= 738 Median=959 Median=4867
*significant compared to Resusix
p<0.001 *
p<0.05 *
n=10 n=6 n=10 n=3
32
RBC counts using FC RBC
Resusix SD FFP LP
0
250
500
750
1000
Plasma Product
RBC
(cel
ls /
uL)
Median=187 Median= 169 Median=234 Median=222
n=10 n=6 n=10 n=3
33
WBC Counts using FC WBC
Resusix SD FFP LP
0
25
50
75
100
Plasma Product
WBC
(cel
ls /
uL)
Median=0 Median= 6 Median=0 Median=3
n=10 n=6 n=10 n=3
34
• Microparticle phenotypes include
– Platelets
– RBC
– Leukocyte,
• Monocyte, neutrophils, T cells
– Endothelial cells
Microparticle Flow Cytometry Methods
35
Flow Cytometry: Methods
• The Small Bead Calibration Kit (Bangs Laboratories, Fisher, IN), measuring 0.2, 0.5 and 0.8 µm of beads, is used to establish appropriate instrument settings for the analyses.
– This beads acquisition allows the setting of cytometer to study MPs within constant region (0.2 µm to 1 µm).
• BD TruCOUNT tubes are used to quantify the absolute cell counts of the microparticles within different plasma products.
36
• Data from 50,000 events acquired at slow rate and analyzed, using an FSC-H threshold of 1000 and an SSC threshold of 4000 for microparticles determination.
• Absolute MP counts were determined by the following formula:
Microparticle Flow Methods
# of events in region containing cell # of events in absolute count bead region
# beads per test* Test volume X
37
Resusix SD FFP LP n=10 n=6 n=10 n=3
CD41a+PS+ (Platelet-derived MP)
D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 28
0
250
500
750
1000
1250
1500
1750
2000
Mic
rop
artic
les/
ul
Median (0.2) (0.4) (0.3) (0.9) (0.9) (140.0) (107.5) (69.3) (935.7) (327.7) (208.0)
(p<0.01)
*
(p<0.01)
* (p<0.002)
#
(p<0.01)
#
PLASMA PRODUCT
* Comparing freshest product to Resusix D 0 # Comparing oldest product to Resusix D 1
38
CD154+PS+ (CD40SL-MP)
D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 28
0
20
40
60
80
100
Mic
ropa
rtic
les/
ul
Resusix SD FFP LP n=10 n=6 n=10 n=3
Median (0.0) (0.0) (0.3) (0.0) (0.6) (0.0) (0.0) (1.1) (0.0) (6.7) (2.0)
PLASMA PRODUCT 39
Platelet Derived – Immune effects 1
1 Horstman, L.L. Keio J Med, 2004. 53(4): p. 210-‐30.
Resusix SD FFP LP n=10 n=6 n=10 n=3
CD235a +PS+ (RBC-derived MP)
D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 28
0
10
20
30
40
50
60
70
80
90
100
Mic
ropa
rtic
les/
ul
Median (0.0) (0.0) (0.3) (0.5) (0.0) (17.9) (43.9) (51.1) (9.0) (35.0) (36.1)
(p<0.02) *
(p<0.001)
#
(p<0.01)
*
(p<0.01)
#
PLASMA PRODUCT 40
Prothrombo+c, impair vasoregula+on and immune modula+on 1-‐5
* Comparing freshest product to Resusix D 0 # Comparing oldest product to Resusix D 1
Spinella, P.C.,Transfusion, 2011. 51(4): p. 894-‐900. Tissot, J.D. Curr Opin Hematol, 2010. 17(6): p. 571-‐7. Sadallah, S. J Leukoc Biol, 2008. 84(5): p. 1316-‐25. Xiong, Z. Transfusion, 2011. 51(3): p. 610-‐21. Belizaire, R.M., J Am Coll Surg, 2012. 214(4): p. 648-‐55.
CD47+PS+
Resusix D 0 Resusix D 1 SD D 0 SD D 3 SD D 5 FFP Day 0 FFP D 3 FFP D 5 LP D 3 LP D 14 LP D 28
0
25
50
75
100
125
150
Plasma Product/ Time Course
Mic
rop
arti
cles
/ ul
p<0.05 *
p<0.05 #
41
(RBC derived)
Immune suppressive – Increased monocyte inhibi+on factor 1 Supports tumor cell growth 1
1. Oldenborg, P.A., CD47: A Cell Surface Glycoprotein Which Regulates MulAple FuncAons of HematopoieAc Cells in Health and Disease. ISRN Hematol, 2013. 2013: p. 614619.
* Comparing freshest product to Resusix D 0 # Comparing oldest product to Resusix D 1
Resusix SD FFP LP n=10 n=6 n=10 n=3
CD45+PS+(Leukocyte-derived MP)
D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 28
0
250
500
750
1000
Mic
rop
arti
cles
/ ul
Median (0.6) (0.8) (0.3) (1.1) (1.1) (5.6) (10.9) (28.9) (11.5) (28.0) (10.7)
(p<0.01)
#
PLASMA PRODUCT
42
* Comparing freshest product to Resusix D 0 # Comparing oldest product to Resusix D 1
Proinflammatory, prothrombo+c, endothelial dysfunc+on 1-‐3
1. Gasser, O Blood, 2004. 104(8): p. 2543-‐8. 2. Mesri, M.. J Biol Chem, 1999. 274(33): p. 23111-‐8. 3. Angelillo-‐Scherrer, A.,. Circ Res, 2012. 110(2): p. 356-‐69.
CD14+PS+ (Monocyte-derived MP)
D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 28
0
20
40
60
80
100
Mic
ropa
rtic
les/
ul
Resusix SD FFP LP n=10 n=6 n=10 n=3
Median (0.0) (0.5) (0.2) (0.9) (1.0) (0.0) (0.8) (2.0) (1.5) (0.0) (1.8)
PLASMA PRODUCT 43
Proinflammatory, promote fibrinolysis 1,2
1. Wang, J.G. Blood, 2011. 118(8): p. 2366-‐74. 2. Lacroix, R. Haematologica, 2012. 97(12): p. 1864-‐72.
CD3+PS+(TCell-derived MP)
Resusix D 0Resusix D 1 SD D 0 SD D 3 SD D 5 FFP D 0 FFP D 3 FFP D 5 LP D 3 LP D 14 LP D 28
0
50
100
150
200
Plasma Product/ Time Course
Mic
ropa
rtic
les/
ul
p<0.05 *
44
* Comparing freshest product to Resusix D 0 # Comparing oldest product to Resusix D 1
Impair vasoregula+on 1,2
1. Simak, J. Transfus Med Rev, 2006. 20(1): p. 1-‐26. 2. Mar+n, S. Circula+on, 2004. 109(13): p. 1653-‐9.
CD142+PS+ (Tissue Factor-MP)
Resusix D 0 Resusix D 1 SD D 0 SD D 3 SD D 5 FFP D 0 FFP D 3 FFP D 5 LP D 3 LP D 14 LP D 28
0
10
20
30
40
50
Plasma Product/ Time Course
Mic
ropa
rtic
les/
ul
45
Prothrombo+c and proinflammatory 1,2
1. Horstman, L.L. Keio J Med, 2004. 53(4): p. 210-‐30. 2. Simak, J. Transfus Med Rev, 2006. 20(1): p. 1-‐26.
Resusix SD FFP LP n=10 n=6 n=10 n=3
CD106+PS+(VCAM Endothelial-derived MP)
D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 28
0
10
20
30
40
50
Mic
ropa
rtic
les/
ul
Median (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (1.2) (0.0) (0.0) (3.5)
PLASMA PRODUCT 46
endothelial stress and inflamma+on 1
1 Horstman, L.L. Keio J Med, 2004. 53(4): p. 210-‐30.
CD105+PS+(Endothelial-derived MP)
Resusix D 0 Resusix D 1 SD D 0 SD D 3 SD D 5 FFP D 0 FFP D 3 FFP D 5 LP D 3 LP D 14 LP D 28
0
20
40
60
80
100
Plasma Product/ Time Course
Mic
ropa
rtic
les/
ul
47 1. Simak, J. Transfus Med Rev, 2006. 20(1): p. 1-‐26
Endothelial injury 1
CD144+PS+ (Endothelial-derived MP)
Resusix D 0 Resusix D 1 SD D 0 SD D 3 SD D 5 FFP D 0 FFP D 3 FFP D 5 LP D 3 LP D 14 LP D 28
0
20
40
60
80
100
Plasma Product/ Time Course
Mic
ropa
rtic
les/
ul
p<0.05 *
p<0.05 #
48
* Comparing freshest product to Resusix D 0 # Comparing oldest product to Resusix D 1
Endothelial injury 1
1. Simak, J. Transfus Med Rev, 2006. 20(1): p. 1-‐26
Coagulation parameters: PT, aPTT, INR and Fibrinogen
• Plasma level for PT, aPTT, INR and Fibrinogen was performed on STAGO instrumentation (STA satellite)
• Fibrinogen level was determined by modified Clauss Method.
49
Coagulation parameters: ELISA
• Native plasma of each of plasma products per time point were collected and stored at -80°C.
• Plasma were be thawed at room temperature,
– Plasmin α2-antiplasmin (PAP), Factor V, Factor VIIa, Factor Xa,
– AT-III, Prothrombin Fragments (F1+2), Peptide A (FPA)
– Total Protein S, Free Protein S, Protein C
50
aPTT
D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 2810
20
30
40
50
60
70
80
90
100
110
120
(sec
onds
)
Resusix SD FFP LP n=10 n=6 n=10 n=3
PLASMA PRODUCT
Median (42.0) (41.8) (35.4) (37.4) (38.1) (35.9) (40.9) (40.1) (39.1) (43.6) (44.7)
(p<0.002)
* (p<0.0001)
* (p<0.001)
#
* Comparing freshest product to Resusix D 0 # Comparing oldest product to Resusix D 1
51
INR
D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 280.5
1.0
1.5
2.0
2.5
Ra
tio
Resusix SD FFP LP n=10 n=6 n=10 n=3
PLASMA PRODUCT
Median (1.3) (1.3) (1.0) (1.0) (1.0) (1.1) (1.2) (1.2) (1.1) (1.2) (1.2)
(p<0.001)
*
(p<0.001)
* (p<0.001)
#
(p<0.0002)
#
* Comparing freshest product to Resusix D 0 # Comparing oldest product to Resusix D 1
52
Fibrinogen
D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 28100
200
300
400
(mg/
dL)
Resusix SD FFP LP n=10 n=6 n=10 n=3
PLASMA PRODUCT
Median (207) (193) (304) (292) (291) (254) (275) (274) (284) (268) (254)
(p<0.001)
* (p<0.001)
# (p<0.001)
#
* Comparing freshest product to Resusix D 0 # Comparing oldest product to Resusix D 1
53
ATIII
D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 280.0
0.1
0.2
0.3
0.4
(mg/
ml)
Median (0.181) (0.179) (0.165) (0.156) (0.187) (0.165) (0.154) (0.133) (0.157) (0.185) (0.180)
Resusix SD FFP LP n=10 n=2 n=10 n=3
PLASMA PRODUCT
54
Total Protein S
D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 2850
75
100
125
150
(%)
Resusix SD FFP LP n=10 n=4-‐5 n=10 n=3
PLASMA PRODUCT
Free Protein S
D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 2850
75
100
125
150
(%)
Resusix SD FFP LP n=10 n=4-‐5 n=10 n=3
PLASMA PRODUCT
p<0.05 *
p<0.05 #
* Comparing freshest product to Resusix D 0 # Comparing oldest product to Resusix D 1
Protein C
D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 280.0
2.5
5.0
7.5
(ug/
L)
* Comparing freshest product to Resusix D 0 # Comparing oldest product to Resusix D 1
p<0.05 #
Resusix SD FFP LP n=10 n=4-‐5 n=10 n=3
Median 3.6 3.7 4.5 4.2 4.1 3.0 2.6 2.4 4.1 3.9 3.6
PLASMA PRODUCT
Thrombin Generation Assay - Methods
• Calibrated Automatic Thrombinoscope (CAT) Device
• During the measurement, the program compares fluorescence in sample to fluorescence in calibrator and computes thrombin concentration.
• Each sample was analyzed three times in triplicate and the data were averaged.
• “Low” in sample name means that thrombin generation reaction was triggered by PPP-Low reagent that produces 1pM TF final concentration in a sample.
58
Resusix
0 10 20 30 40 50
0
50
100
150
200
250
Thro
mbi
n (n
M)
Time (min)
Res-1-30min-PFP-Low Res-2-30min-PFP-Low Res-3-30min-PFP-Low Res-4-30min-PFP-Low Res-5-30min-PFP-Low Res6-30min-PFP-Low- 1 Res7-30min-PFP-Low- 13 Res8-30min-PFP-Low- 1 Res9-30min-PFP-Low- 13 Res10-30min-PFP-Low- 1 Res11-30min-PFP-Low- 13 Res12-30min-PFP-Low- 1 Res13-30min-PFP-Low- 13
0 10 20 30 40 50
0
50
100
150
200
250
Thro
mbi
n (n
M)
Time (min)
Res-1-24h-PFP-Low Res-2-24h-PFP-Low Res-3-24h-PFP-Low Res-4-24h-PFP-Low Res-5-24h-PFP-Low Res6-24h-PFP-Low- 4 Res7-24h-PFP-Low- 16 Res8-24h-PFP-Low- 4 Res9-24h-PFP-Low- 16 Res10-24h-PFP-Low- 4 Res11-24h-PFP-Low- 16 Res12-24h-PFP-Low- 4 Res13-24h-PFP-Low- 16
Time 0 Time 1 day
0 5 10 15 20 25 30 35 40 45 50
0
50
100
150
200
250
Thro
mbi
n (n
M)
Time (min)
FFP-1 FFP-2 FFP-3 FFP-4 FFP-5 FFP-6 FFP-7 FFP-8 FFP-9 FFP-10
0 5 10 15 20 25 30 35 40 45 50
0
50
100
150
200
250
Thro
mbi
n (n
M)
Time (min)
FFP-1 FFP-2 FFP-3 FFP-4 FFP-5 FFP-6 FFP-7 FFP-8 FFP-9 FFP-10
0 5 10 15 20 25 30 35 40 45 50
0
50
100
150
200
250
Thro
mbi
n (n
M)
Time (min)
FFP-1 FFP-2 FFP-3 FFP-4 FFP-5 FFP-6 FFP-7 FFP-8 FFP-9 FFP-10
FFP Time 0 Time 3 day Time day 5
59
Donor Variability for FFP Thrombin generation
0 5 10 15 20 25 30 35 40 45
0
20
40
60
80
100
120
140
Thro
mbi
n (n
M)
Time (min)
FFP-1 FFP-2 FFP-3 FFP-4 FFP-5 FFP-6 FFP-7 FFP-8 FFP-9 FFP-10
0 5 10 15 20 25 30 35 40 45
0
20
40
60
80
100
120
140
Thro
mbi
n (n
M)
Time (min)
FFP-1 FFP-2 FFP-3 FFP-4 FFP-5 FFP-6 FFP-7 FFP-8 FFP-9 FFP-10
0 5 10 15 20 25 30 35 40 45
0
20
40
60
80
100
120
140
Thro
mbi
n (n
M)
Time (min)
FFP-1 FFP-2 FFP-3 FFP-4 FFP-5 FFP-6 FFP-7 FFP-8 FFP-9 FFP-10
Time day 0 Time 3 day Time day 5
60
Day-‐0 Resusix vs Day-‐0 FFP ETP day-0 (median & IQR)
ETP
(nM
x m
in)
FFP
Resus
ix800
1000
1200
1400
1600
Lag Time day-0 (median & IQR)
Lag
Tim
e (m
in)
FFP
Resus
ix3.0
3.5
4.0
4.5
5.0
Peak day-0 (median & IQR)
Pea
k (n
M)
FFP
Resus
ix0
50
100
150
200
250
ttPeak day-0 (median & IQR)
ttPea
k (m
in)
FFP
Resus
ix6
7
8
9
10
11
12
P=0.02 P=0.031
P<0.001 P<0.001
61
day-‐0 FFP vs day-‐5 FFP Lag Time FFP (median & IQR)
Lag
Tim
e (m
in)
Day-0
Day-5
3
4
5
6
7
ETP FFP (median & IQR)
ETP
(nM
x m
in)
Day-0
Day-5
0
500
1000
1500
Peak FFP (median & IQR)
Peak
(nM
)
Day-0
Day-5
0
50
100
150
200
ttPeak FFP (median & IQR)
ttPea
k (m
in)
Day-0
Day-5
8
9
10
11
12
13
14
P=0.05 P<0.0001
P<0.0001 P<0.0002
62
Vasoreactivity Methods
• Samples: • Unprocessed plasma • Platelet Free Plasma
– (centrifuged 2500 g 15 minutes RT) • Microparticle Free Plasma
– (centrifuged 20,000 g 30 minutes 4°C)
• Assays • NO trapping or scavenging • Vascular Rings
• Hypothesis – RBC Microparticles increase NO scavenging and
vasoactive
63
NO Trapping Assay
64
Nitric Oxide (NO) Trapping Assay
• Increased NO trapping or scavenging is indicative of reduced ability to support hypoxic vasodilation – BAD
• Optimal for NO scavenging or trapping to be REDUCED.
65
Total NO Scavenged- Baseline
Native Plasma Platelet Free Plasma Microparticle Free Plasma0
2000
4000
6000
8000
10000Resusix (n=10)SD (n = 5)FFP (n = 7-10)LP (n = 3)
Plasma Product
(AUC)
p<0.05 *
66
Total NO Scavenged in Native Plasma
D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 280
2000
4000
6000
8000
10000
(AUC)
Resusix SD FFP LP n=10 n=5 n=7-‐10 n=3
PLASMA PRODUCT 67
Vascular Ring Assay
• Descending Thoracic aorta dissected from male New Zealand White Rabbit
• Vascular rings cut (sliced like calamari)
• Rings mounted in all glass chamber – on a pair of metal wire hooks
• Chamber filled with Krebs, aerated with 95% oxygen/5% carbon dioxide (for pH), and maintained at 37 degrees)
• Bottom of lower hook fixed in place, top of the upper hook connected by thread to force transducer
68
Force Transducer
Organ Chamber
Rack and Pinion
Vascular Ring Assay
69
Preconditioning - Constriction
• Initial resting tension held at 2 grams (maintained using rack and pinion assembly)
• Once resting tension held steady, we perform preconditioning
• Use alpha 1 adrenergic receptor agonist Phenylepherine to induce vascular ring constriction
• Vessel constriction = increase in gram tension
70
Preconditioning - Relaxation
• To check for an intact endothelium, following the addition of PE to induce a steady constriction response, we inject acetylcholine.
• Induces a relaxation response
• Vessel relaxation results in a decrease force transducer tension = less gram tension
71
Vascular Reactivity in the Freshest Products
NP MPF NP MPF NP MPF NP MPF0
10
20
30
40
50
FFP (n = 7-10)LP (n = 3)
Resusix (n=10)SD (n = 5)
Native Plasma vs Microparticle-Free Plasma
% c
onst
rictio
n (n
orm
aliz
ed to
50n
M
Hb
cons
trict
ion)
p<0.05 *
p<0.05 *
* Significant when compared to Resusix
72
Vascular Reactivity in Native Plasma
D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 280
20
40
60
80
100
Plasma Product
% c
onst
rictio
n (n
orm
aliz
ed to
50n
M
Hb
cons
trict
ion)
Resusix SD FFP LP n=10 n=5 n=7-‐10 n=3
73
Vascular Reactivity in Microparticle-Free Plasma
D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 280
50
100
150
Plasma Product
% c
onst
rictio
n (n
orm
aliz
ed to
50n
M
Hb
cons
trict
ion)
Resusix SD FFP LP n=10 n=5 n=7-‐10 n=3
74
Plasma Quality and Safety Metrics - Conclusions
• Counting cells in plasma products is imprecise – Liquid plasma results from Houston group not generalizable
• SD plasma products vs. FFP/LP – Reduced cellular elements and donor variability – Reduced microparticles from all sources
• May reduce immune modulation or TRIM associated with FFP – Increased thrombin generation
• Plasma is directly vasoactive – Not related to platelets or Microparticles
• Storage time effects quality and safety measures
• Clinical relevance of efficacy and safety measures are unknown, needs exploration
75
Next steps
• Ex vivo analysis of immune function effects secondary to microparticle differences between products
• Mechanistic studies in trials that compare SD treated plasma to thawed FFP/FP24 products
– Immune
– Coagulation
– Endothelial
76
Acknowledgements
• Washington University in St. Louis – Allan Doctor Lab
• Allan Doctor • Stephen Rogers
– Spinella Lab • Ayu Frazier • Dan Martin • Ryan McDonough
– Coagulation Lab • Dennis Dietzen
• Entegrion • Oleg Gorkun
77
78
• FDA approved in early 2013 • Indications
– Replacement of multiple coagulation factors in patients with acquired deficiencies
• Due to liver disease • Undergoing cardiac surgery or liver transplant
– Plasma exchange in patients with thrombotic thrombocytopenic purpura (TTP)
Solvent Detergent Plasma (Octaplas-LG)
79
• Extensive donor screening (serology and NAT) – Screened for non-enveloped viruses and prions
• HAV, HEV, Parvovirus B19
• Donors retested 6 months after plasmapheresis
• Pooling of of 630 to 1500 single units of FFP
• Filtration at 0.2 microns for cell and debris removal
Solvent Detergent Plasma Manufacturing Process
80
• Solvent/detergent treatment - tri(n-butyl)phosphate (TNBP) and octoxynol – Inactivates all lipid enveloped viruses
• (HIV, HBV, HCV, WNV, EBV, CMV….) – Does not inactivate non-enveloped viruses
• (eg, HAV, HEV, parvovirus B19)
• Lipid extraction – Removes solvent/detergent – Removes bioactive lipids, lipoproteins, microparticles
Solvent Detergent Plasma Manufacturing Process
81
• Reduced viral infection risk • Reduced TRALI, TA-GVHD and POSSIBLY TRIM
- Pooling
• Dilution of anti HLA and HNA antibodies
• Immune neutralization of potential pathogens
- Extraction Phase and Filtering • Removal of microparticles, biologic reactive mediators, WBCs
• Consistent dosing (less batch to batch variation) - Pooling
SD Plasma Processing Advantages1-4
1. Flesland O. Intensive Care Med. 2007;33 Suppl 1:S17-‐S21. 2. Krusius T et al. Vox Sang. 2010;99(suppl 1):P-‐1018. 3. Sachs UJ et al. Transfusion. 2005;45(10):1628-‐1631. 4. Sinnou P et al. Eur J Immunogenet. 2004;31(6):271-‐274.
82
Nitric Oxide (NO) Trapping Assay
• Inject nitric oxide (NO) donor into a vacuum tight reaction cell continuously flushed with Helium - linked to nitric oxide analyzer
• NO donor (Dipropylenetriamine NONOate): – spontaneously dissociates in a pH dependent, first order process
(made up in 10mM NaOH and injected into PBS pH 7.4) at 37°C – half-life of three hours at 37°C, pH 7.4 – liberate 2 moles of NO per mole of parent compound
• Once steady NO signal achieved, plasma samples injected and area under curve measured - representing total NO scavenged
83
0.8 µm
0.5 µm 0.2 µm
Size Gating
84
CD41a+ Annexin V+ CD41a+ Annexin V-‐
Determining cell type specific MP
85
Beads
Sample inside the gated area
86
• Briefly, a total of 10 µl platelet free plasma (PFP) from Resusix plasma, S/D plasma, thawed-FFP, or liquid plasma is incubated with 2 µl of lineage-specific monoclonal antibodies, or with and without 2 µl of Annexin V (in the presence of 2.5mM CaCl2) for 30 min at RT in the dark.
• After incubation, samples will be diluted with stain buffer or Annexin V binding buffer up to a total volume of 300 µl.
Microparticle Flow Methods
87
PT
D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 2810
15
20
25
30
(seconds)
Resusix SD FFP LP n=10 n=6 n=10 n=3
PLASMA PRODUCT
Median (16.2) (16.3) (13.7) (13.6) (13.5) (14.5) (14.8) (14.9) (13.4) (14.7) (14.7)
(p<0.001)
* (p<0.001)
#
(p<0.0002)
*
(p<0.0003)
#
* Comparing freshest product to Resusix D 0 # Comparing oldest product to Resusix D 1
88
Plasmin- anti Plasmin (PAP)
D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 280
200
400
600
800
1000
(ng/
ml)
Resusix SD FFP LP n=10 n=4 n=10 n=3
PLASMA PRODUCT
Median (192) (192) (132) (137) (147) (196) (200) (220) (235) (233) (158)
* Comparing freshest product to Resusix D 0 # Comparing oldest product to Resusix D 1
89
Factor V
D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 2810
20
30
40
50
(ng/
ml)
Resusix SD FFP LP n=10 n=4-‐5 n=10 n=3
PLASMA PRODUCT
(p<0.001) *
(p=0.001) #
Median (18.7) (19.0) (17.4) (19.4) (17.0) (27.5) (28.6) (28.0) (17.5) (17.1) (17.5)
* Comparing freshest product to Resusix D 0 # Comparing oldest product to Resusix D 1
90
Factor VIIa
D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 280
50
100
150
200
(ng/
ml)
Resusix SD FFP LP n=10 n=3-‐4 n=10 n=3
PLASMA PRODUCT
(p<0.06) *
(p=0.02) *
Median (48.4) (48.6) (30.3) (54.3) (59.1) (29.5) (37.5) (39.1) (30.4) (63.6) (61.7)
* Comparing freshest product to Resusix D 0 # Comparing oldest product to Resusix D 1
91
PF1+2
D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 280
100
200
300
400
500(p
mol
/L)
Resusix SD FFP LP n=10 n=3 n=10 n=3
PLASMA PRODUCT
(p=0.001) *
(p=0.001) #
Median (73.2) (71.7) (80.3) (79.1) (75.0) (247.5) (263.1) (264.6) (80.3) (82.3) (84.6)
* Comparing freshest product to Resusix D 0 # Comparing oldest product to Resusix D 1
92
Fibrinopeptide A
D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 280
20
40
60
80
100
(ng/
ml)
Resusix SD FFP LP n=10 n=3-‐4 n=10 n=3
PLASMA PRODUCT
(p=0.002) * (p=0.001)
* (p=0.01) *
(p=0.01) # (p=0.001)
# (p=0.01) #
Median (62.0) (62.8) (45.3) (46.1) (48.7) (42.1) (42.2) (41.8) (17.2) (14.5) (15.3)
* Comparing freshest product to Resusix D 0 # Comparing oldest product to Resusix D 1
93
Factor Xa
D 0 D 1 D 0 D 3 D 5 D 0 D 3 D 5 D 3 D 14 D 280
20
40
60
80
100
(ng/
ml)
Resusix SD FFP LP n=10 n=4-‐5 n=10 n=3
PLASMA PRODUCT
(p<0.01) *
Median (27.6) (28.8) (31.5) (31.4) (31.5) (44.3) (42.2) (44.6) (23.9) (21.8) (21.5)
* Comparing freshest product to Resusix D 0 # Comparing oldest product to Resusix D 1
94
day-‐3 FFP vs day-‐5 FFP Lag Time FFP (median & IQR)
Lag
Tim
e (m
in)
Day-3
Day-5
3
4
5
6
7ETP FFP (median & IQR)
ETP
(nM
x m
in)
Day-3
Day-5
600
700
800
900
1000
1100
1200
Peak FFP (median & IQR)
Peak
(nM
)
Day-3
Day-5
0
50
100
150
ttPeak FFP (median & IQR)
ttPea
k (m
in)
Day-3
Day-5
9
10
11
12
13
14
P=0.79 P=0.06
P=0.12 P=0.06
95
day-‐0 FFP vs day-‐3 FFP Lag Time FFP (median & IQR)
Lag
Tim
e (m
in)
Day-0
Day-3
3.0
3.5
4.0
4.5
5.0
5.5
6.0
ETP FFP (median & IQR)
ETP
(nM
x m
in)
Day-0
Day-3
0
500
1000
1500
Peak FFP (median & IQR)
Peak
(nM
)
Day-0
Day-3
0
50
100
150
200
ttPeak FFP (median & IQR)
ttPea
k (m
in)
Day-0
Day-3
8
9
10
11
12
13
P=0.06 P<0.001
P=0.002 P=0.29
96
Day-‐0 Resusix vs Day-‐1 Resusix Lag Time Resusix (median & IQR)
Lag
Tim
e (m
in)
Day-0
Day-1
3.0
3.5
4.0
4.5
5.0
ETP Resusix (median & IQR)
ETP
(nM
x m
in)
Day-0
Day-1
900
1000
1100
1200
1300
1400
1500
Peak Resusix (median & IQR)
Pea
k (n
M)
Day-0
Day-1
100
150
200
250
ttPeak Resusix (median & IQR)
ttPea
k (m
in)
Day-0
Day-1
6
7
8
9
10
P=0.06 P=0.07
P=0.002 P<0.0001
97
Thrombin generation
Upon endothelial damage, +ssue factor (TF) is exposed to the blood and binds factor VII, which is ac+vated to factor VIIa. The TF:VIIa complex enables subsequent ac+va+on of factor X and prothrombin, aier which small amounts of thrombin ac+vate the factor XI-‐IX feedback loop on the platelet surface. Factor IXa will then ac+vate addi+onal factor X. Simultaneously, the trace amounts of thrombin will then ac+vate factors VIII (cofactor to factor IX) and V (cofactor to factor X), which drama+cally enhances cataly+c ac+vity of factors IX and X. Finally, thrombin (factor IIa) ac+va+on leads to fibrin deposi+on. In parallel, local polyphosphate (polyP) release by ac+vated platelets may addi+onally s+mulate ac+va+on of factor XII, factor V, and FXI and inhibit clot lysis. Adopted from Versteeg et al., Physiol Rev (2013) 93:327-‐358
98
Material and Methods
• Frozen plasma samples were thawed at 37C for 10 minutes and 80µl of plasma was added to each of three sample wells containing tissue factor (TF) and phospholipids (PL) enough to produce final concentrations of 1pM TF and 4µM PL.
• In addition, 80µl of same plasma was added to each of two calibrator wells. The use of calibrator (solution containing fixed amount thrombin/α2-macroglobulin complex; Calibrator, Stago) added to the sample plasma allows for correction of inner-filter effect and substrate consumption.
99